History of the Glycosaminoglycan Symposia in the Scientific Context

History of the
Glycosaminoglycan Symposia
in the Scientific Context
Jawed Fareed, Ph.D.
Professor of Pathology and Pharmacology
Loyola University Medical Center
Maywood, IL 60153
Congratulations on the 20th
Anniversary of the Symposium
on Glycosaminoglycans.
An Unparalleled Achievement!
A Dedicated Effort!
Inspirational!
Twentieth Anniversary of the
Glycosaminoglycan Symposium at
Villa Vigoni
• A dedicated scientific forum for the
advancement of science of heparins and
related glycosaminoglycans
• Pioneering research, scientific break
throughs, innovative technology, drug
discovery and lead position in the
understanding of structure and biologic role
• International collaborations
Notable Symposia on Heparins and
Related GAGs
1.
2.
3.
4.
5.
St. Louis symposium organized by Bradshaw and Wessler1974
Heparin and Related Polysaccharides. Structure and
Activities organized by NYAS, Ofosu, Danishefsky and
Hirsh-1987
Uppsala Heparin Symposium- 1991, Lindhal
Glysosaminoglycans and Connective Tissue During the
Rodenian Era, University of Alabama-1994
Other symposia were held in Chapel Hill, Chicago, London,
New York, Boston and Milan at different times
The annual GAG symposia at Villa Vigoni represents the only
sustained yearly update of this growing field
Many other publications
GAG Symposia at Villa Vigoni
Meeting of the Minds
Job Harenberg (Heparin Pharmacology)
Benito Casu (Heparin Structure)
• Initially lead scientists from Italy and Germany joined
forces
• The forum expanded to include other European
countries and later other scientists from around the world
• The forum is unique and multidisciplinary
• Open platform to discuss the structure and biologic
actions of glycosaminoglycans
• Broadened scope to include newer anticoagulant drugs
The forum has an international impact and has served a
consensus organization.
The History of Villa Vigoni
Heinrich (Enrico) Mylius (1769-1854) and Frederike Schnauss
(1771-1851) moved to Milan at the end of the 18th century from
Frankfurt. They fostered cultural exchange between Italy and Germany.
They acquired Villa Vigoni estate for their son and his wife.
Unfortunately the son died and Heinrich transformed the villa into a
place of remembrance with a wide collection of art including sculptures
and paintings. His widow remarried Ignazio Vigoni.
Their grandson Ignazio Vigoni Jr. (1905-1983) bequeathed the property
of Lake Como to the Federal Republic of Germany to commemorate
the friendship of Heinrich Mylius with Goethe and other artists and to
keep the German and Italian cultural exchange alive.
Villa Vigoni
German-Italian Center for
European Excellence
Association founded in 1986 to promote German-Italian
cultural exchange and cooperation in art and science
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Scientific seminars
Graduate schools
Political and Journalistic discussions
Fellowship programs
For the past 20 years is the site of the yearly GAG
symposium organized by Professors Casu and
Harenberg
The gardens are designed by Giuseppe Balzaretto, an architect from
Pavia around the 19th century.
Inside the gardens there is a small temple with neo-classical sculptures and
the work of art by Bertel Thorvaldsen and Pompeo Marchesi.
Clinical Applications of Heparin Enter
the 7th Decade
Structurally Complex, Polypharmacologic, Universal Anticoagulant,
Controversial, Challenging and Academically Rewarding
1917- Accidentally discovered by a medical student J. McLean
1928- Howell recognized that heparin was a carbohydrate
containing uronic acid
1935-36- Bergstrom in Jorpes’s lab showed N- sulfated
glucosamine in heparin. Jorpes with Charles and Scott
produced sufficient amount of heparin and Crafoord used it in
humans
1946- Wolfram identified D-glucuronic acid (sugar alley in Ohio)
1962- Cifonelli and Dorfman identified L-iduronic acid in heparin
1968- Perlan confirmed the L-iduronic acid using NMR
1970s- Casu, Dietrich, McDuffie, Lindahl, Linhardt, Rosenberg,
and many other scientists contributed to the study of heparin
Historic Landmarks in Heparin Research
• Biosynthesis of heparin-Lindahl, Hook
• Structural activity relationship of heparin – Cifonelli,
Barlow
• Synthesis of heparin derivatives – Danishefsky
• Structural studies on heparin – Dietrich, McDuffie,
Perlin, Casu, Conrad, Choay
• Biochemistry and pharmacology of heparin – Jaques,
Estes, Rosenberg, Olivecrona
• Discovery of antithrombin – Seegers, Rosenberg
• Development of heparin fractions and LMWHs –
Johnson, Cifonelli, Choay, Mardiguian, Samama
• Clinical development of heparins – Howell, Wessler,
Salzman, Kakkar, Hirsh, Messmore, Turpie
• The Villa Vigoni GAG symposia era – 1991-2012
Pioneers in Heparin Discovery
McLean
Howell
Jacques
The Swedish Connection
Jorpes
Crafoord
Bergstrom
Toronto Connection -
Connaught Labs.
Best and Benting
Institute
University of Iowa Group: including Brinkhous, Smith, Seegers
Historical Landmarks in Heparin Research
Discovery of Heparin
• Isolation and characterization of heparin
• Clinical use of heparin
• Elucidation of the mechanisms of action of heparin
(AT binding site)
• AT binding site and it’s characterization
• Fractionation of heparin
• Isolation of high affinity oligosaccharides
• Depolymerization of heparin and the development of
LMWH
• Synthesis of pentasaccharide
• Molecular manipulation of heparin
• Administration of heparin
Dietrich
The Brazil
Connection
Stan Wessler
Pioneering work on mechanisms of heparin actions (Anti-FXa effects)
Engelberg
Anti-inflammatory and Cardioprotective Effects of Heparin
Caprini – Optimization of Heparin for Surgical Usage
Sasahara – Clinical Development of
Heparins in Cardiology
Erwin Coyne –
The Living
Legend
Jean Choay
M. Petitou
J.C. Lormeau
The Development
of
Fondaparinux
(Arixtra®)
The First FXa Inhibitor
Drug
Pentasaccharide is a totally synthetic heparin derivative. It is free of
biologic contaminants and does not generate heparin induced
thrombocytopenia associated antibodies.
Jean Mardiguian – The Enoxaparin Innovator
Messmore, Toulemonde, Robbins, Kakkar
Mammen and Walenga
Development of Heparin and Related
GAGs
The Villa Vigoni Era – The First Symposium 1991
Structure Activity Relationship of the Antithrombotic and Haemorrhagic
Properties of GAGs
- Witt, Bianchini, Klauser, Torri and Breddin
- Jann and Jann, Franz
- Preissner, Grulich-Henn, Alisan
- Donati, Harenberg, Stehle
- Casu, Puggi, Markwardt
- Schmitz, Hubner, Coccheri, Pescador
Topics
- Biochemical and Plant derived GAGs
- GAG Protein Interactions
- Neutralization of GAGs
- Endothelial Modulation by GAGs
- GAGs in Cancer
- Oral Absorption of GAGs
Theme
Rational Development of Biologically Active GAG Derivatives and their Biomedical
Applications
Structure – Activity Relationships in Heparin, LMW Heparin and U-LMW Heparin
Lead Clinicians to Validate LMWHs and
New Anticoagulants
Bianchini – Pioneering Contributions in
GAG Development
Coccheri Clinical
Development of
LMWHs and
Dermatans in
Multiple
Indications
Klaus Breddin – Linked Basic Science to Clinical Practice in Heparinology;
Fostered European Collaborations to Lead the Clinical Developments of GAGs
Fritz Markwardt – A Great Mentor and Leader
New Developments of Anticoagulant and NonAnticoagulant GAGs and Other Coagulation Inhibitors
The Villa Vigoni Era – The 20th Symposium 2012
GAG Protein Interactions
Liu, Lindahl, Turnbull, Viskov, Seeberger, Sugahara, Sasisekharan
Molecular Basis for Biological Activities of Heparin and other GAGs
Schneider, Bendes, Borsig, Li, Vlodavsky, Barbieri
GAG in Cancer
Sanderson, Schlesinger, Laux
Advanced Methods for Structural Analysis & Diagnostic Applications
Para, Torri, Kramer, Yates, Hricovini, Vismara
Heparins, LMW Heparins, Biotechnologic Heparins and Other GAGs
Mulloy, Liverani, Seeberger
Past and Future for Heparins, LMWHs, Biotechnological Heparins
Harenberg, Mardiguian and Fareed
Structure Activity Relationship in
Heparin and Glycosaminoglycans
1.
2.
3.
4.
5.
6.
7.
8.
Endogenous protein interactions (glycosylation)
Regulation of enzymes
Endogenous release of mediators (TFPI)
Cellular interactions
Direct hemodynamic effects
Modulation of proteins
Signal transduction mechanisms
Biophysical interactions with membranes and release
of endogenous mediators
9. Structural analysis
10. Development of Heparinomimetics
Fractionation and Depolymerization of Heparin
Anti-Xa/IIa =
1.0
UFH
Anti-Xa/IIa = 2.5 7.5
Anti-Xa/IIa = 10 Heparin derived
15 kDa
- 50
oligosaccharides
< 2.5 kDa
LMWH
4-6 kDa
Ultra LMWH
Anti-Xa/IIa =
> 50
2-4 kDa
Depolymerization inflicts
structural changes
Pentasaccharide
1-7 kDa
Chemical Synthesis of
Heparin Analogues
• Isolation and characterization of heparin
components
• Structural analysis of heparin
components
• Chemical synthesis of heparin
analogues and homologues
• Validation of the biological effects
• Structure-activity relationships in
heparinomimetics
• 1,6 anhydro ring formation
•
Differential structural attributes in depolymerized products.
Depolymerization Inflicted
Structure Modifications to
Heparin
An Artifact not a Attribute!
Do molecular alterations result in
the generation of unique
pharmacophores?
Are all issues resolved?
Differentiation of Low Molecular
Weight Heparins
Not all of the Low Molecular
Weight Heparins are Cleaved
Equally!
Generic Enoxaparins
Variations in Composition and Pharmacologic Profiles
Role of Heparin/Heparan and
Heparinase/Heparanase in
Physiologic Processes
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Inflammation
Thrombosis
Molecular modulation
Pathophysiology of different diseases
Aging
Drug development for multiple
indications
• Topical pharmacology
Adulteration of Heparin
(Molecular Mimicry in Drug Design)
Is the Mystery Solved?
Who Solved the Mystery?
Is There More to Come?
Additional approaches to
contaminate heparin.
Contaminated Heparin from
China in 11 countries
Australia, Canada, China, Denmark, France, Germany, Italy, Japan, the
Netherlands, New Zealand, and the United States
Development of Bioheparins and
Chemoenzymatically
Synthesized Heparin Analogues
• K5 Derived Bioheparins and Low
Molecular Weight Heparins
• Chemoenzymatically synthesized
oligosaccharides
• Additional Chemical and Biological
approaches
Factor IX
Factor VII
Factor X
FVIIa
VKA drugs
FIXa
Anti-Xa drugs
• Tecarfarin
• Warfarin
• Apixaban
• Betrixaban
• Edoxaban
Factor Xa
• Rivaroxaban
• LY 517717
• TAK 442
• YM 150
Antithrombin
Anti-IIa drugs
Factor II
(Prothrombin)
Fibrinogen
•Dabigatran
•Ximelagatran
•AZD 0837
Factor IIa
(Thrombin)
Fibrin
Impact of Scientific Discussions at
Villa Vigoni on GAG Development
1.
2.
Heterogeneity in heparin and related GAGs
Structural characterization of heparin. Structure of
AT binding consensus sequence
3. Differentiation of LMW heparins.
4. Species variations in the structure of heparin
5. Non-anticoagulant effects of heparin
6. Heparin-protein interactions
7. Heparin quality and contaminants
8. Biology of heparan and its regulation
9. Molecular biology of GAGs with reference to
structure-activity relationship
10. Development of newer GAG based drugs
Scientific Impact of GAG Symposia
Villa Vigoni 1991-2012
1.
2.
3.
Over 250 major publications
More than 20 patents
Scientific awareness of the complexity of the structure and
functions of GAGs
4. Regulatory agencies acknowledgment of the need for
structural data for product specifications and quality
5. Differentiation of LMWHs and related products
6. Development of heparin analogues and homologues
7. Development of new drugs
8. Expanding the clinical indications of GAGs beyond
anticoagulants
9. Networking within GAG community and other disciplines
10. Recognition of the overall impact of GAGs by scientific,
medical, pharmaceutical, and academic disciplines
Publications of
manuscripts of the 3rd
(2 volumes),4th, 10th,
and 17th
Glycosaminoglycan
Symposia at Villa
Vigoni, Loveno, Italy
4th Glycosaminoglycan Symposium
1996
ISBN 0-306-45299-5
THANKS TO PROFESSORS CASU and HARENBERG
AND ALL THOSE WHO HAVE CONTRIBUTED TO THIS OUTSTANDING
SERIES OF SYMPOSIA DURING THE PERIOD 1991-2012
GAG Symposia 1991-1995
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Structure activity relationship in GAGs
Antithrombotic and bleeding effects of GAGs
Synthetic and biosynthetic analogues of GAGs
Recognition of GAG science as a moving target in drug
discovery
Analytical methods for characterization (NMR, MS, HPLC, CE,
GCMS and other methods)
Protein interactions with GAGs
Anti-inflammatory actions of GAGs
TFPI release
PK/PD of heparins
Chemically modified heparins
Cellular interactions of GAGs and their relevance to
physiologic and pathologic responses
GAG Symposia 1991-1995
(continued)
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Role of GAGs in cancer
PK/PD of GAGs and related agents
Role of heparan sulfate as a signaling molecule
Growth factor interactions with GAGs
Heparin and angiogenesis
Neuroprotective effects of GAGs
Original discussions on thee development of recombinant
hirudin
• Surface coating of GAGs to develop non-thrombogenic material
• Species specificity in GAGs structure
• Differentiation of porcine and bovine heparins
GAG Symposia 1996-2001
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Structural diversity workshop
Novel clinical applications of GAGs
Antithrombin drugs
Clinical validation of newer anticoagulants
Synthetic bifunctional oligosaccharides
Clinical development of newer antithrombins and
antiplatelet drugs
Newer approaches in DVT and PE management
Cellular biology of GAGs
Unresolved issues in thrombosis
Heparin-induced thrombocytopenia
Standardization of hirudins
GAG Symposia
1996-2001(continued)
• Biotechnology derived heparin oligosaccharides
• Structural components of GAGs and their role in protein and
cellular binding
• The discovery and early development of melagatran
• GAGs in diabetic nephropathy
• Cellular proliferation and growth modulation by GAGs
• Sequencing of specific protein binding GAGs
• Endothelial protection of GAGs
• Synthesis and biologic properties of nitro heparins
• Regioselective structural analysis of GAGs and their
derivatives
• Heparinase-1 as a neutralizing agent for heparin
• Recombinant Heparanase as a tool to develop anticancer drugs
GAG Symposia 2002-2007
Broadened scope to include other areas related to GAGs
• Novel tools to isolate and analyze GAGs
• Heparin oligosaccharides in senial dementia
• TFPI in the control of thrombogenesis
• Glycosaminoglycan protein interactions in the
control and regulation of metastasis
• Argatroban as an anticoagulant for heparin
compromised patients
• GAG mediated modulation by TAFI
• Ultra low molecular weight heparins
• Generic versions of LMWHs
• GAGs in renal disease
• Heparinase biology and pharmacology including
development of inhibitors
GAG Symposia 2002-2007
(continued)
• Role of heparins in cancer
• Engineered heparins
• Ultra LMWHs, octaparin and related agents
• Modulation of osteoporosis and calcification
• Physical methods to prepare LMWHs
• Alternate route to administer heparin and related GAGs
• Heparin in Alzheimer’s disease
• Identification of novel structural sites interacting with AT
and other proteins
• Novel application of NMR and mass spect to analyze
LMWH
• GAGs in the nervous system
GAG Symposia 2008-2011
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Additional biologic effects of heparin
Contaminants in heparin
Biophysical aspects of GAGs
In depth characterization of heparins and LMWHs
Anticoagulants beyond heparin
Focused discussions on heparan sulfate including chemistry,
biochemistry and molecular biology
Inflammation and cancer are targets for GAGs
Conversion approaches to analyze heparin
Pharmacological aspects of heparin anticoagulation
Finding a needle in a haystack! The wonders of new
technologies
Heparinase biology and pathology
GAG Symposia 2008-2011
(continued)
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Adhesion molecules and GAGs
Molecular biology of GAGs with reference to gene transcription
US FDA approval of the generic version of enoxaparin
Oral anti-Xa and anti-IIa drugs
Chemoenzymatic design of heparins
Proteoglycans and GAGs in health and disease
In depth topics on GAGs as anticancer and anti-inflammatory
agents
• Advances approaches in the analysis of GAGs
• Heparins including LMWH, ULMWH, bioheparins and unique
oligosaccharides
• Newer anticoagulants
The production of heparin requires that it be extracted from the
source tissue (mucosa, whole organ), recovered from the
reaction mixture and subsequently purified.
Most commonly, tissue is digested using alkaline pH and/or
proteolytic enzymes.
– Alternately, autolysis or a salt extraction can be performed.
Anion exchange resin is added to the digestate to adsorb
heparin and like GAGs.
Sodium chloride solutions are used to elute the crude heparin
from the resin.
Crude heparin is then purified using filtration and oxidation to
remove potential contaminants (proteins, peptids, amino
acids, nucleutides)
Structural modification occurs around the cleavage point of
heparin
Chemical or Enzymatic - elimination reactions
Double bond formation at the non-reducing end for enoxaparin,
bemiparin, tinzaparin.
• 2-O desulfation:
– In enoxaparin process 2-O desulfation occurs in the same way than
previously developed for heparin.
• Epimerisation in mannosamine
• Mechanism:
– Epimerization reach rapidly a thermodynamic equilibrium which is
dependent of reaction condition (eg: solvent nature, strength of the base).
– In enoxaparin sodium: ~70% glucosamine and 30% mannosamine
FDA Position
Landmarks in the Development of
Heparins
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Biosynthesis of heparin-Lindhal, Hook
Structural activity relationship of heparin – Cifonelli, Barlow
Synthesis of heparin derivatives – Danishefsky
Structural studies on heparin – Dietrich, McDuffie, Perlin, Casu,
Conrad, Choay
Biochemistry and pharmacology of heparin – Jaques, Estes,
Rosenberg, Olivecrona
Discovery of antithrombin – Seeger, Rosenberg
Development of low molecular weight heparins – Lasker, Cifonelli,
Choay, Mardigiuan, Samama, Turpie
Clinical development of heparins – Howell, Wessler, Salzman,
Kakkar, Hirsh, Messmore
The Villa Vigoni GAG symposia era
Heparin
Heparin