Conference Book

Transcription

Conference Book

Sponsors of the DPhG Annual Meeting 2015
MEDI
Conference Book
I3 – Interactions,
Integrations and
Innovations
Annual Meeting of the German
Pharmaceutical Society 2015 – DPhG
Printed by:
Odenkirchener Druck- und Verlags GmbH
Oppelner Straße 6
41199 Mönchengladbach
Tel. +49 (0) 2166 - 610295-0
Fax +49 (0) 2166 - 610295-10
Internet: www.o-d-v.de
Düsseldorf, Germany, September 2015
ISBN 978-3-9816225-2-2
Annual Meeting of the German
Pharmaceutical Society – DPhG
Conference Book
I3 – Interactions,
Integrations and
Innovations
Düsseldorf, Germany
September 23 – 25, 2015
at Heinrich Heine University
www.2015.dphg.de
Institutional Sponsors
Förderer der DPhG-Jahrestagung 2015
Hinweis (FSA-Kodex Fachkreise)
Daiichi Sankyo Deutschland GmbH: Umfang der Unterstützung: 1.000 EUR
Zweck der Unterstützung: Sponsoring.
TABLE OF CONTENTS
CONFERENCE COMMITTEES ................................................................................................................................... ii
WELCOME ADDRESS ............................................................................................................................................... iii
GENERAL INFORMATION ........................................................................................................................................ iv
LOCATIONS ................................................................................................................................................................. vi
CONFERENCE PROGRAM OVERVIEW.................................................................................................................. 1
1 PLENARY LECTURES............................................................................................................................................ 11
2 SCIENTIFIC LECTURES ....................................................................................................................................... 19
2.1
Antiviral Drugs ......................................................................................................................................... 20
2.2
Blood/Brain Barrier ................................................................................................................................. 26
2.3
Regulation of Beta-Cell Function – Implications for Diabetes ...................................................... 29
2.4
GLISTEN – GPCR Medicinal Chemistry ............................................................................................. 33
2.5
Evidence based Medication Management ......................................................................................... 37
2.6
(Bio)Analytics ........................................................................................................................................... 41
2.7
Allosteric Regulation .............................................................................................................................. 46
2.8
Anti-infective Compounds..................................................................................................................... 50
2.9
Poorly Solubles ........................................................................................................................................ 55
2.10 PPP in Drug Development ..................................................................................................................... 59
2.11 Screening Techniques in Pharmacology & Drug Development .................................................... 63
2.12 Signaling in Cell Death........................................................................................................................... 68
2.13 Anticancer and Epigenetic Drugs ........................................................................................................ 72
2.14 Personalized Medicine – Biomarker and Diagnostics ..................................................................... 77
2.15 Focused Pharmaceutical Research....................................................................................................... 80
2.16 Future Molecular Design ....................................................................................................................... 83
2.17 Medication Safety in Special Patient Groups ................................................................................... 87
2.18 Hot Topics in Pharmaceutical Biology – Young Investigators in the Spotlight ........................ 90
3 SHORT POSTER LECTURES ............................................................................................................................... 97
4 POSTERS .............................................................................................................................................................. 111
4.1
Biopharmaceutics (POS.001-POS.006) ........................................................................................... 112
4.2
Pharmacology (POS.007-POS.014).................................................................................................. 115
4.3
Natural compounds (POS.015-POS.022) ....................................................................................... 119
4.4
Pharmaceutical Technology and drug formulations (POS.023-POS.061) .............................. 122
4.5
Analytics (POS.062-POS.077) ........................................................................................................... 138
4.6
GPCR/Ion channels (POS.078-POS.095)......................................................................................... 144
4.7
Cancer/Epigenetics (POS.096-POS.118)......................................................................................... 151
4.8
Drug design/Medicinal chemistry (POS.119-POS.172)............................................................... 159
4.9
Inflammation (POS.173-POS.184)................................................................................................... 178
4.10 Clinical Pharmacy (POS.185-198) .................................................................................................... 183
4.11 Biotechnology (POS.199-POS.204).................................................................................................. 189
4.12 Other topics (POS.205-POS.214) ..................................................................................................... 192
AUTHOR INDEX ..................................................................................................................................................... 195
DPhG Annual Meeting 2015 Conference Book • i
CONFERENCE COMMITTEES
Scientific committee:
Prof. Dr. Stefan Laufer
Prof. Dr. Andreas Link
Dr. Olaf Queckenberg
Prof. Dr. Christoph Friedrich
Prof. Dr. Peter Gmeiner
Prof. Dr. Jochen Klein
Prof. Dr. Peter Langguth
Prof. Dr. Kristina Friedland
Prof. Dr. Angelika Vollmar
Prof. Dr. Hermann Wätzig
Prof. Dr. Thomas Efferth
Prof. Dr. Ulrike Holzgrabe
Prof. Dr. Ulrich Jaehde
Prof. Dr. Heyo Kroemer
Prof. Dr. Irmgard Merfort
Prof. Dr. Klaus Mohr
Prof. Dr. Peter Ruth
Prof. Dr. Manfred Schubert-Zsilavecz
Prof. Dr. Andrea Sinz
Prof. Dr. Holger Stark
Prof. Dr. Dieter Steinhilber
Prof. Dr. Werner Weitschies
Prof. Dr. Gerhard Winter
Organization committee:
Prof. Dr. Jörg Breitkreuz
Dr. German Erlenkamp
Dr. Barbara Gioffreda
Prof. Dr. Holger Gohlke
Dr. Alexandra Hamacher
Dr. Finn Hansen
Prof. Dr. Matthias Kassack
Prof. Dr. Dr.h.c. Peter Kleinebudde
Prof. Dr. Thomas Kurz
Prof. Dr. Stefanie Läer
Prof. Dr. Claus Passreiter
Dr. Christopher Pfleger
Prof. Dr. Peter Proksch
Prof. Dr. Holger Stark
Dr. Aleksandra Zivkovic
Photo: Heinrich-Heine-Universitaet Duesseldorf Jörg Reich
ii • DPhG Annual Meeting 2015 Conference Book
WELCOME ADDRESS
Dear Colleagues,
As President of the German Pharmaceutical Society (Deutsche
Pharmazeutische Gesellschaft, DPhG) and as congress Chairman of this
meeting it is our pleasure to give you a warm welcome in Düsseldorf
attending our Annual Meeting 2015. This year for a very special
occasion we have a double anniversary. 125 years for pharmaceutical
sciences of the DPhG as well as the founding of the Heinrich Heine
University Düsseldorf 50 years ago can be celebrated together with
this Annual Meeting. Many things have changed in this long time, but
still the different aspects on drugs and drug development in life
sciences are in the focus. The motto of this meeting with the important
“three I`s” – Interactions, Integrations and Innovations (I3) – still meets
the highly interdisciplinary fields in life science for the different
pharmaceutical disciplines. The fantastic diversity in subjects ranges
from anti-infective drugs, to new chemical entities or natural
compounds addressing diabetes, cancer or central diseases. Various
topics such as medicinal chemistry on GPCRs, drug design, analytics,
biochemistry, pharmacology, drug formulation as well as medication
management and personalized medicine will be offered as well as
some discussion on regulatory affairs. In this respect, many scientific
sessions may have an overlay with other pharmaceutical topics
handled in other sessions of the compact and exciting programme demonstrating these exciting
“I3”. Beside the science, we hope that you will find some time before or after the congress to
enjoy the city of Düsseldorf and the Rhine area since we are in the fortunate position of being
able to entertain and surprise our visitors with a wide variety of sights to see.
Many thanks to the scientific and organization committees as well as the scientific chairs for
the strong support and to the Pharmaceutical Society of Japan (JPS) for the long-lasting
partnership. We would also like to thank all participants of the meeting for sharing their recent
findings and for their contributions. This abstract book provides all necessary information on
the programme as well as on the abstracts of the scientific contributions of plenary lectures,
scientific lectures, short poster lectures and posters contributing to fruitful scientific exchange
and discussions. Enjoy this meeting, talk to friends as well as to co-operation partners and make
new ones. Have a good time.
Dieter Steinhilber, President
Holger Stark, Chairman
DPhG Annual Meeting 2015 Conference Book • iii
GENERAL INFORMATION
The Annual DPhG Meeting 2015 takes place at the campus of the Heinrich-Heine University, Düsseldorf
in building 23.01.
LANGUAGE
The Conference language is English, no simultaneous translation will be provided.
INSTRUCTIONS FOR USING CONFERENCE WLAN
If your institution is member of the “eduroam” community, you can use the wireless network
“eduroam”. The configuration of your device should be the same as instructed by your home institution.
Please use your account and the domain of your home institution.
If your institution is not member of the “eduroam” community, you can obtain a guest account and a
password at the Conference office.
CONFFERENCE OFFICE
The Conference office is located at the Conference building 23.01.
Opening hours:
Wednesday, September 23rd, 2015: 10:00 – 18:00; on the left of lecture hall 3A
Thursday, September 24th, 2015: 8:00 – 18:00; Building 23.02. Room U1.24
Friday, September 25th, 2015: 8:30 – 12:00; Building 23.02. Room U1.24
LIABILITY
The Organizers of the Conference cannot be held responsible for any loss, theft, damage or injury to
any person or property during the Conference, whatever the cause may be. The liability of persons and
enterprises providing means of transportations or other services remains unaffected. Each congress
participant and accompanying person takes part in all tours at his/her own risk.
HHU App
For more information about the HHU, please also check the HHU App, freely available for Android and
iOS.
(For Android)
iv • DPhG Annual Meeting 2015 Conference Book
(For iOS)
ABSTRACT AND POSTER NUMBERS
Each abstract has a unique identifier, a letter-number combination. Letters refer to the conference topic
a contribution was assigned to (i.e. plenary lectures are identified by the letter “P”, scientific lectures by
the letters “SL”, and poster presentations by the letters “POS”). Please note that in case of poster
presentations the abstract number is identical with the poster number. Poster abstracts, which are
selected for a short poster lectures, were assigned by an additionally letter-number combination and
are identified by the letters “SPL”.
Please refer to the authors index on page 195 for direct access to specific abstracts.
POSTER SESSIONS
Topics: Natural compounds, Pharmaceutical Technology and drug formulations, Cancer/Epigenetics,
Drug design/Medicinal chemistry, Biotechnology, Biopharmaceutics, Clinical Pharmacy, Inflammation,
GPCR/Ion channels, Pharmacology, Analytics, other topics
Posters with even poster numbers are assigned to poster session I, while odd poster numbers are
assigned to poster session II. Presenting authors are asked to be present at their poster during the poster
sessions.
Poster session I
(even poster numbers)
Poster session II
(odd poster numbers)
Session
Wednesday, September 23rd, 2015,
18:00 – 22:00
Thursday, September 24th, 2015,
10:00 – 15:30
Set-up
before 15:00
before 10:00
Dismantling
after 22:00
after 18:00
CONFERENCE DINNER
Separate registration necessary (special fee). Please refer to the Conference office for registration and
details. The Conference dinner will take place at “Im Goldenen Ring”, Burgplatz 21, 40213 Düsseldorf.
BADGES
Badges will be issued to all registered participants and enable access to all scientific sessions.
DPhG Annual Meeting 2015 Conference Book • v
LOCATIONS
Heinrich Heine University campus:
The Heinrich Heine University (HHU) campus is located in the south of Düsseldorf, approx. 3.5 km from
downtown Düsseldorf and the central station.
Arriving by plane:
The Düsseldorf airport is approx. 7 km in the north of the center of Düsseldorf. The S-Bahn train station
is located bellow terminal C. From here take the S11 (destination “Bergisch Gladbach”) to the Düsseldorf
central station. At Düsseldorf central station you can directly travel to the university either by tram (line
707) or Stadtbahn (line U79), destination “Uni-Ost /Botanischer Garten”).
Arriving by train:
Düsseldorf Central station is connected to all international long-distance routes. From here you can
directly travel to the University either by tram (line 707) or Stadtbahn (line U79), destination “UniOst /Botanischer Garten”).
Arriving by car:
From the East: Coming from the interchange “Hildener Kreuz”, follow the A46 to Düsseldorf. Take the
exit toward “D-Zentrum/D-Universität” and follow the B8. At the first traffic lights turn left into
Universitätsstrasse.
From the West: Coming from the interchange “Neuss-Süd”, follow the A46 to Wuppertal/DüsseldorfSüd. After passing the bridge, crossing the river Rhine, take the exit toward “D-Bilk/D-Zentrum/DHafen/D-Benrath”. Stay on the right lane and follow the signs to “D-Benrath” (Münchner Straße). Take
the exit D-Himmelgeist and follow the signage to the University.
The university has a lot of free parking places (except for the clinics). Please note that all parking
offenders on university premises will be towed away.
Heinrich Heine University
Campus
Building 23.01,
Universitätsstr. 1
(how to get to the HHU)
vi • DPhG Annual Meeting 2015 Conference Book
Haus der Universität:
The Haus der Universität is located in downtown Düsseldorf (northern end of the Königsallee), approx.
3.5 km from the Heinrich Heine University (HHU)
Arriving from the Heinrich Heine University by public transport:
• Stadtbahn (line U79): from the station “Uni-Ost /Botanischer Garten” at the University to the
Heinrich-Heine-Allee (downtown)
Arriving by car:
The Haus der Universität is located in the precinct and cannot be reached directly by car. Please take
some extra time for arriving, due to several construction work in this area. Regular parking slots in this
area are rare and it is advisable to use one of the following car parks on your own costs:
• „Schadow Arkaden“; Martin-Luther-Platz 28; daily 7:00 – 1:00
• „Kö-Bogen“; Königsallee 2: daily 6:00 – 1:00
• „Düsseldorfer Schauspielhaus“; Gustaf-Gründgens-Platz 1; daily 6:00 – 24:00
• „Kaufhof an der Kö“; Königsallee 1 – 9; Mon – Sat 9:00 – 20:00
Haus der Universität
(how to get to the “Haus
der Universität”)
DPhG Annual Meeting 2015 Conference Book • vii
CONFERENCE PROGRAM OVERVIEW
Pre-Meeting Program
Tuesday, September 22nd
Bürgersymposium: Geschichte der Pharmazie in Düsseldorf
Ort: Haus der Universität, Düsseldorf Schadowplatz 14
14:00 –
14:15
Begrüßung durch den Vorsitzenden der FG Geschichte der Pharmazie
Prof. Dr. Christoph Friedrich, Marburg
14:15 –
15:00
Zur Entwicklung des Hochschulfaches Pharmazie an der
Heinrich-Heine-Universität Düsseldorf
Prof. Dr. Horst Weber, HHU Düsseldorf
15:00 –
15:45
Zur Entwicklung des Apothekenwesens in Düsseldorf
Dr. Frederik Vongehr, Mörs
15:45 –
16:15
Kaffeepause
16:15 –
17:00
Vesters Archiv – eine besondere pharmaziehistorische Quelle aus Düsseldorf im
Spannungsfeld privater, städtischer und universitärer Interessen
Prof. Dr. Frank Leimkugel, HHU Düsseldorf
17:00 –
17:45
Zur Geschichte des Düsseldorfer Aqua-Zoos und seines Begründers, Apotheker Theodor
Löbbecke (1821–1901)
Sandra Honigs, Aqua-Zoo – Löbbecke-Museum Düsseldorf
19:00 –
21:00
Treffen Arbeitsgemeinschaft Katastrophenpharmazie
Ort: Haus der Universität, Düsseldorf, Schadowplatz 14
20:00 –
21:00
Notfall- und Katastrophenpharmazie - Ein Beitrag der Apotheker
zum Bevölkerungsschutz
Daniel Neuser, Düsseldorf
DPhG Annual Meeting 2015 Conference Book • 1
Wednesday, September 23rd
Meetings der DPhG-Fachgruppen
9:00 –
11:00
11:30 –
13:00
Sitzung VdPPHI e.V. Hochschullehrersitzung, B. Clement (HS 3A)
Fachgruppe
Pharm./Med.
Chemie
P. Gmeiner
(HS 3A)
Fachgruppe
Pharm.
Biologie
A. Vollmar
(HS 3D)
Fachgruppe
Pharmakologie
J. Klein
(HS 3E)
Fachgruppe
Pharm.
Technologie
P. Langguth
(HS 3B)
Fachgruppe
Klinische
Pharmazie
K. Friedland
(HS 3F)
Fachgruppe
Industriepharmazie
C. Küster
(U1.23)
Main Symposium (Congress language English)
13:00 –
13:30
Opening of the Annual DPhG Meeting 2015 (HS 3A)
I3 – Interactions, Integrations and Innovations
13:30 –
14:15
Plenary lecture, Christa E. Müller, Interactions, Integrations and Innovations in medicinal
chemistry of purinergic signaling (HS 3A)
14:15 –
15:00
Plenary lecture, Jörg Breitkreutz, New concepts and products for individual drug dosing (HS 3A)
P.2
15:00 –
15:30
Coffee break - Poster viewing (even numbers)
P.1
SHORT TALKS
15:30 –
17:00
SL1 (HS 3A)
SL2 (HS 3D)
SL3 (HS 3E)
Antiviral Drugs
Chairs: H. Rübsamen-Schaeff,
H. Zimmermann
Blood/Brain Barrier
Chairs: G. Fricker, F. Helm
Regulation of beta-Cell
Function – Implications for
Diabetes
Chairs: G. Drews, E. Oetjen
15:30
SL.01
Thomas Pietschmann:
Directly acting antivirals against
hepatitis C virus – close to
“perfectovir”
15:30
SL.07
Robin Tremmel:
Liposomal delivery of Trientine
for treatment of Wilson’s
Disease in the brain
15:30
SL.10
Gisela Drews:
Role of bile acids in beta-cell
function
15:45
SL.02
Daniela Paulsen:
How to deal with HBV? - State of
the art and new concepts in HBV
therapy
16:00
SL.03
Christoph Stephan:
Advances and challenges in
diagnostics & therapy for
HIV/AIDS
15:50
SL.11
Ingo Rustenbeck:
Mobility of submembrane
SL.08 insulin granules
16:00
Stefan Liebner:
Molecular regulation of
endothelial barrier properties in
the central nervous system
16:15
SL.04
Helga Rübsamen-Schaeff:
The era after nucleosides: Novel
therapeutic approaches against
herpes viruses
16:15
SL.12
Martina Düfer:
Reactive oxygen species:
Pharmacological target or
physiological prerequisite for
beta-cell function?
continued on the next page
2 • DPhG Annual Meeting 2015 Conference Book
16:30
SL.05
Stephan Ludwig:
New avenues in anti-influenza
therapy
16:45
SL.06
Johan Neyts:
Antivirals, a lot has been
achieved, yet a long way to go
16:30
SL.09
Marise Kolter:
Toxicity of poly(n-butylcayanacrylate) (PBCA) nanoparticles
designed to overcome the blood
brain barrier
17:00 –
18:00
Short poster lectures (HS 3A)
17:00 –
18:00
Freunde der DPhG (building 23.02, room U1.23)
18:00 –
22:00
Poster viewing (even numbers) and Welcome Reception
16:35
SL.13
Elke Oetjen:
Regulation of beta-cell function
and mass by the dual leucine
zipper kinase
Thursday, September 24th
8:30 –
9:15
Plenary lecture, Martin Biel, Ebola and more: Endolysosomal cation channels as novel drug targets
(HS 3A)
P.3
9:15 –
10:00
Plenary lecture, Robin Thurmond, The pre-clinical and clinical development of histamine H4
receptor antagonists (HS 3A)
P.4
10:00 –
10:30
Coffee break - Poster viewing (odd numbers)
SHORT TALKS
10:30 –
12:00
SL4 (HS 3A)
GLISTEN - GPCR
Medicinal Chemistry
Chairs: N. Tschammer,
P. Kolb
SL5 (HS 3D)
SL6 (HS 3E)
Evidence Based Medication
Management
Chairs: G. Hempel, S. Läer
10:30
SL.14 10:30
Francine Acher:
Georg Hempel: Introduction
Selective orthosteric agonists in
10:35
SL.18
class C GPCRs
Thilo Bertsche:
Medication management in
clinical care
11:00
SL.15
Hans Bräuner-Osborne:
Identification and pharmacological characterization of
endogenous and surrogate
ligands for orphan G proteincoupled receptors
10:30
SL.22
Kai Stühler:
Protein mass spectrometry –
Methods and applications in
clinical proteomics
10:50
SL.23
Gerhard Scriba:
Capillary electrophoresis-based
SL.19 stereospecific enzyme assay for
methionine sulfoxide reductase
11:00
Olaf Rose:
Medication management in
ambulatory Care: Preliminary
results of the WestGem-Study
11:10
SL.24
Shigeru Ohta:
New approach for drug
discovery and development:
Prediction of human drug
metabolism using chimeric mice
transplanted with human
SL.20 hepatocytes
11:25
Stefan Derix:
11:30
SL.16 AMTS activities of the
Sylwia Gawron:
Pharmacists-Chamber of North
Structure free optimization of
Rhine
fragments for the β2-adrenoreceptor
11:45
SL.17
Matthias Hillenbrand:
Comprehensive analysis of
heterotrimeric G-protein complex
diversity and their interactions
with GPCRs in solution
(Bio)Analytics
Chairs: J. Heilmann,
M. Lämmerhofer
11:45
SL.21
Judith Hildebrand:
Comparison of three lists of
Potentially Inadequate
Medications in old age (PIMs) in
four German long-term care
facilities with special regard to
Adverse Drug Events (ADEs)
(organized by GLISTEN COST
Action CM1207)
11:30
SL.25
Ryoichi Fujiwara:
Importance of extrahepatic UDPglucuronosyltransferase 1A1 in
bilirubin metabolism
11:45
SL.26
Maria K. Parr:
Renaissance of supercritical fluid
chromatography: Fast and
sensitive analysis of polar drugs
and their metabolites by
hyphenated mass spectrometry
12:00 –
13:30
Break for lunch time - Poster viewing (odd numbers)
SHORT TALKS
13:30 –
15:00
SL7 (HS 3A)
SL8 (HS 3D)
Allosteric Regulation
Chairs: K. Mohr,
M. Bünemann
Anti-Infective Compounds
Chairs: U. Holzgrabe,
P. Proksch
Poorly Solubles
Chairs: P. Kleinebudde,
W. Weitschies
13:30
SL.27
Ursula Storch:
Molecular insights into the
mechanosensivity of histamine
H1 receptors
13:30
SL.31
Tanja Schirmeister:
Vinylsulfone-based inhibitors of
rhodesain as new antitrypanosomal compounds
13:30
SL.36
Susanne Page:
The use of screening tools in the
development of amorphous solid
dispersions
13:55
SL.28
Nuska Tschammer:
14:00
SL.32
Biased signaling and probe
Christian
Klein:
dependence at the chemokine
Discovery of dengue protease
receptor CXCR3
inhibitors with nanomolar
affinity
14:00
SL.37
Heike Bunjes:
Colloidal carrier systems for the
formulation of poorly watersoluble drugs
14:15
SL.33
Alexander Titz:
14:20
SL.29 The lectin LecB as target for
anti-infectives against chronic
Wiebke Seemann:
Pseudomonas aeruginosa
Muscarinic M2 receptor
infections
allosterism: Context-sensitive
signaling
14:30
SL.34
Rainer Kalscheuer:
Chlorflavonin inhibits growth of
Mycobacterium tuberculosis by
targeting branched-chain amino
acid biosynthesis
14:45
SL.30 14:45
SL.35
Susanne Hermans:
Georgios Daletos:
Detection of unexplored
Marine natural products as
allosteric pockets using a
potential sources for new
“dummy” ligand approach
antitubercular agents
15:00 –
15:30
SL9 (HS 3E)
14:30
SL.38
Michael Hacker:
Oligomeric cross-linkers for
hydrogel formulation from ECMderived macromolecules
14:45
SL.39
Dominique Lunter:
Contribution of Confocal Raman
Microscopy (CRM) to the
validation of an ex vivo skin
penetration method
SHORT TALKS
15:30 –
17:00
SL10 (HS 3A)
SL11 (HS 3D)
SL12 (HS 3E)
PPP in Drug Development
Chairs: S. Knapp, F. Bracher
Screening Techniques in
Pharmacology & Drug
Development
Chairs: E. Kostenis,
H. Wätzig
Signaling in Cell Death
Chairs: I. Merfort, T. Efferth
15:30
SL.40
Stefan Knapp:
A PPP for the development of
chemical tool compounds
15:30
SL.44
Hermann Wätzig:
Data quality in drug discovery –
The role of analytical
performance in ligand binding
assays
15:30
SL.49
Min Li-Weber:
Sensitization of the anti-cancer
efficacy of the Bcl-2 family
inhibitor ABT-263 by natural
compounds
15:50
SL.45
Ramona Schrage:
Dynamic mass redistribution to
16:00
SL.41 probe the signaling repertoire of 16:00
SL.50
GPCRs
Anke Müller-Fahrnow:
Beatrice Bachmeier:
Public Private Partnership in
miR181b is included by the
lead discovery: Overview and
16:10
SL.46 chemopreventive polyphenol
case study on binding kinetics
curcumin and inhibits breast
Manuel Grundmann:
cancer metastasis via downLabel-free biosensors help to
regulation of the inflammatory
reveal a new mechanism of
cytokines CXCL1 and 2
GPCRs activation
16:20
SL.42
Conrad Kunick:
Development of a selective
DYRK1A inhibitor in a PPP
framework
16:20
SL.47
Manuel Koch:
Assay technologies addressing
GPCRs in drug discovery
16:30
SL.51
Qiaoli Zhao:
Shikonin and its derivatives
inhibit phosphorylation of the
epidermal growth factor
receptor signaling and
synergistically kill glioblastoma
cells in combination with
SL.43
erlotinib
16:40
Olaf Kelber:
The role of adenosine in colonic
inflammation – A study in rat
colon preparations in vitro
16:45
SL.48
Frank Böckler:
Semi-automatic fluorescence
anisotropy titrations (saFLAT)
enhance screening throughput
and data quality
16:45
SL.52
Bertan Bopp:
A novel autodisplay based
screening assay for the
identification of small molecules
that inhibit the dimerization of
human chaperone Hsp90
SHORT TALKS
17:00 –
18:30
SL13 (HS 3A)
Anticancer and Epigenetic
Drugs
Chairs: M. Kassack, M. Jung
SL14 (HS 3D)
SL15 (HS 3E)
Personalized Medicine –
Biomarker and Diagnostics
Chairs: O. Queckenberg,
T. Dingermann
17:00
SL.53 17:00
SL.58
Daniel Rauh:
Reinhard Ortmann:
Targeted cancer therapies
The challenge of leverage
genetic biomarkers from
research level to routine clinical
IVD testing
17:20
SL.54
Sebastian Wesselborg:
Role of apoptosis signaling in
tumorigenesis and therapy
17:30
SL.59
resistance
Frank Kramer:
The potential of biomarkers to
17:40
SL.55 support decision making in
clinical studies in cardiovascular
Stefan Günther:
A novel CREBBP/p300 inhibitor indications
and its molecular effects in
cancer cells
18:00
SL.56
Christian A. Olsen:
Macrocyclic inhibitors of human
histone deacetylase enzymes
18:20
SL.57
Steffen Lüdeke:
Correlation of conformation
with cytotoxic activity of
Focused Pharmaceutical
Research
Chairs: S. Laufer, D. Steinhilber
17:00
SL.61
Dieter Steinhilber:
Project group translational
medicine and pharmacology,
Fraunhofer IME
17:25
SL.62
Christa E. Müller:
Neuroallianz consortium –
Example of an academicindustrial collaboration
17:50
SL.63
Stefan Laufer:
The Interfaculty Centre for
SL.60 Pharmacogenomics and Pharma
Research (ICEPHA)
18:00
Georg Lautscham:
From “omics”-technologies to
stratified medicine in
autoimmune diseases
18:15
General discussion
α-aminoxy oligopeptides: A
circular dichroism study
17:00 –
18:30
Workshop, T. Hotopp, Tipps und Hinweise für eine erfolgreiche Antragstellung (HS 3B)
19:30
Conference dinner (Im Goldenen Ring, Burgplatz 21, 40213 Düsseldorf)
Friday, September 25th
8:30 –
9:15
Plenary lecture, Dieter Häussinger, Liver research in Düsseldorf: Bridging basic and clinical science
HS 3A)
P.5
9:15 –
10:00
Plenary lecture, Karl Broich, Drug approval and regulatory science – Where to go? (HS 3A) P.6
SHORT TALKS
10:30 –
12:00
SL16 (HS 3A)
SL17 (HS 3D)
SL18 (HS 3E)
Future Molecular Design
Chairs: G. Schneider, H. Gohlke
Medication Safety in Special
Patient Groups
Chairs: K. Friedland, U. Jaehde
Hot Topics in Pharmaceutical
Biology – Young Investigators
in the Spotlight
Chairs: S. Alban, A. Vollmar
10:30
SL.64
Andrew Hopkins:
Automated design of bispecific
small molecule drugs
10:30
SL.68
Stephan Scherneck:
Medication safety of pregnant
and lactating women
10:30
SL.71
Kristian Wende:
Cold atmospheric plasma – A
future therapeutic approach?
Bioanalytics as route to
fundamental understanding
10:45
SL.72
Jandirk Sendker:
About the potential of plant
senescence as a new source for
drug discovery
11:00
SL.65
Tina Ritschel:
KRIPO-Protein binding site
similarities for drug design
11:00
SL.69
Stefanie Läer:
Medication for neonates, infants
and children
11:20
SL.66
Johannes Kirchmair:
Predicting the sites and
products of drug metabolism
11:30
SL.70
Ulrich Jaehde:
Medication safety of elderly
11:40
SL.67 patients in nursing homes
Gisbert Schneider:
Enforcing drug discovery by
computational molecular design
11:00
SL.73
Alexander Kristian Apel:
Regulation of the heterologously
expressed novobiocin gene
cluster by the host strain
Streptomyces coelicolor M512
11:15
SL.74
Sonja Keßler:
mRNA binding proteins in
metaflammation and
hepatocarcinogenesis
11:30
SL.75
Timo Niedermeyer:
Cyanobacteria in natural
product research
11:45
SL.76
Till Schäberle:
Analysis of natural product –
Biosynthesis in the post-genomic
era
12:00 –
13:00
Short lunch break
13:00 –
13:45
Plenary lecture, Piet Herdewijn, From synthetic nucleic acids to artificial genes and genomes: Drugs
for the future? (HS 3A)
P.7
14:00 –
15:00
Closing ceremony (HS 3A)
15:15 –
16:30
DPhG Jahreshauptversammlung (HS 3A)
Post-Meeting Program
Saturday, September 26th
15:00 –
18:30
Tag der Offizinpharmazie in Kooperation mit der Deutschen Gesellschaft für Schmerzmedizin,
zentrale Fortbildung der DPhG Regionalgruppe Nordrhein und FG Allgemeinpharmazie mit LAK NR
Ort: Heinrich-Heine-Universität, Universitätsstr. 1, 40225 Düsseldorf, Hörsaal 3D, Gebäude 23.01
PD. Dr. med. Michael Überall, Nürnberg, Präsident der Deutschen Schmerzliga e.V
Erster Teil der Schmerzschulung
1. Informationen zur „Deutschen Gesellschaft für Schmerzmedizin“ und der „Deutschen
Schmerzliga“
Die Versorgungssituation des Schmerzpatienten in Deutschland
2. Grundlagen Schmerz (1)
Definition (akut vs. chronisch, etc.), Strukturen des nozizeptiven Systems, endogene
Schmerzkontrollmechanismen, Pathophysiologie acuter vs- chronischer Schmerzen
3. Grundlagen Schmerz (2)
Epidemioligie, Klassifikation, Schmerz als Symptopm vs. Schmerz als Krankheit, bio-psycho-soziales
Krankheitsmodell, Begleiterscheinungen, Ko-Morbidität, etc.
4. Differentialdiagnostik
Anamnese/ Diagnostik, Verlauf, Chronofizierungsrisiken, Grundlagen der Prävention und Therapie
akuter/chronischer Schmerzen, Schmerzmessung, Selbstauskunftinstrumente, etc.
5. Ziele der (medikamentösen) Schmerztherapie
Schmerzlinderung, Funktionsverbesserung, Teilhabe an den Aktivitäten des (all)täglichen Lebens,
Lebensqualität, individuelle Behandlungs-ziele, etc.
6. Schmerztherapie – WHO-Stufe 1 (Nichtopioide)
Paracetamol, Ibuprofen, ASS, Diclofenac, Coxibe, Metamizol, etc.; jeweils mit WM, UAW, KI
(Schwerpunkt auf apothekenpflichtige Fertigarzneimittel und Beratungs-/ Verkaufssituationen in der
Apotheke)
ANMELDUNG
Anmeldung zum Tag der Offizinpharmazie in Kooperation mit der Deutschen Gesellschaft für
Schmerzmedizin
Kursart: Vortrag mit TED Prüfung
Teilnehmergebühren 1. Teil:
• Kostenfrei
Bitte melden Sie sich bzw. Ihre Apotheke zum Tag der Offizinapotheke online an:
www.dphg.de/apo15
Teilnehmergebühren 2. Teil:
Zum Erwerb des Zertifikats:
• 190 € (zzgl. MwSt)
Anmeldung für den 2. Kurs über die DGS vor Ort oder über:
www.dgschmerzmedizin.de/dgs_campus.html
Möglichkeit und Kosten der Rezertifizierung:
• Jährlich: 38 € (zzgl. MwSt)
• DPhG-Mitgliedsapotheken: 28 € (zzgl. MwSt)
1 PLENARY LECTURES
PLENARY LECTURES
Interactions, Integrations and Innovations in medicinal chemistry of purinergic signaling
P.1
Müller, C. E.1
1
PharmaCenter Bonn, Pharmaceutical Institute, Pharmaceutical Chemistry I, University of Bonn, Bonn, Germany
Membrane receptors activated by purines are subdivided into three distinct families: (i) nucleotide or P2 receptors,
further divided into G protein-coupled P2Y receptors and ATP-gated ion channel P2X receptors; (ii) adenosine or
P1 receptors (A1, A2A, A2B, A3), and (iii) P0 receptors activated by the nucleobase adenine [1,2]. Purine receptors
are widely distributed in the body. Their important role in signal transduction is increasingly recognized and
appreciated, and their potential as drug targets is explored and exploited with growing success. The physiological
ligands of the three classes of receptors, ATP/ADP, adenosine, and adenine, are metabolically linked, and enzymes
interconverting them, in particular ectonucleotidases, are fine-tuning purinergic signalling. Tool compounds for a
wide range of membrane proteins involved in purinergic signalling have been developed in the past decades.
Recent successful efforts of our group have focused on the development of novel assays [3-5] and structure-based
approaches [6-8] to identify and optimize P2 receptor antagonists and ectonucleotidase inhibitors [9-11]. Potent
and selective tool compounds are crucial for elucidating the (patho) physiological roles of purinergic signalling
[12-14].
References:
1. Fredholm, B. B.: Pharmacol. Rev. 2011, 63, 1-34.
2. Müller, C. E.: Curr. Med. Chem. 2015, 22, 929-941.
3. Freundlieb, M. et al.: Anal. Biochem. 2014, 446, 53-58.
4. Lee, S. Y.; Müller C.E.: Electrophoresis 2014, 35, 855–863.
5. Fiene, A. et al.: Analyst 2015, 140, 140-148.
6. Zebisch, M. et al. J. Struct. Biol. 2014, 185, 336-341.
7. Zhang, K. et al.: Nature 2014, 509, 115-118.
8. Zhang, J. et al.: Nature 2014, 509, 119-122.
9. Chang, L. et al. J. Med. Chem. 2014, 57, 10080-100100.
10. Lee, S. et al: Biochem. Pharmacol. 2015, 93, 171–181.
11. Bhattarai, S. et al. J. Med. Chem. 2015, 58(13), 6248-6263.
12. Gnad, T. et al.: Nature 2014, 516, 395-399.
13. Sassi, Y. et al. J. Clin. Invest. 2014, 124, 5385-5397.
14. Kaster, M. P. et al.: Proc. Natl. Acad. Sci. 2015, 112(25), 7833-7838.
PLENARY LECTURES
New concepts and products for individual drug dosing
P.2
Breitkreutz, J.1
1 Institute
of Pharmaceutics and Biopharmaceutics, Heinrich Heine University Düsseldorf, Universitätsstr. 1, 40225 Düsseldorf, Germany
Recent progress in genetic and metabolic profiling, pharmaceutical research and medical knowledge has led to
improved drug therapies with precise dosing recommendations for medicinal products. Many experts see so called
‘Personalized Medicine’ as the future drug therapy for all patients. However, there are no appropriate drug dosage
forms available which match these requirements, e.g. tablets and capsules are mostly provided with only one or
two different dose strengths. Splitting is often no option due to the resulting uneven pieces with incorrect doses,
loss of drug by dust forming, or destroying functional polymer films. Especially for the paediatric and geriatric
patients many medicinal products are inappropriate when taking the specific requirements of these populations into
account [1].
Recent innovations in pharmaceutical technology and medical device industry show exciting advances and promise
to overcome the present gap of patient-adapted, individualized dosing options. Small-sized tablets (< 3 mm in
diameter), so called mini-tablets, can be designed as orodispersible or dispersible drug formulations with very small
dosing steps [2]. Most recently, it has been demonstrated by our group in a series of three clinical studies that these
mini-tablets can be taken as good or even better than a sweet syrup by all paediatric patients, including pre-term
and term neonates [3]. Orodispersible films are another option for precise and individual dosing. Some
prescriptional drug products with this technology has already entered the market, but more innovations are needed
for individual dosing. Film preparation can be performed under the conditions of community or hospital pharmacies
and show some advantages such as better content uniformity over conventional capsules preparation [4]. Another
strategy are innovative medical device enabling the cutting of film strips according to the prescribed dose. Printing
technologies using two- or three-dimensional printers and drug-loaded inks are currently under investigations in
various labs for enabling individualized doses and personalized medicines [5]. It can be foreseen that printed
medicines will replace many conventional dosage forms in industrial processing, but also compounding in hospital
or community pharmacies. Furthermore, individualized printed medicines open completely new treatment options
in surgery, cancer, dental medicine and many more medical disciplines. Scientific pharmacists should quickly
explore these technologies and take the lead regarding drug development, production, quality control and
regulatory sciences in this fascinating area of technology.
References:
1. Breitkreutz, J.; Boos, J.: Exp. Opin. Drug Deliv. 2007, 4: 37-45.
2. Stoltenberg, I.; Breitkreutz, J.: Eur. J. Pharm. Biopharm. 2011, 78: 462-469.
3. Klingmann, V. et al.: J. Pediatr. 2015, in press.
4. Visser, J. C. et al.: Int. J. Pharm. 2015, 478: 155-163.
5. Preis, M. et al.: Int. J. Pharm. 2015, in press.
PLENARY LECTURES
Ebola and more: Endolysosomal cation channels as novel drug targets
P.3
Biel, M.1
1 Center
for Integrated Protein Science Munich CiPSM at the Department of Pharmacy – Center for Drug Research, Ludwig-MaximiliansUniversität München, Butenandtstr. 5-13, 81377 Munich, Germany
Ion channels are transmembrane proteins that confer the flux of ions across lipid membranes of the cell. In the
past, the major focus of ion channel research has been on channels present in the plasma membrane and several
members of these channels have been exploited as drug targets. More recently, ion channels that are localized in
the membrane of intracellular organells have attracted interest. Importantly, a growing number of these channels
has been linked to the pathologies of human diseases making them interesting candidates for drug development.
In my lecture, I will give an overview on our recent work on so-called two-pore channels (TPC1 and TPC2) [1-3].
These channels are exclusively expressed in endolysosomal organells. To facilitate functional characterization of
TPCs in their native environment (i.e. the lysosomal membrane) we have applied novel electrophysiological
techniques. Moreover, knockout mouse models for TPCs have been generated. We found that TPC2 plays a key
role in endolysosomal trafficking and, thereby, is involved in the life cycle control of many cellular receptors including
EGF-R, LDL-R, and Tf-R. Deletion of TPC2 in mice leads to cholesterol overload and the development of fatty liver
hepatitis [1]. We also found that TPCs are essential for the cellular uptake of Ebola viruses (EBOVs) [3]. Disrupting
TPC function by gene knockout, small interfering RNAs or small-molecule inhibitors halted virus trafficking and
prevented infection with EBOVs. Tetrandrine, the most potent small molecule that was tested, inhibited infection of
human macrophages, the primary targets of EBOVs in vivo, and also showed therapeutic efficacy in mice. Taken
together, TPCs represent a novel class of ion channels with significant relevance for future drug development.
Compounds acting on these channels may be considered in the treatment of diseased states affecting homeostatic
control of cellular receptors and cell metabolites and may be effective for antiviral therapy.
References:
1. Grimm, C. et al.: Nature Commun. 2014, 4: 4699.
2. Ruas, M. et al.: EMBO J 2015, 34: 1743-1758.
3. Sakurai, Y. et al.: Science 2015, 347: 995-998.
PLENARY LECTURES
The pre-clinical and clinical development of histamine H4 receptor antagonists
P.4
Thurmond, R. L.1
1
Janssen Research & Development, LLC 3210 Merryfield Row, San Diego, CA 92107
The histamine H4 receptor is a high affinity receptor for histamine and has become an attractive target for the
development of drugs for the treatment of inflammation, pruritus, allergy and asthma. The H4 receptor mediates
chemotaxis and cytokine release of mast cells, eosinophils, monocytes, dendritic cells and T cells. In addition,
histamine released from mast cells or from other cell types can influence T cell polarization via activation of the H4
receptor. The receptor also mediates T cell activity in vivo and has a proinflammatory effect not only in models of
the innate immune response, but also in models of asthma and contact dermatitis, where it mainly affects T cell
responses. Extensive medicinal chemistry and pharmacology efforts have led to the development of modulators
with excellent potency and selectivity for the H4 receptor. This has enabled exploration of the role of the receptor in
human disease and provided candidates for clinical investigation. Several compounds have reached the clinic
including JNJ 39758979 that has progressed into phase II clinical trials. JNJ-39758979 has shown efficacy in
preclinical pruritus and atopic dermatitis models [1,2]. Preclinically it reduces histamine-induced scratching in mice
and it reduces inflammation in a FITC-induced dermatitis model. These findings have been validated in the clinic
with JNJ-39758979 being shown to reduce histamine-induce itch in humans and have effects in patients with atopic
dermatitis [3,4]. This talk will highlight recent clinical and preclinical data supporting a role for the H4 receptor in
human disease, as well as some of the obstacles encountered when advancing compounds with a novel
mechanism into clinical studies.
References:
1. Thurmond, R. L. et al.: J. Pharmacol. Exp. Ther. 2014, 349:176-184.
2. Savall, B. M. et al.: J. Med. Chem. 2014, 57:2429–2439.
3. Kollmeier, A. K. et al.: J. Pharmacol. Exp. Ther. 2014, 350:181-187.
4. Murata, Y. et al.: J. Dermatol. 2015, 42:129-139.
PLENARY LECTURES
Liver research in Düsseldorf: Bridging basic and clinical science
P.5
Häussinger, D.1
1
Department Internal Medicine, Gastroenterology, Hepatology and Infectious Diseases, Heinrich Heine University Düsseldorf
Clinical and experimental liver research has a long-standing tradition at the Heinrich Heine University Düsseldorf.
Over the last two decades, important discoveries were made in the DFG-funded Collaborative Research Centers
SFB 575 “Experimental Hepatology” and SFB 974 “Communication and Systems Relevance of Liver Damage and
Regeneration” and the Clinical Research Group 217 “Hepatobiliary Transport in Health and Disease” and a few
selected achievements of these research networks will be presented in the following. Liver cell hydration was
identified as a dynamic parameter which can change within minutes under the influence of cumulative substrate
uptake into the hepatocyte, hormones, oxidative stress, nerve impulses, urea and hydrophobic bile acids.
Hepatocytes can sense these hydration (cell volume) changes and activate signalling events which link cell
hydration to cell function. ß1 integrins act as osmosensors in response to cell swelling, and trigger a proliferative,
anabolic and choleretic state. On the other hand, hepatocyte shrinkage is sensed via intracellular Cl- and
endosomes and triggers a proapoptotic, catabolic and cholestatic state. Hepatocyte swelling is also an integral part
of insulin signalling and mediates the anti-proteolytic and proliferative effects of the hormone. Bile acid secretion is
the result of a coordinated action of sinusoidal bile salt uptake (e.g. Ntcp) and canalicular secretion (e.g. via Bsep)
and is regulated not only at the level of gene expression, but also at the level of transporter retrieval and insertion,
which trigger cholestasis and choleresis, respectively. Bile acid retention in the hepatocyte induces liver damage
through activation of CD95-dependent apoptosis. The different mutations of the bile salt export pump Bsep can
trigger a broad spectrum of liver pathologies. One of these is the newly discovered autoimmune-Bsep deficiency,
which resembles progressive familial intrahepatic cholestasis (PFIC-2), but cannot be cured by liver transplantation.
Ursodesoxycholate (UDC), which is rapidly conjugated in vivo with taurine (TUDC) is frequently used in the
treatment of cholestatic liver disease due to its choleretic and cytoprotective effects. Recent data showed that
TUDC directly interacts with and activates intracellular ß1 integrins, thereby triggering osmosignalling events
resulting in choleresis, proliferation and cytoprotection. Bile acids are also be sensed by the membrane-bound and
G-protein-coupled receptor TGR5 (Gpbar). The receptor is expressed in many organs, including the brain and in
liver it is found in sinusoidal endothelial cells (SEC), Kupffer cells (KC) and cholangiocytes, but not in liver
parenchymal cells (hepatocytes). TGR5 acts as a bile acid sensor in cilia of cholangiocytes and triggers bile flow,
cholangiocyte protection and proliferation. TGR5 is overexpressed in cholangiocarcinoma and may in view of its
proliferative action be a novel target for anti-tumor therapy. Hepatic stellate cells (HSC) were identified as liverresident mesenchymal stem cells and play an important role in liver regeneration as shown in vitro and in in vivo
HSC transplantation experiments. Hepatic differentiation of HSC is not only achieved by growth factors
(HGF/FGF4), but surprisingly also by TUDC at low concentrations. In addition to the stem cell function of HSC, the
space of Dissé was identified as a stem cell niche in the liver. These findings have considerable impact on our
understanding of liver regeneration, extramedullary hematopoiesis and liver metastasis. In liver, glutamine
synthetase (GS) is restricted to a small perivenous hepatocyte population and acts here as a high affinity ammonia
scavenger before the hepatic blood reaches the systemic circulation. Liver-specific GS knockout triggers
hyperammonemia and cerebral alterations characteristic for hepatic encephalopathy (HE), a neuropsychiatric
syndrome frequently accompanying liver cirrhosis. This complication is the consequence of a low grade cerebral
edema and an oxidative/nitrosative stress response, which can be triggered by ammonia, inflammatory cytokines
and sedatives and results in cerebral protein tyrosine nitration, RNA oxidation, HE-specific alterations in gene
expression and induction of astrocyte senescence. These phenomena were also identified in the brain from patients
dying with liver cirrhosis and HE, but not in cirrhotic patients without HE. Interference with these pathophysiological
events may provide new approaches for the treatment of HE.
PLENARY LECTURES
Drug approval and regulatory science – Where to go?
P.6
Broich, K.1
1 Federal
Institute of Drugs and Medical Devices, 53175 Bonn, Germany
Requirements for marketing authorisation and monitoring of safety of medicinal products is highly harmonized in
the European Union (EU), however, patients increasingly demand earlier access to new and innovative products.
This leads to new concepts and challenges for regulators, e.g. the concept of adaptive pathways is based on a lifespan approach consisting of an early approval of a medicine on a limited database for a restricted patient population
in areas of high unmet medical need. Based on this initial approval the product will be further developed with buyin from multiple stakeholders including HTA/reimbursement bodies, which needs early exchange on study designs,
study endpoints and active comparators for clinical trials. A new clinical trial directive has been introduced and
national implementation is underway.
To fulfil our responsibilities and to adapt to the challenges ahead our own regulatory research activities will be
strengthened. Core areas for these activities have been defined (personalized medicine, pharmacoepidemiology,
safety of medical devices) and together with partners from academia, learned societies, research organisations
and patient representatives we are fostering regulatory science at the BfArM.
The key features of these challenges and activities will be presented.
PLENARY LECTURES
From synthetic nucleic acids to artificial genes and genomes: Drugs for the future?
P.7
Herdewijn, P.1
1 Medicinal
Chemistry, Rega Institute for Medical Research, KU Leuven, Minderbroedersstraat 10, 3000 Leuven, Belgium
With the aim to develop synthetic oligonucleotides, genes and genomes for therapeutic purposes, we have started
a selection procedure of chemically modified nucleotides and nucleic acids that could fulfil the requirements for in
vivo propagation. Several backbone modified nucleic acids (XNA) have been compared for in vivo transliteration
into DNA as well as new base-pair systems to develop an orthogonal genetic alphabet. The implementation of XNA
implies the development of dedicated polymerases to be able to select synthetic aptamers with and without catalytic
activity. Alternatively, implementation of synthetic genes and genomes can be reached by in vivo evolution
technologies and the first unlimited self-reproduction of a microorganism with a chemically modified genome has
been realized.
2 SCIENTIFIC LECTURES
SCIENTIFIC LECTURES
2.1 Antiviral Drugs
Chair: H. Rübsamen-Schaeff, H. Zimmermann
SL.01
Directly acting antivirals for hepatitis C virus – close to “perfectovir”
Pietschmann, T.1
Division Experimental Virology, TWINCORE - Centre for Experimental and Clinical Infection Research, Feodor-Lynen-Str. 7-9, 30625
Hannover, Germany
1
Chronic infection with hepatitis C virus is associated with severe liver disease including hepatitis, cirrhosis and
hepatocellular carcinoma. End stage liver disease due to HCV infection is one of the major indications for liver
transplantation. With an estimated number of 160 million infected individuals world-wide, HCV infection is a global
challenge for public health. Recent advances in HCV drug development have led to the licensing of three different
drug classes including protease and polymerase inhibitors as well as drugs targeting the viral NS5A protein.
Combination therapies including these so called directly acting antivirals (DAAs) cure more than 90% of infected
patients – at least in clinical study populations. However, these regimens are very expensive limiting access to
therapy particularly in countries with highest HCV prevalence. Thus, in the absence of an HCV vaccine, it is unlikely
that these efficacious yet expensive novel drugs will rapidly suppress the HCV disease burden globally.
Therefore, to identify cost effective therapeutics for treatment of HCV infection we screened a compound library
including licensed drugs for treatment of neuronal or heart diseases. Using this approach, we identify a number of
structurally related clinically licensed phenothiazines and diphenylmethylpiperazines that inhibit HCV infection in a
dose dependent fashion. Among our primary hits, we focused our attention on flunarizine, a diphenylmethylpiperazine and T-type calcium channel inhibitor used to treat migraine. This drug specifically inhibited HCV
cell entry (IC50 of 388.2 nM) in Huh-7 cells and in primary human hepatocytes in a genotype-dependent fashion. It
also reduced HCV infection in a genetically humanized mouse model for HCV cell entry. Using a comprehensive
set of experiments including single particle tracking imaging analyses, time of addition studies, viral resistance
selection, and numerous virus chimeras, we provide evidence that this drug specifically inhibits HCV membrane
fusion by targeting the functions of E1 and E2. While flunarizine exclusively targets HCV genotype 2 viruses we
show that closely related licenced drugs like pimozide have an expanded cross-genotype coverage targeting GTs
3, 5 and 7 viruses with an IC50 in the low micromolar range. Although resistance to these drugs can be selected in
cell culture, we show that these mutations involve highly conserved residues and that they enhance viral
susceptibility to neutralizing antibodies. Of note, resistance mutations to these drugs map to a subdomain in the
viral E1 protein which has previously been implicated as viral fusion peptide.
These observations reveal novel details about HCV membrane fusion. Moreover, flunarizine and related
compounds represent first-in-class HCV fusion inhibitors that merit consideration for repurposing as cost-effective
component of HCV combination therapies.
ANTIVIRAL DRUGS
SL.02
How to deal with HBV? - State of the art and new concepts in HBV therapy
Paulsen, D.1
1
AiCuris GmbH & Co.KG, 42117 Wuppertal, Germany
Chronic hepatitis B virus (HBV) infection is a major cause of severe liver disease and premature liver-related
mortality, worldwide. Antiviral therapy has made continuous progress over the past 20 years resulting in two
strategies to treat chronic hepatitis B: Interferon therapy or nucelos(t)idic inhibitors of the HBV polymerase. These
antiviral drugs are effectively suppressing HBV replication, but only a minority of patients is able to mount the
necessary immune control to keep HBV without treatment at bay. Therefore, the current state of the art in HBV
treatment is long-term and perhaps lifelong therapy. New concepts for HBV therapy are underway – either targeting
the virus directly or tackling reconstitution of the immune response via immunomodulatory approaches. Virion
assembly and release inhibitors, entry inhibitors, cccDNA inhibitors, TLR agonists, and therapeutic vaccination
approaches will be discussed in more detail.
SCIENTIFIC LECTURES
SL.03
Advances and challenges in diagnostics & therapy for HIV/AIDS
Stephan, C.1
1
Zentrum der Inneren Medizin / Schwerpunkt Infektiologie Universitätsklinikum Frankfurt, Goethe-Universität, 60590 Frankfurt
HIV-infected patients have reached the daily work of many physicians. Treatment and care of affected individuals
remains to be a challenge, despite the co-morbidity and co-infections’ therapy achievements, but as well because
of an increasingly aging patient group.
However, the guidance and strategic decisions and planning of antiretroviral therapy always belongs to the
specialized HIV-treating physician, in order to control combination therapy and review co-therapies. Nowadays
establishment of HIV diagnosis is still taking place often in advanced stage of HIV-disease and this requires highly
sophisticated treatment standards. Late diagnosis means disadvantages for the individual patient. The missed
diagnosis in prior medical history in a late presenting individual is resulting in avoidable morbidity and subsequent
HIV transmissions.
Current strategies for cure from HIV are fading the efforts to develop a therapeutic vaccination, which had suffered
many setbacks in the past. Actual cure strategies include individual induction of broadly neutralizing antibodies on
the one hand, and gene therapeutic approaches to manipulate lymphocyte function, i.e. regulating the
infectiousness of human cells.
ANTIVIRAL DRUGS
SL.04
The era after nucleosides: Novel therapeutic approaches against herpes viruses
Rübsamen-Schaeff, H.1
1 AiCuris
GmbH &Co KG, Friedrich-Ebert-Strasse 475, 42117 Wuppertal , Germany
Herpesviruses are a large family of viruses and cause chronic infections in humans and animals. While an
immunocompetent individual generally will control these viruses, they can cause severe infections or even be lifethreatening in all conditions of immune-incompetence, e.g. for neonates, transplant recipients or AIDS patients.
The treatment of herpesviruses has so far been confined to inhibitors of the viral polymerases using mainly
nucleoside analogues. However, this approach has two shortcomings: 1) Polymerase inhibitors may cross-react
with human polymerases leading to a number of toxic effects. 2) Polymerase inhibitors are prodrugs and require
for their activation a viral enzyme to conduct the first phosphorylation step, so that they are not active in uninfected
cells and do not protect these from infection.
We, therefore, have attempted to address other targets of herpes viruses and generated a terminase inhibitor,
Letermovir, against the Human Cytomegalovirus (HCMV) and a helicase-primase inhibitor, Pritelivir, against Herpes
Simplex Viruses (HSV 1 and 2). Both drugs stem from non-nucleosidic classes: Letermovir is a 3,4-dihydroquinazoline-4-yl-acetic acid derivative and Pritelivir is a 2-phenyl-N-thiazole-2-yl-acetamide derivative. Both drugs
are active in and protect uninfected cells against infection. Notably, both drugs also have a very steep doseresponse curve for antiviral efficacy. In contrast to polymerase inhibitors they show only small shifts in IC50, when
the multiplicity of the viral infection per cell is increased, meaning that they can very well deal with high viral loads.
These nonclinical observations have also translated into the clinics: In phase II clinical trials, a high efficacy and
good safety profile could be demonstrated for Letermovir (1) as well as for Pritelivir (2). In addition, Pritelivir
demonstrated superiority over the nucleoside analogue valacyclovir in a head to head clinical phase II study in
patients with genital herpes, again with a good tolerability similar to valacyclovir. Letermovir is presently in phase
III clinical testing in patients, who received a stem cell transplantation. Pritelivir is presently on clinical hold due to
so far unexplained observations in a 39 week animal study.
(http://clinicaltrials.gov/ct2/show/NCT02137772?term=letermovir&rank=1).
References:
1. Chemaly, R. F. et al: N Engl. J Med 2014, 370: 1781-1789.
2. Wald, A. L. et al: N Engl. J Med 2014, 370: 201-210.
SCIENTIFIC LECTURES
SL.05
New avenues in anti-influenza therapy
Ludwig, S.1
Institute of Molecular Virology, Westfälische-Wilhelms-University Muenster, Von Esmarch Str. 56, 48149 Muenster,
Germany
1
Influenza virus infection results in the activation of a variety of intracellular signaling responses. Influenza viruses
have acquired the capability to exploit some of these activities to support efficient replication. This dependence of
influenza virus propagation on cellular signaling factors provides opportunities for a novel approach of antiviral
interventions that targets essential host factors instead of viral components.
We have identified several cell signaling targets that are suitable for antiviral intervention, including the classical
mitogenic MAP kinase cascade, that regulates active viral RNP export, or the NF-kappaB pathway, that interferes
with the apoptotic response. In previous work we could demonstrate that inhibition of these pathways efficiently
blocked virus replication in cells and animals. Several inhibitors of the NF-kappaB pathway or the MAP kinase
cascade are now under advanced clinical evaluation or even licensed for clinical use for other diseases. We show
that these novel signaling blockers (a) efficiently inhibit influenza virus replication in cell culture and mouse models,
(b) are broadly active against all influenza A and B viruses analysed so far, (c) showed no tendency to induce
resistant virus variants, (d) are not toxic for cells or animals in the concentration and time line used, and (e) confirm
the postulated and fully unravelled mode of action: blocking of the transport of viral genomes from the nucleus.
Inhibitors of the classical MAP kinase cascade and blockers of the NF-kappaB pathway are promising compounds
against influenza with a broad activity and no tendency to induce resistance. Since there are existing drugs under
clinical evaluation or even licensed compounds available, repurposing of those drugs may be a fast and efficient
way for the development of a completely novel anti-influenza approach.
Accordingly, a phase II clinical trial with a drug that blocks the NF-kappaB pathway is currently ongoing
[www.clinicaltrialsregister.eu/ctr-search/trial/2012-004072-19/DE]. This is the first trial with an anti-influenza drug
that targets a cellular signalling factor and thus represents a paradigm change in anti influenza therapy.
ANTIVIRAL DRUGS
SL.06
Antivirals, a lot has been achieved, yet a long way to go
Neyts, J.1
1 Laboratory
of Virology, Rega Institute for Medical Research, University of Leuven, Belgium
Today, small molecule antiviral drugs are available for the treatment of infections with herpesviruses, HIV, HBV
and HCV as well as with influenza viruses. Ribavirin, a broad-spectrum (but aspecific) antiviral is being used with
very limited success for the treatment of RSV infections. Yet, for many other viruses that cause life-threatening
infections [several of which are considered emerging and/or neglected] there are no drugs available. Ideally, potent
and broad-spectrum (i.e., pan-genus or pan-family virus activity) antiviral drugs should be developed whereby one
drug could be used for the treatment of a number of such viral infections. I will discuss those viral infections with
the greatest need for an antiviral as well as the latest developments in the field.
SCIENTIFIC LECTURES
2.2 Blood/Brain Barrier
Chairs: G. Fricker, F. Helm
SL.07
Liposomal delivery of Trientine for treatment of Wilson’s Disease in the brain
Tremmel, R. 1; Fricker, G.1; Helm, F.1
1 Institut
für Pharmazie und Molekulare Biotechnologie , Ruprecht-Karls Universität Heidelberg, Im Neuenheimer Feld 329, 69210
Heidelberg
Wilson disease, a rare, inherited, autosomal recessive disorder, which leads to copper accumulation in several
organs including the brain. Beside other symptoms it causes neurological and psychiatric disorders, due to the lack
of therapeutics which are able to pass the blood brain barrier (BBB). Therapeutics for a front-line treatment of
Wilson disease are D-Penicillamin and Trientine. Both substances are chelators and forming complexes with copper
to be excreted in the urine. However, both of them show a poor CNS penetration.
One option to overcome the BBB is the encapsulation of API’s in nanosized carriers like nanoparticles or liposomes,
which can be surface modified with vectors targeting the BBB. One possible vector is apolipoprotein-E4 (ApoE),
which is transported across the BBB via transcytosis starting with binding to the low-density lipoprotein receptor.
Other options are the coupling of cationized bovine serum albumin (cBSA) to the liposomal surface in order to utilize
adsorptive endocytosis or the coupling of positively charged cell penetrating peptides.
Here we describe the manufacture of trientine loaded liposomes, their coupling to the above mentioned vectors
and the evaluation of the BBB penetrating properties in vitro in isolated brain capillary endothelial cells (PBCEC)
and in vivo in rats after i.v.-administration.
We reached encapsulation efficiencies of Trientine in liposomes up to 30%. After incubating PBCEC’s with
liposomal formulations, 10-15% of the administered concentration could accumulate inside the cells. In vivo a 12fold
accumulation of liposomal Trientin in brain parenchyma could be observed, compared to Trientine in solution.
BLOOD/BRAIN BARRIER
SL.08
Molecular regulation of endothelial barrier properties in the central nervous system
Guerit, S. 1; Czupalla, C. J.1*; Devraj, K.1; Ziegler, N.1; Gerhardt, H. 2; Plate, K. H.1; Liebner, S.1
1
Institute of Neurology (Edinger-Institute), Goethe University, Heinrich-Hoffmann-Str. 7, 60528 Frankfurt, Germany
Integrative Vaskular Biology, MDC Berlin, Robert-Rössle-Straße 10, 13125 Berlin-Buch
* current address: Stanford University School of Medicine, Department of Pathology, Lane Building, Mailcode 5324, Stanford, CA. 943055324
2
Endothelial Wnt/β-catenin signaling is necessary for developmental angiogenesis of the central nervous system
(CNS) and differentiation, maturation and maintenance of the blood-brain barrier (BBB) [1]. In the adult brain,
pericytes and astrocytes are the closest cellular neighbors of the barrier endothelium in the neuro-vascular unit
(NVU). Although both cell types doubtlessly participate in BBB maintenance and integrity, the contribution of Wnt/βcatenin signaling herein remains obscure.
In order to characterize AC-derived Wnts as BBB maintaining factors, we made use of in vitro (Evilox/lox TAT-Cre
treated ACs) and in vivo (GFAP-Cre:Evilox/lox mice, AC∆Evi) model systems in which ACs do not express the Evi
protein, which is essential for the release of Wnts. Trans-endothelial electrical resistance (TEER) was significantly
decreased when murine brain endothelioma cells (MBE) were co-cultivated with AC∆Evi in comparison to ACwtEvi
controls.
In vivo analysis of the AC∆Evi mice revealed that the AC-specific Evi deletion led to brain edema, indicating a partial
breakdown of BBB structures that however, did not cause lethality of the mice. Additionally, AC∆Evi mice displayed
alterations in vessel remodeling. Together these findings suggest that Wnt growth factors released by ACs play a
role in brain vessel structure and regulation of the BBB phenotype.
Moreover, we currently do not understand how vascular heterogeneity in the CNS is accomplished during
development and how it is maintained in the adult. Besides the contribution of Wnt factors in BBB maintenance,
their function in the differentiation of the leaky vascular phenotype in the circumventricular organs (CVOs),
conferring neurosecretory and -sensory function, is not understood in detail.
Neither during embryonic development, nor at early postnatal stages we detected activation of β-catenin signaling
in CVO vessels of BAT-gal reporter mice. Dominant activation of the β-catenin pathway (gain-of-function, GOF) in
endothelial cells (βCatGOF_EC) during early postnatal development led to expression of claudin-5 in vessels of the
sub-fornical organ (SFO), whereas Meca-32 immunoreactivity was reduced. Moreover, βCatGOF_EC generated a
thinner vascular phenotype within the SFO. Currently, we investigate in detail the circuitry of the Wnt pathway in
the CVOs and its specific role in CVO differentiation.
In summary, we can show that activation of the Wnt pathway in ECs is not only important during early brain
vascularization, but also during later stages of life and particularly in the aged CNS. Herein AC-derived Wnts may
contribute to brain EC differentiation and vascular remodeling. At the same time the lack of Wnt activation from
certain vessels such as those of the CVO is crucial for the formation of their specific phenotype and function.
Further investigations are required to better understand vascular heterogeneity in the brain in general and during
aging in particular.
Acknowledgments: Theodor Kocher Institute Bern, Switzerland, Engelhardt B.
References:
1. Engelhardt, B.; Liebner, S.: Cell Tissue Res. 2014, 355: 687–699.
SCIENTIFIC LECTURES
SL.09
Toxicity of poly(n-butylcyano-acrylate) (PBCA)-nanoparticles designed to overcome the bloodbrain barrier
Kolter, M.1; Fricker, G.1
1 Department
of Pharmaceutical Technology and Biopharmacy, Institute of Pharmacy and Molecular Biotechnology, University of
Heidelberg, Im Neuenheimer Feld 329, 69120 Heidelberg, Germany
The therapy of diseases of the central nervous system is a major challenge, since drugs have to overcome the
blood–brain barrier (BBB), which separates the brain from the peripheral blood stream. A powerful strategy to
circumvent the BBB is the intravenous administration of drug-loaded poly(n-butylcyano-acrylate) (PBCA)nanoparticles coated with polysorbate 80 (PS80), which are able to cross the BBB via receptor-mediated
transcytosis. The aim of this project was the preparation, physicochemical characterization and in particular the
toxicological evaluation of PBCA-nanoparticles at the BBB (in vitro), representing the target organ, in human whole
blood (ex vivo), as the site of administration and in a rat model (in vivo).
A reproducible preparation of endotoxin-free PBCA-nanoparticles, composed of n-butylcyano-acrylate,
poloxamer 188, sodium dodecyl sulfate (SDS) and soybean oil, was developed using a miniemulsion technique [1].
The nanoparticles were approximately 140 nm in size and exhibited a narrow particle size distribution, a negative
surface charge, a spherical shape and a low agglomeration potential.
The toxicological evaluation of PBCA-nanoparticles in an in vitro BBB model, which was composed of porcine brain
capillary endothelial cells, included the effects on metabolic activity, cell viability, cellular oxidative stress and BBB
integrity. Cells were affected in a strongly concentration-, time- and surface-dependent manner, but no cell death
or loss of metabolic activity and only a moderate generation of oxidative stress was observed for nanoparticle
concentrations ≤ 500 μg/ml up to 3 h of treatment. However, the BBB integrity was strongly affected by PBCAnanoparticles even at very low concentrations (≥ 5 µg/ml) resulting in a concentration-dependent, reversible and
repeatable BBB opening. Further experiments indicated that this effect allows a passive and size-dependent
paracellular transport of high molecular weight drugs into the brain.
In human whole blood, PBCA-nanoparticles caused no inflammatory burst. Only IL-8 displayed a significant release
after nanoparticle exposure, whereas other proinflammatory cytokines, which are typically released by
nanoparticles (e.g. IL-1β, IL-6, TNF-α), remained unaffected. In addition, human leukocytes did not suffer major
cell death after incubation with nanoparticles in concentrations up to 500 µg/ml over 3 h. The good blood
compatibility was endorsed by the absence of any hemolytic effect at concentrations of ≤ 500 µg/ml over 24 h.
Finally, the toxicological examination in rats (in vivo) identified neither inflammatory processes nor severe organ
damages indicating an even lower in vivo than in vitro or ex vivo toxicity.
In conclusion, PBCA-nanoparticles are an effective and promising drug delivery system to overcome the BBB with
a low toxic potential demonstrated by hardly any or only moderate cytotoxic and inflammatory effects at therapeutic
concentrations (up to 500 µg/ml) and incubation times (up to 3 h) at the BBB, in human whole blood and in vivo.
Acknowledgments: Deutsche Forschungsgemeinschaft (DFG) (DFG FR1211-17/1)
References:
1. Reimold, I. et al.: Eur. J. Pharm. Biopharm. 2008, 70(2): 627-32.
REGULATION OF BETA-CELL FUNCTION – IMPLICATIONS FOR DIABETES
2.3 Regulation of Beta-Cell Function – Implications for Diabetes
Chairs: G. Drews, E. Oetjen
SL.10
Role of bile acids in beta-cell functions
Drews, G.1
1 Institute
of Pharmacy, Dept. Pharmacology, Toxicology, and Clinical Pharmacy, University of Tübingen
Background: Meanwhile it is well established that bile acids (BAs) play an important role as signaling molecules
besides their role in fat digestion. The most important BA receptors are the G-protein-coupled TGR5 and the nuclear
farnesoid X receptor (FXR). They are involved in the maintenance of glucose homeostasis but also affect lipid
metabolism and energy expenditure by acting on liver, skeletal muscle, and adipose tissue. It has been shown that
the composition of the bile acid pool and the pool size are altered in type-2 diabetes mellitus (T2DM). We have
studied the effects of BAs on pancreatic beta-cells and focused our research on the role of FXR in stimulus secretion
coupling.
Methods: Islet of Langerhans and dispersed beta-cells from wildtype (WT) mice and mice with genetic deletion of
the FXR (FXR-KO) and of KATP channels (SUR1-KO) were used for in vitro experiments to study insulin secretion
(radioimmunoassay), the cytosolic Ca2+ concentration (fluorescence technique), and KATP currents (patch-clamp
technique). In vivo experiments were performed with mice fed a control or high fed diet (HFD) for 12 weeks. Glucose
tolerance and insulin sensitivity were measured after i.p. application of glucose and insulin, respectively.
Results: Chenodeoxycholic acid (CDC) and its conjugates enhance glucose-induced insulin secretion in islets of
WT mice and increase the cytosolic Ca2+ concentration. These effects are lacking in beta-cells of FXR-KO mice,
demonstrating that the effects of these BAs are mediated by FXR activation. Interestingly, activation of glucosedependent insulin secretion by BAs is also absent in islets from SUR1-KO mice, indicating that KATP channels are
involved in the BA effect. Accordingly, the increase in the cytosolic Ca2+ concentration is a result of an indirect effect
of BAs on KATP channels, i.e. closure of KATP channels by BAs only occurs in metabolically intact beta-cells but not
in excised membrane patches. Changes in the cytosolic Ca2+ concentration and KATP channel activity occur within
a few seconds or minutes after drug application, pointing to a non-genomic mode of action of BAs. Consistently
with these in vitro experiments, glucose tolerance is impaired in lean FXR-KO mice whereas insulin sensitivity
remains unchanged, demonstrating that the effect is due to altered beta-cell function. As expected, a HFD diet
induces an impairment of glucose tolerance in WT mice. Remarkably, after a HFD the glucose tolerance of FXRKO mice does not impair but is in the same range as in WT mice under control diet. This clearly shows that the
FXR has different functions in lean and obese mice. It is hypothesized that this shift in function is linked to a
translocation of the FXR from the cytosol to the nucleus.
Conclusions: FXR agonists exert rapid, non-genomic effects in beta-cells. FXR activation in beta-cell clearly
affects glucose homeostasis. FXR stimulation has different effects in lean and obese mice.
SCIENTIFIC LECTURES
SL.11
Mobility of submembrane insulin granules
Rustenbeck, I.1; Brüning, D.1; Schumacher, K.1; Matz, M.2; Baumann, K.2
1 Institute
2 Institute
of Pharmacology and Toxicology, University of Braunschweig, D-38106 Braunschweig, Germany
of Medicinal and Pharmaceutical Chemistry, University of Braunschweig, D-38106 Braunschweig, Germany
Background and aims: The biphasic pattern of insulin secretion is essential for the maintenance of glucose
homeostasis. The currently predominant hypothesis explains this pattern as the consequence of two different pools
of insulin granules. One pool consists of secretion-ready granules attached to the plasma membrane and the other
of granules located further inside the cell. A number of observations have put this hypothesis into question. To
characterize glucose-induced and depolarization-induced insulin secretion, we compared insulin secretion and
parameters of insulin granule mobility in insulin-secreting MIN6 cells and in normal mouse beta-cells.
Methods: The number, mobility and exocytosis of submembrane insulin granules can be visualized by TIRF (total
internal reflection fluorescence) microscopy. For a quantitative evaluation a computerized, observer-independent
evaluation of the video files is required. Insulin granules were fluorescently labeled by transient transfection with
insulin-EGFP in MIN6 cells and by adenoviral transduction in primary mouse beta-cells. The cells were continuously
perifused with oxygenated medium containing the respective stimuli. The TIRF microscopy image files (1 sequence
= 200 images = 25 seconds) were evaluated by an in-house written program. The insulin secretion of MIN6 pseudoislets and mouse islets was measured by perifusion with ELISA of the fractionated efflux.
Results: The perifused islets and pseudo-islets were stimulated by 40 mM K+ and, after a wash-out period, by 30
mM glucose. In MIN6 pseudo-islets 30 mM glucose was much less effective than 40 mM K+. When the K+
depolarization preceded the glucose stimulus, glucose was entirely ineffective. In mouse islets glucose was about
equi-effective with K+ depolarization. Under control condition, the number of granules in the first image of the
sequences was 337 ± 34 per MIN6 cell but only 144 ± 14 per beta cell. In both cell types this parameter remained
essentially stable during imaging. During basal and during stimulated activity of the cells, there was a continuous
arrival of granules at the submembrane space which was closely mirrored by the departure of granules back into
the cell interior. Under basal condition, the total number of granules identified per sequence was 6972 ± 873 in
MIN6 cells and 1966 ± 172 in beta-cells. In MIN6 cells 82% of these granules remained at the plasma membrane
for less than 1 s (short-term residents), and only 1.7% were visible for the entire sequence = 25 s (long-term
residents). The corresponding values for beta-cells were similar, 80% and 3.0%, respectively. Most of the granules
that fused with the plasma membrane were visible from the beginning of the sequence (potential long-term
residents) and had a low lateral mobility, but there were also fusion events a few seconds after arrival at the plasma
membrane.Unexpectedly, the rate of granule fusion was not increased during phases of stimulation, whereas the
vertical mobility (rate of arrival and departures) was significantly increased.
Conclusion: In insulin-secreting cells there is a continuous turnover of granules in the submembrane space. A small
proportion of the granules becomes more firmly attached to the membrane. These are the granules that
preferentially fuse with the plasma membrane. The link between secretion and fusion may be less direct than
currently assumed. Even though a general similarity exists between the situation in MIN6-cells and primary beta
cells, only beta cells are adequate to characterize the response to glucose.
Acknowledgments: Supported by DFG Ru 368/5-2
REGULATION OF BETA-CELL FUNCTION – IMPLICATIONS FOR DIABETES
SL.12
Reactive oxygen species: Pharmacological target or physical prerequisite for beta-cell function?
Düfer, M.1
1
Institute of Pharmaceutical and Medicinal Chemistry, Dept. of Pharmacology, Münster University, Germany
Elevated generation of reactive oxygen species (ROS), e.g. in response to overnutrition, is known to play a pivotal
role for manifestation and progression of type 2 diabetes mellitus. It is well established that key steps for regulation
of insulin release are severely impaired by oxidative stress. As a consequence, therapeutic strategies to reduce
ROS production or accumulation in pancreatic beta-cells are investigated to protect against the deterioration of
beta-cell function in patients suffering from impaired glycemic control. However, there is increasing evidence that
apart from their involvement in beta-cell failure ROS are important signalling molecules necessary for physiological
regulation of insulin release. Acute stimulation of insulin secretion by glucose coincides with an elevation of
hydrogen peroxide and with a decrease in superoxide radical anions. Challenging beta-cell metabolism by high
glucose and lipid concentrations does not cause permanent oxidative stress but drastically reduces the ability of
nutrients to alter intracellular ROS concentrations. This effect is presumably mediated by malfunction of antioxidant
enzymes as activation of the transcription factor Nrf-2, which increases gene transcription of antioxidant pathways,
restores the physiological ROS profile. Based on these observations, the objective of strategies targeting
antioxidant capacity of beta-cells should not be complete prevention of ROS formation but preservation of the
physiological balance between different species of ROS.
SCIENTIFIC LECTURES
SL.13
Regulation of beta-cell function and mass by the dual leucine zipper kinase
Oetjen, E.1
1 Department
of Clinical Pharmacology and Toxicology, University Medical Center Hamburg Eppendorf, Martinistr. 52, 20246 Hamburg;
Institute of Pharmacy, University of Hamburg, Bundesstr. 45, 20146 Hamburg, Germany
The dual leucine zipper kinase DLK induces β-cell apoptosis by inhibiting the transcriptional activity conferred by
the β-cell protective transcription factor cAMP response element binding protein CREB. This action might contribute
to β-cell loss and ultimately diabetes. Within its kinase domain DLK shares high homology with the mixed lineage
kinase (MLK) 3, which is activated by tumor necrosis factor (TNF) α and interleukin (IL)-1β, known prediabetic
signals. In the present study, the regulation of DLK in β-cells by these cytokines was investigated. Both, TNFα and
IL-1β induced the nuclear translocation of DLK. Mutations within a putative nuclear localization signal (NLS)
prevented basal and cytokine-induced nuclear localization of DLK and binding to the importin receptor importin α,
thereby demonstrating a functional NLS within DLK. DLK NLS mutants were catalytically active as they
phosphorylated their down-stream kinase c-Jun N-terminal kinase to the same extent as DLK wild-type but did
neither inhibit CREB-dependent gene transcription nor transcription conferred by the promoter of the anti-apoptotic
protein BCL-xL. In addition, the β-cell apoptosis-inducing effect of DLK was severely diminished by mutation of its
NLS. In two distinct murine models of prediabetes, enhanced nuclear DLK was found. These data demonstrate that
DLK exerts distinct functions, depending on its subcellular localization and thus provide a novel level of regulating
DLK action. Furthermore, the prevention of the nuclear localization of DLK as induced by prediabetic signals with
consecutive suppression of β-cell apoptosis might constitute a novel target in the therapy of diabetes mellitus.
GLISTEN – GPCR MEDICINAL CHEMISTRY
2.4 GLISTEN – GPCR Medicinal Chemistry
Chairs: N. Tschammer, P. Kolb
SL.14
Selective orthosteric agonists in class C GPCRs
Acher F. C.1; Brabet, I.2; Rigault, D.1; Busserolles, J.3; Bertrand, H.-O.4; Eschalier, A.3; Goudet, C.2; Pin, J.-P.2
Université Paris Descartes, UMR8601-CNRS, 45 rue des Saints-Pères, Paris 06, France
Institut de Génomique Fonctionnelle, CNRS UMR5203, INSERM U661, Université Montpellier, 141 rue de la Cardonille, 34094 Montpellier,
France
3 Université d’Auvergne INSERM U 1107, Neuro-Dol, 63001 Clermont-Ferrand, France
4 Biovia Dassault Systems, Parc Club Orsay Université, 20 rue Jean Rostand, 91893 Orsay, France
1
2
G Protein Coupled Receptors (GPCRs) are characterized by a seven helix transmembrane domain where the
binding site of agonists is found. However in class C GPCRs (e.g. metabotropic glutamate receptors, mGluRs) the
orthosteric binding site is located in the large amino terminal domain that folds in two lobes connected by a flexible
hinge building the Venus FlyTrap (VFT) domain. The closed conformation of this domain triggers activation of the
receptors. It has been difficult to discover subtype selective agonists because of the high conservation of the
orthosteric binding site. However by means of a virtual high throughput screening of mGluR subtype 4 VFT
homology model, we have discovered a selective pocket adjacent to the glutamate binding site which is in fact a
critical chloride binding site [1,2]. Binding to this site confers selectivity and higher potency to ligands. Indeed
optimization of the hit, led to the discovery of LSP4-2022 that is mGlu4 receptor selective [3]. Further chemical
modulation provided new agonists with higher potency. These agonists were successfully evaluated for their
antihyperalgesic properties in inflammatory and neuropathic pain models [4].
References:
1. Selvam, C. et al.: J. Med. Chem. 2010, 53, 2797-2813.
2. Tora, A. S. et al.: FASEB J. 2015 [Epub ahead of print]
3. Goudet, C. et al.: FASEB J. 2012, 26, 1682-1693.
4. Vilar, B. et al.: J. Neurosci. 2013, 33, 18951-18965
SCIENTIFIC LECTURES
SL.15
Identification and pharmacological characterization of endogenous and surrogate ligands for
orphan G protein-coupled receptors
Bräuner-Osborne, H.1; Gloriam, D. E.1
Department of Drug Design and Pharmacology, Faculty of Health and Medical Sciences, University of Copenhagen, Fruebjergvej 3, 2100
Copenhagen, Denmark,
1
G protein-coupled receptors (GPCRs) are a large family of cell membrane embedded signal receivers that are
involved in many biological processes and is one of the main target families for approved drugs. GPCRs are
activated by a wide variety of ligands, including neurotransmitters, hormones and food constituents such as amino
acids, sugars and fatty acids. GPCRs activate intracellular G proteins of which four classes exist (Gαq, Gαi, Gαs
and Gα12/13) as well as other intracellular proteins such as ß-arrestins. 358 non-olfactory GPCRs have been
identified in the human genome of which ~1/3 remain orphan receptors without confirmed endogenous agonists.
Given the involvement of GPRCs in many important biological processes and their success as drug targets, there
has been many efforts in academia and industry aiming at identifying the endogenous ligands or developing
surrogate ligands for orphan receptors. Many different strategies have been adopted mainly focusing on
employment of 'generic' pharmacological assays and screening of tissue extracts or libraries of potential
endogenous ligands. This has led to important ligand-receptor matches, but the deorphanization rate has been
slow.
In contrast we have chosen to use a range of computational methods to select ligands for pharmacological testing
to increase the likelihood of hit identification. This has so far led to the following ligand-receptor matches:
1. Identification of basic L-amino acids as potential endogenous agonists for the GPRC6A receptor using receptor
modelling and ligand docking [1].
2. Identification of the first selective GPRC6A antagonists using chemogenomics and 'privileged structures' - multitarget chemical scaffolds [2].
3. Identification of aromatic L-amino acids and dipeptides as potential endogenous agonists for the GPR139
receptor using a pharmacophore based on previously identified surrogate agonists [3].
4. Identification of the first surrogate agonist for the GPR132 receptor using GPCR-directed compound libraries [4].
The present lecture will present these case stories and outline our current strategy for future deorphanizations.
Acknowledgments: Major financial support for this work has obtained from the Danish Council for Independent Research, the Lundbeck
Foundation, the European Research Council and the Hørslev Foundation.
References:
1. Wellendorph, P. et al.: Mol. Pharmacol. 2005, 67: 589-597.
2. Gloriam, D. E. et al.: Chem. Biol. 2011, 18: 1489-1498.
3. Isberg, V. et al.: J. Chem. Inf. Model. 2014, 54: 1553-1557.
4. Shehata, M. A. et al.: RSC Advances. 2015, 5: 48551-48557.
GLISTEN – GPCR MEDICINAL CHEMISTRY
SL.16
Structure free optimisation of fragments for the β2-adrenoreceptor
Gawron, S.1; Aristotelous, T.1; Chevillard, F.2; Hopkins, A. L.1; Kolb, P.2; Hopkins-Navratilova, I.1; Gilbert, I. H.1
1 Division
of Biological Chemistry and Drug Discovery, College of Life Sciences, Sir James Black Centre, University of Dundee, Dundee,
DD1 5EH, UK
2 Philipps-Universität Marburg, Department of Pharmaceutical Chemistry, Marbacher Weg 6, 35032, Marburg, Germany
G-protein coupled receptors (GPCRs) are the primary target class of currently marketed drugs, accounting for about
a quarter of all drug targets of approved medicines. Here we describe a process of structure-free optimisation of
fragments binding to β2-adrenoreceptor in the absence of structure. A decision tree is introduced for optimisation
of both the core scaffold and the different vectors. This approach allowed a generation of ‘virtual’ model of the
binding site. Subsequently it was possible to rationalise the data by docking the fragments into a crystal structure
of the β2-adrenoreceptor.
Acknowledgments: University of Dundee, GLISTEN, Marburg University
References:
1. Aristotelous, T. et al.: ACS Med. Chem. Lett. 2013, 4: 1005-1010.
SCIENTIFIC LECTURES
SL.17
Comprehensive analysis of heterotrimeric G-protein complex diversity and their interactions
with GPCRs in solution
Hillenbrand, M. 1; Schori, C.1; Schöppe, J.1; Plückthun, A.1
1 Department
of Biochemistry, University of Zurich, Winterthurerstrasse 190, 8057 Zurich, Switzerland
Agonist binding to G–protein–coupled receptors (GPCRs) triggers signal transduction cascades involving
heterotrimeric G proteins as key players. A major obstacle for drug design is the limited knowledge of
conformational changes upon agonist binding, the details of interaction with the different G proteins, and the
transmission to movements within the G protein. Although a variety of different GPCR/G protein complex structures
would be needed, the transient nature of this complex and the intrinsic instability against dissociation make this
endeavor very challenging. We have previously evolved GPCR mutants that display higher stability and retain their
interaction with G proteins. We aimed at finding all G-protein combinations that preferentially interact with
neurotensin receptor 1 (NTR1) and our stabilized mutants. We first systematically analyzed by
coimmunoprecipitation the capability of 120 different G-protein combinations consisting of αi1 or αsL and all possible
βγ-dimers to form a heterotrimeric complex. This analysis revealed a surprisingly unrestricted ability of the G-protein
subunits to form heterotrimeric complexes, including βγ-dimers previously thought to be nonexistent, except for
combinations containing β5. A second screen on coupling preference of all G-protein heterotrimers to NTR1 wild
type and a stabilized mutant indicated a preference for those Gαi1βγ combinations containing γ1 and γ11.
Heterotrimeric G proteins, including combinations believed to be nonexistent, were purified, and complexes with
the GPCR were prepared. Our results shed new light on the combinatorial diversity of G proteins and their coupling
to GPCRs and open new approaches to improve the stability of GPCR/G-protein complexes.
References:
Hillenbrand, M. et al.: Proc. Natl. Acad. Sci. USA 2015, 112(11): E1181-1190.
EVIDENCE BASED MEDICATION MANAGEMENT
2.5 Evidence based Medication Management
Chairs: G. Hempel, S. Läer
SL.18
Medication management in clinical care
Bertsche, T.1,2
1 Institute
2 Drug
of Pharmacy, Clinical Pharmacy, Leipzig University
Safety Center, Leipzig University and University Hospital of Leipzig
DRP (Drug-Related Problems) in hospitalized patients have a high risk to cause acute harm and even death to the patient.
Additionally, after discharge from hospital DRP can continuously occur and cause problems in ambulatory care. Vulnerable
patient groups of special interest for strategies to improve patient safety are children and adolescents as well as geriatric
patients (including patients requiring intensive/intermediate or palliative care). Clinical pharmacists join the interdisciplinary
health care team to perform a structured medication management in clinical care in order to prevent DRP before they occur.
Within the hospital setting apart from medication data also laboratory data are regularly used. Additionally, physicians and
nurses can be contacted directly when DRP have been identified. If pharmacists are on duty on the ward, a patient interview
helps to gain a more sophisticated overview of the patients’ situation. Our group perform medication management projects
based on monitoring procedures in the wards with direct patient contact, medication and patient chart reviews including
electronic prescription, regular participation in ward rounds, questionnaire surveys, and (semi-) structured interviews. This way,
we identified DRP and implemented interventional strategies such as information leaflets, algorithm-based decision support or
teaching and training sessions as well as interdisciplinary guidelines. After admission to hospital the medication has regularly
to be switched to the local drug formulary. For a reconciliation management at admission and also during the patients’ hospital
stay the following aspects should be particularly considered in a medication management: indication, effectiveness, dosing,
route of administration appropriate and feasible, drug-drug interactions and interactions with food and comorbidity, double
prescriptions, duration of therapy, pharmaco-economic consideration such as cost/benefit relations. Patients’ medication data
is then to be compared to data from drug information databases, systematic and narrative reviews, original data from RCT
(Randomised Controlled Trials), SmPC (Summary of Product Characteristics) and (inter)national guidelines. However, as we
found, information gained from different sources can highly differ from each other. Besides Rx-medication (requiring
prescription by a physician) also OTC-drugs (Over-The-Counter) and CAM (Complementary and Alternative Medicine) should
be taken into account, because patients frequently continue their drugs during their hospital stay without knowledge of the
hospital physicians. At discharge, the medication recommended in the discharge letter by the physician should be finally
assessed by a pharmacist. Information leaflets directly forwarded to the patient and a direct-to-patient counselling prevent DRP
when the patient takes responsibility of drug administration by himself. Home visits with medicine reviews support a safer drug
administration including the storage of drugs at home. Apart from patient- and medication-related knowledge, communication
skills are essential to gain relevant information as well as to succeed in implementing the recommendations to physicians,
nurses, patients, and relatives. Drug information should be offered in a personalized way and in written and verbal form to the
responsible physician and (if appropriate) to the patient in agreement with the physician. Considering limited resources we are
prioritizing strategies to the most frequent and severe problems according to decision-matrix models. We used particularly
monitoring procedures to identify also knowledge-based DRP that are hardly detectable by CIRS (Critical Incident Reporting
Systems). We found that the total medication process reaching from prescription to administration should be addressed. By
using a feed-back strategy offering information on (in)appropriate drug administration to physicians avoids DRP most
effectively. As we have recently shown, DRP derive not only from health care professionals and patients; also relatives and
other groups such as teachers of children should be trained in save (emergency) drug administration. To reach sustainable
effects we have proven that the pharmaceutical support should be continuously offered. As shown by our group, clinical
pharmacists prevent on the one hand DRP and critical adverse drug events at the patient level. On the other hand, guideline
adherence and effectiveness increase by strategies offered by pharmacists. To summarize, our results confirm that a structured
and systematically performed medication management in routine clinical care is feasible and increases patient safety in drug
therapy substantially.
Acknowledgments: I would like to thank all patients, relatives, physicians, nurses and pharmacists participating in our studies and
supporting our work. Special thanks to the members of my team: Susanne Schiek, Martina P. Neininger, Henriette K. Dumeier, Johanna
Freyer, Claudia Greißing, Almuth Kaune, Dorothee Niemann, Pia M. Schumacher, Monika K. Sluzalek, Dominik Wilke, Janine Zimmer, and
Sandra Paule.
SCIENTIFIC LECTURES
SL.19
Medication management in ambulatory care: Preliminary results of the WestGem-Study
Rose, O.1,6; Schaffert, C.2; Czarnecki, K.2; Mennemann, H. S.3; John, C.6; Mertens-Keller, D.6; Richling, I.6;
Waltering, I.4,6; Hamacher, S.5; Felsch, M.5, Herich, L.5; Köberlein-Neu, J.2
1 Department
of Clinical Pharmacy, University of Bonn, An der Immenburg 4, 53121 Bonn Germany.
of Health Care Management and Public Health, Schumpeter School of Business and Economics, University of Wuppertal,
Rainer-Gruenter-Str. 21 Gebäude FN (1. OG), 42119 Wuppertal, Germany.
3 Muenster University of Applied Science, Robert-Koch-Str. 30, 48149 Muenster, Germany.
4 Department of Pharmacy, University of Muenster, Corrensstr. 48, 48149 Muenster.
5 Institute of Medical Statistics, Informatics and Epidemiology, University of Cologne, Kerpener Str. 62, 50937 Cologne, Germany.
6 Elefanten-Apotheke gegr 1575, Steinstr. 14, 48565 Steinfurt, Germany.
2 Department
Background: Pharmaceutical practice worldwide experiences a shift towards patient oriented services. Medication
Management (MM) is emphasized as the most prominent pharmaceutical care activity. A profound benefit is
expected for the patients, the society and the health care providers. Data for Germany to show the outcomes of a
Medication Management is still scarce.
Purpose: This first RCT on MTM in Germany aims to provide data on the outcomes of a comprehensive MM by
performing repeated PCNE-type-3 Medication Reviews (MR) and interprofessional collaboration.
Methods: The study is designed as a pragmatic cluster-randomized controlled trial, involving 12 general practitioner
(GP) clinics and 165 patients. It follows a stepped wedge design. Pharmacists receive anonymized data by GPs
and home-care specialists. A PCNE type 3 Medication Review (MR) is performed and a SOAP-form is provided to
the GPs. The MR is repeated after 6 months. The primary endpoint is a change in the quality of therapy, measured
by the Medication Appropriateness Index (MAI). Secondary endpoints are changes in the number of drug related
problems (DRPs). Clinical outcomes and interprofessional aspects of the collaboration are evaluated.
Results: Preliminary data shows a significant reduction in the MAI-score, which correlates in time with a stronger
effect in the patients who entered the intervention-phase earlier. Similar effects can be shown on the reduction of
drug related problems (DRPs). A reduction in LDL-Cholesterol can be demonstrated in an eligible subgroup with
the required laboratory data. Kidney function in another subgroup of the study with data on serum-creatinine
declines less than expected from other studies. Pharmaceutical suggestions are accepted by more than 50% by
the GPs.
Conclusions: A collaborative MM in Germany can improve the quality of therapy and clinical endpoints. The effect
of a Medication Management increases over time.
EVIDENCE BASED MEDICATION MANAGEMENT
SL.20
AMTS activities of the Pharmacists-Chamber of North-Rhine
Krüger, M.1; Derix, S.1
1
Linner Apotheke, Appointee for Phamaceutical Care and AMTS, General Secretary North-Rhine Chamber of Pharmacists
Qualification courses and projects, which ensure and enhance the patient oriented medication care in times of
demographic and societal change ever since have belonged to the core strategic competencies of the NorthRhine chamber of pharmacists. AMTS and medication management should ideally become qualified and
reimbursed pharmaceutical services. The multi-professional pharmacotherapeutic management is a central
element of drug safety according to the remarkable and exemplary resolution of the federal state health
conference of Northrhine-Westfalia in 2012.
Thus, the qualification project ATHINA (Drug Safety in Pharmacies) is a priority amongst multiple measures of the
North-Rhine chamber of pharmacists. With focus on geriatric patients the chamber initiated an evaluation study
investigating implementation of pharmaceutical risk management systems in geriatric care units through
specialized pharmacies. Co-operations and grants for several scientific projects (e.g. DIADEMA) have been
established. Results show that AMTS and medication management enhance patient care. Competencies and job
satisfaction of pharmacists are fostered as well.
Better interdisciplinary collaboration and clearer responsibility definitions should be further optimized to ensure
high quality patient care and medication management. The government is requested to ensure the
reimbursement of these additional pharmaceutical services adequately.
SCIENTIFIC LECTURES
SL.21
Comparison of three lists of Potentially Iadequate Medications in old age (PIMs) in four German
long-term care facilities with special regard to Adverse Drug Events (ADEs)
Hildebrand, J.1,2; Hanke, F.1,2; Jaehde, U.3; Thürmann, P. A.1
1 Institute
of Clinical Pharmacology, University of Witten/ Herdecke, Germany
Society of Geriatric Pharmacy - Gero PharmCare Ltd, Cologne, Germany
3 Institute of Pharmacy, Clinical Pharmacy, University of Bonn, Germany
2
Background and aims: During the last 25 years, a variety of PIM lists has been developed in several countries.
Some studies have shown that PIM use is associated with an increased risk for ADEs [1, 2], although this correlation
is still being discussed controversially. We aimed at identifying the applicability and qualification of two international
PIM lists for use in a German nursing home cohort in comparison to the German PRISCUS list [2].
Materials and methods: The medications of 339 nursing home residents living in four long-term care facilities in
Northrhine-Westphalia between July and December 2010 were screened for PIMs. Observation period was 90 days
retrospectively. We compared the German PRISCUS list, the French PIM list [3] and the 2012 Update of the
American Beers list [4]. The prevalence of PIM prescription and the most frequently prescribed PIM substances
were analysed. Additionally, prevalent ADEs were identified and analysed as described recently [5]. Possible
correlations between PIM use and ADE occurrence were analysed using univariate regression analyses (statistical
programme Minitab 17).
Results: The prevalence of PIM prescriptions came to 45.3% (for regular medications and those documented “as
needed”) according to the PRISCUS list and was remarkably higher when applying the French PIM list (50.6%) and
even higher using the Beers list (60.9%). There was no statistically significant difference in PIM use between female
and male nursing home residents (PRISCUS PIMs: p = 0.825, French PIMs: p = 0.802, Beers PIMs: p = 0.419).
The most frequently prescribed PRISCUS PIMs were dimenhydrinate, zopiclone and acetyldigoxin, according to
the French PIM list the most frequent substances were sodium-picosulfate, promethazine and dimenhydrinate, and
the most frequent Beers PIMs were metoclopramide, risperidone and lorazepam. A correlation between PIM use
and ADE occurrence was found for Beers PIMs (p = 0.006), but not for PRISCUS PIMs (p = 0.114) and French
PIMs (p = 0.273).
Conclusions: PIM prescriptions are very common for nursing home residents in Germany. Astonishingly, the
percentage of PIMs was much higher with the Beers and the French PIM list. This comparison shows that there is
variability between PIM lists which can be explained by the fact that all these lists base on expert consensus
assessment rather than on evidence from randomised controlled trials in the elderly population. Apart from the
PRISCUS list, which has been developed specifically for drugs authorised for the German market, international
PIM lists may also offer important information for identifying patients at risk for ADEs. An association could be
shown between the use of PIM drugs (according to the Beers list) and the occurrence of ADEs, which is in line with
findings from other studies in the setting of nursing home residents [6].
References:
1. Dormann, H. et al.: Dtsch. Arztebl. Int. 2013, 110(13): 213–9.
2. Holt, S. et al.: Dtsch. Arztebl. Int. 2010, 107(31-32): 543-51.
3. Laroche, M. L. et al.: Eur. J. Clin. Pharmacol. 2007, 63: 725–31.
4. American Geriatrics Society 2012 Beers Criteria Update Expert Panel: American Geriatrics Society updated Beers Criteria for potentially
inappropriate medication use in older adults, J. Am. Geriatr. Soc. 2012, 60 (4): 616–31.
5. Jaehde, U.; Thürmann, P. A.: Z. Evid. Fortbild. Qual. Gesundhwes. 2012, 106(10):712-6.
6. Lau, D. T. et al.: Arch. Intern. Med. 2005, 165(1):68-74.
(BIO)ANALYTICS
2.6 (Bio)Analytics
Chairs: J. Heilmann, M. Lämmerhofer
SL.22
Protein mass spectrometry – Methods and applications in clinical proteomics
Stühler, K.1,2
1
2
Institut für Molekulare Medizin, Universitätsklinikum Düsseldorf, Düsseldorf, Germany
Molecular Proteomics Laboratory Biologisch-Medizinisches-Forschungszentrum, Heinrich-Heine-Universität, Düsseldorf, Germany
In the last years, mass spectrometry (MS) has rapidly developed and beside other applications in proteomics it is
meanwhile broadly applied for a wide range of protein analytical questions like for instance characterization of
proteins’ primary structure, identification of posttranslational modifications, identification of protein interaction
partners and even absolute protein quantification. Typically electrospray ionization MS instruments coupled with
high performance nano liquid chromatography (nano LC-ESI-MS) systems are used to address especially
quantitative question in biological research. The MS systems are available for high resolution measurements which
are necessary for discovery driven analysis of complex protein mixtures using e.g. label based or label-free MS.
For targeted quantification of proteins triple-quadrupol systems are indispensable tools and are applied in clinical
proteomic. This triple-quadrupol instruments allow the relative and absolute quantification of known proteins by
selected reaction monitoring (SRM)-analysis. Here, current research projects will be presented applying the above
mentioned techniques for protein identification and quantification to exemplify actual applications of protein mass
spectrometry in clinical proteomics.
SCIENTIFIC LECTURES
SL.23
Capillary electrophoresis-based stereospecific enzyme assay for methionine sulfoxide reductase
Scriba, G.1; Zhu, Q.1; Schönherr, R.2; Heinemann, S.2
1 Department
2 Department
of Pharmaceutical/Medicinal Chemistry, Friedrich Schiller University, Philosophenweg 14, 07743 Jena, Germany
of Biochemistry, Friedrich Schiller University, Philosophenweg 14, 07743 Jena, Germany
Under conditions of oxidative stress, L-methionine (Met) residues in peptides and proteins are easily oxidized to
L-methionine sulfoxide [Met(O)] by reactive oxygen species [1]. Protein-bound as well as free Met(O) can be
reduced by methionine sulfoxide reductase (Msr) enzymes, a group of thiol oxidoreductases, protecting cells
against oxidative damage [2]. Because of the chirality of the sulfoxide moiety, Met(O) exists as the pair of
diastereomers L-methionine-(S)-sulfoxide [Met-S-(O)] and L-methionine-(R)-sulfoxide [Met-R-(O)]. For the
reduction of the diastereomers, stereospecific Msr enzymes exist, which share little sequence homology but
possess mirror-like relationships of their active sites. MsrA catalyzes the reduction of either free or protein bound
Met-S-(O). In contrast, MsrB reduces Met-R-(O) in proteins but displays only low activity for free Met-R-(O). In
addition, an enzyme named free methionine-(R)-sulfoxide reductase (fRMsr), which specifically reduces free MetR-(O), has been found in bacteria [2,3].
In order to determine the stereospecific activity of Msr enzymes, capillary electrophoresis-based assays employing
N-acetylated pentapeptides containing a dinitophenyl (Dnp) label at the C-terminus were developed. For example,
the diastereomers of ac-Lys-Ile-Phe-Met(O)-Lys-DNP were separated in a background electrolyte using a dual
elector system consisting of sulfated β-cyclodextrin and 15-crown-5. The optimized conditions were obtained by
experimental design resulting in a 50 mM Tris buffer, pH 7.85, which contained 5 mM 15-crown-5 and 14.3 mg/mL
sulfated β-cyclodextrin. The assay was validated and applied to the determination of the stereospecificity of
recombinant human and fungal Msr enzymes as well as the determination of the Michaelis-Menten kinetic data of
the enzymes. Furthermore, mutations of Aspergillus nidulans MsrA were analysed by using various peptide
substrates.
Acknowledgments: The financial Support of Q. Zhu by the China Scholarship Council is gratefully acknowledged.
References:
1. Voigt, W.: Free Racid. Biol. Med. 1995, 18: 93-105.
2. Broschi-Muller, S.; Gnad, A.; Branlant, B.: Ann. Biochem. Biophys. 2008, 474: 266-273.
3. Lee, B. C.; Gladyshev, V. Y.: Free Racid. Biol. Med. 2011, 50: 211-227.
(BIO)ANALYTICS
SL.24
New approach for drug discovery and development: Prediction of human drug metabolism using
chimeric mice transplanted with human hepatocytes
Sanoh, S.1; Tateno, C.2; Ohta, S.1
1 Graduate
2 R&D
School of Biomedical and Health Sciences, Hiroshima University, 1-2-3 Kasumi, Minami-ku, Hiroshima, 734-8553, Japan.
Dept, PhoenixBio, Co., Ltd., 3-4-1 Kagamiyama, Higashi-Hiroshima, 739-0046, Japan
In drug discovery and development, it is essential to predict human drug metabolism and pharmacokinetics (PK) of new
chemical entity to estimate efficacy and safety in humans. However, it is often difficult to predict these profiles from animal data
because species differences of metabolic activities in drug-metabolizing enzymes are observed between animals and humans.
Screening of metabolic stability of candidates for cytochrome P450 (CYP) has generally been conducted using human liver
microsomes in pharmaceuticals. Human hepatocytes also have been widely used to predict PK parameters such as clearance
(CL) and identify drug metabolites. Hepatocytes are useful to examine both CYP and non-CYP metabolic activities such as
UDP-glucuronosyltransferase (UGT) and aldehyde oxidase (AO).
Chimeric mice with humanized liver which were generated from urokinase-type plasminogen activator/severe combined
immunodeficiency mice transfected with human hepatocytes, are currently used in drug discovery and development. The ratio
of replacement of human hepatocytes is more than 70% in the liver of chimeric mice with humanized liver. The expression of
human specific metabolizing enzymes, transporters has been confirmed in chimeric mice. Therefore, chimeric mice with
humanized liver are expected as in vivo animal model to predict human specific drug metabolism and PK [1,2]. In fact, several
reports show that metabolic profiles of drugs mediated by CYP and non-CYP in chimeric mice reflect those in humans. Six
metabolites of ibuprofen by CYP and UGT were detected in urine of chimeric mice. The urinary excretion profile was similar to
that of humans [3]. Furthermore, metabolic profiles of AO substrates also reflected those of humans although there are
significant species differences of AO metabolism between animals and humans [4,5]. In contrast, chimeric mice transplanted
with rat hepatocytes have also been developed as the control mice for chimeric mice with humanized liver in consideration of
species differences between rats and humans [6]. The comparison of metabolic profiles between rats and chimeric mice with
rat hepatocytes could help to improve predictability of human profiles from data of chimeric mice with humanized liver. The
utility of chimeric mice transplanted with rat hepatocytes was demonstrated [3-5]. Predictability of not only drug metabolism
but also PK profile has been evaluated. Good correlations of hepatic intrinsic CL values between chimeric mice and humans
in several drugs metabolized through multiple pathways by CYP and non-CYP were observed although there are some
limitations [7]. In addition, each relationship of total CL and the volume of distribution at steady state between predicted from
single-species allometric scaling of chimeric mice and observed data of human indicated further good correlations, respectively.
The plasma concentration-time curves in chimeric mice were also generally similar to the profiles in humans. However, the
predicted plasma concentration-time curve of diazepam from chimeric mice was not superimposable with observed data of
humans [8].
In conclusion, chimeric mice with high replacement of human hepatocytes have potential to predict drug metabolism and PK.
Therefore, the approach using chimeric mice with humanized liver may contribute to PK/PD study as part of translational
research. Furthermore, the development of chimeric mice transplanted with some individual human hepatocytes with various
metabolic activities may be helpful to predict inter-individual differences of PK. However, it is necessary to consider the
contribution of extra-hepatic organs not humanized such as intestine and metabolism in mouse residual hepatocytes in the
liver of chimeric mice with humanized liver. Predictability of diazepam could be improved using chimeric mice with higher
replacement exceeding 80% of hepatocytes. Recently, murine cyp3a knockout chimeric mice with humanized liver, which do
not express murine cyp3a in intestine and liver, were developed. The mice are expected to reduce the effects of residual mouse
metabolic contribution by Cyp3a [9]. In future, humanized mice with gene modification could become the useful animal model.
References:
1. Tateno et al.: Am J Pathol 2004, 165(3): 901-912.
2. Sanoh; Ohta: Biopharm Drug Dispos 2014, 35: 71-86.
3. Sanoh et al.: Drug Metab Dispos 2012, 40(12): 2267-2272.
5. Tanoue et al: Xenobiotica 2013, 43(11): 956-962.
6. Utoh et al.: Am J Pathol 2010, 177(2): 654-665.
8. Sanoh et al.: Xenobiotica 2015, 45(7):605-614.
9. Kato et al.: Drug Metab Dispos 2015, 43(8): 1208-1217.
SCIENTIFIC LECTURES
SL.25
Importance of extrahepatic UDP-glucuronosyltransferase 1A1 in bilirubin metabolism
Fujiwara, R.1; Itoh, T.1; Tukey, R. H.2
1 Department
2 Department
of Pharmaceutics, School of Pharmacy, Kitasato University, 5-9-1 Shirokane, Minato-ku, Tokyo, 108-8641, JAPAN
of Pharmacology, University of California San Diego, La Jolla, California, 92093, USA
Background: Accumulating evidence indicates that breast-fed human infants have higher serum levels of bilirubin
than formula-fed infants and are at a higher risk for bilirubin-induced neurological dysfunction. However, the
mechanism underlying the breast milk-induced jaundice has not been clarified completely. While human infants
develop physiological jaundice, experimental mice or rats do not show the induced serum bilirubin level during
periods of developmental change, suggesting that the function of UDP-glucuronosyltransferase (UGT) 1A1, which
is the sole enzyme responsible for bilirubin metabolism, is different between human and mouse.
Methods: We developed humanized UGT1 (hUGT1) mice, in which the original Ugt1 locus was disrupted and
replaced with the human UGT1 locus. We fed human breast milk or infant formula to neonatal hUGT1 mice and
examined the UGT1A1 expression in the liver and small intestine as well as their serum bilirubin levels.
Results: Breast milk-fed hUGT1 mice developed severe hyperbilirubinemia. Overly extreme hyperbilirubinemia
occurs in 5-10% of newborn hUGT1 mice, with the TSB levels often reaching > 20 mg/dL, leading to the
development of seizures and eventual death due to bilirubin accumulation in brain tissue (Figure). In the hUGT1
mice, serum bilirubin levels gradually increased and the peak was at 14 days after birth (Figure). While UGT1A1
was slightly expressed in the liver, a higher expression of UGT1A1 was observed in the small intestine. Interestingly,
the pattern of serum bilirubin during the development correlated well to the expression pattern of intestinal UGT1A1.
Formula-fed hUGT1 mice had lower serum levels of bilirubin. We found that the hepatic UGT1A1 level was similar
between the breast- and formula-fed hUGT1 mice, but intestinal UGT1A1 was significantry higher in the formulafed hUGT1 mice than in breast-fed ones. A higher expression of intestinal UGT1A1 was observed in the preterm
hUGT1 mice, indicating that breast milk is suppressing the expression of UGT1A1 in the small intestine after birth.
Conclusions: The reduction of serum bilirubin correlates to the induction of the intestinal UGT1A1 expression in the
hUGT1 mice, indicating that UGT1A1 expressed in the gastrointestinal tract plays an important role in bilirubin
metabolism [1,2].
Figure: Serum bilirubin levels in neonatal hUGT1 and wild-type mice.
References:
1. Fujiwara, R. et al.: Gastroenterology. 2012, 142(1): 109-118.
2. Fujiwara, R. et al.: Proc. Natl. Acad. Sci. USA. 2010, 107(11): 5024-5029.
(BIO)ANALYTICS
SL.26
Renaissance of supercritical fluid chromatography: Fast and sensitive analysis of polar drugs
and their metabolites by hyphenated mass spectrometry
Parr, M. K.1; Wüst, B.2; Nägele, E.2; Stanic, M.3; Schmidt, A.1,3
1 Institute
of Pharmacy, Freie Universität Berlin, Königin-Luise-Str. 2+4, 14195 Berlin, Germany
Technologies GmbH, Hewlett-Packard-Str.8, 76337 Waldbronn, Germany
3 Chromicent GmbH, Rudower Chaussee 29, 12489 Berlin, Germany
2 Agilent
HPLC is considered as method of choice for the separation of various classes of drugs and their impurities.
Pharmacopoeias show increasing numbers of methods for identification, purity and assay of APIs utilizing HPLC
based methods. However, some analytes are still challenging as HPLC shows limited resolution capabilities and
highly polar analytes interact only insufficiently on the conventional analytical reversed phase columns. Especially
in combination with mass spectrometric detection only limited possibilities for alteration in the selectivity of the
stationary phase are available. Some highly polar sympathetic drugs and their metabolites showed almost no
retention on different reversed phase (RP) columns generally used for HPLC-MS. Even on the more polar RP
phases like phenylhexyl their analysis remained challenging or even impossible.
Supercritical fluid chromatography (SFC) as orthogonal separation technique to HPLC may help to overcome these
issues.
To check for the general potential selected polar drugs and drug metabolites were analysed by SFC separation as
alternative. All compounds showed sharp peaks, good retention and resulted in retention times clearly separated
from the dead time especially for the very polar analytes. Retention times and elution orders using the SFC method
are different to both reversed phase and HILIC separations due to the orthogonality of the SFC technique. Short
cycle times could be realized. As temperature and pressure strongly influence the polarity of supercritical fluids, a
precise temperature and backpressure regulation is required for the stability of the retention times. As CO2 is the
main constituent of the mobile phase in SFC solvent consumption and solvent waste are considerably reduced.
Acknowledgments: The World Anti-Doping Agency is acknowledged for the financial support of this study within their research grant
(14A03KP).
SCIENTIFIC LECTURES
2.7 Allosteric Regulation
Chairs: K. Mohr, M. Bünemann
SL.27
Molecular Insights into the mechanosensitivity of histamine H1 receptors
Storch, U.1; Erdogmus, S.1; Hoffmann, C.2; Gudermann, T.1; Mederos y Schnitzler, M.1
1 Walther
2 Institute
Straub Institute of Pharmacology and Toxicology, Ludwig Maximilians University, Goethestr. 33, 80336 Munich, Germany
of Pharmacology and Toxicology, University of Würzburg, Versbacher Straße 9, 97078, Würzburg, Germany
Mechanosensation plays a role in several physiological processes for example in the vascular system where it is
important for maintaining blood pressure and blood flow. Our previous findings indicate that in vascular smooth
muscle cells Gq/11-protein coupled receptors and especially angiotensin II AT1 receptors are direct mechanosensors
mediating myogenic vasoconstriction without the involvement of endogenous agonists. From all receptors we have
tested, histamine H1 receptors showed the highest mechanosensitivity. Therefore, we aimed to analyse whether H1
receptors adopt an active receptor conformation which might be different from the agonist induced receptor
conformation. For this, the technique of dynamic intramolecular fluorescence resonance energy transfer (FRET)
was employed which allows monitoring of conformational changes of the receptor. Cerulean, a stable cyan
fluorescent protein was attached to the C-terminus of the H1 receptor and a small tetracysteine-binding motif was
inserted at different positions in the third intracellular loop which allows binding of the small yellow fluorescent
molecule FlAsH. Agonist stimulations with histamine (100 µM) and mechanical stimulations with hypotonic bath
solutions with decreasing osmolarities (275, 250, 225, 200, 150 mOsmol) caused FRET signal decreases. When
compared with agonist challenge, mechanical stimulations resulted in significantly larger decreases of the FRET
signal. Furthermore, the amplitude of mechanically induced FRET signals was concentration dependent since
application of different hypoosmotic solutions with decreasing osmolarities evoked stepwise decreases in FRET
amplitudes. Moreover, mechanically induced FRET signals were significantly reduced in the presence of selective
H1 receptor blockers. Altogether, our findings point to the fact that histamine H1 receptors are mechanosensitive
and adopt distinct active receptor conformations upon mechanical stimulation.
ALLOSTERIC REGULATION
SL.28
Biased signaling and probe dependence at the chemokine receptor CXCR3
Tschammer, N.1
1 Department
of Chemistry and Pharmacy, Medicinal Chemistry, Emil Fischer Center, Friedrich Alexander University, Schuhstraße 19,
91052 Erlangen, Germany
Dysfunctions of the chemokine receptor CXCR3 signaling network are linked to a myriad of pathologies including
autoimmune diseases, cancer and vascular diseases. The efforts so far failed to produce therapeutics, which
specifically modulate the activity of CXCR3 and thus contribute to the symptom relieve or cure. In the contrast to
soluble proteins chemokines small synthetic ligands bind to the chemokine receptors at the allosteric site(s) inside
the hydrophobic pocket formed by transmembrane helices. In general it can be seen that all important unique
features of allosteric receptor control (permissivity, saturation of effect, probe dependence) are embodied in the
chemokine receptor system. While this adds a level of complexity to analyses and approaches to drug discovery,
it also introduces a remarkable capacity for pharmacologic control of this physiological system for therapeutic
advantage.
With the use of our allosteric radioligand RAMX3 [1] and novel boronic acid based negative allosteric modulators
[2] we were able to detect complex mechanisms of allosteric modulation in this receptor. We discovered the first
biased negative allosteric modulator of the receptor that preferentially inhibits the CXCL11-mediated recruitment of
β-arrestin 2 over G protein activation [2]. When the second CXCR3 receptor chemokine ligand CXCL10 was used,
the signalling bias was lost, indicating very clear probe dependence on the allosteric modulator effect [3]. Such
probe-dependent allostery may serve to allow fine tuning of chemokine response in the seemingly redundant arena
of multiple chemokine agonists for receptors. Our efforts to develop positive allosteric modulators (PAMs) resulted
in a discovery of a potent biased PAM that promotes solely the CXCR3-mediated recruitment of β-arrestin2.
Furthermore, our most prominent PAM behaves in the presence of endogenous agonists as functional negative
allosteric modulator (NAM), while it causes the receptor desensitation and thus decreases the amount of receptors
available for the interaction with endogenous chemokines. This type of compounds could mean a breakthrough in
the treatment of inflammatory diseases, because they reduce the responsiveness of CXCR3-expressing cells to
endogenous chemokines and thus attenuate chemotaxis, which would ultimately result in reduce recruitment of the
CXCR3+ cells at the site of inflammation.
Our discoveries expand the ways in which the function of CXCR3 can be manipulated. Moreover they provide new
insights into interactions between allosteric ligands, chemokines and their receptor, which could be exploited for
drug discovery.
Acknowledgments: I thank the German Research Foundation for financial support (DFG; grant TS287/2-1,Graduate Training Schools
GRK1910 and GRK1962), and the European COST Action CM1207 (GLISTEN: GPCR–Ligand Interactions, Structures, and Transmembrane
Signalling: a European Research Network).
References:
1. Bernat et al.: ChemMedChem 2012, 7(8): 1481-1489.
2. Bernat et al.: ACS Chem. Biol. 2014, 9(11):2664-2677.
3. Bernat et al.: ChemMedChem 2015, 10(3): 566-574.
SCIENTIFIC LECTURES
SL.29
Muscarinic M2 receptor allosterism: Context-sensitive signalling
Seemann, W. K.1; Mohr, K.2
1 Department
of Pharmacology, University of Cologne, Cologne, Germany
and Toxicology Section, Institute of Pharmacy, University of Bonn, Bonn, Germany
2 Pharmacology
“Conventional pharmacology” seeks to achieve selective G protein-coupled receptor (GPCR) modulation on the
level of binding by discrimination between receptors and receptor subtypes. Here, we shift focus beyond the binding
event towards signaling. For this, we employed the M2 subtype of muscarinic acetylcholine receptors, which signals
into the Gi/o-pathway of guanyl-nucleotide-binding proteins. M2 receptors are widely distributed in the mammalian
body and control a multitude of physiological functions. For example, as previously shown in M2 knock-out mice,
M2 signaling includes reduction of pain, but also depression of cardiac function [1]. As cholinergic Gi/o-signaling and
adrenergic Gs-signaling converge on the level of intracellular cAMP [2], we hypothesized less effective cholinergic
signaling by dualsteric agonists to occur in tissues under conditions of elevated cAMP. In order to probe feasibility
of this concept, we applied a novel type of agonist engineered to transduce receptor activation with down-tuned
efficacy. These “dualsteric” (bitopic orthosteric/allosteric) ligands activate the receptor protein from the orthosteric
transmitter binding site and simultaneously bind to the receptor’s allosteric vestibule [3], thereby limiting the
receptors conformational transition to the fully active state [4]. With the main focus on cardiac function, in-vitro and
in-vivo measurements showed that dualsteric intrinsic efficacy declines in different M2 receptor expression systems
with elevated intracellular cAMP. In conclusion, the dualsteric design concept provides a novel approach for
pharmacological selectivity and might provide a new avenue to generate antinociceptive therapeutics that lack
major muscarinic side effects.
Acknowledgments: W.K.S. was supported by the North-Rhine Westphalia International Graduate Research School BIOTECH-PHARMA at
the University of Bonn.
References:
1. Gomeza, J. et al.: Proc. Natl. Acad. Sci. USA 1999, 96: 1692-7.
2. Brodde, O. E.; Michel, M. C.: Pharmacol. Rev. 1999, 51: 651-90.
3. Antony, J. et al.: FASEB J. 2009,23(2): 442-50.
4. Bock, A. et al.: Nat. Commun. 2012, 3: 1044.
ALLOSTERIC REGULATION
SL.30
Detection of unexplored allosteric pockets using a “dummy” ligand approach
Hermans, S. M. A.1; Pfleger, C.1; Gohlke, H.1
Department of Mathematics and Natural Sciences, Institute for Pharmaceutical and Medicinal Chemistry, Heinrich-Heine-University,
Universitätsstr. 1, 40225, Düsseldorf, Germany
1
Allosteric regulation is the coupling between separated sites in biomacromolecules such that an action at one site
changes the function at a distant site. The identification of novel allosteric pockets is complicated by the large
variation in allosteric regulation, ranging from rigid body motions to disorder/order transitions, with dynamically
dominated allostery in between [1]. Here, we present a new and efficient approach to probe information transfer
through proteins in the context of dynamically dominated allostery that exploits ”dummy” ligands as surrogates for
allosteric modulators.
In a preliminary study to test the general feasibility, the approach was applied to conformations extracted from a
MD trajectory of the holo and apo structures of LFA1. The grid-based PocketAnalyzer program [2] is used to detect
putative binding sites. “Dummy” ligands were generated for each detected pocket along the ensemble. Finally, the
Constraint Network Analysis (CNA) software, which links biomacromolecular structure, (thermo-)stability, and
function, is used to probe the allosteric response by monitoring altered stability characteristics of the protein due to
the presence of the “dummy” ligand [3,4,5]. The results were compared to those of the holo structure with the bound
allosteric ligand to validate the “dummy” ligand approach.
Remarkably, the usage of “dummy” ligands almost perfectly reproduced the results obtained from the known
allosteric effector. Although it turned out that the intrinsic rigidity of the “dummy” ligands over-stabilizes the LFA1
structure, these results are already encouraging. Even for the LFA1 apo structures, where the allosteric pocket is
partially closed, the results are in agreement with known allosteric effectors. Overall, the results obtained from the
validation of the “dummy” ligand approach are encouraging. This suggests that our “dummy” ligand approach for
the characterization of unexplored allosteric pockets is a promising step towards identifying novel drug targets.
References:
1. Motlagh, H. N. et al.: Nature 2014, 508 (7496): 331–339.
2. Craig, I. R. et al.: J. Chem. Inf. Model. 2011, 51: 2666–2679.
3. Pfleger, C. et al.: J. Chem. Inf. Model. 2013, 53: 1007–1015.
4. Krüger, D. M. et al.: Nucleic Acids Res. 2013, 41 (April): 340–348.
5. Pfleger, C.: Ensemble-Based Framework for Analyzing Dynamically Dominated Allostery (Doctoral Thesis, Heinrich Heine University,
Düsseldorf, Germany) 2014
SCIENTIFIC LECTURES
2.8 Anti-infective Compounds
Chairs: U. Holzgrabe, P. Proksch
SL.31
Vinylsulfone-based inhibitors of rhodesain as new antitrypanosomal compounds
Schirmeister, T.1
1 Institut
für Pharmazie und Biochemie, Johannes Gutenberg-Universität Mainz, Germany
Vinylsulfones are a well-known and promising class of irreversible inhibitors of cysteine proteases (see Fig. 1). An
example is K11777, a compound under clinical development for parasitic diseases like Chagas disease [1] or
African trypanosomiasis. The major cysteine protease of the parasite Trypanosoma brucei rhodesiense causing
the African sleeping sickness is rhodesain which is e.g. involved in parasite infiltration into the central nervous
system [2]. Starting from the structure (pdb: 2P7U) of the enzyme-inhibitor complex of rhodesain with K11777[3]
we are developing new vinylsulfones[4] with altered inhibitory properties in terms of chemical reactivity and affinity,
using an iterative design cycle consisting of quantum mechanical/molecular mechanical (QM/MM) computations,
docking, syntheses, and testing.
Figure 1: Mechanism of inhibition of cysteine proteases by K11777.
Acknowledgments: Financial support by the DFG (SFB 630) is gratefully acknowledged.
References:
1. Rhee, S.-W. et al.: J. Label Compd. Radiopharm. 2013, 56: 461-463.
2. Ettari, R. et al.: Med. Res. Rev. 2010, 30: 136-167.
3. Kerr, I. D. et al.: J. Biol. Chem. 2009, 284: 25697-25703.
4. Schneider, T. et al.: New J. Chem. 2015, 39: 5841-5853.
ANTI-INFECTIVE COMPOUNDS
SL.32
Discovery of dengue protease inhibitors with nanomolar affinity
Klein, C. D.1
1 Institut
für Pharmazie und Molekulare Biotechnologie, Universität Heidelberg, Im Neuenheimer Feld 364, D-69120 Heidelberg, Germany
This presentation will summarize research that resulted in the discovery of inhibitors of dengue virus protease with
affinity in the nanomolar range. Infections with dengue virus and related flaviviruses are a major health concern in
the tropical and subtropical climates, and an expansion of the vectors and viruses into temperate zones is to be
expected. In analogy to the closely related hepatitis C virus, the protease of dengue virus represents an attractive
drug target for the discovery of antiviral drugs [1]. Our work started with the development of a robust assay
procedure [2,3], followed by the screening of potential covalent-reversible electrophilic fragments and other small
molecules [4,5], and continued with fragment-merging and optimization based on the preferred substrate
recognition sequence [6,7]. This allowed us to increase the affinity of dengue protease inhibitors by more than three
orders of magnitude, while maintaining ligand efficiency and selectivity towards other serine proteases. A major
factor in this success was the evaluation of non-natural amino acids with modulated or abolished basicity as
replacements for a key arginine residue. Building blocks with potential promiscuity liabilites could be eliminated in
the optimization process. Future tasks are the optimization of PK properties and the antiviral activity in cell culture,
which remain unsatisfying. In this respect, the introduction of electrophilic fragments with covalent-reversible
binding behavior to the catalytic serine appears to be attractive, in that pharmacokinetic factors such as fast
metabolic clearance become less critical.
Acknowledgments: The work summarized here was supported by grants from the Deutsche Forschungsgemeinschaft, Studienstiftung des
Deutschen Volkes and Konrad-Adenauer-Stiftung.
References:
1. Nitsche, C. et al.: Chem. Rev. 2014, 114: 11348–11381.
2. Steuer, C. et al.: J. Biomol. Screening 2009, 14: 1102-1108.
3. Nitsche, C.; Klein, C. D.: Methods Mol. Biol.2013, 1030: 221-236.
4. Nitsche, C.; Steuer, C.; Klein, C. D.: Bioorgan. Med. Chem. 2011, 19: 7318-7337.
5. Steuer, C. et al.: Bioorgan. Med. Chem. 2011, 19: 4067-4074.
6. Behnam, M. A. et al.: ACS Med. Chem. Lett. 2014, 5: 1037.
7. Bastos Lima, A. et al.: Bioorg. Med. Chem. 2015, in press.
SCIENTIFIC LECTURES
SL.33
The lectin LecB as target for anti-infectives against chronic Pseudomonas aeruginosa infections
Titz, A.1
1 Chemical
Biology of Carbohydrates, Helmholtz-Institute for Pharmaceutical Research Saarland, 66123 Saarbrücken, Germany
The opportunistic pathogen Pseudomonas aeruginosa is currently considered as a major threat to public
health care. The Gram-negative bacterium forms biofilms which protects itself from host defense and
antibiotic therapy resulting in chronic infections. The bacterial lectin LecB is a virulence factor and plays
a prominent role in biofilm formation. Inhibition of lectin function with carbohydrate-based ligands was
shown to disrupt bacterial biofilm formation. Based on the crystal structure of the lectin with its glycan
ligands, we dissected the contributions of individual functional groups to protein binding in a biophysicsguided approach. This knowledge was then used for the development of small and drug-like glycan-based
molecules as LecB inhibitors as future anti-biofilm compounds in chronic Pseudomonas aeruginosa
infections.
References:
1. Sommer, R. et al. ChemistryOpen 2015, doi: 10.1002/open.201500162.
2. Hofmann, A. et al.: Carbohydr. Res. 2015, 412, 34-42.
3. Sommer, R.; Exner, T. E.; Titz, A. PLoS ONE 2014, 9(11): e112822.
4. Hauck, D. et al.: ACS Chem. Biol. 2013, 8(8), 1775–1784.
ANTI-INFECTIVE COMPOUNDS
SL.34
Chlorflavonin inhibits growth of Mycobacterium tuberculosis by targeting branched-chain amino
acid biosynthesis
Rehberg, N.1; Akone, H. S.2; Proksch, P.2; Kalscheuer, R.1
1 Institute
for Medical Microbiology and Hospital Hygiene, Heinrich-Heine-University Düsseldorf, Universitätsstr. 1, 40225 Düsseldorf,
Germany
2 Institute of Pharmaceutical Biology and Biotechnology, Heinrich-Heine-University Düsseldorf, Universitätsstr. 1, 40225 Düsseldorf,
Germany
Mycobacterium tuberculosis, the etiologic agent of Tuberculosis (TB), is one of the leading causes of human
mortality and morbidity due to pathogenic microorganisms. In recent years treatment of TB is increasingly facing
serious problems due to the alarming rise of multidrug-resistant (MDR) and extensively drug-resistant (XDR)
Mycobacterium tuberculosis strains, the latter of which are virtually untreatable with currently available anti-TB
drugs. Thus, there is an urgent need for new antibiotics against TB.
Employing bioactivity guided fractionation, chlorflavonin was isolated from an ethylacetate extract obtained from
the endophytic fungus Mucor irregularis, which has been isolated from the Cameroonian medicinal plant Moringa
stenopetala. Chlorflavonin exhibited strong growth inhibitory activity in vitro against Mycobacterium tuberculosis
(MIC90 1.5 μM), which was strictly dependent on presence of the chlorine atom. In contrast to its potent
antitubercular activity, chlorflavonin exhibited no cytotoxicity towards the human fibroblast cell line MRC-5 and
toward the macrophage like human acute monocytic leukemia cell line THP-1 up to concentrations of 100 µM. The
isolation of spontaneous resistant mutants against chlorflavonin, which occurred at a low frequency of 10-7, allowed
us to understand its mechanism of action. Mapping of resistance-mediating mutations employing whole-genome
sequencing as well as chemical supplementation assays revealed that chlorflavonin specifically inhibits the
acetohydroxyacid synthase IlvB1, which mediates the first common step in branched chain amino acids (BCAA)
and pantothenic acid biosynthesis. Thus, chlorflavonin inhibits growth of Mycobacterium tuberculosis by causing
combined auxotrophy to BCAA and pantothenic acid. While exhibiting a bacteriostatic effect in monotreatment,
chlorflavonin displayed synergistic effects with the first-line antibiotic isoniazid leading to a complete sterilization
and no resistance in liquid culture in combination treatment. Using a fluorescent reporter strain, intracellular activity
of chlorflavonin against Mycobacterium tuberculosis inside infected macrophages was demonstrated that was
superior to streptomycin treatment.
Acknowledgments: This work was supported by the Jürgen Manchot Foundation and the Research Commission of the Medical Faculty of
the Heinrich-Heine-University Düsseldorf.
SCIENTIFIC LECTURES
SL.35
Marine natural products as potential sources for new antitubercular agents
Daletos, G.1
1
Institute of Pharmaceutical Biology and Biotechnology, Heinrich-Heine University, Universitaetsstrasse 1, 40225 Düsseldorf, Germany
Tuberculosis (TB) remains one of the world’s lethal infectious diseases. In 2013, the World Health Organization
(WHO) reported 9.0 million new cases and 1.5 million deaths caused by TB worldwide [1]. Marine organisms
represent a rich source of structurally unique compounds that can be utilized for the search for new antitubercular
drugs from nature [2]. Invertebrates, such as sponges, have hitherto provided the largest number of secondary
metabolites with unique structural features and pronounced biological activities [3]. Our ongoing search for new
anti-TB marine natural products already yielded several promising compounds including manzamine A [4],
aerothionin [5], sceptrin [6], and papuamine [7]. Among these active metabolites a group of unusual cyclic peptides
called callyaerins, from the Indonesian sponge Callyspongia aerizusa appeared particularly interesting for further
investigation. The planar structures of the isolated compounds were unambiguously elucidated on the basis of 1D
and 2D NMR spectroscopic data and MS interpretation. The absolute configurations of their constituent amino acid
residues were determined using the Marfey’s method [8]. The basic structural unit of the callyaerins comprises a
cyclic peptide with a linear peptide side chain, both of variable size, linked through a non-proteinogenic (Z)-2,3diaminoacrylic acid (DAA) functional group [9,10]. All compounds were investigated in vitro against Mycobacterium
tuberculosis, as well as against THP-1 (human acute monocytic leukemia), and MRC-5 (human fetal lung fibroblast)
cell lines in order to assess their general cytotoxicity. Callyaerins were found to inhibit M. tuberculosis at low
micromolar concentrations, which highlights the potential of these compounds as promising anti-TB agents.
Acknowledgments: My sincere gratitude to Prof. Dr. P. Proksch (Heinrich-Heine University, Duesseldorf, Germany) for his admirable
supervision and expertise guidance during my work. I wish to thank Dr. R. Kalscheuer (Heinrich-Heine University, Duesseldorf, Germany)
for carrying out the experiments with M. tuberculosis.
References:
1. Global tuberculosis report 2014; World Health Organization: Geneva, Switzerland, 2014.
2. García, A. et al.: Eur. J. Med. Chem. 2012, 49, 1-23.
3. Proksch, P. et al.: Phytochem. Rev. 2010, 9, 475-489.
4. Sakai, R. et al.: J. Am. Chem. Soc. 1986, 108, 6404-6405.
5. Fattorusso, E. et al.: J. Chem. Soc. D 1970, 12, 752-753.
6. Walker, R. P.; Faulkner, D. J.: J. Am. Chem. Soc. 1981, 103, 6772-6773.
7. Baker, B. J.; Scheuer P. J.; Shoolery J. N.: J. Am. Chem. Soc. 1988, 110, 965-966.
8. Marfey, P.: Carlsberg Res. Commun. 1984, 49, 591-596.
9. Ibrahim, S. R. M. et al.: ARKIVOC 2008, xii, 164-171.
10. Ibrahim, S. R. M. et al.: Biorg. Med. Chem. 2010, 18, 4947-4956.
POORLY SOLUBLES
2.9 Poorly Solubles
Chairs: P. Kleinebudde, W. Weitschies
SL.36
The use of screening tools in the development of amorphous solid dispersions
Page, S.1
1 Pharmaceutical
Research and Development, F. Hoffmann-La Roche Ltd, CH-4070 Basel, Switzerland
With the implementation of high-throughput screening in drug development the number of poorly soluble
compounds increased over time. This is not only reflected in the development pipelines in the pharmaceutical
industry, but also in the number of publications in the area of poorly soluble compounds and in particular in the field
of amorphous solid dispersions. Solid dispersions can be classified according to the physical state of their
components as well as the type of stabilizer used. The first generation of solid dispersions contained crystalline
carriers, whereas the second generation of solid dispersions consists of the drug substance and an polymeric
carrier [1]. These polymeric carriers are added to the system in order to stabilize the amorphous form of the drug
and/or modify their dissolution behaviour. In particular the later property led to the third generation of solid
dispersions, which contain a mixture of a polymer and surfactant, a mixture of polymers or surfactants [1].
An initial stage in the development of amorphous solid dispersions is therefore the selection of an appropriate
polymeric carrier and/or surfactant with the aim to obtain a physical stable amorphous system showing a high
degree of supersaturation. A screening assay should therefore focus on two aspects: physical stability and drug
release. Over the years, different screening tools where developed to enable a good and quick selection of
promising prototype formulations which are later manufactured and tested in vivo.
An overview on different screening tools is presented in this talk [2,3,4], which are used for the selection of an
appropriate formulation composition. In addition, examples underlining the suitability of certain analytical tools for
the later development of the process [5,6] are given.
Acknowledgments: The presenter would like to thank Nicole Wyttenbach, Matthias Eckhard Lauer, Monira Siam, Olaf Grassmann, Reto
Maurer, Laurence Jacob, Joseph Tardio, Emmanuel Scheubel, Christine Janas, Christoph Marschall, Jochem Alsenz, Johannes H Kindt and
Andreas Engel for the collaboration in the last years which enabled the development of suitable screening tools, which foster formulation
development of amorphous solid dispersions and solid solutions.
References:
1. Vasconcelos, T.: Drug Discov. Today 2007, doi: 10.1016/j.drudis.2007.09.005.
2. Wyttenbach, N. et al.: Eur. J. Pharm. Biopharm. 2013 84(3):583-98.
3. Lauer, M.-E. et al.: Pharm. Res. 2011 28(3):572-84.
4. Lauer, M.-E. et al.: Pharm. Res. 2013 30(8):2010-22.
5. Lamm, M. S. et al.: AAPS PharmSciTech 2015, doi: 10.1208/s12249-015-0387-9.
6. Marschall, C.´; Maurer, R.; Page, S.: 1st European Conference on Pharmaceutics – Drug Delivery 2015, Reims.
SCIENTIFIC LECTURES
SL.37
Colloidal carrier systems for the formulation of poorly water-soluble drugs
Bunjes, H.1
1
Institut für Pharmazeutische Technologie, TU Braunschweig, Mendelssohnstr. 1, 38106 Braunschweig, Germany
Poorly soluble drugs pose a considerable formulation challenge, in particular with regard to their peroral and
parenteral administration. The need for finding strategies enabling their pharmaceutical administration becomes
increasingly urgent as more and more drug candidates emerging from the development pipeline display poor
aqueous solubility. An interesting option to formulate these substances is the use of colloidal carrier systems which
even allow intravenous administration due to the small size of the particles acting as drug carriers. Among the
different types of colloidal carrier systems available, lipid dispersions are particularly promising since they mainly
consist of components that can be considered to be physiologically well compatible. Accordingly, colloidal lipid
dispersions like fat emulsions or liposomes have proven to be a valuable option for the delivery of poorly soluble
drugs over many years of therapeutic use.
A broad variety of different colloidal lipid carriers systems (emulsions, liposomes, mixed micelles with phospholipids,
solid and liquid crystalline lipid nanoparticles) is available to the pharmaceutical formulation scientist. Thus, the
question arises which of them would be the most promising for a given drug. In order to address this question in an
efficient way, our group has started to develop a screening method that is based on the loading of preformed carrier
dispersions with the drug in the bulk state. After incubation of the drug with the dispersion, excess drug is filtered
off and the amount of drug loaded into the dispersion can be determined analytically [1]. This method has already
been successfully employed to select promising carrier dispersions for an antileishmanial drug candidate [2]. It has
also proven to be very helpful in solving more fundamentally oriented questions as which parameters affect the
loading capacity of carrier particles prepared from a given lipid [3]. As the latter study required the availability of
lipid dispersions with different, very well defined particle size, the newly established method of membrane
emulsification was included in the processes of dispersion preparation [4]. Particle size and physical state of the
particle matrix were found to be of influence depending, however, on the type of drug substance under investigation.
While, for example, curcumin and amphotericin B could better be loaded in dispersions containing solid triglyceride
particles fenofibrate showed a high preference for the corresponding emulsion particles. The use of dispersions
containing smaller particles was always favorable for drug loading albeit to a different extent [3]. Current work on
this method is directed towards understanding the factors that determine the extent of drug loading and the loading
kinetics. Very slow kinetics are probably the reason why this passive loading approach fails for some drugs that are
known to be stably incorporated into the respective dispersions when they are processed in the conventional way
by directly loading them during dispersion preparation.
Acknowledgments: The scientific contributions of Eva Kupetz, Katrin Göke, Karin Rosenblatt, Sonja Joseph, and Sandra Gehrmann are
gratefully acknowledged as is financial support from the Deutsche Forschungsgemeinschaft and the Niedersächsisches Ministerium für
Wissenschaft und Kultur.
References:
1. Rosenblatt, K.; Bunjes, H.: Poster, 6th World Meeting Pharm., Biopharm., Pharm. Technol., Barcelona, April 2008.
2. Kupetz, E. et al.: Eur. J. Pharm. Biopharm. 2013, 85(3A): 511-520.
3. Kupetz, E.; Bunjes, H.: J. Control. Release 2014, 189: 54-64.
4. Joseph, S.; Bunjes, H.: J. Pharm. Sci. 2012, 101(7): 2479-2489.
POORLY SOLUBLES
SL.38
Oligomeric cross-linkers for hydrogel formulation from ECM-derived macromolecules
Hacker, M.1
1
Pharmaceutical Technology, Institute of Pharmacy, Universität Leipzig, Leipzig, 04317, Germany
Hydrogels are well-established delivery devices for APIs of different molecular sizes and properties, including small
molecules, proteins, peptides and nucleic acids. Hydrogel materials have also gained attention as widely adjustable
artificial extracellular matrices (ECM) for applications in regenerative medicine and tissue engineering. In these
applications the control of hydrogel mechanical characteristics, micro- and nano-structural properties and biological
interactions with cells and tissues are of key importance. ECM components or degradation products thereof such
as collagen, hyaluronan, elastin or gelatin, have become interesting hydrogel building blocks due to their availability,
biocompatibility, degradability and biological function. In order to control stability of such ECM component-derived
gels, cross-linking is typically necessary. Despite the availability of numerous cross-linking strategies, one that
achieves high degrees of cross-linking with low toxicity concerns and options for additional functional modifications
is still to be established. We have recently described the synthesis and multi-functionality of maleic anhydride(MA)containing oligoacrylamides that have a high reactivity against primary amines and can be used for the synthesis
of cytocompatible hydrogels from macromolecules with amine functionalities [1]. With these oligomeric MAcontaining cross-linkers (abbreviated oPNMA1), we developed and characterized a platform of gelatin-based
hydrogels and microparticles that can be easily derivatized by pre-cross-linking functionalization of oPNMA with
monovalent amines [2].
This contribution illustrates our current strategies to expand this platform of chemically cross-linked hydrogels
towards different applications and physico-chemical functionalities important for regenerative applications. First, we
made use of the variety of readily available acrylate and acrylamide monomers in order to find suitable comonomers
that impart further functional characteristics to the oligomeric MA-containing cross-linkers and ultimately to the
resulting hydrogels. We have integrated diacetone acrylamide which introduced ketone groups to the oligomer
chains and characterized the ketone reactivity with the objective to immobilize small molecules but also biological
macromolecules to cross-linked hydrogels via biocomjugation using this new functional group. Through the
selection of predominantly hydrophilic acrylates or acrylamides we were able to synthesize MA-containing
oligomers that allowed for the formulation of injectable gelatin-based hydrogels. Second, tubular hydrogel conduits
were fabricated by means of a static mixer and a tube molding procedure. The tubes were extensively characterized
and are envisioned for applications in peripheral nerve regeneration. In these attempts we also focus on hydrogel
derivatization with small molecules to control cell adhesion and differentiation.
Acknowledgments: The authors thank the German Research Council (DFG SFB/Transregio 67 A1) for financial support. MCH gratefully
acknowledges a travel grant by the Galenus Privatstiftung.
References:
1. Loth, T. et al.: React. Funct. Polym. 2013, 73: 1480-1492.
2. Loth, T. et al.: Biomacromolecules. 2014, 15: 2104-2118.
SCIENTIFIC LECTURES
SL.39
Contribution of Confocal Raman Microscopy (CRM) to the validation of an ex vivo skin
penetration method
Lunter, D.1
Department of Pharmaceutical Technology, Institute of Pharmacy and Biochemistry, Eberhard Karls University Tuebingen, Auf der
Morgenstelle 8, 72076 Tuebingen, Germany
1
Skin diseases are conventionally treated with topical formulations. Depending on the disease, the active may be intended to
act on the skin surface (e. g. disinfectants) or within the skin (e. g. antipruritics). It is therefore of interest to evaluate the
concentration of the active within the skin, particularly if an intradermal action is aimed for. To this end, in vivo, ex vivo or in
vitro penetration experiments can be conducted. Conventionally, ex vivo and in vitro experiments rely on the segmentation of
the skin and subsequent extraction and quantification of the active. The stratum corneum usually withholds the highest amount
of the active. In order to prevent bias of the drug amounts detected in the deeper skin layers, it is pivotal to ensure complete
separation of the stratum corneum from the deeper skin layers. This can be done by tape stripping, cyanoacrylates biopsy,
trypsinization or cryo-segmentation.
In this work the effectiveness of tape stripping, cyanoacrylates biopsy and cryo-segmentation to remove the stratum corneum
is evaluated by confocal Raman microscopy (CRM) and an ex vivo skin penetration method is validated on the basis of this
evaluation. As CRM can give spatially resolved information of the chemical composition of a sample, it can be employed to
distinguish between the stratum corneum and deeper skin layers as they exhibit different chemical compositions and therefore
give different Raman signals. In the course of the experiment skin samples were either treated with 30 tape strips, one or five
cyanoacrylates biopsies or cryo-segmentation. Then cross sections of the samples were prepared and average spectra of the
skin surfaces were acquired. For the subsequent analysis, the spectra were normalized. The normalized average spectra were
compared to the normalized average spectrum of the viable epidermis which was extracted from untreated skin samples. This
was done by calculating the root mean square of the differences between the normalized average spectra of the differently
treated samples and the normalized average spectrum of the viable epidermis. The separation method that yielded the lowest
values removed the stratum corneum most effectively and was selected to be used in a penetration experiment. Here, a
hydrophilic procaine HCl containing gel with or without the penetration enhancer POE-23-lauryl ether served as model
formulation [1,2]. The results were validated by CRM. To this end, skin samples were incubated with the model formulations
and cross sections were made with a cryo-microtome. The cross sections were subsequently analysed by CRM. Colour coded
images of the procaine distribution within the skin were calculated. Furthermore, the procaine content in the skin samples was
evaluated by calculating the procaine-peak intensities in the Raman maps.
The analysis of the Raman spectra showed that the deviations from the spectrum of the viable epidermis decreased in the
order: cryo-segmented skin>tape stripped skin>skin biopsied once>skin biopsied five times. Further analysis of the spectra
revealed, that tape stripping did not remove the SC completely and that the use of a cryo-microtome lead to removal of not
only the stratum corneum but also of parts of the viable epidermis. In the case of the cyanoacrylates biopsies, one biopsy did
not lead to complete removal of the SC but five biopsies were found to remove the SC completely. Furthermore, the deviation
of the spectrum of the five times biopsied skin sample was found to be almost as low as the deviations between spectra from
the viable epidermis of different skin samples. Thus, a penetration experiment was conducted with this method. It could be
shown that the method was capable of elucidating the effect of the penetration enhancer on the skin penetration of
procaine HCl. CRM penetration experiments were performed with the same formulations. They yielded similar results and
thereby confirmed the feasibility of the method.
It was thus proven that the method of cyanoacrylates biopsy is most effective in separating the stratum corneum from the
deeper skin layers. The developed method was capable of detecting differences in penetrated amounts of the model active
which were induced by the addition of a penetration enhancer. CRM was found to be a valuable tool in the validation of the
penetration method.
Acknowledgments: Institute of Experimental Medicine, University of Tuebingen, Schenk, M.
References:
1. Shin, C-S. et al.: Int. J. Pharm. 2004, 287: 73-78.
2. Lunter, D.; Daniels, R.: I. Biomed. Opt. 2014, 19: 126015-126027
PPP IN DRUG DEVELOPMENT
2.10 PPP in Drug Development
SL.40
A PPP for the development of chemical tool compounds
Knapp, S.1,2,3
1 Johann
Wolfgang Goethe-University, Institute for Pharmaceutical Chemistry, Building N240 Room 3.03, Max-von-Laue-Str. 9, D-60438
Frankfurt am Main.
2 Buchmann Institute for Molecular Life Sciences, Riedberg Campus, Max-von-Laue-Str. 15, D-60438 Frankfurt am Main
3 University of Oxford, Nuffield Department of Clinical Medicine, SGC and Target Discovery Institute, Oxford, OX3 7DQ, United Kingdom
Selective small molecule inhibitors (chemical probes) have a major impact on our understanding of human biology
and for the validation of novel disease associated targets for the development of new treatment therapies. However,
the development and characterization of chemical probes is a cost intensive multidisciplinary process requiring
significant efforts in medicinal chemistry, structural biochemistry, screening and cell biology that can rarely be
accomplished by an isolated laboratory [1,2]. These complications led to many probe molecules that have initially
been characterized inadequately and have since been proven to be nonselective or associated with poor
characteristics such as the presence of reactive functionality that can interfere with common assay features.
Unfortunately such reagents are still widely using in academic research leading to wrong target-disease association.
To tackle this challenging problem and to combine expertise and resources in different areas of chemical biology
we formed a large multinational group involving academic research laboratories and also currently 8 large
pharmaceutical companies. This consortium was established based on the Structural Genomics Consortium (SGC)
open access model, which distributes and publishes reagents promptly and without constrains imposed by
intellectual property. The consortium has focussed initially on chemical tools developed in the epigenetic target
area but it has now been extended to include protein kinases, protein interaction and potential targets identified by
genetic analysis such as genome wide association studies.
In this presentation I will present the general setup of the project as well as some selected examples (e.g. BET
bromodomain inhibitors [3,4] and protein kinases [5]) that led to comprehensive validation of new disease targets
and to a fast translation into clinical studies.
Acknowledgments: This work was supported by the SGC, a registered charity (number 1097737) that receives funds from AbbVie, Bayer
Pharma AG, Boehringer Ingelheim, Canada Foundation for Innovation, Eshelman Institute for Innovation, Genome Canada, Innovative
Medicines Initiative (EU/EFPIA), Janssen, Merck & Co., Novartis Pharma AG, Ontario Ministry of Economic Development and Innovation,
Pfizer, São Paulo Research Foundation-FAPESP, Takeda, and Wellcome Trust.
References:
1. Arrowsmith, C. H. et al.: Nat. Chem. Biol. 2015, 11(8), 536–541.
2. Knapp, S. et al.: Nat. Chem. Biol. 2013, 9(1), 3-6.
3. Filippakopoulos, P. et al.: Cell, 2012, 49 (1), 214-231.
4. Filippakopoulos, P.; Knapp, S.: Nat. Rev. Drug. Discov. 2014, 13 (5), 337-356.
5. Gammons, MV. et al.: Invest Ophthalmol, 2013, 54 (9), 6052-6062.
SCIENTIFIC LECTURES
SL.41
Public Private Partnerships in lead discovery: Overview and case study on binding kinetics
Müller-Fahrnow, A.1
1
Bayer Healthcare AG, 13353 Berlin
Pharmaceutical industry is faced by significant challenges in its effort to discover new drugs that address unmet
medical needs. Safety concerns and lack of efficacy are the two main technical reasons for attrition. Improved early
research tools including predictive in vitro and in vivo models as well as a deeper understanding of the disease
biology therefore have the potential to improve success rates. In order to foster innovation and to meet the
challenges, we need to combine internal activities with external collaborations in line with the interests and needs
of all partners.
Different types of partnerships ranging from fee-for-service activities to strategic partnerships with significant and
long-term partnerships will be discussed [1].
Kinetics for Drug Discovery (K4DD) is one example for a public private partnership in which Bayer Healthcare is
actively involved. The overall aim of the K4DD project, supported by the Innovative Medicines Initiative (IMI), is to
enable the adoption of drug-target binding kinetics analysis in the drug discovery decision-making process [2].
Together with 19 other partners, we have defined three focus areas, the so-called work packages:
1. Understanding of binding kinetics at a molecular level
 Ability to design appropriate kinetic behaviour rationally
2. Assay technologies
 Information feedback in line with cycle times in drug discovery
3. Understanding translation to complex systems
 Confidence that differences in binding kinetics lead to differentiation in cell-based assays and in vivo
The status of the project including our work on a new database for binding kinetics will be presented.
Acknowledgments: K4DD has received support from the Innovative Medicines Initiative Joint Undertaking under grant agreement no. 115366, resources of
which are composed of financial contribution from the European Union's Seventh Framework Programme (FP7/2007-2013) and EFPIA companies’ in kind
contribution. More info: www.imi.europa.eu
References:
1. Lessl, M. et al.: Angew. Chem. Int. Ed. Engl. 2013, 2684-268
2. Copeland, R. et al.: Nature Reviews Drug Discovery 2006, 5 (9), 730-739
PPP IN DRUG DEVELPOMENT
SL.42
Development of a selective DYRK1A inhibitor in a PPP framework
Kunick, C.; Falke, H.1; Chaikuad, A.2; Becker, A.1; Loaëc, N.3,4; Lozach, O.3,4; Abu Jhaisha, S.5; Becker, W.5;
Jones, P. G.6; Preu, L.1; Baumann, K.1; Knapp, S.2; Meijer, L.3,4
1 Technische
Universität Braunschweig, Institut für Medizinische und Pharmazeutische Chemie, Beethovenstraße 55,
38106 Braunschweig, Germany.
2 University of Oxford, Nuffield Department of Clinical Medicine, Structural Genomics Consortium, Old Road Campus Research Building,
Roosevelt Drive, Headington, Oxford OX3 7DQ, U.K.
3 ManRos Therapeutics, Perharidy Research Center, 29680 Roscoff, Bretagne, France.
4 CNRS, Station Biologique de Roscoff, ‘Protein Phosphorylation & Human Disease’ group, 29680 Roscoff, France.
5 RWTH Aachen University, Institute of Pharmacology and Toxicology, Wendlingweg 2, 52074 Aachen, Germany.
6 Technische Universität Braunschweig, Institut für Anorganische und Analytische Chemie, Hagenring 30, 38106 Braunschweig, Germany.
Dual-specificity tyrosine phosphorylation-regulated kinase 1A (DYRK1A) is a protein kinase which is constitutively
activated by autophosphorylation at Tyr321. After activation, DYRK1A phosphorylates serine or threonine residues
preferentially on substrates with small hydrophobic residues at the P+1 position [1]. Physiological mechanisms
regulated by DYRK1A comprise cell cycling, Notch signaling, apoptosis, and pre-mRNA splicing, etc. The gene
encoding for DYRK1A is located on chromosome 21 which is present in three copies in Down Syndrome (DS, or
trisomy 21), the most frequent congenital reason of mental retardation. DS individuals indeed show increased
DYRK1A expression in the brain [2,3]. Several observations also link DYRK1A to Alzheimer’s disease (AD).
DYRK1A acts as a priming kinase for phosphorylation of Tau protein by glycogen synthase kinase-3 (GSK-3) and
thus may contribute to the formation of hyperphosphorylated Tau aggregates which appear as intracellular
neurofibrillary tangles in the brains of AD patients. By phosphorylating the β-Amyloid Precursor Protein (APP),
DYRK1A increases APP cleavage by γ- and β-secretases. This cleavage produces amyloid peptides which form
extracellular plaques and lead to DYRK1A upregulation. Based on this evidence, DYRK1A has been suggested as
a target for the development of drugs against neurodegenerative diseases [2,3]. For a detailed investigation of
DYRK1A’s physiological role in various test conditions and as starting points for a rational drug design, small
organic DYRK1A inhibitors would be rather helpful. Although up to now more than a dozen of such DYRK1Ainhibitory chemotypes have been published, none has yet exhibited clear selectivity versus protein kinases of the
structurally related cdc-2 like kinase (CLK) family [2,3]. We here report a structure-guided DYRK1A inhibitor
development campaign during which the screening hit 11H-indolo[3,2-c]quinoline-6-carboxylic acid was modified.
X-ray structure determination of three congeners co-crystallized with DYRK1A confirmed the predicted binding
mode within the ATP binding site in the target kinase and led to the design of to KuFal194, a DYRK1A inhibitor
(IC50 = 6 nM) with cellular activity and a two orders of magnitude selectivity over CLK1 [4]. Ideas, services, results
and conclusions were contributed to this collaborative project by a private biotechnology company (ManRos), a
registered charity (SGC), a public research organization (CNRS) and three universities (University of Oxford, RWTH
Aachen and TU Braunschweig).
Acknowledgments: This research was supported by grants from the ‘Fonds Unique Interministériel” (FUI) TRIAD project (LM), the
“Association France-Alzheimer (Finistère)” (LM), the “Fondation Jérôme Lejeune” (LM), the German research foundation (DFG grant Be
1967/3-1, to WB), and an FP7-KBBE-2012 grant (BlueGenics) (LM). SK receives funding from the SGC, a registered charity (number
1097737) that receives funds from AbbVie, Bayer Pharma AG, Boehringer Ingelheim, the Canada Foundation for Innovation, Genome
Canada, GlaxoSmithKline, Janssen, Lilly Canada, the Novartis Research Foundation, the Ontario Ministry of Economic Development and
Innovation, Pfizer, Takeda, and the Wellcome Trust [092809/Z/10/Z]. AC is supported by the European Union FP7 Grant No. 278568
“PRIMES” (Protein interaction machines in oncogenic EGF receptor signalling). We thank Sophie Kaspar for performing cell-based assays
and Dr. Matthias Engel (Saarland University, Saarbrücken, Germany) for providing the HEK293-tau-DYRK1A cell line.
References:
1. Soundararajan, M. et al.: Structure 2013, 21, 986–996.
2. Becker, W.; Soppa, U.; Tejedor, F. J.: CNS Neurol. Disord. – Drug Targets 2014, 13, 26-33.
3. Abbassi, R. et al.: Pharmacol. Ther. 2015, 151, 87-98.
4. Falke, H. et al.: J. Med. Chem. 2015, 58, 3131-3143.
SCIENTIFIC LECTURES
SL.43
The role of adenosine in colonic inflammation – A study in rat colon preparations in vitro
Voß, U.1; Müller, C. E.2; Abdel-Aziz, H.3; Kelber, O.3; Nieber, K.1
1 Institute
of Pharmacy, University of Leipzig, Brüderstr. 34, 04103 Leipzig, Germany
of Pharmacy, Rheinische Friedrich-Wilhems Universität Bonn, An der Immenburg 4, 53121 Bonn, Germany
3 Scientific Department, Steigerwald Arzneimittelwerk GmbH, Havelstr. 5, 64295 Darmstadt, Germany
2 Institute
Adenosine is a purine nucleoside involved in several physiological functions, regulating a wide variety of immune
and inflammatory responses and acting as modulator of gut functions. While it is usually present at low
concentrations in the extracellular space, stressful conditions, such as inflammation, can markedly increase its
extracellular level up to micromolar range. Recent evidence suggests a prominent role of A2A receptors (A2AR) in
the anti-inflammatory effect of herbal preparations as e.g. an Iberis amara extract (STW 6). Also A2B receptors
(A2BR) have been shown to be involved in the pathophysiology of inflammation. In the current study we investigated
the role of A2AR and A2BR to regulate contractility in untreated and inflamed rat colon preparations using specific
receptor agonists and antagonists.
Inflammation was induced by intraluminal instillation of 2,4,6-trinitrobenzenesulfonic acid (TNBS, 0.01/0.1M,
30min). mRNA-expression was determined using RT-PCR. Contractions were measured isometrically in an organ
bath setup. All four adenosine receptor subtypes were expressed in untreated colon preparations. Activation of A1,
A2B, and A3 receptor with specific agonists reduced the acetylcholine (ACh, 10µM)-induced contractions, while
activation of A2BR enhanced it. After incubation with TNBS morphological damages in colonic mucosa and muscle
walls were detectable followed by reduced ACh-contractions. The TNBS-mediated decrease of ACh-contractions
as well as the morphological damages were partially normalized by co-incubation of TNBS with the A2AR agonist
2-p-[carboxyethyl]phenethylamino-5’-N-ethylcarboxamido-adenosine (CGS 21680, 10µM) or the A2BR antagonist
4-(2,3,6,7-tetrahydro-2,6-dioxo-1-propyl-1H-purin-8-yl-benzenesulfonic acid (PSB 1115, 100µM).
In this study using an in-vitro inflammatory model, we demonstrate that the A2AR agonist CGS 21680 or the A2BR
antagonist PSB 1115 effectively counteracted the development of TNBS-induced disturbances in colon
preparations. This model therefore opens new options for uncovering anti-inflammatory mechanisms of action of
herbal medicinal products.
SCREENING TECHNIQUES IN PHARMACOLOGY & DRUG DEVELOPMENT
2.11 Screening Techniques in Pharmacology & Drug Development
SL.44
Data quality in drug discovery – The role of analytical performance in ligand binding assays
Wätzig, H.1; Oltmann-Norden, I.1; Steinicke, F.1; Alhazmi, H. A.1; Nachbar, M.1; Baumann, K.1; Exner, T.2; Böckler,
F. M.2; El Deeb, S.1
1 Institute
of Medicinal and Pharmaceutical Chemistry, TU Braunschweig, 38106 Braunschweig, Germany.
Institute, University of Tübingen, Auf der Morgenstelle 8, 72076 Tübingen, Germany
2 Pharmaceutical
Despite its importance and all the considerable efforts made, the progress in drug discovery is limited. One main
reason for this is the partly questionable data quality. Models relating biological activity and structures and in-silico
predictions rely on precisely and accurately measured binding data. However, these data vary so strongly, such
that only variations by orders of magnitude are considered as unreliable. This can certainly be improved considering
the high analytical performance in pharmaceutical quality control. Thus the principles, properties and performances
of biochemical and cell-based assays are revisited and evaluated. In the part of biochemical assays immunoassays,
fluorescence assays, Surface Plasmon Resonance (SPR), Isothermal Calorimetry (ITC), Nuclear Magnetic
Resonance (NMR) and Affinity Capillary Electrophoresis (ACE) are discussed in details, in addition radiation-based
Ligand Binding Assays (LBA), Affinity Chromatography, Mass Spectrometry (MS), Atomic Force Microscopy (AFM)
and Microscale Thermophoresis (MST) are briefly evaluated. In addition, general sources of error, such as solvent,
dilution, sample pretreatment and the quality of reagents and reference materials are discussed. Biochemical
assays can be optimized to provide good accuracy and precision (e.g. RSD% < 10%). Cell-based assays are often
considered superior related to the biological significance, however, typically they cannot still be considered as really
quantitative, in particular when results are compared over longer periods of time or between laboratories. A very
careful choice of assays is therefore recommended. Strategies to further optimize assays are outlined, considering
the evaluation and the decrease of the relevant error sources. After avoiding the common error sources and
characterizing assay performances by control charts, a simple as possible representative method for the respective
technique needs to be identified and optimized by an experimental design. After reducing the overall variability,
other hidden error sources can be identified more easily. Furthermore, the degree of method complexity can be
subsequently increased, to study the influence of additional parameters. Analytical performance and data quality
are still advancing and will further advance the progress in drug development.
References:
1. Wätzig, H. et al.: J. Comput. Aided Mol. Des. 2015, DOI: 10.1007/s10822-015-9851-6.
SCIENTIFIC LECTURES
SL.45
Dynamic mass redistribution to probe the signaling repertoire of GPCRs
Schrage, R.1; Mohr, K.1
1 Pharmacology
and Toxicology Section, Institute of Pharmacy, University of Bonn, Germany
G protein-coupled receptors are cell surface receptors which are essential for transmitting extracellular signals
across cell membranes and represent one of the largest and most diverse protein families in the human genome
[1,2]. In humans, GPCRs respond to a plethora of different stimuli including hormones, neurotransmitters, and other
sensory inputs like vision, olfaction, and taste [e.g. 3]. More than one third of all marketed drugs target GPCRs [4]
and a lot of effort is still being put into GPCR drug discovery as an emerging number of “undrugged” receptors
display association with various diseases [e.g. 5].
The receptor conformation of a GPCR is sensitive to the shape and physicochemical properties of a bound ligand
as well as to the type of intracellular adaptor protein [6]. As GPCRs exist in ensembles of possible conformations,
different ligands may stabilize distinct subsets of these conformations [7]. Therefore, the cellular response may be
of unique character in case a specific ligand favors certain signaling pathways over others, a phenomenon termed
“biased signaling” or “functional selectivity”.
Unlike classic pharmacological “snapshot assays” that quantify a particular intracellular pathway such as the
generation of second messengers, optical biosensors have the advantage to capture whole cell responses in intact
cells [8]. In case of GPCR drug discovery, the application of specific toxins like pertussis toxin, which specifically
interferes with Gi protein-mediated pathways, allows unraveling which particular pathways contribute to a whole
cell response induced by an agonist. Here, we present the muscarinic M2 receptor as a paradigm for a GPCR that
promiscuously activates Gi- and Gs-dependent signaling pathways, both of which can be quantified in dynamic
mass redistribution (DMR) experiments applying the EPICTM system [9]. Relative to the endogenous messenger
acetylcholine, the oxotremorine M-related compound iperoxo was identified as a non-biased agonist with
outstanding potency and efficacy in DMR experiments [9]. The physicochemical properties and the high affinity of
iperoxo made it the compound of choice to generate a novel muscarinic radioagonist, yielding [3H]iperoxo.
[3H]iperoxo was the first radioagonist to probe all five muscarinic receptors and allowed detection of activationrelated conformational changes in the receptor protein [10].
Taken together, we present DMR experiments as extremely useful in GPCR drug discovery, especially for GPCRs
that promiscuously interact with a variety of intracellular adaptor proteins.
Acknowledgments: We are grateful for the excellent collaboration with Prof. Dr. Evi Kostenis (University of Bonn, Germany), Prof. Dr.
Marco De Amici (Università degli Studi di Milano, Italy), and Prof. Dr. Ulrike Holzgrabe (University of Würzburg, Germany).
References:
1. Millar, R. P.; Newton, C. L.: Mol. Endocrinol. 2010, 24(1): 261-74.
2. Oldham, W. M.; Hamm, H. E.: Nat. Rev. Mol. Cell. Biol. 2008, 9(1): 60-71.
3. Rosenbaum, D. M.; Rasmussen, S. G.; Kobilka, B. K.: Nature. 2009, 459(7245): 356-63.
4. Overington, J. P.; Al-Lazikani, B.; Hopkins, A. L.: Nat. Rev. Drug Discov. 2006, 5(12): 993-6.
5. Garland, S. L.: J. Biomol. Screen. 2013, 18(9): 947-66.
6. Kobilka, B. K.; Deupi, X.: Trends Pharmacol. Sci. 2007, 28(8): 397-406.
7. Nygaard, R. et al.: Cells 2013, 152(3): 532-42.
8. Schröder, R. et al.: Nat. Biotechnol. 2010, 28(9): 943-9.
9. Schrage, R. et al.: Br. J. Pharmacol. 2013, 169(2): 357-70.
10. Schrage, R. et al.: Biochem. Pharmacol. 2014, 90(3): 307-19.
SL.46
Label-free biosensors help to reveal a new mechanism of GPCR activation
Grundmann, M.1; Hudson, B. D.2; Tikhonova, I. G.3; Smith, N. J.4; Mohr, K.5; Ulven, T.6; Milligan, G.2; Kenakin, T.7;
Kostenis, E.1
1 Molecular-,
Cellular- and Pharmacobiology Section, Institute of Pharmaceutical Biology, University of Bonn, Nussallee 6, 53115 Bonn,
Germany
2 Molecular Pharmacology Group, Institute of Molecular, Cell and Systems Biology, College of Medical, Veterinary and Life Sciences,
University of Glasgow, Glasgow, Scotland, UK
3 Molecular Therapeutics, School of Pharmacy, Medical Biology Centre, Queen's University, Belfast, Northern Ireland, UK
4 Molecular Cardiology Division, Victor Chang Cardiac Research Institute, Faculty of Medicine, University of New South Wales, Sydney,
Australia
5 Pharmacology and Toxicology, University of Bonn, Bonn, Germany
6 Department of Physics, Chemistry and Pharmacy, University of Southern Denmark, Odense M, Denmark
7 Department of Pharmacology, University of North Carolina School of Medicine, Chapel Hill, NC, USA
G protein-coupled receptors (GPCRs) are long runners among drug targets and have an outstanding history in
pharmacology. In recent years, high attrition rates in the drug discovery process tarnish the picture of success.
Insufficient preclinical compound evaluation and the development of drug molecules lacking an innovative modeof-action were identified as key issues during this process. The implementation of new holistic cellular readouts into
early phases of drug research raises hopes to overcome the issues and to reinitiate the development of effective
and successful drugs. Label-free assays can provide an important means to achieve a comprehensive compound
profile already in early drug discovery stages including the identification of the mode-of-action.
GPCRs are highly versatile and regulate complex cell signaling. Since a plethora of signal transduction concepts
are mirrored herein, GPCR biology is a treasure trove for ligands with new mechanism of action. To date, ligands
that target GPCRs can be classified either as orthosteric, allosteric or bitopic. In this talk, I will present data that
underpin the capability of cell-based biosensor assays to disclose a novel molecular mechanism of drug-like
compound action. Embedded into an array of classical GPCR signaling readouts and by using pharmacological
and mutational approaches, we made use of the possibility to track cell activation in real time to discover a new
form of bitopic agonism to spatio-temporally modulate a class A GPCR.
Acknowledgments: We are grateful to Ulrike Rick for expert technical assistance. We thank Sunil Pandey for the synthesis of CATPB. This
work was supported by the Danish Council for Strategic Research (grant 11-11619). The authors are grateful to Corning Inc., Perkin Elmer,
and Molecular Devices for their support on the dynamic mass redistribution as well as cellular dielectric spectroscopy biosensors.
SCIENTIFIC LECTURES
SL.47
Assay technologies addressing GPCRs in drug discovery
Koch, M.1
1 Bayer
Pharma AG, Lead Discovery Wuppertal, Aprather Weg 18a, 42096 Wuppertal
Approximately 40% of all approved drugs target G protein-coupled receptors (GPCRs). This large family of
transmembrane receptors transduces extracellular signals into intracellular effector pathways through the activation
of heterotrimeric G proteins. Therefore, they constitute a prominent class of validated pharmacological targets with
an obvious high druggability. Consequently, extensive efforts are under way to explore and validate further
members of the GPCR family as promising drug targets for various therapeutic areas and to identify novel lead
compounds [1,2].
High throughput screening (HTS) is a well-established technology to identify novel starting points for lead
generation out of a large collection of several millions of small molecules. At Bayer Pharma AG we have established
a unique HTS technology platform capable of testing up to 300,000 compounds per day. Furthermore, we select a
suitable set of HTS-compatible functional assays from an assay toolbox fitting to the specific objectives of the
particular target and disease hypothesis.
The identification of compounds with a reasonable profile for further development is a rare event. Most probably,
primary HTS hits are potential artefacts. Therefore, compound characterization with respect to specificity and
selectivity is an essential component in the HTS workflow. Orthogonal readout systems are applied to exclude
unspecific target interactions or assay readout artefacts. Additionally, selectivity tests against closely related
receptors, isoforms and orthologs provide further crucial information to aid the selection of the most promising lead
candidates.
In the post HTS phase, competitive ligand binding assays provide supplementary evidence on the molecular mode
of action, e.g. for differentiation between orthosteric and allosteric modulation of receptor activity [3]. Furthermore,
radiolabeling of selected candidates allows a detailed investigation of ligand binding kinetics, especially of
residence time which is a key parameter for receptor occupancy.
The presentation will exemplify how different assay technologies addressing GPCRs are applied to support lead
discovery and lead optimization towards new molecular entities.
References:
1. Pierce, K. L. et al.: Nature Rev. Mol. Cell Biol. 2002, 3(9): 639-50.
2. Eglen, R. M.; Raisine T.: Methods Mol. Biol. 2009, 552: 1-13.
3. Tschammer N.: Top. Med. Chem. 2015, 14: 87-118.
SL.48
Semi-automatic fluorescence anisotropy titrations (saFLAT) enhance screening throughput and
data quality
Boeckler, F. M.1; Lange, A.1; Heidrich, J.1; Ansideri, F.1.; Koch, P.1
Laboratory for Molecular Design and Pharmaceutical Biophysics, Department of Pharmaceutical and Medicinal Chemistry, Institute of
Pharmaceutical Sciences, Eberhard Karls Universität Tübingen, Auf der Morgenstelle 8, 72076 Tübingen, Germany.
1
Reliable detection of binding constants and improved screening throughput are typically opposing aims in drug
discovery. Still, both are of utmost importance [1] for the hit and lead identification and optimization stages in
academic and industrial labs. Of course, automatization and robotics have strongly contributed to speeding up data
generation and to enhancing data quality. However, such solutions typically require considerable investments,
constituting an obstacle particularly for academic research. Smart solutions for redesigning the experimental setup
and semi-automatic liquid handling systems can help to accelerate research and enhance data quality at a bargain
price.
Based on similar approaches for the measurement of protein-DNA binding [2], we have designed a fluorescence
anisotropy assay which is performed as a titration in microtitre plates [3]. Fluorescein-labelled probe molecules (as
reporters) are displaced from the target binding site by increasing concentrations of competitor ligands. Changes
in fluorescence anisotropy measured using a CLARIOstar plate reader [4] reveal changes in the proportion of bound
to free fluorescein-labelled probe.
Using a VIAFLO 96-channel pipette [5] in a semi-automatic mode, one titration step can be performed in seconds.
The sample volume during the titration is kept constant by aspirating the same volume of the sample prior to addition
of an aliquot of the competitor compound from a source plate. In order to keep the concentration of the fluoresceinlabelled probe and the protein constant, stock solutions of the competitor compounds are mixed with the same
concentration of reporter and protein as in the initial sample. By this process, only the concentration of the
competitor compound changes during the titration. To minimize the errors associated with handling small volumes
and still perform a titration covering several pKD units, different stock solutions of the competitor compounds (in
multiple source plates) can be used subsequently for the titration.
Based on this experimental setup, the KD value of 96 compounds can be determined simultaneously in one single
screening experiment. The number of titration steps and the possible pKD range is predominantly limited by the
solubility of the competitor compounds. Alternatively, the possibility to determine many KD values in parallel can be
used as well to increase the number of replications of the experiment and, therefore, the data reliability.
References:
1. Wätzig, H. et al.: J. Comput. Aided Mol. Des. 2015, in press, DOI: 10.1007/s10822-015-9851-6
2. Veprintsev, D. B.; Fersht, A. R.: C. Nucleic Acids Res. 2008, 36(5): 1589-1598.
3. Vogel, S. et al.: Proc. Natl. Acad. Sci. U. S. A. 2012, 109(42): 16906-16910.
4. BMG Labtech CLARIOstar, http://www.bmglabtech.com/en/products/clariostar/
5. INTEGRA Biosciences VIAFLO 96, http://www.integra-biosciences.com/sites/96_384_channel_pipette.html
SCIENTIFIC LECTURES
2.12 Signaling in Cell Death
SL.49
Sensitization of the anti-cancer efficacy of the Bcl-2 family inhibitor ABT-263 by natural
compounds
Polier, G.1; Giaisi, M.1; Köhler, R.1; Müller, W. W.1; Krammer, H. P.1; Li-Weber, M.1
1
Tumorimmunology Program (D030), German Cancer Research Center (DKFZ), D-69120 Heidelberg, Germany
Tumor initiation, progression and resistance to therapies are tightly associated with over-expression of antiapoptotic proteins Bcl-2, Bcl-xL, Bcl-w and Mcl-1. ABT-263 (Navitoclax), an orally bio-available small-molecule
mimetic of the Bcl-2 homology domain 3, inhibits Bcl-2, Bcl-xL, and Bcl-w and has shown anti-cancer effects mainly
on lymphomas and lymphocytic leukemia. Despite promising results obtained from the clinical trials, the use of
ABT-263 in patients is dose-limited due to induction of death of thrombocytes via inhibition of Bcl-xL and subsequent
thrombocytopenia. Besides, many tumors resist treatment due to high levels of Mcl-1 expression or develop
resistance via up-regulation of Mcl-1 during long-term exposure. These obstacles highlight the demand to improve
the ABT-263-based therapy. Recently, we show that agents that inhibit the translation initiation or transcription
elongation, such as the natural compounds Rocaglamides and flavones, e.g. wogonin, baicalein, apigenin, chrysin
and luteolin, can enhance ABT-263-induced apoptosis and thereby decrease its effective dose in different cancer
cell lines and in primary AML and ALL cells by down-regulation of Mcl-1 expression. Importantly, these natural
compounds do not enhance the toxicity of ABT-263 to proliferating normal T cells and thrombocytes. These
compounds also potentiate the lethality of ABT-263 in cancer cells which have acquired resistance to ABT-263.
Finely, we show that combination of wogonin with ABT-263 promotes in vivo tumor regression in a human T-cell
leukemia xenograft mouse model. Our study demonstrates that the combination therapy can lower the effective
dose of ABT-263 and thereby possibly decreasing the risk of adverse side effects.
Acknowledgments: This work was supported by the Helmholtz Alliance on Immunotherapy of Cancer in the Helmholtz association and
Deutsches Konsortium für Translationale Krebsforschung (DKTK).
References:
1. Ebada, S. S. et al.: Prog. Chem. Org. Nat. Prod. 2011, 94: 1-58.
2. Polier, G. et al.: Cell Death Dis. 2011, 2:e182.
3. Li-Weber, M.: Int. J. Cancer. 2015, 137(8):1791-9.
4. Polier, G. et al.: Int. J. Cancer. 2015, 136(3):688-98.
SINGALING IN CELL DEATH
SL.50
miR181b is induced by the chemopreventive polyphenol curcumin and inhibits breast cancer
metastasis via downregulation of the inflammatory cytokine CXCL1 and 2
Bachmeier, B. E.1; Kronski, E.1; Fiori, M. E.2; Barbieri, O.3,4; Astigiano, S.3; Mirisola, V.5; Killian, P. H.1; Bruno, A.6;
Pagani, A.6; Rovera, F.7; Pfeffer, U.8; Sommerhoff, C. P.1; Noonan, D. M.6,7; Nerlich, A. G.9
1 Institute
of Laboratory Medicine, Ludwig-Maximilians-University, Munich, Germany
of Hematology, Oncology and Molecular Medicine, Istituto Superiore di Sanità, Rome, Italy
3 Department of Experimental Medicine, University of Genoa, Genoa, Italy
4 Embryogenesis and Tumorigenesis in Animal Models, IRCCS AOU San Martino-IST National Cancer Research Institute, Genoa, Italy
5 Integrated Molecular Pathology, IRCCS AOU San Martino-IST National Cancer Research Institute, Genoa, Italy
6 Scientific and Technologic Pole, Fondazione Onlus MultiMedica, Milan, Italy
7 Department of Biotechnologies and Life Sciences, University of Insubria, Varese, Italy
8 Department of Surgical and Morphological Sciences, University of Insubria, Varese, Italy
9 Institute of Pathology, Academic Hospital Munich-Bogenhausen, Munich, Germany
2 Department
Chronic inflammation is a major risk factor for the development and metastatic progression of cancer. We have
previously reported that the chemopreventive polyphenol Curcumin inhibits the expression of the proinflammatory
cytokines CXCL1 and -2 leading to diminished formation of breast and prostate cancer metastases. In the present
study, we have analyzed the effects of Curcumin on miRNA expression and its correlation to the anti-tumorigenic
properties of this natural occurring polyphenol. Using microarray miRNA expression analyses, we show here that
Curcumin modulates the expression of a series of miRNAs, including miR181b, in metastatic breast cancer cells.
Interestingly, we found that miR181b down-modulates CXCL1 and -2 through a direct binding to their 3’-UTR.
Overexpression or inhibition of miR181b in metastatic breast cancer cells has a significant impact on CXCL1 and 2 and is required for the effect of Curcumin on these two cytokines. miR181b also mediates the effects of Curcumin
on inhibition of proliferation and invasion as well as induction of apoptosis. Importantly, overexpression of miR181b
in metastatic breast cancer cells inhibits metastasis formation in vivo in immunodeficient mice. Finally, we
demonstrated that Curcumin up-regulates miR181b and down-regulates CXCL1 and -2 in cells isolated from several
primary human breast cancers.
SCIENTIFIC LECTURES
SL.51
Shikonin and its derivatives inhibit phosphorylation of the epidermal growth factor receptor
signaling and synergistically kill glioblastoma cells in combination with erlotinib
Zhao, Q.1; Efferth, T.1
1 Department
of Pharmaceutical Biology, Institute of Pharmacy and Biochemistry, Johannes Gutenberg University, Staudinger Weg 5,
Mainz, 55128, Germany
Overexpression and mutation of the epidermal growth factor receptor (EGFR) gene play a causal role in
tumorigenesis and resistance to treatment of glioblastoma (GBM) [1]. EGFR inhibitors such as erlotinib are currently
used for the treatment of GBM; however, their efficacy has been limited due to drug resistance [2]. New treatment
strategies are therefore urgently needed. Shikonin, a natural naphthoquinone, induces both apoptosis and
necroptosis in human glioma cells [3,4], but the effectiveness of erlotinib-shikonin combination treatment as well as
the underlying molecular mechanisms is unknown yet. In this study, we investigated erlotinib in combination with
shikonin and 14 shikonin derivatives in parental U87MG and transfected U87MG.∆EGFR GBM cells. Most of the
shikonin derivatives revealed strong cytotoxicity. Shikonin together with five other derivatives, namely
deoxyshikonin, isobutyrylshikonin, acetylshikonin, β,β-dimethylacrylshikonin and acetylalkannin showed synergistic
cytotoxicity toward U87MG.∆EGFR in combination with erlotinib. Moreover, the combined cytotoxic effect of
shikonin and erlotinib was further confirmed with another three EGFR-expressing cell lines, BS153, A431 and DKMG. Shikonin not only dose-dependently inhibited EGFR phosphorylation and decreased phosphorylation of EGFR
downstream molecules, including AKT, P44/42MAPK and PLCγ1, but also together with erlotinib synergistically
inhibited ∆EGFR phosphorylation in U87MG.∆EGFR cells as determined by Loewe additivity and Bliss
independence drug interaction models. These results suggest that the combination of erlotinib with shikonin or its
derivatives might be a potential strategy to overcome drug resistance to erlotinib.
Acknowledgments: Department of Pharmacognosy, Institute of Pharmaceutical Sciences, University of Graz, Graz; Austria Faculty of
Pharmacy, University of Athens, Athens, Greece; Departament de Farmacologia, Facultat de Farmacia, Universitat de Vale`ncia,Valencia,
Spain; Prof. Rudolf Bauer, Prof. Ioanna Chinou, Prof. José-Luis Rios.
References:
1. Furnari, F.B. et al.: Genes & development 2007, 21: 2683-710.
2. Zhu, J. J.; Wong, E. T.: Current molecular medicine 2013, 13: 358-67.
3. Chen, C. H. et al.: Ann. Surg. Oncol. 2012, 19: 3097–106.
4. Huang, C. et al.: Plos One 2013, 8(6): e66326.
SINGALING IN CELL DEATH
SL.52
A novel autodisplay based screening assay for the identification of small molecules that inhibit
the dimerization of human chaperone Hsp90
Bopp, B.1: Ciglia, E.2; Ouald-Chaib, A.2; Groth, G.3; Gohlke, H.2; Jose, J.1
1 Institute
of Pharmaceutical and Medicinal Chemistry, Westfälische Wilhelms-Universität. PharmaCampus. Corrensstr. 48, 48149 Münster,
Germany
2 Institute of Pharmaceutical and Medicinal Chemistry, Heinrich-Heine-University Düsseldorf, Universitätsstr. 1, 40225 Düsseldorf,
Germany
3 Institute for Biochemical Plant Physiology, Heinrich-Heine-University Düsseldorf, Universitätsstr. 1, 40225 Düsseldorf, Germany
Human Hsp90 (Hsp90) is a homodimeric chaperone, essential for the maturation of numerous proteins. Some of
these proteins are involved in tumor formation and growth, which makes Hsp90 an interesting drug target for cancer
treatment [1].
Here, we describe a novel autodisplay based method to screen for small molecules that inhibit PPI. The Autodisplay
technology was used to express the chaperone Hsp90 on the surface of Escherichia coli. Functional folding and
dimerization was confirmed by binding of FITC-labeled p53, a natural client protein of Hsp90 and subsequent
analysis by flow cytometry. By computational analysis hot spots in the C-terminal domain of Hsp90 important for
dimerization were identified [2]. These hot spot predictions were used to design peptides that were supposed to
inhibit Hsp90 dimerization. Because binding of FITC-labeled p53 is dependent on Hsp90 dimer formation, a loss of
fluorescence in the flow cytometer analysis indicates inhibition. This reduction in fluorescence turned out to be
dose-dependent with respect to the inhibitor concentration, and an IC50 of 8.96 µM could be determined for the
best inhibitor H3. By microscale thermophoresis measurement with the purified C-terminal domain of Hsp90 it was
verified that H3 indeed binds the C-terminal domain of Hsp90 with a KD value of 2.19 µM. Up to now, H3 is the first
inhibitor shown to target the C-terminal dimerization domain of Hsp90 with a KD value in the low micromolar range
[3].
References:
1. Wegele, H.; Müller, L.; Buchner, J.: Rev. Physiol. Biochem. Pharmacol. 2004, 151: 1-44.
2. Ciglia, E. et al.: PLoS One 2014, 9: e96031.
3. Bopp, B. et al.: 2015, submitted
SCIENTIFIC LECTURES
2.13 Anticancer and Epigenetic Drugs
Chairs: M. Kassack, M. Jung
SL.53
Targeted cancer therapies
Rauh, D.1
1
Technische Universität Dortmund, Fakultät für Chemie und Chemische Biologie, Otto-Hahn-Strasse 6, 44227 Dortmund (Germany)
The goal of my lab is to develop, synthesize and facilitate desperately needed tools to study target protein function
in cells and organisms to push research to exciting new frontiers. In our research, we employ organic synthesis,
biochemical and cellular compound screening, protein X-ray crystallography, structure-based design as well as
target identification for the development of inhibitors and functional probes to perturb proteins of interest. A strong
focus in the lab is on personalized medicine for the treatment of cancer. Here, we closely collaborate with clinical
oncologists to better understand the mechanisms of drug resistance and to develop compounds to overcome
acquired drug resistance in non-small-cell lung cancer and gastrointestinal stromal tumors. We are motivated that
our techniques and investigations will lead to a better understanding of the molecular and cellular causes of fatal
diseases and stimulate the development of new drugs. Some of the labs’ research initiatives on targeted cancer
therapies will be outlined [1-5].
References:
1. Engel, J. et al.: J. Med. Chem. 2015, in press.
2. Richters, A. et al.: ACS Chem. Biol. 2015,10, 289-298.
4. Fang, Z. et al.: ACS Chem. Biol. 2015, 10, 279-288.
4. Mayer-Wrangowski, S. C. et al.: Angew. Chem. Int. Ed. Engl. 2015, 54, 4379-4382.
5. Weisner, J. et al.: Angew. Chem. Int. Ed. Engl. 2015, 54, 10313-10316.
ANTICANCER AND EPIGENETIC DRUGS
SL.54
Role of apoptosis signaling in tumorigenesis and therapy resistance
Wesselborg, S.1
1 Institute
for Molecular Medicine I, University of Düsseldorf, Universitätsstr. 1, 40225 Duesseldorf, Germany
Activation of the endogenous suicide program (apoptosis) is one of the major mechanisms by that cancer cells are
eliminated by antitumor agents. Though the primary site of action might differ, the basic outcome of most anticancer
drugs is usually the same – induction of the apoptotic program in the cancer cell. Conversely, defects within the
apoptotic signaling machinery convey resistance of tumors to radio- and chemotherapy. Understanding of the
molecular events that regulate the apoptotic machinery induced by anticancer drugs and the measures that the
tumor cell takes to avoid apoptosis might help to develop new therapeutic strategies and drug development.
Theoretically, a cancer cell is nothing but a cell propagating its anarchistic death defying principle of unlimited self
dissemination that – if cellular safeguard mechanisms or the immune system fail to keep under control – is finally
terminated by the demise of the whole organism itself. The basic objective of chemotherapy is therefore to exploit
the different activities that distinguish malignant tumor cells from non-malignant cells in order to specifically
eliminate the 'anarchistic dropouts'. An ideal anticancer drug would accompolish to specifically target the malignant
cancer cell and leave the rest of the non-malignant cells in our body unaffected. Due to detrimental side effects on
non-malignant tissues and the virtue of some tumors not to respond to the respective chemotherapy or to develop
resistance during chemotherapy this ideal concept is unfortunately rarely achieved. However, research of the last
three decades improved our knowledge of tumor biology and enabled the identification of new targets for therapy.
Thus, in addition to uncontrolled cell proliferation, it is well established now that cancer cells also need to induce
their supply with oxygen and nutrients by the induction of angiogenesis of surrounding blood vessels, extravasation
and metastasis by releasing matrix metalloproteases for extracellular matrix degradation and so on. Whereas, the
1980's were dominated by research on the mechanisms of uncontrolled proliferation the 1990's also brought
another feature of cancer cells into focus – that is the counterpart of mitosis i.e. apoptosis. Thus, it turned out that
tumor cells not only propagate their anarchistic desire for unlimited proliferation but they also inactivate external
and internal triggers of the cellular suicide program.
Anticancer drugs are used as therapeutic agents since 1940 with more or less success. Per definition a
chemotherapeutic agent is any drug that contributes to tumor destruction. For decades the major feature of
chemotherapeutic agents had been the infliction of DNA-damage. This kind of therapy basically targets cells with
a high proliferation rate that cannot evade the consequences of DNA-damage by cell cycle arrest or DNA-repair.
Consequently, other benign but highly proliferating tissues such as bone marrow, mucosal membranes, hair follicles
and cells of the gastrointestinal are also affected. Recently, due to the rapidly expanding knowledge of the signal
transduction pathways in tumor biology, a new generation of anticancer drugs evolved in form of ‘targeted therapy’
that complements conventional DNA-damaging radio- and chemotherapy. This new generation of antitumor agents
utilizes genuine features of cancer cells as targets for anti-tumor therapy. Hence, these new approaches target
receptor tyrosine kinases for growth factors (such as EGF, PDGF, TGF-α), inhibit intracellular protein kinases (such
as PKC, PKA, RAF), the proteasome or histone deacetylases (HDACs), and so forth.
SCIENTIFIC LECTURES
SL.55
A novel CREBBP/p300 inhibitor and its molecular effects in cancer cells
Lucas, X.1; Hügle, M.2; Ostrovsky, D.3; Decker, S.4, Gerhardt, S.2; Einsle, O.2; Dierks, C.4; Breit, B.3; Wohlwend,
D.2; Günter, S.1
Institute of Pharmaceutical Sciences, Albert-Ludwigs-University, Hermann-Herder-Str. 9, 79104 Freiburg, Germany
of Biochemistry, Albert-Ludwigs-University, Albertstr. 21, 79104 Freiburg, Germany
3 Institute of Organic Chemistry, Albert-Ludwigs-University, Albertstr. 21, 79104 Freiburg, Germany
4 Department of Hematology/Oncology, University Medical Centre Freiburg, Hugstetter Str. 55, 79106 Freiburg, Germany
1
2 Institute
Proteins that are involved in the regulation of chromatin and transcriptional control represent an important targetclass and provide new opportunities for the development of small molecules as anti-cancer agents. Histone
modification proteins are classified as 'writers', 'erasers' and 'readers'. The last comprise, among others, the wellstudied bromodomain (BRD) family, which specifically recognises acetylated lysine residues. Within the 61 different
BRDs especially the eight members of BET-subfamily have been subjected to several drug discovery campaigns,
and some of the resulting drugs are now in clinical trials for atherosclerosis, acute myeloid leukemia (AML) and
other hematologic malignancies.
Here, we present the results of a model-based drug discovery campaign aiming for the identification of new
inhibitors against CREBBP and p300. Their bromodomains are outside the BET subfamily, and their role in cancer
development is not yet completely understood. An identified high-affinity fragment [1] was further optimized by a
fragment-growing approach. We could show that the resulting inhibitor can successfully inhibit the proliferation of
cells from different cancer types. In vitro screenings with primary blood cells from chronic lymphocytic leukemia
(CLL) patients could show that the observed cytotoxic effects are specific for CLL-cells. Additionally, RNA-seq
analysis could show that the activity of the inhibitor can be related to the down-regulation of important driver genes
involved in CLL progression.
References:
1. Lucas X, et al.: Angew Chem Int Ed Engl. 2013, 52: 14055-14059.
ANTICANCER AND EPIGENETIC DRUGS
SL.56
Macrocyclic inhibitors of human histone deacetylase enzymes
Olsen, C. A.1
1 Center for Biopharmaceuticals and Department of Drug Design & Pharmacology, Faculty of Health and Medical Sciences, University of
Copenhagen, Universitetsparken 2, DK-2100, Copenhagen, Denmark.
A brief introduction to the histone deacetylase (HDAC) enzymes, or perhaps more appropriately the lysine
deacylase (KDAC) enzymes [1], will be given, and the lecture will then be devoted to work on macrocyclic peptidebased inhibitors of this class of enzymes. Several cyclic tetrapeptide and depsipeptide natural products have proven
useful as biological probes and drug candidates due to their potent activities as HDAC inhibitors. These chemotypes
exert their activity through a dual mechanism of action involving binding to the catalytic pocket as well as the surface
of the enzyme where substrate proteins dock. Here, the synthesis of such a class of cyclic tetrapeptide HDAC
inhibitors, the azumamides, will be presented along with structurally modified analogues. We thus achieved total
syntheses of azumamides B–D for the first time, which corroborated the originally assigned structures and enabled
full profiling of the HDAC inhibitory properties of the entire selection of azumamides A–E [2]. Furthermore, an
extended series of analogues containing various structural modifications was evaluated by HDAC profiling, NMR
structure determination, and molecular docking to HDAC crystal structures to reveal insight into the requirements
for potent HDAC inhibition by macrocyclic peptides that disrupt the interaction between HDAC enzyme and
substrate protein [3,4].
References:
1. Olsen C. A. Angew. Chem. Int. Ed. 2012, 51: 3755-3756.
2. Villadsen J. S. et al.: J. Med. Chem. 2013, 56: 6512–6520.
3. Maolanon, A. R. et al.: J. Med. Chem. 2014, 57: 9644–9657.
4. Villadsen, J. S. et al.: Med. Chem. Commun. 2014, 5: 1849–1855.
SCIENTIFIC LECTURES
SL.57
Correlation of conformation with cytotoxic activity of α-aminoxy oligopeptides: A circular
dichroism study
Rüther, A.1; Diedrich, D.2; Rodrigues Moita, A. J. 2; Kurz, T.2; Kassack, M. U.2; Hansen, F. K.2; Lüdeke, S.1
1
Institute of Pharmaceutical Sciences, Albert-Ludwigs-Universität Freiburg, 79104 Freiburg, Germany
of Pharmaceutical and Medicinal Chemistry, Heinrich-Heine-Universität Düsseldorf, 40225 Düsseldorf, Germany
2 Institute
Therapeutic peptides may have active and inactive conformations. The equilibrium between these conformations
depends both on the amino acid sequence and on external parameters, such as pH, or interaction with membranes.
Therefore, in order to understand their influence a therapeutic context, it is critical to identify the conformational
response of peptides or peptidomimetics on environmental stimuli [1].
As circularly polarized light interacts with helically disposed transition dipoles in the peptide backbone, circular
dichroism (CD) spectroscopy is exceptionally sensitive toward conformational changes and provides spectra with
characteristic patterns that are typical for secondary structure [2]. While CD is a widely used tool to study the
conformation of canonical peptides, there is little CD data available for peptidomimetics, such as peptoids [3], or αaminoxy peptides [4]. In particular studies on α-aminoxy peptides are of interest. Due to their proteolytic and
conformational stability they are promising drug candidates [5].
We studied spectra recorded for different cytotoxic α-aminoxy peptides obtained through combined solutionphase/solid-phase synthesis. The α-aminoxy peptides show a remarkable pH-dependent change of their secondary
structure. The presence of liposomes as a model for cell membranes also induces changes in the CD spectrum.
This suggests that membranolytic effects observed in preliminary mode of action studies with cancer cells are
concomitant with conformational changes that occur upon binding of the α-aminoxy peptides to the cell membranes.
References:
1. Sinthuvanich C. et al.: J. Am. Chem. Soc. 2012, 134: 6210–6217.
2. Toniolo, C.; Formaggio, F.; Woody, R. W.: Comprehensive Chiroptical Spectroscopy. (Wiley) 2012.
3. Laursen, J. S. et al.: Nat. Commun. 2015, 6: DOI: 10.1038/ncomms8013.
4. a) Li X.; Yang, D.: Chem. Commun. 2006: 3367–3379. b) Yang D. et al.: Chem. Asian. J. 2010, 5: 1356–1363.
5. a) Draghici, B. et al.: RSC Adv. 2011, 1: 602–606. b) Li X, Wu Y. D.; Yang, D.: Acc. Chem. Res. 2008, 41: 1428–1438.
PERSONALIZED MEDICINE – BIOMARKER AND DIAGNOSTICS
2.14 Personalized Medicine – Biomarker and Diagnostics
Chairs: O. Queckenberg, T. Dingermann
SL.58
The challenge to leverage genetic biomarkers from research level to routine clinical IVD testing
Ortmann, R.1
1
QIAGEN GmbH, Hilden, Germany
During recent years companion diagnostics reached high importance for pharmaceutical development programs
and guidance on clinical treatment decisions. European countries achieved a leading position through early uptake
of companion diagnostics in clinical routine, with the highest patient testing coverage worldwide. Commercial CEin-vitro-diagnostic products and laboratory developed tests, mainly developed by European universities, are applied
side by side. Quality assessment systems, like EQA, are in place. Still, there are some key challenges in
personalized medicine, which will impact future companion diagnostic development and commercialization projects:
1. the increasing complexity of testing, e.g. biomarker panel testing and NGS technologies, 2. the higher throughput
of samples, esp. in liquid biopsy based treatment monitoring, 3. the consolidation of test results to clinically
actionable result reports, through bioinformatics, 4. the EU directive on higher regulatory requirements on
companion diagnostics, and 5. the proof of clinical utility to payers. Previously, clinical drug development projects
guided the evolution of companion diagnostics in general. Now, commercialization success of new drug-companion
diagnostic co-launches increasingly impacts decisions on biomarker strategy in pharmaceutical companies. Further
growth of a strong European position in personalized medicine can be achieved only through a close collaboration
of pharmaceutical and diagnostic companies with clinicians and molecular diagnostic laboratories, as the maxim of
decisions needs to move from early development towards routine clinical application. The consequences of this
change will be addressed during the talk.
SCIENTIFIC LECTURES
SL.59
The potential of biomarkers to support decisions making in clinical studies in cardiovascular
indications
Kramer, F.1
1
Clinical Sciences – Experimental Medicine, BAYER HealthCare AG, Aprather Weg 18a, 42113 Wuppertal, Germany
Cardiovascular (CV) diseases such as heart failure (HF) are complex syndromes, which are driven by multiple
pathomechanisms. Therefore, a comprehensive assessment of the status of different pathomechanisms in every
individual patient is the key to a therapy serving patient`s needs. Due to the complexity of CV disease, a large group
of pharmacotherapies have been established as standard of care (SoC) in CV indications. This comprises, amongst
others, beta-blockers (BB), angiotensin converting enzyme inhibitors (ACEIs), angiotensin receptor blockers
(ARBs), diuretics, calcium channel blockers and mineral corticoid receptor antagonists (MRAs). Use of these agents
has led to a significant improvement of patient care and a decline in deaths from CV diseases [1]. As many of those
therapies target the periphery rather than the heart, there is still a high medical need for therapeutic concepts which
target for example inflammation processes, tissue remodelling or energy metabolism in the myocardium.
Furthermore, the fact that a significant number of patients do not respond to a given therapy stresses the need for
new therapies [2].
To enable successful clinical development of drug candidates in CV indications a comprehensive diagnosis,
including a definition of underlying pathomechanisms in the target population, is essential. Furthermore, the ability
to reliably monitor safety and to detect early signs of efficacy during a clinical study are key elements of modern
drug development. Biomarkers serve as a tool for both: first to stratify patients for a clinical study investigating a
certain mode of action (MoA) and second, to detect early signs of efficacy or undesired side effects. Today the term
“biomarker” is used in a much broader sense and comprises more than just molecular markers to be quantified in
biospecimens, such as blood, urine, saliva or tissue. A comprehensive characterization of patient`s disease status
and drug candidate properties can only be achieved when information provided by molecular biomarkers is
complemented by information derived from a diversity of approaches such as imaging, emerging functional
measurements and medical device-derived data. Especially, the use of data derived from devices such as
implanted cardiac defibrillators (ICDs), cardiac re-synchronization therapy (CRT) devices and pacemakers bear the
potential to allow better surveillance of patient`s disease status upon therapy. Last but not least, data which in the
past could only be generated by implanted devices can nowadays be derived from wearable patches with telemonitoring features. This is the basis for the generation of precious real life data and an increased compliance of
patients in clinical studies because tele-monitoring allows a paradigm shift from an in-hospital to an at-home
diagnosis and thereby reduces frequency of scheduled hospital visits in clinical studies.
Taken together, the availability to have a diversity of biomarker approaches builds the basis for the design of smarter
and potentially smaller clinical studies in well characterized patient populations. A comprehensive assessment of
different pathomechanisms in every individual patient by using different biomarker types allows selection of the
right drug for the right patient and thereby paves the way to a more personalized cardiovascular medicine.
References:
1. Nabel E. G.; Braunwald E.: A Tale of Coronary Artery Disease and Myocardial Infarction. N. Engl. J. Med. 2012; 366:54-63.
2. FDA Report: Paving the Way for Personalized Medicine: FDA’s Role in a New Era of Medical Product Development. October 2013.
PERSONALIZED MEDICINE – BIOMARKERS AND DIAGNOSTICS
SL.60
From “omics”-technologies to stratified medicine in autoimmune diseases
Lautscham, G.1; Budde, P.1; Zucht, H.1; Chamrad, D.1; Telaar, A.1; Vordenbäumen, S.2; Schulz-Knappe, P.1;
Schneider, M.2
1 Protagen
2 Centre
AG, Otto-Hahn-Straße 15, 44227 Dortmund, Germany
of Rheumatology, Heinrich-Heine- University Düsseldorf, Düsseldorf, Germany.
The inherent complexity of clinical manifestations and variety of therapeutic has made treating autoimmune diseases like lupus
especially challenging. Systemic lupus erythematosus (SLE) is an autoimmune disease affecting approximately 5 million
people worldwide, but early diagnosis, differentiation to other autoimmune diseases and prognostic stratification are still great
challenges. Hence, SLE represents an enormous challenge for the clinical development of effective therapies and therapeutic
options for patients remain limited, with the last half a century seeing only Benlysta® approved by the FDA. The degree of
heterogeneity amongst SLE patients is a major obstacle for pharmaceutical development and needs to be overcome before
effective and curative therapies can be identified. In order for this to happen, there is a clear need for both diagnostic biomarkers
(Dx) and assays that enable precise disease characterization, patient stratification and response prediction (CDx).
In SLE, several immune defects have been described, leading to the overproduction of autoantibodies against cellular and
nuclear antigens and immune-mediated damage of several organs and tissues. Although, the process by which self-molecules
become immunogenic is not yet fully understood, specific autoantibodies serve as important diagnostic reporter for SLE. While
most of the current diagnostic autoantibody assays are based on antigens that have been identified in tissue extracts, the
application of “omics” technologies allows to the address the complexity of the disease. The SeroTag® autoantibody profiling
technology enables systematic screening for disease-specific changes in autoantibody reactivities using recombinant proteins
produced from tissue specific expression libraries. Thus, the detection of a broad set of SLE-associated autoantibodies (AABs)
might help to investigate the number, co-prevalence and similarities of AAB reactivities in SLE patients supporting a
personalized disease management approach.
We have recently conducted autoantibody profiling studies of SLE, systemic autoimmune diseases, and healthy controls and
found known diagnostic autoantibodies and novel SLE-associated autoantibodies [1]. Confirmed antigens were employed to
identify clustered autoantibodies and their clinical association in SLE and a multiplexed AAB array was developed. The
discovery phase constituted the broad characterization of serum samples from SLE patients, AID patients (SSc, RA, AS) and
healthy controls. against 6,912 recombinant human proteins using the bead-based Luminex technology. This work resulted in
the development of NavigAID SLE, enabling stratification of SLE into distinct subgroups.
The Protagen NavigAID SLE is a stratification array based on a solid database of more than 700 SLE patients, combining 87
selected known and proprietary biomarkers for the diagnosis and differential diagnosis of SLE, analysis of disease activity,
detection of potential organ involvement, and activity of interferon type I response genes, as well as characterisation of four
homogenous SLE patient subgroups. This array distinguishes between SLE patients ranging from a highly reactive patient
group, who have a high disease activity score and possess broad and homogenous positive autoantibody reactivity, through
to a smaller group of patients who have comparatively low levels of autoantibody reactivity. The co-prevalence of multiple
autoantibodies in SLE patients has rarely been analysed [2,3]. However, this has now been examined in substantially greater
detail and has proven to be invaluable for defining more homogeneous patient groups and solving the problem of heterogeneity
in SLE.
This array forms the basis for the alignment of CDx development with clinical programmes for SLE treatment, thereby
increasing the probability of successful drug development. A greater emphasis on this co-development model, bringing together
CDx and drug development groups, will allow for a much greater understanding of the disease in the earliest possible stages.
This can result in the earlier identification of possible side effects, a shortening of overall trial lengths and numerous
improvements to drug efficacy and safety.
References:
1. Lueking A. et al.: Ann. Rheum. Dis. 2013;72:A535.
2. Voss A. et. al.: J. Rheumatol. 2008, 35(4), 625–630.
3. Meilof J. F. et. al.: J. Autoimmun. 1997, 10(1), 67–75.
SCIENTIFIC LECTURES
2.15 Focused Pharmaceutical Research
Chairs: S. Laufer, D. Steinhilber
SL.61
Project group translational medicine and pharmacology, Fraunhofer IME
Steinhilber, D.1,2; Parnham, M.1; Geisslinger, G.1,3
1 Fraunhofer
IME-TMP, Theodor-Stern-Kai 7, 60596 Frankfurt am Main
für Pharm. Chemie, Max-von-Laue-Str. 9, 60438 Frankfurt am Main
3 Institut für Klinische Pharmakologie, Klinikum der Goethe-Universität, Theodor-Stern-Kai 7, 60596 Frankfurt am Main
2 Institut
Pharmaceutical research promotes the development of new drugs and enhances our understanding of how they
work. However, R&D costs have increased exponentially whereas the number of new drug registrations has
declined steadily over the last 10 years. One critical factor is that the identification of drug targets for inadequately
understood diseases requires more extensive investment in discovery research but has a high attrition rate. This
reflects the lack of validated clinical models for efficacy and safety, and intensive efforts are currently underway to
develop new disease models and preclinical/clinical biomarkers, allowing R&D projects to be translated into benefits
for patients.
The research focus of the project group is drug research, development of predictive preclinical and clinical models
of disease and clinical research. The synergy generated by housing predictive preclinical and clinical models under
one roof will make it easier to take early go/no-go project decisions. We have developed validated disease models
covering the fields of cardiovascular, neurodegenerative and chronic inflammatory gastrointestinal diseases, acute
inflammation and pain (inflammatory, neuropathic, oncological and post-operative), arthritic and skin disorders.
Based on the internal expertise in the field of pathophysiological signalling pathways, we perform research on novel
innovative therapeutic approaches (systems medicine). Drawing on cutting-edge research activities and intellectual
property within Goethe University Frankfurt, we apply the latest technology and research concepts to our
collaborative projects, with pre-competitive research focusing on the treatment of chronic inflammatory joint
disease, pain, neurodegenerative disorders and cardiovascular disease. The project group covers a portfolio of
technologies for drug research and development across the value chain.
FOCUSED PHARMACEUTICAL RESEARCH
Neuroallianz consortium – Example of an academic-industrial collaboration
SL.62
Müller, C. E.1; Pfeifer, A.2
1
PharmaCenter Bonn, 1Pharmaceutical Institute, Pharmaceutical Chemistry I, University of Bonn, An der Immenburg 4, D-53121 Bonn;
of Pharmacology and Toxicology, Biomedical Center, University of Bonn, Sigmund-Freud-Str. 25, D-53105 Bonn
2 Institute
The lengthy process of drug development up to market approval and the associated high risk, rising costs,
increasing complexity, as well as high regulatory demands, require novel strategies. In the framework of the
BioPharma competition by the BMBF (German Federal Ministery of Education and Research) strategic alliances
between pharma-/biotech-companies and academic research institutions are financially supported, which cover the
complete value chain from target identification and validation up to marketing of the developed drugs. Neuroallianz
focuses thematically on the development of drugs for neurodegenerative diseases such as Alzheimer’s and
Parkinson’s. For these indications there is a high, rapidly increasing medical demand due to the ageing population
in our country. The consortium “Neuroallianz” implements a novel strategic partnership model between universities
(Bonn University is the leading university partner), non-university, publicly funded research institutions (e.g.
Research Center Jülich, Fraunhofer Institute St. Augustin), biotech- and pharma-companies. We have currently 20
partners, 10 from academia and 10 from industry; the leading industry partner in the consortium is UCB Pharma,
Monheim.
Neuroallianz is currently working on 14 projects, 6 therapeutic, and 5 diagnostic ones; moreover 2 infrastructure
projects belong to the Neuroallianz portfolio: “compound library”, and “information technology”; in addition we set
up a management project. Importantly, we have a strategy in place to foster public relations activities including
information for the general public, as well as scientific publications and presentations. In addition, we organize
advanced training programs on a regular basis for coworkers and students.
Our optimized model of academic-industrial collaboration is a success story, which results in a win-win situation for
all partners – for the benefit of the patients.
SCIENTIFIC LECTURES
The Interfaculty Centre for Pharmacogenomics and Pharma Research (ICEPHA)
SL.63
Laufer, S.1; Schwab M.2
1 Institute
of Pharmacy, Eberhard-Karls-University Tübingen
Fischer-Bosch-Institute of Clinical Pharmacology (IKP), Stuttgart
2 Margarete
Both Tübingen University and the IKP have a long history in academic drug discovery with several recent success
stories in bringing ideas from bench to bedside. The ICEPHA was found in 2007 to concentrate competences and
capabilities of the faculty of science, the faculty of medicine and the University Hospital Tübingen, the Dr. Margarete
Fischer-Bosch-Institute of Clinical Pharmacology and the Robert-Bosch-Hospital, Stuttgart. The ICEPHA
established an interconnection between chemical and biological sciences and human medicine, thus forming a
dynamic network in focussed research areas across academic expertise and pharmaceutical industry users.
ICEPHA is a research network as well as a service and development center for innovative drugs and therapies.
Such a construction allows to carry out research projects that can not be handled efficiently through one single
institution.
ICEPHA’s main interest is concentrated on genes which (A) affect the susceptibility of patients to drugs or (B) are
associated with the manifestation of disease. Program (A) provides knowledge for defining and investigating targets
for custom-tailored drugs. Program (B) is the basis for a predictable individualized therapy with patient-directed
drugs and dosage. This is of prime importance for the maximum benefit for the patient’s health after therapeutic
intervention, but of equal importance for the economy of the public health system. Seed and basic financing is
provided from the founding organisation. Substantial funding for basic research could be raised as well from industry
and Helmholtz (2 professorships each). BMBF and EU programs are sources for project funding as well.
Beside the research mission, the ICEPHA developed a graduate program as well. Major aim of the ICEPHA
graduate program are “membrane-associated drug targets in personalized cancer medicine” to improve future
outcomes for people with cancer. In order to achieve this goal, the members of the program take advantage of the
synergy achieved through their combined expertise in the basic and clinical sciences.
The following key activities provide a general framework of the research in our consortium:
• Validation of novel drug targets for personalized anti-tumor therapy
• Identification of molecular markers as reliable predictors of treatment response
• Establishment of new drugs for innovative treatment regimes
• Optimization of the transfer of resources, technologies and expertise from academia to healthcare
research and development of clinical trials in pharmaceutical industry
FUTURE MOLECULAR DESIGN
2.16 Future Molecular Design
Chairs: G. Schneider, H. Gohlke
SL.64
Automated design of bispecific small molecule drugs
Hopkins, A. L.1,2
1
2
Division of Biochemical Chemistry and Drug Discovvery, School of Life Science, University of Dundee, Dundee, DD1 3DF
ex scientia Ltd. Dundee Incubator, James Lindsay Place, Dundee, DD1 5JJ
Many effective drugs act via modulation of multiple proteins rather than single targets. Advances in systems biology
are revealing a phenotypic robustness and a network structure that strongly suggests bispecific compounds
targeting two nodes in a network may exhibit superior clinical efficacy. In this presentation I will discuss the
development of our automated drug design platform and its successful application to the discovery and optimization
of novel, bispecific small molecules that deliberately target proteins from distinct gene families. The application of
the design algorithms is demonstrated by the automated invention of bispecific ligands that hits a novel combination
of two enzymes from distinct gene families. Screening and X-ray crystallography experiments confirm the initial
designs exhibit nanomolar potency against both primary targets, as well as high selectivity.
SCIENTIFIC LECTURES
SL.65
KRIPO-Protein binding site similarities for drug design
Ritschel, T.1,2
1 Centre
for Molecular and Biomolecular Informatics (CMBI), Radboud university medical center, 6500 HB Nijmegen, The Netherlands
of Medicinal Chemistry, Faculty of Sciences, Amsterdam Institute for Molecules, Medicines and Systems (AIMMS), VU University
Amsterdam, Amsterdam, The Netherlands
2 Division
Key Representations of Interaction in POckets (KRIPO) is a computational method to detect similar protein binding
sites independent of sequence or structural alignment [1]. The characteristics of binding pockets containing a ligand
are encoded in 3D-pharmacophore fingerprints. The binding site fingerprints were optimized to improve their
performance. A variety of attributes of the fingerprints were considered for the optimization, including the placement
of pharmacophore features, whether or not the fingerprints are fuzzified, and the resolution and complexity of the
pharmacophore fingerprints (2-, 3- and 4-point fingerprints). Finally, fuzzy 3-point pharmacophore fingerprints were
chosen as an optimum representation of localized binding sites in a searchable fragment database.
The KRIPO fingerprint representation is key for fast and efficient processing of PDB scale crystal structure
databases. Therefore, an on-the-fly searching of PDB scale crystal structure database for application in drug design
is feasible.
As application examples bioisosteric replacement, off-target prediction and GPCR binding site analysis will be
presented.
References:
1. Wood, J. D. et al.: J. Chem. Inf. Model. 2012, 52(8):2031-43.
FUTURE MOLECULAR DESIGN
SL.66
Predicting the sites and products of drug metabolism
Kirchmair, J.1; Glen, R. C.2; Tyzack, J. D.3
1 Center
for Bioinformatics, University of Hamburg, Bundesstr. 43, 22763 Hamburg, Germany
for Molecular Informatics, University of Cambridge, Lensfield Road, CB2 1EW, Cambridge, United Kingdom
3 Optibrium Ltd., 7221 Cambridge Research Park, Beach Drive, CB25 9TL, Cambridge, United Kingdom
2 Center
The metabolic system has evolved as the main line of defence of living organisms against foreign, potentially
hazardous substances, by transforming them into readily excretable metabolites [1]. Metabolites can have
physicochemical and pharmacological properties that differ substantially from those of the parent compound [2].
Hence understanding the metabolic fate of xenobiotics is of utmost importance to the development and use of
drugs, cosmetics, agrochemicals, and in fact, any chemicals exposed to biological systems [3].
Today a plethora of computational methods for predicting the sites (atom positions in a molecule where
biotransformations are initiated) and products of drug metabolism are available [3,4]. In this contribution we provide
an overview of the capabilities and limitations of current predictors and present two of our own methods for site of
metabolism prediction: FAME (FAst MEtabolizer) [5] and an approach based on a new probabilistic machine
learning method (RASCAL - Random Attribute Subsampling Classification ALgorithm) [6]. FAME is based on a set
of random forest models that are derived from a set of >100k automatically annotated biotransformations. It covers
phase 1 and phase 2 metabolism of xenobiotics (including drugs and natural products) in various different species
and obtains top-1, top-2 and top-3 rates (i.e. percentage of molecules for which at least one known site of
metabolism is predicted among the top-k highest ranked atom positions) of 71%, 81% and 87% on an external test
set. The second set of models, derived using the RASCAL algorithm, is focused on cytochrome P450-mediated
metabolism. While trained on a much smaller dataset, the method obtains top-1, top-2 and top-3 rates of up to 78%,
91% and 94% (depending on the isozyme).
References:
1. Testa, B.: In Drug Metabolism Prediction (ed. Kirchmair, J.) (Wiley-VCH) 2014.
2. Kirchmair, J. et al.: J. Chem. Inf. Model. 2013, 53(2): 354-367.
3. Kirchmair, J. et al.: Nature Rev. Drug Discov. 2015, 14: 387-404.
6. Tyzack, J. D. et al.: J. Cheminf. 2014, 6(1): 29.
SCIENTIFIC LECTURES
SL.67
Enforcing drug discovery by computational molecular design
Schneider, G.1
1
Department of Chemistry and Applied Biosciences, ETH Zürich
Innovative bioactive agents fuel sustained drug discovery and the development of new medicines. Future success
in chemical biology and pharmaceutical research will fundamentally rely on the combination of advanced synthetic
and analytical technologies that are embedded in a theoretical framework that provides a rationale for the interplay
between chemical structure and biological effect. A driving role in this setting falls on leading edge concepts in
computer-assisted molecular design and engineering, by providing real-time access to a virtually infinite source of
novel tool compounds and lead structures, and guiding experimental screening campaigns. We will present
concepts and ideas for the representation of molecular structure, predictive models of structure-activity
relationships, the de-orphaning of bioactive compounds, automated molecular design, and discuss de novo design
approaches that have proven their usefulness and will contribute to future drug discovery by generating innovative
bioactive agents. Emphasis will be put on the reaction-based construction of potent and selective enzyme inhibitors
and modulators of G-protein coupled receptors. As we are currently witnessing strong renewed interest in bioactive
natural products we will showcase new methods for natural-product inspired molecular design and macromolecular
target prediction.
References:
1. Reker, D. et al.: Nature Chem. 2014, 6: 1072–1078.
2. Reker, D. et al.: Proc. Natl. Acad. Sci. USA 2014, 111: 4067–4072.
3. Reutlinger, M. et al.: Angew. Chem. Int. Ed. 2014, 53: 4244–4248.
4. Rodrigues, T.; Schneider, P.; Schneider, G.: Angew. Chem. Int. Ed. 2014, 53: 5750–5758.
5. Spänkuch, B. et al.: Angew. Chem. Int. Ed. 2013, 52: 4676–4681.
6. Schneider, G.: Nat. Rev. Drug Discov. 2010, 9: 273–276.
MEDICATION SAFETY IN SPECIAL PATIENT GROUPS
2.17 Medication Safety in Special Patient Groups
Chairs: K. Friedland, U. Jaehde
SL.68
Medication safety of pregnant and lactating women
Scherneck, S.1; Schaefer C.2
1 Institute
of Pharmacology, Toxicology and Clinical Pharmacy, Technical University of Braunschweig, Mendelssohnstr. 1, 38106
Braunschweig, Germany
2 Institute for Clinical Teratology and Drug Risk Assessment in Pregnancy, Charité - Universitätsmedizin Berlin, Augustenburger Platz 1,
13353 Berlin, Germany
The use of drugs during pregnancy and lactation is a critical situation for the health care professional. A possible
risk of embryonic or fetal damage caused by a drug has to be weighed against an inadequate treatment of the
mother. For the most pharmaceuticals data are insufficient to precisely assess their risk and safety. Randomized
controlled trials exclude pregnant and breast-feeding women for ethical reasons. In addition, most therapies in
these special patient groups are off-label. For a qualified risk assessment, common reference books like the “Rote
Liste” lack adequate information. Drug labels are often imprecise and tend to overestimate potential risks. Readymade drug risk information/labelling requires detailed information on the magnitude of risks including specification
of the severity of expected toxic effects such as birth defects, if applicable. In case no risk has been found, the
extent of human data should be specified on which safety conclusions were based. Furthermore, detailed
recommendations are required how to counsel and manage in inadvertent drug exposure during an unplanned
pregnancy. In contrast to ready-made risk labelling, Teratology Information Services (TIS) provide individual
counselling and allow studies on drug safety. The largest German TIS, the Institute for Clinical Teratology and Drug
Risk Assessment in Pregnancy, counsels 15,000 information requests per year. Data of drug-exposed pregnant
patients are prospectively ascertained using structured questionnaires. Birth defect rates, effects on prematurity,
miscarriage rates and other pregnancy complications can be investigated in association with prenatal drug
exposure. During recent years several studies based on TIS-data have substantially contributed to risk estimation
of a wide range of pharmaceuticals such as fluoroquinolones, antirheumatic MTX, rivaroxaban, TNF-alpha blockers,
and atypical neuroleptics. These studies improve both the knowledge of drug effects on the unborn and the care of
pregnant women.
Acknowledgments: This work was supported by the German Federal Institute for Drugs and Medical Devices (BfArM).
References:
1. Schaefer C. et al.: Arzneimittel in Schwangerschaft und Stillzeit (Urban & Fischer/Elsevier), 8th ed. 2012.
2. Schaefer C.; Peters P.; Miller R. K.: Drugs during Pregnancy and Lactation (Elsevier), 4th ed. 2015.
SCIENTIFIC LECTURES
SL.69
Medication for neonates, infants and children
Läer, S.1*
Institute of Clinical Pharmacy and Pharmacotherapy. Heinrich-Heine Universitaet Duesseldorf
* on behalf of the LENA Consortium http://www.lena-med.eu/
1
Drug use in children is characterized by a high rate of off-label use of about 30-50%. A systematic drug development
in Europe has not started before 2007 when the European Paediatric Regulation has come into force. Drug
development for children provides systematic data for drugs on dosing, dosing regimens according to safety
measures, efficacy, short term safety and longterm safety. The LENA project acknowledges the current
shortcomings of paediatric drug development and aims to develop an orally administered age-appropriate
formulation of enalapril for use in neonates, infants and children. The development process will include investigatordriven trials that will collectively generate all necessary data for devising a paediatric-use marketing authorization
(PUMA). The project will therefore conclude with a completely tested product ready for broad dissemination to the
pediatric population in the European Union Member States.
Acknowledgments: The research leading to these results has received funding from the EU’s Seventh Framework Programme (FP7/20072013) under grant agreement n°602295 (LENA).
MEDICATION SAFETY IN SPECIAL PATIENT GROUPS
SL.70
Medication safety of elderly patients in nursing homes
Jaehde, U.1; Kulick, M.1; Bitter, K.1
1
Institute of Pharmacy, Department of Clinical Pharmacy, University of Bonn
The multimorbidity of elderly patients often leads to polymedication. Therefore, these patients are at particular risk
to suffer from drug-related problems and adverse drug reactions. Several studies have shown that the incidence of
adverse drug reactions is particularly high in long-term care facilities leading to severe consequences such as
hospitalization and death. In a prospectively designed cross-section analysis in North Rhine-Westfalia the incidence
of adverse drug reactions was found to be 8 per 100 resident-months with a preventability rate of 60%. The majority
of adverse drug events were caused by CNS and cardiovascular drugs [1].
Based on these data, a structured multiprofessional intervention was designed consisting of five measures: (1)
intensive seminars for nurses and pharmacists, (2) advanced training for the prescribing general practitioners, (3)
the implementation of a reminder card summarizing high-risk drugs and monitoring issues, (4) the formation of
medication safety teams in each nursing home consisting of a nurse and a pharmacist, and (5) structured
documentation and communication regarding individual drug therapy. The feasibility of this intervention was studied
in 4 nursing homes in North Rhine-Westphalia [1]. Its efficacy is currently being evaluated in a large interventional
trial (AMTS-AMPEL) in 18 nursing homes in North Rhine-Westphalia and Mecklenburg-Western Pomerania funded
by the German Ministry of Health (BMG).
Another approach to enhance medication safety of nursing home residents is based on a simple medication
analysis by community pharmacists which is currently being evaluated in collaboration with a health insurance
(AOK Rheinland/Hamburg) and the Pharmacists’ Association North Rhine. Based on prescription data of the AOK,
the current medication and further information from the nursing home, the pharmacists detect drug-related problems
(DRP) and propose potential solutions to the prescribing general practitioner. In a feasibility study including five
community pharmacies, the pharmacists detected two DRP per patient in average. 33% of the DRP were drugdrug interactions of which 40% were considered as relevant for the residents’ medication safety. 39% of the patients
took drugs considered as potentially inadequate in the elderly [2]. This new approach has currently been extended
to 17 further community pharmacies and 20 nursing homes in North Rhine-Westphalia.
In conclusion, incidence and severity of adverse drug events indicate serious deficiencies in the health care of
elderly patients living in nursing homes. Medication safety-enhancing interventions are currently being developed
in Germany that have the potential to improve the health status of the residents and to reduce costs, e.g. by avoiding
unnecessary falls and hospitalization.
References:
1. Jaehde U.; Thürmann P.: Z Evid Fortbild. Qual. Gesundh. wesen. 2012, 106: 712-6.
2. Bitter K. et al: DPhG Annual Meeting, September 23rd-25th, Düsseldorf, 2015.
SCIENTIFIC LECTURES
2.18 Hot Topics in Pharmaceutical Biology – Young Investigators in
the Spotlight
Chairs: S. Alban, A. Vollmar
SL.71
Cold atmospheric plasma – A future therapeutic approach? Bioanalytics as route to fundamental
understanding
Wende, K.1; Schmidt, A.1; Bekeschus, S.1; Hasse, S.1; Lalk, M.2; Masur, K.1; von Woedtke, T.1,3
Leibniz Institute for Plasma Science and Technology, INP Greifswald e.V., Felix-Hausdorff-Str. 2, 17489 Greifswald, Germany
EMA University of Greifswald, Institute of Biochemistry, Felix-Hausdorff-Str. 4, 17489 Greifswald, Germany
3 Greifswald School of Medicine Institute for hygiene and environmental health, Walter-Rathenau-Str. 49A, 17475 Greifswald, Germany
1
2
Plasma medicine is an emerging biomedical field in which cold physical plasmas are generated to deliver a delicate mixture of
reactive components to cells and tissues. Cold plasmas are complex gaseous systems, delivering energy to the tissue mainly
as light and chemical entities. Of these, short lived oxygen or nitrogen centred reactive species gained the most attraction due
to their potential ability to interact with biomolecules in a direct or indirect way. While knowledge on their generation and
distribution in gas phase is rich, less is known about the composition of species in liquids [1]. Recent studies suggested a
beneficial role of cold plasmas in the healing of chronic wounds wherein cells of the immune system are assumed to be
responsible for the misled inflammatory response in the area of neoplastic diseases [2]. The mechanisms beyond these
observations remain elusive and require exploration.
Using bioanalytic techniques like molecular biology, metabolomics, transcriptomics and proteomics, the mechanism of plasma
– tissue interaction was investigated. Data show, that cellular processes of human cells could both be stimulated or inhibited.
Accordingly, gene and protein expression was affected by cold plasma treatment. The nuclear erythroid-related factor 2 (Nrf2)
and phase II enzyme-pathway components were found to act as key controllers orchestrating the response [3]. Paracrine
signals for cell-cell communication were detected, e.g. interleukin release. Wound relevant immune cells showed a markedly
increased differentiation and changes in the metabolism [4]. For some conditions, an impact on the p53 – mitogen activated
kinase pathway (MAPK) was found which led to pro- and anti-apoptotic events. No genotoxicity was detected in HRPT1 gene
mutation assay [5].
Cold plasma
Redox balance &
oxidative signaling
Eukaryotic cells & tissues
p53 – MAPK signaling
Nrf2 pathway activation
Protein expression/degradation
Interleukin secretion
Pro- and antiapoptotic signals
Metabolism
Migration
These data show that plasma impacts the cellular redox balance and intra- and extracellular signalling events. By this,
hormesis-like effects bracing the cells against stress from within or without are activated, e.g. by the expression of defence
enzymes, stimulation of cell differentiation, or metabolic changes. The findings spotlight the complex interaction of cold plasma
with eukaryotic tissues and underline the demand for further elucidation. Results obtained so far foster the understanding of
the current clinical observations and contribute to the recognition of the molecular processes leading to cold plasma generated
effects in tissues.
Acknowledgments: This work was founded in part by German Federal Ministry of Education and Research (grant number 03Z2DN11).
References:
1. Wende, K. et al.: Biointerphases 2015, 10(2): 029518
2. Klebes, M. et al: J Biophotonics 2015, 8(5): 382
3. Schmidt, A. et al.: J. Biol. Chem 2015, 290(11): 6731
4. Bekeschus, S. et al.: Ox. Med. Cell. Longevity 2015, article 607969
5. Wende, K. et al.: Mutat. Res-Gen. Tox. En. 2015, submitted
HOT TOPICS IN PHARMACEUTICAL BIOLOGY – YOUNG INVESTIGATORS IN THE SPOTLIGHT
SL.72
About the potential of plant senescence as a new source for drug discovery
Sendker, J.1
1
Institute of Pharmaceutical Biology and Phytochemistry, University of Münster, Correnstrasse 48, 48149 Münster, Germany
In the course of plant senescence, a genetically controlled program is started that accounts for nutrient reallocation
and establishes a strong oxidative environment in the senescing plant tissue. While the impact of senescence
conditions on the proteome and primary metabolites has been intensely investigated, only little effort has been
made towards elucidating senescence-associated changes of the secondary metabolome. Studies on the plant
species of Prununs laurocerasus L., Hedera helix L., Juglans regia L. and Solanum dulcamara L. indicate that
secondary metabolites undergo reproducible alterations in senescing leaves, ranging from subtle modifications to
total decomposition of major compounds giving rise to catabolites hardly detectable in green leaves. Notably,
senescence-associated changes can be biotechnologically provoked using the well-established and cheap
treatment of ethylene-fumigation, which makes senescent plant material a promising source for drug discovery.
Moreover, senescent plant material has proven to effectively aid the identification of bioactive natural products in
metabolomic drug discovery studies: biotesting and LC-MS-profiling of 36 different Juglans extracts has led to the
prediction of hydrojuglonglucoside as antitrypanosomal agent by means of Partial Least Squares (PLS) regression.
The sample set included 9 senescent samples which by their distinct metabolic profiles and bioactivities significantly
added on to sample variability, which is an essential prerequisite for the successful application of PLS or
comparable methods of multivariate statistics. In order to confirm the predicted bioactivity of hydrojuglonglucoside,
activity against Trypanosoma brucei was confirmed by testing the isolated compound [1].
Acknowledgments: Parts of the work were performed as part of the activities of the Research Network Natural Products against Neglected
Diseases (ResNetNPND; http://www.resnetnpnd.org).
References:
1. Ellendorf et al.: Molecules 2015, 20: 10082-10094.
SCIENTIFIC LECTURES
SL.73
Regulation of the heterologously expressed novobiocin gene cluster by the host strain
Streptomyces coelicolor M512
Bekiesch, P.1; Maček, B.2; Forchhammer, K.3; Apel, A. K.1
Pharmaceutical Biology, Pharmaceutical Institute, Eberhard-Karls-Universität Tübingen, Auf der Morgenstelle 8, 72076 Tübingen,
Germany.
2 Proteome Center Tübingen, Eberhard-Karls-Universität Tübingen, Auf der Morgenstelle 15, 72076 Tübingen, Germany
3 Microbiology/ Department of Organismic Interactions, Interfaculty Institute of Microbiology and Infection, Eberhard Karls Universität
Tübingen, Auf der Morgenstelle 28, 72076 Tübingen
1
The fast growing genome databases provide us with a large number of so far unknown secondary metabolite
biosynthetic gene clusters. For actinomycetes these clusters are often heterologously expressed in an engineered
host strain, as is Streptomyces coelicolor M512, to identify and investigate the corresponding compounds. However,
production rates of secondary metabolites often are lower in the heterologous host strain. To better understand
why this is the case, one has to know how the heterologously expressed gene cluster is regulated by the host strain
itself.
As a model system we chose the novobiocin biosynthetic gene cluster expressed in S. coelicolor M512 with a wellstudied operon structure and pathway specific regulation. Performing DNA affinity capturing assays (DACA) [1]
combined with label-free mass spectrometry at three different time points on the promoters of the pathway specific
regulatory genes novE (PnovE) and novG (PnovG), of the first biosynthetic gene novH (PnovH) and of the
vegetative sigma factor gene hrdB (PhrdB) as a negative control we could identify and quantify a total of 2475
captured proteins. The proteins were binding to one or more of the tested promoter regions with intensities reaching
from 103 to 1011. Among them the pathway specific regulator NovG was only captured on its binding site on PnovH,
whereas most of the identified proteins bound to all tested promoter regions, including PhrdB. As positive charges
on the surfaces of the proteins might lead to a low unspecific binding to negatively charged DNA in this very
sensitive assay, we used the lowest NovG binding intensity, in the stationary phase, as threshold to exclude proteins
with lower binding intensities.
Interestingly we identified some well-known global regulators as e.g. NdgR, AdpA, SlbR or WhiA among those
proteins binding with high intensity, but to all tested promoter regions.
We then selected four proteins binding specifically to PnovH or PnovG to study them more in detail. Deletion and
overexpression studies could confirm their regulatory function in novobiocin production. Furthermore for two
proteins binding was confirmed by surface plasmon resonance and binding sites were determined likewise. These
results indicate that DACA combined with label-free mass spectrometry is a very sensitive method and useful to
get a broader view of the regulation of biosynthetic gene clusters.
References:
1. Park, S. S. et al.: Ind. Microbiol. Biotechnol. 2009, 36: 1073-1083.
2. Dangel, V. et al.: Arch. Microbiol. 2008, 190: 509-519.
SL.74
mRNA binding proteins in metaflammation and hepatocarcinogenesis
Keßler, S.1
1
Department of Pharmacy, Pharmaceutical Biology, Saarland University, Saarbücken, Germany
Hepatocellular carcinoma (HCC) is the second most cancer-related death and the sixth most common tumor type
world-wide. Incidence of HCC is rising especially in Western countries due to alcohol abuse and metabolic diseases
leading to fatty liver disease, which displays an important risk factor for HCC development. HCC typically develops
within an inflammatory environment based on chronic inflammatory liver diseases.
Since mRNA binding proteins can bind different transcripts and regulate their translation they are able to interfere
with many different pathways. Therefore, mRNA binding proteins might display key factors in chronic liver disease
and hepatocarcinogenesis, in which metabolism, inflammation, and carcinogensis engage with each other.
The insulin-like growth factor 2 (IGF2) mRNA binding protein 2 (p62/IMP2) was originally isolated as an autoantigen
from an HCC patient and shown to induce a fatty liver in transgenic mice [1]. We could decipher that p62-induced
hepatic lipid accumulation is due to an IGF2-dependent activation of the lipogenic key regulator sterol regulatory
element binding protein 1 (SREBP1) resulting in an induction of the elongase ELOVL6 [2]. During progression of
fatty liver disease p62 is able to promote the manifestation of hepatic inflammation and fibrogenesis [3].
Analysis of human HCC samples revealed a strong overexpression of p62, which was correlated with poor
prognosis and a more aggressive HCC phenotype [4]. Functional studies in p62 transgenic mice confirmed a stem
cell-like and therefore more aggressive phenotype characterized by an increased number of chromosomal
aberrations. In this context p62 induced the imprinted gene DLK1, which is able to activate the small Rho GTPase
Rac1 and thereby leads to the generation of reactive oxygen species (ROS) [5]. Increased ROS levels can cause
genomic instability, which is related to a more stem-like tumor phenotype.
Taken together, the mRNA binding protein p62 displays an important regulator involved in all states of chronic liver
disease. Therefore, p62 might display an interesting target for new treatment options for fatty liver and HCC therapy.
References:
1. Tybl, E. et al.: J. Hepatol. 2011, 54:994-1001.
2. Laggai, S. et al.: J. Lipid. Res. 2014, 55:1087-1097.
3. Simon, Y. et al.: Gut 2014, 63:861-863.
4. Kessler, S. M. et al.: Am. J. Physiol. Gastrointest. Liver Physiol. 2013, 304:G328-G336.
5. Kessler, S. M. et al.: Cell death and disease 2015, in press.
SCIENTIFIC LECTURES
SL.75
Cyanobateria in natural product research – An interesting source for the discovery of new leads
and motivation for developing tools to aid structure elucidation
Niedermeyer, T.1; Kramer, D.2; Strohalm, M.3
1 Institute
for Microbiology and Infection Research, Tübingen University, Auf der Morgenstelle 28, 72076 Tübingen, and German Center for
Infection Research (DZIF), partner site Tübingen, Germany
2 Cyano Biotech GmbH, Magnusstr. 11, 12489 Berlin, Germany
3 Institute of Microbiology, Academy of Sciences of the Czech Republic, v.v.i., Videnska 1083, 142 20 Prague, Czech Republic
Cyanobacteria have in the past been neglected by natural product scientists and received much less attention than
other sources of secondary metabolites. However, nowadays they are acknowledged as a promising yet
underexplored source of natural products with both novel structures and potent bioactivities, and the first drug
product based on a cyanobacterial secondary metabolite has reached the clinic in 2011. Many cyanobacterial
compounds exhibit toxic effects, such as e.g. the well-known microcystins, anatoxins, and saxitoxins. Other
compounds, e.g. the microviridins, anabaenopeptins and microginins, show remarkably potent inhibition of
proteases [1]. The majority of natural products from cyanobacteria are biosynthesised by non-ribosomal peptide
synthetases and polyketide synthases (NRPS/PKS) [2]. Often, these compounds are synthesised as cyclic
peptides. Being of non-ribosomal origin, they often feature uncommon and highly modified amino acids, making
them attractive both from the point of view of their structure elucidation as well as their biosynthesis.
In the first part of this talk I will introduce cyanobacteria as a prolific source of novel natural products and will shortly
present some lead discovery projects I have been involved in.
In the second part of the talk I will present a software tool we developed to aid in the annotation and interpretation
of cyclic peptide tandem mass spectra. The mass spectrometric characterization of non-ribosomal cyclic peptides
is challenging due to the predominant occurrence of non-proteinogenic amino acid monomers and the complex
fragmentation patterns observed. Even though several software tools for the annotation of cyclic peptide tandem
mass spectra have been published, these tools are still unable to annotate a majority of the signals observed in
experimentally obtained spectra. They are thus not suitable for extensive mass spectrometric characterization of
these compounds. This lack of an advanced and user-friendly software tool has motivated us to extend a freely
available open-source software, mMass (http://www.mmass.org), to allow for cyclic peptide tandem mass spectra
annotation and interpretation. The resulting software has been tested on several cyanobacterial and other naturally
occurring peptides and has been found to be superior to all other tools currently available with regard to both
usability and annotation extensiveness. Thus it is highly useful for accelerating the structure confirmation and
elucidation of cyclic as well as linear peptides and depsipeptides [3].
Using this tool, we identified a large number of novel microginin derivatives in the Microsystis aeruginosa strain
HUB 5-3. Microginins are short linear lipopeptides, and potent inhibitors of the angiotensin converting enzyme
(ACE) [4]. Thus we decided to study their biosynthesis in this strain [5]. Although the PKS/NRPS gene cluster
responsible for microginin biosynthesis on a first glance does not possess unique features, we were surprized to
observe microginins that seem to be synthesized by only a part of the biosynthesis machinery.
References:
1. Niedermeyer, T.; Brönstrup, M.: Microalgal Biotechnology: Integration and Economy (de Gruyter) 2012, 169-200.
2. Welker, M.; von Döhren, H.: FEMS Microbiol. Rev. 2006, 30: 530-563.
3. Niedermeyer, T. H. J.; Strohalm, M.: PLoS ONE 2012, 7, e44913.
4. Ishida, K. et al.: Tetrahedron 2000, 56, 8643-8656.
5. Kramer, D.: 2010, US 7,846,686
SL.76
Analysis of natural product – Biosynthesis in the post-genomic era
Schäberle, T. F.1
1
Pharmaceutical Biology, University of Bonn, Bonn/Germany
Natural products have always been and will continue to the best source of lead structures for drug development.
Over the last decades however, industry started doubting the use of natural products for the generation of new
leads, since many known compounds have been rediscovered in bioactivity-based screening approaches. But in
recent years, more and more insights into the molecular mechanisms of natural product synthesis were obtained.
Most of the bioactive molecules are built up by polyketide synthase (PKS) and nonribosomal peptide synthetase
(NRPS) biosynthetic assembly lines. Connecting the products to the genes encoding the biosynthetic machinery of
these modular systems has stimulated new interest in natural product research, since this knowledge gave rise to
genome mining approaches (Figure 1). Using the growing number of available genomic data, it became clear that
the potential for the discovery of ever new structures has been by far underestimated, thus illustrating the lasting
importance of natural products for drug discovery as an endless frontier instead of an ending era.
The elucidation of biosynthetic gene clusters linked to natural products will be exemplified by recent examples from
our laboratory. The gene loci of the antibiotically active compounds teixobactin and corallopyronin A will be
presented [1,2]. From the latter, in detail analysis revealed even the basis for new enzymatic properties of double
bond isomerization and of head to head polyketide fusion [3,4].
Figure 1. Duplex mode of the interconnection between a gene cluster and its corresponding metabolite
References:
1. Ling, L. L. et al.: Nature 2015, 517: 455-459.
2. Erol, O. et al.: ChemBioChem. 2010, 11(9): 1253-65.
3. Lohr, F. et al.: Chem. Sci. 2013, 4: 4175-4180.
4. Zocher, G. et al.: Chem. Sci. 2015, DOI: 10.1039/c5sc02488a.
3 SHORT POSTER LECTURES
SHORT POSTER LECTURES
SPL.001 / POS.018
Antibiotics from predatory bacteria – From discovery to MOA studies
Nett, M.1; Schieferdecker, S.1; König, S.2; Korp, J.1; Werz, O.2
1 Leibniz
Institute for Natural Product Research and Infection Biology/Hans-Knöll-Institute, Beutenbergstr. 11a, 07745 Jena, Germany
and Medicinal Chemistry, Institute of Pharmacy, Friedrich-Schiller-University, Philosophenweg 14, 07743 Jena, Germany
2 Pharmaceutical
Bacteria, which prey on other microorganisms, are commonly found in the environment. While some predatory
bacteria act as solitary hunters, others are known to band together in large, wolf pack-like consortia [1]. Anecdotal
evidence suggests that the wolf pack-forming bacteria utilize antibiotics as part of their feeding strategy and, in fact,
genome sequencing projects unveiled a rich and diverse secondary metabolome in these organisms [2,3].
We recently started to investigate the biosynthetic potential of selected predatory bacteria [4,5]. A Pyxidicoccus
fallax strain, which had previously been isolated from the shores of the river Saale, was found to produce a new
class of macrolide antibiotics with potent activity against staphylococci [6]. The gulmirecins did not exhibit cytotoxic
effects against human cells and they also showed no cross-resistance with established antibiotics. Further analyses
involving label incorporation into DNA, RNA, protein and cell wall biosynthesis revealed the molecular target of
gulmirecin A. Subsequently, we isolated an antiproliferative compound with preferential activity against leukemic
cells from the gulmirecin-producing P. fallax strain. The metabolite was identified as the known natural product
myxochelin A and its antileukemic properties were traced to an inhibition of human 5-lipoxygenase [7].
Acknowledgments: Financial support by the Deutsche Forschungsgemeinschaft within the SFB 1127 “Chemical Mediators in Complex
Biosystems” is gratefully acknowledged.
References:
1. Nett, M.; König, G. M.: Nat. Prod. Rep. 2007, 24(6): 1245–1261.
2. Kiss, H. et al.: Stand. Genomic Sci. 2011, 5(3): 356–370.
3. Nett, M.: Progress in the Chemistry of Organic Natural Products (Springer) 2014.
4. Schieferdecker, S. et al.: J. Antibiot. 2014, 67(7): 519–525.
5. Schieferdecker, S. et al.: Eur. J. Org. Chem. 2015, (14): 3057–3062.
6. Schieferdecker, S. et al.: Chem. Eur. J. 2014, 20(48): 15933–15940.
7. Schieferdecker, S. et al.: J. Nat. Prod. 2015, 78(2): 335–338.
SPL.002 / POS.031
Targeting excipients for individual radiation therapy of cancer – Surface modification of PLGA
polymer and liposome nanoparticles entrapping lanthanides by cholesterol- and PLA-bound
ligands
Nawroth, T.1; Krebs, L.1; Johnson, R.1; Langguth, P.1; Hellmann, N.2; Decker, H.2, Schmidberger, H.3; Goerigk,
G.4; Boesecke, P.5a; Le Duc, G.5b; Bravin, A.5c; Schweins, R.6
Pharmacy & Biochemistry Institute, Pharmaceutical Technology, Johannes Gutenberg University, Staudingerweg, D-55099 Mainz,
Germany
2 Molecular Biophysics Institute, Johannes Gutenberg University, Jakob Welder Weg 26, D-55128 Mainz, Germany
3 Department of Radiooncology and Radiotherapy (Clinics), University Medical Center; Langenbeckstr.1, D-55131 Mainz, Germany
4 HZB, Institute of Soft Matter and Functional Materials , BESSY Synchrotron, ASAXS, D-14109 Berlin, Germany
5 ESRF, European Synchrotron Radiation Facility, a)ID01, b)BioMedical Facility,c)ID17, 71 Avenue des Martyrs, F-38043 Grenoble, France
6 ILL, Institut Laue Langevin, DS / LSS, 71 Avenue des Martyrs, F-38042 Grenoble CEDEX 9, France
1
Nanoparticles can concentrate millions of drug molecules per unit and specifically target them to cancer cells, if
they are recognized by cellular receptors [1,2]. Enhancer radiotherapy drugs increase the radiation cross section
by Lanthanides for photon therapy PT, or Boron and Gadolinium for neutron capture therapy NCT. Case- and
person-specific cell targeting results from surface modifications by signal lipids and anchor-ligands, recognized by
disease specific cellular receptors. Our development, shall deliver a case- and person-specific cancer therapy.
Our therapeutic nanoparticles of 100nm size are liposomes [1,5], polymers [7], including optionally bio-Ferrofluids
[2]. The ligand proteins are bound by a fast click-link technique to anchor-linker constructs, which we synthetize
bearing a terminal binding pre-activated S-group [6]. The ligand SH-group bearing (modified) proteins were bound
in the last step resulting in artificial membrane proteins at the nanoparticle surface. We studied particle structure
[4], drug load and release [5], and surface modifications with SANS, neutron reflectometry (ILL), ASAXS (ESRF,
BESSY), DLS. Radio-therapy tests [3,5,7] were done with photons (PT) at the radiooncology clinics Mainz (linear
accelerator) and the ESRF-synchrotron (ID17 biomedical facility), and with neutrons (NCT) at the ILL, and the
TRIGA reactor Mainz [5]. For therapy tests we developed the EPN-test with kinetic cell cultures as tumor model [3].
Cell-specific nanoparticles for radiation therapy bear target excipients at the surface (artificial membrane protein)
Acknowledgments: We are grateful for the funding by the German Ministry of Science and Education BMBF, grant 05KS7UMA.
References:
1. Nawroth, T.; Rusp M.; May R.: Physica B 2004, 350(1-3): E635–638.
2. Alexiou, C. et al.: Eur. Biophys. J. 2006, 35: 446–450.
3. Buch, K. et al.: Radiation Oncology 2012, 7(1): 1–6.
4. Tenzer, S. et al.: ACS Nano 2011, 5(9): 7155–7167.
5. Peters, T. et al.: Radiation Oncology 2015, 10: 52–64.
6. Krebs, L.: 12/2014, Diploma thesis, Mainz (Bio. Medical. Chemistry BMC / in AK Pharmaceutical-Technology)
7. A particulate system for use in diminishing cell growth / inducing cell killing” EU-Patent 11 007 401.0 ; PCT 13 07 12; Johannes
Gutenberg-Universität Mainz: Buch, K. et al.: (2012) / publication (2014)
SPL.003 / POS.036
Examination of homogeneity of stent coatings produced via fluidized bed process
Wentzlaff, M.1; Senz, V.2; Grabow, N.2; Weitschies, W.1; Seidlitz, A.1
1 Institute
2 Institute
of Pharmacy, C_DAT, Ernst-Moritz-Arndt University of Greifswald, Felix-Hausdorff-Straße 3, 17487 Greifswald, Germany
for Biomedical Engineering, University of Rostock, Friedrich-Barnewitz-Straße 4, 18119 Rostock, Germany
Stents were coated using a Mini-Glatt® fluidized bed apparatus (Mikro-Kit® product container) with the fluorescent
model substance triamterene suspended in a dispersion of ammonium methacrylate copolymer
(Eudragit® RS 30 D). Further components of the spray liquid were formic acid, triethylcitrate and purified water.
Using the same process parameters 9 batches of 50 stents each (length 15 mm, unexpanded diameter 1.6 mm)
were coated under addition of 625 steel springs per batch of comparable measures to fill the product container.
The coating time and used volume of spraying liquid was varied to achieve 3 different coating masses (low,
intermediate, and high coating mass with theoretical model substance deposition of 2.5, 5.0 or 12.5 µg/mm2 surface
area) with 3 batches per mass. The obtained stents were examined regarding the coating process yield (mass gain
of complete batch relative to mass of solid content of the sprayed coating liquid), stent body defect rate (percent
deformed during the process), coating layer mass (differential weighing of coated stents and stents after complete
coating removal), and the model substance content (fluorimetric determination after elution with methanol).
Furthermore, stents were examined via fluorescence and scanning eletron microscopy.
All coating processes were successfully completed with the intended process parameters. The process yield was
42±3% for the high coating mass, 41±3% for the intermediate coating mass, and 47±1% for the low coating mass.
The defect rate of the stent bodies rose with increasing coating time but was overall low with a maximum defect
rate of 8% for the high coating mass (mean processing time 138 min). Results of the determination of coating mass
and model substance content as well as fluorescent microscopic images of stent sections are depicted in Figure 1.
Smooth and form fitting coatings without polymer bridges or webbings were obtained for all batches. Even upon
stent expansion to a diameter of 3 mm with a catheter pump only very few coating defects in the zones of
deformation were observed. Coating split-off or delaminations were not observed during these experiments.
Scanning electron microscopic images revealed a slightly smoother appearance of the coatings with low coating
mass. Standard deviations for the coating mass as well as the model substance content were all below 10% and
an acceptable reproducibility between the batches was obtained. Mean model substance loads of 58±3 µg (low
coating mass), 113±6 µg (intermediate coating mass), and 271±15 µg (high coating mass) were detected.
Drug-eluting stents are often coated individually using spray coating techniques. Such processes have been
reported to yield 5 - 10 coated stents per hour and a deposition rate < 5% [1]. In the process reported here 675
units were coated simultaneously in a time ranging from 30 min (low coating mass) to 138 min with an average
process yield of > 40% and a low defect rate. The variation regarding the model substance content was < 10% of
the mean load which is comparable to drug content variation of commercially available drug-eluting stents [2].
Acknowledgments: Financial support by the federal ministry of education and research (BMBF) within REMEDIS is gratefully
acknowledged.
References:
1. Grabow, N. et al.: Biomed. Tech. (Berl.) 2013, 58(S1): DOI 10.1515/bmt-2013-4383.
2. Seidlitz, A. et al.: Biomed. Tech. (Berl.) 2012, 57(S1): DOI: 10.1515/bmt-2012-4120.
SPL.004 / POS.098
Reversing cisplatin resistance in ovarian carcinoma cells by inhibition of protein disulfide
isomerase 1
Kalayda, G. V.1; Kullmann, M.1; Hellwig, M.1; Kotz, S.2; Hilger, R. A.3; Metzger, S.2,4; Jaehde, U.1
1 Institute
of Pharmacy, Clinical Pharmacy, University of Bonn, An der Immenburg 4, 53121 Bonn, Germany
Biocenter, University of Cologne, Zülpicher Str. 47b, 50674 Cologne, Germany
3 Department of Medical Oncology, West German Cancer Center, University Hospital Essen, University Duisburg-Essen, Hufelandstraße
55, 45147 Essen, Germany
4 IUF-Leibniz Research Institute for Environmental Medicine, Auf’m Hennekamp 50, 40225 Düsseldorf, Germany
2 Cologne
The clinical success of the widely used antitumor drug cisplatin is limited due to the frequently observed
development of resistance in the course of the chemotherapy. Cisplatin resistance is a multifactorial event, which
amongst others includes enhanced intracellular deactivation of the reactive cisplatin species. Recently, several
intracellular binding partners of the fluorescent cisplatin analogue CFDA-cisplatin have been identified, among them
the members of protein disulfide isomerase (PDI) family PDIA1 and PDIA3 [1].
The aim of our work was to elucidate the contribution of PDIA1 and PDIA3 to cisplatin resistance and to enhance
cisplatin sensitivity by modulation of these proteins in A2780 human ovarian carcinoma cell line and its cisplatinresistant variant A2780cis.
Knockdown of PDIA1 performed using the respective small interfering RNA increased cytotoxicity of cisplatin in
resistant A2780cis cells as assessed by the MTT assay. Sensitivity of the parent A2780 cell line to cisplatin was
slightly, however not significantly increased. On the contrary, PDIA3 knockdown had no influence on cisplatin
cytotoxicity in both cell lines. A tendency for enhanced apoptosis (evaluated using the FITC Annexin V Apoptosis
Detection Kit with PI®) was observed after PDIA1 knockdown in the A2780 and A2780cis cell lines. This was not
the case after PDIA3 knockdown. Pharmacological inhibition of PDIA1 with PACMA31 [2] re-sensitized A2780cis
cells to cisplatin treatment. In A2780 cells, the cytotoxicity of cisplatin was only marginally increased. Determination
of the combination index revealed that cisplatin and PACMA31 act synergistically in both cell lines. However,
synergism was much more pronounced in the cisplatin-resistant cells.
Our results warrant further evaluation of PDIA1 as promising target for chemotherapy, and its inhibition by
PACMA31 as a new therapeutic approach. Furthermore, combination of cisplatin with PACMA31 may help to
reverse resistance in ovarian cancer.
Acknowledgments: This project was supported by the Deutsche Forschungsgemeinschaft (JA 817/4-1).
References:
1. Kotz, S. et al.: Electrophoresis 2015, in revision.
2. Xu, S. et al.: Proc. Natl. Acad. Sci. 2012, 109: 16348–16353.
SPL.005 / POS.101
Target-sensitive, vascular directed liposomes with entrapped chemokine receptor antagonists
for a local interference with tumor cell metastasis
Schlesinger, M.1; Roblek, M.2; Calin, M.3; Stan, D.3; Zeisig, R.4; Simionescu, M.3; Bendas, G.1; Borsig, L.2
1 Department
of Pharmacy, Rheinische Friedrich-Wilhelms-University Bonn, 53121 Bonn, Germany
of Physiology, University of Zürich and Zürich Center for Integrative Human Physiology, CH-8057 Zurich, Switzerland
3 Institute of Cellular Biology and Pathology ‘‘N. Simionescu’’ of the Romanian Academy, Bucharest, Romania
4 Experimental Pharmacology & Oncology Berlin Buch GmbH, 13125 Berlin, Germany
2 Institute
Increased levels of chemokines are associated with augmented number of metastases and poor prognosis of
cancer patients. The inflammatory chemokine CCL2 and its receptor CCR2 are known to initiate the early metastatic
niche through recruitment of inflammatory myeloid cells and activation of the endothelium in proximity to cancer
cells [1-4]. Both steps enable tumor cell transmigration and proliferation in the subendothelial tissue. A systemic
blockade of the CCL2-CCR2 axis is demanding due to side effects and poor target specificity. Here we present
PEGylated target-sensitive liposomes (TSL) encapsulated with a CCR2 antagonist (Teijin compound 1) coupled
with a specific peptide recognized by endothelial VCAM-1, which enables a specific delivery to cancer cell-activated
endothelial cells. Binding of TSL to endothelial VCAM-1 triggers the release of the CCR2 antagonist which leads to
a subsequent blockade of endothelial CCR2. A local CCR2 blockade reduces an opening of tight junctions and
finally the vascular permeability. Thus, tumor cells reveal a reduced transmigration and consequently the number
of pulmonary metastatic foci in mice is attenuated. Nevertheless, the number of recruited inflammatory myeloid
cells to the metastatic niche is not reduced. Application of VCAM-1 targeted TSL, loaded with Teijin compound 1,
mitigated the number of pulmonary metastases in a murine (MC-38GFP cells) and a human xenograft (patient’s
derived cells) model. Also in vitro, the transmigration of patient´s derived melanoma cells was strongly decreased.
Hence, here we present a potential therapeutic approach for treatment of pulmonary metastasis without affecting
homeostatic and immune functions.
Acknowledgments: This work was supported by the frame of EuroNanoMed funded under the ERA-NET scheme of the Seventh
Framework Programme of the European Commission.
References:
1. Kitamura, T. et al.: J. Exp. Med. 2015, 212: 1043.
2. Qian, B.-Z. et al.: Nature 2011, 475: 222.
3. Zhao, L. et al.: Hepatol. Baltim. 2013, Md 57: 829.
4. Wolf, M. J. et al.: Cancer Cell 2012, 22: 91.
SPL.006 / POS.106
Rational design and diversity-oriented synthesis of peptoid-based selective HDAC6 inhibitors
with potent anticancer activity
Diedrich, D.1; Syntschewsk, V.1; Hamacher, A.1; Alves Avelar, L. A.1; Gertzen, C. G. W.1; Reiss, G. J.2; Kurz, T.1;
Gohlke, H.1; Kassack, M. U.1; Hansen, F. K.1
Institut für Pharmazeutische und Medizinische Chemie, Heinrich Heine Universität Düsseldorf, Universitätsstr. 1, 40225 Düsseldorf,
Germany
2 Institut für Anorganische Chemie und Strukturchemie, Heinrich Heine Universität Düsseldorf, Universitätsstr.1, 40225 Düsseldorf,
Germany
1
Histone deacetylases (HDACs) catalyze the cleavage of acetyl groups from N-acetyl-lysine residues of histones
and non-histone proteins. These posttranslational modifications (PTMs) are important for the regulation of gene
transcription and protein function [1-5]. Human HDACs have been classified into four groups based on their
homology to yeast histone deacetylases and cofactor dependence [1]. HDAC classes I (HDACs 1-3,8), IIa (HDACs
4,5,7,9), IIb (HDACs 6,10) and IV (HDAC 11) contain zinc-dependent deacetylase domains and are considered as
“classical” HDACs [1-5]. Most of the currently used HDAC inhibitors (HDACi) target multiple HDAC isoforms and
their clinical use may thus cause serious unwanted side effects [1]. Notably, there is increasing evidence that
isoform-selective inhibitors are less toxic than pan-inhibitors [1,2]. Epigenetic drug discovery is therefore shifting
towards the development of class- or isoform-selective HDACi. HDAC6, a class IIb enzyme, is structurally and
functionally unique among the eleven human zinc-dependent histone deacetylases [5]. It is the only HDAC with two
independent functional catalytic domains and a C-terminal zinc finger motif responsible for binding ubiquitinated
proteins. The enzyme was initially described as tubulin deacetylase; however, it also modulates the function of
other non-histone proteins implicated in regulatory processes, including cortactin, peroxiredoxins and Hsp90 [5].
Due to the large number of substrates, HDAC6 is involved in numerous diseases such as autoimmune disorders,
inflammation, neurogenerative diseases and cancer [5]. Consequently, HDAC6 has emerged as an attractive target
for the treatment of the above-mentioned diseases and the search for novel potent and selective HDAC6 inhibitors
is of high importance [5].
We report here on the rational design and diversity-oriented synthesis of a series of selective HDAC6 inhibitors
utilizing peptoid-based cap groups. A synthetic protocol based on an efficient and straightforward multicomponent
approach allowed the rapid generation of a HDACi mini library. The biological evaluation of the target compounds
included cellular HDAC and MTT assays on sensitive and chemoresistant cancer cell lines. The most active
peptoid-based HDACi were investigated for their activity against selected HDAC isoforms and enhancement of
cisplatin-induced cytotoxicity. Molecular modelling, MD simulations and docking studies allowed rationalization of
the observed selectivity profile. Taken together, our data indicate that the peptoid-based HDACi are a new class of
potent and selective HDAC6 inhibitors with remarkable activity against a panel of cancer cells of different
chemosensitivity and tissue origin.
Acknowledgments: This work was supported by funds from the Fonds der Chemischen Industrie (FCI).
References:
1. Witt, O. et al.: Cancer Lett. 2009, 277(1): 8–21.
2. Marek, L. et al.: J. Med. Chem. 2013, 56(2): 427–436.
3. Hansen, F. K. et al.: ChemMedChem 2014, 9(3): 665–670.
4. Hansen, F. K. et al.: Eur. J. Med. Chem. 2014, 82: 204–213.
5. Kalin, J. H.; Bergman, J. A.: J. Med. Chem. 2013, 56(16): 6297–6313.
SPL.007 / POS.118
Inhibition of endothelial Cdk5 reduces tumor growth by promoting non-productive angiogenesis
Merk, H.1; Zhang, S.1; Lehr, T.2, Bibb, J. A.3; Adams R. H.4,5; Zahler, S.1; Vollmar, A. M.1; Liebl, J.1
Department of Pharmacy, Pharmaceutical Biology, Ludwig-Maximilians-University, 81377 Munich, Germany
Clinical Pharmacy, Saarland University, 66123 Saarbrücken, Germany
3 Department of Psychiatry and Neurology and Neurotherapeutics, The University of Texas Southwestern Medical Center, Dallas, Texas
75390-9070, USA
4 Department of Tissue Morphogenesis, Max Planck Institute for Molecular Biomedicine, 48149 Münster, Germany
5 Faculty of Medicine, University of Münster, 48149 Münster, Germany
1
2
Therapeutic success of VEGF-based anti-angiogenic tumor therapy is limited due to resistance. Thus, new
strategies for anti-angiogenic cancer therapy based on novel targets are urgently required. Our previous in vitro
work suggested that small molecule Cdk5 inhibitors affect angiogenic processes such as endothelial migration and
proliferation. Moreover, we recently uncovered a substantial role of Cdk5 in the development of lymphatic vessels.
Here we pin down the in vivo impact of endothelial Cdk5 inhibition in angiogenesis and elucidate the underlying
mechanism in order to judge the potential of Cdk5 as a novel anti-angiogenic and anti-cancer target. By the use of
endothelial-specific Cdk5 knockout mouse models and various endothelial and tumor cell based assays including
human tumor xenograft models, we show that endothelial-specific knockdown of Cdk5 results in excessive but nonproductive angiogenesis during development but also in tumors, which subsequently leads to inhibition of tumor
growth. As Cdk5 inhibition disrupted Notch function by reducing the generation of the active Notch intracellular
domain (NICD) and Cdk5 modulates Notch-dependent endothelial cell proliferation and sprouting, we propose that
the Dll4/Notch driven angiogenic signaling hub is an important and promising mechanistic target of Cdk5. In fact,
Cdk5 inhibition can sensitize tumors to conventional anti-angiogenic treatment as shown in tumor xenograft models.
In summary our data set the stage for Cdk5 as a drugable target to inhibit Notch-driven angiogenesis condensing
the view that Cdk5 is a promising target for cancer therapy.
SPL.008 / POS.141
Design and optimization of N-benzyl benzamides: A novel fused scaffold for orally available dual
sEH/PPARγ modulators for treatment of metabolic syndrome
Proschak, E.1
1 Institute
of Pharmaceutical Chemistry, Goethe-University Frankfurt, Max-von-Laue-Strasse 9, D-60438 Frankfurt a. M., Germany.
The metabolic syndrome (MetS) is a multifactorial disease cluster consisting of dyslipidemia, cardiovascular
disease, type 2 diabetes mellitus and obesity. Pharmacological intervention in the MetS is dependent on numerous
drugs, thus polypharmacy is an obvious problem in the treatment of MetS patients. This study focuses on the dual
target approach to accomplish a more efficient therapy for MetS. The two targets addressed by dual ligand design
are the soluble epoxide hydrolase (sEH) and the peroxisome proliferator-activated receptor type γ (PPARγ). In vivo
studies could demonstrate that even though an inhibitor of sEH or PPARγ agonist have benefits when used
individually, the combination is more beneficial for the multidisease features in cardiometabolic syndrome [1]. Using
a split-and-combine strategy we designed a library of dual sEH/PPARγ modulators and proved that both targets
can be simultaneously addressed by a merged pharmacophore [2]. In a follow-up study, we designed lead-like
merged N-benzyl benzamides which were able to modulate she and PPARγ. Structure activity relationship studies
on both targets were performed resulting in an equipotent submicromolar (sEH IC50 = 0.3 µM / PPAR EC50 = 0.3
µM) propionic acid benzylbenzamide derivative. Evaluation in vitro and in vivo displayed good ADME properties
qualifying the novel dual modulator as pharmacological tool compound for long term animal models of MetS. 8week evaluation in spontaneously hypertensive obese rats (SHROB), a rat model of MetS, demonstrated excellent
efficacy including simultanious reduction of blood pressure and improvement of glucose tolerance.
References:
1. Imig, J .D. et al.: Exp. Biol. Med. 2012, 237(12):1402-12.
2. Blöcher, R. et al.: J. Med. Chem. 2012, 55(23):10771-5.
SPL.009 / POS.167
Development and preclinical characterization of partial farnesoid X receptor agonists for
metabolic disorders
Merk, D.1
1 Institute
of Pharmaceutical Chemistry, Goethe University Frankfurt, Max-von-Laue-Str. 9, 60438 Frankfurt, Germany;
As a member of the nuclear receptor superfamily, farnesoid X receptor (FXR) acts as cellular regulator of bile acid
homeostasis [1]. In this role, FXR controls the expression of several genes involved in bile acid, lipid and glucose
homeostasis [2-4]. The involvement of FXR in several metabolic pathways offers a novel attractive drug target for
the treatment of metabolic disorders, i.e. dyslipidemias and diabetes mellitus and first beneficial effects of
pharmacological FXR activation on hyperglycemia have yet been reported from the clinical development of
obeticholic acid which is the most developed FXR agonist so far [5]. However, the orchestra of nuclear receptors
and their target genes is a very complex network that also bears the risk of severe side-effects as it has e.g. been
observed for the glitazones that are agonists on the peroxisome proliferator-activated receptor γ (PPARγ). A
potential way to reduce the risk of such undesired effects of nuclear receptor over-activation is the development of
partial agonists that only activate the respective receptor to moderate amplitude [6].
Starting from a virtual screening program, we have developed a set of highly potent FXR partial agonists by
systematic structure-activity-relationship studies and repeated rounds of structural optimization [7-9]. Preparation
of co-crystal structures of selected representatives helped the structural optimization process and improved our
knowledge on the molecular mechanism of partial FXR activation.
The resulting agents are selective for FXR, display EC50 values for partial FXR activation in the low nanomolar
range and activate the receptor to an amplitude of around 40% compared to the physiological agonist chenodeoxy
cholic acid. Intensive in vitro pharmacological characterization revealed a direct interaction with the FXR-LBD and
partial agonistic potency on all investigated FXR target genes. Moreover, the compounds showed good metabolic
stability and low toxicity [7-9]. First in vivo data is very promising and warrants further exploration of the strategy
and the compound class. The current optimization addresses the improvement of drug-likeness, stability and
bioavailability to obtain a further optimized model agent for intensive in vivo investigation of partial FXR agonism
as novel strategy to treat metabolic disorders.
Acknowledgments: Financial support by the Else-Kröner-Fresenius-Stiftung and the Vereinigung der Freunde und Förderer der GoetheUniversität is gratefully acknowledged.
References:
1. Makishima, M. et al.: Science 1999, 284(5418): 1362–1365.
2. Kuipers, F.; Bloks, V.; Groen, A.: Nat. Rev. Endocrinology 2014, 10(8): 488–498.
3. Düfer, M. et al.: Diabetes 2012, 61(6): 1479–1489.
4. Fang, S. et al.: Nat. Med. 2015, 21(2): 159–165.
5. Mudaliar, S. et al.: Gastroenterology 2013, 145(3): 574–582.
6. Merk, D.; Steinhilber, D.; Schubert-Zsilavecz, M.: Future Med. Chem. 2012, 4(8): 1015–1036.
7. Merk, D. et al.: Bioorg. Med. Chem. 2014, 22(8): 2447–60.
8. Merk, D. et al.: J. Med. Chem. 2014, 57(19): 8035–8055.
9. Merk, D. et al.: Bioorg. Med. Chem. 2015, 23(3): 499–514.
and unpublished data
SPL.010 / POS.174
Time-resolved in situ assembly of the 5-lipoxygenase / 5-lipoxygenase-activating protein
complex in primary human leukocytes
Garscha, U.1; Gerstmeier, J.1; Werz, O.1
1 Chair
of Pharmaceutical/ Medicinal Chemistry, Institute of Pharmacy, Friedrich-Schiller University Jena, Philosophenweg 14, 07743 Jena,
Germany
5-Lipoxygenase (5-LOX) catalyzes the first two steps in the biosynthesis of pro-inflammatory leukotrienes (LTs)
from arachidonic acid (AA) [1]. Upon cell activation, 5-LOX translocates to the nuclear membrane where cytosolic
phospholipase A2 (cPLA2) liberates AA that is subsequently transferred by 5-LOX-activating protein (FLAP) to 5LOX for efficient metabolism. Although biochemical or fluorescence microscopy data propose an association of 5LOX with FLAP [2,3], the in situ assembly of native 5-LOX/FLAP complexes in primary human cells remains elusive.
The main objective of the present study was the time-resolved visualization of in situ 5-LOX/FLAP interaction at the
nuclear membrane of human primary leukocytes in relation to 5-LOX activity. Immunofluorescence microscopy and
proximity ligation assays [4] were applied to monitor 5-LOX/FLAP co-localization and interaction, respectively. We
show for the first time an in situ 5-LOX/FLAP interaction in human blood monocytes and neutrophils. FLAP inhibitors
(MK886, BAY X-1005) abolished 5-LOX/FLAP complex assembly but failed to block 5-LOX translocation and colocalization with FLAP. Inhibition of cPLA2 by RSC-3388 prevented 5-LOX/FLAP interaction but exogenously added
AA restored it. Interestingly, 5-LOX/FLAP complex assembly appeared delayed at time points when 5-LOX product
formation was already terminated. Though neutrophils and monocytes possess comparable quantities of
catalytically active 5-LO enzyme, 5-LO activity in intact neutrophils is pronounced due to prolonged cellular 5-LO
reaction, accompanied by delayed 5-LO nuclear membrane translocation. Conclusively, our data suggest that FLAP
regulates 5-LOX product synthesis in two ways: by inducing an initial flexible association for efficient 5-LOX product
synthesis, but also by formation of a tight 5-LOX/FLAP complex that terminates 5-LOX activity.
Acknowledgments: Financial support was provided by Deutsche Forschungsgemeinschaft (DFG) within the SFB1127: Chemical Mediators
in complex Biosystems.
References:
1. Rådmark, O. et al.: Biochim. Biophys. Acta. 2015, 1851(4): 331-339.
2. Mandal, A.K. et al.: Proc. Natl. Acad. Sci. U. S. A. 2008, 105(51): 20434-9.
3. Bair, A.M. et al.: Mol. Biol. Cell. 2012, 23(22): 4456-64.
4. Söderberg, O. et al.: Nat. Method. 2006, 3(12): 995-1000.
SPL.011 / POS.203
Defined immobilisation of interleukin-4 (IL-4) for spatial controlled M2 macrophage polarization
Lühmann, T.1; Spieler, V.1; Werner, V.1; Meinel L.1
1 Institute
for Pharmacy and Food Chemistry, University of Würzburg, Am Hubland, 97074 Würzburg, Germany
Introduction: Regulation of macrophage (Mφ) plasticity has broad implications in the treatment of a variety of
inflammatory diseases, including, obesity, impaired wound healing, arteriosclerosis or rheumatoid arthritis. In the
course of the inflammatory process, Mφ are the dominant infiltrating cells into the injured tissue and mediate
temporally defined actions, by polarizing toward a spectrum of different phenotypes, including a classical
(proimflammatory M1) and an alternative (anti-inflammatory, M2) polarization state [1]. Interleukin 4 (IL-4) is one of
the major cytokines to trigger Mφ polarization to the M2 activation state, leading to cellular responses and an
environment that stimulate endogenous tissue repair mechanisms. The therapeutic potential of human recombinant
IL-4 has been previously investigated by subcutaneous administration in Phase II clinical trials but the treatment
was associated with severe toxic side-effects [2]. Therefore novel delivery options for this interesting potent cytokine
need to be explored.
In this study, we aimed to circumvent the current challenges of IL-4 by deploying genetic codon extension to
integrate the non-natural amino acid propargyl-L-lysine (Plk) into the protein backbone at position 42 during protein
translation in E. coli. [3,4]. This approach enables site-specific immobilisation of Plk-IL-4 by biorthogonal chemistry,
avoiding wandering species but rather addressing its Mφ polarization efficiency at specific sites.
Methods: Human IL-4 and human plk-IL4 were expressed in E.coli BL21 (DE3) and were purified using cationic
exchange chromatography as previously described [5]. Characterisation of plk-IL-4 was performed by MALDI-MS,
ESI-LC-MS/MS and HPLC. Bioactivity of IL-4 and Plk-IL-4 was determined by proliferation of TF-1 cells and by
using a secreted alkaline phosphatase (SEAP) STAT-6 reporter gene assay. NHS modified agarose particles were
decorated with azide functionalities and Plk-IL-4 was immobilized using CuAAC chemistry. Monocytes were
isolated from human peripheral blood mononuclear cells obtained from blood buffy coats by a two-step density
gradient centrifugation and by magnetic associated cell sorting. Monocytes were differentiated into Mφ with M-CSF1 (unpolarized, M0) and further treated with a combination of M-CSF-1 and Plk-IL-4 or wild-type IL-4 for M2
polarization or with a cocktail of LPS and IFNγ for M1 polarization, respectively. Degree of Mφ polarization was
assessed by RT-PCR with gene markers described in [6].
Results and Discussion: Plk-IL4 was successfully expressed in the presence of 3 mM Plk and was purified by
cationic exchange chromatography in an analogue manner to wild-type IL-4. The correct incorporation of Plk at
position 42 was confirmed by MALDI-MS and ESI-MS analysis and peptide mapping after trypsin digest. The
soluble Plk-IL-4 was as active as the wild type analogue in respect to TF-1 cell proliferation and SEAP stimulation.
M2 polarization of Mφ as analysed by M2 marker gene upregulation was similarly in the presence of soluble PlkIL-4 compared to the wild type analogue. Plk-IL-4 was successfully immobilized onto azide-functionalized agarose
particles using CuAAC click chemistry. Proliferation of TF-1 cells was stimulated by plk-IL-4 decorated agarose
beads in a dose (particle number) dependant manner and in comparison to controls, in which the immobilisation
was performed without copper sulphate. The potential of immobilized Plk-IL-4 decorated agarose particles on Mφ
plasticity is currently explored, aiming for lasting M2 Mφ polarization.
Site-directed immobilisation of IL-4 is a powerful tool for decoration of material surfaces targeting tissue repair and
wound healing by controlling Mφ plasticity in a spatial controlled manner.
References:
1. Mosser, D.M.; Edwards, J. P.: Nat. Rev. Immunol. 2008, 8(12): 958–69.
2. Whitehead, R. P. et al.; J. Immunother. 2011, 25(4): 352–358.
3. Eger, S. et al.: Methods Mol. Biol. 2012, 832: 589–596.
4. Nguyen, D. P. et al.: J. Am. Chem. Soc. 2009, 131: 8720.
5. Kimmenade, A.V. et al.: Eur. J. Biochem. 1988, 173: 109–114.
6. Jaguin, M. et al.: Cell. Immunol. 2013, 291: 51–61.
SPL.012 / POS.210
Understanding Plasmodium falciparum’s exploitation of the innate immune system aids the
identification of potential novel intervention strategies
Schmidt, C. Q.1; Kennedy, A. T. 2; Harder, M. J. 1; Lim, N. Y. T. 2; Tham, W. H. 2
1 Ulm
University, Institute of Pharmacology of Natural Products & Clinical Pharmacology, Ulm, Germany
University of Melbourne, Department of Medical Biology and The Walter and Eliza Hall Institute of Medical Research, Parkville,
Australia.
2 The
Introduction: Malaria remains one of the world’s deadliest diseases. Of the five human malarias, Plasmodium falciparum inflicts
the most mortality and severe form of human malaria. From initial entry into the human body P. falciparum parasites face the
innate immune system’s first line of defence against invading pathogens, the complement system. This ancient defence system
can directly lyse pathogens via formation of the membrane attack complex or lead to phagocytosis via opsonisation of the
pathogen with C3b. Malaria parasites are exposed to complement when merozoites, the invasive form of the life cycle, are
released from a spent erythrocyte into circulation. Recently, complement receptor 1 (CR1) on erythrocytes has been identified
as an alternative entry receptor for invasion utilized by merozoites during P. falciparum blood stage infection [1,2]. CR1
regulates and protects erythrocytes from self-damage of the host’s complement system, but during merozoite invasion the
parasite adhesin PfRh4 interacts directly with the conserved N-terminal regulatory region of CR1 to mediate successful invasion
[3]. Upon engagement of CR1 by PfRh4 some complement regulatory functions of CR1 are inhibited exposing the merozoite
and invaded red cells to the danger of complement mediated destruction [4].
Objectives: This study aims (i) to identify the amino acids in CR1 that mediate binding to the parasite adhesin PfRh4 and (ii)
to produce soluble domain fragments of CR1 that inhibit the PfRh4 invasion pathway. The third objective (iii) investigates how
the merozoites evade the destruction by the human complement system.
Methods: The first two N-terminal domains of CR1, as well as a panel of rationally designed mutants (homologous substitution)
were expressed recombinantly and tested for biophysical binding characteristics for PfRh4 interaction as well as for their activity
to inhibit erythrocyte entry of merozoites in growth culture. In regards to immune evasion merozoites obtained from parasite
culture were exposed to human serum or complement proteins for subsequent binding studies and functional analysis to
identify routes of complement evasion.
Results: We have produced wildtype CR1 complement control protein (CCP) module pairs 1-2 (CCP1-2) and CCP1-2 variants
bearing specific mutations. All proteins were expressed in the host Pichia pastoris, purified to homogeneity and validated by
mass spectrometry. Using surface plasmon resonance (SPR) we determined affinity constants for PfRh4 binding, thus
shedding more light onto the bimolecular interaction between CR1 and the malaria adhesion PfRh4. We show how these
mutations affect malaria parasite recognition of red blood cells and how they affect parasite growth in a CR1-dependent
manner. We identified the CR1 amino acids 18 and 20 (sequence numbers) as crucial binding partners for PfRh4. In terms of
inhibiting the CR1-dependent invasion pathway, we discovered that the recombinant CR1 protein consisting of CCP1-2
efficiently blocks parasite invasion, in vitro. Finally, we investigated how the merozoites resist clearance by the complement
system. Merozoites were exposed to human non-immune serum and were found to specifically recruit the host complement
regulators Factor H (FH) and FHL-1 protein (FHL-1). Both human complement regulators retained their functional activity when
bound to the merozoite surface. By employing a recombinant FH CCP-domain fragment library, the domains 4 to 6 in FH were
identified to be responsible for the recruitment by the malaria parasites. Co-immunoprecipitation experiments with soluble
parasite lysate identified one merozoite surface protein as the interaction partner for FH/FHL-1 recruitment. Preventing
FH/FHL-1 binding to merozoites enhanced complement-mediated parasite lysis.
Conclusion: We have identified the binding patch on CR1 that mediates erythrocyte invasion via the parasite adhesion protein
PfRh4 and showed that this invasion pathway can be inhibited in vitro with small recombinant protein fragments of CR1. We
also show that FH recruitment to merozoites protects the parasites from complement-mediated lysis and have identified the
merozoite protein responsible for mediating FH/FHL-1 recruitment. These findings have important implications for the future
design of vaccine strategies or the development of peptide inhibitors.
References:
1. Tham, W-H. et al.: Proc. Natl. Acad. Sci. 2010, 107(40): 17327–32.
2. Spadafora, C. et al.: PLoS Pathog. 2010, 6(6): e1000968.
3. Park, H. J. et al.: J. Biol. Chem. 2014, 289(1): 450–63.
4. Tham, W-H.; Schmidt, C.Q. et al.: Blood 2011, 118(7):1923–33.
4 POSTERS
POSTERS
4.1 Biopharmaceutics
POS.001
Comparison of two binding kinetic assays in long term
studies for performance qualification using Biacore X100
Steinicke, F.1; Oltmann-Norden, I.1; Wätzig, H.1
1 Institute
of Medicinal and Pharmaceutical Chemistry, Technical University of Brunswick,
Beethovenstraße 55 in 38106 Braunschweig
Surface Plasmon Resonance (SPR) is a dominant tool for biomolecular
interaction characterization. This technique facilitates label-free binding
analysis studies of biomolecules such as affinity, kinetic,
thermodynamics and specificity analysis in real-time. Therefore SPR is
an important application in drug discovery and proteomics.
In this study the model system for measuring binding kinetics was β2
microglobulin from human urine and the antibody anti-β2 microglobulin
produced in mouse. The antibody was covalently bound by amine
coupling on the surface of the gold chip and the antigen was flowed over
the chip.
Two different assay designs were implemented to determine kinetic rate
constants (kd, ka), dissociation constants (KD), residuals from the
experimental to the optimal fitted curve (RU) and the maximal Response
Units (Rmax) as an indicator for ageing of the chip/antibody.
The first method was a titration series called Single Cycle Kinetic (SCK)
where subsequently five different concentrations of β2 microglobulin (2,
4, 8, 16 and 32 nM) were injected. One regeneration step followed
subsequently to loose off the β2 microglobulin from the antibody. The
second method was a Multi Cycle Kinetic (MCK) where all concentrations
were injected in their own cycle with two regeneration steps after each.
As a further variation the randomization of the injection sequence was
evaluated in MCK. The dilution series was prepared freshly every day
with a HEPES-buffer pH 7.4 and the regeneration solution was glycineHCl 10 mM pH 2.5.
The investigation of this antibody/antigen system by using both SCK and
MCK methods reveals that the parameter that represents best the aging
of the chip was Rmax. In SCK the Rmax was decreasing slightly after 20
runs without affecting the quality of the other kinetic parameters.
Interestingly in contrast to SCK in MCK Rmax was stable over 30 cycles
although there are five times more regeneration steps for one data set
than in SCK. That implies that the ageing of the chip is not only caused
by the harsh regeneration steps but mainly by the time period it is used.
The most stable parameter measured was the kd with a relative percental
standard deviation (RSD%) of 2.5 to 6.2% over a minimum of 30
measurements. In contrast the ka was more unstable within the range of
6.5 to 30%.
Acknowledgments: We thank GE Healthcare Life Sciences for excellent technical support
References:
1. Rich, R. L.; Myszka, D. G.: J. Mol. Recognit. 2011, 24(6): 892-914.
2. GE Healtcare: Biacore X100 Getting Started 2009
3. Olaru, A. et al.: Crit. Rev. Anal. Chem. 2015, 45(2): 97-105.
targeting ligand for folate receptor-mediated uptake and as an anticancer
agent as it is toxic to the target cells by blocking de novo thymidylate and
purine synthesis and consequently DNA and RNA synthesis [4]. Besides,
we exploit a therapeutic siRNA against Eg5 that blocks mitosis to cause
death of rapidly dividing cancer cells. Together we formulate Eg5 siRNA
and MTX-conjugated polymer as a nanosized tissue-specific siRNA
polyplex with synergistic antitumor effect (Figure A).
The sizes of siRNA polyplexes were estimated by atomic force
microscope. The cellular uptake was determined by flow cytometry, and
knockdown of a luciferase reporter gene was used to monitor gene
silencing efficiency. To evaluate the efficacy of Eg5 siRNA, we measured
toxicity by cell viability assay, mRNA expression by qRT-PCR, and aster
formation of cellular DNA. In NMRI-nude mice bearing KB xenograft
tumors, the intratumoral retention of Cy7-labeled siRNA polyplexes was
determined by NIR fluorescence bioimaging to investigate the tissuespecific targeting, and tumor size as well as aster formation in vivo after
the treatments with EG5 siRNA polyplexes were monitored to assess the
therapeutic potency.
The siRNA polyplexes were homogeneous spherical nanoparticles with
6.5 nm of hydrodynamic diameter. These polyplexes were taken up by
KB cells in a MTX-dependent manner, and this attributed to association
with folate receptor. Transfections induced significant silencing of
luciferase expression in KB/eGFPLuc cells. Treatments with MTXconjugated polyplexes containing Eg5 siRNA in KB cells triggered
knockdown of Eg5, resulted in typical aster formation, and caused
enhanced cytotoxicity. in vivo studies indicated that the MTX-based 640
polyplexes showed significantly enhanced intratumoral retention (168 h)
compared to non-targeted alanine-substituted 188 polyplexes (48 h)
(Figure B), and mediated the longer survival time (56 days) than
untreated controls (24 days).
We developed a specific and efficient siRNA carrier system with dualfunctional ligand for cellular delivery and antitumor effect. siRNA
polyplexes successfully delivered cargo into the target cells mainly via
folate receptor. When combined with therapeutic Eg5 siRNA and MTX,
the siRNA polyplexes carried out synergistic cytotoxic activity. This highly
functionalized and molecule-defined carrier system for siRNA delivery
could be a potential strategy for RNAi-based cancer therapeutics.
Acknowledgments: DFG Excellence Cluster Nanosystems Initiative Munich (NIM), Bavarian
Research Foundation, Dr. Rong Zhu
References:
1. Lächelt, U.; Wagner, E.: Chem. Rev. 2015, DOI: 10.1021/cr5006793.
2. Lee, D. J.; Wagner, E.; Lehto, T.: Methods in Molecular Biology (Humana) 2015.
3. Dohmen, C. et al.: ACS Nano. 2012, 6(6): 5198-208.
4. Lächelt, U. et al.: Mol. Pharm. 2014, 11(8): 2631-9.
POS.002
Targeted Co-delivery of Bifunctional GlutamylMethotrexate and Eg5 siRNA Using Nanoplexes for
Combined Antitumoral Potency
Lee, D. J.1,2; Kessel, E.1; He, D.1,2; Klein, P.1; Lächelt, U.1,2; Lehto, T.1;
Wagner, E.1,2
1 Department
of Pharmacy and Center for NanoScience, Ludwig Maximilian University,
Butenandtstraße 5-13, Munich, 81377, Germany
2 Nanosystems Initiative Munich, Schellingstraße 4, Munich, 80799, Germany
The novel strategy that synthetic small interfering RNA (siRNA) can
invoke RNAi responses is expected to be an excellent option for treating
many incurable diseases such as cancer. However, efficient tissuespecific delivery of siRNA remains the major limitation in the development
of RNAi therapy [1]. By solid phase supported synthesis, we have
synthesized a series of sequence-defined polymers which include a
cationic (oligoethanamino)amide core, cysteines (as bioreversible
disulfide-forming units), and polyethylene glycol chain (for shielding
surface charges) coupled to a terminal ligand [2,3]. To recognize the
target cells, the antifolate drug methotrexate (MTX) is employed as both
POS.003
2D separation of proteins: Combining the strengths of SAX
and CGE
Maul, K. J.1,2; Hahne, T.1; Wätzig, H.1,2
1 Institute
of Medicinal and Pharmaceutical Chemistry, Technische Universität Braunschweig,
Beethovenstraße 55, 38106, Germany
2 PVZ – Center of Pharmaceutical Engineering, Franz-Liszt Straße 35a, 38106, Germany
The two techniques HPLC with a strong anion exchanger (SAX) column
and capillary gel electrophoresis (CGE) are both commonly used in
protein analysis. In this work a combination of both techniques is used to
characterize a complex protein mixture such as biopharmaceuticals. This
is still mostly done by using classic two dimensional gel electrophoresis
(2D-GE).
To achieve comparable results the combination of SAX and CGE was
chosen. In the first step the proteins were separated according to their
charge at a certain pH using SAX-HPLC. Afterwards, in the second
dimension CGE was used. The separation of this technique is based on
BIOPHARMACEUTICS
POS.004
A modified disintegration tester for solid dosage forms:
Influence of motion and velocity profiles on disintegration
behaviour
Rach, R.1; Kindgen, S.1; Nawroth, T.1; Abrahamsson, B.2; Langguth, P.1
Institute of Pharmacy and Biochemistry, Department of Pharmaceutical Technology and
Biopharmacy, Johannes Gutenberg University, 55128 Mainz, Germany
2 Pharmaceutical Development, AstraZeneca R&D, 43183 Mölndal, Sweden
1
The disintegration of solid oral dosage forms is determined by the
USP/PhEur disintegration test device. In this compendial method a
basket-rack assembly containing six dosage forms is raised and lowered
in immersion fluid over a distance of 55 mm for 29 to 33 times per minute.
There is no appreciable horizontal motion or movement of the axis from
the vertical and no possibility of velocity adjustment [1].
There are no investigations whether this direction of movement
respectively its velocity imitates physiological conditions. In addition the
physiological conditions vary over the gastrointestinal (GI) tract. For
example the stomach, where immediate release dosage forms mainly
disintegrate, is divided into several parts with different stress conditions.
In the proximal part, the fundus, the mechanical stress conditions are
significantly lower in comparison to the distal part, the antrum [2].
In this project a new designed modified disintegration tester is used to
investigate the influence of movement directions and velocities on
disintegration of tablets.
Acknowledgments: This work has received support from the Innovative Medicines Initiative Joint
Undertaking (http://www.imi.europa.eu) under Grant Agreement No. 115369, resources of which
are composed of financial contribution from the European Union’s Seventh Framework Program
and EFPIA companies' in kind contribution.
100
100
80
80
60
40
Sugar-based 0.1 N HCl
Isomalt-based 0.1 N HCl
20
Isomalt-based SSF
0
10
20
30
60
40
Sugar-based 0.1 N HCl
Isomalt-based 0.1 N HCl
20
Sugar-based SSF
0
Drug release [%]
Acknowledgments: We thank PolymicroTM for providing the capillaries.
To evaluate in vivo parameters relevant for drug release in the oral cavity,
we performed a study in 6 healthy volunteers. The study “medication”
consisted of different types of commercial candies that were either sugarbased or sugar-free or differed in taste, respectively. The volunteers had
to suck a candy at 3 different times of the day over 3 different days and
the following parameters were recorded: overall sucking time,
temperature and pH in the oral cavity before and immediately after
sucking, saliva osmolality before and after sucking. In a second series of
tests, the saliva secretion rate during sucking was recorded. The same
candy formulations were then subject to dissolution experiments with
compendial test methods for lozenges, i.e. experiments were performed
in the paddle apparatus using 500 mL 0.1 N hydrochloric acid or water
as test media. In the final set of in vitro experiments, we wanted to
address essential in vivo parameters such as the presence of small fluid
volumes, continuous saliva flow, saliva composition, agitation and tongue
pressure acting on the dosage form. We thus designed a new test
apparatus consisting of a custom-made flow through cell that allows for
using small media volumes, and an “artificial tongue” which can be
agitated to slide over the dosage form at a predetermined agitation rate.
Simulated Saliva Fluid [1] was used as the test medium.
Drug release [%]
the molecular size. These two steps are very similar to 2D-GE, where
isoelectric focussing is combined with SDS-PAGE.
For this approach a mixture of five proteins was used to determine the
suitability and precision of the combination. These proteins were namely
Myoglobin, β-Lactoglobulin, Ovalbumin, bovine serum albumin and a
monoclonal antibody. The first dimension is conducted at pH 8.5 with
gradient elution. The sodium chloride concentration of the eluent was
increased from 0.0 M to 0.75 M over 40 min. 20 fractions were collected.
As second dimension CGE was used with a SDS-gel buffer. This whole
analysis takes less than 24 hours to get a 2D separation. In both
dimensions a DAD was used to collect the data.
The advantages compared to 2D-GE are the short analysis time, the
possibility to detect in both dimensions and the higher precision.
40
Sugar-based SSF
Isomalt-based SSF
0
0
2
Time [min]
4
6
8
10
12
Time [min]
Figure: Release profiles obtained from two lozenge formulations obtained in 0.1 N HCl and SSF
[1] with the paddle apparatus (500 mL, 75 rpm) (A) and with the new dissolution apparatus for
lozenges (9 mL/min, 180 dpm) (B), mean of n= 3 ± S.D., the grey arrows indicate average in vivo
sucking time.
Results obtained with the compendial dissolution methods available for
testing drug release from lozenge formulations might be useful for quality
control but, as indicated by the time required for total dissolution of the
formulations, did not relate to the real sucking process. It was thus clear
that when the aim is to estimate in vivo sucking time and drug release in
the oral cavity, a different test setup would be required. Results from the
experiments performed with the new dissolution apparatus, in contrast,
are very promising. The new setup will thus be applied to a range of
additional lozenge formulations and with different simulated saliva fluids
with the aim of proposing a set of test parameters that can be applied for
predicting in vivo drug release and potentially also be used for quality
control of lozenges.
References:
1. Mashru, R. C. et al.: Drug Dev. Ind. Pham. 2005, 31(1): 25-34.
References:
1. The International Pharmacopeia, Methods of Analysis, http://apps.who.int/phint/en/p/docf/
(22.05.2015)
2. Koziolek, M. et al.: Eur. J. Pharm. Sci. 2014, 57: 250-256.
POS.006
Application of patient-specific test methods in estimating
in vivo drug release of oral mesalazine formulations
POS.005
Development of a new test methodology for predictive
in vitro dissolution testing of lozenges
Tietz, K.1; Gutknecht, S.1; Klein, S.1
1 Institute
of Biopharmaceutics and Pharmaceutical Technology, Department of Pharmacy,
Ernst Moritz Arndt University, Felix Hausdorff Street 3, Center of Drug Absorption and
Transport, 17489 Greifswald, Germany
A variety of lozenges for local drug delivery to the oral cavity is on the
market since quite a long time. However, when the objective is to
compare drug release from these formulations in a predictive way, no
guideline is available to assist in establishing an appropriate in vitro
dissolution test. The purpose of the present set of experiments was thus
to evaluate critical in vivo parameters for drug release in the oral cavity,
to screen the applicability of compendial dissolution methods in terms of
obtaining results being predictive for the in vitro performance of lozenge
formulations, and finally to evaluate the applicability of a new
methodology in terms of predicting drug release of lozenges in the oral
cavity.
Karkossa, F.1; Krüger, A.1; Klein, S.1
1 Institute
of Biopharmaceutics and Pharmaceutical Technology, Department of Pharmacy,
Ernst Moritz Arndt University, Felix Hausdorff Street 3, Center of Drug Absorption and
Transport, 17489 Greifswald, Germany
There is an ongoing discussion on - apart from clinical trials - how to
demonstrate therapeutic equivalence for locally applied and locally acting
products in the gastrointestinal tract. Possibly, among other alternatives,
in vitro drug release models could be considered surrogates of drug
release and availability at the site of action. However, to date the
conditions in which in vitro models provide valid surrogates of in vivo
release and availability would have to be defined. To demonstrate the
potential applicability of in vitro test methods for screening therapeutic
equivalence of locally acting oral mesalazine formulations and also to get
an idea of which would be the right dosage form for an individual patient,
recently a series of in vitro studies was performed comparing a variety of
in vitro release methods ranging from pharmacopoeial methods through
methods addressing average gastrointestinal transit times and pHconditions to first experiments simulating gastrointestinal conditions in
individual subjects [1]. The objective of the present series of tests was to
further by applying detailed individual pH-gradients as obtained in study
POSTERS
performed with a pH-sensitive capsule [2] and also a set of novel
bicarbonate-based biorelevant media to simulate intestinal passage of
the dosage forms.
Figure: Release profiles obtained when simulating the gastrointestinal passage of Salofalk 500
mg and Claversal 500 mg tablets in three different individuals, paddle apparatus, 500 mL, 75 rpm,
mean of n= 3 ± S.D., the shaded area represents small intestinal residence time.
For this purpose, we selected three individual fasted gastrointestinal
transit profiles representative for essentially different small intestinal
passages [2], i.e. relatively low, average, and relatively high small
intestinal pH conditions and different gastrointestinal transit times.
Experiments were performed with Salofalk 500 mg tablets and Claversal
500 mg tablets in the Paddle apparatus. After simulating gastric
residence in SGFsp, the pHysio-grad [3] device was used to simulate
small and large intestinal transit in bicarbonate-based biorelevant media.
Our results clearly indicate that the two mesalazine formulations will
show an essentially different in vivo performance in individual subjects.
Whereas in the patients with low and high small intestinal pH, drug
release of both formulations is expected to take place in the mid or distal
small intestine, respectively, in the patient with average small-intestinal
pH conditions only the Salofalk formulation will release the active in the
(terminal) small intestine. Drug release from the Claversal tablet will, in
contrast, initiate in the (proximal) colon. Based on the observations made
in the present series of tests we conclude that with a set of
physiologically-based dissolution models taking into account the
particular features in gastrointestinal physiology and typical dosing
scenarios in the target patient group, it should be possible to estimate the
in vivo performance of oral formulations, to discriminate between
formulations and finally to contribute to a safe and effective drug therapy
for the individual patient.
References:
1. Klein, S.: Pharmazie 2015; 70(8).
2. Koziolek, M. et al.: J. Pharm. Sci. 2014, 19 (2014) EPub.
3. Garbacz, G. et al.: Eur. J. Pharm. Sci. 2014, 51: 224-31.
PHARMACOLOGY
4.2 Pharmacology
POS.007
Influence of nuclear receptor ligands on the expression of
metabolizing enzymes and transporter proteins in the
human intestine
Brueck, S.1; Busch, D.1; Martin, P.2; Haenisch, S.2; Cascorbi, I.2;
Siegmund, W.1; Oswald, S.1
1 Department
of Clinical Pharmacology, Center of Drug Absorption and Transport (C_DAT),
University Medicine Greifswald, Felix- Hausdorff-Str. 3, 17487 Greifswald, Germany
2 Institute of Experimental and Clinical Pharmacology, University Hospital Schleswig-Holstein,
Arnold-Heller-Str.3, 24105 Kiel, Germany
Background: Drug transporters and drug metabolizing enzymes located
in the human intestine are crucial determinants in the pharmacokinetics
of orally administered drugs. Nuclear receptor ligands (NRL) like
rifampicin and carbamazepine are known to influence expression of
these proteins and can cause thereby unwanted drug-drug interactions
(DDI). However, the available knowledge is rather limited and was
predominantly focused on gene expression data. Moreover, little is
known about the intestinal regulation of enzymes and transporters
beyond activation of nuclear receptors (e.g. miRNA). Thus, it was the aim
of this study to comprehensively analyse the impact of NRL on gene and
protein expression of clinically relevant enzymes and transporters and
the potential regulatory role of miRNAs in the human intestine.
Methods: Tissue specimen (~2-5 mg) from lower duodenal mucosa were
taken before and after treatment with rifampicin (600 mg, 8 days) [1] or
carbamazepine (600 mg, 18 days) [2], respectively. Protein abundance
of clinically relevant intestinal transporters was quantified using a
validated LC-MS/MS-based targeted proteomics assay. In parallel, gene
expression (mRNA and miRNA) analysis was conducted using TaqMan®
real-time RT-PCR assays.
Results: Rifampicin treatment led to a significant increase in gene
expression (over 1.5-fold) of ABCB1, ABCC2, CYP2B6, CYP2C19,
CYP2C8, CYPC9, CYP3A4, CYP3A5 and UGT1A family and in protein
abundance of ABCB1, CYP3A4, CYP2C9 and UGT1A1. In contrast to
this, pre-treatment with carbamazepine led to no significant changes on
protein levels of the investigated transporters and enzymes, whereas
mRNA expression of ABCB1, ABCC2, ABCG2, SLCO2B1, CYP2C19,
CYP2C9, CYP2D6, CYP3A4, CYP3A5, SULT1A family and UGT1A
family were found to be significantly increased. The observed changes in
gene expression and protein content were correlated to intestinal miRNA
expression.
Conclusion: This study investigated for the first time the impact of
rifampicin and carbamazepine on the gene and protein expression of
clinically relevant enzymes and transporters in the human intestine in a
comprehensive manner. Rifampicin was found to be a strong inducer of
several enzymes and transporters which is in good agreement to
profound DDI observed in clinical studies. In addition to the activation of
nuclear receptors, the expression of intestinal enzymes and transporters
may be influenced by miRNAs.
References:
1. Giessmann, T. et al.: Clin. Pharmacol. Ther. 2004, 76(3): 192-200.
2. Oswald, S. et al.: Clin. Pharmacol. Ther. 2006, 79(3): 206-17.
hyperalgesia after topical application of high-concentration (40%)
menthol in comparison to its solvent ethanol [1,2].
Method: The study was a single-center, randomized, placebo controlled,
double-blind, 2 period cross-over trial in the “balanced placebo design”
(BPD) [3], in a cohort of 16 healthy subjects. The randomization was twofold: firstly, subjects were assigned to the order of substance application
and, secondly, subjects were assigned to the order of information about
the applied substance given by the investigator during the two
applications of each substance. Treatments were topical menthol (40%)
in ethanol as model for cold and mechanical hyperalgesia and topical
ethanol as control condition. Cold and mechanical hyperalgesia and
allodynia were determined by selected parameters of quantitative
sensory testing (QST) [4]. The sensational thresholds and area of
pinprick-hyperalgesia were quantified.
Results: This study was performed with reliable, valid and repeatable
QST measurements. For all parameters no suggestion effect could be
detected in the multivariable models.
Menthol reliably induced cold hyperalgesia. The cold pain threshold
(CPT) was decreased statistically significant (p<0.0001) and a distinct
effect was found for treatment condition and drug condition vs. placebo
and control condition. Minor suggestion effects on cold pain thresholds
could be seen in a subgroup of subjects that have not received menthol
before. This subgroup showed decreased cold pain thresholds in the
placebo condition compared to control condition. However, cold
hyperalgesia seemed to be the most robust parameter. Effect sizes were
similar to those previously published [1,2,5].
Pinprick hyperalgesia was less reliable. Mechanical pain threshold (MPT)
and mechanical pain sensitivity (MPS) only changed marginally after
application of menthol in treatment as well as in drug condition. Effect
sizes differed from previously published data [2,5]. The area size of
pinprick-hyperalgesia was not influenced statistically significant.
Parameters like mechanical pain threshold (MPT) and mechanical pain
sensitivity (MPS) seemed to be influenced by treatment sequence effect
and period effect.
Conclusion: There is no statistically significant effect of suggestion or
expectation on the measured parameters. The menthol model is
therefore a reliable, non-suggestible model to induce cold hyperalgesia.
Pinprick hyperalgesia is not as reliable to induce and may be influenced
by treatment sequence and periodic effect. Performing the model, future
studies should consider a prior demonstration of menthol to familiarize
the subject with the expected effect.
Acknowledgments: The research leading to these results is a part of the Europain Collaboration,
which has received support from the Innovative Medicines Initiative Joint Undertaking, under
Grant Agreement No. 115007, resources of which are composed of funding from the European
Union’s Seventh Framework Programme (FP7/2007–2013) and the kind contribution of EFPIA
companies.
References:
1. Wasner, G. et al.: Brain 2004, 127(5): 1159-1171.
2. Binder, A. et al.: J Pain 2010, 12(7): 764-773.
3. Enck, P.; Klosterhalfen, S.; Zipfel, S.: BMC Med Res Methodol 2011, 11: 90.
4. Rolke, R. et al.: Pain 2006, 123(3): 231-243.
5. Mahn, F. et al.: Eur J Pain 2014, 18(9): 1248-1258.
POS.009
POS.008
Impact of Suggestion and Expectation on a Human
Experimental Model of Cold and Mechanical Hyperalgesia
after Topical Application of High-Concentration Menthol in
Comparison to Ethanol
Barnscheid, L.1; Helfert, S. M.2; Reimer, M.2; Rengelshausen, J.1;
Baron, R.2; Binder, A.2
1 Early
Clinical Science, Translational Science & Strategy, Grünenthal GmbH, 52099 Aachen,
Germany
2 Division of Neurological Pain Research and Therapy, Department of Neurology, University
Hospital Schleswig-Holstein,24105 Kiel, Germany
Background: Human experimental pain models play an important role in
studying neuropathic pain mechanisms. The objective of the present
study was to evaluate the impact of suggestion and expectation on a
human experimental model of cold and mechanical (pinprick)
Characterization of the expression and function of
endogenous transporters in frequently used cellular
models
Otter, M.1; Eriksson, P. O.1; Keiser, M.1; Oswald, S.1
Department of Clinical Pharmacology, Center of Drug Absorption and Transport (C_DAT),
University Medicine Greifswald, Felix-Hausdorff-Str. 3, 17487 Greifswald, Germany
1
Background: Drug transporters are known to be important determinants
in the pharmacokinetics and efficacy of many drugs. In order to
characterize the affinity or inhibitory properties of drugs to transporter
proteins in preclinical drug development, transporter-overexpressing
cellular models based on MDCKII and HEK293 cells are widely used and
recommended by EMA and FDA guidelines. However, there is evidence
that these cells possess a considerable endogenous (“background”)
transporter expression which might affect in vitro transport. In this study,
we compared the expression profile of frequently used cellular models
with the respective tissue of origin. Furthermore, we investigated the
endogenous transporter expression in different MDCKII and HEK293 cell
lines. On this basis, functional consequences of endogenous ABCB1
were examined in cellular uptake studies.
POSTERS
Methods: Renal tissue from human or dog and the following cell lines
have been investigated: HEK293 wild-type cells or stably transfected with
control vector, OATP1A2, OATP1B1, OATP1B3 or OATP2B1; MDCKII
wild-type cells or stably transfected with control vector or ABCB1. The
mRNA expression of clinically relevant human ABC and SLC transporters
(HEK293 cells) and the respective canine transporters (MDCKII cells)
were determined by validated TaqMan® gene expression assays.
Protein abundance was quantified by LC-MS/MS-based targeted
proteomics. The functional study was performed with talinolol and
trospium chloride as probe substrate of OATP1A2 and ABCB1 using
wild-type, stably transfected vector control and OATP1A2overexpressing HEK293 cells. Intracellular accumulation of [3H]-talinolol
and [3H]-trospium chloride was measured by liquid scintillation counting
after cell lysis.
Results: The observed gene expression and protein abundance data
were mostly not correlated. For only few transporters such as ABCB1,
the expression in cellular models and tissue of origin was very similar
while expression of the most transporters (e.g. typical renal transporter
PEPT2) was markedly different in both cellular models. Several drug
transporters could be identified as endogenous transporters in the
different cell lines.
In HEK293 cells, the endogenous ABCB1 protein content was
considerably lower in vector control-, OATP1A2-, OATP1B1-, OATP1B3and OATP2B1-transfected cells compared to wild-type cells. On the
contrary, markedly higher expression has been observed for endogenous
ABCC2 in HEK-OATP1B1 and for endogenous ABCC3 in MDCK-ABCB1
cells.
Uptake studies regarding the functional consequences of endogenous
transporters showed that higher protein abundance of endogenous
ABCB1 in wild-type cells resulted by trend in reduced intracellular
accumulation of ABCB1 probe substrates compared to vectortransfected cells.
Conclusion: There are markedly differences in expression pattern of
endogenous transporters in the investigated HEK293 and MDCKII cells.
These endogenous transporters may affect drug transport and the
estimated transporter affinity to the focused transporter protein.
POS.010
Modulation of the l-arginine-nitric oxide pathway by apelin
in an in vitro model of pulmonary arterial hypertension
Glatzel, A.1; Lüneburg, N.1; Klose, H.2; Böger, R. H.1; Harbaum, L.2
1 Institute of Clinical Pharmacology and Toxicology, University Medical Center HamburgEppendorf, Germany
² Section Pneumology, II. Department of Medicine, University Medical Center HamburgEppendorf, Germany
Background: Idiopathic pulmonary arterial hypertension (IPAH) is a
difficult-to-treat rare lung disease characterised by endothelial
dysfunction with increased vasoconstriction and occlusive vascular
remodeling [1-3]. A dysfunctional endothelial L-arginine-nitric oxide (NO)
pathway is a key pathomechanism of IPAH and can be provoked by
hypoxia in in vitro models [4-5]. The small peptide apelin is involved in
the maintenance of pulmonary vascular homeostasis [6]. However, its
precise mechanism of action is still unclear. Asymmetric dimethylarginine
(ADMA) is an endogenous inhibitor of endothelial NO synthase and is
associated with clinical parameters in IPAH and other cardiovascular
diseases [7-9]. ADMA is degraded by dimethylarginine
dimethylaminohydrolase 1 and 2 (DDAH) enzymes [10].
Objective: To determine the influence of apelin on the L-arginine/NO
pathway in human microvascular pulmonary endothelial cells (HPMECs).
Methods: HPMECs were cultured under normoxic and hypoxic conditions
and treated with apelin. The expression of regulators of the Larginine/NO pathway were analysed using real-time PCR. In addition, the
effect of apelin on the the phosphoinositide-3 kinase (PI3K)/Akt signalling
pathway was determined using an immunoassay and specific inhibitors.
Apelin and ADMA concentrations were measured in cell culture
supernatants and IPAH patients’ serum using an enzyme-linked
immunosorbent assay (ELISA) and a validated high throughput liquid
chromatography–tandem mass spectrometry (LC-MS/MS) assay.
Results: Circulating Apelin was lower in patients with IPAH compared to
age-matched healthy subjects. Its receptor (APLNR) expression was
reduced on HPMECs following treatment with apelin due to a negative
feedback mechanism. Apelin directly influenced the L-arginine/NO
pathway by increasing the expression of DDAH2, but not DDAH1. Thus,
the concentration of ADMA was decreased in HPMECs supernatant by
apelin. This effect was observed under normoxic conditions as well as
under PAH-related hypoxic conditions. Furthermore, apelin induced the
phosphorylation of several proteins playing a role in the PI3K/Akt
signalling pathway. Foremost the glycogen synthase kinase 3 alpha and
beta (GSK-3α/ß) was inactivated as a result of apelin treatment. An
inhibition of GSK-3α/ß by GSK-3 inhibitor I mimicked the effect of apelin
on DDAH2 expression.
Conclusion: Apelin directly modulates the L-arginine/NO pathway and
mediates enhanced degradation of ADMA, which may increase NO
synthesis. The effect of apelin on DDAH2 expression is caused by
inhibition of GSK-3α/ß. An Apelin receptor agonist might be a novel and
promising therapeutic option for treatment of IPAH.
References:
1. Rabinovitch, M. et al.: J. Clin. Invest. 2012, 122(12): 4306-13.
2. Galiè, N. et al.: Eur. Respir. J. 2009, 34(6): 1219-63.
3. Humbert, M. et al.: J. Am. Coll. Cardiol. 2004, 43(12): 13S-24S.
4. Cooper, C. J. et al.: Circulation 1996, 93(2): 266-71.
5. Lüneburg, N. et al.: Biomed. Res. Int. 2014, 2014(2014):501612
6. Andersen, C. et al.: Pulm. Circ. 2011, 1(3): 334-46.
7. Vallance, P. et al.: J. Cardiovasc. Pharmacol. 1992, 20(12): 60S-62S.
8. Kielstein, J. T. et al.: Arterioscler. Thromb. Vasc. Biol. 2005, 25(7): 1414-8.
9. Zoccali, C. et al.: Lancet 2001, 358(9299): 2113-7.
10. Ogawa, T. et al.: Arch. Biochem. Biophys. 1987, 252(2): 526-37.
POS.011
Regulation of mitogen-activated protein kinase kinase
kinase DLK by calcineurin
Duque Escobar, J.1,3; Lemcke, T.2; Hasenpusch, D.2; Oetjen, E.1,2,3
1 Institut
für Klinische Pharmakologie und Toxikologie, Universitätsklinikum Hamburg-Eppendorf,
Martinistr. 52, 20246 Hamburg, Germany
2 Institut für Pharmazie, Bundesstr. 45, 20146 Hamburg, Germany
3 DZHK (German Center for Cardiovascular Research), Partner Site Hamburg/Kiel/Lübeck
Loss of beta-cell mass is the most important factor for the pathogenesis
of type 1 and type 2 diabetes. It is unknown which mechanisms induce
beta-cell apoptosis though different hypotheses are postulated. Inhibition
of the calcium-calmodulin dependent phosphatase calcineurin has been
shown to reduce beta-cell function and induce beta-cell apoptosis. Our
previous results showed that inhibition of calcineurin by the structurally
distinct immunosuppressant drugs cyclosporin A and tacrolimus
decreased insulin gene transcription [1]. Furthermore, both drugs
stimulated the catalytic activity of the mitogen-activated protein kinase
kinase kinase 12 (DLK Dual Leucine zipper kinase) and induced betacell apoptosis [2]. In the present study, the regulation of DLK by
calcineurin was investigated.
In silico analysis revealed two putative calcineurin interaction domains
within DLK [3]. The consensus motifs 273-PNMLIT-278 and 362-LPVP365 were mutated to PNRLKT, APAP and a double mutant, respectively,
by primerless PCR. The expression vectors for these mutants and DLK
wild-type were transiently transfected into the beta-cell line HIT.
Immunoblot analysis using an antibody against the COOH-terminus of
DLK showed similar expression levels of the single mutants, whereas the
double mutant was barely expressed. Both single mutants exhibited no
catalytic activity, measured as phosphorylation of DLK on Ser-302 and
phosphorylation of the downstream kinase JNK.
A DLK homology model was generated, using the crystal structures of
MLK1 and B-Raf as templates:
The model confirmed that 273-PNMLIT-278 interfered with the
conformation of the catalytic domain. Mutation of L362 to A and V364 to
PHARMACOLOGY
A within the second putative calcineurin interaction domain 362-LPVP365 did not reduce DLK protein expression. The L362A DLK mutant
showed no catalytic activity in relation to Ser-302 phosphorylation of DLK
and JNK phosphorylation, whereas the V364A DLK mutant resulted in 3fold higher JNK phosphorylation than DLK wild-type. In addition, in
reporter gene assays this mutant was more potent to inhibit
KCl/Forskolin-induced CRE-dependent gene transcription in a dosedependent manner, than DLK wild-type.
Considering that LxVP represents a calcineurin interaction domain, our
results suggest that the interaction of DLK with calcineurin via its LPVP
motif dephosphorylates and inhibits DLK activity. These findings also
show the importance of calcineurin for the maintenance and survival of
beta-cell function and mass.
The compounds were further investigated at the CB-like orphan G
protein-coupled receptors GPR18 and GPR55, both of which are known
to interact with certain CB receptor ligands [6]. β-Arrestin recruitment
assays were employed [7]. Only few of the studied compounds displayed
a moderate inhibition of GPR55 activation, while none of the compounds
showed any activation or inhibition of GPR18.
References:
1. Pertwee, R. et al.: Pharmacol. Rev. 2010, 62(4): 588-631.
2. Howlett, A. et al.: Pharmacol. Rev. 2002, 54(2): 161-202.
3. Lindigkeit, R. et al.: Forensic. Sci. Int. 2009, 191(1-3): 58-63.
4. Banister, R. et al.: Chem. Neurosci. 2015, DOI: 10.1021/acschemneuro.5b00107.
5. Rempel, V. et al.: J. Med. Chem. 2012, 55, 7967-7977.
6. Henstridge, C. et al.: Mol. Endocrinol. 2011, 25(11): 1835-1848.
7. Rempel, V. et al.: Med. Chem. Comm. 2002, 54(2): 161-202.
References:
1. Oetjen, E. et al.: Mol. Pha. 2003, 63(6): 1289-95.
2. Plaumann, S. et al.: Mol. Pha. 2008, 73(3): 652-9.
3. Rodríguez, A. et al.: Mol. Cell. 2009, 33(5): 616-26.
POS.013
POS.012
Pharmacological evaluation of synthetic cannabinoids
identified as constituents of spice
Schoeder, C.1; Hess, C.2; Madea, B.2; Müller, C. E.1
1 Pharma-Zentrum
Bonn, Pharmazeutisches Institut, Pharmazeutisches Chemie I, An der
Immenburg 4, D-53121 Bonn, Germany
2 Universitätsklinikum Bonn, Institut für Rechtsmedizin, Forensische Toxikologie, Stiftsplatz 12,
D-53111 Bonn, Germany
The cannabinoid (CB) receptor CB1 mediates the main psychoactive
effects of the natural product Δ9-tetrahydrocannabinol (THC) which acts
as a partial agonist at the receptor. The second CB receptor CB2 is mainly
expressed in the immune system [1]. Both CB receptors are Gi proteincoupled mediating inhibition of adenylate cyclase and thereby reducing
intracellular cAMP levels.
In the past decades a wide range of potent synthetic CB receptor
agonists and antagonists has been developed [2]. Natural and synthetic
CB1 agonists are being abused due to their psychoactive, euphoric and
analgesic effects, e.g. as ingredients of products commercialized as
incense called “spice”. Spice has been declared to be a herbal product,
but potent synthetic CB receptor agonists have been found in many spice
preparations, which are not just used as incense, but typically abused by
smoking the dubious mixture of unknown composition. Due to severe
side effects many of the synthetic CB agonists found in spice
preparations are now on the list of controlled substances [3].
However, the drug market is steadily flooded with new synthetic CB
receptor agonists that are not yet subject to control by the authorities. To
obtain compounds with CB1 receptor-agonistic effects, typical
bioisosteric variations of known potent CB receptor agonists are
observed, e.g. replacing hydrogen by fluorine atoms, indole by indazole
residues and naphthyl by quinolone ring systems [4]. The
pharmacological profiles of these compounds are often unknown, and
therefore forensic consequences for producers, traders and consumers
are not enforceable since a scientific basis is lacking.
In the present study we investigated a series of compounds collected by
the Institute of Forensic Medicine and suspected to act as CB1 receptor
agonists.
Neuroprotective effects of monoacylglycerol lipase
inhibition in a 6-hydroxydopamine model of Parkinson’s
disease in mice
Porazik, C.1,2; Witting, A.2; Ferger, B.1
1 CNS
Diseases Research, Boehringer Ingelheim Pharma GmbH & Co. KG, Biberach/Riß,
Germany
2 Department of Neurology, Ulm University, Germany
Monoacylglycerol lipase (MAGL) is the principal enzyme for 2arachidonylglycerol (2-AG) metabolism and plays an important role in the
endocannabinoid system.
Here, we investigate the effects of the commercially available MAGL
inhibitors KML29 and JZL184 in comparison with the cyclooxygenase 2
(COX2) inhibitor rofecoxib in an animal model of Parkinson’s disease
(PD).
6-Hydroxydopamine (6-OHDA) (3.5 µg/µl) was administered in the left
striatum in male C57BL/6JRj mice. KML29, JZL184 and rofecoxib (30
mg/kg/day p.o.) were administered daily for one week starting one day
before 6-OHDA surgery. Motor behaviour indicated by rotarod
measurement was assessed at day 7. Post mortem (day 7), striatal
dopamine depletion and nigral tyrosine hydroxylase positive (TH+) cell
loss were evaluated. Furthermore, the effect of MAGL and COX inhibition
on mechanistic biomarkers was assessed.
6-OHDA led to a 70% dopamine depletion in the striatum and 40% loss
of TH + neurons in the substantia nigra. KML29 treatment significantly
attenuated the 6-OHDA-induced dopamine depletion by 50% and TH+
neuron loss by 30%. Motor behavior was impaired by 6-OHDA but not
restored by MAGL inhibition. Overall, the effects of JZL184 and rofecoxib
treatment were less pronounced. The MAGL inhibitors significantly
increased 2-AG levels and decreased arachidonic acid and
prostaglandins in the brain. Rofecoxib decreased prostaglandins but had
no effect on 2-AG and arachidonic acid levels.
In conclusion, targeting the endocannabinoid system by MAGL inhibition
was found to be superior over COX2 inhibition in the 6-OHDA PD model
and could be a valuable new therapeutic concept for PD in the future.
Acknowledgments: The study was funded by the Boehringer Ingelheim Ulm University
BioCenter (BIU) and supported technically by Johannes Hanselmann and Noemi Pasquarelli.
POS.014
Targeting cardiomyocyte proliferation for heart
regeneration using phenotypic screens in neonatal mouse
cardiomyocytes.
Carrillo García, C.1; Raulf, A.2; Hesse, M. 2; Fleischmann, B. 2;
Schade, D.1
Department of Chemistry and Chemical Biology, TU Dortmund, Otto-Hahn-Str. 6, D-44227
Dortmund, Germany.
2 Intitut für Physiologie 1 - Life and Brain Center, Universität Bonn, Sigmund-Freud Str. 25, D53105 Bonn, Germany.
1
The compounds were investigated in radioligand binding assays for their
interaction with both CB receptor subtypes, CB1 and CB2. Subsequently
they were investigated for their functional properties in cAMP
accumulation assays [5]. Almost all of the investigated compounds were
found to be highly potent CB receptor agonists - some compounds
showing even subnanomolar affinities for the CB1 and CB2 receptor.
There is a great medical need for innovative therapies for the treatment
of heart failure and a true paradigm shift is required which aims at treating
the actual cause instead of rather coping with the symptoms. In this
regard, replacing lost heart tissue with new cardiomyocytes after
myocardial infarction is of particular interest. In contrast to the adult
POSTERS
mammalian heart, the developing heart (embryonic and neonatal)
exhibits a pronounced regenerative capacity. Hence, there is a pressing
need for a detailed understanding of the underlying physiological
mechanisms of cardiomyocyte development during this neonatal period
(e.g. mitotic arrest) to reveal the biological basis of potential regenerative
mechanisms and for successful therapeutic translation [1].
Here, we present a ‘forward chemical genomics’ approach to achieve this
goal. The basis is the establishment of a phenotypic assay that quantifies
cell cycle activity and cytokinesis (along with other phenotypic features)
of neonatal cardiomyocytes via high-throughput-compatible fluorescence
microscopy in 384- and 1536-well plates (high-content analysis). For this,
we isolate and dissect transgenic neonatal murine hearts which express
H2B-mCherry under the control of a cardiac specific promoter (=Myh6),
allowing us to unambiguously detect and quantify cardiomyocyte nuclei
[2]. To date, we could show general feasibility of this primary cells-based
approach for high-throughput application and have characterized the
very few described pro-proliferative compounds and growth factors.
Several key phenotypic endpoints have been addressed including
proliferative activity, cardiomyocyte cell number, cytokinesis and
nuclearity, the latter being of particular importance for cardiomyocytes
and their ability to proliferate.
Therefore, next steps will be to systematically screen focused small
molecule libraries with annotated targets and signaling pathways to
rapidly draw conclusions regarding involved biological factors, pathways
and mechanism. Later, evaluation of compound libraries that cover a
large chemical space will provide “hits” that possibly reveal completely
new biological mechanisms.
References:
1. Schade, D.; Plowright, A.T.: J. Med. Chem. 2015, accepted.
2. Raulf, A. et al.: Basic Res. Cardiol. 2015, 110(3): 33.
NATURAL COMPOUNDS
4.3 Natural compounds
POS.015
Xysmalobium undulatum
everything began
(Uzara®)
research – how
Helmstädter, A.1
1 Institute
of Pharmaceutical Chemistry, Goethe University, Max-von-Laue-Str. 9, 60438
Frankfurt, Germany
Xysmalobium undulatum (Uzara®) is a traditionally used medicinal plant
from South Africa. It found its way into European research in the early
20th century and has been widely used as a remedy against diarrhoea
since then. Circumstances of knowledge transfer and early research into
phytochemistry and therapeutic potential of X. undulatum have been
largely unknown so far. As could be shown, the drug was brought to
Europe by a former soldier, Wilhelm Heinrich Adolph Hopf (1887-1929).
At the age of 16, W. H. A. Hopf joined the German navy, but some years
later he moved to South Africa, where he was engaged in the Boer War.
He married Mary Annie Langham Thomson (1876-1967) December 16,
1906. Most probably in 1909, the family returned to Melsungen (along
with the Uzara roots) and stayed until the end of World War I. Then, Hopf
and his wife moved to England, lived in Rugby and changed their name
from Hopf to Hopford on 19 November 1918. They had two children. He
died december 27, 1929 in Rugby. Details about Hopf’s life are provided
here the first time, as well as some aspects of early research on the drug,
which has mainly been done at the University of Marburg, Germany.
POS.016
The acetyl-CoA carboxylase (ACC) inhibitor soraphen A
blocks the proliferation and migration of primary
endothelial cells
Glatzel, D.1; Müller, R.2; Koeberle, A.3; Werz, O.3; Fürst, R.1
1 Institute of Pharmaceutical Biology, Biocenter, Goethe-University, Max-von-Laue-Str. 9, 60438
Frankfurt am Main, Germany
2 Department of Microbial Natural Products, Helmholtz-Institute for Pharmaceutical Research
Saarland (HIPS), Helmholtz Centre for Infection Research (HZI), Saarland University, Campus
Building C2.3, 66123 Saarbrücken, Germany
3 Chair of Pharmaceutical/Medicinal Chemistry, Institute of Pharmacy, University Jena,
Philosophenweg 14, 07743 Jena, Germany
Acetyl-CoA carboxylase catalyzes the first step in the biosynthesis of
fatty acids in bacterial and eukaryotic cells, i.e. the conversion
(carboxylation) of acetyl-CoA into malonyl-CoA. ACC-generated malonylCoA functions as a substrate for de novo lipogenesis and acts as an
inhibitor of mitochondrial fatty acid β-oxidation. Because of its role in lipid
metabolism this enzyme has become an interesting target in drug
discovery in the field of metabolic diseases and cancer.
Despite the high interest in ACC as pharmacological target, no attention
has as yet been given to the role of ACC in endothelial cells. We aimed
to investigate the role of ACC in two functional key aspects of
angiogenesis: endothelial cell proliferation and migration. To inhibit the
function of ACC, we used the ACC inhibitor soraphen A, a polyketidic
natural compound isolated from the myxobacterium Sorangium
cellulosum, as well as an RNAi-based approach. Primary human
umbilical vein endothelial cells (HUVECs) were used as in vitro model.
First, we analyzed the action of soraphen A on cell viability. The
compound did neither lower the metabolic activity of HUVECs up to a
concentration of 100 µM after 24 and 48 h (CTB assay) nor increase in
the apoptosis rate after 24, 48, or 72 h up to 100 µM. Measuring
adenosine triphosphate (ATP) levels revealed that 30 µM soraphen A
does not alter the ATP levels in HUVECs after 24 h treatment. In contrast,
a 48 h treatment significantly lowers the ATP levels by 12%. Also gene
silencing of ACC1 in HUVECs attenuated the ATP levels by 11%.
Mitochondrial membrane potential (MMP) assays showed 10%
decreased MMP levels in soraphen A-treated cells after 24 h.
Interestingly, the compound inhibited the proliferation of endothelial cells
with an IC50 value of 34 µM. Cell cycle analysis showed that soraphen A
decreases the amount of cells in the G0/G1 phase by 26% and increases
the number of cells in the G2/M phase by 50%. In a wound healing/scratch
assay, 30 µM soraphen A lowered the migration of endothelial cells by
65%. Also gene silencing of ACC1 in HUVECs strongly decreased
endothelial migration. Furthermore, Boyden chamber assays revealed
that soraphen A can also lower chemotactic migration by 34%. Since
actin rearrangement is necessary for migratory processes, we analyzed
the F-actin cytoskeleton (microscopy) and found that soraphen A
decreased the number of filopodia by 60% but did not influence stress
fiber formation. Surprisingly, soraphen A-treated cells did not exhibit
significant alterations in their capacity to form tube-like structures on
Matrigel. In summary, we could gather first hints that inhibiting ACC has
an immense impact on the proliferation and migration of primary
endothelial cells. The mechanistic basis of this phenomenon will be
investigated in future studies by analyzing the lipid profile and the
transcriptome of endothelial cells.
Acknowledgments: This work was supported by the German Research Foundation (DFG, FOR
1406, FU 691/9-2).
POS.017
Does STW 5 have region specific effects in the intestine?
Nieber, K.1; Voß, U.1; Abdel-Aziz, H.2; Okpanyi, S.2; Kelber, O.2
1 Institut
für Pharmazie, Universität Leipzig, Brüderstr. 34, 04013 Leipzig, Germany
Abteilung, Steigerwald Arzneimittelwerk GmbH, Havelstr. 5, 64295
Darmstadt, Germany
2 Wissenschaftliche
STW 5 (Iberogast) is a fixed combination of nine plant extracts. It is
successfully used for treatment of functional dyspepsia and irritable
bowel syndrome (IBS). In this study we compared the effects of STW 5
with the effects of its component Iberis amara (STW 6) and another,
related combination, STW 5-II, which does contain six components, on
tone and acetylcholine (ACh)-induced contractions in intact and inflamed
intestinal preparations. We used 1-1.5 cm long ileum and colon
preparations of male Wistar rats to study region specific differences. The
inflammation was induced by intraluminal instillation of 2,4,6trinitrobenzene sulfonic acid (TNBS, 10 mM, 30 min). Incubation with
STW 5 (512 µg/ml) reduced the tone and decreased ACh-induced
contractions of untreated ileal and colonic preparations concentration
dependently (64-512 µg/ml). The effects of STW 5-II in a concentration
of 533.2 µg/ml were comparable to those of STW 5. STW 6 in equivalent
concentrations (3-24.1 µg/ml) neither affected the tone nor the
contractility. TNBS-induced inflammation was accompanied by a
significant reduction of ACh-induced contractions. Co-incubation of
TNBS with STW 5 (512µg/ml), STW 5-II (533.3 µg/ml) or STW 6 (24.1
µg/ml) partially normalized the TNBS-induced attenuation of tone as well
as of ACh-induced contractions in ileum preparations. In inflamed colon
segments the co-incubation of TNBS with STW 6 in a high concentration
(24.1 µg/ml) revealed protective effects whereas STW 5 as well as STW
5-II had no effects.
In conclusion, STW 5 and the related combination STW 5-II influenced
ACh-induced contractions and tone in untreated ileal and colonic
preparations, whereas their component STW 6 did not contribute to these
effects. In TNBS-inflamed ileum preparations STW 5, STW 5-II as well
as STW 6 normalized contractile disturbances, while in colon
preparations only STW 6 was effective. Our study therefore allows the
conclusion that a region specific action might contribute to the clinically
proven effects in irritable bowel syndrome (IBS).
POS.018
Antibiotics from predatory bacteria – from discovery to
MOA studies
Nett, M. 1; Schieferdecker, S. 1; König, S. 2; Korp, J. 1; Werz, O. 2
1 Leibniz
Institute for Natural Product Research and Infection Biology/Hans-Knöll-Institute,
Beutenbergstr. 11a, 07745 Jena, Germany
2 Pharmaceutical and Medicinal Chemistry, Institute of Pharmacy, Friedrich-Schiller-University,
Philosophenweg 14, 07743 Jena, Germany
For abstract see Short Poster Lecture SPL.001 on page 98.
POSTERS
POS.019
Natural substances as inhibitors of the tumor target human
hyaluronidase hyal-1
Lengers, I.1; Orlando, Z.1; Melzig, M. F.3; Buschauer, A.4; Hensel, A.2;
Jose, J.1
1 Institute
of Pharmaceutical and Medicinal Chemistry
of Pharmaceutical Biology and Phytochemistry, PharmaCampus, Westfälische
Wilhelms-Universität, Corrensstraße 48, 48149 Münster, Germany
3 Institute of Pharmacy, Pharmaceutical Biology, Freie Universität Berlin, Königin Luise Str. 2+4,
14195 Berlin, Germany
4 Institute of Pharmacy, Department of Pharmaceutical/Medicinal Chemistry II, University of
Regensburg, Universitätsstr. 31, 93040 Regensburg, Germany
2 Institute
The negatively charged polysaccharide hyaluronic acid (HA) assembled
from repeating disaccharide units of D-glucuronic acid and
N-acetyl-D-glucosamine. Its chain size triggers different physiological
and pathophysiological functions. Space filling, anti-inflammatory and
antiangiogenic effects are caused by high molecular weight HA (>20
kDa). HA hydrolization by hyaluronidases leads to low molecular weight
HA (<20 kDa), resulting in inflammatory and angiogenic effects [1]. The
degradation of HA is mainly catalyzed by human hyaluronidase Hyal-1.
It has been demonstrated that in prostate or bladder tumour cells the
expression level of Hyal-1 was elevated [2,3]. For this reason Hyal-1 is
an interesting target for drug discovery. The surface display of active
Hyal-1 on Escherichia coli, via Autodisplay, enables the screening for
potential inhibitors in a whole cell system. Based on this technique we
determined the inhibitory effect of different plant extracts and triterpenoid
saponins on human Hyal-1. The IC50 values of the extracts of
Malvae sylvestris flos, Equiseti herba and Ononidis radix were
determined to lay between 1.4 and 1.7 mg/mL. The obtained IC50 values
for the triterpenoid saponins, glycyrrhizinic acid (reference inhibitor),
gypsophila saponin 2, SA1641 and SA1657 were 177 µM, 108 µM, 296
µM and 371 µM, respectively. These natural substances identified can
be used as a starting point for the synthesis of new small molecule
inhibitors targeting human Hyal-1.
References:
1. Stern, R.: Semin. Cancer Biol. 2008, 18: 275-280.
2. Lokeshwar, V.B, et al.: J. Urol. 2000, 163: 348-356.
3. Lokeshwar V.B, et al.: J. Biol. Chem. 2001, 276(15): 11922-11932.
POS.020
Archazolid A influences endothelial processes responsible
for tumor cell adhesion and transmigration
Luong, B.1; Menche, D.2; Müller, R.3; Fürst, R.1
1 Institute of Pharmaceutical Biology, Biocenter, Goethe-University, Max-von-Laue-Str. 9, 60438
2 Kekulé Institute for Organic Chemistry and Biochemistry, University of Bonn, GerhardDomagk-Str. 1, 53121 Bonn, Germany
3 Helmholtz Institute for Pharmaceutical Research Saarland, Department Microbial Natural
Products, Campus, C2.3, Saarland University, 66123 Saarbrücken, Germany
Metastasis is the major cause of death in cancer patients. Archazolid A
is a v-ATPase inhibitor of myxobacterial origin, which has shown antimigratory and cell-death inducing effects on tumor cells. In vivo,
archazolid A reduced metastases formation [1,2]. A key step during the
formation of secondary tumors is the adhesion of tumor cells onto
endothelial cells and their subsequent extravasation through the
endothelium into the underlying tissue. The aim of this study is to
characterize how the interaction between tumor cells and endothelial
cells is influenced by archazolid A.
First, we investigated the impact of archazolid A on the viability of primary
human umbilical vein endothelial cells (HUVECs). Concentrations up to
1 nM did neither significantly influence the metabolic activity (CTB assay)
nor the apoptosis rate (flow cytometry) of HUVECs. Treatment of
HUVECs with 1 nM archazolid A did not affect the confluency of the
endothelial monolayer, but led to a swelling of the cells.
Interestingly, the compound increased the adhesion of MDA-MB-231, a
highly invasive breast cancer cell line, onto the endothelium and
decreased the transmigration of these cancer cells through the
endothelium (Boyden chamber assay). Several adhesion molecules and
also the CXCR4/CXCL12 chemokine system have been postulated to be
involved in the interaction of MDAs and endothelial cells. Quantitative
real-time PCR revealed that the mRNA levels of the potentially involved
adhesion molecules ICAM-1, VCAM-1, E-selectin, N-cadherin, and
galectin-3 were not upregulated in HUVECs upon archazolid A treatment
(1 nM) for 12 h. Also the chemokine system CXCR4/CXCL12 was not
affected by archazolid A. Interestingly, archazolid A exerted a strong
influence on adherens junctions, which are largely composed of VEcadherin. Immunofluorescent stainings as well as a cell surface protein
isolation assay showed that archazolid A leads to a loss of VE-cadherin
at the cell surface, while the total amount of cellular VE-cadherin is not
affected. Despite this breakdown of adherens junctions, neither
endothelial permeability (Transwell assay) nor endothelial contractility
(Western blot) were influenced by archazolid A (1 nM).
Taken together, the anti-metastatic effect of archazolid A seems to be a
consequence of the increased adhesion of tumor cells onto the
endothelium and a reduced transmigration through the endothelium. The
precise underlying mechanisms are still to be resolved. However, there
is no influence on molecules typically involved in tumor-endothelial cell
interaction (adhesion molecules, CXCR4/CXCL12 system). We
speculate that the effect might be associated with an impaired trafficking
of interendothelial VE-cadherin junctions.
Acknowledgments: This work was supported by the German Research Foundation (DFG, FOR
1406, FU 691/9-2).
References:
1. Wiedmann, R. et al.: Cancer Res 2012, 72(22): 5976-87.
2. von Schwarzenberg, K. et al.: J. Biol. Chem. 2012, 288(2): 1385-96.
POS.021
Soraphen A – targeting the membrane composition as an
innovative approach to fight cancer
Stoiber, K.1; Vollmar, A. M.1; Braig, S.1
1
LMU Munich, Department of Pharmacy, Pharmaceutical Biology, Munich, Germany
The fatty acid metabolism is found to play a key role in oncogenic
transformation. First metabolic investigations of human tumors revealed
that malignant cells were characterized by an overexpression of lipogenic
enzymes like the fatty acid synthase and acetyl-CoA carboxylase (ACC),
resulting in a high rate of de novo fatty acid synthesis. We aim to
characterize the impact of Soraphen A, an acetyl-CoA carboxylase
inhibitor isolated from the myxobacterial strain Sorangium cellulosum, on
cancer cells and biophysically disclose its mode of action.
In order to elucidate the influence of ACC inhibition on the phospholipid
composition of cancer cells, phospholipids of Soraphen A treated MDAMB-231 breast cancer cells were extracted and analyzed by liquid
chromatography ESI tandem mass spectrometry. Interestingly, whereas
the total amount of phospholipid content is not modulated, the
composition of phospholipids is strongly altered by Soraphen A
treatment. As phospholipids are the main components of cellular
membranes, we analyzed whether changes in phospholipid composition
induced by stimulation with Soraphen A affect their biophysical
characteristics. We were able to demonstrate that Soraphen A strongly
enhances the rigidity of cellular membranes by generating giant plasma
membrane vesicles (GPMVs). In addition, inhibition of ACC with the
myxobacterial compound impedes the deformability of whole cells as
shown by optical stretcher experiments.
Next, to investigate whether Soraphen A induced alterations in the
biophysical properties of cancer cells have a functional impact on
metastasis, two highly metastatic cancer cell lines were treated for 2
hours with the acetyl-CoA inhibitor and migration and invasion assays
towards FCS and EGF as chemoattractant were performed. Soraphen A
dose-dependently abrogates migratory and invasive potential of MDAMB-231 breast cancer and T24 bladder carcinoma cells.
By using Soraphen A as a chemical tool to target the rate-limiting step of
the fatty acid synthesis, we are able to characterize the impact of
modulated membrane properties in cancer progression. Furthermore,
Soraphen A as a therapeutic lead substance might promote the
development of new cancer targeting drugs affecting the tumorsupportive cellular machinery of membrane features and lipogenesis.
NATURAL COMPOUNDS
POS.022
Crataegus ssp. promotes late-stage cardiac differentiation
and regeneration
Halver, J.1; Carrillo García, C.1; Willems, E.2; Schade, D.1
Department of Chemistry & Chemical Biology, TU Dortmund, Otto-Hahn-Str. 6, D-44227
Dortmund, Germany
Sanford-Burnham Medical Research Institute, 10901 North Torrey Pines Road, La Jolla, CA92037, USA
1
2
Background: The minimal and clearly insufficient ability of the adult heart
to regenerate after ischemic injury is a great appeal for identifying
biological mechanisms, substances and factors that improve this process
[1]. Two main sources for cardiomyocyte renewal and regeneration have
emerged in the field: a) Adult multipotent progenitor cells and b) preexisting cardiomyocytes [2].
Objective: Based on the many positive effects on the myocardium after
infarction and the overall cardiovascular protective activity of Crataegus
ssp. (extract WS®1442) [3], we aimed at studying whether also
mechanisms of cardiac differentiation and regeneration could possibly
play a role.
Results: Here, we show that WS®1442 efficiently stimulated
cardiomyocyte differentiation from murine and human ESCs in a dosedependent manner after mesoderm was formed. This activity was
thoroughly validated in a mESC-based (CGR8-Myh6-GFP) spontaneous
differentiation assay. First bioassay-guided fractionations of the extract
suggested that this activity is reserved for specific compound classes.
Conclusions: According to the observed activity profile, we hypothesize
that the identified active fractions in WS®1442 could possibly target
multipotent progenitors, stimulate their differentiation towards the cardiac
lineage but also expand their pool (proliferation). Further elucidation of
the underlying cellular and molecular mechanisms might lead to novel
targets that can be exploited for ex vivo expansion of cardiac progenitor
cells. Eventually, it will be interesting to see whether (and how) our in
vitro findings translate to in vivo regeneration.
References:
1. Schade, D.; Plowright A. T.: J. Med. Chem. 2015, in press.
2. Harvey, R. P.; Graham R. M.; Pu, W. T.: Stem Cell Res. 2014, 13: 521-714.
3. Koch, E.; Malek, F. A.: Planta Med. 2011, 77(11): 1123-1128.
POSTERS
4.4 Pharmaceutical Technology
and drug formulations
POS.023
In vitro model of infected stratum corneum for the efficacy
evaluation of novel formulations of antifungal ciclopirox
olamine as well as differential scanning calorimetry and
stability studies
Täuber, A.1; Müller-Goymann, C. C.1
1 Institut
für Pharmazeutische Technologie, Technische Universität Braunschweig,
Mendelssohnstraße 1, 38106 Braunschweig, Germany
Introduction: Superficial fungal infections are a common disease and
affect 20-25% of the world’s population [1]. Mostly, the infections are
caused by dermatophyte fungi with Trichophyton rubrum being the main
trigger. In this contribution, the antifungal agent ciclopirox olamine (CPX)
was incorporated into a variety of poloxamer 407-based formulations.
These novel compositions consisted of poloxamer 407 (P407), double
distilled water, propylene glycol (PG), isopropyl alcohol (IPA) and
medium chain triglycerides (MCT) in given ratios. Antifungal efficacy
evaluation against T. rubrum was analysed in a novel in vitro model of
infected stratum corneum (SC). Moreover, stability and differential
scanning calorimetry (DSC) studies were carried out to analyse the
influence of the formulations on the SC.
Methods: The P407-based formulations were automatically stirred at
1440 rpm for 1.5 min with an Unguator® e/s (GAKO Konietzko GmbH,
Bamberg, Germany). Subsequent storage was done for 24 h at
20 ± 1 °C to ensure sufficient equilibration. All the formulations were
given codes reflecting their quantitative composition, e.g. 1P1050
represented a formulation loaded with 1% CPX, while the vehicle itself
contained 10% P407/MCT (4:1), 50% IPA/PG (1:1) and 40% double
distilled water (all w/w). In vitro studies of infected SC were performed
according to Lusiana et al. [2] with slight modifications. In the present
contribution, isolated human SC was used instead of bovine hoof plates
and keratin films, respectively. SC was hydrated in autoclaved, double
distilled water. With a polycarbonate filter as backing, approximately 5
SC were placed in a potato glucose agar. DSC studies were carried out
according to Lusiana and Müller-Goymann [3] with one modification.
Instead of 37 °C in the previous publication, equilibration and incubation
was done at 32 °C. For stability studies, samples were stored in air-tight,
sealed vials at 30 °C under light exclusion. After 1, 3, 6 and 12 months,
CPX contents were determined with high performance liquid
chromatography (HPLC). Approximately 50 mg of the formulation were
weighed in a volumetric flask and dissolved with mobile phase under
vortexing (Vortex-Genie® 2, Scientific Industries, Inc., New York, United
States). 20 µL of each mixture was injected into the HPLC column.
Results: Regarding the infected SC studies, a marketed semi-solid
formulation (Selergo® 1% cream) showed complete fungal growth
(= highest score: 10) after 6 days of incubation. Liquid P407-based
formulations e.g. 1P1050 as well as a marketed nail lacquer (Ciclopoli®)
indicated scores of 0 (= no fungal growth). The semi-solid P407-based
formulations achieved scores of 6-8. 4P4030 was the only P407-based
formulation tested with incorporation of 4% CPX. It showed only slight
fungal growth with a score of 0.5. The scores of all tested formulations
were significantly lower than that of Selergo® 1% cream (p < 0.05).
The DSC measurements gave precise information on the interaction
between applied formulation and SC. Regarding the excipients, water
and PG had only slight impact on the SC, whereas MCT highly influenced
the SC indicated as transition shifts. IPA showed no peak transition shift.
Selergo® 1% cream had slightly influenced the transitions similar to
1P1020 and 1P1030. Increasing IPA/PG concentrations from 1P1020 to
1P1070_21 resulted in higher transition shifts. Furthermore, higher
incorporated CPX amounts such as in 1-4P4030 augmented the
transition shifts.
After 12 months of storage, almost all P407-based formulations still
indicated CPX contents ≥ 95%. Several liquid P407-based formulations
exhibited phase separation. Moreover, slight yellow discolouration
occurred.
Conclusion: An in vitro model of infected SC has successfully been
developed for pretesting novel antifungal formulations. Several liquid
P407-based formulations showed superior T. rubrum growth inhibition in
comparison to a marketed semi-solid reference. DSC studies indicated
rising influence on the SC with increasing CPX- as well as IPA/PG
content. One year stability studies exhibited CPX contents ≥ 95% for the
majority of the P407-based formulations.
References:
1. Havlickova, B.; Czaika, V.A; Friedrich, M.: Mycoses 2008, 51 (Suppl. 4): 2–15.
2. Lusiana; Reichl, S.; Müller-Goymann, C.C.: Eur. J. Pharm. Biopharm. 2013, 84 (3): 599–605.
3. Lusiana; Müller-Goymann, C.C.: Am. Ass. Pharm. Sci. 2011, 12 (2): 496-506.
POS.024
“Nanoparticle Tracking Analysis of Size and Concentration
detection in Particle Suspensions: Influence of
experimental and data evaluation parameters”
Gross, J.1; Karow, A. R.2; Sayle, S.2; Bakowsky, U.1; Garidel, P.2
Department of Pharmaceutics and Biopharmacy, Faculty of Pharmacy, 1 Philipps-University
Marburg, Ketzerbach 63, 35032 Marburg, Germany
2 Boehringer Ingelheim Pharma GmbH & Co.KG, Global Bioprocess & Pharmaceutical
Development, Birkendorfer Straße 65, 88397 Biberach (Riss), Germany
1
Nanoparticle Tracking Analysis (NTA) is an emerging technique for
detecting particle size distributions and particle concentrations in
biologics. This study deals with the performance evaluation for the
detection and characterisation of various particles by NTA. Our
investigation focusses on the NTA measurement parameter set-ups. In
order to achieve this, we used polystyrene standard particles
(monodisperse and polydisperse) as well as protein particles.
On one site, we showed the highly precise and reproducible detection of
particle size and concentration in monodisperse polystyrene particle
systems, under specified and constant parameter settings. On the other
hand, our results exemplify potential risks and errors while setting
parameters with regards to the results and thus interpretation thereof.
Especially changes of the parameters camera level and detection
threshold led to significant changes in the determined particle
concentrations. We defined specified “optimal” camera levels for
monodisperse particle suspension characterizations in the size range of
20 to 1000 nm. We illustrated that the results of polydisperse polystyrene
standard particle solution measurements highly depend on the used
parameter settings. Changes in the settings led to the appearance or
disappearance of “peaks” for polydisperse systems. For the use of NTA
in biopharmaceutical analysis proteinaceous samples were investigated.
We analysed protein particle suspensions and compared unstressed and
stressed protein samples similar to polysterene particle measurements.
We also measured these samples in two measuring modes (general
capture mode and live monitoring mode) that the commercially available
analysis tool is offering. Our results stated the live monitoring mode as
more suitable for protein samples, as the results were more reproducible
and less operator-depending.
In conclusion, NTA is a potential technique and unique in quantitative
evaluation of particle suspensions in the subvisible size range, especially
for monodisperse suspensions. We strongly urge on not underestimating
the influence of the operator, i.e. setting the measuring parameters on
the obtained results. From an industry perspective the documentation of
the parameter settings for each experiment is crucial for data evaluation,
data interpretation and for data comparison.
Acknowledgments: Boehringer Ingelheim Pharma GmbH & Co.KG, Michaela Blech
POS.025
The glass transition temperature as an underestimated
parameter for drug release properties of polymer based
nanoparticles
Lappe, S.1; Langer, K.1
1 Institute
of Pharmaceutical Technology and Biopharmacy, University of Muenster,
Corrensstraße 48, 48149 Münster, Germany
Polymeric nanoparticles are widely used in pharmaceutical research
acting as drug delivery systems. Great efforts were made to understand
the release behaviour of drug loaded nanoparticular delivery systems [1].
PHARMACEUTICAL TECHNOLOGY AND DRUG FORMULATIONS
Nevertheless the subject of the glass transition temperature (Tg), a well
defined parameter for the starting material of nanoparticle preparation, is
underestimated when finished drug delivery systems are characterised.
Therefore the aim of this study was to take a more detailed look on the
correlation between Tg and the drug release behaviour of flurbiprofen
from poly(D,L-lactic-co-glycolic acid) (PLGA), poly(L-lactic acid) (PLA)
and poly(butyl methacrylate-co-(2-dimethylaminoethyl) methacrylate-comethyl methacrylate) (Eudragit® E) nanoparticles.
Nanoparticle preparation was carried out by an emulsion diffusion
method. For the determination of Tg a differential scanning calorimetry
(DSC) method was used. The release kinetics were conducted in
phosphate buffer pH 8.0 or bovine serum albumin solution (5% w/v) at
different temperatures for 48 h.
In comparison to unloaded PLGA and PLA nanoparticles the Tg of
flurbiprofen loaded nanoparticles was lowered. Flurbiprofen forms a solid
solution with the polymer during nanoparticle preparation leading to a
shift in Tg acting as a plasticiser within the polymer matrix [2,3]. Tg of
unloaded Eudragit® E nanoparticles and the flurbiprofen loaded system
were nearly identical suggesting less interactions between drug and
polymer chains.
Drug release kinetics were analysed at temperatures below and above
Tg of the respective nanoparticle system. For the flurbiprofen loaded
PLGA and PLA nanoparticles a reduced release could be observed when
the temperature of the release medium was kept below Tg. An instant
release of almost the entire encapsulated drug was determined
increasing the temperature above Tg [4]. Regarding the flurbiprofen
loaded Eudragit® E nanoparticles a temperature independent burst
release was observed suggesting that the major part of the drug was not
incorporated into the polymeric matrix but was rather adsorbed to the
large nanoparticle surface.
The present study shows the Tg dependent release behaviour of drug
loaded polymeric nanoparticles. A Tg higher than the temperature of the
release medium leads to a reduced burst release of drug substance.
However this assumption is not applicable to any drug-polymer
combination since the type of drug loading is also important. Therefore
the parameter Tg needs to be considered in nanoparticle characterisation
during the development of new drug delivery systems.
References:
1. Sant, S. et al.: J. Control. Release 2005, 107(2): 203-214.
2. Pamujula, S. et al.: J. Pharm. Pharmacol. 2004, 56(9): 1119-1125.
3. Blasi, P. et al.: AAPS PharmSciTech 2007, 8(2): Article 37.
4. Faisant, N. et al.: Int. J. Pharm. 2006, 314(2): 189-197.
POS.026
Nano modified antioxidants for the prevention and
treatment neurodegenerative diseases
Braun, A.1; Law, J. K. Y.1; Scholz, P.2; Keck, C. M.2; Ingebrandt, S.1;
Schaefer, K. H.1
1 Department
of Informatics and Microsystem Technology, University of Applied Sciences,
Zweibrücken, 66482, Germany
2 Department of Applied Logistics and Polymer Sciences, University of Applied Sciences,
Pirmasen, 66482, Germany
Neurodegenerative diseases, including Alzheimer‘s and Parkinson‘s
diseases, are commonly occurring diseases for elderly. Oxidative stress
due to amyloid β (Alzheimer's disease) or α-synuclein (Parkinson‘s
diseases) leads to neuronal death [1]. The symptoms of this diseases
usually take years to develop, unfortunately, there is no effective solution
for the diseases by far. An early stage of identification and prevention of
such diseases will be beneficial. However, it is challenging to obtain
biopsy from the central nervous system (CNS) over time.
Therefore the aim of this project is to develop an effective nutritional
supplement for preventing neurodegenerative diseases. Herbal
substances have high antioxidant contents and studies have shown
promising neuroprotective potential [2]. Moreover, most of these natural
substances have low solubility in water and consequently poor
absorption in human body. In order to enhance the solubility,
nanocrystals of these antioxidants are produced [3].
To study the effect of nano antioxidants on the prevention of the
neurodegenerative diseases, cell culture experiments were performed.
Different kind of nano and micro particles (rutin, hesperidin, β-carotene)
were incubate together with α-synuclein. In the DPPH radical scavenger
assay the nano particles showed much higher antioxidant capacity than
the micro particles. In vito experiments demonstrated that the nanomodified particles have a higher impact on neuronal survival in
pathological condition in comparison to the micro particles.
Acknowledgments: This work is supported by the Federal German Ministry of Education and
Research: “IngenieurNachwuchs2013: Nanomodifizierte Antioxidantien zur Prävention und
Behandlung neurodegenerativer Erkrankungen”. The authors would like to thank Mr. Rainer
Lilischkis for his support for the SEM imaging.
References:
1. Wei, X. et al.: Mol. Cell. Neurosci. 2013, 54: 71-83.
2. Kumiko, I. et al.: Free Radic. Biol. Med. 2001, 30(4).
3. Rachmat, M. et al.: Eur. J. Pharm. Sci. 2009, 36: 502–510.
POS.027
A Slow-Release System of Bacterial Nanocellulose for
Octenidine as Wound Dressing
Alkhatib, Y.1; Dewaldt, M.1; Moritz, S.1; Kralisch, D.1,2; Fischer, D.1
1 Department
of Pharmaceutical Technology, Friedrich-Schiller-University, Jena, 07745,
Germany
2 JeNaCell, Jena, 07745, Germany
The biopolymer bacterial nanocellulose (BNC) has raised a substantial
interest in medical and pharmaceutical research and development due to
its outstanding physicochemical and biological characteristics. Although
BNC has an identical chemical formula like plant cellulose, its unique
structure consisting of a three-dimensional network of nanosized fibres,
a high purity, excellent biocompatibility, and unique mechanical stability
provide an excellent basis as a biomaterial for artificial blood vessels,
scaffolds for tissue engineering and wound dressings. Octenidine was
introduced for skin, mucous membrane, and wound antisepsis more than
two decades ago [1]. The antiseptic exhibits a broad antimicrobial activity
against Gram-positive and Gram-negative, plaque-forming bacteria, and
fungi. In this study, BNC fleeces were functionalized with octenidine to
develop an active wound dressing for infected wounds over one week.
Bacterial nanocellulose fleeces were prepared by strains of
Komagataeibacter xylinus (DSM 14666) in Hestrin-Schramm medium
under static conditions in a 24-well plate, harvested and purified in
alkaline solution [2]. After autoclaving, BNC fleeces, were loaded with
octenidine solution (0.5%, Schülke & Mayr) with and without the addition
of the Poloxamers P338 and P407 (BASF SE) in different concentrations
under shaking (70 rpm) for 48 hours. Release studies were performed in
PBS buffer at pH 7.4 for up to 192 h at 32 °C using the Franz cell diffusion
system (SES GmbH). Octenidine was quantified via UV/Vis
spectrophotometry at 281 nm. Size and zeta potential were measured
using a Zetasizer Nano ZS (Malvern Instruments). Compression and
tensile tests as well as water absorption and water retention values of the
BNC fleeces were determined. Antimicrobial efficacy against S. aureus
and P. aeruginosa was investigated by an agar diffusion test and
fluorescence staining method using the LIVE/DEAD® Bacterial Viability
Kit (Molecular Probes). The biocompatibility of the BNC was tested on an
ex ovo hen’s egg model.
BNC fleeces were successfully loaded by the post synthesis technique.
The addition of 5% Poloxamer P407 or 10% Poloxamer P338 induced
the formation of octenidine loaded micelles (approx. 2-5 nm, zeta
potential -0.8 to 4.3 mV) that retarded the release of octenidine by 2629% in comparison to the release profile of octenidine loaded in BNC
without Poloxamers. The increase of the concentration of Poloxamer
P407 to 18.5% or Poloxamer P338 to 22% resulted in an octenidine
containing thermoreversible hydropolymer gel. The gel formation in the
BNC slowed down the drug release with a continuous profile over up to
192 h. Whereas for octenidine loaded BNC no changes of the mechanical
characteristics could be observed, the incorporation of the Poloxamers
increased the compression stability and decreased the water absorption
and retention values. As demonstrated in agar diffusion and LIVE/DEAD®
tests, octenidine loaded BNC with and without Poloxamers demonstrated
high antimicrobial activity against S. aureus and P. aeruginosa.
POSTERS
In conclusion, bacterial nanocellulose could be loaded effectively with
octenidine. In the presence of different types of Poloxamers in various
concentrations a controlled release over up to one week could be
achieved. Therefore, this system could be used as a wound dressing with
sustained drug release.
Acknowledgments: The authors would like to thank R. Brabetz and E. Pfaff for their excellent
technical assistance. We acknowledge the Thuringian Ministry of Education, Science and
Culture, the EFRE (B714-10032) and the FAZIT Foundation, Gemeinnützige
Verlagsgesellschaft mbH (S. M.) for financial support.
References:
1. Hübner, N.O. et al.: Skin Pharmacol. Physiol. 2010, 23(5): 244-258.
2. Moritz, S. et al.: Int. J. Pharm. 2014, 471(1–2): 45-55.
Figure: SEM image of fleece structure (A), TEM image of nanowhiskers (B)
Acknowledgments: We would like to thank the Deutsche Forschungsgemeinschaft DFG (FI
899/2-1) for funding of the project. Additionally, F.A. Müller and F. Wesarg (IMT, FSU Jena) are
acknowledged for SEM analysis and E. Pfaff and J. Thamm for technical assistance.
References:
POS.028
1. Moritz, S. et al.: Int. J. Pharm. 2014, 471(1–2): 45–55.
2. Müller, A. et al.: J. Pharm. Sci. 2013, 102(2): 579–592.
Bacterial Nanocellulose as a Drug Delivery System for
Nucleic Acids
Pötzinger, Y.1; Rabel, M.1; Ahrem, H.1; Rahnfeld, L.1; Kralisch, D.1,2;
Klemm, D.3; Fischer, D.1
1 Department
of Pharmaceutical Technology, Friedrich-Schiller-University, Otto-Schott-Straße
41, 07745 Jena, Germany
2 JeNaCell GmbH, Winzerlaer Str. 2, 07745 Jena, Germany
3 Polymet Jena e.V., Wildenbruchstraße 15, 07745 Jena, Germany
In the steadily emerging field of natural and renewable materials, the
biopolymer bacterial nanocellulose (BNC) has attracted increasing
importance due to its outstanding physicochemical and biological
properties. BNC is characterized by its high purity, excellent
biocompatibility, and unique mechanical stability favouring the
application as tissue scaffold or wound dressing. Beside this, the
nanosized three-dimensional network (Figure A) offers a huge interface
for the local delivery of antiseptic drugs [1] or larger molecules like
proteins [2]. By acidic hydrolysis, it is also possible to disintegrate the
native BNC fibres in order to generate high-crystalline needle-like
nanowhiskers (Figure B). The anionic surface of these nanowhiskers
allows ionic interactions with charged molecules and opens up a new
field for drug delivery. In the present study the preparation of non-viral
carrier systems for gene therapy based on BNC fleeces on the one hand
and on nanowhiskers on the other was investigated for the first time.
BNC fleeces were produced by strains of Komagataeibacter xylinus
(DSM 14666, DSMZ, Braunschweig) in static culture, harvested and
purified. Resulting nanocellulose hydropolymers were used as neverdried (nd) or freeze-dried (fd) BNC and loaded with nucleic acids using
three different strategies. Loading of nd-BNC with an injection technique
or fd-BNC by reswelling increased the amount of loadable material and
the ease and speed of loading in comparison to a conventional
adsorption technique [2]. In vitro release studies were performed under
agitated conditions. The cumulative release of adsorption loaded fleeces
was highly varying and not reproducible. In contrast, a prolonged release
exhibited a biphasic release profile with a rapid release in the initial 24
hours followed by a slower release rate up to 200h could be
accomplished with fd-BNC loaded by reswelling. Injection loaded ndBNC showed an even more extended release profile represented by an
almost linear curve.
For the preparation of nanowhiskers nd-BNC was disintegrated and
hydrolysed with sulfuric acid, purified and fractionated. By modifying the
hydrolysis parameters it is possible to prepare nanowhiskers with a
controllable size and surface charge, low polydispersity and high purity.
No cytotoxicity of the nanowhiskers was observed in vitro up to a
concentration of 1 mg/mL and 72 h incubation time using L929 mouse
fibroblasts. Hemocompatibility was demonstrated quantitatively and
qualitatively by testing hemoaggregation and hemolysis up to a
concentration of 1 mg/mL. Additionally, no toxic effects of the particles
towards the vascular system of developing chick embryos could be
detected using a shell-less hen’s egg model, neither after topical nor after
systemic administration. An effective DNA loading of the nanowhiskers
was facilitated by modifying the surface with poly(ethylene imine) using
a layer-by-layer technique which could be demonstrated by measuring
size and zeta-potential, performing agarose gel electrophoresis and DNA
quantification using AccuBlueTM.
In conclusion, the results confirmed the suitability of BNC for gene
delivery applications. Both three-dimensional BNC fleeces and BNC
based nanoparticles are highly biocompatible and could be loaded
effectively with nucleic acids. Depending on loading techniques, release
rates could be controlled and release profiles selectively adjusted.
POS.029
Impact of cationic coatings on PLGA nanoparticle
characteristics and cellular uptake
Kunschke, N.1; Loretz, B.1; Lehr, C. M.1,2
1 Helmholtz-Institute
for Pharmaceutical Research Saarland, Department of Drug Delivery,
Saarland University Saarbrücken, 66123, Germany
2 Biopharmaceutics and Pharmaceutical Technology, Department of Pharmacy, Saarland
University Saarbrücken, 66123, Germany
The delivery of therapeutic nucleic acids into the deeper lung implies
certain challenges: to reach the alveolar region, to overcome the noncellular barrier, to avoid the phagocyte clearance and to deliver the cargo
into the cytoplasm to its target. At the same time the drug delivery system
(DDS) has to be non-toxic, biodegradable and biocompatible [1]. A
combined system of PLGA and a cationic coating reduces the required
total amount of cationic material [2], which is leading to a lower
cytotoxicity of the DDS and remains the ability to bind siRNA on the
particle surface. The cationic polymers were selected by differences in
molecular weight and number of primary amine groups which is
significant for transfection efficiency. This may surely influence the entire
particulate system and their ability to enter cells.
Particle preparation was performed by a modification of the emulsiondiffusion-evaporation technique [3]. The model siRNA (21 nucleotides),
provided from GlaxoSmithKline (UK), was loaded on the nanoparticles
via electrostatic attraction. Protamine-, Chitosan- and CatStarch [4]coated PLGA nanoparticles were characterized by Dynamic light
scattering (DLS) and showed a mean diameter from 125 nm to 170 nm.
This value is depending on the MW of the used coating material. With a
higher the MW, the nanoparticles offered an increased size. A positive ζpotential from + 15 mV to + 40 mV supported the assumption of an outer
cationic polymer shell. The monodispersity (DLS: polydispersity index
(PdI) lower 0.200) and spherical shape of the nanoparticles was
confirmed via scanning electron microscopy (SEM). The colloidal stability
could be indicated for at least 21 days at storage conditions (4 °C).
Chitosan-PLGA NPs and CatStarch NPs showed an acceptable viability
(MTT assay) on Calu-3 and A549 cells (> 80%). Protamine-PLGA NPs
could reach at most 60% viability, after 4 h incubation of particle
concentrations lower than 250 mg/ml. Differences in cellular uptake and
localization after a 24h incubation period could be visualized via confocal
laser scanning microscopy (CLSM).
It was shown that a variation of the cationic coating of PLGA
nanoparticles can easily be performed via the emulsion-diffusionevaporation technique. And the resulting particulate systems represent a
promising approach in gene delivery to the lung.
Acknowledgments: COMPACT (Collaboration on the Optimization of Macromolecular
Pharmaceutical Access to Cellular Targets)
References:
1. Kumar, R. et al.: Biomaterials 2004, 25(10): 1771-7.
2. Naffee, N. et al.: Nanomedicine 2007, 3(3): 173-183.
3. Beisner, J. et al.: Lung Cancer 2010, 68(3): 346-54.
4. Yamada, H. et al.: Biomacromolecules 2014, 15(5): 1753-1761.
POS.030
Protein Corona Formation on Polymeric Nanoparticles and its
Impact on Cellular Uptake, Cytotoxicity, and Activation of
Endothelial, Epithelial, or Immune Cells
Obst, K.1; Charbaji, N.1; Miceli, E.2; Dimde, M.2; Balzus, B.3; Bodmeier,
R.3; Calderón, M.2; Haag, R.2; Hedtrich, S.1
Institute for Pharmaceutical Sciences, Freie Universität, Königin-Luise-Str. 2-4, 14195 Berlin,
Germany
2 Institute of Chemistry and Biochemistry, Freie Universität, Takustr. 3, 14195 Berlin, Germany
3 Institute for Pharmaceutical Sciences, Freie Universität, Kelchstr. 31, 12169 Berlin, Germany
1
Nanoparticles have gained great significance in different medical
applications such as drug delivery, diagnostics or therapy.
Nanomaterials, however, undergo a substantial change as soon as they
come in contact with biological fluids such as human blood which leads
to the formation of a protein corona that covers the particle. About 1,000
different proteins are present in blood plasma which readily interact with
the nanoparticles’ surface depending on the particles’ size, surface
charge and composition and which gives the nanomaterial a new
biological identity [1-3]. Comprehensive and systematic studies on
protein corona formation, however, and the impact on interactions
between nanoparticle and biological environment are rather scarce.
Hence, in this project, we systematically evaluated the protein corona
formation (identity and quantity) and subsequently studied its impact on
cellular uptake, cytotoxicity, and activation of endothelial, epithelial or
immune cells. Therefore, nanoparticles (Eudragit RS nanocarriers, ethyl
cellulose nanocarriers, core-multishell (CMS) nanotransporters, dendritic
polyglycerol nanogels, and polyglycerol nanogel with 10%, 30% and 90%
amination) differing greatly in composition, particle size polarity and
surface charge were prepared and subsequently analysed.
Initially, the particle size and zeta potential were assessed. Afterwards,
the nanoparticles were incubated with human plasma for 1 h or 24 h and
afterwards purified with a 0.7 M sucrose cushion and washing steps.
Next, the isolated protein corona was subjected to SDS-Page.
Subsequently, to identify the attached proteins, clear protein bands were
cut out, the gel was digested using trypsin and the samples were
ultimately analysed by mass spectrometry. For protein quantification, a
bicinchoninic acid assay was performed.
To study the effects of the protein corona on biological systems, we used
primary macrophages, keratinocytes and human umbilical vein
endothelial cells (HUVEC) to investigate cell viability, induction of
apoptosis and necrosis, release of pro-inflammatory cytokines, cellular
uptake and the respective uptake mechanism.
The nanoparticles’ size ranged from 30 nm to 200 nm and zeta potential
was between -24 mV and +30 mV. 1 h incubation with blood plasma
already initiated protein corona formation. Incubation for 24 h did not
change the type of attached proteins but significantly increased the
amount. Hereby, most pronounced protein corona formation was
observed with Eudragit RS nanoparticles, whereas with ethyl cellulose,
CMS and nanogels only one dominant adsorbed protein such as
apolipoprotein A-I for ethyl cellulose or serum albumin for CMS and
nanogels was identified. About 10 to 15 proteins adsorbed on Eudragit
RS nanoparticles ranging from 16 to 215 kDa including several
apolipoproteins, inter-alpha-trypsin inhibitors and complement factors.
In terms of cytotoxicity, overall protein corona-coated nanoparticles
showed reduced toxicity and the cellular uptake was slightly diminished.
Preliminary results indicated no change in the cellular uptake
mechanism.
In conclusion, our data suggest that proteins strongly adsorb on cationic
Eudragit RS nanoparticles and significantly less protein corona is formed
on ethyl cellulose and polyglycerol based nanomaterial which might be
advantageous in terms of drug delivery.
Moreover, the results indicate that protein corona formation leads to
reduced cytotoxicity, which might be due to a reduced cellular uptake.
Acknowledgments: This work was supported by a grant from the Helmholtz Virtual Institute. We
thank Dr. Christoph Weise (Institute of Chemistry and Biochemistry, Freie Universität Berlin) for
his help with the mass spectrometry analyses.
References:
1. Rahman et al.: Biophysics (Springer Series) 2013, 15.
2. Tenzer et al.: Nature Nanotechnology 2013, 8: 772-781.
3. Docter et al.: Nat. Protoc. 2014, 9(9): 2030-2044.
POS.031
Targeting Excipients for Individual Radiation Therapy of Cancer –
Surface Modification of PLGA Polymer and Liposome
Nanoparticles entrapping Lanthanides by Cholesterol- and PLAbound Ligands
Nawroth, T. 1; Krebs, L. 1; Johnson, R. 1; Langguth, P. 1; Hellmann, N. 2;
Decker, H. 2; Schmidberger, H. 3; Goerigk, G. 4; Boesecke, P. 5a; Le Duc,
G. 5b; Bravin, A. 5c; Schweins, R. 6
Pharmacy & Biochemistry Institute, Pharmaceutical Technology, Johannes Gutenberg
University, Staudingerweg, D-55099 Mainz, Germany
2 Molecular Biophysics Institute, Johannes Gutenberg University, Jakob Welder Weg 26, D55128 Mainz, Germany
3 Department of Radiooncology and Radiotherapy (Clinics), University Medical Center;
Langenbeckstr.1, D-55131 Mainz, Germany
4 HZB, Institute of Soft Matter and Functional Materials , BESSY Synchrotron, ASAXS, D-14109
Berlin, Germany
5 ESRF, European Synchrotron Radiation Facility, a)ID01, b)BioMedical Facility,c)ID17, 71
Avenue des Martyrs, F-38043 Grenoble, France
6 ILL, Institut Laue Langevin, DS / LSS, 71 Avenue des Martyrs, F-38042 Grenoble CEDEX 9,
France
1
For abstract see Short Poster Lecture SPL.002 on page 99.
POS.032
Perfluorocarbon-Nanoemulsions: Evolution of a marker
agent for in vivo 19F Magnetic Resonance Imaging
Grapentin, C.1; Krämer, W.1; Temme, S.2; Flögel, U.2; Schrader, J.2;
Wang, X.3; Peter, K.3; Schubert, R.1
Department of Pharmaceutical Technology and Biopharmacy, University of Freiburg, Hermann
Herder Str. 9, 79104 Freiburg, Germany
2 Department of Cardiovascular Physiology, University of Düsseldorf, Universitätsstr. 1, 40225
3 Baker IDI Heart and Diabetes Institute, 75 Commercial Road, Melbourne, Australia
1
Perfluorocarbon nanoemulsions (PFC-NE) are disperse systems
consisting of nanoscale liquid perfluorocarbon droplets stabilized by an
emulsifier, usually phospholipids. Perfluorocarbons are chemically inert
and non-toxic substances that are exhaled after in vivo administration.
The manufacture of PFC-NE requires high energy input, suitable
techniques being high pressure homogenization or microfluidization.
Originally investigated as oxygen carriers for cases of severe blood loss,
the application of PFC-NE nowadays is more focused on using them as
marker agents in 19F Magnetic Resonance Imaging (19F MRI). 19F is
scarce in organisms and thus PFC-NE are a promising tool for highly
specific and non-invasive imaging via 19F MRI [1]. Combining 19F images
with classic 1H images, the fluorine signal is set into an anatomic context.
One field of application utilizes the capability of neutrophils,
macrophages and monocytes to phagocytize PFC-NE and the
subsequent migration of these innate immune cells to inflamed tissues.
This technique has proven practicability in the unambiguous visualization
of inflammatory foci in numerous disease models in mice [2]. Recently,
the successful imaging of myocardial infarction in mini pigs has been
reported [3]. The translation to clinical trials in human seems potential,
but requires the development of a stable nanoemulsion whose droplet
size is well characterized over a long storage time. In a study, we used
dynamic light scattering in comparison to a second method, analysis of
transmission electron microscopy images of cryo-fixed samples, to
evaluate stability of PFC-NE [4].
Besides a passive targeting approach towards inflammatory tissue, site
specific delivery of PFC-NE to other pathological foci unaffected by
phagocytic immune cells is of interest. Combining 19F MRI with site
specific targeting of PFC-NE, pathological sites might be visualized
specifically before alterations are observed in classic 1H MRI. Tissue
specific targeting needs the decoration of the nanoemulsion with a
homing ligand, e.g. a peptide or antibody, whose antigen is
overexpressed in the target tissue.
One approach for surface modification of PFC-NE utilizes cholesterolPEGs bound to tissue specific peptides. The cholesterol moiety inserts
into the phospholipid layer of preformed droplets, allowing the
preparation of highly specific marker agents in small scales principally on
demand. This post insertion technique has recently proven feasibility in
the visualization of thrombi using PFC-NE equipped with an α2antiplasmin-based peptide in mice [5].
Small, robust homing ligands like RGD-peptide (bound to a phospholipid
anchor) can be incorporated into NEs during the manufacturing process.
POSTERS
For larger ligands of proteinaceous nature, such as (single chain)
antibodies, high shear forces and elevated temperatures might lead to a
degradation of the protein. A platform technology of PFC-NE bearing a
reactive group for covalent coupling of a targeting ligand is highly
desirable. Such nanoemulsions can be manufactured via
microfluidization using maleimide-PEG2000-phospholipids in a mixture
with standard phospholipids. Our results show that the reactive
maleimide groups stay intact during microfluidization and that ligands
containing a thiol group can be successfully coupled. In an in vitro
attempt, such PFC-NEs are directed to activated platelets via a singlechain variable fragment binding to glycoprotein IIb/IIIa [6]. Ongoing
research focuses on the advancement of this platform technique,
decorating PFC-NE with ligands of different sizes, and thus facilitating
site specific targeting in a variety of disease models both in vitro and in
vivo.
Acknowledgments: The authors gratefully emphasize financial support by Deutsche
Forschungsgemeinschaft (DFG).
References:
1. Temme, S. et al.: WIREs Nanomed. Nanobiotechnol. 2012, 4: 329-343.
2. Floegel, U. et al.: Circulation 2008, 118: 140-148.
3. Boenner, F. et al.: Eur. Heart J. – Cardiovasc. Imaging 2015, 16: 612-620.
4. Grapentin, C. et al.: PLoS ONE 2015, 10(6): e0130674.
5. Temme, S. et al.: Circulation 2015, 131. 1405-1414.
6. Wang, X. et al.: Circulation 2012, 125: 3117-3126.
POS.033
Entrapment of doxorubicin in different nanoparticle
systems – An overview
Pieper, S.1; Langer, K.1
Institute of Pharmaceutical Technology and Biopharmacy, Corrensstr. 48, 48149 Münster,
Germany
1
Doxorubicin-loaded nanoparticles (DOX-NPs) are a promising approach
regarding the therapy of carcinosis. Reported advantages of DOX-NPs
over traditional medication are an improved drug targeting due to EPReffect (enhanced permeability and retention effect), a lower rate of side
effects and good prospects to overcome multi drug resistance in cancer
cells [1]. However, there are several different nanoparticle preparation
techniques and starting materials, which can all influence essential
properties of the nanoparticles in therapy. In order to come up with the
most promising nanoparticle formulation, different materials and
techniques of nanoparticle preparation have been analysed in terms of
their physicochemical properties and their ability to entrap doxorubicin.
The particle size and polydispersity were measured by photon correlation
spectroscopy (PCS), while the amount of entrapped doxorubicin was
calculated via an indirect quantification method. Therefore, the
nanoparticles were centrifuged, the supernatants were collected and
analysed via HPLC using a reversed phase column and a mobile phase
consisting of water and acetonitrile (70%:30%, v/v) containing 0.1%
trifluoroacetic acid [2]. The resulting amount of doxorubicin corresponds
to the non-entrapped part of drug hence affecting the loading efficiency.
The following nanoparticle materials have been investigated: human
serum albumin (HSA), chitosan, poly(lactic-co-glycolic acid) (PLGA) and
PLGA modified with polyethylenimine (PLGA-PEI). For particle
preparation different techniques such as desolvation, emulsion-diffusion
and nanoprecipitation were used. For example, HSA nanoparticle were
formed according to a standard desolvation method, while PLGA and
PLGA-PEI nanoparticles were manufactured by emulsion-diffusion and
nanoprecipitation techniques [3,4]. With this work, we want to present
promising nanoparticle systems, and furthermore, discuss problems and
difficulties that have occurred.
References:
1. Haley, B.; Frenkel E.: Urol. Oncol. 2008, 26: 57-64.
2. Dreis, S. et al.: Int. J. Pharm. 2007, 341: 207-214.
3. Langer, K. et al.: Int. J. Pharm. 2003, 155: 75-82.
4. Astete, C. E.; Sabliov, C. M.: J. Biomater. Sci. Polym. Ed. 2006, 17: 247-289.
POS.034
Archaesomal enwrapped polyplexes (AePPs) for the
expression of luciferase plasmid (pCMV-luc) in various cell
lines
Engelhardt, K. H.1; Pinnapireddy, S.1; Baghdan, E.1; Bakowsky, U.1
1 Department
of Pharmaceutical Technology and Biopharmaceutics, Ketzerbach 63, 35037
Marburg, Germany
Gene therapy is a relatively new and widely researched field with great
potential in treating a wide range of human disorders. Nucleic acid-based
biopharmaceuticals allow treatment of not only symptoms but also the
very cause of an array of diseases by induction and/or inhibition of genes.
Gene therapy is based on the principle of delivering an intact fragment of
genetic material into the cytoplasm or nucleus of a desired cell.
The wide array of non-viral vectors can be broadly classified into
nanoparticles, polymers and lipid vectors. Lipid vectors are the only
synthetic vectors currently used in clinical trials. However, a number of
problems still need to be resolved before liposomes can be used in a
clinical setting. One of the major problems is poor stability of liposomes,
which is partially attributed to the hydrolysis of ester bonds and oxidation
of unsaturated fatty acids [1].
To overcome these stability issues is the use of more stable lipids for the
preparation of transfection reagents. A unique class of lipids, ether lipids,
found exclusively in the cellular membranes of the third domain of life
referred to as archaea, can be considered promising alternatives to
common lipids. Members of the archaea are known to inhabit
environments ranging from ordinary to ones characterised by extremes
of salt, temperature and pH [2]. The presence of ether lipids in
membranes of the archaea is believed to be responsible for their unusual
temperature and pH stability. Lipids from Sulfolobus are characterized by
two different backbones. The first one is called Glycerol-Dialkyl-GlycerolTetraethers (GDGT), where the glycerol is linked with two biphytanyl
ether chains at both sides. The second one is called Glycerol-DialkylNonitol-Tetraethers (GDNT) containing an additional nonitol group [3].
Liposomes containing different kinds of tetraether lipids (archaeosomes)
were first prepared by using the thin-film hydration method and
characterized by AFM (Atomic Force Microscopy) and DLS (Dynamic
Light Scattering). Polyplexes were formed by mixing 25-kDa branched
polyethylenimine (25kDa-bPEI) with pDNA at N/P 15. Archaeosomal
enwrapped polyplexes (AePPs) were finally obtained by mixing
achaeosomes and polyplexes at increasing Lipid/DNA ratios. For
transfection experiments COS-7 and SKOV-3 cells were seeded at a
density of 1 x 104 cells/per well on a 96-well plate 24h before transfection
experiments. After 4 h transfection with pDNA, cells were incubated 48 h
before determination of gene expression efficiency using luciferase
assay.
Polyplexes at N/P 15 and archaeosomes had an overall charge of
approximately +35 mV and -27 mV, respectively. Depending on the
amount of archaeosomes incubated with polyplexes, AePPs had a mean
diameter of 200-300 nm and zeta potential ranged from +35 to +8 mV.
Transfection experiments of aforementioned AePPs revealed successful
transfection of COS-7 and SKOV-3 cells at certain Lipid/DNA ratios. A
reduction of 20-50% of transfection efficiency in comparison with bPEI
alone would be acceptable, considering the much higher stability of
AePPs.
Polyplexes could be enwrapped with archaeosomes resulting in new
transfection reagents that are nanoscaled and stable. AePPs could be
easily prepared by adding positively charged polyplexes to negatively
charged archaesomes. Due to the unique chemical structure of tetraether
lipids, they can resist the acidic environment in the GIT and are less
susceptible to enzymatic degradation. These properties make them more
suitable as drug delivery systems, especially for sensitive compounds
like DNA.
Acknowledgments: The authors would like to thank the Deutsche Forschungsgemeinschaft
(DFG) for the financial support.
References:
1. Felgner, PL. et al.: Proc. Natl. Acad. Sci. 1987, 84: 413-7417.
2. Hanford, J.M.; Peeples, L.T.: Appl. Biochem. and Bio. 2002, 97(1): 45-62.
3. Jacquemet, A.: Biochimie. 2009, 91(6): 711-717.
POS.035
POS.036
Birch bark extract particle deposition from supercritical
solutions and use for stabilisation of semisolid systems
Examination of homogeneity of stent coatings produced
via fluidized bed process
Armbruster, M.1; Wahl, M. A.1
Wentzlaff, M. 1; Senz, V. 2; Grabow, N2; Weitschies, W. 1; Seidlitz, A. 1
1 Department
Germany
of Pharmaceutical Technology, Auf der Morgenstelle 8, 72076 Tübingen,
Introduction: Birch bark dry extract consisting of pentacyclic triterpenes
like for example betulin can be used to stabilize semisolid systems such
as creams and gels [1,2]. One of the crucial parameters for this function
as a pickering emulsifier and a gelling agent is the extract´s surface [3].
Usually birch bark extract is produced by organic solvent extraction (OSE). Birch bark extraction with supercritical fluids can be used as an
alternative extraction method [4], with different types of particle
deposition. Therefore, this method could enable the creation of extract
particles with different surface properties and thereby improve the
production of semisolid systems. The obtained extracts were investigated
by gas adsorption (BET) and inverse gas chromatography (iGC) to
characterize the extract´s surface. To determine the extracts’ ability to
stabilize semisolid systems, gels were made and their rheology studied
by amplitude sweep method.
Materials and Methods: The birch bark (Birken AG) extraction was
performed on a high pressure lab scale extraction unit (Sitec) at 350 bar
and 60 °C with supercritical (sc) carbon dioxide (CO2) as extraction
solvent. Three different types of particle deposition were tested (Figure
1). One way of particle deposition is the generation of a liquid CO2 phase
in a separator, in which the sc solution is expanded at 40 bar. In this liquid
phase the extract accumulates and when the pressure is released
particles deposit and can be removed as a dry extract (SCF-E)
(Figure 1a). Another option is the RESS (rapid expansion of supercritical
solutions) method. Thereby a sc solution is sprayed through a nozzle and
particles deposit directly into the gaseous phase (R-E) (Figure 1b). This
method has been modified by spraying directly into a lipophilic phase
(jojobaoil) to create a gel in a single step production, including extraction,
particle deposition and gel formation (Figure 1c). Other gels were
produced by dispersing 6% extract in jojobaoil with an Ultra-Turrax® (Ika
Labortechnik) for two minutes at 8000 rpm. The amplitude sweep (AS)
was performed at 23 °C with a 25 mm plate-plate setup and a gap of
0.8 mm at a Physika MCR 501 rheometer (Anton Paar). For the AS a
log ramp (deformation 0.1-1000%; frequenzy 1/s) was used.
Figure 1a-c: different types of sc particle deposition; Figure 2: gel formation of OS-E gel and SCF-E gel
Results: The first 6 hours of gelling were investigated for gels made out
of OS-E and SCF-E by performing an AS every 30 min. The mean
storage modulus (G’) in the linear viscoelastic region is used as an
indicator for gel strength and viscosity (Figure 2). For the SCF-E gel the
G’ values start and remain at a very high level compared to the OS-E gel.
Another AS after 24 hours acts as a reference for progressing gelling
process. The SCF-E gel easily reaches this point within the 6 hours
whereas the OS-E gel comes up to only 28% of the reference. Thus, the
SCF-E is able to stabilize the gels faster and better. One possible
explanation is the SCF-E´s higher BET surface area (36.2 ± 2 m2/g)
compared to the OS-E (29.4 ± 0.7 m2/g).
Conclusion: Birch bark particle deposition from supercritical CO2 can
provide extracts with superior gelling properties, compared to organic
solvent extract. Most likely, this is caused by different deposition
conditions and therefore different extract surface properties.
References:
1. Scheffler, A.: United States Patent US2003087789-2003-05-08, 2003.
2. Laszczyk, M.: Triterpentrockenextrakt aus Birkenkork 2007.
3. Grysko, M.: Herstellung und Charakterisierung von halbfesten Systemen auf der Basis
vonTriterpentrockenextrakt aus Birkenkork 2011.
4. Krasutzky, P.: Nat. Prod. Rep. 2006, 23(6): 919-942.
1 Institute
of Pharmacy, C_DAT, Ernst-Moritz-Arndt University of Greifswald, Felix-HausdorffStraße 3, 17487 Greifswald, Germany
2 Institute for Biomedical Engineering, University of Rostock, Friedrich-Barnewitz-Straße 4,
18119 Rostock, Germany
For abstract see Short Poster Lecture SPL.003 on page
100.
POS.037
Lipoproteins as new protein delivery system
Thoma, F.1; Langer, K.1
Institute of Pharmaceutical Technology and Biopharmacy, University of Muenster,
Corrensstraße 48, 48149 Muenster, Germany
1
Proteins are very important pharmaceuticals for the treatment of several
diseases, but in some cases the therapeutic application is limited by
insufficient delivery options. Currently in the field of protein delivery many
promising drug delivery systems such as nanoparticles or liposomes
enter the focus of research. But especially polymer based nanoparticles
have been found to be inadequate for protein delivery due to the high
protein adsorption on hydrophobic surfaces and low entrapment
efficiency [1].
Hence, the aim of this work was the establishment of a new kind of
protein delivery system. Oriented on physiological carriers micelle-like
lipoproteins were built based on soy lecithin, triglycerides, and
cholesterol by an emulsification process using human serum albumin
(HSA) as model protein. The resulting lipoproteins were purified by
extensive dialysis. Furthermore, different mass ratios of the ingredients
were used to allow an enhanced protein loading. The resulting
lipoproteins were characterized with regard to their physicochemical
properties such as hydrodynamic diameter, zetapotential, and
polydispersity index (PdI) by photon correlation spectroscopy (PCS). In
addition, the stability of lipoproteins under physiological conditions was
confirmed by titration with NaCl solution up to the physiological
concentration of 150 mmol/L.
To determine the protein integrity after lipoprotein preparation dissolved
lipoproteins were applied on a sodium dodecylsulfate polyacrylamide gel
electrophoresis (SDS-PAGE). Despite the occurrence of strong shear
forces during emulsification process no additional bands of protein
fragments or aggregates were detectable.
In conclusion we were able to establish lipoproteins as an appropriate
protein delivery system with respect to physical stability and protein
loading.
References:
1. Barichello, J. M. et al.: Drug Dev. Ind. Pharm. 1999, 25(4): 471-476.
POS.038
In vitro dissolution behavior of extended-release
theophylline capsules in media containing alcohol
Knop, K.1; Kremer, M.1; Ridder, S.1; Kleinebudde, P.1
1 Institute
of Pharmaceutics and Biopharmaceutics, Heinrich Heine University Duesseldorf,
Universitaetsstr. 1, 40225 Duesseldorf, Germany
In 2005 severe interactions were reported for extended-release
hydromorphone capsules taken together with alcohol (Palladone case,
FDA alert 7/2005 [1]). Therefore the FDA implemented in vitro dissolution
test media containing up to 40% ethanol in their 'product-specific
recommendations for generic drug development' guidelines for several
drugs [2]. The potential appearance of dose dumping is important for all
drugs with a narrow therapeutic index and can be detected by in vitro
dissolution tests. Aim of the present study was to evaluate the influence
POSTERS
of alcohol on the drug release from extended-release theophylline
capsules purchased on the German market.
Four extended-release capsule preparations (Bronchoretard 350,
Euphylong 300 mg, Theophyllin AL 300 retard, Theophyllin Hexal 300
mg) containing different retardation excipients were purchased in a local
pharmacy. The dissolution tests were performed using the basket
apparatus (Ph.Eur. 2.9.3) and the test conditions described for
'Theophylline Extended-Release Capsules' Test 6 in the USP 37 (0.05 M
pH 6.6 phosphate buffer, 1000 mL, 100 rpm). 10, 20 and 40% of the
buffer solution were substituted with ethanol. Theophylline concentration
was determined spectroscopically at 271 nm. Only the content of the
capsules (pellets) was used for dissolution testing. Disintegration tests
(Ph.Eur. 2.9.1) with the complete capsules were performed in the
different media to ensure that the capsule shell didn't have an influence.
The influence of capsule disintegration on dissolution can be neglected
because all capsules disintegrated within 1 to 2 min in buffer solution and
within 2 to 3 min in buffer with 40% ethanol. All preparations exhibited
extended release behavior in phosphate buffer with MDT (mean
dissolution time) values between 3 h (Euphylong) and 10 h (Theophyllin
Hexal). In ethanolic media all preparations showed dose dumping but
to a different extent. While Bronchoretard released the total dose in the
first two hours in media with 40, 20 and even 10% ethanol (see Figure),
Euphylong and Theophyllin AL underwent dose dumping only in media
with 40% ethanol. Theophyllin Hexal represented a central position with
dose dumping in 40 and 20%, but not in 10% ethanol. The more ethanol
sensitive preparations Bronchoretard and Theophyllin Hexal contain
copolymers of ethyl acrylate and methyl methacrylate or ammonio
methacrylate as retardation excipients. Euphylong and Theophyllin AL
comprise cellulose derivatives and were less affected by alcohol in lower
concentrations. In comparison to extended release theophylline matrix
tablets [3] the capsule preparations were more vulnerable to ethanol in
the dissolution media. As a consequence, extended release theophylline
capsules should not be taken together with alcohol in higher
concentrations.
References:
1. FDA: FDA Alert [7/2005]: Alcohol-Palladone interaction,
http://www.fda.gov/Drugs/DrugSafety/PostmarketDrugSafetyInformationforPatientsandProvi
ders/ucm129288.htm (11.06.2015).
2. FDA: Product-specific recommendations for generic drug development,
http://www.fda.gov/Drugs/GuidanceComplianceRegulatoryInformation/Guidances/ucm07520
7.htm (11.6.2015).
3. Knop, K.; Kleinebudde, P.: In vitro dissolution behavior of extended release theophylline
tablets in ethanolic media,
1st European Conference on Pharmaceutics, Reims 2015.
POS.039
Oral carbon monoxide release system
Steiger, C.1; Meinel, L.1
1 Institute
for Pharmacy and Food Chemistry, University of Würzburg, Germany
Introduction: Carbon monoxide (CO) has therapeutic effects in various
gastrointestinal diseases [1] yet clinical use today is challenged by
inappropriate delivery modes [2]. Consequently, we developed a tablet
referred to as oral carbon monoxide release system (OCORS) providing
precise, controlled and targeted CO delivery for the treatment of
gastrointestinal injury and inflammation, respectively. The model was
downsized to 1 mm and 0.5 mm in diameter for preclinical models
(Figure B).
Methods: OCORS is an oral tablet based on sulfite induced CO release
from the CO releasing molecule 2 (CORM-2) [2]. OCORS performance
was detailed as a function of the presence of buffer within the tablet core
and characteristics of a water-insoluble cellulose acetate coating, forming
a semipermeable shell around the tablet core. Amperometric detection
was deployed for recording CO release profiles throughout 20 hours.
Results and Discussion: OCORS was tuned for environmental pH
insensitivity by appropriate buffer systems blended within the tablet core.
CO release kinetic of 1 mm OCORS is tailorable from 25 min up to 20
hours by variation of coating thickness and coating hydrophobicity
(Figure A).
OCORS is a readily available tablet for oral use in preclinical models. The
controlled release system reliably delivered CO independent of
environmental pH, such that the therapeutic gas can be safely generated
at gastric, intestinal or colonic sites. In vivo experiments of OCORS are
required to demonstrate the pharmacokinetics and clinical potential of
this oral delivery platform for therapeutic gases.
Figure: A) Fast, intermediate, and slow CO release pattern of the 1mm oral carbon monoxide
release system (OCORS). B) 0.5 mm diameter OCORS along with a human hair for size
comparison.
References:
1. Motterlini, R.; Otterbein, L. E.: Nat. Rev. Drug Discov. 2010, 9(9): 728-U724.
2. Steiger, C. et al.: J.Control Release 2014, 189C: 46-53.
POS.040
Vinyl sulfone-poly(vinyl alcohol)-stabilized PLAnanoparticles as reactive particle system for various
surface modifications
Raudszus, B.1; Langer, K.1
Institute of Pharmaceutical Technology and Biopharmacy, University of Muenster, Corrensstr.
48, 48149 Muenster, Germany
1
The functionalization of the surface of nanoparticles plays an important
role as an approach for targeted drug delivery since a specific ligand may
lead to the ability to overcome several physiological barriers like the
blood-brain barrier [1]. The aim of the present project is to obtain
nanoparticles, which feature a surface modification with Apolipoprotein E
in order to allow the particles to cross the blood-brain barrier via receptormediated transcytosis [2].
Poly(vinyl alcohol) (PVA) is a polymer often used for the preparation of
poly(lactic-co-glycolic acid) (PLGA) or poly(lactic acid) (PLA)
nanoparticles, because of its ability to stabilize nanoparticles successfully
because of sterical effects [3]. Due to the high presence of PVA on the
particle surface, the carboxyl groups of the polymers PLGA and PLA are
less accessible for coupling reactions. Therefore the PVA was replaced
by the more reactive vinyl sulfone-poly(vinyl alcohol) (VS-PVA).
The vinyl sulfone-poly(vinyl alcohol) was synthesized by adding an
excess of divinyl sulfone to a solution of PVA in 0.1 M NaOH. The
reaction was stopped by addition of HCl. Afterwards the synthesized vinyl
sulfone-poly(vinyl alcohol) was purified by dialysis, in order to remove
excess divinyl sulfone. The purified product was analysed by 1H-NMR
and IR spectroscopy to prove the covalent linkage of the reaction
partners. The introduced vinyl sulfone-group is amine- and thiol-reactive,
which makes further modifications with molecules, containing these
functional groups, possible.
The investigated nanoparticles were based on PLA and prepared by
emulsion-diffusion method with VS-PVA as stabilizer. The obtained
nanoparticles had a size of about 230 nm, PDI below 0.1, and a negative
zetapotential of about -47 mV.
After nanoparticle preparation different amines like tyramine or PEG
derivatives were coupled by adding a solution of these substance to the
nanoparticle suspension. In order to increase the nucleophilicity of the
amine-containing reagent, the reaction was performed in borate buffer of
pH 9.
In conclusion the obtained particle system offers a starting point for an
effective functionalization of PLGA or PLA particle surfaces. Attached
PEG chains can be used as a linker to introduce further ligands like
proteins, peptides or other macromolecular compounds, which cannot be
attached to the surface directly because of steric hindrance.
References:
1. Kreuter, J.: Adv. Drug Deliv. Rev. 2013, 71: 2-14.
2. Zensi, A. et al.: J. Control. Release 2009, 137: 78-86.
3. Astete, C.; Sabliov C.: J. Biomater. Sci. Polymer Edn. 2006, 17: 247-289.
POS.041
Polyarginine modified human serum albumin (HSA)
nanoparticles for effective cell transfection
sulphate was extracted from the sprayed material with sulphuric acid and
quantified utilising derivatisation with para-dimethylaminobenzaldehyde
followed by photometric determination.
Mesken, J.1; Langer, K.1
Institute of Pharmaceutical Technology and Biopharmacy, University of Muenster,
Corrensstraße 48, 48149 Muenster, Germany
1
Non viral gene therapy has entered the focus of many research groups
in the field of gene therapy in humans. By using a non-viral approach the
safety risks of viral gene delivery systems such as carcinogenicity,
immunogenicity or inflammation can be avoided. However in many cases
an efficient gene transfer to cells is solely achieved by viral vectors.
Hence the aim of the present work was the preparation of biodegradable,
nontoxic, plasmid-loaded human serum albumin (HSA)-based
nanoparticles with a promising transfection efficiency. Therefore the
nanoparticle-surface was modified by the arginine-rich cell-penetrating
peptide nona-arginine.
The plasmid-loaded nanoparticles based on HSA were prepared by
desolvation technique and were stabilised by glutaraldehyde as
described previously [1]. As a model plasmid a vector encoding the
mCherry fluorescent protein was used.
The modification of the surface was performed in a three-step strategy.
After purification of the nanoparticles followed by the introduction of thiol
groups onto the surface [2], nona-arginine was activated by a bifunctional
PEG-based crosslinker, followed by coupling to sulfhydryl groups of the
particle surface.
Dynamic light scattering (DLS) analysis showed a monodisperse size
distribution and the surface modification worked successfully which could
be revealed by C18-RP-HPLC analytics.
Cell culture studies with a focus on transfection efficiency were
conducted in HEK293 and HT-29 cells using fluorescence microscopy
and flow cytometry. Cytotoxicity was evaluated using WST assay and
cellular uptake experiments were performed using fluorescent labelled
HSA-nanoparticles.
In conclusion it was possible to develop a biodegradable, nontoxic
particle system with a potential to transfect cells.
Figure: microscopic image of foam sample generated from a an HFA suspension containing 4%
PLA and 8% solid particles
Generally, samples solidify in minutes after actuation and show
increased material density at specimen’s edges. Microscopic
observations elucidate interconnected pores with a diameter > 200 µm
(Figure). Solutions of 4 to 7% PLA in HFA are emptied to more than
97.5% (standard deviation 0.8%) from the canister, whereas higher
concentrations are not completely expelled due to high viscosity. XRPD
results indicate conversion of calcium phosphates to hydroxyapatite in
< 24 h. This finding is confirmed by scanning electron microscopy which
shows the appearance of typical needle-like crystals. Polymer-containing
samples typically show a decreased brittleness which finds its expression
in an increased deflection.
Acknowledgments: This study is part of a project being funded by the Business Development
and Technology Transfer Corporation of Schleswig-Holstein and Stryker Trauma GmbH.
References:
1. Ginebra, M.-P. et al: Adv. Drug Deliv. Rev. 2012, 64(12): 1090–1110.
2. Salgado, A. J. et al.: Macromol. Biosci. 2004, 4(8): 743–765.
POS.043
References:
The increase of dermal bioavailability of the antioxidant
rutin by new smartCrystal technology
1. Steinhauser, I. et al.: J. Drug Target. 2009, 17 (8): 627-637.
2. Weber, C. et al.: Int. J. Pharm. 2000, 211: 67-68.
Pyo, S. M.1; Keck, C. M. 2; Müller, R. H.1
1 Institute
of Pharmacy; Pharmaceutics, Pharmaceutical Nanotechnology & NutriCosmetics,
Freie Universität Berlin, Kelchstr. 31, 12169 Berlin, Germany
2 PharmaSol GmbH, Stubenrauchstr. 66, 12161 Berlin, Germany
POS.042
Novel foamable cement formulation for application of
antibiotics to bone voids
Arntz, P.1; Nassut, R.2; Scherließ, R.1; Steckel, H.3
1 Department
of Pharmaceutics and Biopharmaceutics, Kiel University, Grasweg 9a, 24118 Kiel,
Germany
2 Stryker Trauma GmbH, Prof.-Küntscher-Strasse 1-5, 24232 Schönkirchen / Kiel, Germany
3 Deva Holding A.S., Halkalı Merkez Mah. Basın Ekspres Cad. No:1, 34303
Küçükçekmece/İstanbul, Turkey
Calcium phosphate cements are recognised as biocompatible and
bioactive materials for bone replacement and are discussed as drug
delivery systems [1]. Disadvantages of currently marketed products are
a laborious manual application procedure and their low flexural strength.
Also, cellular penetration should be granted in an ideal formulation to
allow for vascular ingrowth and remodelling to native bone [2]. These
issues are addressed with a novel foam formulation.
Tetra-calcium phosphate, di-calcium phosphate and tri-sodium citrate
were milled (Labstar, Netzsch, Germany) in ethanol (99.9% V/V) to an
average particle size in the upper nanometre range (Zetasizer Nano ZS,
Malvern, UK) and filled into glass bottles containing poly-D,L-lactic acid
(Resomer R 205) and 1% gentamicin sulphate. Ethanol content is
reduced to 35% (m/m) of dry residue by vacuum drying to minimise the
amount of ethanol per dose but avoid agglomerate formation. After
crimping using continuous valves, PLA is dissolved in 1,1,1,2,3,3,3heptafluorpropane (HFA).
Samples derived from actuation were investigated regarding macro- and
microscopic appearance of foamed specimen, hydroxyapatite formation
using x-ray powder diffraction (XRPD) (STUDI P, STOE & Cie GmbH,
Germany), suspension stability and content delivery. Three-point
bending (BZ2.5/TN1S, Zwick/Roell AG, Germany) was conducted and
compared to results of samples without PLA. Finally, sprayed gentamicin
Although the flavonoid rutin has excellent antioxidant properties on
cellular level, it shows too poor skin penetration and thus an insufficient
dermal bioavailability due to its low solubility and slow dissolution rate.
To enable the dermal use of rutin its solubility was increased by
smartCrystal technology where the µm-sized rutin raw drug powder was
reduced in size to submicron-size range. Whether rutin smartCrystals
bring a real improvement in penetration and antioxidant activity
compared to raw drug powder was investigated by the performance of an
ex vivo penetration study and an in vitro antioxidant activity study.
Rutin smartCrystal suspension was kindly provided by PharmaSol
GmbH. As reference rutin raw drug powder suspension was produced
with identical composition. Particle size distributions of both suspensions
were compared by laser diffraction (LD, Mastersizer 2000, Malvern
Instruments, UK). For the ex vivo penetration study tape stripping test
was performed on pig ears with two hydrogels containing either rutin
smartCrystals or raw drug powder both at 5%. DPPH (2,2-diphenyl-1picrylhydrazyl) assay was performed to compare the antioxidant activity
of rutin smartCrystal contained in intense lifting eye serum (Dr, JK
Cosmeceuticals, Germany) with eight other anti ageing products on the
market having rutin or its derivatives as active. The maximum
discoloration of a methanolic DPPH solution with an absorption of 1.0 at
a wavelength 517 nm was measured over 60 minutes with a PharmaSpec
UV-1700 photometer (Shimadzu Corporation, Japan).
LD diameters 50% of 240 nm and 90% of 860 nm confirmed the
submicron size of the rutin smartCrystals. The penetration depths and
strengths of rutin applied as smartCrystal and raw drug powder hydrogel
were compared. Until the 7th tape strip 1.5 fold higher amount of rutin
could be detected for the raw drug powder hydrogel. This shows a
remaining of active in the upper layers of the stratum corneum standing
for low penetrated amount of active into the deeper layers. Starting from
the 8th tape strip the penetration behaves inversely. In the deeper and
therefore more relevant layers of the stratum corneum rutin
POSTERS
smartCrystal hydrogel shows 2.2 and 2.5 times higher rutin amount for
the 14th and 23th tape strip, respectively, proving the significant
penetration enhancement of rutin smartCrystals compared to the raw
drug powder. The DPPH assay allowed the division of tested products
into three antioxidant activity classes from very strong discoloration of the
methanolic DPPH solution (> 80%, class I) representing very strong
antioxidant activity to medium (60 to 20%, class II) and low (< 10% class
III) discoloration standing for almost no antioxidant activity. Only the
intense lifting eye serum (Dr, JK Cosmeceuticals, Germany) with a
discoloration of more than 85% within 15 minutes counted to class I. The
highest discoloration reached from the reference products was under
60% and three of them even did not shown any discoloration effects on
the DPPH solution. Thus, the antioxidant activity of intense lifting eye
serum (Dr, JK Cosmeceuticals, Germany) with rutin as smartCrystals is
superior towards the tested dermal anti ageing products with rutin or its
derivatives.
Rutin smartCrystals lead to a distinctly deeper and up to 2.5 times
higher penetration into the skin of active compared to raw drug powder
formulation. Also the antioxidant activity of rutin smartCrystals in intense
lifting eye serum (Dr, JK Cosmeceuticals, Germany) proved to be
superior in comparison to other marketed products with rutin formulated
in standard manner. So, the combination of deeper and stronger
penetration with higher antioxidant activity will automatically lead to an
improved dermal bioavailability. Thus, the smartCrystal technology
enables the effective use of poor soluble plant extracts such as rutin in
cosmetic and dermal formulations.
in Milli-Q water. The samples were stored at 25 °C shaking with 100 rpm
in an Innova 4230 shaker for some hours. Then the formulations were
incorporated into 5% hydroxypropyl cellulose (HPC) gel, and were
studied in the pig ear penetration test via tape stripping. High
performance liquid chromatography (HPLC) was performed to determine
the drug amount.
Different actives have different maximum loading in smartPearlsTM, the
range changed from 41% of coenzyme Q10 to 15% of betulin. All of the
loaded smartPearlsTM expressed superior ability in dermal delivery. For
example, the 32% azithromycin loaded Syloid 3D showed 14 times
higher Cs than RDP and 7 times higher Cs than 190 nm sized
nanocrystals. According to the pig ear study, the smartPearls®
demonstrated a higher penetration than their comparison, especially in
deeper skin layers. For instance, the cyclosporine loaded Syloid 3D gel
(1% active in gel) revealed 6 times higher penetration than 5% RDP and
5% 300 nm sized amorphous nanoparticles in the 20th - 30th layer. The
1% rutin Syloid 3D gel represented up to 35 times higher penetration than
250 nm sized nanocrystals (5% in gel) in the 17th - 19th layers.
The smartPearlsTM proved to be an effective dermal delivery system for
poorly soluble actives for cosmetics and pharmaceuticals. smartPearlsTM
are superior due to the increased Cs and skin penetration, and preserve
the amorphous state. In view of the discussion about nanoparticles, they
are not nanoparticles but a micrometer sized delivery system (e.g. 10 to
40 μm porous particles).
Acknowledgments: PharmaSol GmbH Berlin, Germany, Grace GmbH & Co. KG, Worms,
Germany and China Scholarship Council (CSC).
References:
1. Wei, Q.; Keck, C. M.; Müller, R.H.: Int. J. Pharm. 2015, 482(1-2): 11-20.
2. Monsuur, F.; Höfer, H.H.; Keck, C. M.: US patent application 2014.
POS.044
smartPearlsTM: Novel dermal amorphous delivery system
based on porous particles
Keck, C. M.1; Jin, N.2; Du, W. J.2; Staufenbiel, S.2; Monsuur, F.3; Höfer,
H. H.3; Müller, R. H.2
1 PharmaSol
GmbH, Stubenrauchstr. 66, 12161 Berlin, Germany
of Pharmacy, Freie Universität Berlin, Kelchstr. 31, 12169 Berlin, Germany
GmbH & Co. KG, In der Hollerhecke 1, 67547 Worms, Germany
2 Institute
3 Grace
Poorly soluble cosmetic and pharmaceutical actives have often a very
poor dermal penetration and thus low bioavailability, often not reaching
the effective concentration. Approaches such as penetration enhancers
have problems such as tolerability by the skin and lack of regulatory
accepted status. A smarter approach is to increase the saturation
solubility Cs. This leads to an increased concentration gradient, and thus
increased penetration, e.g. by using amorphous µm-sized actives, drug
nanocrystals (small size creating increased dissolution pressure), or the
combination of both, i.e. amorphous nanoparticles (e.g. NanoMorph
concept). However, the problem of the amorphous state is its low stability,
tendency to re-crystallize. In the last decade, increasing interest focussed
in oral drug delivery on porous materials loaded with drug in the
amorphous state (e.g. CapsMorph), using e.g. porous silica. The
confinement of the drug in small pores prevents re-crystallization, which
makes the amorphous state stable up to 5 years [1]. The small size
dimension of the actives in the pores increases solubility, on top of the
effect of the amorphous state.
This delivery principle was transferred to the dermal administration route
(smartPearls) [2]. For example, mesoporous silica Syloid® SP53D-11920
(Syloid 3D, company W. R. Grace & Co., Worms, Germany) was used
for loading with various actives. Both drugs (e.g. azithromycin,
cyclosporine) and cosmetic and consumer care agents (e.g. betulin, rutin,
hesperidin, coenzyme Q10) have been used as model actives to
investigate the performance of smartPearls® as dermal carriers. For
comparison, raw drug powder (RDP), the “gold standard” nanoparticles,
commercial products or effective clinical products were tested in skin
penetration studies.
Each active was dissolved in suitable organic solvent and was sprayed
manually by a spraying nozzle onto certain amounts of silica
(impregnation method). Subsequently, the solvent was evaporated in a
compartment dryer at suitable temperatures. The solvent should
evaporate completely under the temperature in which the active can keep
its activity. In addition, the solvent should be safe in dermal use. The
theoretical maximum loading is relative to the density of the drug, the
density of the solvent and the pore volume of the silica. The practical
maximum loading of silica was monitored by x-ray diffraction (XRD,
Philips PW 1830). To get Cs, the maximum loaded silicas were dispersed
POS.045
Polymorph formation during non-sink dissolution testing
of co-amorphous indomethacin-arginine: spray dried
powder vs. tablet formulation
Lenz, E.1; Jensen, K. T.2; Knop, K.1; Grohganz, H.2; Löbmann, K.2;
Rades, T.2; Kleinebudde, P.1
1 Institute
of Pharmaceutics and Biopharmaceutics, Heinrich Heine University,
Universitaetsstrasse 1, 40225 Duesseldorf, Germany
2 Department of Pharmacy, University of Copenhagen, Universitetsparken 2, 2100 Copenhagen,
Denmark
Co-amorphous drug-amino acid formulations are a promising alternative
to common amorphous formulations which contain polymers as
stabilizers. The co-amorphisation of indomethacin with arginine results in
a physically stable preparation due to ionic interactions, and furthermore
the intrinsic dissolution rate is improved compared to the pure amorphous
drug [1]. Recently, spray dried indomethacin-arginine was successfully
formulated into tablets [2]. In this study, the non-sink dissolution
behaviour of pure spray dried powder was investigated and compared to
the dissolution behaviour of tablets.
Indomethacin-arginine (IND-ARG, 1:1 molar ratio, 1:0.49 weight ratio)
was prepared from a 4% w/v solution in acetone/water (70:30; Büchi B290) [1]. For preparation of tablets (TAB IND-ARG), mannitol
(Parteck® M200), croscarmellose sodium (Ac-Di-Sol® SD-711), colloidal
silicon dioxide (Aerosil® 200) and magnesium stearate
(Parteck® LUB MST) were used. Flat faced tablets were pressed with a
drug load of 50 mg using a rotary die press with 82 MPa. Non-sink
dissolution testing was performed in 900 mL KH2PO4 buffer (100 mM, pH
4.5, 37 °C) for 24 h with a paddle apparatus (n = 3; 50 rpm; HPLC
analysis). After 24 h, media were filtered and the obtained precipitates
were dried. They were characterized using XRPD and DSC. Scanning
electron microscopy was performed after sputter-coating.
Dissolution profiles of spray dried IND-ARG showed an immediate
release of IND resulting in a maximum concentration (cmax) of
42.9±1.3 mg/L (tmax 7 min). The solubility of crystalline IND in the
dissolution medium was thus exceeded by a factor of about 6. This
supersaturation led to a fast crystallization of IND in a needle-like shape,
whereas the stable γ-IND exhibits prism- and plate-like crystals. XRPD
and DSC measurements showed that the precipitates consisted of
α-IND, which is the most commonly observed metastable polymorphic
form [3,4]. TAB IND-ARG slowly eroded within 40 min, resulting in a drug
release with a cmax of 26.1±2.8 mg/L (tmax 35 min) and a supersaturation
by a factor of about 4. The supersaturation was maintained for a longer
period compared to IND-ARG, and as a consequence comparable areas
under the curve were obtained after 24 h. The precipitates of TAB INDARG consisted also of needle-shaped crystals, but according to XRPD
and DSC, another not yet identified polymorph was formed. In
conclusion, co-amorphous IND-ARG crystallizes in different polymorphic
forms in aqueous media depending on the rates of crystallization.
References:
1. Jensen, K. T. et al.: J. Pharm. Pharmacol. 2015, (submitted).
2. Lenz, E. et al.: Tableting of spray dried co-amorphous indomethacin-arginine, AAPS Annual
Meeting & Exposition 2014, San Diego.
3. Lin, S. Y.: J. Pharm. Sci. 1992, 81: 572-576.
4. Surwase, S. A. et al.: Mol. Pharmaceutics 2013, 10: 4472-4480.
resulting in a higher densification factor. Therefore, the density of the
resulting ribbon is higher and its porosity, consequently, lower.
In conclusion, two scales of Freund-Vector compactors were investigated
for different combinations of materials and pressures, and the porosity of
the resulting ribbons was evaluated. It was observed that the increase in
pressure reduces the porosity and higher fraction of MCC leads, in
general, to higher porosities. It was confirmed that the higher the roll
diameter, the lower the porosity of the resulting ribbons.
Acknowledgments: This work was supported by the IPROCOM Marie Curie initial training
network, funded through the People Programme (Marie Curie Actions) of the European Union’s
Seventh Framework Programme FP7/2007-2013/ under REA grant agreement No. 316555. The
authors would like also to thank the University of Surrey (Prof. Charley Wu and his group,
especially Ms. Serena Schiano) and AstraZeneca in Macclesfield (Dr. Gavin Reynolds and Dr.
Andreja Mirtic) both located in U.K., for the materials provided and for allowing the using of their
pieces of equipment.
POS.046
Effect of the scale of two Freund-Vector roll compactors on
the porosity of ribbons
Pérez Gago,
A.1;
Kleinebudde,
P.1
Institute of Pharmaceutics and Biopharmaceutics, Heinrich-Heine-University,
Universitätstrasse 1, 40225 Düsseldorf, Germany
1
In the pharmaceutical industry, the scale-up or transfer of a formulation
from the lab scale to the pilot scale is a necessary step. Roll compaction
is a continuous dry granulation process commonly used and therefore,
understanding its scale-up is highly interesting. Several suppliers of roll
compactors applied diverse design techniques when they developed
their equipment in different scales. One of these manufacturers is the
Freund-Vector Corporation which compactors are characterized by
changing the roll diameter and roll width from one scale to other while
other suppliers keep the roll diameter constant and only change the roll
width.
The aim of this work is to investigate how the change in scale affects to
the porosity of the ribbons produced from MCC, mannitol and five binary
mixtures of 15, 30, 50, 70 and 85% MCC using two different scales of roll
compactors from the Freund-Vector Corporation. These excipients were
chosen as they present opposite behaviours against compaction and
they are widely used in the pharmaceutical industry.
The TFC-Lab Micro and the TF-Mini (Vector Corporation, Marion, U.S.A.)
were used to carry out the roll compaction process using a smooth roll
surface and rim rolls as sealing system in both compactors, ensuring that
only the scale has an effect on the products obtained. The TFC-Lab Micro
is a small lab-scale compactor which roll diameter is 50 mm and width 24
mm while the TF-Mini has rolls of 100 mm diameter and 37 mm width.
The pure materials together to the 50% MCC mixture were roll
compacted in both scales under three different pressures (2, 5 and
8 MPa) while keeping the roll speed in 2 rpm and adjusting the feeding
screw speed in order to reach a gap of 1.5 mm. The roll compaction of
the 15, 30, 70 and 85% MCC mixtures was performed only at 5 MPa
pressure. The resulting ribbons were characterized regarding porosity
using an envelope and T.A.P. density analyser (GeoPyc® 1360,
Micromeritics Instrument Corp., Norcross, U.S.A.).
The porosity of the resulting ribbons was compared in the two scales
considering on the one hand, the effect of the material properties and on
the other hand, the pressure applied during the compaction process. If
the different materials compacted at 5 MPa are compared, the porosity
changes from 20.3% to a maximum of 33.2%, and although some
differences between the two compactors were observed, in general, the
porosity increases while the proportion of MCC rises. However, if the
effect of the pressure is analysed for the pure materials and the 50%
MCC which were compacted at 2, 5 and 8 MPa, as expected, a general
decrease in porosity with higher pressures is observed, although the
change from 5 to 8 MPa was less notable, probably because it was hard
for both pieces of equipment to control this value of pressure. Again, the
porosity tends to be greater as the proportion of MCC increases.
Nevertheless, the most interesting behaviour, which was observed in
both cases, was that the ribbons produced in the smaller-scale
compactor TFC-Lab Micro showed higher porosity than those compacted
on the TF-Mini, and this tendency was observed for all the compacts but
the ones produced from mannitol and 15% MCC. This change on the
porosity with the scale is expected as larger roll diameters result in higher
densities. Two rolls presenting different diameters will have the same nip
angle, which determines where the densification starts. However, for the
larger roll, the volume included between the nip angle and the gap is
higher than for the small roll. This means that if the same material is roll
compacted for a given gap with a large roll, more volume is densified
POS.047
Roll compaction of different grades of alpha-lactose
monohydrate
Grote, S.1; Kleinebudde, P.1
1 Institute
Germany
of Pharmaceutics and Biopharmaceutics, Heinrich-Heine-University, Duesseldorf,
Roll compaction is an important process to improve properties of
pharmaceutical formulations. As an intermediate step in tableting,
properties of the resulting granules like flowability play an important role
due to their effects on the subsequent steps [1]. In this study different
grades of lactose (a milled grade, three primary crystal structures sieved
to different sizes and one agglomerated) are investigated with regard to
their resulting granules by roll compaction. The focus will be on the
particle size and shape and their influence on the granules.
Various formulations based on different grades of lactose (Granulac 200,
Inhalac 250, Inhalac 230, Inhalac 120, Tablettose 80, Meggle,
Wasserburg, Germany) were dry granulated by roll compaction
(Minipactor, Gerteis, Jona, Switzerland) via binary mixtures with MCC
(Vivapur 101, JRS Pharma, Rosenberg, Germany) containing 50% or
80% lactose, respectively. A specific compaction force of 5 kN/cm was
applied and the gap size amounted 2 mm maintained by gap control.
Smooth rolls were used combined with a rim roll sealing system and a
roll speed of 3 U/min.
To characterize the raw materials, particle size distribution (PSD) of the
lactose was measured by laserlight scattering (Mastersizer 3000,
Malvern Instruments, Malvern, Great Britain) and shape was identified by
SEM (Phenom G2 pro, Phenom World, Eindhoven, Netherlands).
Before determination of granules’ PSD via dynamic image analysis
(Camsizer XT, Retsch GmbH, Haan Germany), samples were divided
with a rotating sample divider (Retschmühle, Retsch GmbH, Haan,
Germany). Finally, ffc-values were obtained by a ring shear tester to
characterize flowability (RST-01, Dr. Schulze, Schüttguttechnik,
Wolfenbüttel, Germany)
Results show an influence of raw lactose structure especially on
granules’ PSD. The primary crystals show in both cases, 50% and 80%
Lactose, the highest fraction of fines followed by the milled grade.
However the D(50) values of the 50% mixtures deviate from the fines and
show a range from 599 µm for the milled grade over 608 to 657 µm for
the primary structure up to 676 µm for the agglomerates. These results
are not confirmed by the 80% mixtures where the D(50) of the milled
grade, 553 µm, is placed in between the results of the primary structured
grades (492 to 585 µm). Again the highest value is obtained for the
agglomerated grade (619 µm). Summarising it seems that primary
crystals of alpha-lactose monohydrate can only be roll compacted with
an higher amount of fines although the raw material has higher D(50)
values (54 to 131 µm) in comparison to the milled particles (33 µm). The
agglomerated grade shows the biggest particles as raw material, D(50)
179 µm and also as granules. It may be assumed that a higher porosity
of the starting material lead to harder ribbons and coarser granules.
Evaluation of flowability by ffc-values shows an exceptional position of
the granules produced with milled lactose. They are above the limit of 4,
mixture of 50% 5.6 and mixture of 80% 4.5, which characterize a bulk
material as easy flowing. All the other mixtures, except Inhalac 250 with
an amount of 50% lactose (4.7), are below this limit and therefore
characterized as cohesive.
References:
1. Kleinebudde, P.: Eur. J. Pharm. Biopharm. 2004, 58(2): 317-326.
POSTERS
POS.048
Influence of different cationic lipids and DOPE on the cell
uptake of lipoplexes
Ziller, A.1; Hobernik, D.2; Bros, M.2; Langguth, P.1
Department of Biopharmaceutics and Pharmaceutical Technology, Johannes GutenbergUniversity, Staudingerweg 5, 55099 Mainz, Germany
of Dermatology, University Medical Center of the Johannes Gutenberg- University,
Langenbeckstrasse 1, 55131 Mainz, Germany
1
2 Department
Personalised immunotherapies for cancer treatment are becoming more
and more important. Protein structures which are only expressed on
tumour cells are presented to the human immune system. Hence, the
immune system generates a specific immune response, which leads to
an attack against the tumour cells. One option for presentation of a
protein is DNA, which codes for it and is translated in the target cells. The
transport of DNA to the cells encounters several hurdles. DNases, which
degrade DNA, are omnipresent in the human body. For endocytosis,
DNA has to pass across negatively charged cell membranes. Therefore,
carrier systems can be used to achieve a protected as well as a targeted
transport of DNA [1].
In this study, a carrier system composed of liposomes and DNA, socalled lipoplexes, are tested. For preparation of lipoplexes the Dual
Asymmetric Centrifuge (DAC) is applied [2]. They are composed of
L-α-lysophosphatidylcholine (EPC) as basic ingredient and one kind of
cationic lipid, while DNA concentration is set to 1mg/ml. Various cationic
lipids and their influence on the uptake into DC-cells were studied:
20mol% of 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP), 3ß-[N(N',N'-dimethylaminoethane)-carbamoyl]cholesterol
(DC-Chol),
Dimethyldioctadecylammonium
(DDAB),
1,2-di-O-octadecenyl-3trimethylammonium propane (DOTMA) and 1,2-di-O-octadecenyl-3dimethylammonium propane (DODMA). Since 1,2-di-(9Z-octadecenoyl)sn-glycero-3-phosphoethanolamine (DOPE) as an additional component
influences the uptake of DNA, DOPE is added in a molar ratio 1:1 to the
cationic lipid as well [3]. Additionally, the proportion of different
ingredients is varied. Size and zeta-potential of the formulations were
characterised. The uptake of DNA was measured via the lucerifase assay
at 24h and 72h in comparison to free DNA.
Size and zeta-potential measurements show that formulation of DNA in
lipoplexes is possible by achieving spherical vesicles. While the
formulations with DOTAP, DC-Chol and DDAB show no significant
effects, the formulations with DOTMA and DODMA cause a significant
increase in the uptake in comparison to free DNA. The formulation with
DOTMA and DOPE do not reach a significantly higher uptake than
DOTMA without DOPE, whereas the formulation with DODMA and
DOPE show even higher uptake rates.
In summary, lipoplexes represent interesting delivery systems for the
transport of DNA. Preparation with DAC has shown to be simple and fast,
yielding applicable formulations. Working under DNase-free conditions is
possible as well as very small batch sizes [2]. Hence, DAC supports the
suitability of lipoplexes for individual immunotherapy. It has been shown
that lipoplexes increase the uptake of DNA into cells. Moreover, the
choice of cationic lipid as well as the addition of DOPE influence this
uptake.
Acknowledgments: BionTech AG, An der Goldgrube 12, 55131 Mainz, Germany; Lipoid GmbH,
Frigenstraße 4, 67065 Ludwigshafen, Germany; Deutsche Forschungsgemeinschaft,
Kennedyallee, 53175 Bonn, Germany
References:
1. Wolff, J. A. et al.: Science 1990, 247(4949 Pt 1): 1465-1468.
2. Hirsch, M. et al.: J. Control. Release 2009, 135(1): 80-88.
3. Maitani, Y. et al.: Int. J. Pharm. 2007, 342(1-2): 33-39.
POS.049
Impact of different drying and storage conditions on
mechanical properties of orodispersible films
Thabet, Y.1; Krainitzki, L.1; Dietzel, D.1; Preis, M.2
1 Institute
of Pharmaceutics and Biopharmaceutics, Heinrich Heine University Duesseldorf,
Universitaetsstraße 1, 40225 Duesseldorf, Germany
2 Pharmaceutical Sciences Laboratory, Faculty of Science and Engineering, Åbo Akademi
University, Artillerigatan 6A, 20520 Turku, Finland
The use of orodispersible films (ODF) in pediatric therapy is gaining
interest in the most recent years [1,2]. Mechanical robustness combined
with rapid disintegration are crucial quality aspects of this innovative
dosage form. The present study aimed to investigate whether different
drying (room temperature (RT), 40 °C and 80 °C) and storage conditions
(open, sealed in aluminum sachets, humid conditions) following the
production of solvent-casting films influence the film properties.
Hypromellose (HPMC), hydroxypropylcellulose (HPC) and polyvinyl
alcohol (PVA) were used as film forming agents. Metamizole sodium salt,
an analgetic, antipyretic and antispasmic drug, served as model drug.
Films with and without drug load were prepared and subsequently their
morphological, mechanical and disintegration properties were
investigated.
All produced HPMC films lead to transparent, flexible films. After storage,
HPMC films containing metamizole show a temperature- dependent
recrystallization. The higher the drying temperatures, the less crystals
could be detected (by means of polarized light microscopy). HPC films
dried at 80 °C lead to opaque, yellowish films, which has been described
with regard to drying procedures applied to HPC and could be explained
by the limited water solubility of HPC at higher temperatures [3]. ODFs
with PVA as film forming polymer result in transparent, flexible films which
also show a recrystallization after storage time. All films disintegrated
rapidly within approximately 10-20 s using a modified disintegration tester
with a particular film sample holder [4]. Mechanical testing determining
the puncture strength and elongation of the samples, revealed the
sensitivity to varying drying temperatures. The mechanical strength of the
drug-loaded films decreased with increasing temperatures, e.g. for
HPMC (RT: 1.67±0.41 N/mm2, 40 °C: 1.59±0.37 N/mm2, 80 °C:
0.21±0.06 N/mm2). Subsequent storage at 58.5% relative humidity for
four weeks did not alter the mechanical properties (RT: 1.53±0.16
N/mm2, 40 °C: 1.55±0.19 N/mm2, 80 °C: 0.24±0.05 N/mm2).
Furthermore, higher drying temperature lead to less flexible films. The
drug-load on the one side increased the strength of the films, but
decreased their flexibility on the other side.
This study revealed that depending on polymer and drug properties
certain drying conditions are favorable in the manufacturing of
orodispersible films, but need to be carefully evaluated case by case.
References:
1. Hoffmannn, E .M.; Breitenbach, A.; Breitkreutz, J.: Exp.Opin. Drug Deliv. 2011, 8(3): 299-316.
2. Preis, M et al.: AAPS Pharm Sci Tech. 2015, 16(2): 234-241.
3. Klug, E.D.: J. Polymer Sci.:Partc C 1971, 36(1): 491-508.
4. Beilke, D.; Preis, M.: TechnoPharm. 2014, 4(6): 334-337.
POS.050
Application of numerical simulations in pharmaceutical
tableting
Hildebrandt, C.1; Gopireddy, S. R.2; Scherließ, R.1; Urbanetz, N. A.2
1 Department
of Pharmaceutics and Biopharmaceutics, Kiel University, Grasweg 9a, 24118 Kiel,
Germany
2 Daiichi-Sankyo Europe GmbH, Pharmaceutical Development, Luitpoldstrasse 1, 85276
Pfaffenhofen, Germany
Even though alternative forms of pharmaceuticals exist, tablets remain
the most common and preferred dosage form to date due to its stability
and ease of administration. With the huge volume of tablet production,
analytical tools are in high demand as they are used to control the final
product quality as per the regulatory requirements. Besides process
analytical tools (PAT), the pharmaceutical industry looks for alternative
solutions such as numerical simulations in formulation development and
product quality control. In this study, such a numerical approach is
presented with the focus on the investigation of powder flow within a
tablet press as well as content uniformity in dies thereby increasing the
process understanding of this part of the tableting procedure and to
impact product quality in a positive way.
The powder flow within the tablet press, i.e. from the force feeder to the
dies, depends on material characteristics on the one hand and process
conditions on the other hand. The feed frame often contains a number of
paddle wheels which convey the powder from the hopper outlet over the
die opening (see Figure). In this study, particle micro-dynamics are
computed through the three-dimensional discrete element method
(DEM). The DEM captures individual particle trajectories through
Newton’s equations of motion, which provide dynamic particle scale
information. The considered particle characteristics correspond to
microcrystalline cellulose which is a well-known pharmaceutical tablet
excipient. The numerical model is applied to a lab-scale tablet press force
feeder to predict the effect of feeder paddle wheel speed as well as
rotating speed of the die table on the inter-tablet variability.
The preliminary simulation results show that increasing the paddle wheel
speed at constant die table speed leads to increased inter mixing of
powder within the force feeder and simultaneously, the amount of powder
mass collected in the dies is decreasing. This is due to the fact that with
increased paddle wheel speed the net force acting on particles in the
radial direction within the force feeder increases and it is higher than the
gravitational force. Furthermore, results indicate that at lower paddle
wheel speed the powder tends to segregate in case of poly-disperse
particle size, i.e. the fine particles settle down at the bottom whereas the
large size particles rise to the top of the feeder. This may cause content
variation between the dies when using multicomponent mixtures. This
inter-tablet variability can cause wrong dosing for the patients. Numerical
results also show that the stresses acting on the feeder walls, which apply
shear stress on the powder thereby affecting its flowability and
lubrication, are influenced by the paddle wheel speed.
A
A
B
POS.052
The effect of material properties and process parameters
on roll compacted ribbons
Csordás, K.1; Kleinebudde, P.1
Side view
Top view
high luciferase and EGFP activity, respectively. Moreover biological
activity remains high even after storage up to three months at room
temperature. We also observed PVA microparticles to be dependent on
the manufacturing process. In addition, tested formulations were shown
to be nontoxic. Taken together, this indicates that PVA-MP may
represent a promising system for long term storage and controlled
release of PEI-based polyplexes and lipopolyplexes.
B
A: Force feeder with paddle wheel and hopper
B: Die table
Simulation of powder flow
in the force feeder of a lab
scale tablet prodcution
press (RoTab, kg-pharma).
Poly-disperse,
spherical
particles (in total 1,500,000)
with a mean diameter of
453 µm were simulated.
The material properties
correspond
to
microcrystalline cellulose. The
paddle wheel and die table
speed are both 30 rpm.
The current simulation study helps in visualizing the powder flow from the
feeder to the die, which gives rise to better understanding of the die filling
process. The results show that the particle micro-dynamics are
significantly influenced by the material and process conditions. To fulfil
the requirements of the authorities and to ensure product quality, process
simulations can help in identifying the critical attributes of the tableting
unit operation whereas experimental and analytical approaches could not
consider features such as quantification of the shear stress within the
force feeder. In conclusion such numerical studies will support the
product quality control concurrently with established means of
pharmaceutical development.
POS.051
Encapsulation of PEI-based nanoparticles into a poly(vinyl
alcohol) matrix – a new application for an old excipient
Schulze, J.1; Aigner, A.1
1 Rudolf Boehm Institute for Pharmacology and Toxicology, Clinical Pharmacology, University of
Leipzig, Leipzig, Germany
The nucleic acid based treatment of diseases offers great opportunities,
even in the case of targets considered as otherwise undruggable. Due to
its potential applications, the interest in gene therapy, and consequently
in gene delivery, has grown continuously over the past years. The
development of non-viral delivery systems is under intense investigation
and has led to various lipid and polymeric excipients. Among those,
polyethylenimine (PEI) is a promising candidate due to its high biological
activity. The combination of this cationic polymer with a lipid delivery
system may further improve properties of formed nanoparticles by
combining the beneficial features of a lipid system (low cytotoxicity, high
stability) and the advantages of PEI. The establishment of systems for
their controlled delivery and controlled release remains another
formidable challenge. Here we describe the fabrication and extensive
characterization of a controlled release system for PEI-based
nanoparticles.
The purpose of this study was to evaluate if the combination of nanoscale
polyplexes or lipopolyplexes with a hydrophilic polymer will yield an
appropriate carrier system. To this end, we incorporated PEI-based
nanoparticles into poly(vinyl alcohol) microparticles (PVA-MP). This
feasibility study aimed at investigating whether high transfection efficacy,
zeta-potential and particle size of embedded nanoparticles remain
unchanged during the manufacturing process and storage and upon
release.
We show that polyplexes as well as lipopolyplexes released from PVA
microparticles do not change in size compared to unformulated
nanoparticles, while a profound alteration of the zeta potential was
observed. Furthermore we demonstrate the released nanoparticles to be
taken up by cells and their DNA payload to be transcribed, yielding in
Institute of Pharmaceutics and Biopharmaceutics, Heinrich Heine University, 40225
Duesseldorf, Germany
1
Roll compaction/dry granulation is a widely used agglomeration process
in the pharmaceutical industry. The powder is compacted by applying a
defined force resulting in long shaped or debris ribbons. The ribbons are
milled into granules to achieve desired granule size. Ribbon porosity is
one of the critical quality attributes of roll compaction process, because it
is closely related to the mechanical properties of granules, thus affects
the downstream processes e.g.: tableting [1]. The goal of this work was
to investigate the impact of material properties and process parameters
on roll compacted ribbons. The average ribbon porosity and universal
hardness were determined.
Microcrystalline cellulose (MCC) (Avicel PH 101 FMC BioPolymer, USA),
spray-dried mannitol (Pearlitol 200 SD, Roquette, France) and the 1:1
mixture of them were roll compacted using AlexanderWerk BT 120
(AlexanderWerk AG, Germany) roll compactor. True density of the
starting materials was determined by AccuPyc 1330 Helium Pycnometer
(Micromeritics, USA). Ribbons were manufactured using side-sealing
system and a pair of smooth rolls. The hydraulic pressure (HP) was set
at 18 bar, 24 bar, 36 bar, 48 bar and 60 bar; the gap width (GW) was
adjusted at 1.5 mm and 2.3 mm. The roll speed was kept constant at 3
rpm. For each material 13 batches were prepared in total according to a
full factorial design of experiment (Modde 9.0, Umetrics, Sweden). The
intermediate products, called ribbons were collected and their average
porosity was measured by GeoPyc 1360 (Micromeritics, USA) in a 25.4
mm diameter chamber using a consolidation force of 51.0 N. DryFlo was
used as medium to obtain the measurements (n=3). The universal
hardness was determined using Fischerscope HM 2000 (Helmut Fischer,
Sindelfingen, Germany) excerting 1000 mN indentation force in 20 s in
the middle along the length of the ribbon (n=15).
Good models are observed for ribbon microhardness (R2= 0.88) and
porosity (R2= 0.96). Hydraulic pressure and gap width are found to be
significant quantitative factors for both responses. Ribbon microhardness
increases with increased hydraulic pressure. The contrary is observed in
case of porosity: the higher HP is set, the lower porosity is measured.
Mannitol ribbons represent a higher increase of the microhardness than
MCC ribbons using the same parameter settings. It can be explained by
the breakage of the brittle mannitol particles during feeding into the nip
area and the compaction between the two counter-rotating rolls, thus
more particles could be densified [2]. The microhardness of the 1.5 mm
thin mixture ribbons are between the microhardness values of MCC and
mannitol ribbons, as expected. 2.3 mm thick mixture ribbons are
produced solely using 18 and 36 bar HP, because of the low surface
friction between the powder blend and roll surface. Compacting mannitol
using 48 bar HP and 1.5 mm GW causes the minimum porosity value
(9%), whereas the same parameter setting results in 15% porosity
manufacturing with MCC. This difference is observed due to the plasticelastic recovery of MCC after roll compaction. The most porous ribbons
(34%) are produced using MCC and setting 18 bar and 24 bar HP
regardless of GW. However, compacting with 36 bar and 48 bar HP 5%
increase of porosity is achieved, when the GW is increased from 1.5 mm
to 2.3 mm.
Acknowledgments: ”Roll compaction using AlexanderWerk BT 120 and the envelope density
measurements were supported by Astra Zeneca Ltd. Macclesfield, UK.; This work was
supported by the IPROCOM Marie Curie initial training network, funded through the People
Programme (Marie Curie Actions) of the European Union’s Seventh Framework Programme
FP7/2007-2013/ under REA grant agreement No. 316555.”
References:
1. N. Souihi et al.: Intern. J. Pharm. 2015, 484: 192-206.
2. Y. Teng, Z. Qui, H. Wen: Eur. J. of Pharm. and Biopharm. 2009, 73: 219-229.
POSTERS
POS.053
Excipient effect on colloidal structure and BCS-II drug
solubility in bicarbonate based intestinal model medium
FaSSIFmod6.5 and cholesterol containing biorelevant
intestinal medium (FaSSIF-7C)
Khoshakhlagh, P.1; Johnson, R.1; Nawroth, T.1; Langguth, P.1;
Schmueser, L.2; Hellmann, N.2; Decker, H.2; Szekely, N. K.3
1 Pharmacy and Biochemistry Institute, Division of Pharmaceutical Technology, Johannes
Gutenberg University, Staudingerweg 5, D-55099 Mainz, Germany
2 Molecular Biophysics Institute, Johannes Gutenberg University, Jakob Welder Weg 26, D55128 Mainz, Germany
3 JCNS-FRM-II Outstation at the MLZ, Lichtenbergstraße 1, D-85747 Garching
Surfactants are commonly used in drug formulations for increasing drug
solubility in case of hydrophobic APIs. They can have interaction with
transient colloidal structures of the bile and food in the intestine, which
are develop from micelles (5-10 nm) to liposomes (30-200 nm) and large
cholesteric particles (up to 50 µm). This study investigated the effect of
some model surfactants (Tween 80, Tween 20, decyl sulfate and dodecyl
sulfate) on the solubility of Fenofibrate in bicarbonate model medium
FaSSIFmod6.5 [1] and a novel cholesterol containing biorelevant medium,
FaSSIF-7C [2]. In parallel the structural development was investigated
with dynamic light scattering DLS and neutron small angle scattering
SANS (static and time resolved). The results indicated that the solubility
of Fenofibrate increased by a factor of four with the highest concentration
of Tween 80 to a two-fold reduction with the maximum concentration of
Tween 20. DLS data showed that increasing surfactant concentration
caused destruction of the native liposome structure but only at high
detergent concentration increased the formation of surfactant rich
micelles in the medium, which can resolve the API. In the intermediate
surfactant regime a drug solubility gap was observed. With cholesterol
containing media, the existence range of the liposomes was increased
(stabilized), while the solubility gap was observed at increased surfactant
concentrations (shift). The neutron study indicated a co-existence of
micelles and liposomes, and a developing of the liposome entity during
the development of FaSSIF-C in the first hour after dilution of FeSSIF
with the transfer medium representing the fluid input from gastric
emptying. According to the results only a few surfactants (based on the
type and concentration) have a stabilizing effect on the colloidal structure
and solubility of hydrophobic drugs in biorelevant media.
Lipophilc drug (BCS-II, Feno) incorporates in different nano-structures in
the presence of surfactant excipients.
As small scale feeder a MiniTwin Feeder (DDW-MD1-MT-1HD,
Brabender Pharma, Germany) was used and to compare it to bigger
scales, a KT 20 (K-Tron, Switzerland) and a Flexwall (Brabender
Pharma, Germany) were used. The actual feed-rates, were taken from
the feeder controllers. To evaluate the feeder performance offline,
different feed-rates were set and powder was fed onto an external scale.
Change in weight was recorded with a frequency of 5 Hz and the actual
feed-rates were calculated. Feed-rates from 0.5 to 10 g/min were
investigated. Mean feed-rate and coefficient of variation (CV) were
employed as quality criteria.
For all of the used feeders, feed-rates and materials, the internal
controllers of the powder feeders reported accurate feeding, as they
usually displayed actual feed-rate values, which were close to the set
values. Thus, the results of the feed-rate data, given by the balance was
analysed more in detail.
At feed-rates of 6 and 10 g/min and the use of maize starch the mini-twin
was superior to the bigger scaled feeders regarding precision. At 6 g/min
it featured a CV of only 34% compared to the KT-20 and the Flexwall with
114% and 134%. In the case of accuracy the KT20 feeder showed the
best results. At 6 g/min and 10 g/min it almost exactly met the set value,
independent from the applied powder, compared to 6.18 g/min (Mini twin,
maize starch) and 5.63 g/min (Flexwall, maize starch).
At low feed-rates of 0.5 g/min to 4 g/min the Mini-twin was capable of
feeding the free flowing mannitol very precise with CV of around 40%
compared to CV for the cohesive maize starch from 243% (0.5 g/min) to
94% (4 g/min). However, despite those good precision values, the feeder
was not able to achieve an accurate feeding result for the mannitol, as it
always resulted in values of excessive fed powder. The reason for this
could be seen in the good flowing properties of the mannitol. Additionally
to the feeding, caused by the screw revolutions the powder freely flushed
out of the feeder, resulting in feed-rates higher than the set values. This
problem may be solved by the utilization of a screen, as it was
successfully done by Engisch and Muzzio [1]. The feeding of maize
starch always resulted in accuracy values, which were closer to the set
value, compared to the feeding of mannitol.
The results of this study underline the need of evaluating powder-feeder
performances offline and separated from the data, given by the feeder,
because it is not advisable to trust into these values. Furthermore, it is
important to get control over the feeding systems, by choosing the
appropriate equipment for the set powders and feed-rates.
References:
1. Engisch, W. E.; Muzzio, F. J.: J. Pharm. Innov. 2015, 10: 56-75.
Acknowledgments: We are grateful for the funding by the OrBiTo Eu-project 2013-2018, and
JCNS.
References:
POS.055
1. Buch P. et al.: J. Pharm. Sci. 2010, 99(10): 4427–4436
2. Khoshakhlagh P. et al.: J. Pharm. Sci. 2015, 104(7): 2213-2224.
Elucidating the release mechanism of a poorly soluble
drug from amorphous solid dispersions by in situ Raman
imaging
POS.054
Vukosavljevic, B.1,2; Punčochová, K.3; Štěpánek, F.3; Windbergs, M.1,2,4
Institute of Pharmaceutics and Biopharmaceutics, Heinrich-Heine-University, Universitaetsstr.
1, 40225 Duesseldorf, Germany
of Biopharmaceutics and Pharmaceutical Technology, Saarland University,
Campus A 4.1, 66123 Saarbruecken, Germany
2 Helmholtz Centre for Infection Research (HZI) and Helmholtz-Institute for Pharmaceutical
Research Saarland (HIPS), Department of Drug Delivery, Campus A 4.1, 66123 Saarbruecken,
Germany
3 Department of Chemical Engineering, University of Chemistry and Technology Prague,
Technická 3, 166 28 Prague 6, Czech Republic
4 PharmBioTec GmbH, Science Park 1, 66123 Saarbruecken, Germany
Several continuous manufacturing processes, such as twin screw
extrusion or -granulation, coating and continuous milling require accurate
and precise feeding systems. In early pharmaceutical development such
as preformulation studies, only minor amounts of drugs are available,
which results in the need of small scale equipment. Especially from the
handling of powders the need arises to use gravimetrically controlled
feeders. Thus, inhomogeneities caused by consolidation of the powder
through normal forces, bridge building and other processes that may
cause differences in bulk density can be minimized. These problems get
even worse in small scale processes. The aim of this study was to
compare different powder feeders regarding precision and accuracy,
while feeding a cohesive and a free flowing powder.
Mannitol (Parteck M 100, Merck, Germany) was used as a free flowing
powder and maize starch (CPharm 03406, Cerestar, Germany) as a
poorly flowing powder.
Solid dispersions based on hydrophilic polymers are valuable carrier
systems for the effective delivery of poorly soluble drugs. For rational
development of such systems, the drug release mechanism has to be
elucidated. In this study, we analysed the release behaviour of
aprepitant, a poorly water soluble drug embedded within two different
solid dispersions.
Solid dispersions were fabricated based on either Soluplus® or on
polyvinylpyrrolidone K30 (PVP) by spray drying (Mini Spray Dryer B-290,
Büchi, Switzerland, drug:polymer 1:3 w/w, on a dry basis). In a next step,
the spray-dried particles were compressed to tablets.
We performed modulated temperature differential scanning calorimetry
to determine the glass transitions temperature. One single glass
transition temperature for both formulations confirmed that the drug was
molecularly dispersed, and hence a solid solution was formed. The glass
transition temperatures of pure Soluplus, Soluplus:Aprepitant mixture,
Performance evaluation of powder feeders – small scale
feeding as a challenge
Meier, R.1; Kleinebudde, P.1
1
1 Department
PVP and Aprepitant:PVP mixture were 68.2, 57.8, 159.5 and 142.9 °C,
respectively.
Raman spectra of the amorphous solid dispersions show distinctive
peaks indicating the presence of the drug and polymer, without any
evidence of crystalline aprepitant. Further, CRM allows for distribution
analysis of aprepitant and polymer within the tablets. In addition, we
visualized distribution changes within the amorphous solid dispersions
during dissolution by CRM. Upon dissolution, swelling and gel layer
formation influence diffusion and release of the drug. In order to preserve
intactness of the gel-layer during the analysis, we applied virtual cross
section imaging (x/z scans) with around 100 µm depth through the gellayer.
The Soluplus:Aprepitant formulation exhibits a continuous release with
no phase separation and drug precipitation observed. Based on these
results, Soluplus could be identified as a crucial factor for stabilization of
the amorphous drug. The PVP:Aprepitant formulation shows a different
release mechanism. Firstly, due to the good affinity of PVP to water, the
polymer dissolves very fast from the matrix, thus a highly hydrated gel
layer is formed on the surface. However, after approx. 30 minutes, under
a viscous PVP layer, the drug recrystallizes (depicted in pink) on the
surface of the tablet as depicted in Figure.
New approaches for in situ characterization and elucidation of drug
release from solid dispersions are needed. In this context, CRM is an
upcoming technique for non-invasive and chemically selective
elucidation of release mechanisms of poorly soluble drugs from solid
solutions.
compressive strengths of up to 33±2 MPa and with a pore structure that
allows cells to grow deep into the scaffolds and form mineral deposits.
Compressive moduli between 27±7 MPa and 568±98 MPa were
obtained depending on the hybrid composition and problems associated
with the inherent brittleness of sol-gel glass materials could be overcome.
SaOS-2 cells showed strong mineral accumulation on hybrid glass
scaffolds as early as day 7. On day 14, we also found mineral
accumulation on control scaffolds without cells indicating a positive effect
of the hybrid glass on mineral accumulation.
We fabricated macroporous hybrid glass scaffolds using rapid prototype
templates. By using this templating technique, we were able to produce
hybrids of a wide variety of compositions. In addition, the templating
technique allows for modification of the scaffolds’ pore size and geometry
as well as for an individualization of size and shape of the implant, with a
high reproducibility.
Acknowledgments: The authors thank Prof. Rieger (HTWK Leipzig, Germany) for access to the
compression testing equipment, Dr. Anderegg (University hospital of Leipzig) for support in the
preparation of histosections and Jörg Lenzner (University of Leipzig) for access to the electron
microscope. The authors would also like to thank the AiF Projekt GmbH (Grant no:
KF2734502MU1), the Saxon ministry for science and arts (Grand no: 4-7531.60/64/18) and the
German Research Council (DFG SFB/Transregio 67) for financial support.
References:
1. Jones, J.; Ehrenfried, L.; Hench, L.: Biomaterials 2006, 27(7): 964-973.
POS.057
Towards a new bacterial-epithelial cell co-culture model to
test novel drug delivery systems against Pseudomonas
aeruginosa biofilms
Juntke, J.1; Türeli, N. G.2; de Souza Carvalho-Wodarz, C.1; SchneiderDaum, N.1; Lehr, C. M.1
Drug Delivery, Helmholtz Institute for Pharmaceutical Research Saarland, 66123
Saarbrücken, Germany
2 MJR PharmJet GmbH, 66424 Homburg, Germany
1
Figure: Virtual cross section (x/z scan) of the PVP:Aprepitant tablet formulation during
dissolution after 30 min. Gel layer is depicted in light blue, crystalline drug in pink and inner
tablet in blue.
POS.056
Hybrid glass scaffolds for bone regeneration by indirect
rapid prototyping
Hendrikx, S.1; Kascholke, C.1; Flath, T.2; Schumann, D.3; Gressenbuch,
M.4; Schulze, P.2; Hacker, M. C.1; Schulz-Siegmund, M.1
Pharmaceutical Technology, Institute of Pharmacy, Universität Leipzig, Leipzig, 04317,
Germany
2 Faculty of Mechanical and Energy Engineering, Leipzig University of Applied Sciences,
Leipzig, 04277, Germany
3 Bubbles and Beyond GmbH, Leipzig, 04229, Germany
4 DMG Chemie GmbH, Leipzig, 04347, Germany
1
Implants for bone regeneration have to withstand mechanical stress on
the one hand and, on the other hand, provide high macroporosity to allow
for tissue ingrowth, remodelling and vascularization, making the
fabrication a complex task. Moreover, materials need to be biocompatible and bioactive with regards to the formation of a mechanically
stable bone-implant interface. Last but not least the production costs of
these implants should be reasonable and the technology should allow for
easy up-scaling at reproducible product quality [1].
We present a series of hybrid sol-gel derived bioactive glasses with
oligovalent
organic
cross-linkers
functionalized
with
3Isocyanatopropyltriethoxysilane, which were susceptible to heat
sterilization. The hybrids were processed into pore-interconnected
scaffolds by an indirect rapid prototyping method. A large panel of 2- to
4-armed cross-linkers of different molecular weight were incorporated
and their effect on scaffold mechanical properties was investigated. By
multiple linear regression, ‘organic content’ and the ‘content of ethylene
oxide units in the hybrid’ were identified as the main factors that
determined compressive strength and modulus, respectively. In general,
3- and 4-armed cross-linkers performed better than linear molecules.
Compression tests and cell culture experiments with osteoblast-like
SaOS-2 cells showed that macroporous scaffolds can be produced with
In the lungs of cystic fibrosis (CF) patients, the thick and sticky mucus
offers good growth conditions for bacteria. Especially
Pseudomonas aeruginosa, known as the major pathogen in CF, is almost
impossible to eradicate once a biofilm is formed. An intriguing approach
to overcome drug delivery problems, arising from the thick mucus layer
and the biofilm, is the development of nano-scaled drug delivery systems
(DDS). For this reason, ciprofloxacin-complex-loaded PLGA
nanoparticles (NP) are prepared under controlled conditions by
nanoprecipitation using the novel MicroJet Reactor technology. The NP
should be able to penetrate the mucus and biofilm thus releasing the drug
in a controlled manner.
To mimic disease relevant conditions in vitro, our aim is to establish a coculture model of human bronchial epithelial cells with P. aeruginosa on
top, allowing the formation of a biofilm. The bronchial epithelial cell lines
Calu-3 and CFBE41o- are used, representing the healthy and CFdiseased airways, respectively. Once the cells have formed an epithelial
barrier they are infected with different amounts of P. aeruginosa to
identify a critical seeding density of the bacteria which enables biofilm
formation and still allows the human cells to survive. This model holds
potential for evaluating novel DDS regarding their safety and their ability
to disrupt the bacterial biofilm and to reach P. aeruginosa.
Acknowledgments: Thanks to the BMBF for funding this KMU-innovativ project : „FiDel“ FKZ:
13N12530
POS.058
A unified in vitro test system for tight junction modulators
Saaber, D.1; Reichl, S.1
Institut für Pharmazeutische Technologie, Technische Universität Carolo-Wilhelmina zu
Braunschweig, Mendelssohnstr. 1, 38106 Braunschweig, Germany
1
Tight junctions (TJ) consist of different transmembrane proteins and are
located in the paracellular space. In addition to effects on cell proliferation
and differentiation they contribute to absorption barrier of epithelial and
endothelial cells for many drug substances. Therefore modulation of TJ
POSTERS
barrier function is considered to be a promising approach for drug
permeation enhancement and to improve bioavailability of BCS class
III/IV drugs. Some substances influencing TJ barrier, so-called TJ
modulators of first generation, have been reported over the last two
decades, but based on better knowledge and comprehension of TJ
regulation several mechanism-based (MB) TJ modulators have been
developed in the past few years [1]. In contrast to first generation
modulators they should show less cytotoxic effects and allow
regeneration of barrier properties, determined as transepithelial electrical
resistance (TEER), when application is finished and cells are incubated
with modulator free medium. However, comparison of described TJ
modulators as well as evaluation of usefulness and toxicity is difficult
because most studies include different cell lines and examination
methods.
The purpose of this study was to standardize the characterization of TJ
modulators and to develop a unified test system to assess TJ modulators
for pharmaceutical applications. In our study MDCK I cells were used,
exhibiting high and constant TEER level of 3,000-3,500 Ω·cm² for several
days, 5 days after seeding. Evaluation of the TJ modulating effect was
performed by continuous impedance measurement under standardized
conditions using cellZscope®. Cell viability was tested by MTT assay. For
determination of membrane interference fluorescent micrographs were
taken after 3-fold staining with DAPI, Hoechst 33342 and calcein-AM.
Overall eight different TJ modulators, five of first generation like EDTA,
sodium caprate, sodium nitroprusside, benzalkonium chloride, sodium
fluoride, and three MB modulators like AT-1002, PN159 and labradimil
were tested.
In principle, first generation as well as MB modulators, were able to cause
significant TEER reduction up to 100% within the first 30 min of
incubation. Only for labradimil smaller TEER reductions of 17% for 1 µM
and 27% for 10 µM were detected in our experimental setup. However,
effective doses of first generation modulators induced lower cell viability
in comparison to MB modulators. AT-1002 and PN159 caused fast TEER
reduction within the first 15 min after exposure. Furthermore, the TEER
regeneration after AT-1002 and PN159 incubation was higher and faster
than observed for first generation modulators. In the case of AT-1002
TEER regenerated after 60 min and reached a maximum of 70% in
comparison to the control 15 h after substance depletion. For PN159
regeneration started after 90 min and reaches 100% after 11 h.
Interestingly, MDCK I cells exhibited spontaneous regeneration of TEER
approx. 60 min after incubation with 100 µM sodium caprate and 10 mM
sodium fluoride. For sodium caprate and AT-1002 similar mechanisms of
action have been reported, resulting in an activation of proteinkinase C
[2,3].
A unified test system has been developed to compare efficacy and
toxicity of various TJ modulators. This study showed that MB TJ
modulators can cause significant TEER reduction and exhibit less toxicity
compared to first generation TJ modulators. The test system will be
extended by permeation data and membrane integrity assays. The final
system will offer the opportunity to characterize and evaluate the
potential of new developed TJ modulators.
powder discharge from these containers also depends on the material
characteristics, so the characteristics of discharge may vary between
formulations possibly leading to powder segregation. To evaluate the
variation in the composition of the discharged powder, most commonly
NIR techniques are used, which not only require modification of the
instrument but also a lot of calibration effort. Alternatively, numerical
simulations offer in-depth understanding of the powder dynamics within
these containers. In this study, such simulations are implemented to
investigate the rate of discharge and segregation during discharge from
a production size container.
The simulations are carried out using the three-dimensional discrete
element method, which computes the trajectories of each and every
particle. Usually, the production size container can hold few hundred
kilograms of material, which means several billions of few hundred
micron sized particles to be accounted for in these simulations. Handling
such amounts of material is nearly impossible with the current
computational capacity. To reduce the number of particles for the
simulations, the container is downsized to a scale of 1:10. The effect of
downsizing is computed and validated on the one hand side by simulating
the downsized scale of 1:7.5 and on the other hand side with an
experiment. The materials considered in this study include VIVAPUR
MCC spheres (JRS Pharma GmbH) and Ludipress (BASF
Ludwigshafen), which have poly-disperse particle sizes. The material
properties such as particle size distribution, density and flowability are
measured and entered as input parameters in simulations.
After having established and validated the desired downsized geometry
i.e. 1:10 scale, the rate of powder discharge is computed. One example
of the simulation results is displayed in the figure below. The green line
shows the number of particles which have already exited the container.
The red line is the mean diameter of the remaining particles. The diagram
shows that at the beginning the mean particle size does not change
significantly, but after discharge of about 80% of the initially loaded
material the particle size starts to decrease indicating segregation. This
variation in the particle size with time is of special interest as it is usually
the drug particles having the smallest size in a mixture, meaning that the
discharge of a mixture would lead to a segregation according to the
particle size of the components. The study also investigates the influence
of material properties such as cohesion on the discharge rate, dynamic
flow angles, flow modes (funnel flow or mass flow). In conclusion, the
present study helps in better understanding of the powder dynamics
within a container during discharge, can help in identifying sources of
poor content uniformity and may be used to identify critical material
attributes.
References:
1. Saaber et al.: Expert. Opin. Drug Discov. 2014, 9(4): 367-81.
2. Tomita, M.; Hayashi, M.; Awazu, S.: J. Pharm. Sci. 1996, 85(6): 608-11.
3. Goldblum et al.: FASEB J. 2011, 25(1): 144-58.
POS.059
Numerical investigation of uniformity of powder
discharged from a production container
Rötzer, K. M.1,2; Gopireddy, S. R.2; Schlosser, E.1; Urbanetz, N. A.2
1 Lehrstuhl
fuer Verfahrenstechnik disperser Systeme, Technische Universitaet Muenchen,
Maximus-von-Imhof-Forum 2, 85354 Freising, Germany
2 Daiichi-Sankyo Europe GmbH, Pharmaceutical Development, Luitpoldstrasse 1, 85276
Pfaffenhofen, Germany
In solid dosage form production, similar containers or bins are used for
handling materials in different unit operations as well as in intermediate
steps, e.g., the bin used in mixing the drug and excipients is also used in
tabletting unit operation or vice versa. This interchangeability allows to
have less types of containers, and enables to handle similar scales of
material in every unit operation. When such containers are used in
tableting, they are designed to discharge the bulk solid homogenously
and uninterruptedly. However, besides the container geometry the
POS.060
Biorelevant in vitro dissolution testing of oily suspensions
for intramuscular administration
Probst, M.1; Schmidt, M.1; Seidlitz, A.1; Weitschies, W.1
1 University
Germany
of Greifswald, Institute of Pharmacy, Felix-Hausdorffstr. 3, 17489 Greifswald,
It was the aim of the presented study to test and develop biorelevant in
vitro dissolution test setups for intramuscularly applied oily suspensions.
For the generation of in vivo data an oily suspension of prednisolone
(2.5 mg suspended in 100 µL medium chain triglycerides) was injected
into the muscle tissue of the thigh of rats. The volume and surface area
of the depots were determined and pharmacokinetic data were obtained.
The same formulation was investigated in vitro by adapting the results
from the volume and surface area measurements to the test setups as
possible.
In one test setup, adapted from Bhardwaj and co-workers [1], the
compendial flow through cell (FTC) was combined with a dialysis method.
For this purpose a dialysis tube (MWCO 50 kDa, regenerated cellulose)
was mounted on an adapter which was placed into the FTC. The
suspension was injected into the dialysis tube and the FTC was perfused
with PBS pH 7.4 (Figure A). The dimensions of the adapter and the FTC
were modulated to simulate the obtained in vivo data of the shape of the
depot. This whole setup was either placed in upright or in horizontal
position. Additionally, a new in vitro dissolution test method was
performed. Here, a wire rack was placed in a compendial FTC which was
filled with PBS. The oily suspension was injected into the release
medium. The wire rack prevented the removal of the oily droplet
(Figure B). Subsequently, the cell was perfused with the medium while
the oily droplet was kept in position. The results of these test methods
were compared to the compendial paddle apparatus and the
reciprocating holder. In the latter, the dialysis tube was fixed in an adapter
and reciprocated within the release medium.
The results (Figure C) showed that the testing method had a major
influence on the release of prednisolone. The release of the oily
suspension of prednisolone in the paddle apparatus was by far the
fastest. The dissolution of prednisolone from the oily droplet was faster
as in the test setups using the dialysis bag in the FTC but considerably
slower than in the paddle apparatus. The positioning of the FTC with the
dialysis bag had a major influence on the release behaviour. The
reciprocating cylinder and the dialysis bag in upright position showed a
similar release profile being the slowest compared to the other tested
methods. Compared to the pharmacokinetic profiles observed in the rats
where cmax occurred after 6 h in median, the release from the oily droplet
in the FTC corresponds best to the in vivo observation.
System (DynaMiTES) within the scope of the Center of Pharmaceutical
Engineering (PVZ) of the TU Braunschweig. This system should allow
dynamic donor and acceptor control, continuous impedance
measurement and the application of widely used cell culture inserts.
These advantages would enable the application of the novel dynamic in
vitro test system with various existing cell culture models and facilitate its
transfer to other laboratories. To approach this aim, at first an established
and prevalidated human hemicornea model (classic HC; [2], [3]) was
modified. The classic HC had to be altered into an inverse HC model for
the use in the newly designed DynaMiTES. In the present study various
cell characteristics were compared in both models to prove their
equivalence.
For this comparison the in vitro models were cultivated under serum-free
conditions on permeable polycarbonate filters (Transwells®) using SV40
immortalized human keratocytes (HCK-Ca) and human corneal epithelial
cells (HCE-T). The classic HC was cultivated as described before [2]. In
contrast to this, for the inverse HC the HCE-Ts were seeded on the
external side of the insert’s membrane. After seven days of submerse
cultivation, both models were lifted to air-liquid interface until day ten.
Barrier properties of the HCs were estimated by transepithelial electrical
resistance (TEER) measurements and static permeability studies with
sodium fluorescein. Cell viability was evaluated via MTT testing. Cell
morphology was investigated by cross-sectional slices (Technovit® 7100)
stained with haematoxylin and eosin.
Although the compared models showed different TEER developments,
neither the TEER values nor the apparent permeation coefficients (Papp)
were different at the end of cultivation. The obtained Papp was similar to
the values presented in the prevalidation study [3]. Moreover, cell viability
after cultivation was not statistically different between both groups. The
histologic observations could support this equivalence by morphologic
similarity. The inverse HC developed a multi-layered epithelium similar to
the classic HC and human cornea.
These findings underline that the modification of existing in vitro models
for the DynaMiTES is possible and has been performed successfully for
the prevalidated HC. Thus, it can be assumed that other established and
well characterized in vitro models can also be adapted to the
DynaMiTES. In subsequent investigations the inverse HC shall be used
within the DynaMiTES to simulate tear dynamics and to investigate their
effects. This ocular DynaMiTES will then be one important improvement
of current in vitro models for a better prediction of safety and efficacy
data.
References:
1. Marx, U. et al.: ATLA Altern. Lab. Anim., 2012, 40: 235–257.
2. Hahne, M.; Reichl, S.:Int. J. Pharm. , 2011: 416, 268–279.
3. Hahne, M. et al.: J. Pharm. Sci., 2012, 101: 2976–2988.
References:
1. Bhardwaj, U.; Burgess, D.: Int. J. Pharm. 2010, 388: 287-294
POS.061
Modification of an organotypic human hemicornea model
for the use in a novel Dynamic Micro Tissue Engineering
System (DynaMiTES)
Beißner, N.1,3; Mattern, K.2,3; Dietzel, A.2,3; Reichl, S.1,3
1 Institut für Pharmazeutische Technologie, Technische Universität Carolo-Wilhelmina zu
Braunschweig, Mendelssohnstraße 1,
38106 Braunschweig, Germany
2 Institut für Mikrotechnik, Technische Universität Carolo-Wilhelmina zu Braunschweig, Alte
Salzdahlumer Str. 203,
3 Zentrum für Pharmaverfahrenstechnik, Technische Universität Carolo-Wilhelmina zu
Braunschweig, Franz-Liszt-Straße 35 A,
Preclinical drug absorption studies are frequently performed using ex
vivo tissue or in vitro cell culture models. The bioavailability of topically
applied drugs is often low since at various physiological barriers like the
eye or the nose drugs are drained away within minutes. However, current
in vitro tests for the assessment of pharmacokinetic and toxicological
parameters are mostly performed in static cell culture systems, which
cannot emulate the physiological conditions of drug administration. This
essential disparity may result in misleading interpretation of the outcomes
and wrong prediction of the drug’s safety and efficacy [1]. For this reason,
the Institute of Microtechnology (IMT) and the Institute of Pharmaceutical
Technology (IPhT) developed a Dynamic Micro Tissue Engineering
POSTERS
4.5 Analytics
POS.062
Method development for determination of neurosteroids in
cerebrospinal fluid as potential biomarkers of postoperative cognitive dysfunction
Teubel, J.1; Wüst, B.2; Schipke, C.3; Peters, O.4; Parr, M. K.1
1
2
Institute of Pharmacy, Berlin, Germany, Freie Universität Berlin, Berlin, Germany
Agilent Technologies, R&D and Marketing, Santa Clara, California, USA
3 Charité-Universitätsmedizin Berlin, Experimental and Clinical Research Center, Berlin, Germany
4 Charité
Berlin, Departement of Psychatry and Psychotherapy, Berlin, Germany
Post-operative cognitive dysfunction (POCD) is a condition affecting
mainly elderly patients (> 60 yrs) that undergo surgery and anesthesia. It
may last for days up to weeks and may even persist over time, leading to
lasting health problems.
Since the detailed etiology of POCD and other post-interventional
cognitive deficits is unknown, the project aims to investigate
(neuro-)biological correlates and predictors.
One group of potential correlates are neurosteroids, i.e. steroids that are
synthesized in the central and peripheral nervous system and appear to
have numerous physiological effects on cognition and brain function.
A comprehensive method to quantify specific neurosteroids in
cerebrospinal fluid (CSF), including pregnenolone sulfate that is
emerging as a key substance for cognitive ageing, is developed.
As concentrations in CSF are at the ultra-trace level (generally at a
pg/mL-range) highly sensitive methods have to be used. Further, high
structural similarities of chosen steroids challenges achievement of
chromatographic selectivity. Therefore, different chromatographic
techniques were evaluated.
Detection and separation of 40 selected steroids, including androstanes,
corticoids, estranes and pregnanes, was achieved utilizing mass
spectrometry following chromatographic separation.
POS.063
Determination of phytosterols in herbal medicinal products
for the treatment of lower urinary tract symptoms and food
products marketed in Europe
Müller, C.1; Bracher, F.1
1 Department
für Pharmazie – Zentrum für Pharmaforschung, Ludwig-Maximilians-Universität
München, Butenandtstraße 5-13, 81377 München, Germany
For more than 50 years herbal medicinal products and food products
containing phytosterols have been used for treatment of lower urinary
tract symptoms such as benign prostatic hyperplasia and overactive
bladder in Europe. Mainly products with preparations out of pumpkin
seeds, saw palmetto fruit, and nettle root are used, as well as β-sitosterol
and other poorly defined phytosterol preparations [1]. The Δ7phytosterols, typical constituents of pumpkin seeds, are assumed to be
effective in therapy of lower urinary tract symptoms due to an inhibition
of 5α-dihydrotestosterone binding at cellular androgen receptors in the
prostate [2]. Urinary obstructive symptoms are improved [2], and a
clinical reduction of the International Prostate Symptom Score [3], or at
least a better quality of life are achieved [4]. Due to the heterogeneity of
the phytosterol preparations on the market an analytical tool is needed
for the exact analysis of the sterol compositions as a basis for quality
control and estimation of phytoequivalence and comparability of clinical
data.
We have worked out a convenient extraction and isolation method which,
in combination with sophisticated gas chromatography ion trap mass
spectrometry, is suitable for analysing phytosterols in complex matrices
like unprocessed material (pumpkins seeds, pumpkin seed oil) and
phytosterol-containing preparations (hard and soft gelatine capsules,
tablets) from various medicinal plants [5].
The phytosterols of each sample were separated after alkaline hydrolysis
by liquid/liquid extraction, and analyzed as their corresponding trimethyl
silyl ethers.
We were able to detect ten different phytosterols, the ubiquitary
phytosterol campesterol was detected in 27 samples (n = 38). ΒSitosterol was the quantitatively predominating phytosterol among the
non Δ7-phytosterols (n = 5), and was detected in significant amounts in
most, but not all preparations for which this sterol or “phytosterol” was
declared as a main component. It was detected in 23 samples in a range
from 0.0-53.4 mg/dosage. The content of total non Δ7-phytosterols
ranged from 0.0-58.4 mg/dosage. As expected, Δ7-phytosterols (n = 5)
were only found in pumpkin seed preparations. Surprisingly, a few
pumpkin seed preparations contained only non Δ7-phytosterols. Δ7,25Stigmastadienol was the main Δ7-phytosterol in all samples and ranged
from 0.0-6.1 mg/dosage, and the content of total Δ7-phytosterols per
dosage ranged from 0.0-15.4 mg.
References:
1. Bracher, F.: Urologe 1997, A 36: 10-17.
2. Gossell-Williams, M.; Davis, A.; O'Connor, N.: J. Med. Food 2006, 9: 284-286.
3. Vahlensieck, W. et al.: Urol. Int. 2015, 94: 1-10.
4. Shirvan, M. K et al.: JPMA 2014, 64: 683-685.
5. Müller, C.; Bracher, F.: Planta. Med. in press 2015.
POS.064
LC-ESI-MS method development and validation for
determination of five hypoglycemic agents in mixture using
monolithic silica column
Asmari, M.1; Alhazmi, H. A.1; Wölker, J.1; El Deeb, S.1
Institute of Medicinal and Pharmaceutical Chemistry, TU Braunschweig, D-38106
Braunschweig, Germany.
1
In this work a LC-ESI-MS method has been developed and validated for
the simultaneous determination of metformin, alogliptin, sitagliptin,
vildagliptin and linagliptin in mixture. The method is applicable for the
quality control of a number of pharmaceutical products since metformin
is usually combined in one dosage form with either of the above dipeptidyl
peptidase-4 inhibitors for the treatment of type II diabetes mellitus. The
LC analyses were performed using an Agilent 1100 LC system with
second-generation monolithic silica column Chromolith® HighResolution
RP-18e column (100×4.6 mm, Merck). A mobile phase consisting of
acetonitrile/ ammonium formate buffer (20:80, v/v) at a pH 3 was used at
a flow rate of 0.4 mL/min. A single-quadrupole mass spectrometer
(Agilent 1620) equipped with an electrospray ionization source was used.
After optimization, the best conditions were set with a nebulizer pressure
of 30 psi, drying gas temperature of 250 °C, capillary voltage of 3 kv and
a fragmentor voltage of 70 v under the positive ion mode. After
identification of the individual molecular ions using the scan mode, which
gave excellent mass accuracy, the MS was set on selected-ion
monitoring (SIM) mode using target ions at m/z [M+H+] 130.1 for
metformin, m/z [M+H+] 304.2 for vildagliptin, m/z [M+H+] 340.2 for
alogliptin, m/z [M+H+] 408.1 for sitagliptin and m/z [M+H+] 473.2 for
linagliptin for quantitation. The method was linear over the concentration
range of 0.039-20 µg/ml for metformin and 0.19-100 µg/ml for the others.
The correlation square of the linear regressions were better than 0.99 for
the five quantitated compounds. RSDs% of peak areas and retention
times were less than 0.027% and 0.00079%, respectively. This precise
method would be further tested for stability and bioanalysis
investigations.
POS.065
Child-appropriate high throughput HPLC-MS/MS for
enalapril and enalaprilat in small sample volumes of serum
and urine within a GCLP-compliant environment
Schaefer, J.1; Burckhardt, B. B.1; Tins, J.1; Läer, S.1
Institute of Clinical Pharmacy and Pharmacotherapy, Heinrich-Heine-University Duesseldorf,
1
Background: Heart failure is a life-threatening disease in neonates up to
adolescents. The angiotensin-converting enzyme inhibitor enalapril is a
recommended therapy in paediatric heart failure, although it is not
labelled for patients <20 kg in European countries. The LENA (Labeling
of Enalapril from Neonates up to Adolescents) project aims to generate
data for devising a paediatric-use marketing authorization for an ageappropriate enalapril formulation. For enalapril, pharmacokinetic data
drug assays for serum and urine specifically tailored to paediatric
ANALYTICS
populations were developed and used in the GCLP-compliant setting of
the LENA phase I study.
Objective: Proof of concept of the paediatric tailored assays by
verification of the reliable conduct of sample analysis according to
international bioanalytical guidelines and determination of enalapril and
its active metabolite enalaprilat in the LENA phase I study.
Methods: All LENA phase I samples were analysed applying the
developed high throughput analysis for enalapril and enalaprilat via
HPLC-MS/MS. According to the established LENA GCLP-compliant
quality system, the evaluation of all study samples was conducted by
using freshly prepared quality standards to obtain calibration curves of
enalapril and enalaprilat in serum and urine.
Results: In total, 22 calibration curves in serum and 7 in urine were
required to investigate all samples of the phase I study of LENA. All 29
calibration curves complied with the limits of FDA and EMA bioanalytical
guidelines [1,2] and the applicability of the established high throughput
method in the GCLP environment was proven. About 2100 study
samples were successfully determined within 26 days.
Conclusion: The developed paediatric tailored high throughput HPLCMS/MS analysis proved its applicability in a GCLP-compliant
environment and is suitable for the upcoming phase II and phase III
studies of the LENA project focussing on paediatric patients.
Perspectives: Method development is still ongoing. The LC-separation
achieved for ten substances will be further improved. Parameter settings
for the simultaneous quantification of the drugs from study samples have
to be optimised. The final method will be validated according to the
particular EMA Guideline [1] and may help to improve the medication
safety of the neonates.
Acknowledgments: The research leading to these results has received funding from the
European Union Seventh Framework Programme (FP7/2007-2013) under grant agreement
n°602295 (LENA).
1 Institute
References:
1. EMA. Guideline on Bioanalytical Method Validation. EMA, Committee for Medicinal Products
for Human Use, London, UK 2011.
2. US FDA. Guidance for Industry: Bioanalytical Method Validation. US Department of Health and
Human Services, US FDA, Center for Drug Evaluation and Research, Rockville, MD, USA 2001.
POS.066
Development of an LC-MS/MS method to investigate the
pharmacokinetics of antibiotics and concomitant drugs
given to neonates suffering from sepsis
Fürtig, M.-A.1.; Burau, D.1; Kloft, C.1
1 Dept.
of Clinical Pharmacy and Biochemistry, Institute of Pharmacy, Freie Universitaet Berlin,
Kelchstr. 31, 12169 Berlin, Germany
Background: Safety of drug therapies is usually proven by clinical trials
before administration to patients in clinical routine, but for some special
patient populations e.g. neonates, no clinical data is normally available.
If severe diseases occur in these patients, often the high risk of
administering non-approved drugs needs to be accepted. In a recent
clinical trial, drug concentrations in newborns (n=8) suffering from sepsis
are monitored using microdialysis as sampling technique in the interstitial
fluid of subcutaneous tissue. Microdialysis enables investigations to
unbound drug concentrations in a particular tissue without removal of
blood or any other body fluid which is the main limitation of plasma
sampling in neonates. In addition to vancomycine (VAN), which was
administered as initial antibiotic treatment, 17 concomitant drugs were
given to the patients covering mainly antibiotics, antifungals, diuretics
and sedatives. This trial will give the opportunity to gain an insight into
the pharmacokinetics of several drugs given to neonates.
To gain as much information from the patient samples as possible, we
are developing an LC-MS/MS method for simultaneous determination of
VAN and the concomitant drugs.
Methods: An Agilent 1290 HPLC system coupled with an Agilent 6490
triple quadrupole LC-MS/MS detector with electron spray ionisation (ESI)
and iFunnel technology, which combines the Agilent Jetstream
technology, Hexabore sampling capillary and a dual-stage ion funnel, is
used for method development. VAN and the concomitant drugs are both,
separately and simultaneously injected to find optimal setting for the
simultaneous quantification of all drugs. Different gradients of Milli-Q
water containing formic acid (FA) and acetonitrile with FA are tested to
improve drug separation prior to detection. Ion source parameters,
collision energy, etc. will be optimised to achieve high accuracy and
precision for the substances on the one hand and a very low
quantification limit on the other hand.
Results: Current experiments showed good separation of VAN and nine
concomitant drugs. To further improve the method performance
concerning accuracy, precision, selectivity and sensitivity; detection
needs to be optimised.
References:
1. European Medicines Agency (EMA): Guideline on bioanalytical method validation 2012
(http://www.ema.europa.eu/docs/en_GB/document_library/Scientific_guideline/2011/08/WC500
109686.pdf; 29.06.15)
POS.067
Affinity capillary electrophoresis as an appropriate
technique to investigate the interactions of Pentosan
Polysulfate sodium and related substances with various
proteins
Mozafari, M.1; El Deeb, S.1; Wätzig, H.1
of Medicinal and Pharmaceutical Chemistry, TU Braunschweig, Beethovenstrasse 55,
38106 Brunswick, Germany
Affinity capillary electrophoresis has become a powerful method for
separation of peptides and proteins. Moreover it is a qualified technique
for analysing pharmaceuticals and biopharmaceuticals [1,2,3].
The affinity capillary electrophoresis methods are based on identification
of changes in the electrophoretic mobility of analytes due to changes in
charge through interacting with ligands [1,2,3].
Pentosan polysulfate (PPS) is highly sulfated polysaccharide derived
from beechwood hemicelluloses by sulfate esterification. Beside the well
known antithrombotic activity, other properties such as anti-inflammatory,
anti-angiogenesis and inhibition of cell adhesion are still under
investigation for this substance as well as for heparins [4,5]. Accordingly,
it is important to know how strong PPS interacts with some important
proteins compared to heparin and other heparinoids.
For this purpose affinity capillary electrophoresis methods have been
developed due to its numerous advantages such as short analysis
duration and minute consumption of samples.
In this study we used some model proteins namely HSA, BSA,
myoglobin, ovalbumin, beta-lactoglobulin and two other important
proteins namely vitronectin and P-selectin, which are involved in the
coagulation cascade.
The interactions were calculated using mobility ratios (R) of the EOFmarker, acetanilide and the proteins to avoid effects from the migration
time shifts, which are not related to interactions [1,2,3].
Among the investigated proteins myoglobin, ovalbumin and beta
lactoglobulin, showed no significant interactions with the heparinoids as
expected.
HSA and BSA showed stronger interactions with PPS than heparin and
other heparinoids.
In case of HSA, the shape of the peak obtained by electrophoresis was
changed and splitted into two peaks after interaction with PPS. The
higher the concentration of PPS, the more distinct this effect was.
P-selectin showed very strong interaction with the capillary wall so that
the use of coated capillaries and a special rinsing procedure were
necessary. As well, p-selectin showed strong interactions with PPS and
heparinoids, which was probably dependant on the presence of Ca2+
ions.
With appropriate ACE methods we could demonstrate that heparinoids
exhibit strong affinity to some of these important proteins.
Acknowledgments: bene pharmaChem for financial support of this project, Polymicro for the
donation of capillary material
References:
1. Redweik, S. et al.: Electrophoresis 2012, 33: 3316-3322.
2. Alhazmi, H. A. et al.: J. Pharm. Biomed. Analysis 2015, 107: 311-317.
3. El Deeb, S. et al.: Electrophoresis 2015, 35: 170-189
4. Abdel-Haq, H. et al.: J. Chrom. Analysis 2012, 1257: 125-130
5. Degenhardt, M. et al.: Arch. Pharm. Pharm. Med. Chem. 2001, 334: 27-29.
POSTERS
POS.068
A novel fluorogenic probe for the investigation of free thiols:
Application to kinetic measurements of acetylcholinesterase
activity
Mertens, M. D.1; Bierwisch, A.2; Li, T.1; Gütschow, M.1; Wille, T.2;
Elsinghorst, P. W.1,3
1 Pharmaceutical
Institute, Pharmaceutical Chemistry I, University of Bonn, An der Immenburg
4, 53121 Bonn, Germany
2 Bundeswehr Institute of Pharmacology and Toxicology, Neuherbergstraße 11, 80937
München, Germany
3 Supervisory Agency for Public Law Tasks of the Bundeswehr Medical Service South,
Dachauer Straße 128, 80637 München, Germany
In vitro measurements of acetylcholinesterase (AChE) activity are usually
carried out following the method of Ellman [1], where a chromogenic
reagent (DTNB) is applied to detect the thiocholine liberated from acetylthiocholine by action of AChE. However, spectrophotometric detection of
the reaction product can be impaired in highly coloured solutions or in
samples of low AChE activity. These drawbacks can to a reasonable
extent be overcome by use of a fluorogenic substrate providing improved
signal selectivity and sensitivity.
Several fluorescent probes for in vitro and in vivo detection of free thiols
have been developed and in rare cases have also been applied to detect
AChE activity [2]. However, when used for determination of AChE activity
only end-point measurements were carried out without further kinetic
analysis [3,4].
variety of N-hydroxylated compounds including amidines, guanidines,
amidinohydrazones, aromatic amines as well as N-hydroxylated
nucleosides and nucleobases. In addition, the three component Nreductive enzyme system is able to reduce N-oxides [2-4]. According to
these findings, TMAO may also serve as a substrate for mARC. So far,
the physiological role of mARC is unknown though it contributes both to
the activation of prodrugs and to the metabolism of xenobiotics [5]. The
mARC mediated reduction of TMAO would be a hint to a participation of
the molybdo enzyme in prevention of CVD.
To investigate whether TMAO is reduced through mARC it is necessary
to develop a specific assay and analytical method to quantify the
metabolite TMA whereupon the volatile character as well as the lack of a
chromophore of TMA poses particular challenge for this task. The
derivatization of TMA to a non-volatile compound displays a promising
opportunity to quantify the metabolite via LC/MS.
References:
1. Wang, Z. et al.: Nature 2011, 472 (7341): 57-63.
2. Havemeyer, A. et al.: J. Biol. Chem. 2006, 281(46): 34796-34802.
3. Gruenewald, S. et al.: J. Med. Chem. 2008, 51(24): 8173-8177.
4. Jakobs, H. et al.: ChemMedChem 2014, 9: 2381-2387
POS.070
The GAPAGPLIVPY - Ca2+-Complex – An example for
comparing differences in observations of different
techniques used for peptide-metal ion binding studies
Nachbar, M.1; Mozafari, M. 1; Alhazmi, H. A. 1; Preu, L.1; Redweik, S.1;
El Deeb, S.1; Lehmann, W. D.2; Wätzig, H.1
1 Institute
Figure: A nosyl-protected coumarin (top) was developed to allow for fluorescence detection of
free thiols, e.g., in kinetic measurements of acetylcholinesterase activity using acetylthiocholine
(right).
Here, we describe the preparation of a nosyl-protected/quenched
coumarin (Figure, left) designed as a probe for free thiols based on the
well-known susceptibility of the nosyl-group towards thiol-mediated
cleavage. We subsequently used this probe to develop a fluorimetric
assay of AChE activity that allows for kinetic measurements rather than
end-point observations (Figure, right). Details of this assay and
associated kinetic analyses are presented.
Acknowledgments:This study was funded in part by the German Ministry of Defence.
References:
1. Ellman, G. L. et al.: Biochem. Pharmacol. 1961, 7: 88–95.
2. Peng, H. et al.: Sensors 2012, 12(11): 15907–15946.
3. Maeda, H. et al.: Angew. Chem. Int. Ed. 2005, 44(19): 2922–2925.
4. Gainullina, E. T. et al.: Bull. Exp. Biol. Med. 2006, 142(6): 751–752.
POS.069
mARC and its potential role in prevention of cardiovascular
disease
Schneider, J.1; Girreser, U.1; Clement, B.1
Department of Pharmaceutical Chemistry, Christian-Albrechts-University Kiel, Gutenbergstraße
76, 24118 Kiel, Germany
1
Cardiovascular disease (CVD) is the leading cause of morbidity
worldwide. It is therefore of great interest to reveal pathogenic as well as
diagnostic factors to establish valid diagnostic tests and therapeutic
approaches [1].
It was discovered that the metabolic profile of trimethylamine N-oxide
(TMAO) in plasma predicts the risk for CVD, potentially through TMAOrelated promotion of atherosclerosis. TMAO is a physiological metabolite
that derives from dietary choline. It is well accepted that gut microbiota
converts dietary choline and its precursors to trimethylamine (TMA).
Hepatic flavin monooxygenase metabolizes the TMA to TMAO which is
subject to renal excretion [1]. The recently in our lab discovered
mitochondrial amidoxime reducing component mARC forms along with
the heme-containing cytochrome b5 and its flavin-containing cytochrome
b5 reductase an N-reductive enzyme system which is able to reduce a
2 Core Facility Molecular Structure Analysis, German Cancer Research Center (DKFZ),
Heidelberg, Germany
Quantitative structure-activity relationships (QSARs) are an important
tool in generating new bioactive compounds. However, the quality of
these models is strongly depending on the used empirical data [1]. The
choice of the used technique and method has a big impact on the results
of binding studies. Direct electrospray ionization mass spectrometry
(ESI-MS) as technique for binding essays, for example, has some pitfalls
described in literature e.g. in-source dissociation, ESI-induced changes
in solution pH and temperature, non-uniform response factors and nonspecific binding [2]. However, there are also other obstacles related to
this technique. Lehmann et al. reported that the peptide GAPAGPLIVPY,
derived from galectin-3 by a combined chymotrypsin/AspN digest,
showed a strong, sequence specific affinity towards alkaline earth metal
ions in nanoESI-MS experiments [3]. The relevance of these
GAPAGPLIVPY-complexes for aqueous solutions was not quite certain,
since Dunbar et al. described differences in the binding mode between
the gas and aqueous phase [4]. For this purpose, the binding behaviour
of the peptide GAPAGPLIVPY towards various metal ions was also
investigated in aqueous phase using mobility-shift affinity capillary
electrophoresis (ACE) and compared to the reported results. The
outcome of the ACE experiments showed no significant interactions in
aqueous phase, except for Cu2+- and Mn2+-ions.
To explain these contradictions, computational methods were used to
make a GAPAGPLIVPY-Ca2+-complex for the gas phase. Afterwards, its
behaviour in vacuum phase and aqueous solution was investigated by
molecular dynamics simulations. These investigations showed that the
complex was only stable in the gas phase due to differences in the gas
phase binding mode compared to the aqueous solution. In vacuum, the
complex is stabilized by the Ca2+-ion, since the complex then exhibits a
hydrophobic surface and solvates highly charged species like ions inside.
Typical interactions of polar groups like hydrogen bonds, dipole-dipole
interactions and ligand exchanges are more favourable in aqueous
solution and therefore destabilize the GAPAGPLIVPY-Ca2+-complex,
which obviously results in a fast disassembly of the complex.
Acknowledgments: The authors thank Polymicro Technologies for providing the capillaries used
in this work.
References:
1. Wätzig, H. et al.: J. Comput. Aided Mol. Des. 2015, DOI: 10.1007/s10822-015-9851-6
2. Kitova, E. N. et al.: J. Am. Soc. Mass Spectr. 2012, 23(3): 431–441.
3. Lehmann, W.D.; et al.: Rapid Commun Mass Sp. 2006, 20(16): 2404–2410.
4. Dunbar, R.C.; et al.: Int. J. Mass Spectrom. 2013, 354-355: 356-364.
ANALYTICS
POS.071
Identification of photodegradation products of ketoprofen
utilizing online photoreactor-HPLC, GC/MS and isotopic
labeling
Assaf, J.1; Zulkiewicz Gomes, D.1,2; Schulze, T.1; Wuest, B.3; Parr, M.
K.1
1 Freie
Universität, Institute of Pharmacy, Königin-Luise-Straße 2+4,14195 Berlin.
IPT – Institute for Technological Research LAQ - Chemical analysis Laboratory, Av. Prof.
Almeida Prado, 532 Cidade Universitária, 05508-901 São Paulo, SP, Brazil.
3 Agilent Technologies, 5301 Stevens Creek Blvd, Santa Clara, CA 95051, United States.
2
Photochemical properties of drug substances and their associated
phototoxic properties are becoming more important in the drug
characterisation as well in drug developments.Therefore pharmaceutical
industries are also requested by the regulation agencies to provide data
for new or renewed licenses of medicines.
The aim of this work is to study the photostability of Ketoprofen by using
a modified HPLC system, which consists of a tailored online photoreactor
with back-flush and two column system. Identification was performed
using a mass selective detector and confirmed by isotopically labelled
(deuterated) ketoprofen.
Irradiation of the nonsteriodal anti-inflamatory agent ketoprofen with UVA
resulted in decreasing amounts of the target compound. First order
kinetics was found as best model. Several photoproducts were detected
after irradiation in aqueous solution (pH 7.0), when maintaining the
temperature in the reactor at 25-30 °C. Mass spectrometric
characterization supported the structures reported from literature.
Ketoprofen and one of its proposed photoproducts are shown in Figure.
These data showed the potential for the use of the new device for fast
and easy photostability studies that may help to reduce time consuming
in vitro experiments and animal trials. Targeted MS/MS-based methods
may be generated using the results obtained by these online-irradiations
for use in vitro and in vivo. Scale-up may also be realized for the
generation of reference material for quantification as well as for toxicity
testing.
Figure: Ketoprofen (left) and one of its proposed photoproducts 2-ethylbenzophenon (right).
Acknowledgments: Katholischer Akademischer Ausländer-Dienst (KAAD) is thankfully
acknowledged for financial support.
References:
Methods: The investigations were performed consecutively with CMA71
probes (100 kDa cut-off) and a mixture of Ringer`s solution (RS) and
human serum albumin solution (HSA) (0.5%, v/v) as perfusate and
probe-surrounding medium. The AFG concentration in the probesurrounding medium was 1 µg/mL for (i) uncoated probes and (ii) CFG
(50 µg/mL in perfusate) coated probes. Microdialysate (µDialysate) was
collected (nprobes=3) in 40 min intervals over 8 h (flow rate of 1 µL/min).
Probes in (ii) were coated 16 h prior to and during the actual recovery
investigation with the CFG containing perfusate. Quantification of AFG
was performed with a previously developed and validated HPLC assay
(LiChrospher 100 RP-18 column (125 x 4 mm, 5 µm), isocratic method
with eluent: methanol and ammonium dihydrogen phosphate 85:15 (v/v),
concentration range: 0.1–20 µg/mL, accuracy and precision of the LLOQ
(0.1 µg/mL) were RE ≤ ±19.6% and CV ≤ 10.6%, respectively). The
influence of coating the probe with CFG containing perfusate was
investigated with regard to the relative recovery (RR) values of AFG.
Recovery investigation with (i) was compared with (ii) over time. All
results of RR were volume-corrected by their volume of µDialysate.
RR, %=
cdialysate
*100
cmedium
Results: One µDialysate sample after the first sampling interval of (i) and
another of (ii) had concentrations of AFG < LLOQ and were not included
in the calculations. All µDialysate and medium sample concentrations
were quantified by the HPLC assay and afterwards RR was calculated
from these concentrations.
RR showed a steep increase in the first 3 h for both approaches.
Afterwards RR was stable for the following 5 h with max. RR ranging from
23.2%-26.9% and 27.0%-30.1% for (i) and (ii), respectively. The
comparison of approach (i) and (ii) led to very similar results with regard
to the time necessary to reach stable RR values.
Conclusions: AFG displayed substantial adsorption to µD probe material
for both approaches. CFG was considered to block the AFG binding sites
on the probe material to prevent AFG from adsorption. It was however
observed that AFG was still adsorbed on the probe material which was
displayed by the increase of RR within the first 3 h of investigation.
Further research is required to reduce the initial increase in RR due to
adsorption and obtain constant recovery of AFG in µD as this is the
prerequisite for reproducible and reliable in vivo µD to gain profound
knowledge of target-site pharmacokinetics and ultimately evaluate
dosing regimens.
References:
1. Weiser et al.: 25th ECCMID, Copenhagen, Denmark. 2015, P-0222.
2. Traunmüller et al.: J. Chromatogr. 2006, B 843: 142-146.
1. Salgadoa, R. et al.: J.Hazard. Mater. 2013, 244-245: 516– 527.
2. Schulze, T.: Entwicklung einer In-Vitro-Methoden zur Beurteilung des photosensibilisierenden
Potentials von Arzneistoffen, Freie Universität Berlin, Universitätsbibliothek 2013.
POS.073
POS.072
A new approach for in vitro microdialysis of anidulafungin
Weiser, C.1; Zeitlinger, M.2; Kloft, C.1
1 Dept.
Kelchstr.31, 12169 Berlin, Germany
2 Dept. of Clinical Pharmacology, Medical University of Vienna, Währinger Gürtel 18-20, 1090
Vienna, Austria
Objectives: Anidulafungin (AFG), approved in Germany in 2007, is a
lipophilic echinocandin antifungal drug and a treatment option for
invasive candidiasis in non-neutropenic adult patients. Microdialysis
(µD), a minimally invasive technique, can serve in clinical settings to
determine the drug concentrations directly at the target-site e.g. in the
interstitial fluid of organ and muscle tissue. Since only unbound drug
molecules are able to pass the semipermeable membrane of the µD
catheter solely the pharmacologically active fraction of the drug is
collected and subsequently quantified. Because AFG shows high
adsorption on probe material (membrane, tubing) [1] caspofungin (CFG),
also known to be highly adsorbing on µD probes, was chosen to
overcome AFG adsorption by coating the probes with CFG prior to the
actual investigation [2]. Therefore CFG was selected to adsorb on and
thus block the potential AFG binding sites on the probe material, so that
AFG would be prevented from adsorption.
Handling small sample volume for capillary electrophoresis
investigations
Mozafari, M.1; Nachbar, M.1; El Deeb, S.1
1 Institute
Capillary electrophoresis is an appropriate technique for analysing
pharmaceuticals and biopharmaceuticals [1,2] for which using small
volume samples is advantageous.
There are numbers of benefits involved in handling small sample volume
in capillary electrophoresis, among which using less material, generating
less waste, and applying high concentrations of valuable samples which
are all very important from an economic perspective stand out [3,4].
Most of the conventional CE instruments need approximately 50 µL of
the sample in the injection vial to perform the analysis, even though the
injected sample volume in capillary electrophoresis is only in the nanoliter
range.
Hence, in order to fully profit from the low injection volumes, smaller vial
volumes are required.
Furthermore the immersion depth of the capillary of most CE instruments
is not properly adjustable. Even if it is, one cannot ensure that the
capillary does not break at the end or at the detection window if it comes
against the bottom of the vial in lower settings. Therefore it is important
to fill the dead volume of the sample vials, which is inaccessible for the
capillary with a chemically inert liquid.
POSTERS
Thus experiments were performed using silicone oil as a filler of the vial
dead volume and the results were compared to those performed without
this filling.
As study example five standard proteins namely beta-lactoglobulin, BSA,
HSA, Myoglobin and Ovalbumin, and one of the coagulation cascade
involved proteins called vitonectin were investigated using capillary
electrophoresis. The experiments were performed once without the
addition of silicone oil in the sample vial and once in the presence of
silicone oil in the sample vial. The equation of migration times of EOFmarker and proteins (mobility ratios) as well as peak areas were
compared. However no significant changes were observed (RSDs% for
mobility ratios and peak areas were better than 0.9% and 5.8%
respectively).
Afterwards an affinity capillary electrophoresis method was used to
investigate the interactions of two proteins, namely HSA and vitronectin,
with three ligands namely enoxaparin sodium, unfractionated heparin
and pentosan polysulfate sodium (PPS). Mobility shift precision results
over 12 hours analysis showed that the employment of the filling has no
noticeable effect on any of the protein-ligand interactions. Accordingly,
the employed silicone oil is suitable as a water immiscible and inert liquid
for filling the dead volume of sample vials. Using a commercial instrument
and an autosampler the required sample volume is reduced down to 10
µL, and almost this complete volume can be subsequently injected
during repeated experiments.
Acknowledgments: bene pharmaChem for financial support of this project, Polymicro for the
donation of capillary material
References:
1. Redweik, S. et al.: Electrophoresis 2012, 33: 3316-3322.
2. Alhazmi, H.A. et al.: J. Pharm. Biomed. Analysis 2015, 107: 311-317.
3. Walter, R. et al.: Anal. Chem. 2002, 74: 3575-3578.
4. Beutner, A. et al.: Microchim Acta 2015, 182: 351-359.
POS.074
Towards selective CK2α and CK2α’ inhibitors: A novel
screening assay by Autodisplay of heterotetrameric human
protein kinase CK2
evaluation of the relative inhibition of a set of known ATP-competitive
CK2 inhibitors. Additionally the system was used to screen a small library
of CK2 inhibitors with a indeno[1,2-b]indole scaffold [3] in order to identify
a selective inhibitor of CK2α or CK2α’. Our results demonstrate that this
assay allows the discovery of CK2 inhibitors with a distinct effect on the
two different catalytically active CK2 isoforms, thus enabling the
identification of inhibitors with a selective affinity towards either CK2α or
CK2α’.
References:
1. Cozza, G.; Bortolato, A.; Moro, S.: Med. Res. Rev. 2010, 30: 419-462.
2. Gratz, A. et al.: Microb. Cell Fact. 2015, 14: 74.
3. Gozzi, G. J. et al.: J. Med. Chem. 2015, 58: 265-277.
POS.075
Site specific labelling of human protein kinase CK2 for
drug discovery applications
Nienberg, C.1; Becher, K.2; Mootz, H. D.2; Jose, J.1
Institut für Pharmazeutische und Medizinische Chemie, PharmaCampus, Westfälische
Wilhelms-Universität Münster, Corrensstraße 48, D-48149 Münster, Germany.
2 Institut für Biochemie, Westfälische Wilhelms-Universität Münster, Wilhelm-Klemm-Straße 2,
D-48149 Münster, Germany.
1
Human CK2 is a heterotetrameric constitutively active serine / threonine
protein kinase, phosphorylating and regulating a variety of cellular
processes. In tumor and rapidly proliferating cells the activity of CK2 is
increased. Downregulating the activity of the protein kinase in the
affected tumor cells initiates apoptosis [1]. Thus, CK2 represents a
promising target in current cancer research.
The kinase is composed of two catalytic CK2α subunits and two
regulatory CK2β subunits. Most protein-protein interaction (PPI) studies
or screening assays are based on fluorescence detection and require the
labelling of the target enzyme by a fluorophore. The catalytic subunit
CK2α loses activity after labelling by commercial applications.
Furthermore, the labelling ratio of the protein sample differs and is not
exactly reproducible.
Bollacke, A.1; Le Borgne, M.2; Jose, J.1
Institut für Pharmazeutische und Medizinische Chemie, PharmaCampus, Westfälische
Wilhelms-Universität Münster, Corrensstraße 48, 48149 Münster, Germany.
2 Université de Lyon, Université Lyon 1, Faculté der Pharmacie-ISPB, EA 4446 Biomolécules
Cancer et Chimiorésistances, SFR Santé Lyon-Est CNRS UMS3453-INSERM US7, Lyon
Cedex 8, 69373, France.
1
Novel inhibitors that target the human protein kinase CK2 are of great
interest in the development of new therapeutics for the treatment of
cancer. The human protein kinase CK2 commonly occurs as a
heterotetrameric enzyme, the so-called CK2 holoenzyme, and is
composed of two catalytically active α- and/or α’-subunits attached to a
dimer of non-catalytically active β-subunits [1]. We recently reported the
successful co-expression of CK2 subunits α and β on the cell surface of
Escherichia coli via Autodisplay and demonstrated the interaction of the
surface presented CK2 subunits, as well as the possible application for
the purpose of inhibitor screening by a CE-based assay [2].
The solution for this problem was an incorporation of an unnatural amino
acid into the CK2α subunit followed by a Strain-Promoted Alkyne-Azide
Cycloaddition (SPAAC) [2]. Therefore, a suitable position in the
sequence of CK2α was selected and mutated to the amber nonsense
DNA codon, TAG. By suppression of the mutation with an amber
suppressor tRNA, the unnatural amino acid para-acidophenylalanine
(pAzF) could be incorporated at this position [3]. Performing the SPAAC
click reaction by the use of dibenzylcyclooctyne-fluor 545 (DBCO 545)
led to a specifically labelled CK2α and CK2 holoenzyme.
This specific kind of labelling does not impair the phosphorylation activity
of the CK2α subunit alone nor the holoenzyme, which was evaluated by
capillary electrophoresis. The innovatively labelled kinase in combination
with the Autodisplay technology could be a significant advancement for
inhibitor screening assays by flow cytometry and for CK2α/CK2β
interaction studies [4].
References:
1. Wang, G. et al.: Mol. Cell. Biochem. 2005, 274(1-2): 77-84.
2. Mbua, N. E. et al.: ChemBioChem. 2011, 12(12): 1912-21.
3. Chin, J. W. et al.: J. Am. Chem. Soc. 2002, 124(31): 9026-7.
4. Jose, J.; Meyer, T. F.: Microbiol. Mol. Biol. Rev. 2007, 71(4): 600-19.
POS.076
We herein describe the successful Autodisplay of a paralogous isoform
of the catalytically active CK2 subunit, the CK2α’-subunit. Analysis of the
kinase activities at different NaCl concentrations for the individually
expressed α’-subunit and for the α’-subunit and the non-catalytically
active β-subunit co-expressed, confirms the interaction of the subunits on
the cell surface. We further illustrate the potential application of the two
surface presented CK2 holoenzymes as a novel inhibition assay by the
Dried Blood Spot Analysis for Therapeutic Drug Monitoring
of Antipsychotics
Weber, J.1; Hempel, G.1
1 Department
of Pharmaceutical and Medical Chemistry – Clinical Pharmacy, University of
Münster, Corrensstrasse 48, 48149 Münster, Germany
Background: Dried blood spot (DBS) analysis is an innovative sample
technique to collect and analyse very small volumes of whole blood. The
blood obtained from the finger pulp is spotted on a filter paper. After
ANALYTICS
drying, the analyte can be extracted and analysed. The main benefit of
DBS analysis is the possibility of long storage, simple transfer and noninvasive blood sampling [1]. Increasingly sensitive analytics enable
quantitative measurements of very small volumes of blood and thereby
facilitate DBS analysis for Therapeutic Drug Monitoring (TDM) [2].
Various antipsychotics and antidepressants show a correlation between
drug effect and blood concentration, such as risperidone, paliperidone,
mirtazapine and citalopram [3]. These substances are commonly
prescribed in nursing homes where physicians are mostly absent.
Collecting DBS by nurses or even by the patient himself at any time offers
an excellent possibility to perform TDM. Thus, the aim is to develop a
DBS method for several antipsychotics and antidepressants for TDM.
Methods: Blank whole blood was spiked with the analytes risperidone,
paliperidone, mirtazapine and citalopram. By using a heparinized
capillary a defined volume of spiked blood was spotted on treated and
non-treated DBS cards (FTATM DMPK-C Cards and Whatman 903 paper,
GE Healthcare, Freiburg, Germany). After a minimum drying period of
two hours the spots were cut out and transferred into an Eppendorf cap.
The internal standard (IS) clozapine was added and the analytes were
extracted by several organic solvents using an ultrasonic bath. After
drying under nitrogen gas the filter paper were removed. The samples
were subsequently purified by liquid-liquid extraction (LLE) in order to
prevent matrix effects and to achieve a high selectivity. After evaporating
and dissolving with methanol the extracted samples were analysed on a
Shimadzu LCMS-2020 system (Shimadzu, Duisburg, Germany).
Chromatographic separation was achieved using a Kinetex C18 reversed
phase column (3 x 100 mm, particle size 2.6 µm, Phenomenex,
Torrance, CA, USA) and a gradient elution with a flow rate of 0.5 ml/min.
Results: The best selectivity especially with regard to matrix effects was
achieved by non-treated Whatman 903 paper cards. Extracting the
analytes from the DBS in an ultrasonic bath, the best results were
achieved by using methanol. In order to improve the purity level of the
samples a LLE is well suited because the hydrophilic substances were
separated from the analytes. The best recovery for all four analytes was
achieved by using hexane/butyl acetate and a sodium hydroxide solution
as liquid phases. The calibration curve showed an excellent fit over a
concentration range of 2.5-300 µg/l for risperidone and paliperidone and
a concentration range of 10 – 300 µg/l for mirtazapine and citalopram.
Conclusion: This investigation shows a successful method to combine
DBS analysis with LLE for preventing matrix effects and to ensure
selectivity. It sets the basis to develop a quantifying DBS method for
risperidone, paliperidone, mirtazapine and citalopram which can be used
for TDM.
Method: For separation, Chromabond® C18 50 mg solid phases
(Machery-Nagel, Germany) and a SPE 12G glass column processor (J.T.
Baker®, Avantor Performance Materials, USA) were used. Conditioning
of the columns was done with 300 µL methanol, 750 µL phosphate buffer
pH 7.4 and 100 µL of a mixture of blank plasma and 5% glucose solution.
50 µL of spiked plasma were mixed with 50 µL internal standard and 100
µL 5% glucose solution and applied to the solid phase. To remove
plasma components and liposomes, 100 µL of a mixture of blank plasma
and 5% glucose solution, 500 µL phosphate buffer pH 7.4 and 100 µL
demineralized water was used for rinsing. An additional washing step
with 200 µL 80% methanol was implemented in order to purify the eluate.
Afterwards, the columns were dried under vacuum for 5 minutes and 400
µL elution solvent was applied. All samples were evaporated under
nitrogen stream and dissolved in mobile phase for HPLC analysis.
Samples were analysed using a Shimadzu Prominence HLPC System
(Shimadzu, Germany) with a RF-20Axs fluorescence detector (excitation:
495 nm, emission: 580 nm) and a Kinetex® 2.6 µm C18 100 Å, 100 x 3
mm (Phenomenex, USA) LC column for chromatographic separation.
Results: Three different attempts to improve elution were carried out and
the best result of each attempt was implemented in the next one.
Elution with additional 2% formic acid compared to pure methanol
increased peak area of DAUN by 54% (±10%) and DAUNol by 24%
(±9%). In comparison to Chromabond® C18 50 mg, Bond Elut C18 50
mg and Bond Elut Plexa 30 mg solid phases increased peak area of
DAUN by 105% (±3%) and 121% (±1%) and DAUNol by 98% (±3%) and
115% (±2%), respectively, and also lowered the variance between
different samples. Separating the elution solvent into two portions of 200
µL showed an increase of DAUN and DAUNol peak areas by 3% (±1%)
each.
Conclusion: Best results for improving the amount of free DAUN and
DAUNol being eluted were achieved with Bond Elut Plexa 30 mg solid
phases and 400 µL methanol containing 2% formic acid. Double elution
did not show any difference, but was more time-consuming and thus was
not implemented in the advanced method.
References:
1. Bellot, R.; Pouna, P.; Robert, J.: J. Chromatogr. B 2001, 757(2): 257-67.
2. Griese, N. et al.: J. Chromatogr. A 2002, 979(1-2): 379-88.
References:
1. Wagner, M. et al.: Mass Spectrom. Rev. 2014, doi: 10.1002/mas.21441
2. Edelbroek, P.M.; van der Heijden, J.; Stolk L.M.: Ther Drug Monit. 2009, 31: 327–336
3. Hiemke, C. et al.: Pharmacopsychiatry 2011, 44: 195–235
POS.077
Improved separation of free daunorubicin and its
metabolite daunorubicinol from liposomal daunorubicin in
plasma using solid phase extraction
Liebich, M.1; Hempel, G.1
1 Department
of Pharmaceutical and Medical Chemistry - Clinical Pharmacy -, University of
Münster, Corrensstraße 48, 48149 Münster, Germany
Background: Liposomal daunorubicin (DaunoXome®) is used in
induction therapy for the treatment of acute myeloid leukemia in pediatric
patients. The liposomal form is designed to reduce side effects,
especially cardiotoxicity, and to increase therapeutic response. After
administration, daunorubicin (DAUN) is slowly released from the
liposomes and free DAUN is metabolized to daunorubicinol (DAUNol).
Separating the liposomal encapsulated from the free drug in plasma is
challenging. The method should avoid high mechanical stress, organic
solvents, strong acids or bases to prevent destruction of the liposomes.
Solid phase extraction is a method to separate the liposomal and the free
drug, because the liposomes show no adsorption to the solid phase and
are eluted by buffer solutions while free DAUN and DAUNol adsorb to the
solid surface and can be eluted by an organic solvent. The developed
method is based on two publications by Bellot et al. [1] and Griese et al.
[2], however, aiming at increasing the amount of free DAUN and DAUNol
being eluted from the solid phase and reducing the required sample
volume.
POSTERS
4.6 GPCR/Ion channels
POS.078
GluN2B selective NMDA receptor antagonists with 3benzazepine scaffold: The deconstruction reconstruction
approach
Dey, S. 1,2; Wünsch, B.1
1 Institut
für Pharmazeutische und Medizinische Chemie, Corrensstraße-48, D-48149, Münster,
Germany.
Graduate School of Chemistry, University of Münster, Wilhelm-klemm-str., D-48149, Münster,
Germany
2
A functional NMDA receptor is built from at least one GluN1 and one
GluN2 subunit. The simultaneous binding of two agonists, (S)-glutamate
at the GluN2 subunit and glycine at GluN1 or GluN3 subunit activates the
NMDA receptor. The NMDA receptor has received significant attention
due to its unique properties and therapeutic potential. It is considered as
an important therapeutic target for a variety of conditions like epilepsy [1],
ischemia [2], depression [3] and chronic neurodegenerative diseases like
Alzheimer’s disease and Parkinson’s disease [4]. Without affecting the
main pharmacophore, the deconstruction approach of known ligands can
provide a clear rationale of essential structural elements. According to
this deconstruction-reconstruction approach a lead compound is
deconstructed by removal of structural elements and functional groups
and subsequently reconstructed to produce a diverse set of compounds
with different functional groups [5]. (Figure 1)
essential pharmacophore could be derived from its phloroglucinol moiety.
The first mono- and diacylated phloroglucinol compounds were tested for
their hyperforin-like activity profile and it turned out that the active ones
were also specific for TRPC6 but did not induce CYP3A4 [1,2]. Because
of this more derivatives were synthesized and investigated for their
structure-activity-relationships. It was shown that for the activation of
TRPC6 two acyl side chains are necessary but their symmetry is of no
importance. However, it seems that the activity increases only slightly
along with the lipophilicity of the compounds. The molecules were tested
in a calcium sensitive fluorescence assay in different concentrations
using PC12 cells as a model for neuronal cells. Additionally the assay
was performed with calcium-free extracellular buffer to differentiate
between intracellular and extracellular calcium release and barium influx
was measured to investigate TRPC6-like properties. We could also
demonstrate that the known CYP3A4-inducing antibiotics doxycycline,
erythromycin, griseofulvin, nafcillin and rifampicin have no activity
towards TRPC6.
Acknowledgments: We thank ERA-Net NEURON for funding the HypZiTRP consortium.
References:
1. Leuner, K. et al.: Mol. Pharmacol. 2010, 77: 368–77.
2. Kandel, B.A. et al.: J. Pharmacol. Exp. Ther. 2014, 348: 393–400.
POS.080
Selective activation of different pathways by dualsteric
compounds in muscarinic M1 acetylcholine receptors
Bödefeld, T.1; Messerer, R.2; Dallanoce, C.3; De Amici, M.3;
Holzgrabe, U.2; Mohr, K.1; Schrage, R.1
1 Pharmacology
The 3-benzazepine 1 is a conformationally restricted analogue of the
flexible ligand ifenprodil with high GluN2B affinity [6]. In this project
deconstruction of the lead compound 1 is envisaged, resulting in
simplified compounds 3, which will be decorated with different functional
group at the phenyl ring to obtain the compounds 4. This concept will
allow the identification of essential structural elements essential for high
GluN2B Affinity.
Acknowledgments: A special thanks to Graduate School of Chemistry for the funding of S.D.
during his PhD.
References:
1. Löscher, W.; Rogawski, M. A.: Selected Works of Micheal Rogawski; 2002.
2. Di, X. et al.: Stroke 1997, 28: 2244-2251.
3. Mony, L. et al.: Br. J. Pharmacol. 2009, 157: 1301-1317.
4. Wessell, R. H. et al.: Neuropharmacol. 2004, 47: 184-194.
5. Chen, H. et al.: Drug Discov. Today 2015, 20: 105-117.
6. Wünsch, B. et al.: ChemMedChem 2010, 5: 687-695.
POS.079
2, 4-Diacylphloroglucinols as classic transient receptor
potential-6 activators
Fritz, N.1; Eberle, J.1; Bouron, A.2,3,4; Nowak, G.5; Heinrich, M.6;
Friedland, K.1
1 Department of Chemistry and Pharmacy, Friedrich-Alexander-Universität Erlangen-Nürnberg,
Cauerstraße 4, 91058 Erlangen, Germany
2 Université Grenoble Alpes, LCBM, F-38054 Grenoble, France
3 CNRS, UMR 5249, F-38054 Grenoble, France
4 CEA, LCBM, 17 rue des Martyrs, F-38054 Grenoble, France
5 Department of Neurobiology, Institute of Pharmacology, Polish Academy of Sciences, Smetna
12, 31-343 Krakow, Poland
6 Department of Chemistry and Pharmacy, Friedrich-Alexander-Universität Erlangen-Nürnberg,
Schuhstraße 19, 91052 Erlangen, Germany
Hyperforin, the major antidepressant component of St. John´s wort, is a
specific activator of canonical transient receptor potential-6 (TRPC6) and
its antidepressive properties are mediated by activation of this
nonselective cation channel. But there are a few disadvantages like its
minor stability and its ability to induce Cytochrome P450 3A4 (CYP3A4),
therefore leading to pharmacokinetic interactions with other drugs. As it
is an acylated phloroglucinol derivative we hypothesized that the
and Toxicology Section, Institute of Pharmacy, University of Bonn, GerhardDomagk-Straße 3, 53115 Bonn, Germany;
of Pharmaceutical Chemistry, University of Würzburg, Am Hubland, 97074 Würzburg,
Germany;
3 Department of Pharmaceutical Sciences, University of Milan, Via Mangiagalli 25, 20133 Milan,
Italy
2 Institut
G protein-coupled receptors (GPCRs) respond to a broad range of
different extracellular stimuli, thereby evoking intracellular signaling.
Dualsteric compounds bind simultaneously to the receptor’s orthosteric
transmitter binding site and its allosteric vestibule [1]. In the muscarinic
acetylcholine M2 receptor (M2 mAChR) these dualsteric compounds
were shown to impair the activation-related conformational change of the
receptor protein by spatial restriction. This can eventually control the
signaling pattern of a GPCR that promiscuously activates a variety of
different signaling pathways [2].
In the present work, we checked whether this concept could be
transferred to the M1-subtype of muscarinic receptors (M1 mAChR)
which preferentially signals into Gq/11-dependent pathways, but can also
activate Gs and Gi proteins [3]. Therefore, M1 receptor-mediated signaling
induced by the orthosteric full agonist iperoxo and several dualsteric
compounds were investigated. The latter consist of iperoxo as the
orthosteric building block linked to an allosteric phthalimide (phth) or
naphthalimide (naph) moiety via alkyl chains of different length. To
distinguish between Gq/11- and Gs-dependent signaling pathways, CHO
cells stably transfected with the human M1 muscarinic receptor were
applied in IP1 and cAMP accumulation assays, respectively. Our findings
show that the bulky allosteric naph residue impaired both signaling
pathways to a greater extent than the smaller substituent phth. In
particular, the hybrid iper-6-naph completely lost intrinsic activity at the
M1 mAChR, although M2 mAChR activation by this compound had been
demonstrated in a previous study [2]. In contrast to iper-6-naph, the less
spacious congener iper-6-phth only lost intrinsic activity for Gs-activation,
while it was still a partial agonist for Gq/11-dependent signaling.
All other hybrids tested were able to activate both the Gq/11 protein and
the Gs protein. Remarkably, iper-7-phth had a significantly higher efficacy
for Gs protein activation than all the other compounds under investigation.
However, there was no significant difference between iper-7-phth and
iper-8-phth for Gq/11-dependent signaling. Furthermore, iper-7-naph and
iper-8-naph showed no significant differences in both Gq/11- and Gsassays.
Our data indicate that it might be possible to gain subtype selectivity by
exploiting the allosteric moiety or the length of the linker chain. In
particular, the bitopic derivative iper-6-naph was shown to be a partial
GPCR/ION CHANNELS
agonist at the M2 mAChR, but was not able to activate the M1 mAChR
[2].
Taken together, these data demonstrate that, in comparison to Gq/11mediated signaling, activation of the Gs protein in M1 mAChR is more
sensitive to spatial restriction in the allosteric vestibule. Thus, it is
possible to control signaling of the M1 mAChR by allosteric constraint of
the conformational flexibility.
References:
1. Antony, J. et al.: FASEB J. 2009, 23:442-450.
2. Bock, A. et al.: Nat. Commun. 2012, 3:1044 doi: 10.1038/ncomms2028.
3. Gregory, K.J.; Sexton P.M.; Christopoulos A.: Curr. Neuropharmacol. 2007, 5:157-167.
POS.081
mAChR - G protein dissociation kinetics reflect coupling
efficiencies and allow quantification of G protein subtype
selectivity.
receptors (ADOR). The ADORA3 is a potential drug target [1,2], and
several antagonists have been developed and pharmacologically
evaluated [3,4]. Apparently this receptor is involved in the progression of
inflammatory diseases, and shows ameliorating effects on plasma
extravasation [4], asthma bronchiale, chronic obstructive pulmonary
disease and idiopathic pulmonary fibrosis [5]. Moreover, ADORA3
antagonists may be useful for the treatment of cancer, either as
monotherapy are in combination with chemotherapy [6]. The A3 receptor
is coupled to Gi proteins mediating inhibition of adenylate cyclase [7].
In the present study quinazolines were modified by nucleophilic
substitution at position 4 of the quinazoline scaffold. The potency
increased considerable by the introduction of 3-aminopyrazole residues.
Elongation with alkyl groups led to a dramatic increase in subtypeselectivity for the ADORA3. Additional substitution at position 6 and 7 of
the quinazoline core led to extraordinarily potent and selective
derivatives. Compound 1 was found the most potent derivative of the
present series at human ADORA3 displaying affinity in the subnanomolar
range (Ki = 0.248±0.017 nM) combined with high selectivity.
Prokopets, O. S.1; Buenemann, M.1
1 Department of pharmacology and clinical pharmacy, Philipps-University of Marburg, Karl-vonFrisch Str.1, 35043 Marburg, Germany
Although recent structural research gave an idea about the GPCR
structure, the degree of selectivity of receptor-G protein coupling and its
underlying mechanisms are still unclear. Here we establishing a
quantitative method based on Fluorescence resonance energy transfer
(FRET) for measuring the affinity of Gα-subunit towards muscarinic
receptors and characterize dynamics of Go/i - and Gq/11-proteins binding
to activated M1-, M2- and M3-AChRs and their subsequent dissociation
in single permeabilized HEK293T cells under conditions of GTPdepletion.
In accordance with currently accepted models of ternary complex
formation of agonist, receptor Gα and Gβγ subunits we tested for Gαsubtype dependent stabilization of these complexes. As a measure of
affinity we measured receptor G protein dissociation after agonist
withdrawal and found fast Go and slow Gq proteins dissociation kinetics
(>10-fold slower) from M3- and M1-AChRs during agonist withdrawal
under the GTP depletion conditions. Furthermore, we observed a
significant shift (> 15 fold) of concentration-response curves of Go
proteins binding to M3-AChR in comparison to Gq. In order to relate these
findings to the coupling efficiency of these receptors towards these G
proteins we determined concentration response curves for G protein
activity in intact cells by means of FRET. Our results show a > 10 fold
higher sensitivity of Gq towards M3-AChR receptors compared to Go.
To test for subtype selectivity of M2-AChR towards Gi/o proteins, we have
determined the kinetics of Gi1-, Gi2-, Gi3-, and Go-proteins dissociation
from M2-AChR. We found two-fold faster kinetics for Gi1 and Gi3 in
comparison to Gi2 and Go proteins, indicating the existence of a subtype
selectivity of M2-AChR for Gαi2 and Gαo containing G proteins.
Taken together our study illustrates a suitable method for quantification
of the affinity of receptor-G-protein interactions and shows that the affinity
of this interaction reflects coupling efficiency and G protein selectivity for
a given receptor.
Acknowledgments: Heinrich-Heine University, Department of Lasermedicine, Universitätsstr. 1,
40225 Duesseldorf, Germany, Prof. Dr. Horst Lemoine
POS.082
Synthesis of quinazoline derivatives as adenosine A3
receptor antagonists
Acknowledgments: Thanks are due to J. Ortwein for HPLC analysis, Dr. L. Hennig for recording
and analysis of NMR data and C. Vielmuth for the biological testing.
References:
1. Fredholm, B. B. et al.: Pharmacol. Rev. 2011, 63(1): 1–34.
2. Gessi, S et al.: Pharmacol. Ther. 2008, 117: 123–140.
3. Lee, J. et al.: Am. J. Pathol. 2013, 183: 1488–97.
4. Mikus, E.G. et al.: Eur. J. Pharmacol. 2012, 699(1–3): 62–66.
5. Della Latta, V. et al.: Pharm. Res. 2013, 76: 182–189.
6. Merighi, S. et al.: Pharmacol. Ther. 2003, 100: 31–48.
7. Birnbaumer, L.: Biochim. Biophys. Acta 2007, 1768(4): 772–93.
POS.083
CRTC1-deficient mice show cardiac hypertrophy and
reduced cardiac function
Morhenn, K.1,2; Geertz, B.3; Eschenhagen, T.2,3; Cardinaux, J.-R.4;
Lutz, S. 5,6; Oetjen, E.1,2,7
1 Department
of Clinical Pharmacology and Toxicology, Cardiovascular Research Center,
University Medical Center Hamburg Eppendorf, Martinistraße 52, 20246 Hamburg, Germany
(German Center for Cardiovascular Research), Partner Site Hamburg/Kiel/Lübeck
3 Department of Experimental Pharmacology and Toxicology, Cardiovascular Research Center,
University Medical Center Hamburg Eppendorf, Martinistraße 52, 20246 Hamburg, Germany
4 Center for Psychiatric Neuroscience, Site Cery, 1008 Prilly-Lausanne, Switzerland
5 Department of Pharmacology, University Medical Center Göttingen, Robert Koch Straße 40,
37075 Göttingen, Germany
6 DZHK (German Center for Cardiovascular Research), Partner Site Göttingen
7 Institute of Pharmacy, University of Hamburg, Bundesstraße 45, 20146 Hamburg, Germany
2 DZHK
Cardiac hypertrophy leads to heart failure, one of the common causes for
hospitalization. Chronic β-adrenergic signaling contributes to the
pathogenesis of cardiac hypertrophy, as evidenced by the therapeutic
success of β-adrenoceptor antagonists. The cAMP Regulated
Transcriptional Coactivator 1 (CRTC1) is regulated by increases in cAMP
and calcium/calcineurin, as elicited by β-adrenergic signaling, both
known to participate in the development of cardiac hypertrophy [1]. Our
previous data showed that the protein content of CRTC1 is elevated in
hearts of mice and humans under conditions of maladaptive hypertrophy.
Mice globally deficient in Crtc1 show signs of hypertrophy indicated by a
higher ratio of heart weight to tibia length as well as increased myocyte
size.
Lang, M.1; Hinz, S.2; Schäke, F.1; Kubicova, L.3; Müller, C. E.2; Briel, D.1
1 University of Leipzig, Institute of Pharmacy, Bruederstraße 34, 04103 Leipzig, Germany
2 Rheinische Friedrich-Wilhelms-Universität Bonn, Pharmazeutisches Institut, An der
Immenburg 4, 53121 Bonn, Germany
3 University of Vienna, Division of Molecular Systems Biology, Faculty of Life Sciences,
Althanstrasse 14, Vienna, A-1090, Austria
Quinazolines that were modified at position 2, 4, 6 and 7 were
synthesized to evaluate their structure-activity relationships at the four
subtypes (A1, A2A, A2B and A3 [1]) of G-protein-coupled adenosine
POSTERS
To study the role of CRTC1 in the pathogenesis of cardiac hypertrophy,
in the present study Crtc1-deficient mice were further investigated.
Echocardiographically assessed, the ejection fraction, the fractional area
shortening and the cardiac output were reduced by 47±8%, 49±12% and
42±9%, respectively, in Crtc1-deficient mice compared to their wild-type
littermates, indicating a systolic dysfunction of the heart. Meanwhile,
Serca and Phospholamban protein contents remained unchanged, as
measured by immunoblot. As measured by RT-qPCR, no differences in
mRNA levels of the markers for cardiac hypertrophy Nppa, Nppb, Acta1
and Myh7, as well as the pro-fibrotic Ctgf were observed. It is known that
the Regulator of G-Protein Signaling 2 (RGS2) reduces hypertrophy via
reduction of Gαq-protein induced signaling and that Rgs2 gene
transcription is induced by CREB [2,3]. We previously showed a
stimulation of the transcriptional activity of the Rgs2 promoter by CRTC1
in HEK cells and decreased Rgs2 mRNA and protein levels in Crtc1deficient mice. By chromatin immunoprecipitation, we now showed the
recruitment of endogenous CRTC1 to the Rgs2 promoter in cardiac
tissue. mRNA levels of Rgs3, Rgs4, Rgs5 and Rgs6 did not differ in
Crtc1-deficient mice.
In conclusion, our data indicate that increased CRTC1 protein content in
maladaptive cardiac hypertrophy is a compensatory mechanism to delay
disease progression, in part by upregulation of RGS2. Thus, CRTC1
might represent a novel player in cardiac hypertrophy.
References:
1. Bittinger, M. et al.: Curr. Biol. 2004, 14(23): 2156-61.
2. Xie, Z. et al.: J. Biol. Chem. 2011, 286(52): 44646-58.
3. Zhang, P.; Mende, U.: Trends Cardiovasc. Med. 2014, 24(2): 85-93.
POS.084
A homogenous A2B adenosine receptor fluorescence
binding assay based on flow cytometry
POS.085
GPR17: Understanding its signaling and physiological
function
Simon, K.1; Hennen, S.1; Merten, N.1; Blättermann, S.1; Gomeza, J.1;
Kostenis, E.1
1
Institute for Pharmaceutical Biology, University of Bonn, Nussallee 6, 53115 Bonn, Germany
Oligodendrocyte differentiation and maturation, ultimately leading to
formation of myelin sheaths, is regulated temporally during postnatal
development but also during repair of demyelinating lesions [1]. Recent
studies suggest G protein-coupled receptors (GPCRs) as key players in
the development of proliferating oligodendrocyte precursor cells to
myelinating oligodendrocytes [1,2,3]. Activation of the orphan GPCR
GPR17, which is predominantly expressed in the oligodendrocytelineage cells of the central nervous system (CNS), has been linked to
arrest of primary mouse oligodendrocytes in an immature, less
myelinating stage [1,3]. Accordingly, GPR17 inhibition emerges as
therapeutic strategy to enhance repair of lost myelin sheaths in
demyelinating diseases, such as multiple sclerosis (MS). However, the
intracellular signaling pathways linking activated GPR17 to
oligodendrocyte differentiation block are poorly understood at present.
To further delineate these downstream components, we took advantage
of the recently identified first surrogate GPR17-agonist MDL29,951 [3]
and applied this molecule in Oli-neu cells, an immortalized cell line
derived from primary murine oligodendrocytes [4], and primary rat
oligodendrocyte cultures isolated from brain tissue. We anticipate that
detailed understanding of cellular mechanisms governing GPR17mediated inhibition of oligodendrocyte differentiation will aid to develop
novel therapies for demyelinating pathologies.
References:
1. Chen, Y. et al.: Nat. Neurosci. 2009, 12: 1398–1406.
2. Ackerman, S. D. et al.: Nat Commun 2015, 6: 6122.
3. Hennen, S. et al.: Sci. Signal 2013, 6: ra93.
4. Jung, M. et al.: Eur. J. Neurosci. 1995, 7: 1245–1265.
Köse, M.1; Karcz, T.2; Gollos, S.1; Fiene, A.1; Heisig, F.1; Spanier, C.1,
Kieć-Kononowicz, K.2; Müller, C. E. E.1
1 PharmaCenter Bonn, Pharmaceutical Chemistry I, University of Bonn, An der Immenburg 4,
53121 Bonn, Germany
2 Department of Technology and Biotechnology of Drugs, Faculty of Pharmacy, Jagiellonian
University Medical College, Medyczna 9, 30-688, Kraków, Poland
Homogenous binding assays based on fluorescence measurement
represent attractive alternatives to the traditional radioligand binding
assays as they are faster, cheaper and less hazardous. Recently the first
fluorescence binding assay for the A3 adenosine receptor (AR) was
described [1,2]. So far, no fluorescence binding assay is available for the
human A2B AR.
In the present study, we developed a new flow cytometry-based
fluorescence binding assay for the human A2B AR using a newly
synthesized fluorescent antagonist PSB-12105. In radioligand binding
assays PSB-12105 was shown to display high affinity at the human A2B
receptor with a Ki value of 15.0 nM. It was also found to be selective
versus the A1, A2A and A3 receptor subtypes. CHO cells stably transfected
with A2B ARs were preincubated with adenosine deaminase (ADA) for 2
h at 37 °C before adding the fluorescent ligand PSB 12105 (1 nM).
Nonspecific binding was determined in the presence of the potent and
selective A2B antagonist PSB-603 (100 nM). The cell suspension was
incubated for 1 h at 37 °C. The fluorescence was subsequently measured
using flow cytometry.
We determined an IC50 value of 0.543 nM for the standard A2B antagonist
PSB 603 which is in good agreement with the Ki value determined in
radioligand binding assays (0.553 nM) [3]. The results indicate that our
assay is robust and reproducible and provides a simple, convenient
alternative method to radioligand binding assays adaptable to highthroughput screening. To our knowledge, this is the first fluorescence
binding assay for the human A2B AR reported to date. This new method
may serve as an important pharmacological tool for the development and
investigation of new A2B AR ligands.
Acknowledgments: Partly supported by Polish National Science Center founding, project DEC2012/04/M/NZ4/00219 and GLISTEN COST Action CM1207
References:
1. Kozma, E. et al.: Biochem. Pharmacol. 2013, 85: 1171-1181.
2. Kozma, E. et al.: Biochem. Pharmacol. 2012, 83: 1552-1561.
3. Borrmann, T. et al.: J. Med. Chem. 2009, 52: 3994-4006.
POS.086
Novel 6-benzamidochromen-4-one-2-carboxylic acids as
pharmacological tools for investigating the orphan
receptor GPR35
Meyer, A.1; Funke, M.1; Thimm, D.1; Müller, C. E.1
1
Pharmaceutical Institute, University of Bonn, An der Immenburg 4, 53121 Bonn, Germany
G protein-coupled receptors (GPCRs) are the largest protein family in the
human genome with about 800 different sequences. about one third of
all marketed drugs and also those in (pre)clinical development are
targeting GPCRs. GPR35 is an orphan GPCR whose endogenous ligand
is still unknown. It is expressed in the gastrointestinal tract and in liver, in
the immune, cardiovascular and the central nervous system. GPR35
agonists have potential for the treatment of inflammation and pain
whereas antagonists might be useful for treating cardiovascular
diseases. We identified and developed a novel class of potent GPR35
agonists based on the chromen-4-one scaffold [1]. One of our most
potent agonists, PSB 13253, was obtained in tritium-labeled form with a
specific activity of 36 Ci (1.33 TBq) / mmol. [3H]PSB-13253 displayed a
KD value of 5.27 nM, and its binding was saturable and reversible.
[3H]PSB 13253 is the first radioligand for GPR35 and represents a useful
tool for investigating this poorly studied orphan receptor. Based on
radioligand binding studies we further optimized the series developing
agonists with subnanomolar affinity, e.g. 6 bromo-8-(2,6-difluoro-4methoxybenzamido)-4-oxo-4H-chromene-2-carboxylic acid (PSB13007). Its Ki value of 0.589 nM makes it the most potent GPR35 agonist
known to date [2]. With a calculated clogP of 2.6 and a molecular weight
of 454 g/mol it meets the criteria for drug-likeness. In summary, a new
series of 8 benzamidochromen-4-one-2-carboxylic acid derivatives was
developed resulting in valuable tools for the characterization of the so far
poorly studied GPR35, which may represent a new drug target.
References:
1. Funke, M. et al.: J. Med. Chem. 2013, 56: 5182-5197.
2. Thimm, D. et al.: J. Med. Chem. 2013, 56: 7084-7099.
GPCR/ION CHANNELS
POS.087
Binding mode prediction and validation of bile acid and
neurosteroid agonists of the G-Protein coupled receptor
TGR5
Gertzen, C. G. W.1; Spomer, L.2; Häussinger, D.2; Keitel, V.2; Gohlke,
H.1
1 Institute
for Pharmaceutical and Medicinal Chemistry, Universitätsstr. 1, 40225 Düsseldorf,
Germany
2 Clinic for Gastroenterology, Hepatology, and Infectious Diseases, Heinrich-Heine-University,
Moorenstr. 5, 40225 Düsseldorf, Germany
The structurally unknown G-protein coupled bile acid receptor (GPCR)
TGR5 is the first bile acid sensing GPCR and directly interacts with
several G-protein subtypes [1]. Tissues with high expression levels of
TGR5 include the brain, the liver, and the gastrointestinal tract. TGR5 is
an emerging target for the treatment of metabolic diseases [2-4].
Therefore, developing selective and potent agonists of TGR5 is of high
importance [5]. However, without an experimentally determined binding
mode, the rational design of compounds is difficult. Recently,
Macchiarulo et al. [6] proposed a binding mode of natural and synthetic
bile acids in TGR5 based on single template homology modeling,
molecular docking, and mutational analysis. However, this binding mode
does not interact with transmembrane helices (TM) 5 and 6, which are
crucial for GPCR activation [7, 8]. Additionally, the binding mode of
Macchiarulo et al. does not address E169 in TM 5, which the authors
found to be important for receptor activation and is a conserved residue
among the TGR5 family [6].
Here, we present an experimentally validated binding mode of 68 TGR5
agonists, including natural and synthetic bile acids and neurosteroids.
Our strategy consisted of multi-template homology modeling, molecular
docking, and structure-based 3D-QSAR with subsequent mutational
analysis and molecular dynamics simulations. After two cycles of this
strategy, the binding mode model of the TGR5 agonists results in a good
3D-QSAR model (q² = 0.50), thus indicating that differences in the
agonist structures correlate with differences in experimentally determined
pEC50 values in the predicted binding mode. Based on this binding mode,
nine mutants of binding site residues were suggested that should either
influence agonist binding or TGR5 activation. Activity analysis using
cAMP reporter gene assays and FACS analysis for membrane
localization confirmed these predictions in all cases. In particular, through
the Y240F mutant we could demonstrate the importance of hydrogenbonding interactions between the tyrosine and TGR5 agonists.
Additionally, we identified the stereoselectivity-determining residue Y89
for hydroxyl-groups in position seven on the cholane scaffold. This
provides strong support to the validity of the binding mode. Our binding
mode differs from the binding mode by Macchiarulo et al. in three
important aspects: I) The cholane moiety is rotated by 180°; II) the
sidechains of bile acids bind to R79, which is 12 Å away from the
respective interaction partner postulated in [6]; III) agonists address
residues in TM 5 and 6, which are essential for receptor activation. Our
binding mode could facilitate the structure-based design of new TGR5
agonists.
Acknowledgments: We are grateful to the ‘‘Zentrum für Informations und Medientechnologie’’
(ZIM) at the Heinrich Heine University for computational support, Stefanie Lindner und Waltraud
Kuß for technical assistance, and to Dr. Nadine Homeyer, Yasemin Bilgic, and Alina Völz for help
with the molecular modeling. This work was supported by the Deutsche Forschungsgemeinschaft
through the Collaborative Research Center SFB 974 (‘‘Communication and Systems Relevance
during Liver Damage and Regeneration’’, Düsseldorf) and the Clinical Research Group KFO 217
(‘‘Hepatobiliary Transport in Health and Disease’’, Düsseldorf).
References:
1. Hannah, M.: International Journal of Interferon, Cytokine and Mediator Research 2014, 6:
27-38.
2. Pols, T. W. H. et al.: Cell Metab. 2011, 14: 747-757.
3. Pols, T. W. H. et al.: J. Hepatol. 2011, 54: 1263-1272.
4. Perino, A. et al.: The Journal of Clinical Investigation 2014, 124: 5424-5436.
5. Sato, H. et al.: J. Med. Chem. 2008, 51: 1831-1841.
6. Macchiarulo, A. et al.: ACS Med. Chem. Lett. 2013, 4: 1158-1162.
7. Zimmerman, B. et al.: Sci. Signal. 2012, 5: ra33.
8. Xu, F. et al.: Science 2011, 332: 322-327.
POS.088
Differential modulation of Gβγ effectors by ligands
targeting GPCRs
Büllesbach, K.1; Bautista, O.2; Schröder, R.1; Gütschow, M.2;
Kostenis, E.1
1 Molecular-,
Cellular- and Pharmacobiology Section, Institute of Pharmaceutical Biology,
University of Bonn, Bonn, Germany
2 Pharmaceutical Institute, Pharmaceutical Chemistry I, University of Bonn, Bonn, Germany
G protein-coupled receptors constitute the largest family of membrane
signaling proteins. They respond to a wide array of stimuli and serve to
translate extracellular information to the inside of the cell via coupling to
heterotrimeric αβγ G proteins [1]. G proteins are central to signal
transduction because they connect GPCRs to diverse intracellular
effectors whose activities they regulate via both Gα and Gβγ subunit
complexes. A common dogma in G protein signaling stated that G protein
heterotrimers are regulated uniformly by activated GPCRs, whereby
receptors discriminate between individual G protein isoforms depending
on the inherent bias of each ligand used to stimulate the receptor. We
recently challenged this dogma demonstrating that the small molecule
Gue1654 acts as a non-competitive inhibitor of OXE-R, a GPCR
responding to the endogenous lipid mediator 5-oxo-eicosatetraenoic acid
[2]. Specifically, Gue1654 exclusively inhibited Gβγ but not Gαi signaling
triggered upon activation of OXE-R in both recombinant and human
primary cells. The question therefore arose whether biased perturbation
of G protein heterotrimers can extend to effectors further downstream of
Gβγ.
To this end we investigated the ability of Gue1654 to discern between
effectors of Gβγ such as extracellular regulated kinases 1 and 2
(ERK1/2), phospholipase Cβ (PLCβ), and G protein inward-rectifying
potassium (GIRK) channels [3] in human embryonic kidney (HEK293)
cells forcibly expressing OXE-R. We compared the resulting profile to
that obtained with the small molecule Gβγ inhibitor Gallein [4], which
targets a hotspot on the Gβ protein thereby preventing Gβγ from
interacting with its downstream effector molecules in a receptorindependent fashion.
We find that both Gue1654 and Gallein completely blunt IP1 production,
yet differ in their inhibition profile concerning activation of ERK1/2 and
GIRK channels. This pattern of inhibition is distinct from that observed
with canonical competitive OXE-R inhibitors [5] which blunt Gβγ
downstream signaling equally. Our data therefore suggest capacity to
selectively engage or disrupt individual Gβγ downstream effects in a
receptor-specific manner demonstrating another layer of regulation within
the GPCR-G protein signaling axis.
Acknowledgments: K.B. is a member of the DFG-funded Research Training Group RTG 1873
References:
1. Rosenbaum, D. M. et al.: Nature. 2009, 459(7245):356-63.
2. Blättermann, S. et al.: Nat. Chem. Biol. 2012, 8(7): 631-8.
3. Clapham, D. E.; Neer E. J.: Annu. Rev. Pharmacol. Toxicol. 1997, 37: 167-203.
4. Lehmann, D. M. et al.: Mol. Pharmacol. 2008, 73(2): 410-8.
5. Gore, V. et al.: J. Med. Chem. 2014, 57(2): 364-77.
POS.089
A mechanistic view on GPCR modulation
Bermudez, M.1; Wolber, G.1
Computer-Aided Drug Design, Institute of Pharmacy, Department Pharmaceutical Chemistry,
Freie Universität Berlin, 14195 Berlin
1
G-protein coupled receptors (GPCRs) enable the transmission of signals
into cells, which makes them highly interesting drug targets. Recent
achievements in GPCR crystallography provide us with new structural
data on GPCRs in distinct activation states [1]. However, these structures
represent only a single static view on highly flexible proteins that display
their functionality by recognizing extracellular stimuli, subsequent
adaption of their conformation and hence allows for an intracellular
response [2]. The combination of crystallographic data with state-of-theart computer-driven simulations allows for a mechanistic view on the first
two steps of GPCR function: ligand binding and the resulting
conformational change. Taking muscarinic acetylcholine receptors
(MAChRs) as representative examples we explained how GPCR can be
modulated in a predictable fashion.
POSTERS
Our mechanistic models comprise both inactive and active-like receptor
states (Figure). After a characterization of the orthosteric binding side,
we focused on dualsteric ligand binding [3,4]. These kinds of ligands
simultaneously bind to the orthosteric and the allosteric binding site and
combine the high affinity of orthosteric ligands with the high specificity of
the allosteric binding site. Here we report the structural basis for a specific
modulation of MAChRs focusing on dualsteric ligand binding. This
includes subtype selectivity, biased signaling and a novel concept for
partial agonism. Our dynamic models illustrate how distinct
conformational states can be stabilized in a ligand-dependent manner.
Finally, our structural and functional models proved their high explanatory
power in biological experiments and offer the possibility to rationally
design specific modulators for MAChRs but also for other GPCRs.
comparing the binding modes of known antagonists for MCH1R and H3R
will allow us to identify potent dual antagonists of H3R and MCH1R.
Acknowledgments: Financial support for David Schaller by the Elsa-Neumann-Scholarship is
gratefully acknowledged.
References:
1. Kopelman, P. G.: Nature 2000, 404: 635-43.
2. Kim, G. W. et al.: Clin. Pharmacol. Ther. 2014, 95(1): 53-66.
3. Singh, M.; Jadhav, H. R.: Mini Rev. Med. Chem. 2013, 13(1): 47-57.
4. Szalai, K. K. et al.: Recent. Pat. CNS Drug Discov. 2014, 9(2): 122-40.
5. Parks, G. S. et al.: J. Physiol. 2014, 592(10): 2183-2196.
6. Thorsen, T. S. et al.: Structure 2014, 22(11): 1657-1664.
7. Chien, E. Y. et al.: Science 2010, 330(6007): 1091-5.
8. Shimamura, T. et al.: Nature 2011, 475(7354): 65-70.
POS.091
Histamine H4 receptor affinity of 1,3,5-triazine derivatives
Kamińska, K.1; Więcek, M.1; Adami, M.2; Stark, H.3; Kieć-Kononowicz,
K.1
1 Department
of Technology and Biotechnology of Drugs, Jagiellonian University Medical
College, Medyczna 9, 30-688 Cracow, Poland.
2 Department Neuroscience, University of Parma, Via Volturno 39, 43125 Parma , Italy.
3 Institute of Pharmaceutical and Medicinal Chemistry, Heinrich-Heine-University,
Universitaetsstr. 1, 40225 Duesseldorf, Germany.
Figure: Superimposition of homology models for all subtypes of muscarinic receptors in different
activation states.
References:
1. Venkatakrishnan, A. J. et al.: Nature 2013, 494(7436): 185–194.
2. Bermudez, M.; Wolber, G.: Bioorg. Med. Chem. 2015, 23(14): 3907-3912.
3. Schmitz, J. et al.: J. Med. Chem. 2014, 57(15):6739-50.
4. Bermudez, M. et al.: Mol. Inf. e-pub ahead of print, 2015.
POS.090
Rational multi-target drug design by ligand-guided
homology modeling
Schaller, D.1; Wolber, G.1
1 Institut
für Pharmazie, Freie Universität Berlin, Königin-Luise-Str. 2+4, 14195 Berlin
Obesity has become a major human health risk and is associated with
many serious diseases including type 2 diabetes, stroke and cancer [1].
Although being studied for decades, anti-obesity drugs still lack efficacy
and show serious or unpleasant side effects [2]. Using a chemogenomics
approach we identified histamine H3 receptor (H3R) and melaninconcentrating hormone receptor 1 (MCH1R) as promising target pair for
the development of multi-target directed ligands. Targeting these single
receptors alone did not yet result in an effective obesity treatment [3,4].
Recent studies suggest that antagonizing H3R and MCH1R at the same
time may induce a synergistic effect [5].
This study aims at generating homology models that are able to explain
the structure activity relationship of known antagonists of H3R. To
achieve our goal we implemented a ligand-guided homology modeling
workflow that is able to sample the conformational space of side chains
in the orthosteric binding pocket of H3R. The increasing number of crystal
structures of G protein-coupled receptors (GPCRs) allows the
identification of comparable interaction patterns between bound ligands
and receptors. In particular an ionic interaction between the tertiary or
quaternary amine of ligands and the conserved residue ASP3.32 of
GPCRs can be observed in many crystal structures including muscarinic
receptors (4U15), dopamine receptors (3PBL) and histamine H1 receptor
(3RZE) [6,7,8]. The crystal structure 3RZE of H1R was used as template
to create 1000 homology models of H3R. A specific set of tertiary aminecontaining H3R antagonists was docked against these models.
Subsequently, the homology models were ranked by calculating the
distance between the tertiary amine of the docked ligands and the key
amino acid ASP3.32 of H3R. Analyzing the resulting models revealed
distinct side chain conformations that favor the interaction of the tertiary
amines with ASP3.32 of H3R. This knowledge was employed to create
homology models that explain the structure activity relationship of known
ligands in a highly consistent way. Several steps of minimization and
redocking were performed to ensure an optimized geometry.
This methodology is adaptable to other GPCRs like MCH1R that show
the described ionic interaction with bound ligands. Elucidating and
Histamine plays its function through binding with four already known
histamine receptors, designed as H1-H4. It is assumed, that the youngest
member of the family – histamine H4 receptor (H4R), which was
discovered and cloned in 2000/2001 by several independent research
groups [1], is involved in inflammatory processes and immune
responses, because of its mainly expression in various cells of the
immune system (monocytes, mast cells, dendritic cells, eosinophils and
basophils) [2]. Potential therapeutic effects of H4R antagonists/inverse
agonists in animal models of acute inflammations, allergic rhinitis,
asthma or pruritus were confirmed [3]. As physiological role of H4R is not
clear - new, potent and selective ligands are required to investigate its
action. Among H4R ligands already described in the literature and patent
data there can be found a large group of triazine derivatives [4,5].
The aim of this study was to evaluate in vivo activity of five 4-(4methylpiperazin-1-yl)-1,3,5-triazine derivatives (KB-4, KB-30, JN-38, TR11 and TR-40), Compounds were tested in croton oil-induced ear edema
model and ear pruritus model in vivo in mice. Compounds examined in
the presented studies were selected from the library of compounds
synthesized in our Department.
The obtained results showed that pre-treatment with KB-4, KB-30 or TR11 has strong to moderate influence on ear edema and pruritus. Among
the tested compounds, KB-4 and TR-11 seem to have the most favorable
profile, combining a good affinity at the human histamine H4 receptor with
a high efficacy in the intact animal. The results in detail will be presented
and discussed.
Acknowledgments: This work was kindly supported by National Science Center DEC2011/02/A/NZ4/00031 and GLISTEN: COST Action CM1207.
References:
1. Nguyen, T. et al.: Mol. Pharmacol. 2001, 59(3): 427-433.
2. Walter, M. et al.: Eur. J. Pharmacol. 2011, 668(1-2): 1-5.
3. Tiligada, E. et al.: Expert Opin. Investig. Drugs. 2009, 18(10): 1519-1531.
4. Kiss, R.; Reserű, G.M.: Expert Opin. Ther. Pat. 2012, 22(3): 205-221.
5. Stark, H.: H4 receptor: A Novel Drug Target in Immunomodulation and Inflamation , 2012
(Versita).
POS.092
Multiple targeting with histamine H3 receptor antagonists:
Epilepsy
Schwed, J. S.1,2; Sadek, B.3; Khan, N.3; Subramanian, D.3; Weizel, L.2;
Walter, M.2; Stark, H.1,2
1 Institute
of Pharmaceutical and Medicinal Chemistry, Heinrich Heine University,
2 Biocenter, Institute of Pharmaceutical Chemistry, Goethe University, Max-von-Laue-Str. 9,
60438 Frankfurt, Germany
3 Department of Pharmacology and Therapeutics, College of Medicine & Health Sciences, P.O.
Box 17666, Al Ain 0097, United Arab Emirates University, United Arab Emirates
Epilepsy is a chronic disorder of the brain and characterised by an
enduring predisposition to generate recurrent epileptic seizures and by
the neurobiological, cognitive, psychological and social consequences of
GPCR/ION CHANNELS
this condition [1]. For medical therapy different antiepileptic drugs (AEDs)
are in use, but are hampered in clinical usage by either having an
effectiveness within a maximum of 60-80% of all patients and diverse
unwanted side effects like headache, nausea, cognitive impairment etc.
[2]. Central histamine was found to play an important role in epilepsy [3],
especially the third histamine receptor subtype (H3R) has by
neurotransmitter modulating abilities raised hopes for novel AEDs [3,4].
Pitolisant (Wakix®), a non-imidazole H3R antagonist/inverse agonist
submitted to EMA for market authorisation, was found to be effective in
different in vitro and in vivo animal seizure models [3] and also in
photosensitivity epilepsy in human patients [5]. Safinamide (Xadago®), a
recently marketed anti-parkinson MAO B inhibitor, was primarily
designed as an AED [6] and shown to have good affinity at voltage-gated
Na+ and Ca++ channels as well as good inhibitory potency on glutamate
release. As a multi-targeting or polypharmacological approach we
combined the pharmacophore elements of H3R antagonist to that of
safinamide or phenytoin with different spacer moieties.
All compounds showed moderate to good affinity at hH3R (pKi values
between 7.36 and 8.21) with good selectivity profile among the other
histamine receptor subtypes tested (hH1R and hH4R, pKi values < 5). The
ligands were further characterized in vivo in different convulsion models
in rats using chemical-induced (pentylenetetrazole (PTZ)- or strychnine
(STR)-induced seizure) or in maximum electroshock-induced seizure
models (MES). The safinamide related derivatives with bulky elements
showed significantly and dose-dependently reduced seizures or
exhibited full protection in MES and PTZ convulsions model with some
stereochemical preference for the R-configured isomer.
Acknowledgments: This work was partly supported by the DFG (INST 208/664-1) as well as by
the European COST Actions CM1103 and CM1207. Grants are from CMHS, UAE University.
References:
1. Fisher, R. S. et al.: Epilepsia 2005, 46: 470-472.
2. Sadek, B. et al.: Eur. J. Med. Chem. 2014, 77: 269-279.
3. Bhowmik, M. et al.: Br. J. Pharm. 2012, 167: 1398-1414.
4. Sander, K. et al.: Biol. Pharm. Bull. 2008, 31: 2163-2181.
5. Bialer, M. et al.: Epilepsy Res. 2015, 111: 85-141.
6. Fariello, R. G.: Neurotherapeutics 2007, 4: 110-116.
POS.093
Derivates of imidazolylpropylguanidine (SK&F-91486):
Synthesis and pharmacological in vitro activities at
histamine receptors (hH1,2,3,4R and gpH1,2R)
Pockes, S.1; Buschauer, A.1; Elz, S.1
1 Institute of Pharmacy, University of Regensburg, Universitätsstraße 31, 93053 Regensburg,
Germany
3-(1H-Imidazol-4-yl)propylguanidine (SK&F-91486 [1]) is the long-known
prototypic pharmacophore of highly potent histamine H2-receptor (H2R)
agonists of the guanidine class of compounds including, e.g.,
arpromidine [2] and the recently discovered acylguanidines [3]. In order
to gain more insight into the structure-activity relationship of simple
analogues of SK&F-91486, we started a project aiming at the synthesis
and in vitro characterisation of alkylated imidazolylpropylguanidines and
synthesised alkylated, aminoalkylated, ureidoalkylated and
guanidinoalkylated derivatives of SK&F-91486 as well as the
corresponding dimers, equipped with different spacer lenghts (C3-C12)
(1). Furthermore, we picked out the most promising molecules of each
group and replaced the imidazol-4-yl group by the bioisosteric moieties
imidazol-1-yl, 2-amino-4-methylthiazol-5-yl and 2-aminothiazol-5-yl,
respectively, to study their influence on histamine receptor subtype
selectivity.
In addition, we created a batch of simple alkylated
imidazolylpropylcyanoguanidines (2) belonging to the imidazol-4-yl and
the imidazol-1-yl series. To finalise the project, we also synthesised a few
derivatives by heteroatomic exchange at the guanidine group to give, for
example, (acylated) urea or thiourea analogues of SK&F-91486.
We analysed the in vitro properties of 78 compounds in the guinea-pig
ileum assay (gpH1R), the guinea-pig right atrium assay (gpH2R), in
radioligand binding assays (hH1,2,3,4R), and in the [35S]GTPγS assay
(hH1,2,3,4R). There were just a small number of compounds with
mentionable affinity at gp/hH1R. A longer alkyl spacer length afforded a
higher (agonistic) affinity at the gpH2R as well as at the hH2,3,4R. Dimeric
compounds turned out to have highest (agonistic) affinity at these
receptors (gpH2R: pEC50 ≈ 8.5, hH2,3,4R: pKi ≈ 7.5-8.0). Bioisosteric
exchange with imidazol-1-yl decreased the affinity at each receptor.
Replacement with amino(methyl)thiazoles gave only decreased affinities
at hH3,4R, affording selective ligands for gp/hH2R. Surprisingly, two
compounds of the imidazol-1-ylpropylcyanoguanidines proved to be
strong agonists at hH3,4R (hH3,4R: pEC50 ≈ 10.0-11.4, pKi ≈ 9.3-9.4),
while being nearly inactive vis-à-vis the gp/hH1,2R.
References:
1. Parsons, M. E. et al.: Agents Actions 1975, 5: 464.
2. Buschauer, A.: J. Med. Chem. 1989, 32: 1963-1970.
3. Igel, P. et al.: J. Med. Chem. 2009, 52: 2623-2627.
POS.094
Multiple targeting with histamine H3 receptor antagonists:
Morbus Parkinson
Affini, A.1; Walter, M.2; Schwed, J. S.1,2; Esteban, G.3; Unzeta, M.3;
Stark, H.1
1 Institute
Universitaetsstr. 1, 40225 Düsseldorf, Germany
2 Biocenter, Institute of Pharmaceutical Chemistry, Goethe University, Max-von-Laue-Str. 9,
3 Universitat Autonoma de Barcelona, Institut de Neuroscìencies, Department de Bioquimica i
Biologia Molecular, 08193 Barcelona, Spain
Parkinson’s disease (PD) is among the most common neurodegenerative
diseases and a major health problem in the aging society. Dopaminergic
therapy including L-DOPA is highly effective during the early stages of
the treatment, but after several years of medications is complicated by
motor fluctuations and the onset of dyskinesia [1,2]. Safinamide
(Xadago®) has recently been introduced as a reversible monoamine
oxidase B (MAO-B) inhibitor (IC50= 9.0 nM) with some additional
properties for Parkinson add-on therapy [3]. In the non-dopaminergic
therapy development line, pitolisant (Wakix®) is in late stage of clinical
premarket development for the treatment of excessive daytime
sleepiness with Parkinson patients [4]. This histamine H3 receptor (H3R)
antagonist (Ki = 0.3-1.0 nM) possess procognitive and wake-enhancing
properties [5].
Since both approaches produce different effects in vivo on different
targets for the same disease, the combination of both pharmacophore
elements in one molecule may produce additional or synergistic effects
with simple interaction and pharmacokinetic profiles. This multi-targeted
or polypharmacological [6] approach may be promising on the H3R in
combination with MAO-B inhibition for novel therapeutics for Parkinson´s
disease. Here, we have combined the basic H3R pharmacophore with
the main elements of safinamide and tested this small series of
compounds for hH3R affinity as well as for MAO-A and MAO-B inhibitory
potencies. All compounds showed affinities in the nanomolar
concentration range at hH3R and moderate inhibitory potency at MAO-B
isoenzyme in the micromolar concentration range. The compounds
showed slight stereochemical discrimination with MAO-A and –B
inhibition as also observed with the parent safinamide structure. So far,
it is unclear what would be the best balance of H3R affinity and MAO-B
inhibitory potency. The number of derivatives prepared is too small to
drive any general conclusion, but as a proof-of-concept the combination
of the specific G-protein coupled receptor affinity and the enzyme
inhibitory property has simultaneously been shown for both targets. The
more we understand the molecular mechanism of multifactorial diseases,
the better we can design on this polypharmacological approach novel
compounds with designed profiles for a potentially improved medical
therapy.
the European COST Actions CM1103 and CM1207.
References:
1. Meissner, W.-G. et al.: Nat. Rev. Drug Discov. 2011, 10: 337-393.
2. Kassel, S. et al.: Eur. Neuropsychopharmacol. 2015, in press.
3. Borgohain, R. et al.: Mov. Disord. 2014, 29, 1273-1280.
4. Schwartz, J.- C.: Br. J. Pharmacol. 2011, 163: 713-721.
5. Sander, K. et al.: Biol. Pharm. Bull. 2008, 31: 2163-2181.
6. Nikolic, K. et al: J. Taiwan Inst. Chem. Eng. 2015, 46: 15-29.
POSTERS
POS.095
Studies on biased signaling of novel dopamine D2 and D3
receptor ligands
Kassel, S.1; Saur, O.2; Kottke, T.1,2; Stark, H.1
Institute of Pharmaceutical and Medicinal Chemistry, Heinrich-Heine-University,
Biocenter, Institute of Pharmaceutical Chemistry, Goethe University, Max-von-Laue-Str. 9,
1
2
The knowledge on the structural and functional properties of G-protein
coupled receptors as one of the most important targets for drugs has
tremendously increased within the last decade. Ligands acting at the
same G-protein coupled receptor can stabilize multiple and distinct
receptor conformations linked to different signaling pathways and
functional outcomes, termed as biased signaling [1]. The investigation of
biased signaling pathways for dopamine D2 and D3 receptor ligands
provides the opportunity to separate wanted therapeutic effects from
unwanted side effects in the treatment of dopamine-related diseases [2],
e.g. biased signaling behavior was described for the antipsychotic agent
aripiprazole approved for the treatment of schizophrenia [1,3].
Aripiprazole was assumed as a partial agonist at the dopamine D2
receptor explored for cAMP inhibition or β arrestin recruitment, but
appears as an antagonist of Gβγ signaling and in GTPγS binding assay
at dopamine D2 receptors. Aripiprazole´s unique signaling profile is
considered to be responsible for its antipsychotic efficacy producing only
minimal extrapyramidal symptoms, leading to its consideration as third
generation antipsychotic [4]. In our study a N-propyl etrabamine-like
derivatives were combined via an alkyl spacer of different length to an
most probably orthosteric 2,3-dichlorophenylpiperazine substructure like
the key element of aripiprazole´s pharmacophore [4]. Determinants for
affinity, selectivity and functional activities of synthesized compounds
were examined. Biased signaling behavior and dependencies on the
linker length were investigated evaluating G-protein activation and
ERK1/2/CREB phosphorylation. The compounds showed high affinity at
dopamine D2 and D3 receptors in the nanomolar concentration ranges
with slight preference for the D3 receptor. Biased signaling on D2
receptors could be obtained after introduction of an extended alkyl linker,
whereas connection via an ethyl linker showed no biased signaling. Our
studies may give new impetus in investigation of novel and high affine
D2/D3 receptor agonists [5] with unique signaling patterns, hoping to
disclose and discriminate the therapeutically most relevant pathways in
dopamine-related disorders like schizophrenia or Parkinson´s disease.
the European COST Actions CM1103 and CM1207.
References:
1. Shonberg, J. et al.: Med. Res. Rev. 2014, 4: 1286−1330.
2. Beaulieu, J.-M.; Gainetdinov, P. R.: Pharmacol. Rev. 2011, 63: 182−217.
3. Brust, T.: Biochem. Pharmacol. 2015, 93: 85-91.
4. Stark, H. et al.: International Patent PCT WO 2009 056805 A1. 2009 (07.05.2009).
5. Kassel, S. et al.: Eur. Neuropsychopharmacol. 2015, in press.
CANCER/EPIGENETICS
4.7 Cancer/Epigenetics
POS.096
Inhibition of HSP90 as a tool to increase chemosensitivity
towards cisplatin in the human ovarian cancer cell line
A2780
Rodrigues Moita, A. J.1; Hamacher, A.1; Spanier, L.1; Ciglia, E.1;
Hansen, F. K.1; Gohlke, H.1; Kurz, T.1; Kassack, M. U.1
Institute of Pharmaceutical and Medicinal Chemistry, Heinrich-Heine-University Düsseldorf,
Germany
1
Heat shock protein 90 (HSP90) is an abundantly found molecular
chaperone of 90 kDa, induced by stress conditions such as infection,
inflammation, starvation, hypoxia and cancer. Folding and maturation of
client proteins such as steroid hormone receptors, protein kinases, and
transcription factors are the main functions of HSP90. To fulfill that task,
HSP90 forms a homo-dimer and assembles with several co-chaperones
to form the so called HSP90 machinery. The C-terminus contains the
dimerization domain whereas the N-terminus harbors the classical ATP
binding domain. Given that HSP90 has over 280 identified client proteins,
many of which being present in pathways leading to cancer, HSP90
inhibitors are promising anti-cancer drugs. Geldanamycin and radicicol
were the first HSP90 inhibitors both binding to the N-terminal ATP pocket.
Both compounds show antiproliferative and antitumor effects. NVPAUY922 is another small molecule inhibitor of the N-terminal ATPbinding site of HSP90 and has entered phase I clinical trials. Novobiocin
is an HSP90 inhibitor binding to the C-terminal domain of HSP90 and
thus inhibiting the dimerization of the monomers. This is a different mode
of action leading to inhibition of dimerization and disrupted interactions of
HSP70 with HSP90 [1].
The aim of this study was to test a recently designed inhibitor (compound
A) of the C-terminal domain for its inhibitory effect on HSP90 function and
anticancer effects with a focus on reverting chemoresistance. Compound
A was able to increase chemosensitivity of the ovarian cancer cell line
A2780 towards cisplatin. This effect was also observed at the resistant
cell line A2780CisR but to a smaller extent. Furthermore, compound A
inhibited HSP90-dependent refolding of luciferase. Cytotoxicity
measured by MTT assay was similar in the sensitive A2780 (IC50 of
compound A: 5.84 µM) and its resistant subclone A2780CisR (IC50 of
compound A: 6.54 µM). In addition, effects of compound A on apoptosis
and cell cycle were investigated. To prove activity of compound A via
HSP90 inhibition, the occurrence of oligomers of HSP90 was
investigated via EGS-mediated crosslinking and Western blot analysis.
Compound A reduced formation of oligomers compared to blank and a
negative reference control.
In conclusion, compound A is a novel inhibitor of HSP90, increases
chemosensitivity towards cisplatin similar to NVP-AUY922, and may thus
constitute a new therapeutic option against chemoresistant cancers.
Recently, in the parasite Schistosma mansoni we have identified a class
I zinc-dependent HDAC subtype, HDAC8 (smHDAC8), as a standalone
target to control infection [5]. These small flat worm parasites are one of
the causative agents of a neglected human parasitic disease called
bilharzia or schistosomiasis; infect around 200 million people worldwide
and cause at least 300 000 deaths yearly, with about 800 million people
further at risk of infection [6,7]. Increased risk of resistance and reduced
efficacy of the only drug of treatment, praziquantel [8], has evoked the
search for potential novel drug candidates against schistosomes for
which vaccination is still not available.
Here, we discuss several assays employed as a screening tool to identify
novel inhibitors of smHDAC8 and to determine their selectivity against
human HDACs. These assays are non-isotopic, include the use of
fluorescence-labeled substrates enabling convenient and reproducible
measurement of enzyme activity [9]. Our in-house synthetic small
molecule substrates ZMTFAL (Z-Lys(F 3 Ac)- AMC) [10] and ZMAL (ZLys(Ac)-AMC) [11] are easy to synthesize, cheap and can be used in a
trypsin-based set-up. In addition to ZMTFAL, a commercial substrate
based on the p53 tetra-peptide {RHK(Ac)K(Ac)} has also been used to
determine inhibition of schistosomal or human HDAC8 [12]. We have
shown that in many cases IC50 values are very similar but may differ with
different substrates up to 20 fold in potency for certain inhibitors [13]. For
selectivity testing with human counterparts HDAC1 and 6, ZMAL is our
substrate of choice [11]. Using a combination of these assays we have
identified novel inhibitors of smHDAC8 with in vivo efficacy [5,14].
Currently, such assay-based approaches to screen Trypanosoma cruzi
HDAC inhibitors are also in progress in our lab.
Acknowledgments: The authors have been supported by funding from the European Union's
Seventh Framework Programme for research, technological development and demonstration
under grant agreements nos. 241865 (SEtTReND) and 602080 (A-ParaDDisE).
References:
1. Seto, E.; Yoshida, M.: Cold Spring. Harb. Perspect. Biol. 2014, 6(4): a018713.
2. de Ruijter, A. J. et al.: Biochem. J. 2003, 370 (Pt 3): 737-749.
3. Haberland, M. et al.: Nat. Rev. Genet. 2009, 10(1): 32-42.
4. Chakrabarti, A. et al.: Trends. Pharmacol. Sci. 2015, doi: 10.1016/j.tips.2015.04.013.
5. Marek, M. et al.: PLoS. Pathog. 2013, 9(9): e1003645.
6. Gray, D.J. et al.: BMJ. 2011, 342: d2651.
7. Dömling, A.; Khoury, K.: ChemMedChem. 2010, 5(9): 1420−1434.
8. Doenhoff, M.J.; Cioli, D.; Utzinger, J.: Curr. Opin. Infect. Dis. 2008, 21(6): 659−667.
9. Hauser, A. T.; Gajer Née Wagner, J. M. , Jung, M.: Methods. Mol. Biol. 2013, 981: 211-227.
10. Heltweg, B. et al.: J. Med. Chem. 2004, 47(21): 5235–5243.
11. Heltweg, B. et al.: Anal. Biochem. 2003, 319(1): 42-48.
12. http://www.enzolifesciences.com/BML-KI178/fluor-de-lys-hdac8-deacetylase-substrate/
13. Kannan, S. et al.: J. Chem. Inf. Model. 2014, 54(10): 3005-3019.
14. Stolfa, D.A. et al.: J. Mol. Biol. 2014, 426(20): 3442-3453.
References:
1. Eckl, J. M.; Richter, K.: Int. J. Biochem. Mol. Biol. 2013, 4(4):157-165.
POS.098
POS.097
In vitro screening assays for discovery of novel inhibitors
of parasitic histone deacetylases
Chakrabarti, A.1; The A-ParaDDisE consortium; Sippl, W.2; Jung, M.1
1 Institute
of Pharmaceutical Sciences, University of Freiburg, 79104 Freiburg, Germany
of Pharmaceutical Chemistry, University Halle-Wittenberg, 06120 Halle/Saale,
2 Department
Germany
Histone deacetylases (HDACs), a widely studied group of epigenetic
enzymes, cleave acetylated ε-amino groups of lysines in histones (and
other proteins) resulting in global chromatin condensation and repression
of transcription [1]. Human HDACs comprise of four different classes (I–
IV); class I, II, and IV, are zinc-dependent amidohydrolases or “classical”
HDACs, whereas class III, called sirtuins requires NAD+ [2]. Aberrant
expression of HDACs is associated with development of several
diseases such as cancer, making these enzymes interesting therapeutic
targets in humans [3] but also for parasitic diseases [4].
Reversing cisplatin resistance in ovarian carcinoma cells
by inhibition of protein disulfide isomerase 1
Kalayda, G. V.1; Kullmann, M.1; Hellwig, M.1; Kotz, S.2; Hilger, R. A.3;
Metzger, S.2,4; Jaehde, U.1
1 Institute
of Pharmacy, Clinical Pharmacy, University of Bonn, An der Immenburg 4, 53121
Bonn, Germany
2 Cologne Biocenter, University of Cologne, Zülpicher Str. 47b, 50674 Cologne, Germany
3 Department of Medical Oncology, West German Cancer Center, University Hospital Essen,
University Duisburg-Essen, Hufelandstraße 55, 45147 Essen, Germany
4 IUF-Leibniz Research Institute for Environmental Medicine, Auf’m Hennekamp 50, 40225
101.
POSTERS
POS.099
Simultaneous inhibition of EGFR and PI3K/AKT/mTOR can
overcome cisplatin resistance in a triple-negative breast
cancer cell line with activated growth factor receptors.
Gohr, K.1; Hamacher, A.1; Engelke, L. H.1; Kassack, M. U.1
1 Institut für Pharmazeutische und Medizinische Chemie, Heinrich-Heine-Universität,
Universitätsstr. 1, 40225 Düsseldorf, Germany
Intrinsic or acquired resistance against chemotherapy is a common
problem in tumor treatment often leading to therapeutic failure. Cisplatin,
a commonly used cytostatic agent, is under investigation for the
treatment of triple negative breast cancer. Previously, in the breast
cancer cell line MCF-7 activation of EGFR has been demonstrated to
play a crucial role in the development of resistance [1]. In this study, we
wanted to investigate the underlying mechanisms of cisplatin resistance
in the triple-negative breast cancer cell line HCC38 CisR which was
generated by weekly exposure to cisplatin from the sensitive subclone
HCC38 over a period of 37 weeks. The examination of both cell lines via
phospho-receptor tyrosine kinase array and western blotting showed an
increased activation of insulin-like growth factor 1 receptor (IGF-1R) and
epidermal growth factor receptor (EGFR, ErbB-1) in the cisplatin resistant
subclone. The doubling time was lower in HCC38 CisR than in HCC38
underlining the activation of growth factor receptors. The highly activated
IGF1-R in HCC38 CisR is also reflected in the sensitivity of HCC38 CisR
for IGF1-R inhibition. The IC50 of NVP-AEW-541 determined in MTT
assay after 120h of treatment was 4-fold lower in HCC38 CisR than in
HCC38.
We showed that simultaneous treatment with NVP-AEW541 and
Lapatinib heavily reduces growth in HCC38 CisR whereas this effect was
not present when inhibiting only one of the receptors. Dual inhibition
could also induce apoptosis in HCC38 CisR.
Although this treatment reduced cell viability it had no influence on
cisplatin cytotoxicity in HCC38 CisR. Therefore we combined the
receptor inhibitors with NVP-BEZ235 as an inhibitor of the downstream
acting kinases PI3K and mTOR. The combination of Lapatinib and NVPBEZ235 led to a complete resensitization in the cisplatin resistant cell
line.
We showed that the effect of dual inhibition of IGF1-R and EGFR is
superior to the use of single inhibitors in HCC38 CisR containing
activated growth factor receptors. Furthermore combining Lapatinib with
an inhibitor of downstream acting kinases can overcome cisplatin
resistance in this cell line. In conclusion our studies showed that
combining targeted therapies may be a useful tool in cancer therapy.
References:
1. Eckstein, N. et al.: J. Bio.Chem. 2008, 283(2): 739-750.
site, termed PIF-pocket, for mediating the regulation on the enzymatic
activity. Along the process of regulation, N- or C-terminal regions are
modified by different signaling and these act directly on the residues
forming the PIF-pocket which in turn affect the intrinsic activities of the
kinases, allosterically. Current state of the art in the drug development to
protein kinases involves the targeting of the ATP-binding site (type I
inhibitors), and sites directly extending from the ATP-binding site (type II
inhibitors). However, the development of drugs targeting sites which are
distant from the ATP-binding site (type III inhibitors) are still
unconventional. We previously described biochemically and structurally
how small compounds binding to the PIF-pocket could allosterically
inhibit the activity of aPKCs. Here we describe the development and
characterization of a follow-up series represented by PS432, an allosteric
inhibitor that specifically targets the PIF-pocket of aPKCs, inhibits
proliferation of lung cancer cells in culture and tumour progression of lung
cancer in tumour xenograft model.
POS.101
Target-sensitive, vascular directed liposomes with
entrapped chemokine receptor antagonists for a local
interference with tumor cell metastasis
Schlesinger, M. 1; Roblek, M. 2; Calin, M. 3; Stan, D. 3; Zeisig, R. 4;
Simionescu, M. 3; Bendas, G. 1; Borsig, L. 2
1 Department
of Pharmacy, Rheinische Friedrich-Wilhelms-University Bonn, 53121 Bonn,
Germany
2 Institute of Physiology, University of Zürich and Zürich Center for Integrative Human
Physiology, CH-8057 Zurich, Switzerland
3 Institute of Cellular Biology and Pathology ‘‘N. Simionescu’’ of the Romanian Academy,
Bucharest, Romania
4 Experimental Pharmacology & Oncology Berlin Buch GmbH, 13125 Berlin, Germany
102.
POS.100
Allosteric inhibition of atypical PKC for the treatment of
cancer
D.1;
Odadzic,
Arencibia, J.
Fröhner, W. ; Schulze,
Neimanis,
S.2; Proschak, E.4; Engel, M.3; Zeuzem, S.2; Stark, H.5; Biondi, R. M.2
M.2;
3
J. 2;
1 German
Cancer Research Center(DKFZ), Im Neuenheimer Feld 280, 69121 Heidelberg,
Germany
2 Research Group PhosphoSites, Medizinische Klinik 1, Universitätsklinikum Frankfurt, TheodorStern-Kai 7, 60590 Frankfurt, Germany.
3 Pharmaceutical and Medicinal Chemistry, University of Saarland, 66041 Saarbrücken,
Germany.
4 Institute for Pharmaceutical Chemistry, J.W. Goethe University,60438 Frankfurt, Germany.
5 Pharmaceutical and Medicinal Chemistry, Heinrich-Heine-University, 40225 Düsseldorf,
Germany
Protein kinases play key regulatory roles in cells and organisms by
responding to specific stimuli and selectively phosphorylating protein
substrates. To achieve their specific functions, protein kinases have
evolved very sophisticated mechanisms of regulation. Protein kinases
possess a conserved catalytic domain, consisting of two lobes, a small
lobe and a large lobe, with the ATP-binding site located in the cleft
between the lobes. To achieve the required exquisite selective
regulation, protein kinases frequently possess additional N- and Cterminal domains. The AGC group of protein kinases, named after its
representatives Protein kinase A, Protein kinase G and Protein kinase C,
are regulated by very different stimuli but often converge in a conserved
POS.102
The class IIa preferential HDAC inhibitor LMK235 enhances
the efficacy of cisplatin in HNSCC cell lines.
Hamacher, A.1; Schulte, M.1; Hansen, F. K.1, Kurz, T.1, Kassack, M. U.1
Institut für Pharmazeutische und Medizinische Chemie, Heinrich Heine Universität Düsseldorf,
1
Cisplatin-based chemotherapy is part of the standard treatment of head
and neck squamous cell carcinoma (HNSCC). However, many patients
develop cisplatin resistance during treatment. The mechanism of
cisplatin resistance is multifactorial and includes genetic and epigenetic
alterations such as DNA methylation and histone acetylation.
Furthermore, aberrant HDAC expression was found in many tumors to
be associated with poor response to chemotherapy. Epigenetic therapies
including pan-HDAC inhibitors (HDACi) such as vorinostat have shown
suppressive effects against various tumor cells by inhibiting cell
proliferation and inducing cell cycle arrest as well as apoptosis. Up to
now, it is unknown which HDAC subtype may be the most promising for
anticancer effects. Further, pan-HDACi show numerous side effects.
Subtype selective HDACi may thus be a promising class of epigenetic
anticancer drugs.
CANCER/EPIGENETICS
A set of 30 hydroxamate-based HDACi was previously evaluated at the
HNSCC cell lines Cal27 and Kyse510 and their cisplatin resistant
sublines. LMK235 was identified as the most potent compound
concerning HDAC inhibition and cytotoxicity [1]. In this study, LMK235
was selected for further characterization of its anticancer effects and
reversal of cisplatin chemoresistance.
LMK235 showed a concentration-dependent inhibition of cellular
proliferation in all 4 HNSCC cell lines. HDAC isoform profiling of LMK235
revealed a preference for HDAC4 and HDAC5 with IC50 values of 11.9 nM
and 4.22 nM in contrast to vorinostat. Incubation with LMK235 48 h prior
to cisplatin resulted in a 46-fold (Cal27) and 60-fold (Kyse510)
enhancement of cisplatin-induced cytotoxicity. Furthermore, complete
reversal of cisplatin resistance was observed for both cisplatin resistant
sublines Cal27CisR and Kyse510CisR. In addition, preincubation with
LMK235 increased the sensitivity of the HNSCC cell lines to cisplatinmediated apoptosis.
In summary, our study has identified a potent HDACi with a new class IIa
selectivity profile. LMK235 is a promising tool to investigate the function
of HDAC4 and HDAC5 concerning cancer development and to improve
the treatment of HNSCC.
References:
1. Marek, L. et al.: J. Med. Chem. 2013, 56(2): 427-436.
POS.103
Interference with chemokines as potential antitumor
strategy: Sulfated glycans inhibit the CXCL12-induced
activation of both CXCR4 and CXCR7.
Alban, S.1; Ehrig, K.; Liewert, I.; Schneider, T.1
1 Pharmaceutical
Germany
Institute, Christian-Albrechts-University, Gutenbergstr. 76, 24146 Kiel,
Introduction: Chemokines mediate leukocyte trafficking during
homeostasis and inflammation and are essential for linking innate and
adaptive immunity. The chemokine CXCL12 is the main factor to home
hematopoietic progenitor cells to the bone marrow but it also plays an
important role in cancer cell proliferation, invasion and metastasis.
Therefore, blocking of this signaling axis is a promising strategy for tumor
therapy [1-3]. Heparins are known to interact with CXCL12 and various
other chemokines and they also exert antimetastatic effects in vivo.
Objectives: The aim of this study was to elucidate whether algae-derived
glycans interact with CXCL12. Furthermore, the potencies of algaederived glycans and heparins concerning the inhibition of CXCL12induced activation of CXCR4 and CXCR7 should be evaluated.
Materials and Methods: Sulfated glycans of the brown algae Saccharina
latissima and Fucus vesiculosus as well as unfractionated heparin and
tinzaparin were selected as test compounds. The approved CXCR4anatgonist plerixafor (AMD3100, Mozobil™) served as reference
compound. CXCR4 and CXCR7 activation in a human Burkitt’s
lymphoma cell line was monitored by flow cytometry via receptor
internalization upon ligand binding. The binding of sulfated glycans to
CXCL12 was determined with a competitive sulfated polysaccharide
coating-ELISA. As exemplary downstream effects cell migration was
quantified and secretion of matrix metalloproteinases was investigated
by zymography.
Results: In contrast to plerixafor, algae-derived glycans bound CXCL12
and thereby blocked the CXCL12-induced activation of CXCR4 as well
as CXCR7. Furthermore, treatment with these glycans or heparins
impaired downstream effects in human Burkitt’s lymphoma cells like
migration and secretion of matrix metalloproteinase 9. Concerning the
inhibition of CXCL12-signaling algae-derived glycans proved to be more
active than heparins. Their strong inhibition of CXCL12-signaling and low
anticoagulant properties, which reduce the risk of bleeding, make them
promising candidates for tumor therapy.
Conclusion: Sulfated glycans bind CXCL12 and thereby inhibit the
activation of both CXCR4 and CXCR7 in human Burkitt’s lymphoma cells.
The inhibitory potency of algae-derived glycans on this axis is
significantly superior to that of heparins.
Acknowledgments: This work was supported by the Federal Ministry of Research and Education
(BMBF) within the national research project “Algae Against Cancer” (0315812F).
References:
1. Cojoc M. et al.: Onco. Targets Ther. 2013 6: 1347–1361.
2. Domanska, U. M. et al.: Eur. J. Cancer. 2013 49(1): 219-230.
3. Chatterjee, S.; Behnam Azad, B.; Nimmagadda, S.: Adv. Cancer Res. 2014 124: 31–82.
POS.104
Development of 3-amido-benzhydroxamic acids as small
molecule inhibitors against smHDAC8 for the treatment of
schistosomiasis
Heimburg, T.1; Melesina, J.1; Chakrabarti, I.2; Walter, A.2; Hauser, A.-T.2;
Schmidtkunz, K.2; Marek, M.3; Lancelot, J.4; Romier, C.3; Pierce, R.4;
Schmidt, M.1; Jung, M.2; Sippl, W.1
Institut für Pharmazie, Martin-Luther-Universität Halle-Wittenberg, Wolfgang-Langenbeck –
Straße 4, 06120 Halle (Saale), Germany
2 Institut für Pharmazeutische Wissenschaften, Albert-Ludwigs-Universität Freiburg, Albertstr. 25,
79104 Freiburg, Germany
3 IGBMC, Universite de Strasbourg, 1 rue Laurent Fries, 67404 Illkirch Cedex, France
4 Center for Infection and Immunity of Lille (CIIL), Université Lille Nord de France, Institut Pasteur
de Lille, 1 rue Professeur Calmette, 59019Lille Cedex, France
1
Schistosomiasis is one of the major human neglected parasitic diseases
[1], which is caused by small flat worm parasites from the genus
Schistosoma. At the moment, there are no licensed vaccines available
against Schistosomiasis and for Praziquantel, the one drug which is
effective against all schistosome species, reduced efficiency and drug
resistance are reported [2]. Currently inhibitors of human epigenetic
enzymes are investigated as novel anti-cancer drugs and have the
potential to be used as new anti-parasitic agents [3]. Here, we report that
Schistosoma mansoni histone deacetylase 8 (smHDAC8), the most
expressed class I HDAC isotype in this organism, is a functional acetylL-lysine deacetylase that plays an important role in parasite infectivity. A
combination of virtual screening and biological testing resulted in
linkerless aromatic-hydroxamic acidsas novel smHDAC8 inhibitors which
induce apoptosis and mortality in schistosomes. Crystal structures of
smHDAC8 with the linkerless inhibitors could be solved which showed
the accuracy of the applied modeling approach [4,5]. Consequently
several 3-amido-benzhydroxamic acids were synthesized and tested
against smHDAC8 and optimized in potency and selectivity.
Acknowledgments: This work and the authors received funding from the European Union's
Seventh Framework Programme for research, technological development and demonstration
under grant agreements nos. 241865 (SEtTReND) and 602080 (A-ParaDDisE).
References:
1. Hotez, P. J.; Pecoul, B.: PLoSNegl Trop. 2010, Dis 4: e718.
2. Doenhoff, M. J.; Cioli, D.; Utzinger, J.: CurrOpin Infect. 2008, Dis 21: 659-667.
3. Andrews, K. T.; Haque, A.; Jones, M. K.: Immunol Cell Biol. 2012, 90: 66-77.
4. Marek, M. et al.: PLOS Pathogens. 2013.
5. Kannan, S.; Melesina, J.; Hauser, A. et al.: J. Chem. Inf. and Mod. 2014.
POS.105
Synthesis and in vitro characterization of hydroxamic acids
as small molecule inhibitors for protozoal targets
Bayer, T.1; Melesina, J.1; Chakrabarti, A.2; Walter, A.2; Marek, M.3;
Romier, C.3; Schmidt, M.1; Jung, M.2; Sippl, W.1
1 Institut
für Pharmazie, Martin-Luther-Universität Halle-Wittenberg, Wolfgang-Langenbeck –
Straße 4, 06120 Halle (Saale), Germany
2 Institut für Pharmazeutische Wissenschaften, Albert-Ludwigs-Universität Freiburg, Albertstr.
25, 79104 Freiburg, Germany
3 IGBMC, Université de Strasbourg, 1 rue Laurent Fries, 67404 Illkirch Cedex, France
Schistosomiasis, also known as bilharzia is caused by a blood-dwelling
fluke of the genus Schistosoma. It uses a fresh water snail as an
intermediate host and is transmitted through contaminated water. Within
the human host the parasite passes through several life-cycle stages and
causes various severe symptoms [1]. Taking into account that an
estimate of 200 million people are infected worldwide among who
280,000 die annually and another 800 million are at risk of infection
schistosomiasis is one of the most important parasitic diseases [2].
Praziquantel is an anthelminthic which is effective against all human
forms of schistosomiasis. Without any effective vaccine available and the
excessive use of praziquantel for the treatment of infected individuals as
well as for preventive treatment the problem of resistant schistosome
strains is arising [1].
POSTERS
Histone deacetylases (HDACs) take an important part in epigenetics
since the state of acetylation of the histones correlates with
transcriptional control [3]. The schistosome histone deacetylase 8
(smHDAC8) was recently identified as a potential target for antiparasitic
therapy [4]. The screening hit J1075 (a 3-chloro-benzothiophen
derivative) has proven effective in terms of smHDAC8 inhibition, mortality
and unpairing of adult schistosomes (and therefore the disability of
reproduction) and consequently is an interesting lead structure for further
derivatisation and optimization [5]. Here we present different classes of
compounds derived from the starting point J1075.
nanomolar range and a comprehensive DSF screening showing
remarkable selectivity over other bromodomains.
References:
1. Filippakopoulos, P. et al.: Nat. Rev. Drug Discov. 2014, 13: 337-356.
2. Filippakopoulos, P. et al.: Nature 2010, 468: 1067-1073.
3. Rooney, T. P. C. et al.: Angew. Chem. 2014, 126: 1–6.
4. http://www.thesgc.org/chemical-probes/ICBP112
5. Hay, D. A. et al.: J. Am. Chem. Soc. 2014, 136: 9308-9319.
Acknowledgments: SEtTReND, A-ParaDDisE
POS.108
References:
1. Deribew, K. et al.: Int. J. Med. Med. Sci. 2013, 5(3): 131-139.
2. Steinmann, P. et al.: Lancet Infect. Dis. 2006, 6(7): 411-425.
3. KrennHrubec, K. et al.: Bioorg Med Chem Lett. 2007, 17(10): 2874 – 2878.
4. Kannan, S. et al.: J. Chem. Inf. Model. 2014, 54(10): 3005–3019.
5. Marek, M. et al.: PLoS pathogens, 2013, 9(9): e1003645.
α-Aminoxy oligopeptides: Solid-phase synthesis,
conformational investigation and anticancer activity
Diedrich, D.1; Rodrigues Moita, A. J.1; Rüther, A.2; Kurz, T.1; Lüdeke, S.2;
Kassack, M. U.1; Hansen, F. K.1
1 Institute
POS.106
Rational design and diversity-oriented synthesis of
peptoid-based selective HDAC6 inhibitors with potent
anticancer activity
Diedrich, D.1; Syntschewsk, V.1; Hamacher, A.1; Alves Avelar, L. A.1;
Gertzen, C. G .W.1; Reiss, G. J.2; Kurz, T.1; Gohlke, H.1; Kassack, M.
U.1; Hansen, F. K.1
2 Institut für Anorganische Chemie und Strukturchemie, Heinrich Heine Universität Düsseldorf,
Universitätsstr.1, 40225 Düsseldorf, Germany
1
103.
POS.107
Design of new, selective benzoxazepine-type inhibitors for
the CBP/EP300 bromodomains
Popp, A. T.1; Fedorov, O.2; Tallant, C.2; Knapp, S.3; Bracher, F.1
Department Pharmazie, Ludwig-Maximilians-Universität, Butenandtstraße 5-13, 81377
Munich, Germany
Structural Genomics Consortium, University of Oxford, Old Road Campus Research Building,
Roosevelt Drive, Headington, Oxford, OX3 7DQ, United Kingdom
3 Institut für Pharmazeutische Chemie, Goethe Universität, Max-von-Laue-Str. 9, 60438
1
2
Reversible acetylation of histones and other proteins is involved in the
transcriptional regulation and DNA repair. Bromodomains, the “readers”
of N-ε-acetylated lysine, are an interesting target for research and
pharmaceutical industry [1,2]. Most of the work was focused on
bromodomains of BET sub-family such as BRD4, but recently inhibitors
of other proteins also emerged. Several inhibitors are now available for
phylogenetically closely related BRDs CBP and EP300 [3-5]. Their
utilization as research tools may further accelerate and yield better
understanding and potential avenues of intervention for diseases such
as Rubinstein-Taybi syndrome, leukemia, ovarian, breast and lung
cancers, and systemic lupus erythematosus [1,4]. Here we report novel
CBP/EP300 inhibitors based on I-CBP112 design. We synthesized and
characterized in biochemical assays approximately 50 compounds with
scaffold A. This effort gave further insight into the SAR of benzoxazepine
type inhibitors and resulted in a CBP/EP300 inhibitor with an IC50 in the
of Pharmaceutical and Medicinal Chemistry, Heinrich-Heine-Universität Düsseldorf,
40225 Düsseldorf, Germany
2 Institute of Pharmaceutical Sciences, Albert-Ludwigs-Universität Freiburg, 79104 Freiburg,
Germany
Cancer is a leading cause of death in economically developed countries.
The global burden of cancer continues to increase significantly in
particular due to the aging and growth of the world population.
Chemotherapy using cytotoxic agents is one major approach to combat
cancer. Unfortunately, cancer cells, which are initially suppressed by
established antitumor drugs, may develop a resistance to standard drug
therapy. Thus, there is a need for new anticancer agents preferably
acting with unique modes of action. Over the years, anticancer peptides
(ACPs) have been identified as promising class of cytotoxic agents [1].
However, the high production cost and the intrinsic instability of peptides
towards proteases have resulted in extensive research in
peptidomimetics and the field of foldamers has received massive interest
[2].
Despite their very attractive properties such as increased proteolytic and
conformational stability [3,4], α-aminoxy peptides have not received as
much attention as other foldamers such as β-peptides or peptoids. One
important reason for this is the difficult synthetic access to longer
oligomers. We herein present an improved synthetic route to α-aminoxy
oligopeptides by the straightforward use of a combination of solutionphase and solid-phase supported methods. Although the field of ACPs is
considered as a rapidly emerging research area, no anticancer properties
have been reported for α-aminoxy peptides thus far. In this context, we
report here the remarkable cytotoxic activity of α-aminoxy oligopeptides
against cancer cells. CD spectroscopy was employed to get a more
profound understanding of the folding properties of α-aminoxy
oligopeptides and to investigate possible bioactive conformations.
Preliminary mode of action studies revealed that membranolytic and
proapoptotic effects may contribute to the anticancer activity of αaminoxy oligopeptides.
Acknowledgments: This work was supported by funds from the Strategischer Forschungsfonds
of the Heinrich-Heine-Universität Düsseldorf and Fonds der Chemischen Industrie.
References:
1. Gaspar, D.; Veiga, A. S.; Castanho M. A. R. B.: Front. Microbiol. 2013, 4: 294.
2. Martinek, T. A.; Fülöp, F.: Chem. Soc. Rev. 2012, 41(2): 687–702.
3. Li, X.; Wu, Y-D.; Yang, D.: Acc. Chem. Res. 2008, 41(10): 1428–1438.
4. Draghici, B. et al.: RSC Adv. 2011, 1(4): 602–606.
CANCER/EPIGENETICS
POS.109
6‐Arylamino‐3,4‐dihydroisoquinolin‐1(2H)‐ones as new
pharmacophores for linear hinge binders inducing the
glycine-flip
A convenient synthetic methodology to access the scaffold was
developed employing an intramolecular Heck reaction as the key step for
building up the tricyclic core. A small library of inhibitors was prepared
and characterized using in vitro biochemical as well as cellular assays.
Potencies down to picomolar range could be achieved along with
enhanced selectivity for JAK3 compared to Tofacitinib [4].
Praefke, B. A.1; Laufer, S. A.1
1 Faculty
of Science, Pharmaceutical and Medicinal Chemistry, University of Tuebingen, Auf der
Disregulation in kinase activity can be related to various diseases such
as cancer, diabetes and inflammatory disorders [1]. We developed
dibenzosuberones, dibenzoxepinones and benzosuberones as highly
potent and selective p38α MAPK inhibitors with a carbonyl based hinge
binding motif. When binding the p38α MAP kinase these scaffolds induce
a “glycine-flip”, a rotation of Gly110 in the hinge region, resulting in the
formation of two hydrogen bonds from the carbonyl oxygen to the amideNH of Gly110 and Met109 [2,3].
Only 46 of the known 518 protein kinases possess a glycine in the
corresponding position, making the induction of a glycine-flip a promising
way to increase selectivity [4]. Based on our previous scaffolds we
developed a new pharmacophore, replacing the ketone by an amide
group. We expected the increased electron density of the amide oxygen
to strengthen the bidentate hydrogen bonds to the flipped glycine and the
adjacent amino acid. The lipophilicity and electron density of the
arylamino moiety was varied by different substituents (R2) to explore the
hydrophobic region I of kinases bearing a glycine in the hinge region.
References:
1. Cornejo, M. G.; Boggon, T. J.; Mercher, T.: Int. J. Biochem. Cell B 2009, 41(12): 2376-2379.
2. O'Shea, J. J.: Annals of the rheumatic diseases 2004, 63 Suppl 2: ii67-ii71.
3. Gehringer, M. et al.: ChemMedChem 2014, 9: 2516-2527.
4. Gehringer, M. et al.: ChemMedChem 2014, 9(2): 277–281.
POS.111
New insights into the pro-apoptotic activity of P8-D6, a
potent dual topoisomerase inhibitor for antineoplastic
therapy
Meier, C.1; Koburg, M.2; Bischoff, F.2; Vollmar, A. M.2; Clement, B.1
Department of Pharmaceutical and Medicinal Chemistry, Institute of Pharmacy, CAU Kiel,
Gutenbergstraße 76, 24118 Kiel – Germany
2 Department of Pharmacy – Pharmaceutical Biology, LMU Munich, Butenandtstraße 5-13,
81377 Munich – Germany
1
Topoisomerase inhibitors constantly remain an indispensable instrument
in antitumor therapy due to their effectiveness and a considerable clinical
experience regarding therapy safety and combined administration with
other cytostatics [1,2,3]. Nevertheless, resistance mechanisms like the
upregulation of the expression of one topo-isomerase form by selective
inhibition of the other as well as a membrane-triggered decrease of
intracellular concentrations of approved topo agents are frequently
described [4,5,6]. Consequently, the design of dual topoisomerase I/II
inhibitors that exhibit a high cytotoxicity and a strong affinity to both
enzyme classes remains a highly desirable goal in antitumor drug
development to date [1,7].
We recently developed P8-D6, a small molecule with strong equipotent
topoisomerase I /IIα/IIβ inhibitory activity and an excellent broad
spectrum cytotoxicity in the nanomolar range. Besides its
physicochemical advantages, e.g. a solubility in the millimolar region, a
high tolerability towards P8-D6 was shown in first maximum tolerated
dose studies (athymic nude mice) encouraging its further development in
in vivo efficacy models.
References:
1. Manning, G. et al.: Science. 2002, 298(5600): 1912-1934.
2. Koeberle, S. C. et al.: J. Med. Chem. 2012, 55(12): 5868-5877.
3. Baur, B. et al.: J. Med. Chem. 2013, 56(21): 8561-8578.
4. Martz, K. E. et al.: J. Med. Chem. 2012, 55(17): 7862-7874.
POS.110
A Comprehensive Structure-Activity Relationship Study on
Tofacitinib Bioisosteres to improve the Selectivity of JAK3
Inhibitors
Pfaffenrot, E.1; Gehringer, M.1; Bauer, S. M.1; Laufer, S. A.1
Department of Pharmaceutical and Medicinal Chemistry, Pharmaceutical Institute, University
of Tuebingen, Auf der Morgenstelle 8, 72076 Tuebingen, Germany
1
JAK3 is a cytosolic non-receptor tyrosine kinase belonging to the Janus
kinase (JAK) family. The four family members JAK1, JAK2, JAK3 and
TYK2 (Tyrosine kinase 2) are involved in various signaling cascades
initiated by cytokines and growth factors mediating immune response
and proliferation. JAK3 plays a key role in the development of
lymphocytes such as T cells, B cells and NK cells and represents an
attractive therapeutic target for the treatment of inflammatory diseases.
Selective inhibition of JAK3 is suggested to provide immunosuppression
with less side effects due to the exclusive expression of JAK3 in
hematopoietic tissue [1,2].
In an extensive structure-activity relationship study we synthesized a
library of Tofacitinib bioisosteres and tested their ability to inhibit JAK3
[3]. A novel class of tricyclic Janus kinase (JAK) inhibitors was designed
by applying a rigidization approach to the FDA-approved drug Tofacitinib.
Figure: P8-D6 as promising dual topoisomerase inhibitor.
Herein presented studies are dealing with the anti-leukemic activity of P8D6 in Jurkat ALL cells, focussing on pro-apoptotic impact. Furthermore,
we investigated the growth-inhibitory effects of P8-D6 in combination with
the protein disulfide isomerase (PDI) inhibitor PS89. It was shown that
this substance could sensitize various cancer cells towards Etoposide
treatment [8]. The results were also compared with effects on healthy
human lymphocytes to give first information about the selectivity towards
tumor cells.
References:
1. van Gijn, R. et al.: J. Oncol. Pharm. Pract. 2000, 6: 92–108.
2. Pommier, Y.: Nat. Rev. Cancer 2006, 6: 789–802.
3. Deweese, J. E.; Osheroff, N.: Nucl. Ac. Res. 2009, 37: 738–748.
4. Felix, C. A.; Kolaris, C. P.; Osheroff, N.: DNA repair 2006, 5: 1093–1108.
5. Lewis, L. J. et al.: Anti-cancer drugs 2007, 18: 139–148.
6. Whitacre, C. M. et al.: Cancer Res. 1997, 57: 1425–1428.
7. Salerno, S. et al.: Curr. Med. Chem. 2010, 17: 4270–4290.
8. Eirich, J.; Braig, S. et al.: Angewandte Chemie Int. Ed. 2014, 53: 12960-12965.
POSTERS
POS.112
Tetra-substituted pyridinylimidazoles as dual inhibitors of
p38α mitogen-activated protein kinase and c-Jun Nterminal kinase 3 for potential treatment of
neurodegenerative diseases
Muth, F.1 ; Günther, M.1; Bauer, S. M.1; Döring, E.1; Fischer, S.1; Maier,
J.2; Drückes, P.3 ; Köppler, J.3; Trappe, J.3; Rothbauer, U.2; Koch, P.1;
Laufer, S. A.1
1 Department of Pharmaceutical and Medicinal Chemistry, Institute of Pharmaceutical Sciences,
Eberhard-Karls-University Tuebingen, Auf der Morgenstelle 8, 72076 Tuebingen, Germany.
2 Natural and Medical Sciences Institute at the University of Tuebingen, Markwiesenstrasse 55,
72770 Reutlingen, Germany.
3 Novartis Pharma AG, CH-4002 Basel, Switzerland.
Multi-target drugs inhibiting several key drivers involved in
neurodegeneration may exhibit additive or even synergistic effects in
vivo. For the treatment of complex diseases like neurodegeneration,
therapies that act at multiple targets and providing symptomatic as well
as neuroprotective effects may thus be more effective. Recently, Taylor
et al. reported enhanced levels of MKP-1 are neuroprotective via
dephosphorylation and therefore deactivation of JNK3 and p38α MAP
kinase [1].
Tetra-substituted imidazoles were designed as dual inhibitors of c-Jun Nterminal kinase (JNK) 3 and p38α mitogen-activated protein (MAP)
kinase. A library of 45 derivatives was prepared and evaluated in a kinase
activity assay for their ability to inhibit both kinases, JNK3 and p38α MAP
kinase. Dual inhibitors with IC50 values down to the low double-digit
nanomolar range at both enzymes were identified. Two inhibitors of
series 6 (6l and 6o) show an excellent selectivity profile. The best
balanced dual JNK3/p38α MAP kinase inhibitors are 6m (IC50 JNK3: 18
nM, p38α: 30 nM) and 14d (IC50 JNK3: 26 nM, p38α: 34 nM) featuring
both, excellent solubility and metabolic stability. They may serve as
useful tool compounds for preclinical proof-of-principle studies in order to
validate the synergistic role of both kinases in the progression of
Huntington’s disease.
Acknowledgments: We thank Peter Keck for the help with the processing of the raw data. We
are grateful to Matthias Gehringer for contributing Figure 3. Daniela Müller and Katharina Bauer
are also acknowledged for skillful technical assistance in compound testing.
References:
1. D. M. Taylor, et al.: J. Neurosci. 2013, 33: 2313-2325.
POS.113
Multicomponent synthesis and anticancer activity of a
novel class of peptoid-based HDAC inhibitors
Syntschewsk, V.1; Hamacher, A.1; Kurz, T.1; Kassack, M. U.1; Hansen,
F. K.1
1
Proteins that are able to regulate epigenetic processes became
interesting targets for the development of drugs. In this context, histone
deacetylases (HDACs) play an important role, due to their ability to
remove acetyl groups from N-acetyl-lysine residues of histones and nonhistone proteins. These posttranslational modifications of chromatin,
caused by HDACs, have an influence on the gene expression [1-4]. Thus
far, 18 HDAC isoforms have been identified and divided into different
classes based on their homology to their corresponding yeast
orthologues. HDAC classes I (HDACs 1-3,8), IIa (HDACs 4,5,7,9), IIb
(HDACs 6,10) and IV (HDAC 11) contain zinc-dependent deacetylase
domains, whereas class III are NAD+ dependent [2]. There are several
pathways which are addressed by HDAC inhibitors (HDACi) to inhibit
tumor cell growth such as apoptosis, differentiation and cell cycle arrest
[2]. HDACi therefore emerged as interesting targets for the development
of anticancer drugs. Currently, four HDACi have been approved by the
FDA for the treatment of cutaneous/peripheral T-cell lymphoma or
multiple myeloma (vorinostat, romidepsin, belinostat and panobinostat).
However, there is still an urgent need for the development of novel
HDACi with finetuned pharmacokinetic and -dynamic properties.
In this poster, we report on the multicomponent synthesis and biological
evaluation of a new class of potent HDACi bearing peptoid-based cap
groups. An Ugi four-component reaction (U-4CR) was utilized for the
straightforward and systematic variation of the cap moiety. A series of
post-Ugi transformations allowed the introduction of different zincbindung groups (ZBGs). All novel peptoid-based HDACi were screened
for their biological activity in cellular HDAC and MTT assays using
different sensitive and chemoresistant cancer cell lines. Notably, the
most potent HDACi exceeded the activity of the reference compounds
vorinostat and cisplatin, respectively. Selected HDACi were further tested
for their enhancement of cisplatin-induced cytotoxicity. Based on these
preliminary data, this series of compounds represents a valuable starting
point for further elaboration and the results reported here will help to
guide future efforts toward developing novel HDACi with optimized
anticancer properties.
References:
1. Cai, J. et al.: Bioorg. Med. Chem. 2015, 23(13): 3457-3471.
2. Witt, O. et al.: Cancer Lett. 2009, 277(1): 8–21.
3. Minucci, S.; Pelicci, P. G.: Nat. Rev. Cancer 2006, 6(1): 38-51.
4. Bertrand, P.: Eur. J. Med. Chem. 2010, 45(6): 2095-2116.
POS.114
Pt(II) complexes – potential inhibitors of seleniumdependent redox enzymes
Lemmerhirt, H.1; Behnisch, S.1; Lillig, C. H.2; Bednarski, P. J.1
1 Ernst-Moritz-Arndt-University of Greifswald, Institute of Pharmacy, Friedrich-Ludwig-JahnStraße 17, DE 17489 Greifswald, Germany
2 Ernst-Moritz-Arndt-University of Greifswald, University Medicine, Institute for Medical
Biochemistry and Molecular Biology, DZ7, Ferdinand-Sauerbruchstraße, DE 17475 Greifswald,
Germany
Platinum complexes (e.g. cisplatin, carboplatin, oxaliplatin) are
established drugs in cancer therapy. They induce DNA damage, cell
cycle arrest and apoptosis in proliferating cancer cells. Since the
discovery of cisplatin, a wide range of platinum complexes has been
developed with novel mechanisms of action.
Hydrogen peroxide is known as a secondary messenger for apoptosis.
Glutathione peroxidase (GPx), an enzyme that detoxifies H2O2, is
believed to be involved in the development of resistance of cancer cells
treated with anti-cancer drugs [1,2]. Due to the overexpression of this
enzyme, it is assumed that it acts to increase the degradation of H2O2.
Another selenocysteine-dependent redox enzyme is thioredoxin
reductase (TrxR), which is targeted by cisplatin and is also a promising
target enzyme for anticancer therapy.
We have synthesized a library of Pt(II) complexes derived from cisplatin
and the inactive isomer transplatin, coordinated to hydrazone ligands
known to weakly inhibit GPx (Figure) [1]. It was envisioned that the
compounds would interact irreversibly by way of their Pt(II) atom with the
active site selenium atom of the enzymes while the organic ligand would
increase both selectivity and binding affinity to the enzyme active site.
GPx and TrxR inhibitors might find use in cancer treatment to re-sensitize
resistant cancer cells to anti-cancer therapy. Furthermore, selectively
inhibiting GPx or TrxR could be useful to investigate and to better
understand their distinct roles in cell physiology.
B
A
OH
O
N
H3 C
N
N
H
N
NH3
Cl
N+
Pt
NH3
H3C
OH
O
N
N
H
N
Figure A) hydrazone inhibitor of GPx; B) corresponding trans-Pt(II) complex
Here, we present the results of experiments designed to measure the
inhibition of GPx and TrxR by these cis- and trans-Pt(II) complexes, their
effects on cellular levels of reactive oxygen species (ROS) and their
ability to initiate apoptosis. Cell cytotoxicity and interactions of the novel
platinum compounds with DNA will also be presented.
References:
1. Schulz, R. et al.: Bioorg. Med. Chem. Lett. 2012, 22(21): 6712–6715.
2. Wilde, F. et al.: Mo.l Divers. 2014, 18(2): 307–322.
CANCER/EPIGENETICS
POS.115
Gold(I) Complexes with N-Heterocyclic Carbene,
Phosphane and Alkyne Ligands show Promising
Anticancer Properties – An Overview
Prochnicka, A.1,2; Andermark, V.1,2; Kankowski, S.1; Meyer, A.1;
Rubbiani, R.1; Göke, K.2,3; Bunjes, H.2,3; Ott, I.1,2
1 Institut
für Medizinische und Pharmazeutische Chemie, Technische Universität Braunschweig,
Beethovenstraße 55, D-38106, Braunschweig, Germany,
2 Zentrum für Pharmaverfahrenstechnik (PVZ), Franz-Liszt-Straße 35A,Technische Universität
Braunschweig, D-38106 Braunschweig, Germany
3 Institut für Pharmazeutische Technologie, Technische Universität Braunschweig,
Mendelssohnstraße 1, D-38106 Braunschweig, Germany
Organometallic gold(I) complexes are an interesting group of drugs,
which show activity against tumor relevant enzymes and have good
antiproliferative activities [1,2]. They are part of the attempt to develop
new anticancer agents, which show fewer side effects, good tumor cell
selectivity and no drug resistance. Especially N-heterocyclic carbenes
(NHC), phosphanes and alkynes as ligands of gold(I) offer a huge
potential as anticancer agents [3,4].
This work gives an overview of our different projects that engage with
these complexes (see Figure 1) with a closer look at complexes with
alkynyl ligands.
We measured good antiproliferative effects in vitro for all combinations of
gold(I) with NHCs, phosphanes and alkynes. Cellular uptake studies
indicated that such gold(I) complexes can accumulate in cancer cells.
Many of these gold(I) complexes also showed selective activity against
thioredoxin reductase (TrxR), which is one of the overexpressed
enzymes in cancer cells. A decrease in respiration suggested
mitochondria as further possible targets for NHC-gold(I)-phosphanes.
The group of alkynyl-gold(I)-phosphanes triggered anti-angiogenic
effects, as shown in former work with zebrafish embryos [5]. Other targets
like the G-quadruplex DNA, which is involved in oncogenetic regulation,
and the DNA repairing enzyme poly(ADP-ribose)polymerase (PARP-1)
are under discussion for gold(I) complexes (see Figure 2) [6,7].
Taking together previous and new data, this work makes a good step
towards an improved overview of possible targets, biodistribution and
structure-activity-relationships for the different gold(I) complexes.
New fluorinated NHC gold(I) complexes (Figure) of the phenylimidazole
type were synthesized, purified and characterized via 1H-/13C-/19F-NMR,
MS-ESI/MS-EI and elemental analysis. They were screened against
various tumorigenic cell lines as well as a non-tumorigenic human kidney
cell line. Their antiproliferative potential varies depending on the lipophilic
moieties.
Thioredoxin reductase (TrxR) is a well-known target of gold complexes
due to their high affinity to selenocysteine in the flexible N-terminus of the
enzyme, which is easily accessible. TrxR is upregulated in several
carcinoma cells, is involved in cell protection processes and prevents
cells from oxidative stress and apoptosis [2,3]. Tests with the isolated
enzyme confirm the inhibition by the NHC gold(I) complexes. Beside the
enzyme inhibition, the suppression of mitochondrial respiratory activity is
also discussed as a possible mode of action [4].
These results were investigated in more detail through cellular uptake
studies in MCF-7 breast carcinoma cells using high resolution continuum
source atomic absorption spectroscopy (HR CS-AAS) technique for the
quantification of the intracellular amount of the metal. Higher
accumulations in the cells are accompanied by lower IC50 values in the
proliferation inhibition assay.
The affinity to transport proteins, such as albumin, transferrin or fetuin
can have a huge influence on the biodistribution of the complexes [5].
Protein binding studies with isolated serum albumin as well as fetal calf
serum (FCS) show an interesting correlation between the accumulation
rate of intracellular gold and the protein binding behaviour.
Further studies include in-vitro permeability tests with a Caco-2 cell layer
model to demonstrate the tracking of apical and basal transport through
intestinal epithelial cells to investigate possible routes of application.
References:
1. Oehninger, L.; Rubbiani, R.; Ott, I.: Dalton Trans. 2013, 42: 3269–3284.
2. Fritz-Wolf, K. et al.: Nat. Commun. 2011, 2(383): 1–8.
3. Pratesi, A. et al.: J. Inorg. Biochem. 2014, 136: 161–169.
4. Holenya, P. et al.: Metallomics 2014, 6: 1591–1601.
5. Zheng, X. et al.: Biotechnol. Prog. 2006, 22(5): 1294–1300.
Acknowledgments: Financial support by Deutsche Forschungsgemeinschaft (DFG) and the
state of Lower Saxony (graduate program SynFoBiA) is gratefully acknowledged.
References:
1. Ott, I.: Coord. Chem. Rev. 2009, 253: 1670-1681.
2. Bertrand, B.; Casini, A.: Dalton Trans. 2014, 43: 4209-4219.
3. Oehninger, L.; Rubbiani, R.; Ott, I.: Dalton Trans. 2013, 42: 3269-3284.
4. Rubbiani, R. et al.: Med. Chem. Commun. 2013, 4: 942-948.
5. Meyer, A. et al.: Angew. Chem. 2012, 51: 8895-8899.
6. Bertrand, B. et al.: Inorg. Chem. 2014, 53: 2296-2303.
7. Rubbiani, R. et al.: ChemMedChem 2014, 9: 1205-1210.
POS.117
Studies on the cytotoxicity of a novel family of synthetic
pentathiepins
Behnisch, S.1; Zubair, M.2; Lemmerhirt, H.1; Schulzke, C.3; Bednarski,
P. J.1
1 Institute
of Pharmacy, University of Greifswald, F.-L.-Jahn-Straße 17, 17489 Greifswald,
Germany
2 School of Chemistry, Trinity College Dublin, College Green, Dublin 2, Ireland
3 Institute of Biochemistry, University of Greifswald, F.-Hausdorff-Straße 4, 17489 Greifswald,
Germany
POS.116
Fluorinated gold(i) n-heterocyclic carbene complexes with
anticancer properties – investigation of cytotoxicity,
enzyme inhibition, biodistribution and intestine
permeability
Schmidt, C.1; Reichl, S.2,3; Ott, I.1,3
1 Institut
Beethovenstraße 55, 38106 Braunschweig, Germany
2 Institut für Pharmazeutische Technologie, Technische Universität Braunschweig,
Mendelssohnstraße 1, 38106 Braunschweig, Germany
3 Zentrum für Pharmaverfahrenstechnik, Technische Universität Braunschweig, Franz-LisztStraße 35 A, 38106 Braunschweig, Germany
Auranofin represents a well investigated lead structure for gold Nheterocyclic carbene (NHC) complexes. It is established in the current
antirheumatic therapy, shows antiproliferative effects and a remarkable
inhibition of enzymes associated with the thioredoxin system, which has
the ability to detoxify cell damaging reactive oxygen species (ROS) [1].
To assess the potential anticancer activities of a novel family of synthetic
tetra-cyclic pentathiepins [1], we studied the ability of the compounds to
induce apoptosis and initiate the formation of reactive oxygen species
(ROS) in human cancer cells in culture. Natural pentathiepin derivates
such as varacin (Figure, left) are known for their anticancer, antifungal
and antibacterial activity. Varacin is also known to cause DNA cleavage
in supercoiled plasmid DNA in vitro through a mechanism of hydroxyl
radical formation in presence of thiols [2,3].
We have recently found that synthetic tetracyclic pentathiepins such as
ZZ-QME2-Pn (Figure, right) have strong cytotoxic activities on a broad
range of human cancer cell lines in vitro, with IC50-values ranging
between 2.0-7.5 µM. Cancer cells treated with ZZ-QME2-Pn already
show massive changes in morphology after an incubation time of only 6
h. These cells undergo apoptosis, as measured by flow of cytometry by
the Annexin-V / propidium iodide method and cleavage of PARP, as
measured by western blotting. Following a 30 min incubation of cells with
ZZ-QME2-Pn, we observed by flow cytometry a stark increase in
intracellular ROS levels, which could be dependent on cellular
POSTERS
glutathione (GSH) [4]. After preincubation of ZZ-QME2-Pn with GSH
before application to cells, we found a significant decrease in ROS
formation compared to cells without GSH preincubation. Thus, GSH
would appear to protect cells from ROS formation. We also observed by
flow cytometry an increase in cellular lipid peroxidation, which may be a
direct result of ROS formation. These findings suggest that tetracyclic
pentathiepins influence strongly the redox homeostasis of cells. Evidence
has also been collected indicating that ZZ-QME2-Pn moderately inhibits
isolated glutathione peroxidase (GPX1), but not the thioredoxin
reductase (TxR). Further studies will be aimed at understanding the
importance of GPX inhibition in cells.
Figure: Varacin (left); ZZ-QME2-Pn (right)
References:
1. Zubair, M. et al.: Chem. Commun. (Camb) 2013, 49: 4343-5.
2. Chatterji, T. et al.: Bioorg. Med. Chem. Lett. 1998, 8: 535-8.
3. Lee, A. H. et al.: Chem. Commun. (Camb) 2002, 2112-3.
4. Chatterji, T. et al.: Bioorg. Med. Chem. Lett. 2003, 13: 1349-52.
POS.118
Inhibition of endothelial Cdk5 reduces tumor growth by
promoting non-productive angiogenesis
Merk, H.1; Zhang, S.1; Lehr T.2; Bibb, J. A.3; Adams R. H.4,5; Zahler, S.1;
Vollmar A. M.1; Liebl, J.1
Department of Pharmacy, Pharmaceutical Biology, Ludwig-Maximilians-University, 81377
Munich, Germany
Clinical Pharmacy, Saarland University, 66123 Saarbrücken, Germany
3 Department of Psychiatry and Neurology and Neurotherapeutics, The University of Texas
Southwestern Medical Center, Dallas, Texas 75390-9070, USA
4 Department of Tissue Morphogenesis, Max Planck Institute for Molecular Biomedicine, 48149
Münster, Germany
5 Faculty of Medicine, University of Münster, 48149 Münster, Germany
1
2
104.
DRUG DESIGN/MEDICINAL CHEMISTRY
4.8 Drug design/Medicinal
chemistry
POS.119
Does FXR antagonism account for liver toxicity of nonsteroidal anti-rheumatic drugs (NSAIDs)?
Schmidt, J.1; Klingler, F.1; Proschak, E.1; Steinhilber, D.1; SchubertZsilavecz, M.1; Merk, D.1
1 Institute
of Pharmaceutical Chemistry, Goethe University Frankfurt, Max-von-Laue-Str. 9,
60438 Frankfurt a. M., Germany;
The nuclear farnesoid X receptor (FXR) is a ligand-activated transcription
factor, which acts as cellular sensor for bile acids [1-3]. It takes part in
the self-regulation of bile acids with the result that bile acid synthesis is
blocked and their metabolism is enhanced when high levels of toxic bile
acids occur. Hence, FXR is a highly important liver protector [4] but is
also involved in metabolic systems such as glucose or lipid homeostasis
and seems to have anti-inflammatory effects as well. The most potent
bile acid on FXR is chenodeoxycholic acid (CDCA) [3] whereas a number
of synthetic FXR agonists and antagonists were discovered in the past
years [5]. The 6α-ethyl derivate of CDCA, obeticholic acid (OCA), shows
a 100-fold greater agonistic activity on FXR than its natural prototype. A
number of studies showed the capacity of OCA to increase insulin
sensitivity and to regulate glucose homeostasis. OCA also modulates
lipid metabolism, and exerts anti-inflammatory as well as antifibrotic
effects in the FXR-expressing organs, liver, kidney and intestine [6]. The
most widely used non-steroidal FXR agonist is the synthetic isoxazole
GW4064 which is not suitable as drug for its toxicity and poor
bioavailability, however.
While FXR agonism is interesting for potential therapeutic applications in
the treatment of several liver disorders and metabolic diseases, little is
known about the pharmacological effects of FXR antagonism. So far,
antagonism on FXR does not seem to have broad therapeutic value. FXR
seems to be overexpressed in some cancer cells, especially in pancreatic
and colon cancer, Barett’s Esophagus and adenoma. These
observations hold a limited therapeutic potential for FXR antagonism and
on the other hand it might be a treatment strategy for cholestasis.
However, in intestinal and hepatic cancer reduced FXR activity is
connected with tumor growth and FXR antagonism might furthermore
disturb adipogenesis [7].
A recent report by Lu et al. [8] described non-steroidal anti-rheumatic
drugs (NSAIDs) as FXR antagonists and correlated the liver damage
caused by chronic intake of these drugs with antagonistic activity on FXR.
We were interested in this statement because of the possibility to design
safer NSAIDs by generating cyclooxygenase inhibitors which spare FXR.
On the other hand FXR antagonists are not common but they are
required to evaluate the physiological and pathophysiological roles of
FXR. The small and drug-like NSAIDs might therefore serve as lead
structures for the development of potent FXR antagonists. However, we
were confounded by the low maximum FXR activation in the test systems
Lu et al. reported and the fact that NSAIDs with such different structures
as diclofenac and indomethacin create a comparable activity on FXR.
We therefore planned to further characterize the reported antagonistic
activity and to scrutinize diclofenac, ibuprofen and indomethacin due to
their high structural variety. We trialed the three NSAIDs in several test
systems including a full-length reporter gene assay, a hybrid reporter
gene assay, quantitative real-time PCR and thermal shift experiments [9].
Our results contradict the reported antagonistic effects of the NSAIDs on
FXR as we found no evidence for FXR activity of NSAIDs alone or in
competition with the well-known FXR agonists GW4064, OCA or CDCA.
In contrast, we discovered that the activity reported by Lu et al. is due to
misinterpreted toxicity. We therefore conclude that reduced FXR
signaling in presence of NSAIDs is merely a consequence than a cause
of hepatotoxicity
POS.120
Identification of aza-stilbenes as inhibitors of the cellular
checkpoint kinase Myt1
Platzer, C.1; Decker, C.1; Gershkovich, M.2; Erdmann, F.1; Sippl, W.1;
Schmidt, M.1
1 Institute
of Pharmacy, Department of Medicinal Chemistry, Martin-Luther-University-HalleWittenberg, Wolfgang-Langenbeck-Str. 4, 06120 Halle (Saale), Germany
2 Institute of Biochemistry and Biotechnology, Department of Enzymology, Martin-LutherUniversity-Halle-Wittenberg, Kurt-Mothes-Str. 3, 06120 Halle (Saale), Germany
Human Myt1 kinase is a negative regulator of Cdk1/Cyclin B complex
and maintains the G2/M transition in the cell cycle especially in cancer
cells. Many cancer cell lines only depend on G2 checkpoint because of
G1 defect caused by p53 mutation [1]. Therefore, the inhibition of G2
checkpoint represents a promising concept for preferential damaging of
cancer cells without affecting normal cells [2]. Investigations of Chow and
Poon revealed that Myt1 seems to be more essential for cancer cells than
for healthy cells [3], making Myt1 an interesting target for anticancer
therapy. At the moment only few compounds are known to inhibit Myt1.
In the current work, we established several in vitro assays and tested a
library of 800 known kinase inhibitors available from GlaxoSmithKline
(Kinase Inhibitor Set I and II). In our screen we identified several hits
including aza-stilbenes as new class of Myt1 inhibitors. We investigated
the binding affinity and inhibition of the compounds at Myt1 full length and
kinases domain75-362 with a fluorescence anisotropy-based binding assay
and a short-peptide based activity assay. The obtained results can be
used for establishing structure-activity relationships as well as for the
structure-based optimization.
Acknowledgments: GlaxoSmithKline LLC
References:
1. Levine, A.: Cell 1997, 88(3): 321-331.
2. Kawabe, T.: Mol. Cancer Ther. 2004, 3(4): 513-519.
3. Chow, J.P.; Poon, R.Y.C.: Oncogene 2013, 32(10): 4778-4788.
POS.121
Identification of new potential inhibitors of GyrB ATPase
Schieback, P.1; Lemcke, T.1
1 University
of Hamburg, Institute of Pharmacy, Bundesstr. 45, 20146 Hamburg, Germany
Since multi drug resistance is present in pathogenic bacteria due to
increasing use of antibiotics worldwide, the design of potential new
antibiotics becomes more important to deal with bacterial infections like
respiratory and urinary tract infections [1]. One very well-known target for
antibiotics, such as fluoroquinolones, is DNA gyrase. It is a target that
only exists in bacteria and not in human cells, and it has a high degree of
sequence conservation throughout many bacterial species [2]. DNA
gyrase is divided into two subunits and only drugs inhibiting subunit A
(GyrA) are actually in clinical use. In addition to the development of multiresistant strains in bacteria, subunit A inhibitors exhibit also several side
effects.
Inhibition of the ATPase binding pocket of Gyrase subunit B (GyrB) is
getting more important although Novobiocin, which is an
aminocoumarine derivate and a highly potent GyrB inhibitor, was
unsuccessful in clinic due to severe side effects.
A virtual screening campaign [3] of a subset of the ZINC database
resulted in a collection of potential ligands of the GyrB ATP-binding site
of E.coli. For the final hit ranking and compound selection, a 3D QSAR
model [4], generated using literature known GyrB ATPase inhibitors, was
used.
Acknowledgments: We thank Dr. Krister Bamberg, AstraZeneca R&D Mölndal, Sweden, for the
generous gift of the recombinant FXR-LBD.
References:
1. Seol, W.; Choi, H. S.; Moore, D. D.: Mol. Endocrinol. 1995, 9(1): 72–85.
2. Forman, B. M. et al.: Cell 1995, 81(5): 687–693.
3. Parks, D. J. et al.: Science 1999, 284(5418): 1365–1368.
4. Kuipers, F.; Bloks, V. W.; Groen, A. K.: Nat. Rev. Endocrinol. 2014, 10(8): 488–498.
5. Merk, D.; Steinhilber, D.; Schubert-Zsilavecz, M.: Future Med. Chem. 2012, 4(8): 1015–1036.
6. Luciano Adorini, L.; Pruzanski, M.; Shapiro, D.: Drug Discov. Tod. 2012, 17(17): 988-997.
7. Lamers, C.; Schubert-Zsilavecz, M.; Merk, D.:Curr. Top. Med. Chem. 2014, 14(19): 2188-2205.
8. Lu, W. et al.: Sci. Rep. 2015, 5: 8114.
9. Schmidt, J. et al.: Sci. Rep.2015, submitted.
Figure: Docking pose of hit A in the GyrB ATPase binding site of E. coli
POSTERS
23 structures were purchased and tested in a fluorescence quenching
assay for gyrase inhibiting activity. A gyrase supercoiling assay was used
to verify the inhibitory activity of all hits. Finally, three structurally divers
substances were identified as potential GyrB inhibitors.
To expand the hit set and to get a preliminary SAR, five additional
derivatives of hit A were docked into the GyrB ATP binding site, evaluated
using the validated 3D QSAR model and finally purchased and tested. All
substances show inhibitory activity in the micro molar range.
The results of the hit expansion and biological testing of these new
potential GyrB ATPase inhibitors will be presented on this paper.
The relevance of CLKs in the onset of the abovementioned diseases and
the lack of selectivity of current inhibitors emphasises the need for novel
small molecules as tools for biological experiments or as lead
compounds in the drug discovery process. We here present the synthesis
and biological evaluation of a structurally new class of CLK inhibitors.
Representatives of the 6,7-dihydropyrrolo[3,4-g]indol-8-ones selectively
inhibit CLKs and are not active on a panel of related serine threonine
kinases including DYRK1A.
References:
1. Hagiwara, M.: Biochim. Biophys. Acta. 2005, 1754: 324-331.
2. Liu, Y. et al.: Nucleic Acids Res. 2013, 41: 4949-4962.
3. Glatz, D.C. et al.: J. Neurochem. 2006, 96: 635-644.
References:
1. Nakaminami, H. et al.: Int. J. Antimicrob. Ag. 2014, 43(5): 478-479.
2. Tari, L.W.; et al.: PLoS ONE 2013, 8(12): e84409.
3. Münsterberg, M.et al.: Presentation EFMC-ISMC Lisbon. 2014.
4. Schieback, P.; Lemcke, T.: Presentation EFMC-ISMC Lisbon. 2014.
POS.124
POS.122
Development and characterization of a compound series
based on a hit structure obtained from the Tres Cantos
Antimalarial Compound Set
Weidner, T.1; Nasereddin, A.2; Preu, L.1; Grünefeld, J.1; Dzikowski, R.2;
Kunick, C.1
1 Institut
2 Department of Microbiology and Molecular Genetics, IMRIC, Hebrew University, Hadassah
Medical School, P.O. Box 12272, Jerusalem 91220, Israel
Publicly available substance libraries provide various starting points for
the development of new drugs against malaria parasites. The Tres
Cantos Antimalarial Compound Set (TCAMS), containing 13,533
structures with activity against Plasmodium falciparum [1], includes the
compound TCMDC-137332, which was suggested as one of “47 quality
starting points for malaria drug discovery” [2]. TCMDC-137332 appears
to be one of the most potent structures of this collection and, in addition,
it matches with the requirements of the Lipinski rules for orally available
drugs [3]. Analogues of TCMDC-137332, which are based on a 2phenoxyanilide scaffold, were synthesized by standard amide coupling
reactions. The poster reports the synthesis of overall 19 derivatives,
which were completely characterized regarding identity and purity by
spectroscopic as well as chromatographic methods and elemental
analyses. Furthermore, the results of the biological evaluation of all new
congeners against P. falciparum erythrocyte stage parasites will be
presented.
Acknowledgments: This project was funded by the German Federal Ministry of Education and
Research (BMBF BioDisc 7; 13GW0024)
References:
Computer-based studies of Bisamidines as PRMT1 and
PRMT6 inhibitors
Robaa, D.1; Sauer, B.1; Wagner, T.2; Jung, M.2; Sippl, W.1
1
2
Department of Pharmaceutical Chemistry, Martin-Luther University Halle-Wittenberg
Institute of Pharmaceutical Sciences, Albert-Ludwigs University Freiburg
Protein arginine methyltransferases (PRMTs) are a conserved family of
proteins which catalyze the posttranslational N-methylation of arginine
residues. They constitute an important family of epigenetic enzymes,
which can regulate gene expression through histone methylation.
Besides histones, PRMTs have numerous non-histone proteins as
substrates, including RNA-binding proteins, signal transducers and
transcriptional coregulators [1]. PRMTs appear to play a role in the
pathogenesis of cancer [2], diabetes [3] and cardiovascular diseases [4]
and to be important for viral replication [5], suggesting that these
enzymes are interesting targets for drug discovery.
Virtual screening approaches of various compound databases were
successfully deployed in our research group and led to the identification
of several lead structures as PRMT1 inhibitors, including bisamidines
[6-8]. To study the binding of bisamidines to PRMT1 and PRMT6, docking
and molecular dynamic studies were performed on generated homology
models of both enzymes. Additionally, structural optimization of these
lead compounds, guided by in silico studies, was carried out.
References:
1. Lee, Y.; Stallcup, M.R.: Mo.l Endocrinol. 2009, 23(4): 425–433.
2. Yoshimatsu, M. et al.: Int. J. Cancer. 2011, 128(3): 562–573.
3. El-Osta, A. et al.: J. Exp. Med. 2008, 205(10): 2409–2417.
4. Vallance, P.; Leiper, J.: Arterioscler. Thromb. Vasc. Biol. 2004, 24(6):1023–1030.
5. Xie, B. et al.: J. Virol. 2007, 81(8): 4226–4234.
6. Spannhoff, A. et al.: J. Med. Chem. 2007, 50(10): 2319–2325.
7. Spannhoff, A. et al.: Bioorg. Med. Chem. Lett. 2007, 17(15): 4150–4153.
8. Heinke, R. et al.: ChemMedChem 2009, 4(1): 69–77.
1. Gamo, F.-J. et al.: Nature 2010, 465(7296): 305–310.
2. Calderón, F. et al.: ACS Med. Chem. Lett. 2011, 2(10): 741–746.
3. Lipinski, C. A. et. al.: Adv. Drug Delivery Rev. 1997, 23(1-3): 3–25.
POS.125
POS.123
Novel selective inhibitors of the splicing regulatory CDC2like kinases (CLKs)
Walter, A.1; Helmer, R.1; Loaëc, N.2; Preu, L.1; Meijer, L.2; Kunick, C.1
1 Institut
2 ManRos Therapeutics, Hôtel de Recherche, Centre de Perharidy, 29680 Roscoff, France
CLK family kinases (CDC2-like kinases) play an important role in the
regulation of pre-mRNA splicing by phosphorylating several splicing
factors [1]. Alternative splicing, occurring after transcription of DNA into
pre-mRNA, is an essential process in eukaryotes and an origin of protein
diversity. Strict control of the splicing mechanism is required as a change
might potentially have significant effects on the cellular proteome or even
on the whole organism. Any dysregulation may alter or abolish the
original function of an affected protein thus contributing to the
development of diseases. Abnormal splicing influenced by the
phosphorylation of splicing factors by CLKs was observed in the
pathogenesis of frontotemporal dementia and ovarian cancer [2,3].
Development of new Dengue Virus NS2B-NS3 protease
inhibitors and the investigation on their mode of action
von Hammerstein, F.1; Gellert, A.1; Wu, H.1; Holloway, S.1; Kiefer, W.1;
Bodem, J.2; Kanitz, M.3; Steuber, H.3; Diederich, W.3; Hellmich, U.1;
Schirmeister, T.1
Institute of Pharmacy and Biochemistry, University of Mainz, Staudingerweg 5, D-55099
Mainz, Germany
2 Institute of Virology and Immunology, University of Würzburg, Versbacher Strasse 7, D-97078
Würzburg, Germany
3 Institute of Pharmaceutical Chemistry, University of Marburg, Marbacher Weg 6-10, D-35032
Marburg, Germany
1
Dengue fever is a mosquito-borne tropical disease caused by the dengue
virus (DENV). It is transmitted to humans by the Aedes aegypti and
Aedes albopictus mosquitoes [1]. This viral infection is becoming
continually a global threat, as there are nearly 3.6 billion people living in
the areas, tropical and subtropical regions of the world (predominantly in
Southeast Asia, Africa and the Americas), where the infection is common
[2,3]. The DENV infection can result in classic dengue fever, dengue
hemorrhagic fever (DHF) or dengue shock syndrome (DSS) [4].
Worldwide there are over 50 million infections reported annually and the
infection causes over 20,000 deaths each year [5]. DENV is a single
positive-stranded RNA virus of the family Flaviviridae with four distinct
serotypes [6,7]. The dengue virus genome encodes a serine protease
with a classical catalytic triad (His51, Asp75 and Ser135) [8,9], which is
responsible for the post-translational proteolytic processing of the
polyprotein precursor and essential for the viral replication [10,11],
making it an important and attractive therapeutic target [12].
Our projects [13] include synthesis, characterization and testing of
DENV2/3 NS2B-NS3pro inhibitors based on the structure of variously
substituted diaryl (thio)ethers. The synthesized compounds are screened
both in vitro in fluorometric enzyme assays using different fluorogenic
AMC-derived substrates or by microscale thermophoresis (MST) [14] and
in cell culture. The possible binding modes are analyzed by docking
studies and are being examined by NMR spectroscopy.
Acknowledgments: University of Mainz for financial support.
References:
1. Yildiz, M. et al.: ACS Chem. Biol. 2013, 8: 2744-2752.
2. Murray, N. E. A. et al.: Clinical Epidemiology 2013, 5: 299-309.
3. Guzman, M. G. et al.: Nat. Rev. Microbiol. 2010, 8: S7-S16.
4. Martina, B. E. et al.: Clin. Microbiol. Rev. 2009, 22: 564-581.
5. Wilder-Smith, A. et al.: Arch. Med. Res. 2002, 33(4): 330-342.
6. Li, H. et al.: J. Virol. 1998, 73(4): 3108-3116.
7. Chambers, T. J. et al.: Annu. Rev. Microbiol. 1990, 44: 649-688.
8. Bazan, J. F.; Fletterick, R. J.: Virilogy 1989, 171: 637-639.
9. Nitsche, C. et al.: Chem. Rev. 2014, 114(22): 11348–11381.
10. Falgout, B. et al.: J. Virol. 1991, 65: 2467-2475.
11. Zhang, L.; Mohan, P. M.; Padmanabhan, R.: J. Virol. 1992, 66: 7549-7554.
12. Zheng, Y. et al.: Bioorg. Med. Chem. Lett. 2006, 16: 36-39.
13. Wu, H. et al.: Antimicrob. Agents Chemother. February 2015, 59(2): 1100-1109.
14. Jerabek-Willemsen, M. et al.: Assay Drug Dev. Technol. 2011, 9(4): 342-353.
POS.126
Novel purine-based inhibitors of anaplastic lymphoma
kinase (ALK)
Schlütke, L.1; Preu, L.1; Totzke, F.2; Schächtele, C.2; Kubbutat, M. H. G.2;
Kunick, C.1
1 Institut für Medizinische und Pharmazeutische Chemie, TU Braunschweig, Beethovenstraße 55,
2 ProQinase GmbH, Breisacher Straße 117, 79106 Freiburg, Germany
Chromosomal rearrangements of anaplastic lymphoma kinase (ALK)
have been described in several human cancer diseases [1]. Deregulation
of ALK was first identified in anaplastic large cell lymphoma (ALCL),
which is associated with NPM (nucleophosmin) ALK fusion gene [2]. The
similar fusion gene EML4-ALK plays an important role in the
development in 5% of all non-small cell lung carcinoma (NSCLC) [3]. In
2011 the first dual ALK/cMet inhibitor Crizotinib was approved. Although
Crizotinib shows a high response rate, emerging resistances are
observed with a median of 10.5 months [3]. Second-generation ALK
inhibitors like Ceritinib and Alectinib address some of the mutations
underlying these resistances [4]. Further development of structurally
unrelated inhibitors remains a challenging problem. Design by dockingstudies, syntheses, and kinase inhibitory activities of the title compounds
will be presented in the poster.
Acknowledgments: L.S. is grateful for a stipend funded by the Cusanuswerk, Bonn, Germany.
References:
1. Bossi, R. et al.: Biochemistry 2010, 49: 6813-6825.
2. Morris, S. W. et al.: Science 1994, 263: 1281-1284.
3. Roskoski, R. et al.: Pharmacol. Res. 2013, 68: 68-94.
4. Kinoshita, K. et al.: Biorg. Med. Chem. 2012, 20: 1271-1280.
POS.127
Design and synthesis of 2-ureidothiophene-3-carboxylic
acids as dual bacterial RNAP and HIV-1 RT inhibitors for
treatment of patients co-infected with MRSA and HIV-1
Elgaher, W. A.1; Sharma, K. 2; Haupenthal, J. 1; Mély, Y. 2;
Hartmann, R. W.1
Department of Drug Design and Optimization, Helmholtz Institute for Pharmaceutical
Research Saarland, Campus C2.3, Saarland University, 66123 Saarbrücken, Germany,
2 Laboratory of Biophotonics and Pharmacology, Faculty of Pharmacy, Strasbourg University,
74 route du Rhin, 67401 Illkirch, France
1
Human immunodeficiency virus type 1 (HIV-1) is a retrovirus that targets
and destroys the CD4 cells of the immune system. Consequently, it
causes the acquired immune deficiency syndrome (AIDS), which is one
of the biggest killer infectious diseases worldwide. Even worse, HIV
patients are easily attacked by bacteria, e.g. methicillin-resistant
Staphylococcus aureus (MRSA) [1]. Current antiretroviral therapy (ART)
is a combination of at least three antiretroviral drugs to allow efficient
suppression of virus replication. However, in this context viral resistance
is presenting a major problem. The treatment of an HIV-1/MRSA coinfection is even more critical. Administration of antibacterial agents
should consider the ongoing prevalence of resistant bacteria in HIV
patients [2], as well as potential interactions between ART drugs and the
antibacterial agents [3]. This prompted us to develop novel compounds
with both antiretroviral and antibacterial activities. This was achieved via
exploiting, for the first time, the mechanistic function similarity between
the RNA polymerase (RNAP) switch region and the non-nucleoside
reverse transcriptase inhibitor (NNRTI) binding site. Starting from our
bacterial RNAP inhibitors [4,5], we succeeded in developing potent RT
inhibitors while retaining or enhancing the RNAP inhibitory activity
following a structure-based drug design approach. A SAR study revealed
structural features necessary for RT inhibition. The novel compounds
showed good antiviral (in cellulo assay) and antibacterial activities
(against MRSA) in the low micromolar range, along with low or no
cytotoxicity. Furthermore, the mode of action of our novel inhibitors was
studied regarding RT, revealing a non-competitive inhibition mechanism
similar to nevirapine.
References:
1. Hidron, A. I. et al.: Infect. Drug Resistance 2010, 3: 73−86.
2. Diep, B. A. et al.: Ann. Intern. Med. 2008, 148: 249−257.
3. Piscitelli, S. C.; Gallicano, K. D.: N. Engl. J. Med. 2001, 344: 984−996.
4. Sahner, J. H. et al.: Eur. J. Med. Chem. 2013, 65: 223−231.
5. Elgaher, W. et al.: RSC Adv. 2014, 4: 2177−2194.
POS.128
A direct Enzyme-Linked Immunosorbent Assay (ELISA) for
the quantitative evaluation of Janus Kinase 3 (JAK3)
inhibitors
Bauer, S. M.1; Gehringer, M.1; Laufer, S. A.1
1 Department
of Pharmaceutical and Medicinal Chemistry, Institute of Pharmaceutical Sciences,
Eberhard Karls University of Tuebingen, D-72076 Tuebingen, Germany
Janus kinases (JAKs) are a group of non-receptor tyrosine kinases
involved in the signal transduction processes of cytokines, growth factors
and interleukins. Thus, the four JAK family members JAK 1,2,3 and TYK
2 are potential targets for treatment of inflammatory diseases as well as
oncological disorders [1-3].
Being restricted to the hematopoietic lineage, JAK 3’s limited tissue
expression makes it an attractive drug target for the treatment of
inflammation with limited side effects. Several JAK inhibitors are currently
under investigation in clinical trials and the pan-JAK inhibitor Tofacitinib
has recently been approved by the FDA [4]. With Ruxolitinib a JAK 1 and
JAK 2 selective inhibitor for the treatment of myelofibrosis was approved
by the EMA [5]. Even though being a promising target, no selective JAK
3 inhibitor is available for therapy until now.
In an academic environment, the availability of a robust, precise and costefficient test system is the prerequisite for the performance of any
medicinal chemistry program. For identification of novel JAK 3 inhibitors
and determination of structure activity relationships, a direct enzymelinked immunosorbent assay was developed. Being applicable in a
standard laboratory, the main advantage over existing methods is the
basic equipment requirement as this assay does not need special
handling originating from the use of cell cultures or radiometric readout.
POSTERS
In contrast to earlier methods relying on two-step procedures, our assay
uses a single peroxidase conjugated antibody to quantify and detect
substrate phosphorylation. This optimized assay features a limit of
detection of 0.032 µg/mL, a wide dynamic range of 0.638 OD units and
an appropriate Z’ factor of 0.66. The determined IC50 value for Tofacitinib
was 3.5±0.6 nM (n = 18).
For investigation of structure activity relationships, the assay is readily
discriminating the effects on kinase inhibition even within a series of
structurally similar molecules and is thus suited for lead optimization.
When screening our Tofacitinib-derived compound library, the reported
ELISA allowed the determination of inhibitory potencies in the low
nanomolar range which were subsequently confirmed by commercial
radiometric and cellular assays. As a result of our research in this area,
we recently described a new class of highly potent tricyclic JAK inhibitors
with superior selectivity compared to Tofacitinib [6].
The developed ELISA primarily addresses researchers within the
academic setting facing the task to identify lead structures and explore
structure activity relationships of JAK 3 inhibitors at reasonable costs.
Acknowledgments: The authors want to thank L. Fischer and K. Bauer for successful laboratory
co-work during the establishment of this assay. Special thanks to Jun.-Prof. Dr. Pierre Koch and
Dr. Peter Keck for their help, support and advice.
References:
1. Pesu, M. et al.: Immunol. Rev. 2005, 203: 127 - 142
2. Kim, B.H. et al.: Br. J. Haematol. 2010, 148: 132-143.
3. Haan, C.; et al.: Chem. Biol. 2011, 18: 314-323.
4. O'Shea, J.J.: Ann. Rheum. Dis. 2013, 72: 111-115.
5. Tefferi, A. et al.: Blood 2012, 119: 2721 – 2730.
6. Gehringer, M. et al.: ChemMedChem. 2014, 9: 277-281.
POS.130
Thieno[3,2-d]pyrimidin-2-amine derivatives as inhibitors of
anaplastic lymphoma kinase
Immer, M.1,2; Wölfel, S.1; Preu, L.1; Totzke, F. 3; Schächtele, C. 3;
Kubbutat, M. H. G.3; Kunick, C.1,2
1 Institut für Medizinische und Pharmazeutische Chemie, TU Braunschweig, Beethovenstraße 55,
2 Center of Pharmaceutical Engineering (PVZ), TU Braunschweig, Franz-Liszt-Straße 35A, 38106
Braunschweig, Germany
3 ProQinase GmbH, Breisacher Straße 117, 79106 Freiburg, Germany
The tyrosine kinase anaplastic lymphoma kinase (ALK) is a well known
target in various cancer diseases. ALK was identified for the first time as
a part of the nucleophosmin (NPM)-ALK fusion protein, which was
detected in 60% of anaplastic large cell lymphoma (ALCL) patients [1].
The fusion gene echinoderm microtubule-associated protein like 4
(EML4)-ALK was identified in approximately 5% of all non-small-cell lung
cancer (NSCLC) patients [2]. In 2011, Crizotinib was approved by the
FDA as the first ALK inhibitor. In 2014 two other ALK inhibitors, Alectinib
and Ceritinib, were approved [3,4]. Due to numerous mutations of ALK
and thereby acquired resistance the further development of new ALK
inhibitors is of great interest. In this poster, we present results of docking
experiments of thieno[3,2-d]pyrimidin-2-amine derivatives in the ATP
binding pocket of ALK, as well as the syntheses and the ALK inhibitory
activity of these compounds.
Acknowledgments: M.I. is grateful for a stipend in the framework of „SynFoBiA“, a cooperative
research progam funded by “VW-Vorab”.
References:
POS.129
Probing the preferred binding orientation of the axially
oriented phenyl moiety of 1,3-dioxane NMDA receptor
antagonists by conformational restriction
1. Morris, S. W. et al.: Science 1994, 263: 1281-1284.
2. Shaw, A. T. et al.: N. Engl. J. Med. 2013, 36: 2385–2394.
3. Kinoshita, K. et al.: Biorg. Med. Chem. 2012, 20: 1271-1280.
4. Shaw, A. T. et al.: N. Engl. J. Med. 2014, 370: 1189-1197.
Asare-Nkansah, S.1; Wünsch, B.1
Institut für Pharmazeutische und Medizinische Chemie, Corrensstraße-48, D-48149 Münster,
Germany.
POS.131
1
The N-methyl-D-aspartate (NMDA) receptor is a ligand-gated ion channel
that plays a prominent role in various central nervous system (CNS)
events such as development of neurons, synaptic plasticity, memory and
learning [1]. The NMDA receptor is highly permeable to Ca2+ ions, and
under pathological conditions, elevated intracellular Ca2+ ion
concentrations up to cytotoxic levels contribute partly to neuronal death
(excitotoxicity. The NMDA receptor is consequently implicated in
diseases of the CNS (Alzheimer’s disease, Parkinson’s disease,
Huntington’s disease and epilepsy), which are associated with
excitotoxicity [2]. Therefore, the NMDA receptor represents a potential
therapeutic target for the development of innovative drugs for the
treatment of neurological disorders.
The 1,3-dioxolane derivatives dexoxadrol (1) and etoxadrol (2) have high
NMDA receptor affinity (Figure) with non-tolerable side effects including
retrograde amnesia and psychotomimetic effects [3]. Studies performed
on 1 and 2 have led to the development of 1,3-dioxane analogs 3 with
even higher affinity towards the phencyclidine (PCP) binding site of the
NMDA receptor [3,4]. The phenyl moiety in axial orientation at the acetalic
C-atom of 3 is postulated as essential for binding with high affinity to the
NMDA receptor. The conformationally restricted ligands 4 should
therefore give insight into the preferred binding orientation of the phenyl
moiety (parallel (3a) or perpendicular (3b)). In this project, the phenyl
moiety is fixed in an axial position perpendicular to the 1,3-dioxane ring
(4).
Figure: Development of novel 1,3-dioxane based NMDA receptor antagonists
References:
1. Ono, S. et al.: Chem. Pharm. Bull. (Tokyo) 2002, 50: 966–8.
2. Flores-Soto, M. E. et al.: Neurologia 2012, 27: 301–10.
3. Aepkers, M.; Wünsch, B.: Bioorg. Med. Chem. 2005, 13: 6836–49.
4. Utech, T.; Köhler, J.; Wünsch, B.: Eur. J. Med. Chem. 2011, 46: 2157-69.
The Trypanothione Synthetase ATP Binding Site:
Flexibility and Similarity to Other ATP Binding Proteins
Ehrt, C.1; Brinkjost, T.1,2; Koch, O.1
1 Faculty
2 Faculty
for Chemistry and Chemical Biology, TU Dortmund, Germany
of Computer Science, TU Dortmund, Germany
Trypanothione synthetase (TryS) catalyzes the two-step biosynthesis of
trypanothione, which is a key intermediate in trypanosomatid parasites of
the species Trypanosoma and Leishmania. Thus, TryS is an attractive
drug target to cope with neglected diseases like Chagas disease,
leishmaniasis, or African trypanosomiasis that affect approximately 15 to
20 million people worldwide. Interestingly, paullones, a chemical class of
potent kinase (GSK-3β and different cyclin-dependent kinases)
inhibitors, were shown to inhibit TryS [1]. Based on a comparison of
available structures, the binding of paullones to TryS seems to be in
accordance with the principle of ligand-sensing cores [2]. That is, the
spatial arrangement of secondary structure elements around the ATP
binding sites of TryS and kinases is quite similar, independent of the
overall fold, which indicates binding of similar scaffolds.
Unfortunately, the available Leishmania major TryS X-ray structure (pdbid 2vps) was solved without substrates and an important loop region of
the ATP grasp fold is missing. The structure of a related GSP synthetase
from Escherichia coli (pdb‑id 2io7) led to a complete model of LmTryS,
containing the substrate ATP. Exhaustive MD simulations confirmed this
model and revealed that the presence of ATP leads to a partial closure
of an associated β-sheet over the bound triphosphate. This validated
model gave us the possibility to model structures of TryS from other
pathogenic species of Trypanosoma and Leishmania and to compare
their ATP binding sites with respect to rational drug design. Furthermore,
the conformational space of this highly flexible binding site was
elucidated with the help of accelerated MD simulations.
As we recently developed a reasonably fast method to search for
common ligand-sensing cores in a set of different protein structures
based on graph comparisons, we used this method to compare the ATP
binding sites of different kinases and our modelled TryS structures from
various species. We searched for ATP binding proteins in the Protein
Data Bank and compared them not only against representative TryS
structures, but also against each other. Structural similarities between
different TryS structures and certain kinases can be shown by only taking
into account the protein backbone. A structural comparison of the
involved secondary structure elements and the residues which are part
of the common ligand-sensing core revealed that, although the backbone
architecture is quite similar in both proteins, the underlying residues are
quite different regarding physicochemical properties.
Known highly potent inhibitors of kinases with the highest similarity to
TryS were extracted from the ChEMBL database, clustered and
afterwards docked into different TryS ATP binding sites from the MD
simulations to find potential candidates for biochemical testing and
further modification with regard to selectivity. Here, we present this
workflow as a new method to exploit the huge knowledge about kinase
inhibitors for the treatment of neglected diseases and facilitate drug
discovery for novel targets. We will also show the results of the
comparison of ATP binding protein structures against each other and
discuss how this can help to extend our knowledge with regard to offtarget effects and structural classification.
References:
1. Koch, O.: Drug Discovery in Infectious Diseases (Wiley-VCH) 2013: 429-443.
2. Koch, O. et al.: Future Med. Chem. 2011, 3: 699-708.
POS.132
A lovely Liaison: Privileged Scaffolds and conserved
Structural Elements in Proteins
Humbeck, L.1; Koch, O.1
1
TU Dortmund, Otto-Hahn-Straße 6, 44221 Dortmund, Germany
The term “privileged scaffolds” is often used for multiple molecules that
show bioactivity on different targets but consist of the same scaffold [1].
Within proteins, conserved structural elements can also be found in
different proteins, ranging from conserved motifs that interact with
specific functional groups to similar spatial arrangements of secondary
structure elements around the ligand binding site (the “ligand-sensing
core”) in proteins with different folding patterns that can bind similar
scaffolds [2]. Information about similar ligand-sensing cores can be
useful for rational identification of new lead structures [3] or predicting
polypharmacology [2].
Chemical compound databases like DrugBank (http://www.drugbank.ca/)
or ChEMBL (https://www.ebi.ac.uk/chembl/) contain a huge amount of
data about molecules and their bioactivity on different protein targets.
Therefore, we decided to develop a Python based tool for knowledge
discovery to get new insights into the relationship of privileged scaffolds
and conserved structural elements in proteins. The main idea of this data
mining approach is the identification of scaffolds that bind to different and
unrelated protein targets for analyzing potential conserved structural
elements.
In a first step, a command line version of Scaffold Hunter [4] is used to
reduce all molecules in a database to their containing scaffolds. The
second step analyzes the sequence similarity of protein targets of all
molecules sharing a common scaffold. Only protein targets with identity
below 40% are regarded as unrelated. The last step visualizes the results
for an in-depth analysis.
We will present the overall workflow and the result of an exhaustive
chemogenomics analysis of the DrugBank. Around 1500 scaffolds were
identified that are active against different protein targets. An analysis of
one example already ended up in a new ligand-sensing core that is
shared between four different protein targets and can help to identify new
lead structures for the respective targets. To prove the hypothesis of
relationships between privileged scaffolds and conserved structural
elements in proteins (the ligand-sensing cores) we also present the
results of a high-throughput screen (HTS) against new inhibitors of one
protein of the novel ligand-sensing core, which were similar to known
ligands of one of the other proteins of the ligand-sensing core.
Acknowledgments: We thank the BMBF (Grant No. 1316053) and DFG (“Algorithms for Big
Data” SPP 1736) for funding.
References:
1. Welsch, M. E.; Snyder, S. A.; Stockwell, B. R.: Curr. Opin. Chem. Biol. 2010, 14(3): 347-361.
2. Koch, O.: Fut. Med. Chem. 2011, 3(6): 699-708.
3. Willmann, D. et al.: Int. J. Cancer. 2012, 131(11): 2704-2709.
4. Klein, K. et al.: Mol. Inf. 2013, 32: 964–975.
POS.133
Discovery of the first small-molecule CsrA-RNA interaction
inhibitors as potential anti-infectives using surface
plasmon resonance and fluorescence polarization-based
screening
Maurer, C. K.1,*; Fruth, M.1,*; Empting, M.1; Herrmann, J.1,2; Müller, R.1,2;
Dersch, P.3; Hartmann, R. W. 1,4
1 Helmholtz
Institute for Pharmaceutical Research Saarland (HIPS), Saarland University,
Campus C2.3, 66123 Saarbrücken, Germany
2 German Centre for Infection Research (DZIF), Partner Site Hannover-Braunschweig, 30625
Hannover, Germany
3 Helmholtz Centre for Infection Research (HZI), Inhoffenstraße 7, 38124 Braunschweig,
Germany
4 Department of Pharmaceutical and Medicinal Chemistry, Saarland University, Campus C2.3,
66123 Saarbrücken, Germany
*These authors contributed equally to this work.
Bacteria use global regulators to adapt gene expression in response to
environmental changes. For instance, the regulator protein CsrA affects
translation and stability of messenger RNA by binding to the ribosome
binding site of target trancripts [1]. CsrA is widespread among bacteria
and is highly conserved in structure and RNA recognition [2]. As global
regulator, it controls numerous physiological processes involved e.g. in
virulence, metabolism, motility, and biofilm formation [3]. Notably, a csrA
knockout mutant of the enteropathogen Yersinia pseudotuberculosis
exhibited attenuated virulence in a murine infection model (P. Dersch,
unpublished data). Therefore, we considered CsrA as promising target
for development of novel anti-infectives.
To date, no low-molecular-weight inhibitor of the CsrA-RNA interaction
has been described. Thus, for the discovery of such inhibitors, we
followed two strategies, a screening and a ligand-based approach.
During primary screening, compounds binding in a concentrationdependent manner to Yersinia pseudotuberculosis CsrA should be
identified using surface plasmon resonance technology (SPR).
Secondary screening based on a fluorescence polarization (FP)-based
competition assay should filter out CsrA binders that could inhibit the
CsrA-RNA interaction. From a library comprising 712 small molecules,
76 CsrA binders were identified, 4 out of which inhibited the CsrA-RNA
interaction. Additionally, screening of a library consisting of 259
myxobacterial metabolites yielded 4 CsrA-RNA interaction inhibitors. Hit
validation with the FP assay revealed dose-dependent inhibition with low
micromolar Ki values for the 7 studied screening hits. The most promising
compound exhibited a Ki of 2 µM. For the ligand-based strategy, short
oligonucleotides of varying length and nucleic acid backbone derived
from the highly conserved RNA binding motif were systematically studied
[4]. In the FP assay, a GGA-motif with RNA-backbone turned out to be
the most active ligand-derived inhibitor so far with a Ki value of 48 µM.
The identified hits will be further characterized regarding their binding
behaviour by SPR and regarding their cellular activity in virulence assays
with different bacterial species such as Yersinia pseudotuberculosis and
Pseudomonas aeruginosa.
References:
1. Romeo, T.: Mol. Microbiol. 1998, 29(6):1321-30.
2. Dubey, A. K.et al.: RNA 2005, 11(10):1579–1587.
3. Timmermans, J.; Van Melderen, L.: Cell Mol Life Sci. 2010, 67(17):2897–2908.
4. Lapouge, K. et al.: RNA Biol. 2013, 10(6): 1030–1041.
POS.134
Shifting Selectivity: From JAK1/2 to JAK3 inhibition via
click chemistry
Forster, M.1; Gehringer, M.2; Bauer, S. M.1; Laufer, S. A.1
1 Institute
of Pharmacy, Medicinal Chemistry, Eberhard-Karls-Universitaet Tuebingen, Auf der
2 Institute of Pharmaceutical Sciences, Department of Chemistry and Applied Biosciences, ETH
Zurich, Vladimir-Prelog-Weg 1-5/10, 8093 Zurich, Switzerland
The Janus kinase family (JAK) is consisting of four members, JAK1-3
and TYK2. Belonging to the non-receptor tyrosine kinases they associate
with cytokine receptors and mediate extracellular signals via the
phosphorylation of STAT-proteins (signal transducer and activator of
transcription). Whereas the other members of the JAK family are
expressed ubiquitously, JAK3 is predominantly found in cells of the
haematopoietic system where it has a crucial function in the maturation
of T-cells, B-cells and natural killer cells. Patients with a malfunction of
JAK3 develop the phenotype of severe combined immunodeficiency
(SCID) and are lacking T-lymphocytes and NK-cells. The restriction of its
POSTERS
function to the immune system suggests that JAK3 inhibition is a
promising therapeutic strategy for the treatment of autoimmune diseases,
inflammation and allograft rejection [1]. On the other hand it is not finally
proven that solely JAK3 inhibition results in an immunosuppression [2].
Therefore highly selective JAK3 inhibitors are needed to further
investigate this question.
POS.136
Novel ´butterfly` derivatives of FTY720 reduce symptoms in
active experimental autoimmune-induced
encephalomyelitis (EAE) in mice
Zivkovic, A.1; Imeri, F.2; Fallegger, D.2; Schwalm, S.3; Blankenbach, K.3;
Engelhardt, B.4; Meyer zu Heringdorf, D.3; Pfeilschifter, J.3; Huwiler,
A.2,3; Stark, H.1
1 Institute
Universitaetsstrasse 1, 40225 Duesseldorf, Germany
2 Institute of Pharmacology, University of Bern, CH-3011 Bern, Switzerland;
3 Pharmazentrum Frankfurt/ZAFES, University Hospital, Goethe University Frankfurt am Main,
Germany;
4 Theodor-Kocher Institute, University of Bern, CH-3012 Bern, Switzerland.
Ruxolitinib is a JAK-Inhibitor with preference for JAK1/2, which is
approved for the treatment of myelofibrosis [3]. In our attempt to create
new JAK-inhibitors with an improved selectivity towards JAK3, we used
Ruxolitinib as scaffold and made a bioisosteric replacement of the
pyrazole ring by a triazole. While the synthesis and derivatization of the
pyrazole moiety would be a multi-step procedure, the corresponding
triazoles are readily accessible via the copper catalyzed azide-alkyne
cycloaddition [4] (CuAAC) and can be synthesized in an almost
combinatorial fashion. In this study a library of triazole-containing
Ruxolitinib derivatives were synthesized and tested for their ability to
inhibit JAK3. A chosen subset of the synthesized inhibitors was also
tested in a selectivity assay including JAK1-3 and TYK2 to further
investigate the selectivity within the Janus kinase family [5].
References:
1. Cornejo, M. G.; Boggon, T.J.; Mercher, T.: Int. J. Biochem. Cell. Biol. 2009, 41: 2376-2379.
2. Haan, C. et al.: Chem. Biol. 2011, 18(3): 314-323.
3. Mesa, R. A.; Yasothan, U.; Kirkpatrick, P.: Nat. Rev. Drug Discov. 2012, 11: 103-104 .
4. Rostovtsev, V. V. et al.: Angew. Chem. Int. Ed., 2002, 41: 2596-2599.
5. Gehringer, M.; Forster, M.; Laufer, S.A.: ACS Comb. Sci. 2015, 17(1): 5-10.
POS.135
Synthesis and Antiparasitic Activity of Aromatic
Bisamidines
Sauer, B.1; Robaa, D.1; Schmidt, M.1; Pierrot, C.3; Bouchut, A.3;
Andrews, K.2; Khalife, J.3; Sippl, W.1
1 Martin-Luther-University
Halle-Wittenberg, Wolfgang-Langenbeck-Str.4, 06120 Halle,
Germany
Tropical Parasitology Lab, Eskitis Institute for Drug Discovery, Don Young Road, Griffith
University, Nathan, Queensland, Australia 4111
3 U1019-CNRS UMR 8204, Univ. Lille Nord de France, Institut Pasteur de Lille, 1, Rue du
professeur Calmette, 59019 Lille, France
2
Malaria belongs to the most important tropical diseases and is subject of
various scientific activities concerning drug development. However, there
is a need of new drug scaffolds due to resistance problems. In 1977 the
aromatic bisamidine furamidine was initially developed as an
antiprotozoal agent against trypanosomes [1]. Subsequently, furamidine
and its prodrug pafuramidine (DB289) were subjected to clinical trials for
the treatment of the sleeping sickness but liver and kidney toxicity led to
their discontinuation[2]. Despite this fact the potency against Plasmodium
falciparum (IC50: 15.5 nM) [3] identifies furamidine as a promising lead
scaffold. The present work aimed for the synthesis of a compound series
based on the furamidine core structure. A set of furamidine based
bisamidines was prepared introducing modifications on the furan core
structure as well as on the aromatic amidine moiety. Subsequently the
toxicity of derived compounds against Plasmodium falciparum and
different human cell lines was determined ex vivo uncovering structureactivity relationships.
References:
1. Das, B. P.; Boykin, D. W.: J. Med. Chem. 1977, 20: 531–536.
2. Wenzler, T. et al.: Antimicrob. Agents Chemother. 2009, 53: 4185–92.
3. Soeiro, M. N. et al.: Expert Opin. Investig. Drugs 2005, 14: 957–972.
The sphingosine-1-phosphate (S1P) receptor subtype 1 (S1P1) is a
novel therapeutic target for the treatment of multiple sclerosis. FTY720
(fingolimod) is the first functional modulator of S1P1 that is approved for
oral treatment of relapsing-remitting multiple sclerosis. In this study we
have developed two novel derivatives of FTY720, ST-968 and ST-1071,
having an oxazolo-oxazole structure, named as butterfly motive, which
proved to be active in intact cells without the necessity of prodrug
activation. A straightforward synthesis route for the butterfly derivatives
has been developed [1,2]. Both compounds triggered S1P1 and S1P3,
but not S1P2, receptor internalisation as a result of receptor activation
when using a GFP-coupled receptor constructs transfected into HEK293
cells. Furthermore, in the human endothelial cell line EA.hy 926, ST-968and ST-1071-triggered within minutes a rapid and dose-dependent
activation of the classical p42/p44-MAPK cascade which was blocked by
pertussis toxin and the S1P1/S1P3 antagonist VPC23019, but not by the
S1P2 receptor antagonist JTE-013, suggesting the involvement of a Go/i
protein and the S1P1 or S1P3 receptor subtypes.
Functionally, ST-968 and ST-1071 acted similar to FTY720 to abrogate
S1P-triggered chemotaxis of various immune cells including mouse
splenocytes, mouse T cells and of the human monocytic cell line U937.
Additionally, endothelial cell permeability, which was enhanced by the
pro-inflammatory stimuli TNFα and LPS, was normalized in a dosedependent manner by either S1P, FTY720, ST-968 or ST-1071. All
compounds also reduced TNFα-induced ICAM-1 and VCAM-1 mRNA
expression, but restored TNFα-mediated downregulation of PECAM-1
mRNA expression. In the in vivo setting, application of ST-968 or ST1071 to mice at a single dose of 1mg/kg resulted in a significant reduction
of blood lymphocytes by 70% after 24h. The Multiple Sclerosis (MS)
mouse model on active experimental autoimmune encephalomyelitis
(EAE) showed that both compounds significantly reduced the clinical
symptoms of EAE comparable to that of FTY720 either by prophylactic
or therapeutic treatment.
References:
1. Stark, H. et al.: PCT WO 2012 131096 A1 (publ. date: October 4, 2012).
2. Zivkovic, A.; Stark, H.: Tetrahedron. Lett. 2010, 51: 3769-3771.
POS.137
Antiseizure properties of chlorophenoxy aminoalkyl
derivatives
Łażewska, D.1; Kuder, K.1; Stark, H.2; Kieć-Kononowicz, K.1
1 Department
of Technology and Biotechnology of Drugs, Jagiellonian University Medical
College, ul. Medyczna 9, 30-688 Krakow, Poland
2 Institute of Pharmaceutical and Medicinal Chemistry, University Düsseldorf, Universitätsstraße
1, 40225 Düsseldorf Germany
,
Epilepsy is the most common brain disorder and affects about 1% of the
world human population. Despite the availability of many various
antiepileptic drugs, about 30% of all seizures are resistant to these drugs.
Therefore there is still strong need for new anticonvulsants with higher
level of potency and lower levels of toxicity.
This work is a part of our research for potential anticonvulsant agents
among histamine H3 receptor ligands. A series of chlorophenoxy
aminoalkyl derivatives were evaluated for anticonvulsant activity and
neurotoxicity according to the standard protocols within Anticonvulsant
Screening Program at the NIH/NINDS Bethesda (USA). Pharmacological
tests included maximal electroshock (MES) and subcutaneous
pentetrazole seizure threshold (scMet) assays as well as neurotoxicity
(TOX) evaluation in mice and/or rats after intraperitoneal (i.p.)
administration and/or in rats after oral (p.o.) administration. For some
compounds good protection against MES induced seizures was
observed. However neurotoxicity was also detected. The most promising
results were obtained for 4-chlorophenoxyhexyl derivatives.
Acknowledgments: This project was financed by a grant from the National Science Center
based on decision No DEC-2011/02/A/NZ4/00031.
of (auto)inflammatory diseases [1], cancer [2] and neurodegenerative
diseases [3]. Although, many p38α MAP kinase inhibitors have been
developed, there is still an urgent need to generate successful clinical
candidates with improved in vivo efficacies and reduced side effects [4].
We recently published series of dibenzosuberone and dibenzooxepinone
inhibitors with outstanding activity in biological systems and low ATP
competitiveness, combined with excellent selectivity against p38α MAP
kinase [5,6].
POS.138
Characterization of the HPA-1 polymorphism by MD
simulations and FRET measurements
Pagani, G.1; Ventura Pereira, J. P.2; Homeyer, N.1; Stoldt, V. R.2;
Scharf, R. E.2; Gohlke, H.1
1 Dept.
of Pharmaceutical and Medicinal Chemistry, Heinrich Heine University, Universitätsstr. 1,
40225 Düsseldorf, Germany
2 Dept. of Experimental and Clinical Hemostasis, Hemotherapy, and Transfusion Medicine,
Heinrich Heine University Medical Center, Moorenstr. 5, 40225 Düsseldorf, Germany
The human platelet integrin αIIbβ3 mediates platelet adhesion and
aggregation; it is essential for hemostasis but can also foster thrombus
formation [1]. αIIbβ3 is polymorphic; its β3 subunit carries the human
platelet antigen (HPA)-1. The HPA-1 polymorphism of αIIbβ3 arises from
a leucine-to-proline mutation resulting in HPA-1a (Leu33) or its variant
isoform HPA-1b (Pro33). This mutation is clinically relevant, since
patients with coronary artery disease, who carry the HPA-1b allele,
experience their myocardial infarction 5.2 years earlier than HPA-1a/1a
patients [2]. HPA-1b is also known as the prothrombotic variant of αIIbβ3,
since it was shown to activate platelets enhancing their adhesion and
thrombus formation as a consequence of increased classical outside-in
signaling. However, the underlying mechanism by which the mutation
contributes to the prothrombotic properties of the variant integrin has
remained elusive so far.
Integrins exist in two main and mutually exclusive conformations: the
bent, closed form and the unbent, open structure. Local and global
structural rearrangements are required in changing the closed to the
open form, thereby leading to integrin activation. In the present study, a
combined strategy, integrating large-scale all-atom molecular dynamics
(MD) simulations with FRET measurements, was used to characterize
the consequences of the Leu33→Pro33 exchange on the structural
dynamics of αIIbβ3 at an atomic level. MD simulations of the two αIIbβ3
variants starting from the closed conformation of in total 3 µs length were
carried out. For FRET, cyan or yellow fluorescent proteins had been
cloned to the C-termini of the αIIb or β3 subunits and expressed on
HEK293 cells.
Comparative analyses of the MD trajectories revealed that the Pro33Leu
mutation gives rise to a local instability that propagates and gradually
affects the entire structure, thus leading to the system being globally less
stable. FRET measurements confirmed the conformational changes
observed in our MD simulations and occurring in the cytoplasmic tails
upon integrin activation. MD results supported by experimental findings
reveal how a single-point mutation located more than 90 Å away from
any ligand binding site in αIIbβ3 can allosterically influence the fine-tuned
conformational equilibrium of this protein.
References:
1. Kunicki, T.J.; Newman, P.J.: Blood, 1992, 80: 1386-1404.
2. Zotz, R.B. et al.: J. Thromb. Haemost. 2005, 3:1522-1593.
POS.139
p38α MAP kinase inhibitors with excellent enzyme and
whole blood activity - enhancement of binding interactions
between hydrophobic region II and deep pocket
Wentsch, H. K.1; Walter, N.1; Mayer-Wrangowski, S. C.2; Rauh, D.2;
Laufer, S. A.1
1 Institute
of Pharmacy, Department of Pharmaceutical and Medicinal Chemistry, Eberhard Karls
Universitaet Tuebingen, Auf der Morgenstelle 8, 72076 Tuebingen, Germany
2 Faculty of Chemistry and Chemical Biology, Technische Universitaet Dortmund, Otto-HahnStrasse 6, 44227 Dortmund, Germany
The fundamental role of p38 mitogen-activated protein kinases (MAP
kinases) in the biosynthesis of proinflammatory cytokines like Il-1β and
TNFα underlines their importance as therapeutic targets for the treatment
Figure: Binding mode of compound 1 in p38α MAP kinase (PDB code: 3UVQ)
Our lead compound 1 (Figure) showed an excellent IC50 value (IC50 = 1
nM) with respect to p38α MAP kinase but unfortunately a moderate
inhibitor activity in a whole blood TNFα release assay (IC50 = 280 nM).
One reason might be a short target residence time due to a high ATP
concentration and competitive inhibition mechanism. Based on the X-ray
structure (PDB code: 3UVQ) [6] we synthesized compounds which form
the same interactions to the enzyme and can moreover enhance the
interactions between hydrophobic region II and the deep pocket of the
enzyme. Both are valid strategies to improve the shortcomings
mentioned above. Furthermore we pursued the strategy of a parallel
synthesis of a dibenzooxepinone and a dibenzosuberone scaffold both
to compare their affinity to the enzyme and their activity in whole blood
tests as well as to avoid potential metabolism issues of the
dibenzosuberone scaffold. Finally we synthesized a wide range of
dibenzepinones following the intentional binding mode and could improve
the IC50 value on the isolated enzyme down to the picomolar range and
IC50 values in whole blood tests below 50 nM.
References:
1. Player, M. R.: Curr. Top Med. Chem. 2009, 9: 598.
2. Tsai, C. J.; Nussinov, R. Semin. Cancer Biol. 2013, 23: 235-42.
3. Anton, R. et al.: PLoS One 2014, 9: e95641.54.
4. Zhang, J. et al.: T. in Pharm. Science 2007, 28: 286-295.
5. Baur, B. et al.: J. Med. Chem. 2013, 56: 8561-8578.
6. Fischer, S. et al.: J. Med. Chem. 2013, 56: 241-53.
POS.140
Synthesis of a modified deuterated ω-Acyl Ceramide [EOS]
Species and the Impact to the Nanostructure of Stratum
Corneum Lipid Model Membranes
Sonnenberger, S.1; Eichner, A.1; Lange, S.1; Langner, A.1; Neubert, R.
H. H.1; Dobner, B.1
Institute of Pharmacy, Martin-Luther-University Halle-Wittenberg, Wolfgang-Langenbeck-Str.
4, 06120 Halle (Saale), Germany
1
The mammalian skin covers an area of approximately 10% of the body
and is, with approximately 10% of the body mass, the largest organ of
human being. Most important for the organism is the skin´s barrier
function. The skin is divided into three main layers: the subcutis, as the
innermost layer, followed by the dermis and epidermis, as the outer layer.
The epidermis is additionally divided into further sublayers, in which the
Stratum corneum (SC), the outermost layer, plays the crucial role for the
skin barrier properties. The SC consists of flattened and keratin-filled
dead cells (corneocytes) which are embedded into a multilamellar lipid
matrix. The matrix consists of cholesterol (CHOL) and its derivatives such
as cholesterol sulfate (ChS), free fatty acids (FFA) and ceramides (CER).
As the main fraction of the SC lipids, ceramides play a fundamental role
in organisation of the lipid matrix. The ceramides are structurally very
heterogeneous. In general, they consist of a fatty acid bonded to the
amino group of different sphingoid bases. The fatty acid can exhibit
hydroxyl group in alpha or in omega position, whereas the omega
hydroxyl can be esterfied to another fatty acid, mainly linoleic acid. The
omega hydroxyl fatty acid is, with a chain length about 30 carbon atoms,
significantly longer than the non- or alpha-hydroxyl fatty acids with chain
length of about 18-26 carbon atoms. Due to this unusual chain length,
this ceramide (CER[EOS]) is mainly contributed to the SC´s barrier
function. Therefore it is necessary to receive a detailed insight into the
molecular arrangement of the lipid matrix organisation. Via neutron
POSTERS
scattering experiments it is possible to get structural and dynamical
information of biological membranes. Neutrons interact with the atomic
nucleus and penetrate deep into the membrane. Furthermore, it is
possible to distinguish between different isotopes, i.e. hydrogen and
deuterium, because of their varying neutron scattering lengths. However,
using extracted native lipids, it is difficult to get detailed information
because of their complex composition. So, model membranes, based on
the native lipid composition were prepared.
In the present work we describe the synthetic approaches of a modified
deuterated CER[EOS] species (Figure). The derivate consist of
sphingosine, 30-hydroxytriacontanoic acid and 10-methylhexadecanoic
acid instead of the native bonded linoleic acid. Earlier works already
showed, that such a modified ceramide is comparable in its phase
behaviour to the native CER[EOS] [1]. Via deuterium labelling,
conclusions can be made about the ceramide´s molecular arrangement
within the model membrane. For this purpose, 10-D3methylhexadecanoic acid was prepared, coupled with triacontan-1,30diol and after oxidation coupled with sphingosine. First results of neutron
scattering experiments show that for the labelled fatty acid varying
arrangements are possible.
Figure: deuterated methyl branched Ceramide EOS derivative
Acknowledgments: We would like to thank Helmholtz-Zentrum-Berlin (HZB, Berlin, Germany)
for the allocation of beam time for the neutron scattering investigations. Further we thank Evonik
Industries AG (Essen, Germany) for provision of ceramide [AP] C-18. This work was supported
by grants of the Deutsche Forschungsgemeinschaft (DFG).
References:
1. Engelbrecht et al.: Soft Matter 2011, 7(19): 8998-9011.
omega-hydroxy acid can be esterified (EO), e.g. with linoleic acid. The
chain length of the acids and the sphingoid bases varies mainly between
C18 and C26.
Figure: deuterated Ceramide [NP] and [NS]
To investigate the arrangement and the composition of the different
ceramide species in the lipid matrix, neutron scattering and LC/ESI-MS
are commonly used methods [1-3]. Therefore, specifically labelled
ceramides with a well-defined structure are needed. For our experiments
we used ceramides with a chain length of 18 carbon atoms in the
sphingoid base and changed the length of the fatty acid (18, 22 and 24
carbon atoms). In this work we will describe a way to synthesise
deuterium labelled non-hydroxy fatty acids at the end of the alkyl chain
via the use of LiAlD4. Furthermore, we tried to optimise the coupling of
the fatty acid with the sphingoid base and therefore tested different
coupling reagents. These results will be additionally presented here.
Acknowledgments: We would like to thank Evonik Industries AG (Essen, Germany) and the
Deutsche Forschungsgemeinschaft (DFG) for the financial support.
References:
1. Sahle, F. F. et al.: Journal of Pharmaceutical and Biomedical Analysis 2012, 60: 7-13.
2. Engelbrecht, T. et al.: Soft Matter 2011, 7: 8998-9011.
3. Schröter, A. et al.: Biophysical Journal 2009, 97: 1104-1114.
POS.141
Design and optimization of N-benzyl benzamides: A novel
fused scaffold for orally available dual sEH/PPARγ
modulators for treatment of metabolic syndrome
Proschak, E. 1
1 Institute
of Pharmaceutical Chemistry, Goethe-University Frankfurt, Max-von-Laue-Strasse 9,
D-60438 Frankfurt a. M., Germany.
For abstract see Short Poster Lectures SPL.008 page 105.
POS.142
Synthesis of specific deuterated ceramide species [NP]
and [NS] with different chain lengths
Lange, S.1; Sonnenberger, S.1; Eichner, A.1; Langner, A.1; Neubert, R.
H. H.1; Dobner, B.1
1 Institute of Pharmacy, Martin Luther University Halle-Wittenberg, Wolfgang-Langenbeck-Str. 4,
06120 Halle (Saale), Germany
The stratum corneum (SC), as the outermost layer of human skin, plays
an important role in the homeostasis of mammalians (e.g. regulation of
water and the thermal balance). Normally, phospholipids are the main
lipid part of biologically membranes, which have to be penetrable for a
bearing transport process through them. In contrast to the lipid matrix of
the SC, where they are totally lacking, phospholipids are known to keep
the membranes fluidity. Compared to that, the ceramides are highly
discussed to play a key role to the rigidity of the SC barrier function and
further to protecting functions of the skin. For this reason, there is a high
interest in the detailed structure of the lipid layers, ceramides are
arranging.
Next to free fatty acids, cholesterol and its salts, ceramides are the main
ingredients of the SC lipid mixture, as they are a group of structurally very
heterogeneous sphingolipids. They consists of a long-chain fatty acid
(alpha-hydroxy (A), non-hydroxy (N) or omega-hydroxy (O)), which is
bond to a sphingoid base (sphingosine (S), dihydrosphingosine (DS),
phytosphingosine (P) or 6-hydroxysphingosine (H)). Furthermore, an
POS.143
Determining the molecular consequences of clinically
relevant glutamine synthetase mutations
Frieg, B.1; Görg, B.2; Homeyer, N.1; Keitel, V.2; Häussinger, D.2; Gohlke,
H.1
1 Institute
for Pharmaceutical and Medicinal Chemistry, Heinrich-Heine-University,
2 Clinic for Gastroenterology, Hepatology, and Infectious Diseases, Heinrich-Heine-University,
Moorenstr. 5, 40225 Düsseldorf, Germany
Glutamine synthetase (GS) catalyzes the ligation of ammonia and
glutamate to glutamine under the use of ATP and, thus, essential for
nitrogen metabolism [1,2]. Loss of hepatic GS activity has been linked to
serious clinical conditions [3]. In particular, two mutations of human GS
(R324C and R341C) were connected to congenital glutamine deficiency
with severe brain malformations resulting in neonatal death [4]. In a single
case known to date, another GS mutation (R324S) was identified in a
neurologically compromised patient [5]. However, the underlying
molecular mechanisms of GS deactivation by these mutations have not
been understood yet.
By molecular dynamics simulations, free energy calculations, and rigidity
analyses we found that all three mutations influence ATP binding, the first
step of GS glutamine formation cycle. In the case of the R324S and
R324C mutations, we found a loss of direct salt-bridge interactions with
the substrate ATP that cause a deterioration of GS catalytic activity.
Remarkably, for the R324S mutant, we observed water-mediated
interactions with ATP that reduce this effect and may explain the
suggested higher GS residual activity [6]. The R341C mutation
destabilizes residue R340 that is important for ATP binding. Furthermore,
we predicted the R341C mutant to result in a significant destabilization of
helix H8, which should hamper glutamate binding. In order to provide
evidence for this, we introduced an additional GS variant through alanine
mutagenesis of amino acids interacting with R341, mimicking the loss of
interactions in the R341C mutant. After GS overexpression in HEK293
cells, dot-blot analyses revealed that the structural stability of H8 was
impaired in the case of the newly introduced GS mutant. This results in a
loss of masking of the epitope in the glutamate binding pocket for a
monoclonal anti-GS antibody by L-methionine-S-sulfoximine; in contrast,
cells transfected with wild type GS did show the masking.
Our analyses reveal complex molecular effects underlying GS
deactivation in three clinically relevant mutants. Furthermore, since there
is currently no adequate therapy available [6] to treat a glutamine
deficiency caused by the R324S mutant, our findings could stimulate the
development of ATP binding-enhancing molecules by which the R324S
mutant can be “repaired”.
Acknowledgments: We are grateful to the ‘‘Zentrum für Informations und Medientechnologie’’
(ZIM) at the Heinrich Heine University for computational support. This work was supported by the
Deutsche Forschungsgemeinschaft through the Collaborative Research Center SFB 974
(‘‘Communication and Systems Relevance during Liver Damage and Regeneration’’, Düsseldorf).
References:
1. Häussinger, D.: Eur. J. Biochem. 1983, 133(2): 269-275.
2. Häussinger, D.: Biochem. J. 1990, 267(2): 281-290.
3. Qvartskhava, N. et al.: P. Natl. Acad. Sci. USA 2015, 112(17): 5521-5526.
4. Häberle, J. et al.: New. Engl. J. Med. 2005, 353(18): 1926-1933.
5. Häberle, J. et al.: Mol. Genet. Metab. 2011, 103(1): 89-91.
6. Häberle, J. et al.: Orphanet J. Rare. Dis. 2012, 7(48): 1-10.
analgesic flupirtine, which on the one hand is a less active opener of
potassium channels but on the other hand a clinically valuable drug.
However, in some patients even the use of the drug flupirtine rises some
concerns related to drug-induced liver injury, most probably because of
metabolic toxification and the formation of reactive metabolites. This
poses the question whether there is a pattern of cellular reactivity and/or
toxicity related to physicochemical properties, i.e. a hierarchy of activity
associated with reversible or irreversible covalent interactions with
KV7.2/3 channels or other proteins. In order to investigate structureactivity-relationships for this target we synthesized multiple merged
structures with the goal of identifying essential moieties of the active
molecules aiming for agents with clean toxicity profile and retained
activity of the ICA lead compound. The synthesised structures involve
carbamate-derivatives of ICA-027243 as well as amide analogs of
flupirtine with shifted positions of the pyridine-nitrogen atom.
As recent results by our group showed a potential correlation between
the oxidizability and the activity of flupirtine and its derivatives [2], the
developed compounds are to be tested by cyclic voltammetry. To verify
the postulated association the anodic peak potentials are compared to
the activities in further studies.
POS.144
TopModel: A multi-template meta-approach to Homology
Modeling
Mulnaes, D.1; Gohlke, H.1
1 Mathematisch-Naturwissenschaftliche
Fakultät, Institut für Pharmazeutische und Medizinische
Chemie, Heinrich-Heine-Universität, 40225 Düsseldorf, Germany
Knowledge of a protein structure is essential to understand its function
[1], evolution [2], dynamics [3], stability [4], interactions [5,6] and for datadriven protein- or drug-design [7]. Experimental structure determination
rates however are far exceeded by that of next-generation sequencing,
resulting in less than 1/1000th of proteins having a known structure.
Computational structure prediction seeks to alleviate this problem, and
many different methods were developed in the last decade alone. The
CASP experiments [8] are widely considered the golden standard for
structure prediction benchmarks and have shown the value of
consensus- and meta-methods that utilize complementary algorithms.
We present a multiple-template meta-approach to structure prediction
that combines a broad range of state-of-the-art methods to provide a
versatile work-flow and toolbox for structure prediction. Our automated
evaluation shows an accuracy competitive with state-of-the-art
automated procedures and enables easy re-evaluation/parametrization
on any dataset. We anticipate that future integration of new methods will
increase the accuracy, sensitivity and applicability of our consensus
methods, and that our workflow can be used to evaluate threading
software and software for multiple sequence alignments in a consistent
manner based on the quality of models produced by the generated
alignments.
References:
1. Amato, G. et al.: ACS Med. Chem. Lett. 2011, 2(6): 481–484.
2. Lemmerhirt, C. J. et al.: ChemMedChem 2014, 10(2): 368–379.
POS.146
Acknowledgments: We thank the Ministry of Innovation, Science and Research of NRW and
HHU Düsseldorf for scholarships within the CLIB-Graduate Cluster Industrial Biotechnology. We
are grateful to our coauthors in refs. 1 & 5 for fruitful collaborations.
Development of a combinatorial chemistry strategy for the
synthesis of DNA-encoded libraries
References:
Škopić, K. M.1; Bugain, O.1; Kalliokoski, T.2; Brunschweiger, A.1
1. Widderich, N. et al.: J. Mol. Biol. 2014, 426(3): 586-600.
2. Prieto, A .I. et al.: Structure 2013, 21(9): 1690-1697.
3. McCammon, J. A.; Gelin, B. R.; Karplus, M.: Nature 1977, 267(1): 585-590.
4. Rathi, P.C.; Hoeffken; H. W.; Gohlke, H.: J. Chem. Inf. Model. 2014, 54(2): 355-361.
5. Gohlke, H. et al.: J. Chem. Inf. Model. 2013, 53(10): 2493-2498.
6. Janin, J.: Protein Science 2005, 14(2): 278-283.
7. Moult, J. et al.: Proteins 2014, 82(S2): 1-6.
POS.145
Synthesis and characterization of potassium channel
openers with N-benzyl-aminopyridine moiety
Bock, C.1; Lemmerhirt, C.1; Link, A.1
1 Institute
of Pharmacy, Ernst-Moritz-Arndt-University Greifswald, Friedrich-Ludwig-Jahn-Str. 17,
17489 Greifswald, Germany
The small molecule ICA-027243 is a very potent modulator of KV7.2/3
channels but its reactivity-related toxicity in a rat model impedes
therapeutic usage [1]. The structure bears some similarities with the
1 Faculty
of Chemistry and Chemical Biology, Technische Universität Dortmund, Otto-HahnStraße 6, 44227 Dortmund, Germany
2 Lead Discovery Center GmbH, Otto-Hahn-Straße 15, 44227 Dortmund, Germany
The identification of small molecule ligands for proteins of interest is a
crucial first step in small molecule probe or drug development programs.
The screening of large, pooled DNA-encoded small molecule libraries
(DELs) by a conceptually simple selection assay represents a validated
technology for the target-based identification of bioactive compounds [1].
We synthesize DNA-encoded screening libraries on “privileged scaffolds“
such as the pyrazolopyrimidine 1. The scaffolds display a
functionalization pattern allowing for coupling of these compounds to 5´aminolinker modified DNA and subsequent combinatorial substitution by
amide synthesis and Cu(I)-catalyzed alkyne-azide cycloaddition. We
selected a set of 150 carboxylic acid and a set of 110 azide building
blocks with the help of chemoinformatics in order to optimize diversity of
the library (Tanimoto fingerprint analysis), and to control the calculated
physicochemical properties of the 16.500-membered library. The aim of
the present study was to develop a procedure for parallel, combinatorial
synthesis of the library. Whereas 100 of the 110 azides showed excellent
conversion (> 90%) to the target triazoles, the coupling efficiencies of the
carboxylic acids were very variable. Thus, we decided to couple scaffold
POSTERS
1 to 5’-amino-linker modified DNA on solid support, remove the Fmoc
group, and introduce the carboxylic acid building blocks in parallel
manner on solid support. The DNA-conjugates were then purified by RPHPLC, encoded by T4 ligation, and immobilized on DEAE
(diethylaminoethanol) sepharose, an ion exchange resin with excellent
DNA bind and release properties [2]. We then introduced the azide
building blocks by Cu(I)-catalyzed cycloaddition and were able to
extensively wash the resin with EDTA solution to remove copper
contaminants prior DNA release. In summary, we have developed an
efficient and high-yielding protocol for the combinatorial synthesis of
DNA-encoded screening libraries.
a)
Figure: Development of an encoding strategy for small molecules based on DNA ligation and of
the selection assay with encoded model substances and their target proteins.
References:
1. Manocci, L. et al.: Chem. Commun. 2011, 47: 12747-12753.
2. Buller F. et al.: Bioconjugate Chem. 2010, 9(21): 1571–1580.
POS.148
From stem cell screening towards small molecule tools for
in vivo heart regeneration
b)
Längle, D.1; Flötgen, D.1; Duburs, G.2; Strohmann, C.1; Koch, O.1;
Werner, T.3; Hirt, M.3; Schade, D.1
1 Department
of Chemistry & Chemical Biology, TU Dortmund, Otto-Hahn-Straße 6, D-44227
Dortmund, Germany
2 Latvian Institute of Organic Synthesis, Aizkraukles 21, Riga LV-1006, Latvia
3 Institute of Experimental & Clinical Pharmacology and Toxicology, University Hospital
Hamburg-Eppendorf, Martinistr. 52, D-20246 Hamburg, Germany
Figure: Evaluation of combinatorial synthesis strategies to a kinase-directed DEL.
References:
1. Buller, F. et al.: Bioconjugate Chem. 2010, 21(8): 1571-1580.
2. Halpin, D. R. et al.: PLoS Biology 2004, 2(7): 1031-1038.
POS.147
Encoding small molecule libraries with DNA - and
development of a selection assay for DNA-encoded small
molecule libraries
Jung, K.1; Bugain, O.1; Brunschweiger, A.1
1 Faculty
(DELs) represents a promising technology for the discovery of small
molecule ligands at target proteins [1,2] Protein ligands can be identified
from these libraries by selection. We use double strand (ds)DNA to
encode our small molecule libraries. dsDNA is a rather rigid rod-like
structure less likely forming secondary structures that might interfere with
the selection assay. We developed an efficient encoding strategy based
on T4 DNA ligation in order to record the chemical steps employed in
library synthesis. Tetramer overhangs gave high ligation yields, also in
two consecutive ligations that are required to encode two building blocks,
whereas longer overhangs yielded multiple side products in our hands.
To our astonishment a PEG4-linker used as spacer between the small
molecule and the DNA inhibited the ligation reaction under standard
conditions (16 °C; 16h). To overcome this effect different conditions
(temperature, incubation-time, lengths of overhangs, additives to the
buffer) were tested. We found out that efficient ligation can be achieved
by ligating DNA at 20 °C, for 16h and adding PEG6000 to the ligation
buffer. We used the optimized encoding strategy to synthesize small
molecule-dsDNA conjugates that we employ to establish a selection
assay protocol: desthiobiotin (1), binding to streptavidin; 4sulfamoylbenzoic acid (2), binding to carbonic anhydrase IX; and N1-(2aminophenyl)-N4-terephthalamide (3) binding to HDAC 1. Each DNA has
a unique primer region so that for development of a selection assay for
DNA-encoded small molecule libraries we can assess conveniently
various selection assay conditions by PCR, such as different incubation
times, number of washing cycles, different temperatures, ionic strengths
of the wash buffer. The newly established selection assay will be used to
identify bioactive small molecules from in-house synthesized DNAencoded small molecule libraries.
Background: The development of small molecules that control stem cell
fate is of tremendous interest for various regenerative medicine
applications as it opens up the druggable space [1]. From a high-content
screen of 17,000 compounds in mESCs we discovered in the past a novel
class of TGF-β inhibitors in the context of cardiac differentiation [2]. A
specific subclass of 1,4-dihydropyridines (DHPs) stimulated
cardiomyogenesis from murine and human ESCs in stages when
uncommitted mesoderm specifies towards a cardiac fate [2,3].
Objective: Since TGF-β is also involved in cardiac remodeling and
fibrosis, we propose a dual mode of action for these DHPs as
regenerative agents. However, in order to demonstrate proof-of-concept
in animal models later in development, the compounds require hit-to-lead
optimization. Here, a translational case study illustrates earliest steps in
a preclinical drug development campaign for phenotypic screeningderived small molecules.
Results: We describe the multidisciplinary medicinal chemistry workflow
from ‘screening hit’ to in vivo-suitable pharmacologic tool candidates.
Ligand-based (quantitative) structure-activity relationships (SARs), X-ray
crystal structure analysis, pathway selectivity and in vitropharmacokinetic profiling provided a solid basis for the development of
selective, potent and drug-like lead candidates [4]. Key obstacles that
typically limit in vivo applicability, such as poor compound solubility and
stability, could be addressed. Moreover, we demonstrated efficacy in an
engineered heart tissue (EHT, from neonatal rat cardiomyocytes) as a
model of hypertrophy and fibrosis. Additionally, utilizing our ligand-based
pharmacophore model, a virtual screening approach gave rise to new
chemotypes of TGF-β inhibitors (= scaffold hopping).
Conclusions: Systematic, medicinal chemistry-driven efforts led to highly
attractive small molecules as novel in vivo pharmacology tool candidates
to study heart regeneration and remodeling after myocardial infarction.
References:
1. Längle, D. et al.: ACS Chem. Biol. 2014, 9(1): 57-71.
2. Willems, E. et al.: Cell Stem Cell 2012, 11(2): 242-252.
3. Schade, D. et al.: J. Med. Chem. 2012, 55(22): 9946-9957.
4. Längle, D. et al.: Eur. J. Med. Chem. 2015, 95: 246-266.
POS.149
3,4-Bis(indol-3-yl)cyclobut-3-ene-1,2-diones as novel
potential antimalarial drugs
Lande, D. H.1; Nasereddin, A.2; Dzikowski, R.2; Preu, L.1; Grünefeld, J.1;
Kunick, C.1
1 Institut
2 Department of Microbiology and Molecular Genetics, IMRIC, Hebrew University, Hadassah
Medical School, P.O. Box 12272, Jerusalem 91220, Israel
Owing to the restricted arsenal of antimalarial drugs and the increasing
resistance of the parasite, malaria caused by protozoal parasites of the
genus Plasmodium remains one of the most deadly infectious diseases
of humans [1]. Annually, more than half a million people of the global
population, particularly young children and pregnant women in tropical
African countries, are killed by this disease [2]. The urgent need for
developing novel potential antimalarial drugs is therefore obvious.
Herein we report the syntheses and characterization of 3,4-bis(indol-3yl)cyclobut-3-ene-1,2-diones and their activity profiles against
erythrocytic stages of P. falciparum. Furthermore, molecular docking
studies in a homology model of PfGSK3, a putative intracellular target for
the new compound class, were conducted to explain the observed
structure activity relationships.
Acknowledgments: This project was funded by the German Federal Ministry of Education and
Research (BMBF BioDisc 7; 13GW0024).
References:
1. Murray, C. J. L. et al.: J.-Lancet 2012, 379(9814): 413–431.
2. WHO, World Malaria Report 2014.
POS.150
Synthesis of functionalized “privileged” scaffolds for DNAencoded libraries
Bugain, O.1; Klika Škopić, M.1; Brunschweiger, A.1
1 Faculty
(DELs) represents a validated technology for the target-based discovery
of bioactive compounds [1]. DELs are composed of small organic
molecules covalently coupled to DNA sequences serving as PCRamplifiable identification bar codes. Small molecule protein ligands can
be identified from these libraries by selection. Our aim is to generate
DNA-encoded compound libraries based on “privileged scaffolds“ [2].
These scaffolds are overrepresented among bioactive small molecules
and therefore attractive core structures for library synthesis. We required
the scaffolds to be endowed with a functionalization pattern allowing for
coupling to 5´-aminolinker modified DNA and subsequent combinatorial
substitution by amide synthesis and alkyne-azide cycloaddition. We
designed synthesis strategies that yielded the xanthine (1),
pyrazolopyrimidine (2), and benzodiazepine (3, 4, 5) ring systems
displaying a carboxylic acid, an Fmoc-protected amine, and a terminal
alkyne. Currently, we are using these five compounds to synthesize
DNA-encoded libraries by split-and-pool combinatorial synthesis. Each
scaffold will yield a final pooled DNA-encoded library encompassing
16,500 compounds.
POS.151
Alkoxyurea-based HDAC Inhibitors with quinoline cap
groups possess improved activity against chemoresistant
cancer cells
Stenzel, K.1; Hamacher, A.1; Hansen, F. K.1; Kassack, M. U.1; Kurz, T.1
1 Institut
für Pharmazeutische und Medizinische Chemie, Heinrich Heine Universität,
Universitätsstrasse 1, 40225 Düsseldorf, Germany
Cancer research remains to be one of greatest challenges of our time.
Histone deacetylases (HDACs) are linked with different types of cancer
and HDAC inhibitors (HDACi) modulate the expression of genes
associated with cell cycle, cell differentiation and in addition induce
apoptosis in many tumor cells via cell death pathways. Many HDACi
demonstrate pronounced cytotoxicity against tumor cells and weaker
toxicity on normal cells [1]. Currently four HDACi have been approved for
cancer therapy by the FDA. Variation of epigenetic regulation is one of
the known causes of abnormal gene expression in a malignant tumor
resistant to chemotherapy. Experimental data suggest that the
combination of HDACi with established anticancer drugs (e.g. cisplatin)
provides synergistic effects in the treatment of hematological and solid
tumors, probably generated through HDACi-mediated increased
accessibility of DNA [2,3,4].
Isoform-selective HDACi could provide a more effective chemotherapy
compared to pan-inhibitors, with fewer side effects and more precise
therapeutic properties [5]. Starting from LMK235, a HDACi with HDAC4
and 5 preference [6], we reasoned that the enlargement of the cap group
and the connecting unit should lead to a novel type of HDACi with HDAC6
preference. The isoform profiling of the prototype compound 1a
(LMK214) confirmed our hypothesis and revealed a pronounced
preference for HDAC6 and no inhibition of HDAC4 up to a concentration
of 10 µM. A microwave-assisted protocol allowed the systematic
variation of the cap moiety. In addition, selected alternative zinc-binding
groups (ZBG) were introduced instead of the hydroxamic acid. The
synthesized novel inhibitors are characterized by a substituted quinoline
or naphthalenyl cap group and an alkoxyurea connecting unit linker
region.
Figure: Strategy and target compounds
The biological evaluation of the target compounds included cellular
HDAC and MTT assays on the HNSCC cell line Cal27 and the ovarian
cancer cell line A2780 and their cisplatin resistant sublines. Some of the
compounds showed similar or improved eﬀects compared to vorinostat
on inhibition of cellular HDACs in a whole-cell HDAC assay. Furthermore,
incubation with these compounds 48h prior to cisplatin resulted in an
enhancement of cisplatin-induced cytotoxicity in the cisplatin resistant
sublines.
References:
1. Witt, O. et al.: Cancer Lett. 2009, 277: 8–21.
2. Ong, P.-S. et al.: Int. J. Oncol. 2012, 40: 1705–1713.
3. Eckstein, N. et al.: J. Biol. Chem. 2008, 283: 739–750.
4. Mueller, H. et al.: J. Biomol. Screen. 2004, 9: 506–515.
5. Balasubramanian, S.; Verner, E.; Buggy, J. J.: Cancer Lett. 2009, 280: 211–221.
6. Marek, L. et al.: J. Med. Chem. 2013, 56: 427–436.
Figure: “Privileged” scaffolds functionalized to furnish DNA-encoded libraries
References:
1. Stockwell B. R.: Nature 2004, 432(7019): 846-854.
2. Evans, B. et al.: J. Med. Chem. 1988, 31(12): 2235-2246.
POSTERS
POS.152
Fighting the Human African Trypanosomiasis – Optimizing
an aza-analogous Furamidine by a new prodrug principle
elastase, bovine chymotrypsin, bovine trypsin, human factor Xa, human
thrombin, human cathepsin L and B) was evaluated by different activity
assays and analyzed e.g. with the slow-binding equation. To verify the
ability of the probe to target HLE, it was incubated with HLE, subjected
to SDS-PAGE and evaluated by in-gel fluorescence detection.
Polosek, P.1; Girreser, U.1; Clement, B.1
Department of Pharmaceutical and Medicinal Chemistry, Institute of Pharmacy, CAU Kiel,
Gutenbergstraße 76, 24118 Kiel, Germany
1
The Human African Trypanosomiasis (HAT) is known since the beginning
of the 20th Century with the first documented case in 1902 [1]. In the
1960’s the HAT seemed to be controlled, but at the beginning of the 21st
Century the reported cases increased significantly due to the negligence
of the disease [1]. The infection can be separated into 2 stages: the first
stage named haemo-lymphatic stage is possible to cure by an early
treatment. The second stage called the meningo-encephalitic stage
remains hard to treat mainly due to insufficient therapy options (strong
adverse effects, difficult application) with an inadequate therapy referring
to adverse effects and difficult appliance [1]. The pipeline of available
substances to cure the patients is very small and since the admission of
nifurtimox and eflornithine as a combination for the second stage of the
HAT no substance has been approved for around 20 years [1]. In 2001
pafuramidine maleate (DB 289) began its clinical trials as the first oral
applicable substance against HAT [1]. Unfortunately the substance
missed its approval due to liver and kidney toxicity in an additional phase
Ι trial [1].
Figure: Inactivation of a serine protease (Ser-OH) by the fluorescence-labeled sulfonyloxyphthalimide derivate.
Results: A novel probe was designed, synthesized and its spectroscopic
properties were determined. The probe was shown to be selective for
HLE with an kobs/[I] value of 8360 M-1s-1 and an IC50 value of 12 nM
(determined from a reaction time of 1 hour), whereas the other enzymes
were inhibited with IC50 values of more the 1 µM. The analysis of the ingel fluorescence revealed a higher detection limit than the common
coomassie staining.
Conclusion: We obtained a selective and sensitive probe for HLE which
is expected to be a powerful tool to study the activity of this enzyme in
cell or tissue samples.
References:
1. Korkmaz, B. et al.: Pharmacol. Rev. 2010, 62(4): 726-759.
2. Kasperkiewicz, P. et al.: Proc. Natl. Acad. Sci. USA 2014, 111(7): 2518-2523.
3. Neumann, U.; Gütschow, M.: J. Biol. Chem. 1994, 269(34): 21561-21567.
Figure: Pentamidine, Furamidine (DB 75), Aza-analogue of Furamidine (DB 829)
Based on that structure many new substances were synthesized heading
to aza-analogous structures with promising potentiality. So far only
substances including the pyridine ring with the nitrogen atom in o-position
to the furane ring revealed remarkable results in the cure rate of mice
and monkeys in the first stage as well as in the second stage of the HAT
[2]. However, the results only appeared by intramuscular application and
did not occur by the oral treatment. Therefore it is necessary to exchange
the prodrug principle used so far. The prodrug principle reported in the
published articles is an O-methylation of the amidoxime [2]. Other
prodrug principles are improving the activation of the prodrug, the water
solubility and the oral bioavailability [3]. Due to this information a new
prodrug of an aza-analogue pentamidine derivation has been developed
for oral application. The chosen prodrug-principle is an amidoximeester
with a very good solubility which is currently being tested in a mouse
model for the second stage of the HAT.
References:
1. WHO Technical Report Series 984 2013
2. Thuita et al.: PLoS Negl. Trop. Dis. 2015, 9(2):e0003409. doi: 10.1371/journal.pntd.0003409
3. Kotthaus et al.: ChemMedChem 2011, 6: 2233–2242. doi: 10.1002/cmdc.201100422
POS.154
Synthesis of fluorine-containing PDE10A-Inhibitors as
potential Ligands for Positron Emission Tomography (PET)
Franz, L.1; Scheunemann, M.2 ; Wagner, S.2; Lang, M.1; Brust, P.2; Briel,
D.1
Institute of Pharmacy, University of Leipzig, Bruederstraße 34, 04103 Leipzig, Germany
Dresden-Rossendorf, Institute of Radiopharmaceutical Cancer Research,
Permoserstraße 15, 04318 Leipzig, Germany
1
2 Helmholtz-Zentrum
Phosphodiesterases (PDE´s) are second messenger hydrolysing
enzymes and important regulators of signal transduction mediated by
these molecules. PDE10A, a cAMP and cGMP sensitive hydrolase, is
primarily expressed in the striatum and was identified as drug target for
the therapy of diverse disorders in the central nervous system (CNS) [1]
like schizophrenia or chorea huntington [2]. Recently, 1-arylimidazo[1,5a]quinoxalines have been reported to be potent and selective inhibitors
of PDE10A [3]. In terms of a potential use as 18F-labelled PET imaging
agent new substituted derivatives were synthesized. It has been shown
that the methoxy substituted inhibitors are prone to metabolic oxidation,
which leads to a loss of inhibitory potency or ability to cross the blood
brain barrier [3,4].
POS.153
A Sulfonyloxyphthalimide Derivate as an Activity-based
Probe for Elastase
Schulz-Fincke, A. C.1; Tikhomirov, A. S.1,2; Gütschow, M.1
1 Pharmaceutical
Institute, Pharmaceutical Chemistry I, University of Bonn, An der Immenburg 4,
D-53121 Bonn, Germany
2 Present address: Gause Institute of New Antibiotics, 11B. Pirogovskaya Street, Moscow 119021,
Russia
Objective: Aberrant expression of human leucocyte elastase (HLE) can
lead to degradation of endogenous proteins and extracellular matrix
components. Such a scenario is associated with pulmonary diseases and
lung cancer [1]. Fluorescence-labeled probes are expected to be very
useful for investigating the cell biology and (patho)physiology of HLE [2].
Materials and Methods: The design of the probe is based on a
phthalimide structure as a new reactive warhead. It is expected that the
nucleophilic active site serine residue of HLE attacks the phthalimide
warhead, following by a ring opening and a subsequent Lossen
rearrangement. This reaction leads to a covalent modification of the
target HLE (Figure) [3]. A polyethylene glycol-derived linker connects the
warhead with coumarin 343 which was introduced to visualize the active
enzyme. The inactivation of several proteases (HLE, porcine pancreatic
To improve the metabolic stability of inhibitors the methoxy function in
position 6 was exchanged by chlorine. In the first synthesis step chlorine
was introduced at position 6 by electrophilic aromatic substitution. An
electron deficient system was generated in step 2 by oxidation of the
amine to a nitro function to allow the nucleophilic aromatic substitution of
fluorine by 4-methylimidazole in step 3. Afterwards, the amine was
recovered by acidic reduction with elementary iron in step 4 and
acetylated in step 5. Cyclisation in step 6 was realized by a BischlerNapieralski reaction. The derivatization of the 1-arylimidazo[1,5a]quinoxaline was focused on position 1 and 8. Finally, the fluoropyridinyl-group was introduced by Suzuki-coupling with the
corresponding boronic acid at the brominated positions to afford the
mono- or disubstituted pyridinyl derivatives. All compounds were
characterized by high performance liquid chromatography, nuclear
magnetic resonance spectroscopy and mass spectrometry.
It is expected that the new chlorinated derivatives have the same
pharmaceutical effects as their methoxy analogues.
A 3D-Diverse Fragment Library for Crystallographic
Screening and Drug Discovery
Acknowledgments: Thanks are to J. Ortwein for HPLC analysis, Dr. L. Hennig for recording and
analysis of NMR data and Dr. J. Preidl for LC-MS analysis.
Metz, A.1; Huschmann, F. U.1,2; Schiebel, J.1; Mueller, U.2; Weiss,
M. S.2; Heine, A.1; Klebe, G.1
References:
1. Liras, S.; Bell, A. S.: Phosphodiesterases and Their Inhibitors (Wiley-VCH) 2014.
2. Schmidt, C. J. et al.: J. Pharm. Exp. Ther. 2008, 325: 681-690.
3. Malamas, M. et al.: J. Med. Chem. 2011, 54: 7621–7638.
4. Schwan, G.: Synthese und in vitro Metabolismus fluorhaltiger PDE10A-Inhibitoren als
potentielle PET-Liganden, 2011, University of Leipzig, Germany.
POS.155
Structure characterization of 17β-hydroxysteroid
dehydrogenase Type 14 and
identification of the first inhibitors
Bertoletti, N.1; Braun, F.1; Hartmann, R. W. 2; Möller, G.3; Adamski, J.3;
Heine, A.1; Klebe, G.1; Marchais-Oberwinkler, S.1
1 Institute
for Pharmaceutical Chemistry, Philipps University Marburg, Marbacher Weg 6, 35037
Marburg, Germany
2 Helmholtz Institute for Pharmaceutical Research Saarland (HIPS), 66123 Saarbrücken,
Germany
3 Helmholtz Zentrum München, German Research Center for Environmental Health, 85764
Neuherberg, Germany
17β-Hydroxysteroid dehydrogenase type 14 (17β-HSD14) is a recently
characterized enzyme [1,2], which is mainly located in the brain, liver and
placenta. 17β-HSD14 catalyses the oxidation in vitro of estradiol,
5-androstene-3β, 17β-diol and testosterone to estrone,
dehydroepiandrosterone and androstedione, respectively, using NAD+ as
cofactor. The functional role of this enzyme remains unclear.
Administration of estradiol is efficient in the treatment of neuronal
diseases like migraine, schizophrenia or Alzheimer disease but is related
to severe side effects. We expect that inhibition of 17β-HSD14 will lead
to a local increase of estradiol in the brain, which might be beneficial for
the treatment of these neuronal disorders or for the prevention of
Alzheimer disease. Potent and selective inhibitors are also useful tools
to study the role of enzymes in vivo. The goal of this study was to identify
the first inhibitors of 17β-HSD14 and optimized their substitution pattern
using the 3D structure of the protein/ligand complex.
First of all the pure protein was produced in high amount after
heterologous expression in E. coli and the purification protocol was
established. Then a competitive enzymatic assay was established using
the purified enzyme (fluorescence-based assay, following cofactor
fluorescence changes). The most interesting inhibitor identified so far in
our laboratory was advanced to crystallographic study.
The purified enzyme was crystallized as apoenzyme, holoenzyme with
NAD+ and in complex with the most interesting inhibitors. The 3D
structures of different complexes were resolved and will be presented.
These structures will be useful to get insight in to the active cleft and
improve the inhibitor potency by structure-based design.
References:
1. Lukacik, P. et al.: Biochem. J. 2007, 402(3): 419–427.
2. Marchais-Oberwinkler, S. et al.: J. Steroid Biochem. Mol. Biol. 2011, 125(1-2): 66–82.
POS.156
Institute of Pharmaceutical Chemistry, Philipps-University Marburg, Marbacher Weg 6, 35032
Marburg, Germany
2 Helmholtz-Zentrum Berlin, Institute Soft Matter and Functional Materials, Macromolecular
Crystallography (HZB-MX)
Electron Storage Ring BESSY II, Albert-Einstein-Str. 15, 12489 Berlin, Germany
1
Ligand binding pockets differ regarding their shape and arrangement of
potential interaction sites. Thus, we design a general-purpose 3D-diverse
fragment library for crystallographic fragment screening that can address
a great variety of binding sites while providing particularly suitable
starting points for subsequent drug discovery efforts. To facilitate
fragment-to-lead evolution after hit identification, this library must
adequately cover the accessible chemical space of fragments including
substructures of drug-like ligands.
A reasonable coverage of chemical space aims at fully exploiting the fact
that already a small selection of appropriate fragments (~103 cpds.)
covers a much larger proportion of the overall chemical fragment space
(~107 cpds.) than a typical high-throughput screening collection (105–106
cpds.) with respect to the drug-sized chemical space (~1063 cpds.,
MW < 500 Da). Despite the relatively low affinity of fragments to their
target, their high ligand efficiency (ΔGbind. divided by number of nonhydrogen atoms) makes them excellent starting points for drug discovery.
In addition, fragments often bypass the strict steric requirements for the
binding of larger drug-like ligands, thus leading to high hit rates up to
15%.
Customarily, different biophysical pre-screening methods are applied,
often serially, to limit the number of fragments to a selection manageable
by routine X-ray crystallography. However, a comparative study with our
361-fragment library [1] confirmed a limited overlap amongst prescreening methods [2] that also missed many of direct crystallographic
screening hits [1]. In contrast, we strive for an appropriate library design
and use state-of-the-art virtual screening to equally increase
effectiveness and enrichment at no cost of materials. In conjunction with
its increased capacity and level of automation this suggests direct
crystallographic screening by crystal soaking of a small and preferably
target- or structure-based selection of 102–103 fragments as a gold
standard.
To complement our established in-house fragment library, we now
compile a set of 1000 high-quality fragments with properties particularly
suited for crystal soaking, immediate fragment-to-lead evolution,
prepared for computer-aided subset selection and fragment-to-lead
evolution. This expanded library will be part of the Frag2Xtal service
facility for crystallographic fragment screening, which will be available at
the semi-automated crystallographic BL14.2 at the BESSY II storage ring
of the Helmholtz-Zentrum Berlin [3].
Aiming at a representative coverage of chemical space, all sufficiently
available and biophysically suitable fragments (> 250,000 cpds. adopting
> 1.4·106 conformational and molecular states) were clustered in groups
of 3D-similar compounds. To this end, we calculated > 1012 pairwise
molecular similarities based on the 3D overlap of volume and interaction
features (charges, hydrogen bond donors/acceptors, aromatic ring, etc.)
using the ROCS (rapid overlay of chemical structures) method [4] and
used these similarities for a hierarchical clustering with the SPARSEHC
algorithm [5] at the Marburg HPC cluster MaRC2. Selecting favorable
representative fragments from each cluster will allow covering the
available chemical space with fragments that are particularly suited for
crystallographic fragment screening and as starting points for drug
discovery projects.
References:
1. Köster, H. et al.: J. Med. Chem. 2011, 54(22): 7784-7796.
2. Schiebel, J. et al.: ChemMedChem 2015, DOI: 10.1002/cmdc.201500267.
3. Mueller, U. et al.: J. Synchr. Rad. 2012, 19(3): 442-449.
4. FastROCS. OpenEye Scientific Software. Santa Fe, NM, USA, URL: www.eyesopen.com.
5. Nguyen, T.-D.; Schmidt, B.; Kwoh, C.-K.: Procedia Comput. Sci. 2014, 29: 8-19.
POSTERS
POS.157
Convergent synthesis of a linker-connected fluorescent
ebselen-coumarin heterodimer
Küppers, J.1; Palus, J.2; Giurg, M.2; Skarżewski, J.2; Gütschow, M.1
1 Pharmaceutical
Institute, Pharmaceutical Chemistry I, University of Bonn, An der Immenburg
4, D-53121 Bonn, Germany
2 Department of Organic Chemistry, Wrocław University of Technology (A2), Wyspiańskiego 27,
50-370 Wrocław, Poland
Objective: Ebselen (1) is a multifunctional drug with a wide range of
pharmacological effects that are predominantly due to its interaction with
selenoenzymes, e.g. glutathione peroxidase, thioredoxin reductase [1,2].
Such enzymes play an important role in protecting biomembranes and
other cellular components from oxidative stress [2]. Fluorescencelabeled probes containing ebselen can be suitable for further biological
and medicinal studies, to profile enzyme activities, identify target
enzymes and characterize their functions [3].
Synthesis: The synthetic route starts with the procedure for synthesizing
ebselen [4]. Anthranilic acid as the starting material was converted into a
diazonium salt, which was treated with disodium diselenide to obtain the
2,2´-diselenobisbenzoate (2). After treating 2 with thionyl chloride, a
reaction with an appropriate para-substituted aniline derivative with a
protected primary aliphatic amine group was performed to give the first
component, compound 3. To obtain the PEG linker for the connection of
the two components, the amino group of 2-(2-aminoethoxy)ethanole was
first Cbz-protected. Afterwards, the hydroxyl group was alkylated with
tert.-butyl bromoacetate followed by a Cbz deprotection. The resulting
primary aliphatic amine was coupled with the second components, the
fluorescent coumarin 343 (4). This was synthesized by submitting 8hydroxyjulolidine-9-carboxaldehyde to a Knoevenagel condensation. In
the final steps, both the ebselen derivative 3 and the PEG-coumarin 343
building block were deprotected and the desired probe 5 was assembled.
Application: The new probe will be provided to biochemical and
pharmacological studies.
performed in two steps. First, a clique-detection algorithm is computed
which only relies on the information about the pseudocenters that were
stored in the database. Second, the cavities are aligned and a score is
calculated based on the overlap of the surfaces of the two aligned cavities
[2]. There are three applications for binding site comparisons. First the
design of novel ligands by retrieving similar ligands (scaffold-hopping) or
ligand portions (bioisosteric replacements) from similar pockets. Second
the prediction of protein function based on the analysis of active sites by
matching well-known sites onto other proteins of unknown properties.
The main use, however, is to study ligand selectivity by identifying
possible off-targets.
In order to characterise similarities of binding sites on a more local level,
we developed a strategy to split the cavities into smaller subpockets.
Therefore the information about the bound ligands are exploited to
generate such subpockets automatically. This is done by decomposing
the original ligand into fragments. Every pseudocenter, which is coincides
within a predefined radius about the fragment, is selected to define the
subpocket. After this process is accomplished, each individual subpocket
is used as a separate query for a Cavbase retrieval. In order to validate
this approach a well-known example of cross-reactivity has been studied:
the approved drug Celecoxib binds to COX-II and CA-II [3]. The result
was as follows: (I) an improved similarity score ranked both proteins
prominently, (II) among the retrieved proteins another known target of
Celecoxib (the PDK-1 protein) was ranked high, (III) and the binding to a
previously unknown target was predicted, PPARγ, which was
subsequently confirmed experimentally, with an EC50 ≈ 30 µM. This
demonstrates that we are able to predict cross-reactivities.
Acknowledgments: We thank Prof. Dr. Schubter-Zsilavecz and Christina Lamers (GoetheUniversity Frankfurt, Institute of Pharmaceutical Chemistry) for measuring the PPARγ assay
and the Center for Synthetic Microbiology, Marburg (SYNMIKRO) for financial support.
References:
1. Hendlich, M.; Rippmann, F.; Barnickel, G.: J. Mol. Graphics Modell. 1997, 15(6): 359–363.
2. Schmitt, S.; Kuhn, D.; Klebe, G.: J. Mol. Biol. 2002, 323(2): 387–406.
3. Weber, A. et al.: J. Med. Chem. 2004, 47(3): 550–557.
References:
1. Stoyanovsky, D. A. et al.: ACS Med. Chem. Lett. 2014, 5(12): 1304-1307.
2. Azad, G. K.; Tomar, R. S.: Mol. Biol. Rep. 2014, 41(8): 4865-79.
3. Xu, K. et al.: Chem. Sci. 2013, 4(3): 1079-1086.
4. Palus, J.; Młochowski, J.; Juchniewicz, L.: Polish J. Chem. 1998, 72(8): 1931-1936
POS.159
Development of novel prodrugs for the nitric oxide (NO)
precursor Nω-hydroxy-L-arginine (NOHA): A carbohydratebased approach
Litty, F.-A.1; Gudd, J.1; Girreser, U.1; Clement, B.1; Schade, D.2
Pharmaceutical Institute, Christian-Albrechts University, Gutenbergstr. 76, 24118 Kiel,
Germany
2 Department of Chemistry & Chemical Biology, TU Dortmund, Otto-Hahn-Str. 6, 44227
Dortmund, Germany
1
POS.158
Cross-reactivity prediction using binding site
fragmentation
Rickmeyer, T.1; Klebe, G.1
Institute of Pharmaceutical Chemistry, Philipps-University Marburg, Marbacher Weg 6-10,
35032 Marburg, Germany
1
Cavbase is a widely used method to quantify the similarity of binding sites
across proteins. Surface-exposed binding sites of proteins are detected
by the Ligsite algorithm [1]. By use of the aforementioned algorithm a
grid-based approach is applied to the protein to identify buried regions on
the surface. Subsequently, the physicochemical properties of the cavityflanking residues are encoded into 3D descriptors, the so-called
pseudocenters. There are seven different types of pseudocenters
covering the following properties: H-bond donor, acceptor, mixed
donor/acceptor, aliphatic, pi-pi interactions, aromatic, and metal ion.
Following the described procedure, binding sites are stored in a
database. The estimation of the similarity between binding sites is
Background: Nω-Hydroxy-L-arginin (NOHA) is formed during the
physiological generation of nitric oxide (NO) from L-arginine, a process
catalysed by nitric oxide synthases (NOSs). In addition to being the most
potent substrate for NOSs, NOHA releases NO only at that site where it
is ultimately needed as opposed to other NO donors (e.g., nitrates).
Furthermore, NOHA is a potent inhibitor of arginases whose
overexpression and activity is closely linked to atherosclerotic changes.
Hence, NOHA is a promising NO modulator for the treatment of
cardiovascular diseases [1,2].
Objective: Since NOHA exhibits a poor chemical and metabolic stability
we have devised prodrug concepts for the problematic Nhydroxyguanidine moiety that successfully overcame these liabilities [3].
N- and O-substitution greatly increased the hydrolytic and oxidative
stability of the hydroxyguanidine moiety, but in vitro assays revealed that
N-carbamates are not bioactivated. Building on our findings that Oacetalic substitution with carbohydrates (R1) furnished surprisingly stable
prodrugs, we next aimed at designing these without carbamate groups.
Here, we present both a novel synthetic route and a cell-based assay to
quantify cytosolic NO release by automated fluorescence microscopy.
Results: We successfully established an orthogonal protecting group
(PG) strategy that enabled installing the desired carbohydrate-based
prodrug groups and subsequent, gentle deprotection of PGs. Introduction
of an allyloxycarbonyl (Alloc)-PG at Nω-position was the key concept for
this carbamate-free approach. PG cleavage was performed palladiumcatalyzed without compromising the labile glyosidic bond. Moreover,
esterification of the α-carboxylic moiety (R3) was possible to increase the
lipophilicity. In addition, we established a 96-well-plate, cell-based
fluorescence microscopic imaging assay to demonstrate cellular uptake
of the new NOHA prodrugs along with in-cell bioactivation resulting in the
release of NO. Briefly, NO was quantified in murine macrophages using
DAF-FM-DA after stimulation with LPS.
Conclusions: In conclusion, we were able to synthesize a great number
of diverse carbamate-free and carbohydrate-based NOHA prodrugs
employing an orthogonal PG concept. By fluorescence-based imaging
we could show that NOHA as well as most prodrugs entered the cells
and increased intracellular NO levels. In fact, we observed a tendency
that higher substituted glucose-based prodrugs released NO most
efficiently. Moreover, we could show a high chemical stability of the new
acetalic NOHA prodrugs further underlining their drug candidate qualities
and utility as novel cardiovascular agents.
References:
1. Schade, D.; Kotthaus J.; Clement, B.: Pharmacol. Ther. 2010, 126: 279-300.
2. Moncada, S.; Higgs E. A.: Br. J. Pharmacol. 2006, 147: 193-201.
3. Schade, D. et al.: Org. Biomol. Chem. 2011, 9: 5249-5259.
POS.160
Canthin-4-one alkaloids and related compounds as a novel
chemotype of DYRK1A inhibitors
Tremmel, T.1; Meijer, L.2; Bracher, F.1
1 Department
2 ManRos
of Pharmacy – Center for Drug Research, LMU Munich, 81377 Munich, Germany
Therapeutics, Perharidy Research Center, 29680 Roscoff, Bretagne, France
The canthin-4-one alkaloids represent a small class of natural products,
with tuboflavine, isotuboflavine and norisotuboflavine being the only
known constituents. In previous investigations we worked out the first
efficient approach to this ring system, including the total syntheses of
tuboflavine and norisotuboflavine, starting from appropriate 1-acyl-βcarbolines and amide acetals [1]. In subsequent investigations we found
significant antimicrobial activities of the parent canthin-4-one and 5substituted as well as annulated analogues [2].
This prompted us to work out a novel approach to the canthin-4-one ring
system which allows for the introduction of variable substituents at C-6.
Readily accessible 1-bromo-β-carboline [3] is converted to 1-ethynyl-βcarboline under Sonogashira conditions and subsequent 1,3-dipolar
cycloaddition with nitrile oxides provides 1-isoxazolyl-β-carbolines.
Reductive ring cleavage leads to intermediate primary enaminoketones,
followed by ring closure to give 6-substituted canthin-4-ones in high
yields. Further substitution in position 5 can be achieved easily by
iodination followed by palladium catalyzed cross-coupling reactions [4].
Selected canthin-4-ones and some of the synthetic precursors showed
interesting inhibitory properties on the protein kinase DYRK1A.
POS.161
Biological evaluation of a dual-target PQS-quorum sensing
inhibitor that hinders biofilm formation in Pseudomonas
aeruginosa
Thomann, A.1,+; de Mello Martins, A. G.1,+; Brengel, C.1; Weidel, E.1;
Plaza, A.2; Börger, C.3; Empting, M.1; Hartmann, R. W.1,3,4,*
Helmholtz Institut for Pharmaceutical Research Saarland, Department of Drug Development
and Optimization, Campus C23, 66123 Saarbrücken, Germany
2 Helmholtz Institut for Pharmaceutical Research Saarland, Department of Microbial Natural
Products, Campus C23, 66123 Saarbrücken, Germany
3 PharmBioTec GmbH, Sciencepark 1, 66123 Saarbrücken, Germany
4 Pharmazeutische und Medizinische Chemie, Campus C2 , Universität des Saarlandes, 66123
3
Saarbrücken, Germany
* Corresponding author; + Authors equally contributed to this work
1
Emergence of Pseudomonas aeruginosa (PA) as a leading cause of
nosocomial infections and morbidity in immunocompromised patients
has consolidated it in the race for novel antimicrobial compounds [1]. PA
infections are notoriously difficult to eradicate due to intrinsic resistance
to a variety of available antibiotics. Its distinguished ability to form biofilms
amplifies resistance and promotes immune response evasion.
The PA quorum-sensing (QS) is a sophisticated network of genome-wide
regulation triggered in response to population density. A major
component is the pseudomonas quinolone signal (PQS) QS system that
regulates the production of several non-vital virulence and biofilm-related
determinants [1]. Hence, this QS circuitry is an attractive target for antivirulence agents with lowered resistance development potential. We
have developed a dual-inhibitor compound (cmpd. VI) of low molecular
weight and high solubility that targets PQS transcriptional regulator
(PqsR) and PqsD, a key enzyme in the biosynthesis of PQS-QS signal
molecules HHQ and PQS [2,3].
In vitro, cmpd. VI markedly reduced virulence factor production and
biofilm formation accompanied by a diminished content of extracellular
matrix components. Additionally, co-administration with ciprofloxacin
increased susceptibility of PA14 to antibiotic treatment under biofilm
conditions. Finally, disruption of pathogenicity mechanisms was also
assessed in vivo, with significantly increased survival of challenged
larvae in a Galleria mellonella infection model [4].
Favourable physicochemical properties and effects on virulence/biofilm
establish a promising starting point for further optimization. In particular,
the ability to address two targets of the PQS autoinduction cycle at the
same time with a single compound holds great promise in achieving
enhanced, synergistic cellular effects while potentially lowering rates of
resistance development.
References:
1. Tashiro, Y. et al.: Microbes Environ. 2013, 28(1): 13-24.
2. Storz, M. P. et al.: J. Am. Chem. Soc. 2012, 134(39): 16143-16146.
3. Lu, C. et al.: Chem. Biol. 2012, 19(3): 381-390.
4. Hill, L. et al.: Int. J. Antimicrob. Agents. 2014, 43(3): 254-261.
POS.162
Allosteric signaling deduced from in silico perturbations
on biomacromolecules
Pfleger, C.1; Gohlke, H.1
References:
1. Puzik, A.; Bracher, F.: J. Heterocyclic Chem. 2009, 46: 770-773.
2. Puzik, A.; Bracher, F.: Lett. Org. Chem. 2013, 10: 568-572.
3. Bracher, F.; Hildebrand, D.: Tetrahedron 1994, 50: 12329-12336.
4. Tremmel, T.; Bracher, F.: Tetrahedron, 2015, 71: 4640-4646.
Institute of Pharmaceutical and Medicinal Chemistry, Heinrich Heine-University,
1
Understanding allosteric regulation in biomacromolecules is of great
interest for current drug design efforts. Conventionally, models that
explain allostery involve a conformational change upon binding of an
POSTERS
allosteric effector molecule [1,2]. Over the last decades, the view of
allostery has been extended to cover the role of dynamics [3]. This type
of dynamically dominated allostery can occur in the absence of
conformational changes, and thus, is difficult to deduce from static X-ray
structures alone. Here, allosteric effectors partially rigidify the
biomacromolecule, which then percolates through a network of
interactions to distant sites [4]. With respect to this dynamically
dominated mechanism, it should be possible to predict putative residues
that form an allosteric regulation pathway for signaling between distant
sites.
For studying dynamically dominated allostery, mechanical perturbation
free energies were deduced from rigidity analyses using a constraint
network representation of biomacromolecules [5]. The CNA (Constraint
Network Analysis) software package has been developed by us to infer
biologically relevant characteristics from rigidity analyses, which are
essential for understanding the relationship between biomacromolecular
structure, (thermos-)stability, and function [6]. Here, CNA was used to
analyse changes in stability upon in silico perturbations on ensembles of
network topologies, which was quantified by the mechanical perturbation
free energies. In order to identify pathways for allosteric signaling, a perresidue decomposition of the mechanical perturbation free energy was
performed to predict putative residues that mediate the allosteric
signaling between distant sites.
We applied CNA on conformational ensembles of PTP1B, LFA-1, and
β2AR. Ensembles were derived from MD trajectories of 300 ns length
starting from the effector bound state. Each ensemble was perturbed in
silico by removing the allosteric effector. We identified multiple residues,
which have pronounced mechanical perturbation free energies.
Remarkably, these residues form continuous pathways for allosteric
signaling connecting residues in the orthosteric site or functionally
important residues for allosteric regulation.
Being able to identify allosteric signaling and to predict the mechanism of
allosteric regulation provides an excellent tool for studying systems with
yet unknown allosteric mechanisms. Furthermore, direct assessment of
flexibility and rigidity characteristics requires no filtering of spurious
correlations, which is needed in the analysis of correlated motions.
impermeable cell wall. An analysis of calculated polar surface area and
logP indicated an influence of these properties on the minimum inhibitory
concentration. Based on computational molecular design, compounds
with improved psychochemical properties were designed and
synthesized. In fact, these compounds showed an improved activity on
mycobacterial growth which underlines the assumption regarding
optimized properties. Permeability measurements using a PAMPA-assay
also proved an increased permeability and showed the usability as a
model system for mycobacterial cell wall permeability.
We will present and discuss the design and the results of our improved
compounds. In addition, further experiments towards a detailed
biochemical characterisation are in progress, and toxicity experiments
and tests on infected macrophages are being planned.
References:
1. World Health Organization Tuberculosis Programme; http://who.int/tb/
2. Jaeger, T.; Flohé, L.: Biofactors. 2006; 27: 109-120.
3. Koch, O. et al.: J. Med. Chem. 2013; 56(12): 4849-4859.
4. Koch, O.: A New Player in the Fight against Tuberculosis: Thioredoxin Reductase Inhibitors
with High Bioactivity on M. tuberculosis. Talk at “Frontiers in Medicinal Chemistry”,
Saarbrücken, Germany, 2011.
POS.164
Looking for multi-target acting xanthine derivatives
Karcz, T.1; Drabczyńska, A.1; Olejarz, A.1; Köse, M.2; Hinz, S.2; DorozPłonka, A.1; Kubas, B.1; Müller, C. E. E.2; Kieć-Kononowicz, K.1
Department of Technology and Biotechnology of Drugs, Jagiellonian University Medical
College, Medyczna 9, 30-688 Kraków, Poland
2 PharmaCenter Bonn, Pharmaceutical Institute, Pharmaceutical Chemistry I, An der
1
Bioactive M. tuberculosis thioredoxin reductase inhibitors:
An update
Triple active compounds that inhibit the monoamine oxidase B (MAO-B),
block A2A adenosine receptors (A2A AR) and A1 adenosine receptors (A1
AR) are expected to show synergistic effects in the treatment of
Parkinson’s disease or Alzheimer’s disease. Such drugs may
simultaneously influence several symptoms of these complex
neurodegenerative diseases [1-3].
Previously we described a group of annelated xanthine derivatives which
showed good A2A AR and/or A1 AR antagonistic properties [4,5]. Here we
focused on compounds with multifunctional activity. New tricyclic
xanthine derivatives containing a dopamine moiety were designed and
synthesized. In addition analogs lacking the third annelated ring were
investigated.
All compounds were evaluated in radioligand binding assays for their
affinity towards the adenosine receptor subtypes: A1, A2A, A2B and A3.
Additionally, compounds were tested at the human MAO-B.
In summary, potent dual-target-directed A1/A2A adenosine receptor
antagonists which showed MAO-B inhibitory properties in the
submicromolar range were identified.
Bering, L. 1; Pretzel, J.1; Rudo, A.1; Schneefeld, M.2; Hölscher, C.3;
Bange, F.-C.2; Koch, O. 1
Acknowledgments: Partially supported by Polish National Science Center funds, granted on the
basis of decision; No: UMO-2012/04/M/NZ4/00219 and COST Action CM1207 (GLISTEN),
financed by EU-FP7.
References:
1. Monod, J. et al.: J. Mol. Biol. 1965, 12(1): 88-118.
2. Koshland, D. E. et al.: Biochemistry 1966, 5(1): 365-385.
3. Cooper, A.; Dryden, D. T.: Eur. Biophys. J. 1984, 11(2): 103-109.
4. Gohlke, H. et al.: Proteins 2004, 56(2): 322-337.
5. Pfleger, C.: Ensemble-Based Framework for Analyzing Dynamically Dominated Allostery
(PhD thesis), Düsseldorf, 2014.
6. Pfleger, C. et al.: J. Chem. Inf. Model. 2013, 53(4): 1007-1015.
POS.163
1 TU
Dortmund University, Otto-Hahn-Straße 6, 44221 Dortmund, Germany
School Hannover, Carl-Neuberg-Straße 1, 30625 Hannover, Germany
Center Borstel, Parkallee 22, 23845 Borstel, Germany
2 Medical
3 Research
The resurgence of tuberculosis, caused primarily by Mycobacterium
tuberculosis, and the appearance of multi-drug resistant and extensively
drug resistant M. t. strains strengthen the need for new drugs with
alternative modes of action [1]. The interaction between the
mycobacterial thioredoxin reductase (TrxR) and its substrate thioredoxin
(Trx) is a promising new drug target for the treatment of tuberculosis,
since M.t. lacks the common glutathione system and the M.t. TrxR shows
a substantial difference in sequence, mechanism and structure to human
TrxRs. The TrxR is part of the M.t. antioxidant system that reduces
hydroperoxides, contributes to ribonucleotide reduction, and thus
guarantees the survival within macrophages [2]. Although the target
mechanism is a protein-protein interaction, the first known inhibitors with
different scaffolds could be identified using an exhaustive highthroughput docking based on the available TrxR X-ray structures [3]. By
means of structure-based design, the activity of the most promising
candidate could be increased to an IC50 up to the low nanomolar range
that also showed an influence on the growth of M. tuberculosis [4].
In order to further improve the bioactivity of the promising compounds we
focused on optimizing the physicochemical properties that are important
for permeability, since M. tuberculosis shows an unusual thick and
References:
1. Van der Schyf, C.J.: Expert. Rev. Clin. Pharmacol. 2011, 4(3): 293-298.
2. Petzer, J. P. et al.: Neurotherapeutics. 2009, 6(1): 141-151.
3. Pisani, L. et al.: Curr. Med. Chem. 2011, 18(30): 4568-4587.
4. Drabczyńska, A. et al.: Purinergic Signal. 2013, 9(3): 395-414.
5. Drabczyńska, A. et al.: Eur. J. Med. Chem. 2011, 46(9): 3590-3607.
POS.165
New P2Y11 ligands: Synthesis and biological activity
Müller, D.1; Hongwiset, D.2; Kassack, M. U.1
1 Institut
für Pharmazeutische und Medizinische Chemie, Heinrich-Heine Universität Düsseldorf,
Universitätsstr. 1, 40225 Düsseldorf
2 Faculty of Pharmacy, Chiang-Mai University, Suthep Rd, Mueang Chiang Mai District, Chiang
Mai 50200
The P2Y11 receptor is a GPCR belonging to the nucleotide family of
purinergic receptors. During the last decade several pathophysiological
processes could be identified, in which the P2Y11 receptor participates.
P2Y11 is associated with narcolepsy or different inflammatory diseases
like acute myocardial infarction [2,3]. Furthermore, interactions of this
receptor subtype can influence cell proliferation or sensitivity of tumor cell
lines against cytotoxic agents [4]. Despite its participation in a variety of
pathophysiological processes, only few potent agonists and antagonists
of the P2Y11 receptor are known. In addition, there is a lack of selective
ligands [5]. To drive the development of potent and selective P2Y11ligands further, our research focused on new non-nucleotide P2Y11
receptor ligands. We synthesized a series of symmetrical and
asymmetrical arylureas, starting from commercial and non-commercial
building blocks in a four-step sequence. The biological activity of the
compounds was determined with a functional calcium assay using
1321N1 cells recombinantly expressing P2Y11 receptors.
POS.167
Development and preclinical characterization of partial
farnesoid X receptor agonists for metabolic disorders
Merk, D. 1
1 Institute
of Pharmaceutical Chemistry, Goethe University Frankfurt, Max-von-Laue-Str. 9,
60438 Frankfurt, Germany;
POS.168
Design, synthesis and preliminary evaluation of chemical
tools for target identification of novel TGFβ signaling
inhibitors
Flötgen, D.1; Längle, D.1; Carrillo García, C.1; Schade, D.1
References:
1 Department
of Chemistry & Chemical Biology, TU Dortmund, Otto-Hahn-Str. 6, D-44227
Dortmund, Germany
1. Abbracchio, M. P. et al.: Pharmacol. Rev. 2006, 58(3): 281-341.
2. Mignot, E. et al.: Nat. Genet. 2011, 43(1): 66-71.
3. Erlinge, D. et al.: European Heart Journal 2007, 28(1): 13-18.
4. Hu, J. et al.: J. Cell. Biochem. 2011, 112(9): 2257-2265.
5. Jacobson, K. A. et al.: Drug Discov. Today 2010, 15(13-14): 570-578.
POS.166
Development of ligands for the cannabinoid-like orphan
receptors GPR18 and GPR55
Kieć-Kononowicz, K.1; Rempel, V.2; Karcz, T.1; Schoeder, C.2; DorozPłonka, A.1; Kaleta, M.1; Müller, C. E.2
Department of Technology and Biotechnology of Drugs, Jagiellonian University Medical
College, Medyczna 9, 30-688 Kraków, Poland
2 PharmaCenter Bonn, Pharmaceutical Institute, Pharmaceutical Chemistry I, An der
1
GPR18 and GPR55 are orphan G protein-coupled receptors (GPCRs)
that interact with certain cannabinoid receptors (CBRs) ligands. GPR18
was reported to be activated by Δ9-tetrahydro-cannabinol (THC). GPR55
was found to be activated by cannabinoid receptor antagonists, including
the CB1R inverse agonist - rimonabant. GPR18 was found be one of the
most highly upregulated receptors in metastatic melanoma [1]. Activation
of GPR18 was reported to induce migration of human endometrial cells.
Therefore, GPR18 receptor antagonists may be useful novel therapeutics
for the treatment of endometriosis and cancer [2]. On the other hand,
GPR55, highly expressed in adrenal glands, central nervous system,
certain cancer cell lines and immune cells, was postulated to play a role
in mediating inflammatory and neuropathic pain, promoting cancer cell
migration and proliferation. Thus, GPR55 receptor antagonists may be
useful for the treatment of cancer, neuropathic pain [3].
Recently, several bicyclic imidazole-4-one derivatives were discovered
as ligands of GPR18 and GPR55 [4] using β-arrestin translocation
assays. Selectivity of these compounds with respect to CB1 and CB2
receptors was also evaluated.
In the present study, 3-benzyloxybenzylideneimidazo[2,1-b][1,3]thiazin3-ones (group I) and related 3-benzyloxybenzylideneimidazo[2,1b][1,3]thiazepin-3-ones (group II) were investigated for their interaction
with GPR18, GPR55 and CBRs. Selective antagonists for GPR18 as well
as for GPR55 were found in group I. In contrast, in group II compounds
showed higher affinity for CB1 and CB2 receptors. One of the new
compounds exhibited agonistic activity at GPR55 indicating that
antagonists for this receptor may easily be converted to compounds that
can activate the receptor.
Acknowledgments: Partially supported by Polish National Science Center funds, granted on the
basis of decision; No: UMO-2013/11/B/NZ7/04865 and COST Action CM1207 (GLISTEN),
financed by EU-FP7.
References:
1. Qin, Y.; et al.: Pigment Cell Melanoma Res. 2010, 24(1): 207-217.
2. Alexander, S.P.: Br. J. Pharmacol. 2012, 165(8): 2411-2413.
3. Shore, D. M.; Reggio, P.H.: Front. Pharmacol. 2015, 6: 69.
4. Rempel, V. et al.: Med. Chem. Commun. 2014, 5: 632-649.
Background: Target identification of small molecules derived from
phenotypic screens (forward chemical genetics) is a demanding task but
also very rewarding as it may reveal unknown (druggable) targets, thus
representing a crucial step in drug development [1]. In our case, a
subclass of 1,4-dihydropyridines (DHPs) was discovered from a screen
for stimulators of cardiogenesis in murine embryonic stem cells.
Mechanistic studies revealed a new mode of TGFβ signaling inhibition
via proteasomal degradation of the TGFβ type II receptor [2].
Objectives: Since we have established a sound structure-activity
relationship (SAR), 3D-QSAR and pharmacophore model, [3, 4] we had
a good picture of where to incorporate reactive functionalities for an
affinity-based approach. The desired DHP tool compounds should be
potent, selective and carry functional groups for photocrosslinking to the
unknown target as well as for fluorescence-based detection. Here, we
summarize 1) synthetic routes that were explored to furnish a diverse set
of these DHP derivatives, 2) bioactivity as TGFβ inhibitors and 3) initial
data on their utility as molecular tools to study cellular processes.
Results: We chose to incorporate small-sized, aliphatic diazirines for
intracellular photocrosslinking and alkynes for click reaction with azidecontaining fluorophores to enable their detection. DHPs with these
groups in 2-, 3- and 7-position were successfully synthesized. In this
regard, using a small, iodine-containing diazirine as a building block
proved particularly valuable. Importantly, most of these DHP derivatives
still potently inhibited TGFβ signaling (IC50 = 0.5-2.0 µM). Moreover,
>100 conditions were systematically explored to optimize the click
reaction of DHP alkynes with FAM-azide in HEK cells for confocal
microscopy, allowing the analysis of the subcellular localization of the
labelled DHPs (= click products).
Conclusions: A diverse set of DHP tool compounds for target
identification could be synthesized and were active as TGFβ inhibitors.
According to our preliminary cellular data, they can be used to study colocalization of DHPs with TGFβ type II receptor (mCherry fusion protein).
Next steps will include their use to ‘fish’ the target protein by
photocrosslinking and proteomic analyses, potentially unravelling a novel
(maybe universal) regulatory circuitry of receptor dynamics within the
TGFβ superfamily of ligands.
References:
1. Ziegler, S. et al.: Angew. Chem. Int. Ed. 2013, 52: 2744–2792.
2. Willems, E. et al.: Cell Stem Cell 2012, 11: 242–252.
3. Schade, D. et al.: J. Med. Chem. 2012, 55: 9946–9957.
4. Laengle, D. et al.: Eur. J. Med. Chem. 2015, 95: 249–266.
POSTERS
POS.169
α5β1 Integrins are Receptors for Bile Acids with a
(Nor-)Ursodeoxycholane Scaffold
Bonus, M.1; Sommerfeld, A.2; Häussinger, D.2; Gohlke, H.1
1 Institute
for Pharmaceutical and Medicinal Chemistry, Heinrich-Heine-University Düsseldorf,
Universitätsstr. 1, 40225, Germany
2 Clinic for Gastroenterology, Hepatology and Infectious Diseases, Heinrich-Heine-University
Düsseldorf, Moorenstr. 5, 40225, Germany
Integrins are ubiquitously expressed cell adhesion receptors and the
most prevalent bidirectional signaling molecules on the cell surface. They
are involved in osmosensing in the liver [1,2,3] and facilitate the
communication between components of the extracellular matrix and
cytoplasmic proteins. Upon activation, integrins undergo large-scale
conformational changes from a compact, bent state to an open, extended
state [4]. A recent study combined immunofluorescence staining (IFS)
experiments and molecular dynamics (MD) simulations to identify
tauroursodeoxycholic acid (TUDC) as potent agonist of α5β1 integrins in
hepatocytes [5]. Activation of α5β1 leads to choleresis by FAK/cSrc/MAPK dependent signaling events [6,7,8]. TUDC-induced integrin
activation and subsequent signaling is sensitive to inhibition by the
trihydroxylated taurocholic acid (TC), which tightly binds to α5β1 in MD
simulations. However, effects of other bile acids on α5β1 integrin
activation have not been investigated at the molecular level.
Here we report on combined IFS experiments and MD simulations that
indicate that α5β1 integrins are not exclusively activated by TUDC. 24nor-ursodeoxycholic acid (norUDCA), a side chain-shortened homologue
of UDCA, induces conformational changes in the βA domain of α5β1 that
are similar to the one evoked by TUDC, but overall less pronounced.
Conformational changes in simulations of integrin ectodomains bound
with the taurine conjugate of norUDCA (TnorUDCA) and
glycoursodeoxycholic acid (GUDC) were significantly less pronounced.
Unconjugated UDCA, similar to the inhibitory TC, only showed
insignificant alterations in the structure of the integrin ectodomain and
was considered fully inactive. A ranking based on the extent of structural
changes observed during the MD simulations correlates with results from
IFS experiments on the efficacy of the bile acids.
These results indicate that norUDCA activates α5β1 integrins and that MD
simulations are able to predict different degrees of bile-acid induced
integrin activation. Minor structural changes in the bile acids strongly
influence their efficacy. This holds true for a comparison of TUDC
(activating) vs. TC (inhibitory) and norUDCA (activating) vs. UDCA
(nonactivating).
Acknowledgments We are grateful to the ‘‘Zentrum für Informations und Medientechnologie’’
(ZIM) at the Heinrich Heine University for computational support. This work was supported by the
Deutsche Forschungsgemeinschaft through the Collaborative Research Center SFB 974
(‘‘Communication and Systems Relevance during Liver Damage and Regeneration’’, Düsseldorf).
References:
1. Häussinger, D. et al.: Gastroenterology 2003, 124(5): 1476-1487.
2. vom Dah,l S. et al.: J. Biol. Chem. 2003, 278(29): 27088-27095.
3. Schliess et al.: J. Biol. Chem. 2004, 279(20): 21294-301.
4. Xiong, J. P. et al.: Science 2001, 294(5541): 339–345.
5. Gohlke, H. et al.: Hepatology 2013, 57(3): 1117–1129.
6. Schliess, F. et al.: Gastroenterology 1997, 113(4): 1306–1314.
7. Beuers, U. et al.: Hepatology 2001, 33(5): 1206–1216.
8. Häussinger, D. et al.: Gastroenterology 2003, 124(5): 1476–1487.
POS.170
C2-Linked Dimeric Strychnine Analogs as Bivalent Ligands
Targeting Glycine Receptors
Zlotos, D. P.1; Banoub, M.1; Holzgrabe, U.2; Breitinger, H.-G.; Breitinger,
U.1; Villmann, C.3
Dept. of Pharmaceutical Chemistry, The German University in Cairo, New Cairo City, 11835
Cairo, Egypt
2 Institut für Pharmazie and Lebensmittelchemie, Universität Würzburg, Am Hubland, 97074
Würzburg, Germany
3 Institut für Klinische Neurobiologie, Universitätsklinikum Würzburg, Versbacherstr. 5, 97078
Würzburg, Germany
1
Strychnine (1), the major alkaloid from the plant Strychnos nux vomica,
exhibits pharmacological activity at several neurotransmitter receptors.
Its most pronounced pharmacological action is a strong antagonistic
activity at glycine receptors (GlyRs), which are anionic chloride channels
composed of five subunits and linked to hyperpolarisation and inhibition
of neuronal firing [1-3]. The glycine receptors are one of the major
mediators of rapid synaptic inhibition in the mammalian brainstem, spinal
cord, and higher brain centers. Dysfunction of the receptors is associated
with motor disorders such as hypereflexia, or some forms of spasticity [4].
To date, strychnine is the ligand displaying the highest activity at
recombinant and native GlyRs in binding and functional assays [2,3].
Here, we report the synthesis and pharmacological evaluation of the first
series of bivalent ligands 4a-d targeting glycine receptors. Designed as
strychnine dimers, the target compounds should be able to bind
simultaneously to two subunits of the pentameric receptors causing a
possibly stronger inhibition than the monomeric strychnine.
N
N H
O
N
O2N
H
H
H
O
N H
2. H2SO4
O
H
H
H
N H
EtOH
O
O
H
N
H
H
O
H
H
H
H
H
O
3
N
H2OC-(CH2)n-CO2H
n = 2,4,6,8
H
2
strychnine (1)
CH2Cl2, EDCI HCl
N
H2N
SnCl2
H
1. HNO3
H
H
N
(CH2)n
N
H
O
O
N H
H N
O
4a
4b
4c
4d
O
n=2
n=4
n=6
n=8
H
H
H
O
Acknowledgments: Deutscher Akademischer Austauschdienst (DAAD), Bundesministerium für
Bildung und Forschung (BMBF)
References:
1. Lynch, J.W.: Physiol. Rev. 2004, 84: 1051.
2. Laube, B. et al.: Trends Pharmacol. Sci. 2002, 23: 519.
3. Jensen, A.A.; Kristiansen, U.: Biochem. Pharmacol. 2004, 67: 1789.
4. Breitinger, H.-G.; Becker, C.-M.: Curr. Pharm. Des. 1998, 4(4): 315-34.
POS.171
Human induced pluripotent stem cell (hiPSC)-derived
cardiomyocytes: Chemically-defined derivation, genetic
manipulation and applications in drug development
Rathmer, B.1; Carrillo García, C.1; Greber, B.2; Schade, D.1
Department of Chemistry & Chemical Biology, TU Dortmund, Otto-Hahn-Str. 6, D-44227
Dortmund, Germany
2 Max Planck Institute for Molecular Biomedicine, Von-Esmarch-Str. 54, 48149 Münster,
Germany
1
The Nobel Prize awarded discovery that somatic cells can be
reprogrammed to an induced pluripotent stem cell-like state and the
ability to generate cardiomyocytes (CMs) from these iPSCs using
directed differentiation methods have provided a fertile ground for many
applications. Such applications are not only of therapeutic nature (=
regenerative medicine) but also include technologies for the drug
discovery process (= safety pharmacology and toxicology) and disease
modeling approaches (= disease-in-a-dish) for basic and applied
research in cardiac biology [1-3].
Although current differentiation protocols enable the derivation of CMs
from human stem cells in high yields under chemically-defined
conditions, there are still several obstacles for the above outlined
applications. These include the heterogeneous constitution of hiPSCCMs as they typically consist of a mixture of ventricular, atrial and
pacemaker cells. Moreover, the level of maturity of hiPSC-CMs is lower
than that of native, adult CMs which may limit some applications. Notably,
not much is known about the underlying developmental processes and
suitable small molecule modulators are lacking [2,3].
Here, we provide an overview on our hiPSC-based approaches to the
discovery and characterization of new small molecular modulators of
cardiac differentiation and regeneration. These efforts build on using a
robust and efficient chemically-defined differentiation protocol that allows
us to establish a platform of new high-content assays [4]. In this regard,
we are exploring different methods for generating hiPS reporter cell lines
harboring fluorescence reporter cassettes (e.g., bacterial artificial
chromosome transgenesis) to determine CM yields, constitution and their
proliferative capacity. One aim is, for example, the identification of proproliferative small molecules by ‘forward chemical genetics’. This
approach would not only allow the expansion of early, immature CMs for
transplantation but also increase our knowledge of mechanistic cues that
direct the postnatal loss of CM proliferation.
Moreover, we are using the assay platform to study mechanistic aspects
of (new) small molecule Wnt/β-Catenin inhibitors as this pathway is a key
player within cardiopoietic differentiation. These studies might be useful
to enable cell context-specific Wnt inhibition (safety for in vivo
applications) and improving the quality of hiPSC-derived CMs.
References:
1. Takahashi, K.; Yamanaka, S.: Cell 2006, 126: 663-676.
2. Schade, D.; Hansen, A.; Greber, B.: Drug Target Review 2015, 2(2): 34-38.
3. Schade, D.; Plowright, A. T.: J. Med. Chem. 2015, in press.
4. Zhang, M. et al.: Stem. Cells 2015, 33: 1456–1469.
POS.172
The Crystal Structure of the Nisin Resistance Protein
Reveals the Underlying Mechanism of Lantibiotic
Resistance
Khosa, S.1; Frieg, B.2; Mulnaes, D.2; Kleinschrodt, D.3; Hoeppner, A.4;
Gohlke, H.2; Smits, H. J. S.1
Institute of Biochemistry, Heinrich Heine University, Universitätsstr. 1, 40225 Düsseldorf,
Germany
of Pharmaceutical and Medicinal Chemistry, Heinrich Heine University, Universitätsstr.
1, 40225 Düsseldorf, Germany
3 Protein Production Facility, Heinrich Heine University, Universitätsstr. 1, 40225 Düsseldorf,
Germany
4 Crystal and X-ray Facility, Heinrich Heine University, Universitätsstr. 1, 40225 Düsseldorf,
Germany
1
2 Institute
Lantibiotics are potent antimicrobial peptides and often considered as
next generation antibiotics [1]. Some pathogenic bacteria, however,
express membrane-associated resistance proteins, which proteolytically
inactivate lantibiotics [2].
The most prominent member of lantibiotics is nisin, which contains five
specific and crucial lanthionine rings that are the hallmark for its activity
as well as recognition [3,4]. We report the first three-dimensional
structure of the lantibiotic resistance protein superfamily, namely the nisin
resistance protein from Streptococcus agalactiae (SaNsr) at 2.2Å, which
is active against nisin [5,6]. It contains three domains, a N-terminal helical
bundle, a protease cap domain and a protease core domain, which also
harbors the highly conserved TASSAEM region. The active center and
the binding site of nisin within SaNsr have been characterized via sitedirected mutagenesis and molecular modeling, respectively. Due to the
presence of the lanthionine rings, nisin itself is highly unsusceptible to
proteolytic degradation. However, SaNsr takes advantage of the methyllanthionine rings present in nisin for substrate recognition and specificity.
This structural information would pave way for designing small molecular
compounds inhibiting lantibiotic resistance proteins by which the potency
of these fascinating small peptides can be fully explored.
Acknowledgments: We thank Lutz Schmitt for fruitful discussions, encouragement, support and
invaluable advice. We are grateful to Phillip Ellinger for initiating the project and Andre Abts for
stimulating discussions, constant support and ideas. We thank Michael Lenders for help with the
MALS measurement. We are grateful to the staff of ESRF ID23-2 and ID29 for support during
crystal screening and data collection, especially Christoph Mueller-Dieckmann for his enormous
patience, assistance and support. We are thankful to Heinrich Heine International Graduate
School of Protein Science and Technology (iGRASPseed) for providing a scholarship to S.K. We
are grateful to the “Zentrum für Informations- und Medientechnologie” (ZIM) at the HeinrichHeine-University Düsseldorf for providing computational support.
References:
1. Hancock, R. E.; Sahl, H.-G.: Nat. Biotechnol. 2006, 24: 1551-1557.
2. Nawrocki, K. L.; Crispell, E. K.; McBride, S. M.: Antibiotics 2014, 3: 461-492.
3. Sahl, H.-G.; Bierbaum, G. Annu. Rev. Microbiol. 1998, 52: 41-79.
4. Wiedemann, I. et al.: J. Biol. Chem. 2001, 276: 1772-1779.
5. Sun, Z. et al.: Antimicrob. Agents Chemother. 2009, 53: 1964-1973.
6. Khosa, S.; AlKhatib, Z.; Smits, S. H.: Biol. Chem. 2013, 394: 1543-1549.
POSTERS
POS.175
4.9 Inflammation
POS.173
BMP2K is a crucial mediator of inflammatory and
angiogenic processes in the human endothelium
Bischoff,
1 Institute
I.1;
Dai,
B.2;
Strödke,
B.3;
Bracher,
F.3;
Fürst,
R.1
of Pharmaceutical Biology, Goethe-University Frankfurt, Max-von-Laue-Str. 9, 60438
Biology, Center for Drug Research, University of Munich, Butenandstraße 513, 81377 München, Germany
3 Department of Pharmacy - Center for Drug Research, University of Munich, Butenandstraße 7,
81377 München, Germany
2 Pharmaceutical
Chronic inflammation is characterized by constant leukocyte infiltration
and angiogenesis in the inflamed tissue, which is the case in various
diseases such as rheumatoid arthritis or chronic inflammatory bowel
diseases. As permanent leukocyte extravasation leads to severe tissue
damage, there is a demand for compounds that promote the inhibition of
inflammation and angiogenesis and the discovery of new drug targets.
The bone morphogenetic protein-2 (BMP-2)-inducible kinase (BMP2K)
might be a novel target of such substances. The goal of this study is to
elucidate the role of BMP2K in the endothelium and in leukocytes during
inflammation and angiogenesis. In this project C81, an inhibitor of
BMP2K, as well as RNAi-based gene silencing was applied to inhibit
BMP2K and, therefore, to clarify the role of this kinase during
inflammatory processes.
Different from prechondroblastic cells, the BMP2K seems not to be
regulated by BMP-2 in human umbilical vein endothelial cells (HUVECs)
(q PCR). In addition, pro-inflammatory stimuli such as LPS or TNFα did
not show any effect on BMP2K regulation on the mRNA level.
The inhibition of BMP2K by C81 or gene-silencing reduced the migratory
capacity of a human microvascular endothelial cell line (HMEC-1). Also
the proliferation of HMEC-1 was reduced after C81 treatment with an IC50
of 6.9 µM. The performance of a tube formation assay using Matrigel
demonstrated that the inhibition of the BMP2K by C81 significantly
impaired the formation of capillary-like structures in a dose-dependent
manner. Interestingly, the analysis of signaling molecules in HUVECs
that play a crucial role in cell proliferation (ERK, Akt, JNK, p38) revealed
that these pathways are not influenced by C81 treatment or genesilencing of BMP2K (Western blot).
Rising concentrations of C81 reduced the TNFα-triggered expression of
cell adhesion molecules (ICAM-1, VCAM-1, E-selectin) on the surface of
HUVECs (flow cytometry) and on mRNA levels (qPCR). Similar results
for surface expressions were detected in BMP2K-silenced endothelial
cells. Based on these findings a cell adhesion assay using the monocytic
cell line THP-1 and C81-treated or BMP2K-silenced HUVECs was
performed. THP-1 showed a significantly decreased adhesion to
activated HUVECs. Most notably, C81 or knock-down of BMP2K did not
lead to the reduction of TNF-induced IκBα degradation or p65
translocation into the nucleus.
The results indicate that BMP2K is crucially involved in the proinflammatory and pro-angiogenic activation of human endothelial cells.
Furthermore, these data highlight C81 as a promising tool to interrupt
BMP2K-mediated signaling events. Further studies are needed to
elucidate the precise role of BMP2K during inflammation and
angiogenesis and to clarify the regulatory mechanisms that underlie
those processes.
Regulatory role of 5-Lipoxygenase-activating protein
(FLAP) in 5-Lipoxygenase substrate acquisition, turnover
and interaction
Gerstmeier, J.1; Newcomer, M.2; Werz, O.1; Garscha, U.1
1 Chair of Pharmaceutical/Medicinal Chemistry, Institute of Pharmacy, Friedrich-SchillerUniversity, 07743 Jena, Germany; 2Department of Biological Sciences, Louisiana State
University, Baton Rouge, Louisiana 70803
Leukotriene (LT) biosynthesis from arachidonic acid (AA) is catalyzed by
5-Lipoxygenase (5-LOX), and proceeds via the intermediate 5hydroperoxyeicosatetraenoic acid (5-HPETE) to LTA4. Among all LOXs,
solely 5-LOX catalysis from endogenous substrate requires an essential
translocation process from the cytosol or perinuclear region to the
nuclear membrane-embedded helper-protein 5-LOX-activating protein
(FLAP), upon cell activation. Although structures for both FLAP and 5LOX have been solved, the molecular basis for efficient AA transfer from
FLAP to 5-LOX remains elusive, and the “corked” cavity of 5-LOX has
led to speculation on how AA can enter the active site. We used sitedirected mutagenesis, coupled with 5-LOX product analysis and
fluorescence microscopy techniques in stable transfected HEK293 cells
[1] to reveal the entry portal for AA. Our results support a model in which
AA gains access to the active site through the plugged “FY” cork. Upon
5-LOX translocation and association with the membrane, the two bulky
side chains of the “FY” cork swing out for membrane insertion to uncork
the active site. This “uncorking” however must be reversible for full
cellular 5-LOX activity as a permanently open cavity, mimicked by
alanine mutants of both “corking” residues, completely precludes LT
biosynthesis. Of great interest, co-expression of FLAP rescues the 5LOX activity of “cork” mutations. The ability of FLAP to restore AA
metabolism to an “uncorked” 5-LOX strongly suggests that a physical
protein-protein interaction between 5-LOX and FLAP occurs to complete
the 5-LOX active site for efficient substrate acquisition and turnover. In
particular, LTA4 formation at the expense of 5-HPETE is aided by the
“FY” cork. Finally, distinct proof for the true existence of 5-LOX/FLAP
complexes was accomplished by proximity ligation assay (PLA). Unlike
GFP-based FRET approaches, PLA probes capture the interacting
proteins in their native form. Although the “FY” cork itself is not an
interaction site with FLAP, both residues are pivotal for complete
translocation capacity of 5-LOX and LTA4 formation. Our findings
highlight the significance of FLAP in 5-LOX substrate acquisition and
conversion, and offer novel perspectives to control of subcellular
localization and activity of 5-LOX.
References:
1. Gerstmeier, J. et al.: Biochim. Biophys. Acta. 2014, 1840(9): 2961-2969.
POS.176
In situ forming gel devices as local depot therapeutic for
Rheumatoide Arthritis
Mohammadi, M.1; Abebe, D.2; Li, Y.1; Kandil, R.1; Xie, Y.1; Fujiwara, T.2;
Merkel, O. M.1
1 Department
of Pharmaceutical Sciences, Wayne State University, Detroit, MI 48201
of Chemistry, The University of Memphis, 213 Smith Chemistry Building,
Memphis, Tennessee, 38152, USA
2 Department
POS.174
Time-resolved in situ assembly of the 5-lipoxygenase / 5lipoxygenase-activating protein complex in primary human
leukocytes
Garscha, U. 1; Gerstmeier, J. 1; Werz, O. 1
1 Chair
of Pharmaceutical/ Medicinal Chemistry, Institute of Pharmacy, Friedrich-Schiller
University Jena, Philosophenweg 14, 07743 Jena, Germany
More efficient, novel anti-inflammatory therapies with reduced side
effects are needed to treat Rheumatoid arthritis (RA), a chronic and
disabling autoimmune condition that affects about 1% of the population
in developed countries [1]. Even though a multitude of cell types is
involved in RA
inflammation, it is
agreed upon that
macrophages play
a central role in the
pathophysiology of
RA [2]. Locally
implantable,
targeted, macrophage-specific RNA interference (RNAi)-based therapies
could therefore revolutionize RA therapy.
INFLAMMATION
Three-layered
micelles
(3LM)
entrapping
and
encapsulating
nucleic acids were
formed from triblock
copolymers of PLLAPEI-PLLA and PLLA-PEG-PLLA in a three-step procedure outlined in
upper Figure [3]. Their structure and DNA entrapment in the core were
determined by staining DNA with silver nitrate and TEM detection [4].
Hydrodynamic diameters and zeta potentials were measured by dynamic
light scattering and laser Doppler anemometry. DNA release in neutral
and acidic pH was detected by modified SYBR Gold assays [3]. For
targeting of activated
macrophages, folic
acid
(FA)
was
attached to the PEGchain of a PLLA-PEG
diblock
affording
PLLA-PEG-FA.
Subsequently, 3LM
were formed with
PLLA-PEG-FA in the outer polymer shell. RAW264.7 cells were activated
with LPS [5] or left resting. One day after the activation, the cells were
treated with targeted and non-targeted 3LM loaded with fluorescently
labeled DNA. The uptake of 3LM or PEI/DNA polyplexes was quantified
by flow cytometry. Thermoresponsive hydrogels were obtained by
stereocomplexing 3LM which contain PLLA-PEG-PLLA in the outer core
with PDLA-PEG-PDLA which is shown in lower Figure [6].
The core-corona structure and efficient DNA entrapment in the core were
confirmed by TEM. The sizes were found to be less than 200 nm, and
the encapsulation efficiency of DNA was optimized based on the ratio of
the PEI block in PLLA-PEI-PLLA per DNA [3]. 3LM were stable at neutral
pH but released DNA in an acidic environment [3]. 3LM were efficiently
targeted to activated macrophages by blending PLLA-PEG-FA and
PLLA-PEG-PLLA in the outer layer, while non-targeted micelles or PEI
polyplexes were not efficiently taken up. Stereocomplexes of 3LM form
hydrogels above their phase transition temperature.
References:
1. WHO, Chronic diseases and health promotion in: Chronic rheumatic conditions 2015,
Geneva.
2. Gordon, S.;Taylor, P. R.: Nat. Rev. Immunol. 2005, 5: 953-964.
3. Abebe, D. G. et al.: Macromolecular Bioscience 2015, 15(5): 698-711.
4. Zheng, M. et al.: ACS Nano 2012, 6: 9447-9454.
5. Funk, J. L. et al.: Atherosclerosis 1993, 98: 67-82.
6. Abebe, D.G.; Fujiwara, T.: Biomacromolecules 2012, 13: 1828-1836.
POS.177
Establishment of a cell-based model to study LTC4synthase inhibitors independent on exogenous LTA4
supply
Liening, S.1; Scriba, G.1; Werz, O.1; Garscha, U.1
1 Chair of Pharmaceutical/Medicinal Chemistry, Institute of Pharmacy, Friedrich-SchillerUniversity of Jena, Philosophenweg 14, 07743 Jena, Germany
Cysteinyl-leukotrienes (cys-LT) are powerful pro-inflammatory mediators
that cause bronchoconstriction in anaphylaxis and asthma. They are
formed via the 5-lipoxygenase (5-LOX)/leukotriene C4-synthase (LTC4S) pathway in immunocompetent cells. 5-LOX metabolizes arachidonic
acid to the unstable leukotriene A4 (LTA4) that is subsequently conjugated
with glutathione (GSH) to leukotriene C4 (LTC4) by LTC4-S, an integral
membrane protein that belongs to the superfamily of membraneassociated proteins in eicosanoid and glutathione metabolism (MAPEG).
The tripeptide side chain of LTC4 is cleaved in two successive steps to
form LTD4 and LTE4. Cys-LTs are recognized by GPCRs and cys-LT
receptor antagonists have mainly been developed to diminish the
inflammatory action of cys-LTs. Besides the receptors, LTC4-S might be
an additional interesting target to intervene with cys-LTs. However,
structural similarities to other MAPEGs and the lack of an appropriate test
system have hampered the possibility to reveal and develop selective
LTC4-S inhibitors.
The main objective of our work was to establish a cell-free and a cellbased assay system for LTC4-S to evaluate putative LTC4-S inhibitors.
Therefore HEK293 cells that lack expression of 5-LOX and other
MAPEGs were transfected with cDNA of LTC4-S, and stably LTC4-Sexpressing colonies were selected. From these cells, LTC4-S containing
microsomes were prepared and incubated with LTA4 methyl ester (LTA4me). LTC4-me was analyzed and quantified by LC-MS/MS and 2.5 µg of
microsomes formed around 7 ng of LTC4-me. MK886 as a known LTC4S inhibitor reduced LTC4-me synthesis with an IC50 value of 3.5 µM.
However, a major drawback of the cell free assay is the need to supply
expensive and unstable LTA4-me. In order to circumvent this problem,
we stably co-expressed the LTA4-forming 5-LOX together with LTC4-S in
HEK293 cells. Stimulating these cells with Ca2+-ionophore and 20 µM
exogenous arachidonic acid, around 300 ng of LTC4 could be detected.
MK886 inhibited the LTC4 synthesis with an IC50 value of 3.1 µM. Since
HEK293 cells are void of other MAPEG enzymes like 5-LOX-activating
protein (FLAP) or microsomal prostaglandin E2 synthase-1 (mPGES-1),
the reduced formation of LTC4 by putative inhibitors can be exclusively
deduced from LTC4-S intervention.
Summarizing, we established a cell-free and cell-based HEK293 system
in order to evaluate putative LTC4-S inhibitors. Especially, the cellular
system is advantageous since LTA4 as LTC4-S substrate is generated in
situ. Additionally, this cell system can be applied to investigate LTC4-S
functionality, as site-directed modifications can be studied in a cellular
environment.
POS.178
Structure-dependent potentiation of the complement
inhibiting C1 inhibitor by glycosaminoglycans and other
sulfated glycans
Alban, S.1; Schoenfeld, A.-K.1
1 Pharmaceutical
Germany
Institute, Christian-Albrechts-University, Gutenbergstr. 76, 24146 Kiel,
Introduction: Activation of the human complement system as part of the
innate immunity is associated with many diseases like rheumatoid
arthritis or age-related macular degeneration. Hence, reduction of
overwhelming activity displays a promising therapeutic strategy. Heparin
and other sulfated glycans (SG) are known inhibitors of complement
activation. One assumed mechanism is the potentiation of C1 inhibitor
(C1inh), an endogenous regulator of the complement system [1].
Objectives: We examined series of SG differing in their degree of
sulfation (DS) and their molecular weight (MW) for their C1inh
potentiating effect to evaluate structure-activity relationships. The latter
could be helpful to develop optimized complement modulators.
Materials and Methods: The effect of C1inh in presence and absence of
SG on the activity of C1s esterase was measured by a chromogenic
substrate assay. We examined the C1inh modulating effects of following
SG: (1) seven heparins with MW ranging from 3 to 15 kDa, (2) further
partially chemically modified glycosaminoglycans, e.g. heparan sulfates
and danaparoid, (3) two series of high-MW (≈ 160 kDa) and low-MW
(≈ 19 kDa) semisynthetic β 1,3 glucan sulfates, and (4) three algaederived SG.
Results: None of the tested SG inhibited C1s esterase directly. The
heparins (DS ≈ 1.1) and the algal SG (DS ≈ 0.6) exhibited a C1inh
potentiating effect depending on their MW. On the contrary, the MW
turned out to be not important for compounds with a DS above 1.3. For
the low-MW β-1,3-glucan sulfates and glycosaminoglycans a DSdependent potentiation of C1inh could be observed, whereas the highMW β-1,3-glucan sulfates led to a potentiation independent of their DS.
Even in case of a low DS (≈ 0.7) the high-MW compound exhibited a
strong C1inh potentiating effect, whereas the low-MW β 1,3 glucan
sulfate was inactive.
Conclusion: The C1inh potentiating effect of SG correlated with both
increasing DS and MW, whereby a high DS showed to compensate a low
MW and, inversely, a high MW a low DS.
Acknowledgments: The study was financially supported by the European Fishery Fund (EFF) of
the European Union and the Ministry of Agriculture, Environment and Consumer Protection
Mecklenburg-Vorpommern
References:
1. Rajabi, M. et al.: Biochim. Biophys. Acta. 2012, 1820(1): 56-63.
POSTERS
POS.179
Design, synthesis and structure-activity relationship of Nphenylbenzenesulfonamides as dual 5-lipoxygenase and
microsomal prostaglandin E2 synthase-1 inhibitors
As lead structures aspirin, a covalent COX inhibitor, and indomethacin, a
competitive COX inhibitor, have been selected. The cluster substituted a
(chloro)phenyl ring.
Cheung, S.-Y.1; Hanke, T.1; Fischer, K. 2; Listing, M. 2; Temml, V.3;
Schuster, D.3; Werz, O.2; Schubert-Zsilavecz, M.1
1 Goethe
University of Frankfurt am Main, Max-von-Laue-Str. 9, 60438 Frankfurt am Main,
Germany
University of Jena, Philosophenweg 14, 07743 Jena, Germany
3,4 Institute of Pharmacy/Pharmacognosy3 or Pharmaceutical Chemistry4, Leopold-FranzensUniversity of Innsbruck, Innrain 80/82, 6020 Innsbruck, Austria
2 Friedrich-Schiller
Prostaglandins (PGs) and leukotrienes (LTs) are powerful bioactive lipid
mediators that have a large number of biological actions in the human
body [1,2]. The common precursor of PGs and LTs is arachidonic acid
(AA). The 5-lipoxygenase (5-LO) and the microsomal prostaglandin E2
synthase-1 (mPGES-1) are both enzymes which are involved in the AA
cascade. The 5-LO is the initial enzyme which catalyzes the
transformation of AA to LTs; whereas the mPGES-1 is responsible for the
conversion of PGH2 into PGE2 which is one of the most prominent
mediators of inflammation, pain, and fever. A novel pharmacological
approach for anti-inflammatory therapy is the dual inhibition of 5-LO and
mPGES-1. In contrast to the traditional NSAIDs, the dual inhibition of PGs
and LTs might be superior over single interference with PGs in terms of
anti-inflammatory effectiveness as well as regarding reduced side effects
[3].
In this study we wanted to explore the structure-activity relationship of Nphenylbenzensulfonamide derivatives as dual 5-LO/mPGES-1 inhibitors.
The lead structure of this series was 4-(N-octyl-4-methylbenzenesulfonamido)-benzoic acid (compound 1, see Figure below),
which was originally identified in a virtual screening approach by
Waltenberger, B. et al. [4]. For this compound, a facile three-step
synthesis was developed and structural optimization was carried out in
three
directions,
while
maintaining
the
central
Nphenylbenzenesulfonamide scaffold (see Figure below). Molecular
docking studies supported the importance of the sulfonamide moiety.
Furthermore, we were able to identify moieties with beneficial or
detrimental impact on the overall potency.
Asborin, the carborane analogue of aspirin showed a reduced COX
inhibition, but proved to be a potent aldo/keto reductase 1A1 inhibitor
(IC50=1.4µm) instead. The carborane moiety increased the acetylating
activity followed by decreased selectivity [2,3,4]. Indoborin, the ocarborane analogue of indomethacin, remained a potent COX inhibitor,
but only against the COX-2 isoform (IC50=3.7µm), which is the favorite
COX drug target [5]. Further esterification to the corresponding indoborin
methyl ester gave a highly active and selective COX-2 inhibitor
(IC50=0.08µm).
Acknowledgments: I thank the Beck-Sickinger group (Leipzig), Gust group (Innsbruck), HeyHawkins group (Leipzig), Marnett group (Nashville) for the opportunity to perform the
experiments and the Studienstiftung des deutschen Volkes for financial support.
References:
1. Scholz, M. et al.: Chem. Rev. 2011, 111: 7035-7062.
2. Scholz, M. et al.: ChemMedChem 2009, 4: 746-748.
3. Scholz, M. et al.: ChemMedChem 2011, 6: 89-93.
4. Scholz, M. et al.: Eur. J. Med. Chem. 2011, 46: 1131-1139.
5. Scholz, M. et al.: Bioorg. Med. Chem. 2012, 20: 4830-4837.
POS.181
Structure-Activity Relationships and Development of Novel
Aminothiazole-Comprising 5-Lipoxygenase Inhibitors
Kretschmer, S. B. M.1; Woltersdorf, S.1; Rödl, C. B.1; Vogt, D.1; Stark,
H.1,2; Steinhilber, D.1; Hofmann, B.1
1 Goethe
University, Institute of Pharmaceutical Chemistry, Max-von-Laue-Str. 9, 60438
Frankfurt am Main, Germany;
2 Heinrich Heine University, Institute of Pharmaceutical and Medicinal Chemistry,
Universitätsstraße 1, 40225 Düsseldorf, Germany;
References:
1. Funk, C. D.: Science 2001, 294(5548): 1871−1875.
2. Samuelsson, B.; Morgenstern, R.; Jakobsson, P.J.: Pharmacol. Rev. 2007, 59(3): 207–224.
3. Koeberle, A.; Werz, O.: Curr. Med. Chem. 2009, 16(32): 4274–4296.
4. Waltenberger, B. et al.: J. Med. Chem. 2011, 54(9): 3163–3174.
POS.180
ortho-Carborane as Pharmacophore in Cyclooxygenase
Inhibitors
Scholz, M. S.1
1
Pharmaceutical Institute, University of Bonn, An der Immenburg 4, 53121 Bonn, Germany
Artificial building blocks, such as boron clusters, are innovative moieties
in extending the chemical space used in drug discovery. Boron forms
various cluster types which are often unstable under aqueous conditions.
However, C2B10-carborane-clusters, with two carbon atoms at selected
vertex positions, are sufficiently stable fragments. Among those, orthocarborane reveals a unique electronic composition and a spherical
geometry which occupies double the volume of a rotating phenyl ring [1].
In order to study the applicability of ortho-carborane as pharmacophore
it was integrated into well-investigated cyclooxygenase (COX) inhibitors.
Acute inflammation is a physiological response to injury, aimed at
removing the noxae to restore homoestasis. In contrast, excessive
inflammation can be seen as a unifying component in many chronic
diseases [1]. Leukotrienes (LTs) herein play an important role, as these
lipid mediators are chemotactic for leukocytes (namely LTB4) and
increase vasopermeability of small blood vessels (LTC4) [2]. In this
respect, LTs are associated with diseases like asthma, allergic rhinitis,
rheumatoid arthritis, atherosclerosis and certain types of cancer [3].
5-Lipoxygenase (5-LO) as the key enzyme of LT biosynthesis is a viable
and well studied target. Nonetheless, currently there is only one
approved drug on the market (Zileuton, Zyflo®, FDA approval) which
usage is limited, partly due to hepatotoxic adverse effects [4]. 2Aminothiazoles may fill this gap, since certain derivatives present well
balanced compounds with high potency and appropriate ADMET
characteristics.
Previous studies focused on the impact of small variations preserving a
basic scaffold (ST-1083) on 5-LO inhibition and related targets [5,6]. In
this study, however, we focused at assessing the underlying structureactivity relationships on 5-LO inhibition while extending the 2aminothiazole scaffold in order to achieve novel superior lead structures.
ST-1853 represents such a novel lead structure with a high potency
(IC50 = 50 nM in isolated intact polymorphonuclear leucocytes),
appropriate specificity as well as non-cytotoxic behavior. We present a
successful lead optimization for further investigations as novel antiinflammatory drug.
Acknowledgments: This work was supported by Else Kröner-Fresenius-Stiftung, TRIP, LOEWE,
OSF, Fonds der Chemischen Industrie and DFG INST 208/664-1.
References:
1. Han, J.; Ulevitch, R. J.: Nat. Immunol. 2005, 6(12): 1198–1205.
2. Rådmark, O. et al.: Biochim. Biophys. Acta 2015, 1851(4): 331-339.
3. Peters-Golden, M.; Henderson W. R.: N. Engl. J. Med. 2007, 357(18): 1841-1854.
4. Steinhilber, D.; Hofmann, B.: Basic Clin. Pharmacol. Toxicol. 2014, 114(1): 70–77.
5. Suh, J. et al.: Chem. Biol. Drug. Des. 2012, 80(1): 89-98.
6. Rödl, C. B. et al.: Eur. J. Med. Chem. 2014, 84: 302-311.
INFLAMMATION
POS.182
Filaggrin Deficiency Triggers the Invasion of CD4+ T cells
via TSLP Secretion
Wallmeyer, L.1; Hedtrich, S.1
1 Institute of Pharmacy, Pharmacology and Toxicology, Freie Universität Berlin, Königin-LuiseStraße 2+4, 14195 Berlin, Germany
Introduction: Loss-of-function mutations in the filaggrin gene (FLG) are a
major predisposing factor for atopic dermatitis (AD), but also
immunologic mechanisms are involved in its pathophysiology. The
release of cytokines such as IL-4, IL-13, IL-31, TNF-alpha and cellular
crosstalk between keratinocytes and lymphocytes are important
promoters for the manifestation and maintenance of AD. Moreover,
thymic stromal lymphopoietin (TSLP) is highly expressed by
keratinocytes in lesional skin of AD patients and stimulates the
differentiation of naïve CD4+ cells into Th2 cells which contribute to the
induction of allergic inflammation [1]. Hence, to study the effects of CD4+
T cell migration caused by a lack of FLG in more detail, a FLG knockdown
skin model (FLG-) was established [2,3]. After T cell supplementation,
the expression of TSLP and skin barrier proteins as well as the barrier
function of the skin models was investigated.
Methods: Normal (FLG+) and FLG deficient (FLG-) skin models were
generated according to previously published procedures [2]. Naïve CD4+
T cells were generated from human peripheral blood mononuclear cells
by negative selection, activated with anti-CD3/CD28-beads and
thereafter, added to the skin models for 2 days at day 12 of tissue
cultivation. Subsequently, the skin models were analyzed for protein
expression of FLG, involucrin (IVL) and TSLP using immunofluorescence
staining. To analyze the influence of Th2 cytokines on T cell migration
and differentiation, the skin models were pre-incubated with IL-4 and IL13 (30 ng/ml) for 2 days before T cell supplementation. To investigate the
effect of TSLP secretion on T cell invasion, skin models were preincubated with TSLP neutralizing antibody (20 µg/ml). Skin absorption
studies were performed with the radioactive labeled standard compound
testosterone to assess the skin barrier function.
Results and Discussion: Interestingly, FLG deficiency alone triggered the
migration of CD4+ T cells into the dermal equivalent of the skin models.
In contrast, no immune cell migration was observed in FLG+, but the
stimulation with IL-4 and IL-13 stimulated migration of T cells into FLG+
and FLG-. Immunofluorescence staining showed significantly increased
TSLP levels in FLG-, FLG- supplemented with CD4+ cells and in FLG+
supplemented with CD4+ cells, although in the latter no immune cell
migration was detected. A compensatory upregulation of IVL in FLG- was
observed, which was leveled out after T cell contact. Furthermore, only
due to the presence of CD4+ T cells underneath the construct, without
direct contact, the expression of FLG and IVL in FLG+ were reduced. The
influence of T cells led to a reduced barrier function of the skin models.
Interestingly, after inhibition of TSLP secretion no T cell migration was
observed.
Our data show, that an increase of TSLP due to the lack of FLG plays a
potential role for the migration of T cells into dermal tissue. As there are
no dendritic cells presenting in the skin model, our data suggest a direct
link between TSLP and stimulation of T cells without involvement of
dendritic cells.
Acknowledgments: Financial support by the foundation SET (Foundation for the Promotion of
Alternate and Complementary Methods to Reduce Animal Testing) is gratefully acknowledged.
References:
1. Soumelis, V. et al.: Nat. Immunol. 2002, 3: 673-80.
2. Küchler, S. et al.: Altern. Lab. Anim. 2011, 39: 471-80.
3. Vávrová, K. et al.: J. Invest. Dermatol. 2014, 134: 746-53
predisposing factor for the manifestation of AD [1]. Aside from barrier
deficiencies, AD is characterized by over-shooting Th2-mediated
inflammatory processes and impaired innate immunity such as altered
expression of antimicrobial peptides (AMP) [2]. The Th2 cytokines IL-4 and
IL-13 significantly contribute to the pathogenesis of AD, but their effects on
the skin barrier and particularly the interdependencies with FLG deficiency
are not yet fully understood. A deficient skin barrier appears to upregulate
AMPs [3] and absence of FLG increases the expression of other structural
proteins such as involucrin [4]. In this study, the influence of FLG
knockdown on the expression of the human β-defensins 1-3 and skin barrier
proteins under inflammatory conditions was evaluated.
Methods: Normal (FLG+) and FLG deficient (FLG-) skin models were
generated according to previously published procedures [5]. In primary
human keratinocytes gene knock down was induced by transfection with
FLG specific siRNA. Starting at day 10 of cultivation, the skin models were
exposed to IL-4 and/or IL-13 (30 ng/ml) for 4 days. Subsequently, protein
expression was determined using western blot analysis and
immunostaining; gene expression was quantified via RT-PCR. The skin
models were further investigated for morphology (H&E staining) and
release of the proinflammatory cytokines IL-6 and IL-8 via ELISA. To
unravel the mechanism of increased AMP expression, primary human
keratinocytes were pre-incubated with IL-1R antagonist (10 µg/ml), IL-6 and
TLR-2 neutralizing antibodies (5 µg/ml), respectively.
Results and Discussion: Histological examination revealed a thickening of
the viable epidermis in the skin models following IL-4 and IL-13 treatment.
FLG knockdown amplified this effect (FLG+ 91.0 ± 13.8 µm vs. FLG+/IL4/13 138.1 ± 10.7 µm and FLG- 108.7 ± 14.4 µm vs. FLG-/IL-4/13 164.2 ±
11.0 µm). Cytokine specific increased levels of IL-8 and IL-6 in FLG- models
indicate altered susceptibility to inflammatory stimuli. Additionally, we
observed a compensatory upregulation of involucrin in the FLG- models,
which was considerably disturbed by IL-4 and IL-13 exposure. Furthermore,
cytokines significantly reduced the expression of FLG and IVL in normal
skin models. Most interestingly, we detected significantly higher expression
of HbD2 and HbD3 in FLG- models. This was especially interesting because
HbD2 and HbD3 are known to be upregulated through bacteria or
inflammation but not by a genetic defect. Since HbDs stimulate the release
of proinflammatory cytokines, these results might explain the more severe
AD phenotype in patients with FLG mutations [6]. Pre-incubation with an IL1R antagonist significantly diminished the upregulation of HbD2 and HbD3
indicating an IL-1 mediated mechanism. Levels of the constitutively
expressed HbD1 were unaffected.
In conclusion, FLG deficiency leads to a counterregulation of skin barrier
proteins, which is disturbed by Th2 derived cytokines, and, secondly, to
an altered immune response indicated by an upregulation of HbD2 and
HbD3.
Acknowledgments: Financial support by the Collaborative Research Center 1112 for the project
C02 is gratefully acknowledged.
References:
1. Palmer, C.N. et al.: Nat. Genet. 2006, 38(4): 441-6.
2. Kopfnagel, V. et al.: Curr. Opin. Allergy Clin. Immunol. 2013, 13(5): 531-6.
3. Ahrens, K. et al.: J. Invest. Dermatol. 2011, 131(2): 443-52.
4. Presland, R.B. et al.: J. Invest. Dermatol. 2000, 115(6): 1072-81.
5. Küchler, S. et al.: Altern. Lab. Anim. 2011, 39(5): 471-80.
6. McAleer, M.A.; Irvine, D.A.: J. Allergy Clin. Immunol. 2013, 131(2): 280-91.
POS.184
Impact of torsion of N,4-diaryl-1,3-thiazol-2-amines on 5lipoxygenase inhibitory potency
Woltersdorf, S.1; Kretschmer, S. B. M.1; Rödl, C. B.1; Vogt, D.1;
Steinhilber, D.1; Hofmann, B.1; Stark, H.1,2
1 Institute
POS.183
Filaggrin deficiency alters the innate immune response in a
3D skin model
Hönzke, S.1; Schäfer-Korting, M.1; Hedtrich, S.1
1 Institute
for Pharmaceutical Sciences, Pharmacology & Toxicology, Freie Universität Berlin,
Königin-Luise-Straße 2+4, 14195 Berlin, Germany
Introduction: Atopic dermatitis (AD) is a chronic, relapsing inflammatory skin
disease which is characterized by an impaired skin barrier function. In 2006,
mutations in the filaggrin gene (FLG) were identified as a major
of Pharmaceutical Chemistry, Goethe University Frankfurt, Max-von-Laue-Str. 9, D60438 Frankfurt am Main, Germany
2 Institute of Pharmaceutical and Medicinal Chemistry, Heinrich Heine University Düsseldorf,
Universitätsstrasse 1, D-40225 Düsseldorf, Germany
Leukotrienes (LTs) are important lipid mediators derived from
polyunsaturated fatty acids playing a key role as regulators in immunity
and inflammatory processes and thereby influencing acute as well as
chronic diseases, e.g. asthma, allergic rhinitis, cardiovascular disease
and certain types of cancer [1]. The LT biosynthesis is initiated by the 5lipoxygenase enzyme (5-LO). It catalyzes the conversion of free
arachidonic acid to LTA4 which is subsequently converted into further LT
subtypes [2]. Up to date, there is only one approved direct 5-LO inhibitor
in clinical use: Zileuton. It acts by chelating the catalytic iron in the active
POSTERS
site of the enzyme. However, this drug exhibits non-optimal
pharmacodynamic and pharmacokinetic profiles. Therefore, novel potent
5-LO inhibitors are of great interest for an anti-inflammatory therapy [3].
Starting from the well-known 5-LO inhibitor SKI-II [4], we prepared a
series of N,4-diaryl-1,3-thiazol-2-amines with bulky substituents
preferring a twisted conformation at 4-position of the heterocycle (R2) and
investigated their influence on inhibition of human 5-LO activity. The
impact of the torsion was addressed by additional substitution at the 5position (R1) influencing especially the cytotoxicity profile. The chemical
structure of the thiazole-2-amine scaffold was further optimized at
positions R3 maintaining high 5-LO inhibitory activity and selectivity.
OH
R3
S
NH
R1
N
SKI-II
R3
S
NH
R2
Cl
OH
N
Substituents R1 - R2 for torsion
Substituens R3 for efficacy
From this series we could deduce the importance of bulky substituents
and torsion on 5-LO inhibitory potency. With compound ST-1906 (4-((4(2,4-dichlorophenyl)-5-methylthiazol-2-yl)amino)-2,6-dimethylphenol) we
present the most potent derivative of this series. It blocks the 5-LO activity
with an IC50 value of 0.05 µM (0.033 - 0.067 µM) and demonstrates no
signs of cytotoxicity.
Acknowledgments: This work was supported by Else Kröner-Fresenius-Stiftung, TRIP, LOEWE,
OSF and Fonds der Chemischen Industrie.
References:
1. Gualde, N et al.: Trends. Mol. Med. 2008, 14(10): 461-9.
2. Dennis, EA et al.: J. Lipid. Res. 2009, 50(6): 1015-38.
3. Steinhilber, D.; Hofmann, B.: Basic Clin. Pharmacol. Toxicol. 2014: 114(1): 70–77.
4. Suh, J et al.: Chem. Biol. Drug. Des. 2012, 80(1): 89-98.
.
CLINICAL PHARMACY
4.10 Clinical Pharmacy
POS.185
Investigation of altered organ blood flow on carvedilol
disposition in adult and paediatric chronic heart failure
patients by using PBPK modelling
Rasool, M. F.1,2; Khalil, F.1; Läer, S.1.
1 Institute
of Clinical Pharmacy and Pharmacotherapy, Heinrich-Heine University, 40225
of Pharmacy, Bahauddin Zakariya University Multan, 60800, Pakistan.
2 Department
Background: The reduction of organ blood flows in chronic heart failure
(CHF) can significantly affect the clearance of drugs with high hepatic
extraction. Physiologically based pharmacokinetic modelling (PBPK),
due to its ability to incorporate the pathophysiological changes in different
diseases can be used in CHF to predict clearance of drugs with high
hepatic extraction as carvedilol.
Methods: A PBPK model based on cytochrome-P450 clearances was
developed. The Developed model was evaluated in healthy adults and
after incorporation of reduced organ blood flows in adult CHF patients,
the evaluated adult CHF model was scaled to paediatric CHF patients
using population based simulator Simcyp®. A two-fold error range for the
ratios(Obs/Pred) of the pharmacokinetic parameters was used for model
evaluation.
Results: The prediction results were within the 2-fold error range. The
CL/F ratio(Obs/Pred) was clearly improved after incorporation of reduced
organ blood flows in adult CHF patients. In paediatrics CHF patients,
improvement in predictions were seen only in adolescents above 17
years of age, staged with NYHA system of classification.
Conclusion: There was a clear link between reduced organ blood flows
and reduced carvedilol clearance in adult patients with CHF. It was
suggested that Ross scoring system in paediatrics was not well
correlated with organ blood flow reductions as the NYHA classification
system. Due to the mechanistic nature of the developed PBPK model, it
can be extended to other drugs with high hepatic extraction.
n°602295 (LENA) and from the Faculty Development Program Bahauddin Zakariya University
Multan, 60800, Pakistan.
POS.186
Evaluation of a regulatory-compliant bioanalytical setting
suitable to determine pharmacokinetics and
pharmacodynamics of enalapril in Phase II/III studies in all
paediatric age groups
Burckhardt, B. B.1; Tins, J.1; Läer, S.1
1 Institute
of Clinical Pharmacy and Pharmacotherapy, Heinrich-Heine-University,
Universitaetsstr.1, 40225 Düsseldorf, Germany
Background: Clinical investigations on pharmacokinetics and
pharmacodynamics are highly required to improve paediatric
pharmacotherapy. The trial-related blood loss should not exceed 3% of
the total blood volume within 4 weeks [1]. Prior to this investigation
bioanalytical assays in serum, urine and saliva were developed to meet
the challenging ethical and analytical burdens especially in neonates and
infants. The applicability of the tailored paediatric assays has not been
shown and is recommended prior the assays are applied to the
vulnerable paediatric population.
Objective: To evaluate the applicability and reliability of a new tailored
bioanalytical setting for pharmacokinetic and pharmacodynamic
determination of drugs acting on the RAA system in paediatric patients.
Invasive and non-invasive collection procedures need to be considered.
Methods: A proof-of-concept study in 22 healthy adults was conducted
by applying the developed low-volume bioanalytical setting. Utilizing
HPLC-MS/MS, the bioanalytical assays determined enalapril and
enalaprilat concentrations in 50 µL serum as well as 100 µL urine and
saliva. Changes of humoral parameters of the RAA system were
measured by five immunological assays. Drug concentrations in serum
were determined 31 times during the first 24 hours after drug
administration while blood for humoral parameters were sampled 10
times. Additionally, the applicability of the low-volume assays was
assessed by a regulatory-compliant Phase I study in 24 healthy
volunteers analyzing urine and serum concentrations of enalapril and
enalaprilat.
Results: The proof-of-concept study made very dense and reliable
individual concentration-time profiles of the drugs and the humoral
parameters available. Using the low-volume assays revealed
comparable pharmacokinetic results of enalapril and enalaprilat in serum
and urine when compared to data published in literature. Changes in the
humoral parameters are also similar and prove the applicability of the
bioanalytical platform. Applying the developed low-volume bioanalytical
assays allows to determine 6 drug concentrations and 2 times the
measurements of changes in the renin angiotensin aldosterone system
in newborns at a single day without infringing ethical recommendations.
The conducted investigations on non-invasive sampling approaches in
saliva appeared inappropriate to replace invasive pharmacokinetic
sampling of enalapril.
Conclusion: The applicability of the child-appropriate bioanalytical
platform was proven in adults and it can be applied in upcoming
paediatric Phase II and Phase III studies of the LENA (Labeling of
Enalapril from Neonates up to Adolescents) project. Meaningful
concentration-time profiles of the cardiovascular drug and their effect on
humoral parameters can be obtained even in neonates without infringing
ethical recommendations on trial-related blood loss. The obtained
pharmacokinetic and pharmacodynamic dataset within the proof-ofconcept study allows to validate physiological-based computer models
that are of increasing interest for an optimal planning and conduction of
paediatric clinical trials.
Acknowledgments: The research leading to these results has received funding from the European
Union Seventh Framework Programme (FP7/2007-2013) under grant agreement n°602295
(LENA).
References:
1. European Commission, Ethical considerations for clinical trials on medicinal products
conducted with the paediatric population 2008.
POS.187
Thrombosis prophylaxis in high-risk patients: Is the
recommended dosage sufficient? An observational study
on pharmacokinetics of Enoxaparin
Beheiri, S. N.1,2; Wähnert, D.2; Schulz, D.3; Völler, S.1; Hempel, G.15
WWU Münster Institut für Pharmazeutische und Medizinische Chemie- Klinische Pharmazie,
Corrensstraße 48, 48149 Münster, Germany
2 Universitätsklinik Münster- Klinik für Unfall-, Hand- und Wiederherstellungschirurgie, AlbertSchweitzer-Campus 1, 48149 Münster, Germany
3 Universitätsklinik Münster- Klinik für allgemeine Orthopädie und Tumororthopädie, AlbertSchweitzer-Campus 1, 48149 Münster, Germany
1
Background: Deep vein thrombosis is one of the most serious
complications in musculoskeletal surgery. The prevalence in critically ill
patients is reported up to 60% [1]. Patients after major operations like
spinal surgery, endoprosthesis as well as pelvic fractures exhibit a
significant elevated risk. Obese patients also belong to the high-risk
collective without reference to the type of surgery. As opposed to this,
the presence of renal failure entails the danger of accumulation of the
anticoagulative agent and raises the risk of bleeding complications.
In clinical routine, all patients receive a fixed dose of 40 mg enoxaparin,
as recommended in the prescribing information. We hypothesized that
distinct patients may have insufficient thrombosis protection by the actual
dose recommendations. The aim of this study is to evaluate the
effectiveness of the recommended dosage of enoxaparin using antifactor-Xa activity as a surrogate-parameter.
Materials and Method: A prospective case controlled study was
designed. 117 patients were included and divided in into three groups
(Group I (n=43): high- risk patients, <100 kg, eGFR >60 ml/min; group II
(n=62): >100 kg, eGFR >60 ml/min; group III (n=12): <100 kg, eGFR 3050 ml/min). Postoperatively, all patients received enoxaparin 40 mg once
daily. Anti-factor-Xa activity was measured four and twelve hours (peak
and trough level) after application of the third dose to verify if the target
area of 0.1-0.4 I.E./ml was reached. Dose adaption was conducted in
case of insufficient anti-factor-Xa activity and the activity was checked
again later. Furthermore, a population pharmacokinetic model using
NONMEM was applied using the anti-factor-Xa activity data of all
patients. This model was used to prove that dose adaptation results in
reaching target range.
POSTERS
Results: After 4 h, adequate anti-factor-Xa activity was found only in
group I (median 0.20 I.E./ml). 28.5% of all anti-factor-Xa peaks of the
obese patients (body weight 114 kg) reached the level of 0.1 I.E./ml
(median 0.13 I.E./ml). In group III (median eGFR 45.2 ml/min) all peak
levels reached the target area but were significantly increased (median
0.29 I.E./ml). The median activity of <0.1 I.E./ml after 12 h in all groups is
not sufficient for prophylaxis.
In patients with peak levels of <0.1 I.E./ml after 4 h, the dosage was
increased. If the trough level after 12 h was <0.1 I.E./ml, enoxaparin was
administered twice daily. In all patients with adapted dose, anti-factor-Xa
activity was controlled again. Measured trough levels do not indicate
accumulation of enoxaparin (group I: Figure). There was no increase in
bleeding complications after adaptation of the dosage schedule.
A two-compartment model was adequate for describing the enoxaparin
kinetics. Body weight, body mass index and renal clearance were the
most influencing covariates for enoxaparin clearance (25.6 ml/h/kg).
Interindividual variability was found to be 25.9%. The simulation of antifactor-Xa activities for the adapted dose schedule demonstrates that
activities in the target range are achieved.
final concentration of 2∙106 CFU/mL in the CC. Next (t=0 h), LEV was
added to the CC mimicking an i.v. bolus injection. Samples (350 µL) were
drawn at predefined points of time(-2, 0, 0.5, 1, 1.5, 2, 3, 4, 6, 20, 24 h).
The experiments were performed at least in duplicate and included a
growth control without addition of antibiotic as positive control. The
quantification of LEV was performed with a developed, validated
fluorimetric assay and for the bacteria the droplet plate assay was used
[3]. The simulations of LEV concentration-time profiles were realised with
Berkeley Madonna™.
Figure: Schematic illustration of the dynamic IVIM [2]
Conclusion: Our study demonstrates that anticoagulation with
enoxaparin once daily without consideration the patients’ weight and
renal function does not achieve sufficient anti-factor-Xa levels in all
patients. Consequently, a possible increased risk of thrombosis may exist
despite guideline-oriented anticoagulation with enoxaparin.
Acknowledgments: Klinik für Unfall-, Hand- und Wiederherstellungschirurgie der
Universitätsklinik Münster, Klinik für allgemeine Orthopädie und Tumororthopädie der
Universitätsklinik Münster
References:
1. Geerts, W. H. et al.:. N Engl J Med. 1994, 331: 1601-6.
POS.188
Dynamic in vitro infection model enables mimicking of in
vivo levofloxacin PK profiles showing eradication of E. coli
infection
Bartels, I. K.1; Goebgen, E. B.1; Kloft, C.1
Results: After single i.v. bolus administration the measured maximum
LEV concentrations were 8.74±2.08 µg/mL (n=6). The shape of the
curve showed an exponential decrease with a half-life of 7.0 h.
Concentrations below 1.0 µg/mL were reached after 20 to 22 h. The
profile of the measured data were comparable to the previously simulated
concentration-time profiles of LEV. For time-kill curves with E. coli (n=1)
bacterial samples >3 h after start of LEV exposition were below the limit
of quantification. The growth control, however, shows a logarithmic
increase of bacteria up to 6 h followed by a constant conc. of bacteria of
2∙109 CFU/mL over time.
Discussion/Conclusion: The dynamic IVIM allowed to mimic in vivo
concentration-time profile in plasma of LEV after i.v. bolus administration.
Therefore, implementation of the PK properties of drugs in in vitro
investigations on the PD in clinically relevant scenarios are feasibible to
e.g. to detect subtherapeutic antiinfective therapies before clinical trials
are performed. Especially investigations on the development of
resistance, induced by e.g. drug concentrations below the minimum
inhibitory concentration (MIC) at the end of dosing intervals, could be
impeded. Due to the neglected immune system the results of the
presented IVIM can give a first idea of the PD what can be expected in
vivo and need further investigations in e.g. clinics. As proof-of principle,
the investigated drug concentration could successfully eradicate E. coli
after 3 hours treatment. Further investigations should include bacteria
with higher MICs in which sub-therapeutic concentrations at the target
side are more likely.
References:
1. http://www.rxlist.com/levaquin-drug/clinical-pharmacology.htm (Accessed: 23.06.2015)
2. Michael, J.: Pharmacodynamic in vitro studies contributing to the rational use of linezolid in
infections by vancomycin resistant Enterococcus faecium. 2011.
3. Schwalbe, R.; Steele-Moore, L.; Goodwin, A. C.: Antimicrobial susceptibility testing protocols
(CRC Press) 2007.
Dept. of Clinical Pharmacy and Biochemistry, Institute of Pharmacy, Freie Universitaet Berlin,
1
Objectives: In order to determine the pharmacodynamic (PD) effect of
antibiotics against clinically relevant bacterial strains, often the simpler
static in vitro infection models (IVIMs) with a constant unchanging drug
concentration are used. As a possibility to allow for a better
characterisation of the PD considering in vivo pharmacokinetic (PK)
properties of the drug, dynamic IVIMs were developed, which include
changing drug concentrations over time. For the antibiotic levofloxacin
(LEV), a treatment option for e.g. urosepsis caused by Escherichia coli
(E. coli), the maximum plasma concentration after single i.v.
administration of 750 mg is 11.5±4.0 µg/mL. With the half-life of
7.5±1.6 h a simulation of a concentration-time profile is possible [1]. The
aims of these investigations were (i) mimicking the in vivo plasma
concentration-time profile of LEV with a dynamic IVIM [2] and (ii)
determining the PD resulting from the applied PK in time-kill experiments
with E. coli.
Methods: The dynamic IVIM used for the investigations was previously
presented [2]. Briefly, pumps managed in- and outflow to the central
heatable compartment (CC) (see Figure).The flow rate of the pumps was
calculated based on the in vivo half-life of LEV and the volume of the CC
(90 mL) and set to 0.15 mL/min. In a pre-incubation step (2 h) E. coli
(ATCC 25922) was grown in cation-adjusted Mueller Hinton Broth to a
POS.189
Analysis and optimization of the medication of multimorbid
patients on acute geriatric wards - A prospective
intervention study (OptiMe)
Nachtigall, A.1; Heppner, H. J.2; Thürmann, P. A.1
1 Clinical
Pharmacology, Faculty of Health, Department of Human Medicine, HELIOS Clinical
Center, 42283 Wuppertal, Germany
2 Geriatric Medicine, Faculty of Health, Department of Human Medicine, HELIOS Clinical
Center, 58332 Schwelm, Germany
Background: Demographic change is a frequently discussed topic due to
the challenges that health care professionals have to face whilst dealing
with elderly patients. Geriatric patients are characterized by multiple
morbidities like congestive heart failure, diabetes, osteoarthritis,
hypertension etc. The guideline-adapted treatment of numerous
diseases inevitably leads to polypharmacy [1]. This causes drug related
problems (DRP) and drug-drug interactions (DDI), which are often
unpredictable and can therefore contribute to adverse outcomes such as
hospitalizations. As only few clinical trials exist regarding elderly people
taking multiple drugs, evidence-based decision-making is often
CLINICAL PHARMACY
impossible. Therefore the cooperation between health care professionals
like pharmacists and physicians is essential to provide an effective and
adequate health care.
Objective: The aim of this project is the optimization of pharmacotherapy
during hospitalization on a geriatric ward in order to improve the elderly
patients’ outcomes.
The short-term objective is the implementation of a clinical medication
management by a pharmacist to detect drug related problems (DRP).
Primary endpoint is the percentage of patients having a DRP defined as
a) prescription of potentially inappropriate medication (PIM, PRISCUSlist), b) drug-drug interaction (DDI) from “contraindicated” to “concurrent
application not recommended”, c) presence of an adverse drug reaction
and d) prescription without indication.
Methods: For this prospective, controlled, interventional study 400
patients will be enrolled: 200 patients in the intervention ward (A) and
another 200 in the control ward (B) will be recruited on the Geriatric ward
(HELIOS clinic Schwelm, North Rhine-Westphalia, Germany) during a
one year period (January – December 2015). The allocation to ward (A)
or (B) is done study-independently in the administration department of
the clinic, resulting in a quasi-randomization. Inclusion criteria are the
prescription of at least five medications, age of 70 years or above and
signed informed consent. Each patient from ward (A) will be paired with
one patient from side (B) having the same sex and age ±5 years. Data
are recorded in an ACCESS database (Microsoft 2013) specifically
developed for this project, where drugs are coded according to the ATC
code, adverse drug reactions according to the WHO SOC and their
severity using CTCAE criteria. Analysis and screening of prescribed
drugs will be performed by using the Medication Appropriateness Index
(MAI) [2] and the PRISCUS-List [3] (screening for potentially
inappropriate medication (PIM)). Drug-drug interactions will be checked
using the ABDA module for DDIs. Functional scores for mobility and
cognition and the preferences of the patient - identified by a questionnaire
– complete the tools for decision-making. In both groups, the patient’s
medication will be documented at admission and discharge, but only in
the intervention group drug related problems will be communicated to the
physicians. All suggestions of the pharmacist will be documented and
coded as well as their acceptance by physicians.
Results: The prevalence of DRP is expected to be reduced from approx.
50% to 20% in the intervention group and - considering the learning bias
and the routine work - to 30% in the control group.
The study protocol was approved by the ethics committee of the
University Witten/Herdecke. So far, 106 patients in the intervention and
103 patients in the control group have been included. First experience
shows the feasibility of the intervention. Acceptance rate and
cooperativeness are high, whereas the daily lack of time of physicians is
a limiting factor.
Acknowledgments: This study was supported by HELIOS Kliniken GmbH, grant ID 063614 and
by Robert Bosch Stiftung.
References:
1. Boyd, C. M. et al.: JAMA. 2005, 294(6): 716-24.
2. Hanlon, J. T. et al.: J Clin Epidemiol. 1992, 45 (10): 1045–51.
3. Holt, S.; Schmiedl, S.; Thürmann, P. A.: Dtsch Arztebl Int. 2010, 107(31-32): 543–51.
POS.190
Development of a drug-drug interaction registry based on
notifications collected in german community pharmacies
Braun, C. A.1; Woltersdorf, R.1; Simons, S.2,3; Klahn, D.4; Jaehde, U.1
1 Institute
Bonn, Germany
2 MVDA e.V., Kirschbaumweg 23, 50996 Cologne, Germany
3 Apotheke am Stadttor, Werdohler Straße 4-6, 58809 Neuenrade, Germany
4 LINDA AG, Emil-Hoffmann-Straße 1a, 50996 Cologne, Germany
Background: In their daily practice community, pharmacists deal with a
multitude of drug-drug interaction (DDI) notifications.
Purpose: The aim is to develop a DDI register that continuously collects
all DDI notifications in German community pharmacies including how
pharmacy staff manages DDI. This can help to estimate the incidence
and relevance of DDI in community pharmacies. A DDI register also
provides a basis for advanced education of the pharmacy staff in order
to improve the quality of DDI management.
Methods: We perform our evaluation in cooperation with the LINDA AG,
a network of community pharmacies in Germany. In participating
pharmacies the pharmacy software automatically generates datasets of
all detected DDI notifications, which are sent to a DDI register. A
statistical analysis is performed regarding the total amount of DDI
notifications, the distribution of the degree of severity, and the incidence
of distinct DDI.
Results: In a pilot study, we detected more than 490,000 DDI notifications
in 74 community pharmacies within four months. Preliminary analysis
revealed that about 5% (23,000) of these notifications were due to severe
DDI. QT interval prolonging drugs, psychotropic drugs,
sympathomimetics, and antihypertensives represent classes of drugs
most often involved in severe DDI notifications.
Conclusions: Our preliminary analysis indicates the high frequency of
detected potential DDI in German community pharmacies but does not
reflect their actual relevance. Therefore, we developed a standardized
electronic system for documentation of DDI management for further
analysis.
POS.191
The standardized medication plan – First results from the
MMP16 consortium
Abbas, Z.1; Schächtele, S.2; Griebel, L.3; Prokosch, H. U.3; Dörje, F.4;
Maas, R.2; Fromm, M.2; Dormann, H.5; Friedland, K.1
1 Molecular
and Clinical Pharmacy, Department of Chemistry and Pharmacy, FriedrichAlexander-University Erlangen/Nürnberg, Cauerstr.4, 91058 Erlangen, Germany
2 Clinical Pharmacology, Institute of Experimental and Clinical Pharmacology and Toxicology,
Friedrich-Alexander-University/ Erlangen-Nürnberg, Fahrstr. 17, 91054 Erlangen, Germany
³ Institute for Medical Informatics, Department of Medical Informatics, Friedrich-AlexanderUniversity Erlangen-Nürnberg, Wetterkreuz 13, 91058 Erlangen-Tennenlohe, Germany
4 Pharmacy Department, Erlangen University Hospital, Erlangen, Palmsanlage 3, 91054
Erlangen, Germany
5 Department of Emergency Medicine Hospital Fürth, Jakob-Henle-Straße 1, 90766 Fürth,
Germany
Side effects and medication errors are frequent causes for hospital
admissions. One reason for these mistakes are information gaps in the
actual medication of the patient. As an answer to solve these problems,
a standardized medication plan was developed by the German ministry
of health. This plan is currently tested in three model regions regarding
comprehensibleness and acceptance by patients but also by health care
providers such as physicians and pharmacists. Our consortium MMP16
consists of 10 medical practices and 10 corresponding community
pharmacies in Nürnberg and Fürth which will use the standardized
medication plan over 2 years. Our consortium also involves clinicians,
clinical pharmacologists, clinical pharmacists, scientists in health
economy and medical computer scientist. Together, we developed
questionnaires to address the acceptance of patients of the standardized
medication plan. In addition, we will analyze the completeness and the
update of the medication plans by health care providers using the
computer platform MediTalk. Our questionnaires are divided in different
parts, they start with demographic questions about the age, sex,
education and mother tongue. Then questions regarding the general
attitude to medication are included, which are mostly about the
importance of medication and the patients’ knowledge about their
medication. Adherence is measured using the 8-item-Moriskyquestionnaire. Finally, the questionnaire contains several questions
about the medication plan itself as well as the patient’s expectations
regarding the effect of having a medication plan for himself but also for
the health care providers.
To evaluate the ease of use and the comprehensibility, a pretest including
13 patients was conducted in a community pharmacy. Interestingly, 23%
of the asked patients do not know the name of the active compound of
their medication. Therefore, the question was changed asking for the
drug name and not for the active compound. Several other points were
also changed improving the ease of use of the questionnaires. In a next
step, the questionnaires will be filled in by 120 patients using the
standardized medication plan.
Acknowledgments: We thank the BMG for funding the MMP16 consortium.
POSTERS
POS.192
Medication review for geriatric patients in nursing homes
Bitter, K.1; Pehe, C.2; Krüger, M.3; Heuer, G.3; Quinke, R.3; Jaehde, U.1
1 Institute
Bonn, Germany
2 AOK Rheinland/Hamburg, health insurance, Kasernenstr. 61, 40213 Düsseldorf, Germany
3 Pharmacists’ Association North Rhine, Tersteegenstr. 12, 40474 Düsseldorf, Germany
Background: Drug-related problems (DRP) are common in nursing home
residents due to polymedication [1]. Community pharmacies supplying
drugs to nursing homes may play an important role in detecting and
solving DRP in nursing home residents.
Objectives: This project aims to evaluate whether a simple medication
review, solely based on the patient’s medication and performed by
community pharmacists, can enhance the medication safety of nursing
home residents.
Methods: At the beginning, participating pharmacists attended a special
training focusing on pharmacotherapy and adverse drug events in the
elderly. Patients at the minimum age of 65 years insured by AOK
Rheinland/Hamburg (AOK) and regularly taking at least five drugs per
day were invited to participate. The AOK provided prescription data of
these patients to the pharmacies where the current medication and
further information from the nursing home, e.g. dose regimens, were
added. If necessary, unclear or false medication data of the nursing
homes was corrected. Based on the medication list, pharmacists
performed a simple medication review according to a specific guideline.
The detected and solved DRP were counted.
Results: So far, we tested the feasibility of this intervention in a pilot study
including five community pharmacies. The medication of 28 patients was
surveyed. In 89% of the cases, the pharmacists added further medication
data to the provided prescription data. Concerning the nursing homes’
medication data, 18 DRP were detected including e.g. poor
documentation of dosage forms or dose regimens or even missing
documentation of drugs. In average, the pharmacists identified two DRP
per patient. Most frequent were drug-drug-interactions (33%) of which
40% were considered as relevant for the residents’ medication safety.
39% of the patients took drugs considered as potentially inadequate in
the elderly. The pharmacists gave recommendations to the physicians if
necessary. However, the acceptance rate by the general practitioners
was 15% only.
Conclusions: A simple medication review performed by pharmacists
seems to be feasible and can be performed on the basis of a complete
drug anamnesis.
Acknowledgments: We thank all participating pharmacies for the excellent collaboration.
References:
1. Gurwitz, J. H. et al.: Am. J. Med. 2005, 118: 251-8.
In an ongoing clinical trial, microdialysis is performed in the ISF of
subcutaneous tissue of neonates treated with VAN. Prior to clinical trials,
an in vitro microdialysis characterisation of the particular drug is crucial
in order to set optimal settings for a consistent in vivo relative recovery
(RR); being a prerequisite for calculation of ISF concentration.
Methods: In vitro characterisation of VAN was performed with a
standardised microdialysis system [2] and as in the clinical trial with CMA
63 catheters (membrane length 10 mm, molecular weight cut-off 20 kDa).
VAN concentration ranged from 10 to 100 µg/mL; the surrounding
medium was heated to 37 °C to mimic body temperature. Three different
settings were investigated: (i) Ringer’s solution (RS) as perfusate and
surrounding medium, (ii) RS as perfusate and phosphate buffered saline
(PBS) as medium and (iii) PBS for both, medium and perfusate. The
surrounding medium mimicked the ISF. RR was determined for a flow
rate of 1.0 µL/min and pH values of 7.0 and 7.4 (for PBS) in both, delivery
(i.e. VAN diluted in RS or PBS in the perfusate) and recovery (i.e. VAN
diluted in PBS or RS in the surrounding medium) experiments, to quantify
and assess equality of RR in both permeation directions. Five replicates
were taken in each experiment. An HPLC assay for the quantification of
VAN from microdialysate samples was developed and validated
according the EMA guideline [3].
Results: RS used as perfusate and PBS as medium (ii) resulted at pH
7.0 in a consistent RR in both experiments over the entire concentration
range. At pH 7.4, RR showed lower recovery values compared to delivery
but less variable than if PBS was used for both. Setting (i) leads to higher
RR in delivery than in recovery and so the RR values were not equal in
both directions. In setting (iii) the RR for delivery was consistent over the
investigated concentrations and pH range, recovery experiments showed
high variations (CV ≤ 23.2%).
Conclusion: Based on RR findings, use of RS as perfusate and PBS as
surrounding medium led to a consistent RR in in vitro investigations. This
setting is also the most similar to the setting in the clinical trial. Equality
of RR in both directions could not be proven in vitro and reasons have to
be further investigated. In upcoming investigations alternative buffers to
PBS will be examined to improve the setting.
References:
1. Plock et al.: Biomed. Chromatogr. 2005, 19: 237–44.
2. Simmel, F.; Kloft, C.: Int. J. Clin. Pharmacol. Ther. 2010, 48(11): 695–704.
3. European Medicines Agency (EMA): Guideline on bioanalytical method validation 2012
(http://www.ema.europa.eu/docs/en_GB/document_library/Scientific_guideline/2011/08/WC500
109686.pdf; 29.06.15)
POS.194
Variability of meropenem serum concentrations in
intensive care patients
Ehmann, L.1,2; Zander, J.3; Zoller, M.4; Minichmayr, I. K.1,2, Kloft, C.1
1 Dept.
Graduate Research Training program PharMetrX, Germany
3 Institute of Laboratory Medicine, Hospital of the Ludwig-Maximilans-University of Munich,
Marchioninistr. 15, 81377 Munich, Germany
4 Dept. of Anaesthesiology, Hospital of the Ludwig-Maximilans-University of Munich,
Marchioninistr. 15, 81377 Munich, Germany
2 and
POS.193
Optimisation of microdialysis of vancomycin in neonates
by in vitro experiments
Burau, D.1; Fürtig, M.-A.1; Schröpf, S.2; Kloft, C.1
1 Dept.
2 Dept. of Pediatrics, Dr. von Haunersches Kinderspital, Lindwurmstraße 4, 80337 Munich,
Germany
Background: Vancomycin (VAN) is used as an antibiotic agent against
infections with gram-positive bacteria especially methicillin resistant
Staphylococcus aureus (MRSA) e.g. sepsis. In order to achieve
therapeutic VAN concentrations, the pharmacokinetics (PK) has to be
well characterised. Extrapolation of PK parameters from adults to other
patient populations, e.g. neonates, may increase the risk of therapy
failure (underdosing) or adverse effects (overdosing). But especially in
neonates, in whom infections may easily be life-threatening, PK studies
are difficult to perform due to the ethical reasons and limited availability
of blood volume. An alternative, minimally invasive method is the
microdialysis technique which benefits from sampling without collecting
body fluids. In the particular interstitial fluid (ISF) of the target site, a
catheter consisting of a semipermeable membrane is inserted, allowing
measurements of unbound and therefore effective drug concentrations
continuously [1].
Background and Objectives: Meropenem (MER) is a broad spectrum
carbapenem antibiotic frequently used for the treatment of severe
nosocomial infections in intensive care patients. In these patients, high
pharmacokinetic variability has been shown, which increases the risk of
overdosing on the one hand and underdosing or resistance development
on the other hand, potentially leading to therapy failure [1]. MER
undergoes primarily renal elimination and its activity is linked to time
above minimum inhibitory concentration (MIC) [2]. The objective of the
present work was to assess serum concentrations after standard doses
of MER in a critically ill patient population with respect to effective
concentrations.
Methods: A monocentric prospective observational study was conducted
in an ICU at the University Hospital of Munich in intensive care patients
with severe infections. For this analysis, a subpopulation of 39 patients
without renal replacement therapy and extracorporeal membrane
oxygenation was chosen. 1000 mg (n=38) or 2000 mg (n=1) MER were
administered every 8 hours as intravenous short-term infusions (30 min).
Multiple serum samples were taken over 4 days and quantification of
MER was performed using liquid chromatography tandem mass
spectrometry (LC-MS/MS). Besides, different patient factors including
e.g. age, weight, BMI, disease scores (APACHE II Score, SOFA Score)
CLINICAL PHARMACY
and laboratory values (e.g. IL-6, CRP, CD64, creatinine clearance
(CLCR)) were determined. An exploratory statistical and graphical data
analysis was performed using R 3.2.0. In order to determine the variability
of MER serum concentrations, minimal concentrations (Cmin) were
compared. To assess whether effective serum concentrations were
reached, minimal concentrations of all 4 study days were investigated
and compared to non-species-related MIC breakpoints (EUCAST: S/I
breakpoint: 2 mg/L, I/R breakpoint: 8 mg/L [3]).
Results: Patient characteristics of the examined population were shown
to be highly variable. 22 male and 17 female patients with a median age
of 58 years (range: 29-84 years) and a median body weight of 70 kg
(range: 44.0-140 kg) were analysed. The majority of the patients suffered
from sepsis (82.1%), which was most frequently caused by pneumonia
(78.1% of sepsis patients) or peritonitis (15.6% of sepsis patients). The
patients showed considerable variability in the creatinine clearance
(median: 81.0 mL/min, range: 19-229 mL/min) and in IL-6 serum
concentrations (median: 100 pg/mL, range: 24.0-10096 pg/mL). High
inter-patient variability was also observed for MER minimal
concentrations, which differed up to a factor of 1000 (Cmin: median: 2.52
mg/L range: 0.03-30.4 mg/L). Not only variability between the patients
was detected, but also within individuals, between the single study days.
During the 4 study days, 79.5% of the patients showed at least one Cmin
measurement below the S/I breakpoint. In 25.8% of those, even all Cmin
values were below 2 mg/L. Comparing the Cmin measurements with the
I/R breakpoint, 87.2% of the patients showed at least one Cmin
measurement lower than 8 mg/L during the study period. In 67.6% of
those, Cmin was always below this breakpoint. In a first investigation to
explain the high observed variability in the MER concentrations, a
hyperpolic relationship between Cmin concentrations of MER and CLCR
was found.
Conclusion: High variability of MER serum concentrations was observed
in intensive care patients, potentially exposing them to the risk of
subtherapeutic levels. Based on the clinical data, a population PK model
will be developed to describe the concentration-time profiles in the
population as well as variability between patients and study days.
Ultimately, a covariate analysis will be performed to identify further
patients factor explaining the large variability in the population and to
assess whether dose adjustments are required in intensive care patients.
References:
1. Gonçalves-Pereira, J.; Póvoa, P.: Crit. Care 2011, 15(5): 1-17.
2. Nicolau, D .P.: CID 2008, 47: 32-40.
3. http://www.eucast.org/clinical_breakpoints (date of access: 25 June 2015)
POS.195
Population pharmacokinetics of Voriconazole in pediatric
cancer patients, taking the inflammatory status into
account
München, S. E.1; Pieper, S.2; Kirchhefer, U.3; Müller, C.4; Moskovits, J.5;
Lehrnbecher, T.5; Groll, A. H.2; Hempel, G.1
Institut für Pharmazeutische und Medizinische Chemie, Klinische Pharmazie, Westfälische
Wilhelms-Universität Münster, Corrensstr. 48, 48149 Münster, Germany
2 Klinik für Kinder- und Jugendmedizin, Pädiatrische Hämatologie und Onkologie,
Universitätsklinikum Münster, Albert-Schweizer-Str. 33, 48149 Münster, Germany
3 Medizinische Fakultät, Institut für Pharmakologie und Toxikologie, Westfälische WilhelmsUniversität Münster, Domagkstr. 12, 48149 Münster, Germany
4 Institut für Pharmakologie, Universitätsklinikum Köln, Gleueler Str. 24, 50931 Köln, Germany
5 Zentrum der Kinder und Jugendmedizin, Hämatologie und Onkologie, Universitätsklinikum
Frankfurt am Main, Theodor-Stern Kai 7, 60590 Frankfurt, Germany
1
Due to underlying condition and treatment, immunocompromised
pediatric patient populations such as pediatric cancer patients are at high
risk to develop invasive fungal infections (IFIs). One option to treat and
prevent IFIs is the antifungal triazole voriconazole. The pharmacokinetic
properties of voriconazole show a high inter-individual variability as well
as a high intra-individual variability, which is even more pronounced in
the pediatric population [1].
Furthermore, pharmacokinetic properties differ in pediatrics and adults.
Previous studies demonstrated that voriconazole is eliminated in adult
patients following Michaelis-Menten type pharmacokinetics. However, at
adult standard doses of 3-4 mg/kg BID, the elimination of voriconazole in
pediatric patients seems to be linear with a lower voriconazole exposure
at similar weight-based doses. Thus, to reach adequate plasma
concentrations in pediatric patients, doses have to be increased,
eventually leading to non-linear pharmacokinetics [2].
One reason for the high intra-patient variability might be the acute level
of inflammation which is reflected by using C-reactive protein (CRP)
plasma concentrations. Statistical evaluation has confirmed a linear
relationship of CRP plasma concentrations and voriconazole trough
concentrations in adults [3].
We therefore analyzed the correlation of voriconazole trough
concentrations with the CRP in a cohort of immunocompromised
pediatric patients.
A linear regression analysis with 14 pediatric cancer patients, 13 years
old or younger, confirmed the linear relationship of CRP plasma
concentrations and voriconazole trough concentrations (p<0.001). A 1
mg/L increase in CRP resulted in an 0.018 mg/L increase in voriconazole
trough concentrations (95% CI: 0.011 – 0.026 mg/L). Thus, 49.7% of the
variance in the trough concentration can be explained by CRP plasma
concentrations.
For this study, patient data from therapeutic drug monitoring of
voriconazole has been collected since 2002 and collection is ongoing.
With this data a pediatric model for voriconazole using a nonlinear mixedeffect modelling approach (NONMEM) was created regarding certain
covariates such as CRP levels representing the inflammatory status. A
two-compartment model with combined linear and non-linear elimination
is used.
The aim of this study is to better characterize pharmacokinetic processes
of voriconazole in pediatric patients, with special regards to those
patients under 2 years of age and to determine if current dosing regimens
are the most appropriate and also applicable for this very young patient
group.
References:
1. Pieper, S et al.: J. Antimicrob. Chemother. 2012, 67(11):2717-24.
2. Karlsson, M. O. et al.: Antimicrob. Agents Chemother. 2009, 53(3):935-44.
3. Van Wanrooy, M. et al.: Antimicrob. Agents Chemother. 2014, 58(12):7098-101.
POS.196
High predictability of plasma lacosamide and no
differences by different age and gender through
normalization processes
Schaefer, C.1,2; Cawello, W.1; Andreas, J. O.1
1 UCB
Pharma, Monheim, Germany
of Düsseldorf, Institute Clinical Pharmacy and Pharmacotherapy, Düsseldorf,
Germany
2 University
Rationale: Lacosamide (LCM) is a newer antiepileptic drug indicated as
adjunctive therapy for partial-onset seizures (POS) in adults. The
objective of this analysis was to assess the effect of age and gender on
the pharmacokinetic (PK) parameters of LCM through normalization
processes and to evaluate if PK of LCM is different over age or influenced
by gender and enforces considerations about therapeutic regimes.
Methods: Data were extracted from two Phase I clinical pharmacology
studies in healthy subjects. First study included healthy Caucasian
women (18-40 years, oral LCM 400mg/day) whereas the second study
included healthy male and female subjects (>65 years, 200mg/day) and
healthy male subjects (18-45 years, 200mg/day). The PK parameters,
area under the concentration time curve over a dosing interval at steady
state (AUCtau,ss) and maximum measured concentration in a dosing
interval at steady state (Cmax,ss) were determined by non-compartmental
methods. AUCtau,ss and Cmax,ss values were normalized by body weight,
height , fat free mass (FFM), lean body weight (LBW), and by volume of
distribution (Vd). FFM, LBW and Vd were approximated through empiric
equations.For AUCtau,ss and Cmax,ss statistical comparison of different
groups were performed after log-transformation based on an analysis of
variance (ANOVA). Results were compared to results of a population
pharmacokinetic (POP PK) study of LCM in patients with POS.
Results: A total of 66 healthy subjects were included and stratified by age
group and gender. AUCtau,ss and Cmax,ss tended to show higher values in
females compared to males and also in elderly vs. young subjects.
Differences between groups were smaller when data were normalized by
the above mentioned parameters.
Before normalization higher AUCtau,ss and Cmax,ss values in females
compared with males in both age groups (older and younger) were found.
The relative bioavailability for AUCtau,ss and Cmax,ss concerning age and
gender effects showed that normalization by Vd led the 90% confidence
intervals of the ratio for AUCtau,ss and Cmax,ss for age and gender
comparisons to fall within the range 80-125%. Same was true for Cmax,ss
normalized by LBW or FFM. The POP PK study in adults with POS
POSTERS
conformed these results and concluded that inter-individual variability of
Vd can be explained to a large extent by differences in gender and body
composition.
Conclusion: Pharmacokinetic evaluations in healthy subjects and in
patients with POS revealed that almost all differences in LCM PK
parameters observed between young and elderly or between male and
female subjects can be explained by body weight or differences in Vd.
Results are confirmed by the results of the POP PK study.
Disclosure: Work was funded by UCB. All authors are employees of UCB
Pharma.
References:
The poster was presented at the AES in Seattle 2014. The corresponding abstract is published
in Epilepsy.Curr. 2015 15(s1):319.
POS.197
Reliability of a European survey on the pharmacological
management of paediatric heart failure
Makowski, N.1; Castro Diez, C.1; Khalil, F.1; Läer, S.1
1 Institut
für Klinische Pharmazie und Pharmakotherapie Heinrich-Heine-Universität Düsseldorf,
Universitätsstraße 1, 40225 Düsseldorf, Germany
Background: Little evidence is at present available to help clinicians
guide decisions when tackling the pharmacological management of
paediatric heart failure (HF). As a consequence, therapeutic strategies
are largely supported by adults’ data extrapolation and own expertise.
The variability in drug treatment routines across Europe is expected to
be high. Nevertheless, there are no epidemiological data that describe
the current situation.
Survey research is a form of scientific inquiry that merits rigorous design
and analysis in order to allow gathering reliable and unbiased data [1]. In
this regard, different strategies need to be implemented during the design
and conduction of the study. Making sure that the questions are designed
in a clear and unambiguous way is a crucial aspect of the survey
development procedure and a key point to guarantee the reliability of the
survey instrument.
Aim: To assess the reliability of the questionnaire used for a survey
developed in the context of the LENA (Labeling of Enalapril from
Neonates up to Adolescents) project to characterise the different
therapeutic strategies for the management of paediatric HF that are
currently practiced across Europe.
Methods: A test-retest strategy was chosen to assess the reliability of the
questionnaire. This method is used to evaluate whether the same
question posed to the same individuals yields consistent results at
different times [1]. The answers of a panel of six experts of 3 countries
were analysed for this purpose. The questionnaire was tested in October
2014 and retested in April 2015. Individual answers given by each
participant were compared for consistency. Consistency was defined for
single choice questions (SQC) and open questions (OQ) as an identical
answer in test and retest. For multiple choice questions (MCQ)
consistency was calculated as percentage of identical answer options
selected by each participant per MCQ.
Results: Reliability was assessed for 44 questions (considering
subquestions). Twenty-two were SCQ, 10 OQ and 12 MCQ. For the 22
SCQ, on average 76% (33-100%) of the participants gave a consistent
answer. For 6/22 questions, all participants were consistent. For the OQ
on average 65% (25-100%) of the participants gave a consistent answer.
In two out of ten questions, all experts gave a consistent answer. For the
MCQ, physicians gave a consistent answer with an average of 83% (6892%).
Conclusions: No formal cut-off values exist to judge the acceptable level
of reliability of questionnaires within the scientific community. The
calculated values for the SCQ (76%), OQ (65%) and MCQ (83%) seem
to be similarly high. It has to be taken into consideration that the long
period of time (6 months) between the test and retest, rapidly changing
knowledge in medical science, and also increased awareness to the
subject triggered by the survey could have contributed to the fact that full
consistency was not reached.
Acknowledgments:The research leading to these results has received funding from the European
Union Seventh Framework Programme (FP7/2007-2013) under grant agreement n°602295
(LENA)
References:
1. Burns, K. et al.: CMAJ 2008, 179(3): 245-252.
2. Andres, L.: DESIGNING & DOING SURVEY RESEARCH (SAGE) 2012.
POS.198
Compatibility analysis of propofol – Optimization of the
drug treatment safety
Gersonde, F.1; Kunze, T.1; Eisend, S.2, Haake, N.3
1 Department
of Clinical Pharmacy, Pharmaceutical Institute, Christian-Albrechts-University Kiel,
Gutenbergstraße 76, 24118 Kiel, Germany
2 Dispensary University Hospital Schleswig-Holstein, Campus Kiel, Arnold-Heller-Straße 3,
24105 Kiel, Germany
3 Department of Intensive Care, imland Hospital Rendsburg, Lilienstraße 20-28, 24768
Rendsburg, Germany
Infusion therapy is an essential part in the treatment of intensive care
patients. Due to increasingly complex therapy standards a simultaneous
application of multiple drugs through a central multi-lumen catheter is
unavoidable [1]. This entails the risk of physicochemical and chemical
incompatibility reactions.
One standard sedative used on the intensive care unit is propofol.
Because of its physicochemical and optical properties propofol poses a
special risk in identifying stability problems and incompatibilities.
Additionally, only limited compatibility data are available [2,3].
The purpose of this work is the optimization of the drug treatment safety
in a cardiovascular intensive care unit in preventing drug incompatibilities
of propofol with other analgetic and sedative drugs.
On the cardiovascular intensive care unit documented propofol drug
combinations were narrowed down to practice-oriented combinations of
propofol 2% with clonidine, midazolam, sufentanil, remifentanil,
piritramid, (S)-ketamine, lormetazepam, γ-hydroxybutyric acid and
dexmedetomidine which were diluted with sodium chloride 0,9% to
standardized concentrations. Mixtures at a ratio of 1:1, 1:10 and 10:1
were stored at room temperature for 7 days. Samples were taken at
defined points of time.
The physical and the emulsion stability in particular were determined by
pH value, zeta potential and globule size distribution measurements
using light backscattering as well as dynamic light scattering and laser
diffraction. Analyses on crystal and microbiological growth give additional
information about the stability.
The chemical stability determination is carried out by high performance
liquid chromatography (HPLC).
The light backscattering and zeta potential analyses resulted in three
stability groups for the 1:1-mixtures in which the group with the most
destabilization phenomena consisted of propofol, remifentanil and
lormetazepam. All other mixtures remained stable over a defined period
of time.
The results of the globule size distribution measurements of the 1:1, 1:10
and 10:1 mixtures using dynamic light scattering and laser diffraction
showed that each combination of propofol with remifentanil, piritramid
and γ-hydroxybutyric acid is instable.
No crystal and bacterial growth as well as no stability-relevant pH-shift
could be detected.
The HPLC data indicate a chemical stability of all previously tested
propofol drug (1:1) combinations.
Evidence for incompatibilities and compatibilities of propofol with
analgetic and sedative drugs could be obtained. Through further
investigations the drug treatment safety should be increased.
References:
1. Kanji, S. et al.: Crit. Care Med. 2010, 38(9): 1890-1898.
2. Michaels, MR.; Stauffer, GL.; Haas DP.: Ann. Pharmacother. 1996, 30: 228-232.
3. Lilley, E. M. M. et al.: Anaesthesia 1996, 51: 815-818.
BIOTECHNOLOGY
4.11 Biotechnology
POS.199
Modification of glyco-engineered living cells by copper-free
(SPAAC) and copper catalyzed azide alkyne cycloaddition
(CuAAC)
Gutmann, M.1; Memmel, E.2; Braun, A.1; Seibel, J.2; Meinel, L.1;
Lühmann, T.1
1 Institute
2 Institute
for Organic Chemistry, University of Würzburg, Germany
Introduction: The surface of cells consists of a variety of functional
elements, including a cell and site specific glycocalyx. These glycan
structures impact the function, stability and localization of proteins on the
cell surfaces or critically control cell-cell recognition processes [1]. Azide
modified monosaccharides can be incorporated into the glycocalyx, and
are thereby accessible for e.g. Cu(I)-catalyzed azide alkyne cycloaddition
(CuAAC) and strain-promoted alkyne-azide cycloadditions (SPAAC)
[2,3]. This study addresses (i) the temporal design space within which the
chemistries can be performed without jeopardizing cell toxicity, (ii) the
effectiveness of cell surface decoration through the azide functionlized
glycocalyx and (iii) the cellular sequestration of fluorescence dyes
coupled onto the cell surfaces by means of these chemistries.
Materials and Methods: An azide-functionality was introduced onto the
surface by means of a glyco-engineering step using tetraacylated-Nazidoacetylglucosamin (Ac4GlcNAz) [2]. Cell toxicity of the CuAAC was
assessed by FACS measurements of propidium iodide (PI) and
fluorescein diacetate (FDA) stained cells and qPCR analysis of apoptotic
and anti-apoptotic maker genes. The Intensity of CuAAC compared to
SPAAC was measured after surface staining with the fluorescent dye
sulfo-Cy5-alkine by flow cytometry (FACS) [4]. The sequestration of the
surface coupled fluorescent dye was assessed by following the residence
time through confocal laser scanning microscopy (CLSM).
Results and Discussion: qPCR analysis of all conditions did not induce
mRNA levels of apoptotic (Caspase-2, Bcl 2 associated X protein,
apoptotic protease activating factor I) and anti-apoptotic (B cell
lymphoma 2) maker genes after 5 and 20 minutes using best conditions
(50 µM CuSO4; 250 µM Tris[(1-benzyl-1H-1,2,3-triazol-4yl)methyl]amine (THPTA); 2,5 mM C6H7NaO6) verified by FACS analysis.
In a series of experiments, the cell surface of azido-modified NIH3T3 and
Freestyle 293-F cells was decorated by means of glycoengeneering
followed by CuAAC or SPAAC reaction using alkyne-functionalized
fluorescent dyes. Images of fluorescence labelled cells show a high
degree labelling of the cell membranes (Figure A) with decrease in the
fluorescence over time (Figure B) (12 hours). Flow cytometry analysis of
Freestyle 293-F cells shows an increased degree of labelling with a
fluorescent dye over time in both reaction-types (CuAAC and SPAAC)
with reduced background noise and efficiency regarding SPAAC.
CuAAC is an acceptable chemistry when applied to living cells in a strictly
controlled time frame. Exposure to Cu+ as required for the reaction may
not exceed 20 minutes and covalently coupled molecules (as assessed
for a fluorescent dye) remain detectable on cellular surfaces for 12 hours
following the coupling. SPACC shows advantages in form of lower
toxicity and less unspecific binding but leads to lower coupling efficacy.
POS.200
Spatially controlled decoration of biologics using
enhanced green fluorescent protein as model protein
Wurzel, J.1; Lühmann, T.1; Meinel, L.1
1 Institute
Introduction: Replacing existing amino acids (AA) by unnatural AA (uAA)
allows for an efficient introduction of one or more novel and unique
functional group(s) into a protein. The resulting proteins can be decorated
solely at the site of the uAA with small chemical drugs, other peptides or
proteins, or polymers with unprecedented spatial control and
homogenous product outcome. Therefore, this genetic engineering
strategy is instrumental in addressing the heterogeneous product
outcome of current, chemical decoration strategies of biologics.
In this study, we demonstrate two different methods for spatially
controlled N-terminal decoration using eGFP as a model protein, (#1) a
copper-catalyzed azide-alkyne cycloaddition (CuAAC) or (#2) by factor
XIIIa (FXIIIa) mediated acyl-transfer. Biotin (as an example for a low
molecular weight molecule) and a 10 kDa polyethylene glycol (PEG)
polymer were deployed as decorating molecules.
Materials and Methods: eGFP analogues were profiled for subsequent
CuAAC by introducing the uAA propargyl-L-lysine (Plk) into the protein at
position 4 (L4Plk) deploying E. coli with an expanded genetic code [1,2].
In an alternative approach, a specific amino acid sequence
(NQEQVSPL), derived from alpha-2-plasmin inhibitor (a2PI) serving as a
high affinity target for the transglutaminase FXIIIa, was integrated [3,4].
The factor XIIIa mediated acyl-transfer requires a second peptide (FXIIIapeptide: Ac-FKGG-PEG6-C-NH2) [5], which was synthesized by solid
phase peptide synthesis. Azido functionalized Biotin or PEG (required for
decoration strategy #1; CuAAC click chemistry) and
maleimide/acrylamide functionalized Biotin or PEG (required for
decoration strategy #2; the biotin/PEG was coupled to cysteine of the
FXIIIa-peptide through a Michael addition) were purchased. A2PI-eGFP
and Plk-eGFP were expressed in E.coli BL21 (DE3) and purified by ion
affinity chromatography. All species were characterized by MALDI-MS,
ESI-LC-MS/MS and HPLC and fluorometric characterization.
Results and Discussion: A2PI-eGFP and Plk-eGFP were expressed in
reasonable yields (30 and 2 mg/L, respectively) and purities of
approximately 95%. Each expressed mutant displayed typical fluorescent
properties identical to wild-type eGFP. The synthesis of FXIIIa-peptide,
A2PI-eGFP-biotin/PEG and Plk-eGFP-biotin/PEG constructs was
successfully performed and characterized.
Both strategies (#1 CuAAC; #2 Transglutaminase) proved successful for
efficient, rapid, and site directed decoration of eGFP. The technique is
introducing tight spatial control for the decoration of biologics translating
into unprecedented homogenous product outcome of the decorated
species for future pharmaceutical application.
References:
1. Eger, S. et al.: Methods Mol. Biol. 2012, 832: 589-596.
2. Nguyen, D. P. et al.: J. Am. Chem. Soc. 2009, 131: 8720.
3. Patterson, J. et al.: Mater. Today 2010, 13(1-2): 14–22.
4. Früh, S. M. et al.: Chembiochem. 2014, 15(10): 1481-6.
5. Hu, B. H.; Messersmith, P. B.: J. Am. Chem. Soc. 2003, 125(47): 14298-9.
POS.201
Figure: 5 min CuAAC reaction (50 µM CuSO4; 250 µM THPTA; 2,5 mM C6H7NaO6) of NIH 3T3
cell membrane with a red fluorescent refers to Sulfo-Cy5-alkyne and blue fluorescent to Dapi.
(A) Image after 30 minutes (B) Image after 12 hours
References:
1. Freeze, H. H. et al.: Nature Rev. 2006, 7: 537-551.
2. Homann, A. et al.: Beilstein J. Org. Chem. 2010, 6: 24.
3. Memmel, E. et al.: ChemComm (Cambridge, England). 2013, 49(66): 7301–3.
4. Hong, V. et al.: Bioconjugate Chem. 2010, 8(8): 1620-34.
Site-directed immobilization and bioresponsive release of
anticatabolic agents for muscle regeneration
Braun, A.1; Ritzer, J.1; Gutmann, M.1; Ebert, R.2; Jakob, F.2; Lühmann,
T.1; Meinel, L.1
1 Institute
Center for Musculoskeletal Research, Würzburg, Germany
2 Orthopedic
Introduction: Progressive loss of skeletal muscle mass, strength and
function during aging (sarcopenia), poses a major threat to independence
POSTERS
and quality of life in the elderly. Therefore, developing therapeutic
interventions to stop its progression is of great interest. In our strategy
we target myostatin, a potent negative regulator of myogenesis that is
highly enriched in skeletal muscle of sarcopenic patients, with peptide
antagonists [1]. Intending to control the release of this biologic in case of
local tissue inflammation during the progression of the disease, we
immobilize the peptides by means of protease-degradable linkers on the
surface of nanoparticles. In this way, we create a bioresponsive release
system that is sensitive to the upregulation of matrix metalloproteinases
in case of local inflammation triggering the release of the biologic from
the particulate carrier.
Materials and Methods: The myostatin inhibitors and protease-sensitive
linker sequences were manufactured by solid phase peptide synthesis
[2] with a propargyl-modified glycine analogue introduced into the
sequence in case of the myostatin inhibitor and an azido-homoalanine in
case of the linkers for the conjugation via click chemistry [3]. RP-HPLC
and MALDI-MS were deployed for verification of purity, and potency was
profiled by a luciferase-based reporter gene assay and by C2C12
myoblast differentiation ability. Particle preparation was performed by
reacting alkyne-functionalized poly methyl methacrylate (PMMA)
nanoparticles with the bi-azido-functional MMP-sensitive linker in
presence of 0.25 mM copper(I)-catalyst and 0.5 mM THPTA, followed by
reaction with the fluorescent dye DBCO-PEG4-carboxyrhodamine and
fluorescence-labelled anticatabolic peptide, respectively. Release
experiments were performed using 900 ng/mL of MMP-1, -8 and -9 at
37 °C for a period of 20 hours, during which several readings were
conducted in order to obtain a release profile [4].
Results and Discussion: Successful synthesis and purification of the
functionalized peptides was demonstrated by mass spectrometry and
HPLC. The luciferase assay and C2C12 differentiation assay confirmed
the inhibiting activity on myostatin signalling and comparison of the
potency of alkyne-functionalized and original peptides resulted in similar
outcome. The protease-sensitive linker functionalized for conjugation
with the anticatabolic peptides was optimized accordingly to ensure a
high percentage of proteolytic cleavage (≥ 96%) by incubation with
elevated concentration of each of the proteases MMP-1, -8 and -9. The
successful coupling of the linker to alkyne-functionalized PMMA particles
was indicated by labelling with the fluorescent dye DBCO-PEG4carboxyrhodamine. Fluorescence intensity of the supernatant after
cleavage with MMPs was analyzed by RP-HPLC with fluorescence
detection and confirmed the successful cleavage of the proteasesensitive linker from particle surface.
In conclusion, a potent site-specifically modified myostatin inhibitor with
retained activity was developed for advanced controlled release
approaches. The immobilization through protease-sensitive linkers on
nanoparticles demonstrated the ability for surface decoration and created
a system responding to local tissue inflammation. This approach is
further profiled for boosting muscle function and regeneration in ongoing
studies e.g. by combining the anticatabolic agent with growth factors like
IGF-1.
Acknowledgments: The financial support from the Bavarian Research Foundation (FORMOsA
grant) is gratefully acknowledged.
References:
1. Tsuchida, K. et al.: Current Opinion in Drug Discovery & Development, 2008, 11: 487-94.
2. Han, H. et al.; 2004, US 2004/0181033 A1.
3. Hong, V. et al.: Angewandte Chemie- International Edition. 2009, 48: 9879-83.
4. Steinhagen, M. et al.: ACS Applied Materials & Interfaces. 2014, 6: 5891-99.
POS.202
MiR-CLIP – a chemical biology strategy for the direct
identification of microRNA targets enabled by chemically
synthesized microRNA probes
Brunschweiger, A.1,2,5; Imig, J.1,5; Brümmer, A.3; Guennewig, B.1; Mittal,
N.3; Kishore, S.3; Tsikrika, P.1; Gerber, A. P.4; Zavolan, M.3; Hall, J.1
1 Institute
of Pharmaceutical Sciences, ETH Zurich, Zurich, Switzerland;
of Chemistry and Chemical Biology, TU Dortmund University, Dortmund, Germany;
3 Computational and Systems Biology, Biozentrum, University of Basel, Basel, Switzerland;
4 Department of Microbial and Cellular Sciences, Faculty of Health and Medical Sciences,
University of Surrey, Guildford, Surrey, UK.
5 Contributed equally.
2 Faculty
Identifying the interaction partners of noncoding RNAs is essential for
elucidating their functions. The chemical synthesis of site-specifically
modified microRNAs [1,2] enabled a chemical biology strategy for the
direct identification of their RNA targets, their “targetome”. We
synthesized (pre-)miRNAs modified with a psoralen photo linker and
biotin affinity handle to capture their targets in cells in an approach,
termed microRNA crosslinking and immunoprecipitation (miR-CLIP) [3].
Photo-crosslinking coupled to a tandem purification strategy consisting
of Argonaute 2 immunopurification followed by streptavidin affinity
purification of mirCLIP probe-linked RNAs provided selectivity in the
capture of targets, which were identified by Illumina sequencing. miRCLIP with pre-miR-106a, a miR-17-5p family member, identified
hundreds of putative targets in HeLa cells, many carrying conserved
sequences complementary to the miRNA seed but also many that were
not predicted computationally. MiR-106a overexpression and inhibition
experiments confirmed that miR-CLIP captured functional targets,
including H19, a long noncoding RNA that is expressed during skeletal
muscle cell differentiation. We showed that miR-17-5p family members
bind H19 in HeLa cells and myoblasts. During myoblast differentiation,
levels of H19, miR-17-5p family members and mRNA targets changed in
a manner suggesting that H19 acts as a ‘sponge’ for these miRNAs. The
miR-CLIP protocol was successfully applied to identify targets of further
microRNAs, e.g. let-7g.
Figure: Schematic presentation of miR-CLIP, a chemical biology approach for the identification
of interaction partners of microRNAs.
Acknowledgments: This work was supported with two grants by the Swiss National Science
Foundation: CRS205321_124720 and CRSII3_127454 (joint Sinergia grant).
References:
1. Rebhan, M. A.; Brunschweiger, A.; Hall, J.: Chembiochem 2013, 14(16): 2091-2094.
2. Pradère, U. et al.: Angew. Chem. Int. Ed. 2013, 52(46): 12028-12032.
3. Imig, J. et al.: Nat. Chem. Biol. 2015, 11: 107-115.
POS.203
Defined immobilisation of interleukin-4 (IL-4) for spatial
controlled M2 macrophage polarization
Lühmann,T.1; Spieler V. 1; Werner, V. 1; Meinel, L. 1
1 Institute
for Pharmacy and Food Chemistry, University of Würzburg, Am Hubland, 97074
Würzburg, Germany
108.
POS.204
Screening of nanoparticles for drug-delivery across the
blood-brain barrier using autodisplayed LRP1 IV-domain
on E. coli
Fenoy, C. G.1; Raudszus, B.2; Nienberg, C.1; Langer, K.2; Jose, J.1
1 Institute
of Pharmaceutical and Medicinal Chemistry, PharmaCampus, Westfälische WilhelmsUniversität Münster, Corrensstraße 48, Münster, 48149, Germany
2 Institute of Pharmaceutical Technology and Biopharmacy, PharmaCampus, Westfälische
Wilhelms-Universität Münster, Corrensstraße 48, Münster, 48149, Germany
The blood-brain barrier (BBB) surrounding the central nervous system
(CNS) protects it from widely different potential hazards. Due to its
complex structure the pass of compounds across the BBB is highly
restricted. This is an important drawback for the therapy of diseases like
brain tumors or Alzheimer´s disease. The high molecular weight drugs
used are normally not able to reach its target specifically and in large
amounts, without altering the integrity of the BBB [1]. Many specific
transport systems, like LPR1 (Low-density lipoprotein receptor-related
protein-1), contribute to the flow of substances between bloodstream and
the CNS. LRP1 has diverse biological functions, including ApoE binding
BIOTECHNOLOGY
and endocytosis. Nanoparticles modified with ApoE, loaded with a drug
of interest, could bind to LRP1. The whole system will be internalized and
the drug could pass though the BBB, thereby reaching its target [2].
Previous studies showed the high binding affinity of ApoE to the fourth
binding domain of LRP1 (LRP1-IV) [3]. In this work, LRP1-IV was
expressed on the surface of E. coli using Autodisplay technology. For this
purpose an artificial gene for a fusion protein was constructed. The fusion
protein consisted of an N-terminal signal peptide directing the protein
across the inner membrane of E. coli, the LRP1-IV domain, and the Cterminal autotransporter facilitating the transport of LRP1-IV to the
surface of the cell. Once LRP1-IV is expressed on the surface, whole
cells can be used for binding studies with ApoE, avoiding cumbersome
protein purification [4]. Surface display of LRP1-IV was verified by
western blot of outer membrane preparations and flow cytometry of
whole cells with a specific LPR1 antibody. Flow cytometric analysis also
indicated that cells displaying LRP1-IV bind purified ApoE3 and ApoE3
labeled with PromoFluor-NHS 633 Our next step will be to test the binding
of ApoE3 modified nanoparticles to surface displayed LPR1-IV. These
nanoparticles could be used as carriers across the BBB for drugs which
normally are not able to cross this barrier [5,6]. The assay established in
this study allows further investigation relevant for the development of
nanoparticles, since binding affinities between ApoE3 variants and
LRP1-IV or variants thereof could be easily quantified.
References:
1. Abbott, N. J.: J. Inherit. Metab. Dis. 2013, 36: 437-449.
2. Wagner, S. et al.: PLoS One 2012, 7: e32568.
3. Neels, J. G. et al.: J. Biol. Chem. 2009, 274: 31305-31311.
4. Jose, J.; Meyer, T. F: Microbiol. Mol. Biol. R. 2007, 71: 600-619.
5. Michaelis, K. et al.: J. Pharm. Exp. Ther. 2006, 317: 1246-1253.
6. Zensi, A. et al.: J. Control. Release 2009, 137: 78–86.
POSTERS
POS.206
4.12 Other topics
POS.205
Rapid bacterial cell counting method: Proof-of-principle
with a batch culture of E. coli
Goebgen, E.
1 Dept.
B.1;
Wicha, S.
G.1;
Kloft,
C.1
Objectives: Quantification of bacteria is one of the key steps in in vitro
infection models and performed for different purposes, e.g. determination
of antibiotic treatment efficacy or bacterial resistance. The objective of
the current investigations was to use a recently developed and validated
electronic cell counting method [1] to quantify bacterial concentrations of
a growing culture of Escherichia coli (E. coli). Furthermore, the droplet
plate counting method as the “gold standard” was to be performed in
parallel to enable a comparison between the two methods.
Methods: The in vitro experiments were performed under static
conditions in a cell-culture flask (batch culture), filled with 9.5 mL cationadjusted Mueller Hinton Broth (CA-MHB). Colonies from a freshly
prepared subculture of E. coli (ATCC 25922) dispensed in 0.9% NaCl
and adjusted to McFarland 0.5 were added to the culture flasks to obtain
an inoculum of 106 CFU/mL after a pre-incubation step of 2 hours.
Samples were taken either directly after the start of the incubation “lagexperiments” or after the pre-incubation step of 2 hours “logexperiments”. The predefined sampling time points were the first sample
(t=0), 1, 2, 4, 6, 8, 10 and 24 hours after the first sample. Bacterial
concentrations of the samples were measured using both the electronic
cell counting and the droplet plate counting method; both methods being
explained elsewhere [1, 2]. All experiments were performed at least in
triplicate. For reasons of comparability the results of the electronic cell
counting method were transformed from count/mL to colony forming units
(CFU)/mL, using a strain-specific defined calibration function
(y=0.166x+1951).
Results: Bacterial concentrations in CFU/mL versus the time after start
of incubation (TASI) showed a comparable curve shape for both cellcounting methods, with a slightly higher bacterial concentration
(<1 decade) for the electronic cell counting method. This difference was
visible until TASI=6 hours or TASI=4 hours for lag- or log-experiments,
respectively. The delay in bacterial replication in the first 2 hours, visible
as a slight increase of half a decade in the first 2 hours, was more
pronounced when performing the “gold standard” as quantification
method (“gold standard” vs. electronic cell counting: 5∙105 to 106 CFU/mL
vs. 9∙105 to 9∙107 CFU/mL). From the maximum bacterial concentration
109 to 1010 CFU/mL at TASI=6 hours, the bacterial concentration
stabilised in a “plateau phase” between 109 to 1010 CFU/mL until
TASI=12 hours. At the end of the incubation period (TASI=24 or 26 hours
for lag- or log-experiments, respectively), the bacterial concentration
reached a value of ≤109 CFU/mL.
Conclusions: The newly validated electronic cell counting method for E.
coli successfully quantified samples from a batch culture. Comparison
with results determined with the “gold standard” of bacterial quantification
showed good agreement between the two different measurement
methods. Furthermore, the shape of the bacterial concentration-time
profile showed the expected shape with the different phases typical of
batch cultures: delay in bacterial growth (lag-phase), exponentially
growing bacteria (log-phase), dynamic equilibrium between growing and
less viable bacteria (stationary phase), decline of viable cells (net death
phase) [3]. Therefore, the electronic quantification method seemed to be
a promising alternative to the time-consuming “gold standard”, which
includes an incubation step of at least 20 hours for E. coli. With a
measuring time of approximately 10 s, the electronic cell-counting
method could streamline the investigations on antibiotic efficacy in in vitro
infection models if a quantification step of bacteria is included. Further
investigations on the influence of the diameter evolvement, especially in
the first two hours of incubation (lag-time), on the number of measureable
cells shall be the next steps.
References:
1. Goebgen, E.B.; Wicha, S.G.; Kloft, C.: 25th ECCMID, Copenhagen, Denmark. 2015: EV0532
2. Schwalbe, R.; Steele-Moore, L.; Goodwin, A.C.: Antimicrobial susceptibility testing protocols
(CRC Press) 2007.
3. Monod, J.: Selected Papers in Molecular Biology (Academic Press, Inc.) 1978.
Animal substances in the materia medica of the 18th
century
Krafczyk, K.1; Friedrich, C.1
Institute for the History of Pharmacy, Universität Marburg, Roter Graben 10, 35037 Marburg,
Germany
1
Animal derived remedies played an important role in the history of the
materia medica from ancient times until the end of the 18th century.
In the 17th century the pharmaceutical use of animals reached heyday,
because of the ‘Signaturenlehre’ and the publication of the ‘Heylsame
Dreck-Apothecke […]’ (Healing dirt pharmacy) by Christian Franz Paullini
(1634–1712) in 1696. During our studies we examined three important
pharmacopoeias of the 18th century to depict the postulated decline of
the pharmaceutical use of animals. In the Dispensatorium
Brandenburgicum (1698) 54 different animals were used to obtain
remedies. Because of the lexical scope of the Pharmacopoea
Wirtenbergica (1741) we could even prove use of 78 animals. In the
Pharmacopoea Borussica (1799) only 21 animal derived remedies were
detected.
Consequently, as it is shown by the above numbers, various animals
have been pharmaceutically used. One of them was the red coral
(Corallium rubrum), also referred to as the precious coral. We present
preliminary results of our study about the typical therapeutic indications
of this cnidarian in the 18th century. It was used for example as an
antiepileptic, heart strengthening and hemostatic remedy. Furthermore,
it was applied against stomach complaints, which can be explained by its
high content of calcium carbonate, absorbing excess gastric acid. This
poster will illustrate the great variety of animals used as remedies and
describe the pharmaceutical use of the red coral in the 18th century.
References:
1. Friedrich, C.; Müller-Jahncke, W.-D.: Geschichte der Pharmazie. Bd. 2 - Von der Frühen
Neuzeit bis zur Gegenwart (Govi-Verlag) 2005.
2. Schneider, W.: Lexikon zur Arzneimittelgeschichte. Sachwörterbuch zur Geschichte der
pharmazeutischen Botanik, Chemie, Mineralogie, Pharmakologie, Zoologie. Bd. 1 - Tierische
Drogen (Govi-Verlag) 1968.
3. Schindler, H.; Frank, H.: Tiere in Pharmazie und Medizin. 50 Einzeldarstellungen
(Hippokrates-Verlag) 1961.
POS.207
Implementation of a GxP compliant quality system
encompassing all consortium members of the FP-7 funded
LENA Project
Ciplea, A. M.1; Kleine, K.2; Burckhardt, B. B.1; Läer, S.1; Breitkreutz, J.3;
Špatenková, L.4; Klingmann, I.4
1 Department
of Clinical Pharmacy and Pharmacotherapy, Heinrich-Heine-University,
Universitätsstr.1, 40225 Düsseldorf, Germany
2 Simply Quality – Dr. Karl Kleine, Johannes-Damrich-Str.4, 82362 Weilheim i. OB, Germany
3 Ethicare GmbH, Am Fliederbusch 2, 45721 Haltern am See, Germany,
4 Pharmaplex bvba, Avenue Saint-Hubert 51, 1970 Wezembeek-Oppem, Belgium
Background: The LENA (Labeling of Enalapril from Neonates up to
Adolescents) project has been initiated to improve the healthcare of
children with heart failure by an enalapril orodispersible mini-tablet. The
LENA consortium combines academic clinical research centres, SMEs
(small and medium-sized enterprises) and a patient/parent advocacy
organisation. The objective of the project requires to comply with
respective GxP regulations like Good Manufacturing Practice (GMP),
Good Clinical (Laboratory) Practice (GCP/“GCLP” [1]) and Good
Vigilance Practice (GVP). The project team is comprised of sub-teams
experienced in paediatric clinical practice, medicines development,
clinical research and project management, but not all team members
work in comprehensive quality framework.
Aim: To establish a well-documented, efficient quality system applying a
new approach for ensuring quality in all trial aspects by combining
existing organisation-related quality system elements of the project
partners with newly developed SOPs and overarching, integrating trialspecific elements to ensure a reliable quality environment for the LENA
Phase I clinical trial.
Methods: Based on the network-structure of the project organisation, a
strategy based on a team approach with joint responsibilities for the
quality conduct of the project was pursuit, forming a QM Team consisting
of the project leader, the leaders for pharmaceutical and clinical
OTHER TOPICS
development and an external quality expert. The team compiled a quality
manual and an organisational chart displaying the sub-teams and their
responsibilities. Another responsibility of the team was the integration of
existing SOPs and Work Instructions as well as the creation of studyspecific procedures presented in “Manuals” and furthermore the
verification of appropriate qualification of all staff involved in the project
through CVs, job descriptions and training records.
Results: For the Phase I study, a thorough analysis of all existing relevant
SOPs and Work Instructions, forms and other quality elements was
performed, uncovered trial-related processes were identified and a work
plan was established to fill the gaps with the smallest possible number of
newly developed organisation-related SOPs/Work Instructions and by
preparing trial-specific process manuals. Demonstration of the trial team
members was ensured by completing documentation concerning CVs,
job description and training records. Among the sub-teams, the “GCLP”
environment of the bioanalytical laboratory was started from scratch and
could adequately support the LENA Phase I study by “GCLP” compliant
quality work and sample logistics.
Conclusion: The consortium’s approach enabled the preparation of a
comprehensive, reliable GxP compliant quality system within a short
timeframe and with the limited resources of a publicly funded project.
n°602295 (LENA).
References:
1. GCP Inspectors Working Group, Reflection paper for laboratories that perform the analysis or
evaluation of clinical trial samples (EMA/INS/GCP/532137/2010), 2012.
These results have been presented at the ESDPPP Congress 2015.
POS.208
State exam in pharmacy according to the German
regulations for pharmacist training: Performance profiles
in written (MCQ) exams and frequency of highest scores in
part 1/3
POS.209
Pharmaceutical innovation in the early 20th century –
Salvarsan and its derivatives
Odenweller, S.1, Helmstädter, A.1
1 Institute
of Pharmaceutical Chemistry, Goethe Universität Frankfurt, Max-von-Laue-Straße 9,
60438 Frankfurt am Main, Germany
Arsphenamine (Salvarsan), developed by Paul Ehrlich and launched in
1910 on the german market, was the first drug with significant effect
against the syphilitic disease [1]. It was the first drug of the so called
“arsenophenamines” and the breakthrough innovation in this class. It
was, however, not easy in handling, showed several side effects and
faced severe criticism in public. This is why Paul Ehrlich and the
Hoechster Farben AG continued to develop additional compounds of
the same class. Between 1912 and 1931 nine derivatives reached the
market:
- Neosalvarsan (Neoarsphenamine, 1912)
- Kupfer-Salvarsan (Copper Arsphenamine, 1914)
- Salvarsan-Natrium (Arspenamine Sodium, 1915)
- Sulfoxylsalvarsan (1919)
- Silbersalvarsan (Silver Arsphenamine, 1920)
- Neosilbersalvarsan (Neosilver Arsphenamine, 1920/21)
- Myosalvarsan (Sulpharsphenamine, 1926)
- Solusalvarsan (1931)
- Isosalvarsan
As a first step in a research project about pharmaceutical innovation in
the 20th century, it was investigated which Salvarsan derivative, and why,
eventually earned greatest success. As could be shown,
Neoarsphenamine was the most successful compound preparation with
a market share of 94% between 1927 and 1942 [2], although being
neither the most potent nor the easiest to handle derivative in
retrospective. It represented the first major improvement after Salvarsan
had prepared the ground for chemotherapy of syphilis.
References:
1. Helmstädter, A.: Pharmazeutische Zeitung 2010, 155: 4844-4851.
2. Rothermundt, M.: Zeitschrift für Hygiene und Infektionskrankheiten 1942, 124: 366-400.
Shahla, H. 1; Spahn-Langguth, H.2
1 Institut für Medizinische und Pharmazeutische Prüfungsfragen – IMPP, Departments of Data
Processing, Statistics, and Documentation Große Langgasse 8, D-55116 Mainz
2 Institut für Medizinische und Pharmazeutische Prüfungsfragen – IMPP, Departments of
Pharmacy, Große Langgasse 8, D-55116 Mainz
According to the regulations in Germany (AAppO), the pharmaceutical
examination consists of 3 different segments, P1, P2, and P3. During the
past 40 years, the regular 1st pharmacy state exam (P1) has been carried
out as a cluster of four separate written MCQ tests in 4 different topics or
subject groups (P1-I to -IV), including e.g., pharmacy-relevant basic
natural sciences (physical science and life science), introductory drug
formulation, and drug analysis*. Between 2400 and 2850 candidates per
year (= numbers throughout the recent 15 years) are taking the written
tests. Grades for the tests range from 1 (very good) to 5 (failed). Failure
rates have been ranging from 3 to 26%. It is well-known that performance
in these tests largely correlates with school grades of the students.
The purpose of the current investigation was to evaluate the overall
performance profiles over the past four decades and the predictive value
of the MCQ tests’ outcome for the subsequent oral exams (= 2nd state
exam P2 and 3rd exam P3). In order to generate an overview on the
detailed results, the four P1 exams were summarized, and numbers
given below include the arithmetical mean of all candidates in P1-I, -II, III, and -IV.
The overall mean of the candidates’ P1 grades amounts to 3.13.
Approximately five percent of the grades (5.11%) are above or at 2.0,
and only 0.33% of all candidates reach the best possible grade in all four
P1 tests (mean = 1.0).
For P2 vs. P3, the respective numbers are as follows: arithmetical means
of the overall grades, 2.43 vs. 2.53; fraction of candidates with grades
>1.0 and <2.0, 32.8 vs. 31.5%; fraction with highest possible grade of
1.0, 2.51 vs. 7.2%.
From grades obtained in P1, P2, and P3, an average grade is generally
calculated according to the weighting scheme given in the AAppO
(weighting factors P1 – P2 – P3: 2 – 3 – 2).
The overall analysis yielded a very small group of students/candidates
with an overall grade of 1.0 in P1-3 (i.e. grade/score of 1.0 in all single
state exam parts), which is represented by a candidate fraction as small
as approximately 0.07% on average.
(*AAppO, Anlage 13)
POS.210
Understanding Plasmodium falciparum’s exploitation of
the innate immune system aids the identification of
potential novel intervention strategies
Schmidt, C. Q.1; Kennedy, A. T.2; Harder, M. J.1; Lim, N. Y. T.2; Tham,
W. H.2
1 Institute
of Pharmacology of Natural Products & Clinical Pharmacology, Ulm University, Ulm,
Germany
2 Department of Medical Biology and The Walter and Eliza Hall Institute of Medical Research,
The University of Melbourne, Parkville, Australia.
POS.211
Ethnopharmacological information from the botanical
correspondence of Berthold Seemann (1825-1871) – a pilot
study
Helmstädter, A.1
1 Institute
of Pharmaceutical Chemistry, Goethe University, Max-von-Laue-Str. 9, 60438
Frankfurt, Germany
Historical research may be able to contribute to the exploration of
traditional knowledge about medicinal plants and promising attempts
have been made investigating byzantine texts, early modern herbals, and
writings of christian missionaries. In this pilot study it should be explored
if publications, travel reports, diaries or correspondence of the botanical
explorers of the 19th and early 20th centuries may serve a source of
ethnopharmacological information as well and may be able to guide
modern phytopharmacological research. Writings of Berthold Seemann
(1825-1871), a german investigator exploring the botany of Middle
POSTERS
America, the Fiji islands and other regions, are investigated as a first
example. It could be shown that Seemann’s heritage mainly kept at Kew
Garden Archives, does contain ethnopharmacological information which
in part has already been confirmed by recent study results indicating
some reliability of his observations. However, there are also reports
about traditional medicinal plants scarcely investigated so far, including
Schultesia stenophylla Mart. (syn. S. guainensis (Aubl.) Malme), Trixis
inula Crantz, Waltheria glomerata Presl., Gonophlebium attenuatum
(Humb. & Bonpl. Es Willd) C. Presl., or Pseudoelephantopus spicatus
(Juss ex Aubl.) C.F. Baker. It is suggested to further explore their
potential as medicinal plants. In general, as Seemann’s example has
shown, publications and correspondence of botanical explorers of the
past seem to be a valuable and hitherto almost neglected source of
information to be considered in further historical and
ethnopharmacological research.
POS.212
Displaced German pharmacist after World War II – The
pharmacist Joseph Krause from East Prussia
Schröder,
J.1;
Friedrich,
C.1
Institute for the History of Pharmacy, Philipps University Marburg,Roter Graben 10, 35032
Marburg, Germany
1
The fate of the expulsion struck about 14 million Germans, who had to
leave their region of origin in the former German territories and settlement
areas in Central, Southeast and Eastern Europe. In the general refugee
literature pharmacists are not addressed.
Because of the existing system of concessions at that time no larger
number of pharmacies could initially arise, despite the massively
increased population due to the influx of millions of displaced persons.
Displaced pharmacists were not substituted for their lost operating rights.
The poster shows the professional positions in the curriculum vitae of the
pharmacist Joseph Krause, who was born in 1906 in East Prussia and
graduated in pharmacy at Wroclaw University in 1933. Since 1936, he
was manager and later owner of a pharmacy in Olsztyn / East Prussia.
Sent to the front in Italy as a soldier, he met his family again in April 1946
in Flensburg, after his captivity. Until 1952 he worked as a senior
pharmacist in army hospitals, which served as hospitals for returnees
from Russia. He got a leading position in a public pharmacy from 1952 to
1958. After the Federal Constitutional Court established the unrestricted
freedom of pharmacy establishment as a legal principle on the 11th June
1958, Joseph Krause founded a pharmacy in Göttingen in 1958.
POS.213
Professor Erika Cremer (1900–1996) – A pioneer in gas
chromatography
Michler, V.1; Friedrich, C.1
Institut für Geschichte der Pharmazie, Institute for the History of Pharmacy, Universität
Marburg, University of Marburg, Roter Graben 10, 35037 Marburg, Germany
1
Professor Cremer is one of the real pioneers in gas chromatography: in
fact, one can surely say that she is the real pioneer of this discipline.
Despite all troubles during World War II, she conceived gas
chromatography in 1944, when this technique did not yet exist. One may
say that this work occurred after the 1941 publication of A.J.P. Martin
(1910–2002) [2] and R.L.M. Synge (1914–1994) [3] on liquid-liquid
partition chromatography, in which the possibility of using a gas as the
mobile phase was predicted [4]. But however, it is important to note that
during the war the issues of the Biochemical Journal, where Martin and
Synge had published their results, were not available in Germany [4].
We would like to emphasize here that all activities from Erika Cremer
happened years before James and Martin published the first report
dealing with gas chromatography in 1952. Thus, the priority of professor
Erika Cremer’s work in carrying out the first actual gas chromatographic
experiments and doing it on her own intuition, definitely without any
outside suggestions, is beyond any dispute.
In this poster, we will present life and work of Erika Cremer. Looking
finally back over more than sixty years to this exciting period when all
these pioneering activities of chromatography were undertaken, it is
amazing to see under what primitive conditions Professor Erika Cremer
and all her students had to work. Scientists belonging to the post-war
generation cannot imagine the hardships they had to face at that time.
This fact still underlines more the importance of her work: even under
these circumstances and difficulties she could achieve great findings and
was one the first to publish gas-chromatographic results [5].
References:
1. Beneke, K.: Beiträge zur Geschichte der Kolloidwissenschaften 1999, 8: 311–334.
2. Adlard, E.: LC-GC Europe 2002, 15(9): 610–611.
3. Gordon, H.: Biographical Memoirs of the Fellows of the Royal Society 1996, 42: 455–479.
4. Martin, A.; Synge, R.: Biochemical Journal 1941, 35(12): 1359.
5. Bobleter, O.: Chromatographia 1996, 43(11/12): 581–582.
7. Ettre, L.S.: Chromatographia 1990, 29(9/10): 413–414.
8. Bobleter, O.: Chromatographia 1990, 30(9/10): 471–476.
POS.214
Heinrich Hörlein – A life between science and economy
Zummersch, M.1; Friedrich, C.1
Institut für Geschichte der Pharmazie der Philipps-Universität Marburg / Institute for the
History of Pharmacy, University of Marburg, Roter Graben 10, 35032 Marburg, Germany
1
Heinrich Hörlein (1882–1954) belonged to the kind of chemists who were
not contented with their research work in the industrial area, but also
successful in the economic field. As chemist, physicist and economist he
combined his natural-scientist expertise with specialist knowledge in
economy which was imperative for his work in the industry, making him
much better informed than he would have been if he had been restricted
to the area of specialised chemistry in his long-term employment with the
paint manufacturers, formerly Friedrich Bayer & Co.
Furthermore, he supported the development of science by being active
over decades in numerous scientific societies and advisory boards,
serving the chemical-medical research, the support of German high
schools, care for the young generation and scientific literature.
Thanks to personal contacts and friendships with former university
chemists and significant physicians along with insights into human
nature, Hörlein was able to employ suitable staff for pharmaceutical
research. Hörlein was active in creating the development of modern
pharmaceutical chemistry and medication synthesis.
The profiling of pharmaceutical research, especially the conversion of the
Elberfeld laboratories to the biggest pharmaceutical research facility has
to be listed as Hörlein’s life’s work.
Acknowledgments: References and picture credits: Bayer AG Corporate History and Archives
OTHER TOPICS
AUTHOR INDEX
A
Abbas, Z. .................................................................................. 185
Abdel-Aziz, H. .................................................................. 62, 119
Abebe, D. ................................................................................. 178
Abrahamsson, B. .................................................................... 113
Abu Jhaisha, S. .......................................................................... 61
Acher F. C. .................................................................................. 33
Adami, M. ................................................................................ 148
Adams R. H.............................................................................. 104
Adamski, J................................................................................ 171
Affini, A. ................................................................................... 149
Ahrem, H.................................................................................. 124
Aigner, A. ................................................................................. 133
Akone, H. S. ................................................................................ 53
Alban, S. ..........................................................................153, 179
Alhazmi, H. A. ........................................................ 63, 138, 140
Alkhatib, Y............................................................................... 123
Alves Avelar, L. A. .................................................................. 103
Andermark, V. ........................................................................ 157
Andreas, J. O. .......................................................................... 187
Andrews, K. ............................................................................. 164
Ansideri, F. ................................................................................. 67
Apel, A. K. ................................................................................... 92
Arencibia, J. M. ....................................................................... 152
Aristotelous, T. .......................................................................... 35
Armbruster, M........................................................................ 127
Arntz, P..................................................................................... 129
Asare-Nkansah, S. .................................................................. 162
Asmari, M. ............................................................................... 138
Assaf, J...................................................................................... 141
Astigiano, S. ............................................................................... 69
B
Bachmeier, B. E. ........................................................................ 69
Baghdan, E............................................................................... 126
Bakowsky, U. ..................................................................122, 126
Balzus, B. .................................................................................. 125
Bange, F.-C. .............................................................................. 174
Banoub, M. .............................................................................. 176
Barbieri, O. ................................................................................. 69
Barnscheid, L. ......................................................................... 115
Baron, R. ................................................................................... 115
Bartels, I. K. ............................................................................. 184
Bauer, S. M. ................................................ 155, 156, 161, 163
Baumann, K. ................................................................ 30, 61, 63
Bautista, O. .............................................................................. 147
Bayer, T..................................................................................... 153
Becher, K. ................................................................................. 142
Becker, A. ................................................................................... 61
Becker, W. .................................................................................. 61
Bednarski, P. J. .............................................................. 156, 157
Beheiri, S. N. ............................................................................ 183
Behnisch, S. ................................................................... 156, 157
Beißner, N. ............................................................................... 137
Bekeschus, S. ............................................................................. 90
Bekiesch, P. ................................................................................ 92
Bendas, G. ................................................................................ 102
Bering, L. .................................................................................. 174
Bermudez, M. .......................................................................... 147
Bertoletti, N. ............................................................................ 171
Bertrand, H.-O. .......................................................................... 33
Bertsche, T. ................................................................................ 37
Bibb, J. A. .................................................................................. 104
Biel, M. ........................................................................................ 14
Bierwisch, A. ............................................................................ 140
Binder, A. .................................................................................. 115
Biondi, R. M. ............................................................................ 152
Bischoff, F. ............................................................................... 155
Bischoff, I. ................................................................................ 178
Bitter, K. .............................................................................89, 186
Blankenbach, K. ...................................................................... 164
Blättermann, S. ....................................................................... 146
Bock, C. ..................................................................................... 167
Böckler, F. M. ............................................................................. 63
Bödefeld, T. ............................................................................. 144
Bodem, J. .................................................................................. 160
Bodmeier, R. ............................................................................ 125
Boeckler, F. M............................................................................ 67
Boesecke, P. ............................................................................... 99
Böger, R. H. .............................................................................. 116
Bollacke, A................................................................................ 142
Bonus, M. ................................................................................. 176
Bopp, B. ....................................................................................... 71
Börger, C................................................................................... 173
Borsig, L. ................................................................................... 102
Bouchut, A. .............................................................................. 164
Bouron, A. ................................................................................ 144
Brabet, I. ..................................................................................... 33
Bracher, F. .................................................. 138, 154, 173, 178
Braig, S...................................................................................... 120
Braun, A. ......................................................................... 123, 189
Braun, F..................................................................................... 171
Bräuner-Osborne, H. ................................................................ 34
Bravin, A. .................................................................................... 99
Breit, B. ....................................................................................... 74
AUTHOR INDEX
Breitinger, H.-G....................................................................... 176
Breitinger, U. ........................................................................... 176
Breitkreutz, J. ................................................................... 13, 192
Brengel, C. ............................................................................... 173
Briel, D. ............................................................................145, 170
Brinkjost, T. ............................................................................. 162
Broich, K...................................................................................... 17
Bros, M. .................................................................................... 132
Brueck, S. ................................................................................. 115
Brümmer, A. ............................................................................ 190
Brüning, D................................................................................... 30
Bruno, A. ..................................................................................... 69
Brunschweiger, A...................................... 167, 168, 169, 190
Brust, P. .................................................................................... 170
Budde, P. ..................................................................................... 79
Buenemann, M. ...................................................................... 145
Bugain, O. ............................................................. 167, 168, 169
Büllesbach, K. ......................................................................... 147
Bunjes, H. .......................................................................... 56, 157
Burau, D. .........................................................................139, 186
Burckhardt, B. B. ................................................. 138, 183, 192
Busch, D. .................................................................................. 115
Buschauer, A. .................................................................120, 149
Busserolles, J. ............................................................................. 33
C
Calderón, M............................................................................. 125
Calin, M. ................................................................................... 102
Cardinaux, J.-R. ....................................................................... 145
Carrillo García, C....................................... 117, 121, 175, 176
Cascorbi, I................................................................................ 115
Castro Diez, C. ........................................................................ 188
Cawello, W. ............................................................................. 187
Chaikuad, A. ............................................................................... 61
Chakrabarti, A. ..............................................................151, 153
Chakrabarti, I. ......................................................................... 153
Chamrad, D. ............................................................................... 79
Charbaji, N. ............................................................................. 125
Cheung, S.-Y. .......................................................................... 180
Chevillard, F. .............................................................................. 35
Ciglia, E. ............................................................................ 71, 151
Ciplea, A. M. ............................................................................ 192
Clement, B. ................................................. 140, 155, 170, 172
Csordás, K................................................................................ 133
Czarnecki, K. .............................................................................. 38
Czupalla, C. J. ............................................................................. 27
D
Dai, B......................................................................................... 178
Daletos, G.................................................................................... 54
Dallanoce, C. ........................................................................... 144
De Amici, M. ........................................................................... 144
de Mello Martins, A. G. ......................................................... 173
de Souza Carvalho-Wodarz, C. ........................................... 135
Decker, C. ................................................................................. 159
Decker, H. ..........................................................................99, 134
Decker, S. ................................................................................... 74
Derix, S. ...................................................................................... 39
Dersch, P................................................................................... 163
Devraj, K. .................................................................................... 27
Dewaldt, M............................................................................... 123
Dey, S. ....................................................................................... 144
Diederich, W. .......................................................................... 160
Diedrich, D. ............................................................. 76, 103, 154
Dierks, C. .................................................................................... 74
Dietzel, A. ................................................................................. 137
Dietzel, D. ................................................................................. 132
Dimde, M.................................................................................. 125
Dobner, B. ...................................................................... 165, 166
Döring, E. .................................................................................. 156
Dörje, F. .................................................................................... 185
Dormann, H. ............................................................................ 185
Doroz-Płonka, A. ........................................................... 174, 175
Drabczyńska, A. ...................................................................... 174
Drews, G. .................................................................................... 29
Drückes, P. ............................................................................... 156
Du, W. J. ................................................................................... 130
Duburs, G. ................................................................................ 168
Düfer, M. .................................................................................... 31
Duque Escobar, J. ................................................................... 116
Dzikowski, R. ................................................................. 160, 169
E
Eberle, J..................................................................................... 144
Ebert, R. .................................................................................... 189
Efferth, T. ................................................................................... 70
Ehmann, L. ............................................................................... 186
Ehrig, K. .................................................................................... 153
Ehrt, C. ...................................................................................... 162
Eichner, A. ...................................................................... 165, 166
Einsle, O...................................................................................... 74
Eisend, S. .................................................................................. 188
El Deeb, S. ........................................... 63, 138, 139, 140, 141
Elgaher, W. A. ......................................................................... 161
Elsinghorst, P. W. ................................................................... 140
Elz, S. ......................................................................................... 149
Empting, M. ................................................................... 163, 173
Engel, M.................................................................................... 152
Engelhardt, B. .......................................................................... 164
Engelhardt, K. H. .................................................................... 126
Engelke, L. H. ........................................................................... 152
Erdmann, F. ............................................................................. 159
Erdogmus, S. ............................................................................. 46
Eriksson, P. O. ......................................................................... 115
Eschalier, A. ............................................................................... 33
Eschenhagen, T....................................................................... 145
AUTHOR INDEX
Esteban, G. ............................................................................ 149
Exner, T. ....................................................................................63
F
Falke, H. ....................................................................................61
Fallegger, D. .......................................................................... 164
Fedorov, O. ............................................................................ 154
Felsch, M. .................................................................................38
Fenoy, C. G. ........................................................................... 190
Ferger, B. ............................................................................... 117
Fiene, A. ................................................................................. 146
Fiori, M. E. ................................................................................69
Fischer, D. .....................................................................123, 124
Fischer, K. .............................................................................. 180
Fischer, S. .............................................................................. 156
Flath, T. .................................................................................. 135
Fleischmann, B. .................................................................... 117
Flögel, U. ................................................................................ 125
Flötgen, D. ....................................................................168, 175
Forchhammer, K. ....................................................................92
Forster, M. ............................................................................. 163
Franz, L. ................................................................................. 170
Fricker, G. .................................................................................28
Friedland, K. .................................................................144, 185
Friedrich, C...................................................................192, 194
Frieg, B. .........................................................................166, 177
Fritz, N. .................................................................................. 144
Fröhner, W. ........................................................................... 152
Fromm, M.............................................................................. 185
Fruth, M. ................................................................................ 163
Fujiwara, R. ..............................................................................44
Fujiwara, T. ........................................................................... 178
Funke, M. ............................................................................... 146
Fürst, R. ............................................................... 119, 120, 178
Fürtig, M.-A. .................................................................139, 186
G
Garidel, P. .............................................................................. 122
Garscha, U. ......................................................... 107, 178, 179
Gawron, S. ................................................................................35
Geertz, B. ............................................................................... 145
Gehringer, M...................................................... 155, 161, 163
Geisslinger, G...........................................................................80
Gellert, A................................................................................ 160
Gerber, A. P. .......................................................................... 190
Gerhardt, H. .............................................................................27
Gerhardt, S. ..............................................................................74
Gershkovich, M. ................................................................... 159
Gersonde, F. .......................................................................... 188
Gerstmeier, J. ...............................................................107, 178
Gertzen, C. G. W. ........................................................ 103, 147
Giaisi, M. ...................................................................................68
Gilbert, I. H. ............................................................................. 35
Girreser, U. ......................................................... 140, 170, 172
Giurg, M. ................................................................................ 172
Glatzel, A. ............................................................................... 116
Glatzel, D................................................................................ 119
Glen, R. C. ................................................................................ 85
Gloriam, D. E. .......................................................................... 34
Goebgen, E. B. ............................................................. 184, 192
Goerigk, G. ............................................................................... 99
Gohlke, H. ...... 49, 71, 103, 147, 151, 165, 166, 167, 173,
176, 177
Gohr, K. .................................................................................. 152
Göke, K. .................................................................................. 157
Gollos, S. ................................................................................ 146
Gomeza, J. .............................................................................. 146
Gopireddy, S. R........................................................... 132, 136
Görg, B.................................................................................... 166
Goudet, C. ................................................................................ 33
Grabow, N. ............................................................................ 100
Grapentin, C. ......................................................................... 125
Greber, B. ............................................................................... 176
Gressenbuch, M. ................................................................... 135
Griebel, L................................................................................ 185
Grohganz, H. ......................................................................... 130
Groll, A. H. ............................................................................. 187
Gross, J. .................................................................................. 122
Grote, S. ................................................................................. 131
Groth, G.................................................................................... 71
Grundmann, M. ...................................................................... 65
Grünefeld, J. ................................................................ 160, 169
Gudd, J.................................................................................... 172
Gudermann, T. ........................................................................ 46
Guennewig, B. ....................................................................... 190
Guerit, S. .................................................................................. 27
Günter, S. ................................................................................. 74
Günther, M. ........................................................................... 156
Gutknecht, S. ......................................................................... 113
Gutmann, M. ......................................................................... 189
Gütschow, M. ........................................... 140, 147, 170, 172
H
Haag, R. .................................................................................. 125
Haake, N................................................................................. 188
Hacker, M................................................................................. 57
Hacker, M............................................................................... 135
Haenisch, S. ........................................................................... 115
Hahne, T................................................................................. 112
Hall, J. ..................................................................................... 190
Halver, J. ................................................................................. 121
Hamacher, A.................................... 103, 151, 152, 156, 169
Hamacher, S. ........................................................................... 38
Hanke, F. .................................................................................. 40
Hanke, T. ................................................................................ 180
Hansen, F. K. .................. 76, 103, 151, 152, 154, 156, 169
AUTHOR INDEX
Harbaum, L.............................................................................. 116
Harder, M. J. ............................................................................ 109
Hartmann, R. W. ....................................... 161, 163, 171, 173
Hasenpusch, D. ....................................................................... 116
Hasse, S. ...................................................................................... 90
Haupenthal, J. ......................................................................... 161
Hauser, A.-T. ........................................................................... 153
Häussinger, D. ............................................. 16, 147, 166, 176
He, D. ........................................................................................ 112
Hedtrich, S......................................................................125, 181
Heidrich, J. .................................................................................. 67
Heimburg, T. ........................................................................... 153
Heine, A.................................................................................... 171
Heinemann, S. ........................................................................... 42
Heinrich, M.............................................................................. 144
Heisig, F. .................................................................................. 146
Helfert, S. M............................................................................ 115
Hellmann, N. .................................................................... 99, 134
Hellmich, U. ............................................................................. 160
Hellwig, M. .............................................................................. 101
Helmer, R. ................................................................................ 160
Helmstädter, A. .............................................................119, 193
Hempel, G. ............................................................ 142, 143, 187
Hendrikx, S. ............................................................................. 135
Hennen, S. ............................................................................... 146
Hensel, A. ................................................................................. 120
Heppner, H. J........................................................................... 184
Herdewijn, P............................................................................... 18
Herich, L. ..................................................................................... 38
Hermans, S. M. A. ..................................................................... 49
Herrmann, J. ............................................................................ 163
Hess, C. ..................................................................................... 117
Hesse, M................................................................................... 117
Heuer, G. .................................................................................. 186
Hildebrand, J. ............................................................................. 40
Hildebrandt, C. ....................................................................... 132
Hilger, R. A. ............................................................................. 101
Hillenbrand, M. ......................................................................... 36
Hinz, S. ............................................................................145, 174
Hirt, M. ..................................................................................... 168
Hobernik, D. ............................................................................ 132
Hoeppner, A. ........................................................................... 177
Höfer, H. H. ............................................................................. 130
Hoffmann, C. ............................................................................. 46
Hofmann, B. ...................................................................180, 181
Holloway, S. ............................................................................ 160
Hölscher, C. ............................................................................. 174
Holzgrabe, U. .................................................................144, 176
Homeyer, N. ...................................................................165, 166
Hongwiset, D. ......................................................................... 174
Hönzke, S. ................................................................................ 181
Hopkins, A. L....................................................................... 35, 83
Hopkins-Navratilova, I. ............................................................ 35
Hudson, B. D. ............................................................................. 65
Hügle, M...................................................................................... 74
Humbeck, L. ............................................................................ 163
Huschmann, F. U. ................................................................... 171
Huwiler, A. ............................................................................... 164
I
Imeri, F...................................................................................... 164
Imig, J. ....................................................................................... 190
Immer, M. ................................................................................ 162
Ingebrandt, S. ......................................................................... 123
Itoh, T.......................................................................................... 44
J
Jaehde, U. ........................................................ 40, 89, 101, 186
Jakob, F. .................................................................................... 189
Jensen, K. T. ............................................................................. 130
Jin, N.......................................................................................... 130
John, C......................................................................................... 38
Johnson, R. ........................................................................99, 134
Jones, P. G. ................................................................................. 61
Jose, J ......................................................................................... 120
Jose, J. ....................................................................... 71, 142, 190
Jung, K....................................................................................... 168
Jung, M. ................................................................ 151, 153, 160
Juntke, J. ................................................................................... 135
K
Kalayda, G. V. .......................................................................... 101
Kaleta, M. ................................................................................. 175
Kalliokoski, T. .......................................................................... 167
Kalscheuer, R............................................................................. 53
Kamińska, K. ............................................................................ 148
Kandil, R. .................................................................................. 178
Kanitz, M. ................................................................................. 160
Kankowski, S. .......................................................................... 157
Karcz, T. ................................................................ 146, 174, 175
Karkossa, F. .............................................................................. 113
Karow, A. R. ............................................................................. 122
Kascholke, C. ........................................................................... 135
Kassack, M. U. .......76, 103, 151, 152, 154, 156, 169, 174
Kassel, S.................................................................................... 150
Keck, C. M. ........................................................... 123, 129, 130
Keiser, M. ................................................................................. 115
Keitel, V. ......................................................................... 147, 166
Kelber, O. ..........................................................................62, 119
Kenakin, T. ................................................................................. 65
Kennedy, A. T.......................................................................... 109
Kessel, E.................................................................................... 112
Keßler, S. .................................................................................... 93
Khalife, J. .................................................................................. 164
Khalil, F. .......................................................................... 183, 188
Khan, N. .................................................................................... 148
AUTHOR INDEX
Khosa, S. .................................................................................. 177
Khoshakhlagh, P. ................................................................... 134
Kieć-Kononowicz, K. ...................... 146, 148, 164, 174, 175
Kiefer, W. ................................................................................ 160
Killian, P. H. ................................................................................ 69
Kindgen, S. .............................................................................. 113
Kirchhefer, U........................................................................... 187
Kirchmair, J. ............................................................................... 85
Kishore, S................................................................................. 190
Klebe, G. ..........................................................................171, 172
Klein, C. D. .................................................................................. 51
Klein, P. .................................................................................... 112
Klein, S. .................................................................................... 113
Kleine, K. .................................................................................. 192
Kleinebudde, P. ............................... 127, 130, 131, 133, 134
Kleinschrodt, D. ...................................................................... 177
Klemm, D. ................................................................................ 124
Klika Škopić, M. ..................................................................... 169
Klingler, F. ............................................................................... 159
Klingmann, I. .......................................................................... 192
Kloft, C. ............................................. 139, 141, 184, 186, 192
Klose, H. ................................................................................... 116
Knapp, S..................................................................... 59, 61, 154
Knop, K. ...........................................................................127, 130
Köberlein-Neu, J. ....................................................................... 38
Koburg, M................................................................................ 155
Koch, M. ...................................................................................... 66
Koch, O........................................................ 162, 163, 168, 174
Koch, P............................................................................... 67, 156
Koeberle, A. ............................................................................. 119
Köhler, R. .................................................................................... 68
Kolb, P. ........................................................................................ 35
Kolter, M. .................................................................................... 28
König, S. ...................................................................................... 98
Köppler, J. ................................................................................ 156
Korp, J. ......................................................................................... 98
Köse, M. ..........................................................................146, 174
Kostenis, E. ............................................................. 65, 146, 147
Kottke, T. ................................................................................. 150
Kotz, S. ..................................................................................... 101
Krafczyk, K. ............................................................................. 192
Krainitzki, L. ............................................................................ 132
Kralisch, D. .....................................................................123, 124
Kramer, D. .................................................................................. 94
Kramer, F. ................................................................................... 78
Krämer, W. .............................................................................. 125
Krammer, H. P. .......................................................................... 68
Krebs, L. ...................................................................................... 99
Kremer, M................................................................................ 127
Kretschmer, S. B. M. ....................................................180, 181
Kronski, E. ................................................................................... 69
Krüger, A.................................................................................. 113
Krüger, M.......................................................................... 39, 186
Kubas, B. .................................................................................. 174
Kubbutat, M. H. G.........................................................161, 162
Kubicova, L. ............................................................................. 145
Kuder, K. ................................................................................... 164
Kulick, M. ................................................................................... 89
Kullmann, M. ........................................................................... 101
Kunick, C. ............................................ 61, 160, 161, 162, 169
Kunschke, N............................................................................. 124
Kunze, T.................................................................................... 188
Küppers, J. ................................................................................ 172
Kurz, T. ............................ 76, 103, 151, 152, 154, 156, 169
L
Lächelt, U. ................................................................................ 112
Läer, S. ................................................. 88, 138, 183, 188, 192
Lalk, M. ....................................................................................... 90
Lancelot, J................................................................................. 153
Lande, D. H. ............................................................................. 169
Lang, M. .......................................................................... 145, 170
Lange, A. ..................................................................................... 67
Lange, S. ......................................................................... 165, 166
Langer, K. ................................ 122, 126, 127, 128, 129, 190
Langguth, P. .................................................. 99, 113, 132, 134
Längle, D......................................................................... 168, 175
Langner, A. ..................................................................... 165, 166
Lappe, S. ................................................................................... 122
Laufer, S. ........................................................................ 155, 161
Laufer, S. A. ......................................................... 156, 163, 165
Lautscham, G............................................................................. 79
Law, J. K. Y. .............................................................................. 123
Łażewska, D. ............................................................................ 164
Le Borgne, M. .......................................................................... 142
Le Duc, G. ................................................................................... 99
Lee, D. J. .................................................................................... 112
Lehmann, W. D. ...................................................................... 140
Lehr, C. M....................................................................... 124, 135
Lehr, T. ...................................................................................... 104
Lehrnbecher, T........................................................................ 187
Lehto, T..................................................................................... 112
Lemcke, T. ...................................................................... 116, 159
Lemmerhirt, C. ........................................................................ 167
Lemmerhirt, H............................................................... 156, 157
Lengers, I. ................................................................................. 120
Lenz, E. ...................................................................................... 130
Li, T. ........................................................................................... 140
Li, Y............................................................................................ 178
Liebich, M. ............................................................................... 143
Liebl, J. ...................................................................................... 104
Liebner, S. .................................................................................. 27
Liening, S. ................................................................................ 179
Liewert, I. ................................................................................. 153
Lillig, C. H. ................................................................................ 156
Lim, N. Y. T.............................................................................. 109
Link, A. ...................................................................................... 167
Listing, M. ................................................................................ 180
Litty, F.-A. ................................................................................. 172
Li-Weber, M............................................................................... 68
AUTHOR INDEX
Loaëc, N. ........................................................................... 61, 160
Löbmann, K. ............................................................................ 130
Loretz, B. .................................................................................. 124
Lozach, O. ................................................................................... 61
Lucas, X. ...................................................................................... 74
Lüdeke, S. ......................................................................... 76, 154
Ludwig, S .................................................................................... 24
Lühmann, T. ...................................................................108, 189
Lüneburg, N. ........................................................................... 116
Lunter, D. .................................................................................... 58
Luong, B. .................................................................................. 120
Lutz, S....................................................................................... 145
M
Maas, R. .................................................................................... 185
Maček, B...................................................................................... 92
Madea, B. ................................................................................. 117
Maier, J. .................................................................................... 156
Makowski, N. .......................................................................... 188
Marchais-Oberwinkler, S. .................................................... 171
Marek, M.................................................................................. 153
Martin, P. ................................................................................. 115
Masur, K. ..................................................................................... 90
Mattern, K................................................................................ 137
Matz, M. ...................................................................................... 30
Maul, K. J. ................................................................................ 112
Maurer, C. K. ........................................................................... 163
Mayer-Wrangowski, S. C. .................................................... 165
Mederos y Schnitzler, M......................................................... 46
Meier, C. ................................................................................... 155
Meier, R. ................................................................................... 134
Meijer, L. ................................................................. 61, 160, 173
Meinel, L. .............................................................. 108, 128, 189
Melesina, J. .............................................................................. 153
Mély, Y. .................................................................................... 161
Melzig, M. F............................................................................. 120
Memmel, E. ............................................................................. 189
Menche, D................................................................................ 120
Mennemann, H. S. .................................................................... 38
Merk, D. ...........................................................................106, 159
Merk, H..................................................................................... 104
Merkel, O. M. .......................................................................... 178
Merten, N................................................................................. 146
Mertens, M. D. ........................................................................ 140
Mertens-Keller, D. ..................................................................... 38
Mesken, J. ................................................................................ 129
Messerer, R.............................................................................. 144
Metz, A. .................................................................................... 171
Metzger, S. .............................................................................. 101
Meyer zu Heringdorf, D. ...................................................... 164
Meyer, A..........................................................................146, 157
Miceli, E.................................................................................... 125
Milligan, G. ................................................................................. 65
Minichmayr, I. K. ................................................................... 186
Mirisola, V.................................................................................. 69
Mittal, N. .................................................................................. 190
Mohammadi, M. ..................................................................... 178
Mohr, K. .............................................................. 48, 64, 65, 144
Möller, G. .................................................................................. 171
Monsuur, F............................................................................... 130
Mootz, H. D. ............................................................................. 142
Morhenn, K. ............................................................................. 145
Moritz, S. .................................................................................. 123
Moskovits, J. ............................................................................ 187
Mozafari, M. ........................................................ 139, 140, 141
Mueller, U................................................................................. 171
Müller, C. ........................................................................ 138, 187
Müller, C. E. ................ 12, 62, 81, 117, 145, 146, 174, 175
Müller, D................................................................................... 174
Müller, R. .................................................... 119, 120, 129, 163
Müller, R. H. ............................................................................. 130
Müller, W. W. ........................................................................... 68
Müller-Fahrnow, A. .................................................................. 60
Müller-Goymann, C. C........................................................... 122
Mulnaes, D. .................................................................... 167, 177
München, S. E. ........................................................................ 187
Muth, F. .................................................................................... 156
N
Nachbar, M. ............................................................ 63, 140, 141
Nachtigall, A. ........................................................................... 184
Nägele, E. ................................................................................... 45
Nasereddin, A. .............................................................. 160, 169
Nassut, R. ................................................................................. 129
Nawroth, T.............................................................. 99, 113, 134
Neimanis, S. ............................................................................ 152
Nerlich, A. G. ............................................................................. 69
Nett, M. ....................................................................................... 98
Neubert, R. H. H. .......................................................... 165, 166
Newcomer, M.......................................................................... 178
Neyts, J........................................................................................ 25
Nieber, K. ..........................................................................62, 119
Niedermeyer, T......................................................................... 94
Nienberg, C.................................................................... 142, 190
Noonan, D. M. ........................................................................... 69
Nowak, G. ................................................................................. 144
O
Obst, K. ..................................................................................... 125
Odadzic, D................................................................................ 152
Odenweller, S.......................................................................... 193
Oetjen, E. ................................................................. 32, 116, 145
Ohta, S. ....................................................................................... 43
Okpanyi, S................................................................................ 119
Olejarz, A.................................................................................. 174
Olsen, C. A. ................................................................................ 75
AUTHOR INDEX
Oltmann-Norden, I. ........................................................ 63, 112
Orlando, Z. .............................................................................. 120
Ortmann, R................................................................................. 77
Ostrovsky, D............................................................................... 74
Oswald, S. ................................................................................ 115
Ott, I. ......................................................................................... 157
Otter, M.................................................................................... 115
Ouald-Chaib, A. ......................................................................... 71
P
Pagani, A. .................................................................................... 69
Pagani, G. ................................................................................. 165
Page, S. ........................................................................................ 55
Palus, J. ..................................................................................... 172
Parnham, M................................................................................ 80
Parr, M. K. ............................................................... 45, 138, 141
Paulsen, D. .................................................................................. 21
Pehe, C...................................................................................... 186
Pérez Gago, A. ........................................................................ 131
Peter, K. .................................................................................... 125
Peters, O................................................................................... 138
Pfaffenrot, E. ........................................................................... 155
Pfeffer, U. ................................................................................... 69
Pfeifer, A. .................................................................................... 81
Pfeilschifter, J. ........................................................................ 164
Pfleger, C. ......................................................................... 49, 173
Pieper, S. .........................................................................126, 187
Pierce, R. .................................................................................. 153
Pierrot, C. ................................................................................. 164
Pietschmann, T.......................................................................... 20
Pin, J.-P......................................................................................... 33
Pinnapireddy, S. ..................................................................... 126
Plate, K. H. .................................................................................. 27
Platzer, C. ................................................................................. 159
Plaza, A. .................................................................................... 173
Plückthun, A. .............................................................................. 36
Pockes, S. ................................................................................. 149
Polier, G. ...................................................................................... 68
Polosek, P. ................................................................................ 170
Popp, A. T. ............................................................................... 154
Porazik, C. ................................................................................ 117
Pötzinger, Y............................................................................. 124
Praefke, B. A. ........................................................................... 155
Preis, M. ................................................................................... 132
Pretzel, J. .................................................................................. 174
Preu, L......................................... 61, 140, 160, 161, 162, 169
Probst, M. ................................................................................ 136
Prochnicka, A. ......................................................................... 157
Prokopets, O. S. ...................................................................... 145
Prokosch, H. U. ....................................................................... 185
Proksch, P. .................................................................................. 53
Proschak, E. .......................................................... 105, 152, 159
Punčochová, K. ....................................................................... 134
Pyo, S. M. ................................................................................. 129
Q
Quinke, R. ................................................................................. 186
R
Rabel, M.................................................................................... 124
Rach, R. ..................................................................................... 113
Rades, T. ................................................................................... 130
Rahnfeld, L. .............................................................................. 124
Rasool, M. F. ............................................................................ 183
Rathmer, B. .............................................................................. 176
Raudszus, B.................................................................... 128, 190
Rauh, D. .............................................................................72, 165
Raulf, A. .................................................................................... 117
Redweik, S. .............................................................................. 140
Rehberg, N. ................................................................................ 53
Reichl, S. ............................................................... 135, 137, 157
Reimer, M................................................................................. 115
Reiss, G. J.................................................................................. 103
Rempel, V. ................................................................................ 175
Rengelshausen, J. ................................................................... 115
Richling, I. .................................................................................. 38
Rickmeyer, T. .......................................................................... 172
Ridder, S. .................................................................................. 127
Rigault, D.................................................................................... 33
Ritschel, T. ................................................................................. 84
Ritzer, J. .................................................................................... 189
Robaa, D. ........................................................................ 160, 164
Roblek, M. ................................................................................ 102
Rödl, C. B. ....................................................................... 180, 181
Rodrigues Moita, A. J. .......................................... 76, 151, 154
Romier, C. ................................................................................ 153
Rose, O........................................................................................ 38
Rothbauer, U. .......................................................................... 156
Rötzer, K. M. ............................................................................ 136
Rovera, F..................................................................................... 69
Rubbiani, R. ............................................................................. 157
Rübsamen-Schaeff, H.............................................................. 23
Rudo, A. .................................................................................... 174
Rustenbeck, I. ............................................................................ 30
Rüther, A. ..........................................................................76, 154
S
Saaber, D. ................................................................................. 135
Sadek, B. ................................................................................... 148
Sanoh, S. .................................................................................... 43
Sauer, B........................................................................... 160, 164
Saur, O. ..................................................................................... 150
Sayle, S. .................................................................................... 122
Schäberle, T. F. ......................................................................... 95
Schächtele, C. ................................................................ 161, 162
Schächtele, S. .......................................................................... 185
AUTHOR INDEX
Schade, D. ............................... 117, 121, 168, 172, 175, 176
Schaefer, C. ............................................................................. 187
Schaefer, J. .............................................................................. 138
Schaefer, K. H. ........................................................................ 123
Schäfer-Korting, M. ............................................................... 181
Schaffert, C. ............................................................................... 38
Schäke, F. ................................................................................. 145
Schaller, D. .............................................................................. 148
Scharf, R. E. ............................................................................. 165
Scherließ, R. ...................................................................129, 132
Scherneck, S. ............................................................................. 87
Scheunemann, M. .................................................................. 170
Schieback, P. ........................................................................... 159
Schiebel, J. ............................................................................... 171
Schieferdecker, S. ..................................................................... 98
Schipke, C. ............................................................................... 138
Schirmeister, T. ............................................................... 50, 160
Schlesinger, M. ....................................................................... 102
Schlosser, E. ............................................................................ 136
Schlütke, L. .............................................................................. 161
Schmidberger, H....................................................................... 99
Schmidt, A. .......................................................................... 45, 90
Schmidt, C. .............................................................................. 157
Schmidt, C. Q. ........................................................................ 109
Schmidt, J. ............................................................................... 159
Schmidt, M. ................................................ 136, 153, 159, 164
Schmidtkunz, K. ..................................................................... 153
Schmueser, L. ......................................................................... 134
Schneefeld, M......................................................................... 174
Schneider, G............................................................................... 86
Schneider, J. ............................................................................ 140
Schneider, M.............................................................................. 79
Schneider, T. ........................................................................... 153
Schneider-Daum, N. .............................................................. 135
Schoeder, C. ...................................................................117, 175
Schoenfeld, A.-K. ................................................................... 179
Scholz, M. S. ........................................................................... 180
Scholz, P................................................................................... 123
Schönherr, R. ............................................................................. 42
Schöppe, J................................................................................... 36
Schori, C...................................................................................... 36
Schrader, J. .............................................................................. 125
Schrage, R......................................................................... 64, 144
Schröder, J. .............................................................................. 194
Schröder, R.............................................................................. 147
Schröpf, S. ............................................................................... 186
Schubert, R.............................................................................. 125
Schubert-Zsilavecz, M. ................................................159, 180
Schulte, M. .............................................................................. 152
Schulz, D. ................................................................................. 183
Schulze, J. .......................................................................133, 152
Schulze, P. ............................................................................... 135
Schulze, T. ............................................................................... 141
Schulz-Fincke, A. C. ............................................................... 170
Schulzke, C. ............................................................................. 157
Schulz-Knappe, P. ..................................................................... 79
Schulz-Siegmund, M. ............................................................ 135
Schumacher, K. ......................................................................... 30
Schumann, D. .......................................................................... 135
Schuster, D............................................................................... 180
Schwab M. ................................................................................. 82
Schwalm, S. ............................................................................. 164
Schwed, J. S. .................................................................. 148, 149
Schweins, R. .............................................................................. 99
Scriba, G. ...........................................................................42, 179
Seemann, W. K. ........................................................................ 48
Seibel, J. .................................................................................... 189
Seidlitz, A. ...................................................................... 100, 136
Sendker, J. .................................................................................. 91
Senz, V. ..................................................................................... 100
Shahla, H. ................................................................................. 193
Sharma, K................................................................................. 161
Siegmund, W. ......................................................................... 115
Simionescu, M. ....................................................................... 102
Simon, K. .................................................................................. 146
Sippl, W.............................................151, 153, 159, 160, 164
Skarżewski, J. .......................................................................... 172
Škopić, K. M. ........................................................................... 167
Smith, N. J.................................................................................. 65
Smits, H. J. S............................................................................ 177
Sommerfeld, A........................................................................ 176
Sommerhoff, C. P..................................................................... 69
Sonnenberger, S. .......................................................... 165, 166
Spahn-Langguth, H. ............................................................... 193
Spanier, C................................................................................. 146
Spanier, L. ................................................................................ 151
Špatenková, L. ......................................................................... 192
Spieler, V. ................................................................................. 108
Spomer, L. ................................................................................ 147
Stan, D. ..................................................................................... 102
Stanic, M. ................................................................................... 45
Stark, H. ......................... 148, 149, 150, 152, 164, 180, 181
Staufenbiel, S.......................................................................... 130
Steckel, H. ................................................................................ 129
Steiger, C. ................................................................................. 128
Steinhilber, D. .............................................. 80, 159, 180, 181
Steinicke, F. ......................................................................63, 112
Stenzel, K. ................................................................................ 169
Štěpánek, F. ............................................................................. 134
Stephan, C.................................................................................. 22
Steuber, H. ............................................................................... 160
Stoiber, K. ................................................................................ 120
Stoldt, V. R............................................................................... 165
Storch, U. ................................................................................... 46
Strödke, B. ................................................................................ 178
Strohalm, M............................................................................... 94
Strohmann, C. ......................................................................... 168
Stühler, K. .................................................................................. 41
Subramanian, D...................................................................... 148
Syntschewsk, V. ........................................................... 103, 156
Szekely, N. K. .......................................................................... 134
AUTHOR INDEX
T
Tallant, C.................................................................................. 154
Tateno, C. ................................................................................... 43
Täuber, A. ................................................................................ 122
Telaar, A. ..................................................................................... 79
Temme, S. ............................................................................... 125
Temml, V. ................................................................................ 180
Teubel, J. .................................................................................. 138
Thabet, Y. ................................................................................ 132
Tham, W. H............................................................................. 109
Thimm, D. ................................................................................ 146
Thoma, F. ................................................................................. 127
Thomann, A. ........................................................................... 173
Thürmann, P. A. .............................................................. 40, 184
Thurmond, R. L. ........................................................................ 15
Tietz, K. .................................................................................... 113
Tikhomirov, A. ........................................................................ 170
Tikhonova, I. G. ......................................................................... 65
Tins, J. ..............................................................................138, 183
Titz, A. ......................................................................................... 52
Totzke, F. ........................................................................161, 162
Trappe, J. ................................................................................. 156
Tremmel, R................................................................................. 26
Tremmel, T. ............................................................................. 173
Tschammer, N. .......................................................................... 47
Tsikrika, P. ............................................................................... 190
Tukey, R. H. ................................................................................ 44
Türeli, N. G. ............................................................................. 135
Tyzack, J. D................................................................................. 85
U
Ulven, T. ...................................................................................... 65
Unzeta, M. ............................................................................... 149
Urbanetz, N. A. ..............................................................132, 136
V
Ventura Pereira, J. P. ............................................................. 165
Villmann, C. ............................................................................ 176
Vogt, D. ...........................................................................180, 181
Völler, S.................................................................................... 183
Vollmar, A. M. ..................................................... 104, 120, 155
von Hammerstein, F.............................................................. 160
von Woedtke, T. ....................................................................... 90
Vordenbäumen, S..................................................................... 79
Voß, U. .............................................................................. 62, 119
Vukosavljevic, B. .................................................................... 134
W
Wagner, E. ............................................................................... 112
Wagner, S. ............................................................................... 170
Wagner, T. ............................................................................... 160
Wahl, M. A. .............................................................................. 127
Wähnert, D. ............................................................................. 183
Wallmeyer, L. .......................................................................... 181
Walter, A. ....................................................................... 153, 160
Walter, M. ...................................................................... 148, 149
Walter, N.................................................................................. 165
Waltering, I................................................................................ 38
Wang, X.................................................................................... 125
Wätzig, H. ..................................................... 63, 112, 139, 140
Weber, J.................................................................................... 142
Weidel, E. ................................................................................. 173
Weidner, T. .............................................................................. 160
Weiser, C. ................................................................................. 141
Weiss, M. S. ............................................................................. 171
Weitschies, W............................................................... 100, 136
Weizel, L................................................................................... 148
Wende, K. .................................................................................. 90
Wentsch, H. K. ........................................................................ 165
Wentzlaff, M. .......................................................................... 100
Werner, T. ................................................................................ 168
Werner, V. ............................................................................... 108
Werz, O. .................................... 98, 107, 119, 178, 179, 180
Wesselborg, S. .......................................................................... 73
Wicha, S. G. ............................................................................. 192
Więcek, M. ............................................................................... 148
Wille, T. .................................................................................... 140
Willems, E. ............................................................................... 121
Windbergs, M. ........................................................................ 134
Witting, A. ............................................................................... 117
Wohlwend, D. ........................................................................... 74
Wolber, G. ...................................................................... 147, 148
Wölfel, S................................................................................... 162
Wölker, J................................................................................... 138
Woltersdorf, S. ............................................................. 180, 181
Wu, H. ....................................................................................... 160
Wuest, B. .................................................................................. 141
Wünsch, B. ..................................................................... 144, 162
Wurzel, J................................................................................... 189
Wüst, B. .............................................................................45, 138
X
Xie, Y. ........................................................................................ 178
Z
Zahler, S. .................................................................................. 104
Zander, J. .................................................................................. 186
Zavolan, M. .............................................................................. 190
Zeisig, R. ................................................................................... 102
Zeitlinger, M. ........................................................................... 141
Zeuzem, S. ............................................................................... 152
AUTHOR INDEX
Zhang, S. .................................................................................. 104
Zhao, Q........................................................................................ 70
Zhu, Q.......................................................................................... 42
Ziegler, N. ................................................................................... 27
Ziller, A. .................................................................................... 132
Zivkovic, A. .............................................................................. 164
Zlotos, D. P............................................................................... 176
Zoller, M. .................................................................................. 186
Zubair, M.................................................................................. 157
Zucht, H. ..................................................................................... 79
Zulkiewicz Gomes, D. ............................................................ 141
Zummersch, M........................................................................ 194
Geschäftsführer und Leiter der Geschäftsstelle
Apotheker Dr. Michael Stein
DPhG Geschäftsstelle
Varrentrappstr. 40 – 42
60486 Frankfurt
Tel.: 069-7191596-0
Fax: 069-7191596-29
Email: info@dphg.de
http://www.dphg.de
Univ.-Prof. Dr. Holger Stark
Heinrich-Heine-Universität Düsseldorf
Institut für Pharmazeutische and Medizinische Chemie
Universitätsstr. 1
40225 Düsseldorf, GERMANY
Phone: +49-(0)211–81 1 0478
Fax:
+49-(0)211–81 1 3359
E-mail: stark@hhu.de
15.09.2015
NOTES
NOTES
NOTES
SHORT PROGRAM
Annual Meeting of the German Pharmaceutical Society 2015- DPhG
Tuesday, September 22nd
14:00 – 17:45
Bürgersymposium: Geschichte der Pharmazie in Düsseldorf
(Haus der Universität, Düsseldorf Schadowplatz 14)
19:00 – 21:00
Treffen/Vortrag Arbeitsgemeinschaft Katastrophenpharmazie (Haus der Universität, D, Schadowplatz 14)
9:00 – 11:00
Sitzung VdPPHI e.V. Hochschullehrersitzung, B. Clement
11:30 – 13:00
DPhG Fachgruppen Meetings
13:00 – 13:30
(HS 3A)
Opening of the Annual DPhG Meeting 2015 (HS 3A)
I3 – Interactions, Integrations and Innovations
13:30 – 14:15
P.1 – C. E. Müller: I3 in Medicinal Chemistry of purinergic signaling
(HS 3A)
14:15 – 15:00
P.2 – J. Breitkreutz: New concepts and products for individual drug dosing
(HS 3A)
15:00 – 15:30
Coffee break - Poster viewing (even numbers)
15:30 – 17:00
SL1 - Antiviral drugs
17:00 – 18:00
Short poster lectures
17:00 – 18:00
Freunde der DPhG-Stiftung
18:00 – 22:00
Poster viewing (even numbers) and Welcome Reception
SL2 - Blood/brain barrier
SL3 - Regulation of beta-cell
function – Implications for
(HS 3E)
(HS 3D) diabetes
(HS 3A)
(HS 3A)
(building 23.02, room U1.23)
8:30 – 9:15
P.3 – M. Biel: Ebola and more: Endolysosomal cation channels as novel drug targets
(HS 3A)
9:15 – 10:00
P.4 – R. Thurmond: The pre-clinical and clinical development of H4R antagonists
(HS 3A)
10:00 – 10:30
10:30 – 12:00
SL4 - GLISTEN - GPCR
Medicinal Chemistry
SL5 - Evidence based
medication management
SL6 - (Bio)Analytics
(HS 3D)
(HS 3A)
(HS 3E)
12:00 – 13:30
Break for lunch time - Poster viewing (odd numbers)
13:30 – 15:00
SL7 - Allosteric regulation
SL8 - Anti-infective compounds SL9 - Poorly solubles
(HS 3A)
(HS 3D)
15:00 – 15:30
15:30 – 17:00
SL10 - PPP in drug development SL11 - Screening techniques in SL12 - Signaling in cell death
pharmacology & drug
(HS 3A) development
(HS 3D)
(HS 3E)
17:00 – 18:30
SL13 - Anticancer and
epigenetic drugs
17:00 – 18:30
Workshop, T. Hotopp, Fördermaßnahmen der DFG – Tipps und Hinweise
19:30
Conference dinner
(HS 3E)
SL14 - Personalized Medicine – SL15 - Focused pharmaceutical
Biomarker and diagnostics
research
(HS 3A)
(HS 3D)
(HS 3E)
(HS 3B)
Friday, September 25th
8:30 – 9:15
P.5 – D. Häussinger: Liver research in Düsseldorf: Bridging basic and clinical science
(HS 3A)
9:15 – 10:00
P.6 – K. Broich: Drug approval and regulatory science – Where to go?
(HS 3A)
10:30 – 12:00
SL16 - Future molecular design
SL17 - Medicaton safety in
special patient groups
SL18 - Hot topics in Pharmaceutical Biology – Young
investigators in the spotlight
(HS 3D)
(HS 3E)
(HS 3A)
12:00 – 13:00
Short lunch break
13:00 – 13:45
P.7 – P. Herdewijn: From synthetic nucleic acids to artificial genes and genomes: …
(HS 3A)
14:00 – 15:00
Closing ceremony
(HS 3A)
15:15 – 16:30
DPhG Jahreshauptversammlung
(HS 3A)
Saturday, September 26
th
15:00 – 18:30
Tag der Offizinpharmazie – Schmerzschulung (1. Teil)
(HS 3D)

Conference Book

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