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Discover Clarity in Transporters
Transporter Reference Guide
3rd Edition
3rd Ed
with 20ition
FDA & 12
EMA
Guidan
ce
Translating cellular science into human outcomes
with the most definitive assay systems
Transporter
Reference Guide
• Overview
• Intestinal Transporters
• Hepatic Transporters
• Renal Transporters
• Concentration Selection
• FAQs
• Glossary
Exton, PA 19341
Tel: +1 610-280-7300
Scotland, UK
Tel: +44 7403 713697
San Diego, CA 92111
Tel: +1 858-560-9000
absorption.com
© 2012 Absorption Systems
Panama City, Panama
Tel: +1 484-576-6143
Discover Clarity in Transporters:
Transporter Reference Guide
3rd Edition, October 2012
Produced and Published by
Absorption Systems
Exton, Pennsylvania, 19341
+1.610.280.7300
absorption.com
© 2010, 2011, 2012, Absorption Systems
All Rights Reserved.
CellPort Technologies ® is a registered
trademark of Absorption Systems.
Any unauthorized reprint or use of this
material is prohibited. No part of this book
may be reproduced or transmitted in any form
or by any means, electronic or mechanical,
including photocopying, recording, or by any
information storage and retrieval system
without express written permission from
Absorption Systems. Every reasonable
attempt has been made to identify owners
of copyright. Errors or omissions will be
corrected in subsequent editions.
Electronic version available online:
absorption.com/cellport_ebook
Written requests for reprint or other
permission should be emailed to:
marketing@absorption.com
About Absorption Systems
Absorption Systems assists pharmaceutical and medical device
companies in identifying and overcoming ADMET (Absorption,
Distribution, Metabolism, Excretion, and Toxicity) barriers in the
development of drugs and medical devices. The company’s mission is to
continually develop innovative research tools that can be used to accurately
predict human outcomes or to explain unanticipated human outcomes when
they occur. The CellPort Technologies® platform, a suite of human cell-based
test systems for drug transporter characterization, exemplifies Absorption
Systems’ commitment to innovation. The company’s facilities are strategically
located on both US coasts and Panama, and encompass nearly 65,000 sq. ft.,
servicing hundreds of customers throughout the world. For more information
on the company’s comprehensive contract services and applied research
programs, please visit absorption.com.
Preclinical Services & Capabilities
Lead Optimization
Physicochemical Properties
Permeability
Stability
Metabolism and Transporters
Binding and Distribution
Formulation Assessment
Bioavailability and Exposure | rodent, non-rodent
Candidate Selection
Formulation Development
PK and Biodistribution | multiple species and dose routes
Barriers to Bioavailability
In Situ and Isolated Organ Perfusion | rat: brain, liver, intestine
Ex Vivo Tissue Permeability | intestinal, dermal, ocular, buccal, nasal, vaginal
IND- and NDA-Enabling
Transporter-Based Drug Interactions
BCS Permeability and Solubility
Classification for Biowaivers
Metabolism-Based Drug Interactions
Metabolite ID and Production
Medical Device Testing
Toxicology
Bioanalysis
Bioanalytical Support from Discovery through Clinical
GLP and Non-GLP
Services Portfolio for Substrate Assessments
Transporter
CellPort Test System
Caco-2 vs. CPT-B1;
proprietary BCRP knockdown Caco-2 cells
BCRP (ABCG2)
Caco-2; human colon adenocarcinoma
BCRP-MDCK vs. MDCK; canine kidney cells
transfected with human BCRP
Caco-2 vs. CPT-M1;
proprietary MRP2 knockdown Caco-2 cells
ABC
MRP2 (ABCC2)
Caco-2 vs. CPT-P1;
proprietary P-gp knockdown Caco-2 cells
P-gp (MDR1, ABCB1)
SLC
MDR1-MDCK vs. MDCK; canine kidney cells
transfected with human P-gp
4
BSEP (ABCB11)
hBSEP-expressing vesicles; membrane vesicles
derived from recombinant baculovirus-infected
insect cells
OATP1B1 (SLC01B1)
OATP1B1-HEK293; human embryonic kidney cells
transfected with OATP1B1*1A or OATP1B1*1B
OATP1B3 (SLC01B3)
transfected with OATP1B3*1 or OATP1B3*2
PepT1 (SLC15A1)
Caco-2; induced human colon adenocarcinoma
MCT (SLC16A)
OCT2 (SLC22A2)
OCT2-HEK293; human embryonic kidney cells
transfected with OCT2
OAT3 (SLC22A8)
OAT3-HEK293; human embryonic kidney cells
transfected with OAT3
OAT1 (SLC22A6)
OCT1 (SLC22A1)
OATP1A2 (SLC21A3)
OATP1A2-HEK293; human embryonic kidney cells
transfected with OATP1A2
OATP2B1 (SLC02B1)
transfected with OATP2B1
MATE1 (SLC47A1)
MATE1-HEK293; human embryonic kidney cells
transfected with MATE1
MATE2K (SLC47A2)
MATE2K-HEK293; human embryonic kidney cells
transfected with MATE2K
NTCP (SLC10A1)
NTCP-HEK293; human embryonic kidney cells transfected
with NTCP
absorption.com/cellport
© 2012 Absorption Systems. All rights reserved.
CellPort Advantage
• Specific transporter knockdown eliminates dependence on non-specific
chemical inhibitors
• Enables waiver of clinical DDI studies based on drug-specific factors
• Highly sensitive test system
chemical inhibitors
chemical inhibitors
• Highly sensitive test system
• Also predicts intrinsic brain permeation potential
• Inside-out vesicles increase sensitivity of the test system through direct access
to the transporter
•Robust expression of the transporter in vesicles from over-expressing cells
• Prevalent clinically relevant genetic variants
• Highly sensitive test system with induced PepT1 expression
• Optimize oral exposure of peptide pro-drugs
• Optimize oral absorption of carboxylic acids
• Physiologically relevant human kidney cell line • Stably transfected with well-characterized phenotype
• Physiologically relevant human kidney cell line
• Stably transfected with well-characterized phenotype
• Human cell line
• Human cell line
• Human cell line
• Human cell line
5
Services Portfolio for Inhibitor Assessments
Transporter
CellPort Test System
CPT-P1; proprietary P-gp knockdown Caco-2 cells
ABC
BCRP (ABCG2)
BCRP-MDCK; canine kidney cells transfected with
human BCRP
CPT-B1; proprietary BCRP knockdown Caco-2 cells
P-gp (MDR1, ABCB1)
SLC
MDR1-MDCK; canine kidney cells transfected with
human P-gp
6
BSEP (ABCB11)
hBSEP-expressing vesicles; membrane vesicles
derived from recombinant baculovirus-infected
insect cells
OATP1B1 (SLC01B1)
transfected with OATP1B1*1A or OATP1B1*1B
OATP1B3 (SLC01B3)
transfected with OATP1B3*1 or OATP1B3*2
PepT1 (SLC15A1)
Caco-2; induced human colon adenocarcinoma
Amino Acid (ASC
System A&B,0,+ LAT)
OCT2 (SLC22A2)
OAT3 (SLC22A8)
OAT1 (SLC22A6)
OCT1 (SLC22A1)
OATP1A2 (SLC21A3)
OATP1A2-HEK293; human embryonic kidney cells
transfected with OATP1A2
OATP2B1 (SLC02B1)
transfected with OATP2B1
MATE1 (SLC47A1)
MATE1-HEK293; human embryonic kidney cells
transfected with MATE1
MATE2K (SLC47A2)
MATE2K-HEK293; human embryonic kidney cells
transfected with MATE2K
NTCP (SLC10A1)
NTCP-HEK293; human embryonic kidney cells
transfected with NTCP
CellPort Advantage
• More definitive test system with less interference from P-gp
• Specific clinically relevant probe combined with proprietary definitive test system
• Clinically relevant probe
• More definitive test system with less interference from BCRP
• Inside-out vesicles allow low permeability compounds direct access to the
transporter
•Validated using clinically relevant inhibitors
• Highly sensitive test system with induced PepT1 expression
• Optimize oral absorption of amino acid pro-drugs
• Probes established for small, cationic and large neutral amino acids
• Physiologically relevant human kidney cell line • Stably transfected with well-characterized phenotype
• Human cell line
• Human cell line
• Human cell line
• Human cell line
7
Overview
Drug Transporters Are Challenging
Researchers studying drug transporters continue to grapple with a lack
of definitive tools, unlike drug metabolism, which has a plethora of highly
specific pharmacologic reagents and assay systems. Nearly all available
transporter probe substrates and inhibitors interact with multiple transporters.
Some transporters have multiple binding sites, which can result in substratedependent inhibition as has been reported for drug-metabolizing CYP
enzymes and some transporters. Therefore, reliance on probe substrates and
inhibitors can potentially lead to false negatives.
In recognition of this, the new FDA Draft Guidance on drug interaction studies
is less reliant on chemical inhibitors to identify the transporters mediating drug
efflux than was the previous (2006) draft guidance. The new EMA Guideline
even provides a non-inclusive list of assay formats that could be considered:
“(over-)expressing the transporter in vector systems and comparing to normal
vectors, using selective inhibitors to inhibit one specific transporter, inhibiting
transporter expression either by knocking out the gene or through the use
of silencing mRNA, etc”. This acceptance of alternative test systems by both
regulatory agencies demonstrates that the industry is in pursuit of definitive
solutions like CellPort Technologies® to make drugs safer by predicting DDIs
reliably before they happen.
Why Do the FDA and EMA Care about Transporters?
Two reasons: pharmacokinetics and drug interactions. Presumably the latter
is more important. You need to know what potential drug interactions to watch
for and which ones you might have to test for in a clinical trial. For example,
any new drug that inhibits members of the OATP uptake transporter family will
probably have to be tested alongside a statin in a clinical trial.
BCRP
MRP2
Understanding
different phases of
drug transport
and metabolism is
essential for drug
safety evaluation.
Phase 3
Gut Lumen
P-gp
Drug
Phase 0
Drug
Nuclear
Receptors
Phase 2
Metabolism
Phase 1
Metabolism
Adapted from Gut 2003; 52: 1788-95
Portal Blood
9
We Understand What the FDA and EMA Expect
with Regard to In Vitro Transporter Data.
That brings us to CellPort Technologies, the suite of transporter assay
systems from Absorption Systems:
• Unique, patented human cell lines in which we have achieved stable
knockdown of one efflux transporter at a time: BCRP, P-gp, MRP2.
We use them in the vectorial transport format in two different ways:
1. Efflux ratio in control vs. knockdown cells identifies which efflux
transporter(s) handle a test compound.
2. Using the probe substrate, cladribine, in P-gp knockdown cells, we
have a “cleaner” BCRP inhibition assay with less interference by P-gp.
• Human cell lines in which important transporters are over-expressed:
• OATP1B1
• OCT1 • OAT1 • P-gp
• OATP1B3
• OCT2
• OAT3
• BCRP
Why Evaluate Transporters?
Transporters can have an enormous influence on the pharmacokinetics of
drugs that are substrates, either limiting their intestinal absorption, mediating
their excretion via bile or urine, or determining how much gets into the brain,
cancer cells, or placenta. By influencing drug concentration levels in plasma or
at pharmacological sites of action, drug transporters may significantly impact
both safety and efficacy.
10
If your compound is a substrate of an uptake or efflux transporter, it could
be susceptible to drug-drug interactions with co-administered drugs that are
either substrates or inhibitors of the same transporter. This could require dose
adjustment, particularly for a drug with a narrow therapeutic index.
If your compound is a substrate or inhibitor of a transporter, it could alter
the pharmacokinetics of co-administered drugs that are substrates of the
same transporter.
The evaluation of drug transporters is of critical importance in
drug development, and is part of the FDA’s and EMA’s regulatory
review process.
The FDA and EMA now expect to see in vitro data on drug interactions with
transporters in NDA filings. Data from in vitro drug interaction studies can help
determine the likelihood of clinical drug-drug interactions, design appropriate in
vivo drug interaction studies (if necessary), and establish appropriate labeling.
Examples of clinical drug-drug interactions mediated by transporters:
Interacting
Drugs
Affected
Drug
Consequence
Effects on Transporter(s)
Quinidine
Digoxin
Digoxin Exposure 1.7-fold h
P-glycoprotein (P-gp, MDR1)
Inhibition
Rifampin
Digoxin
Digoxin Exposure 30% i
P-gp Induction
Dronedarone
Digoxin
Digoxin Exposure 2.6-fold h
P-gp Inhibition
Probenecid
Cephradine
Cephradine Exposure
3.6-fold h
Organic Anion Transporter
(OAT) Inhibition
Cimetidine
Metformin
Metformin Exposure 1.4-fold h
Organic Cation Transporter
(OCT) Inhibition
Cyclosporine
Rosuvastatin
Rosuvastatin
Exposure 7-fold h
Organic Anion Transporting
Polypeptide (OATP) Inhibition
& Breast Cancer Resistance
Protein (BCRP) Inhibition
Lopinavir/
Ritonavir
Rosuvastatin
Rosuvastatin Exposure 2-fold h OATP Inhibition
Table adapted from “Membrane Transporters in Drug Development: Comment on the Report from the
International Transporter Consortium (ITC)”, Lei Zhang, Ph.D., Office of Clinical Pharmacology, Office of
Translational Sciences; Center for Drug Evaluation and Research, Food and Drug Administration; AAPS
Webinar; July 29, 2010
Besides evaluating liabilities for drug-drug interactions, transporters are
also studied as targets for drug delivery. The broad substrate specificities
of intestinal uptake transporters such as PepT1, OATP2B1, and amino acid
transporters, provide great potential for targeted delivery of amino acid-based
drugs and pro-drugs. See Intestinal Transporters section for additional details.
11
Localization of Human Transporters
Brain
P-gp, BCRP, MRP2,
OATP2B1, ASC/LAT
Liver (Canalicular)
P-gp, BCRP,
MRP2, BSEP
Liver (Sinusoidal)
OCT1, OATP1B1,
OATP1B3, NTCP
Kidney (Basolateral)
OCT2, OAT1, OAT3
Intestinal Lumen
P-gp, BCRP,
MRP2, OCT1,
OATP2B1, PepT1,
ASC/LAT
Kidney (Apical)
P-gp, BCRP, MRP2,
OATP2B1, PepT1,
ASC/LAT, MATE
12
Regulatory Perspective on Transporters
Since the issuance of the new FDA Draft Guidance on Drug Interaction
Studies (February 2012) and European Medicines Agency Guideline on
the Investigation of Drug Interactions (June 2012), it has been made
apparent that these two groups share a much more harmonized view on
what transporters are of greatest importance as related to drug-drug
interactions. Recommendations from these organizations are summarized
in the following table:
P-gp
BCRP
MRP
BSEP
OATP1B1
OATP1B3
OCT1
OCT2
OAT1
OAT3
MATE1/2
FDA Guidance
2012
EMA Guideline
2012
a
a
a
a*
a
a
a
a*
a
a
a
a*
a
a
a*
a*
a
a
a
a
Absorption Systems
Portfolio
a
a
a
a
a
a
a
a
a
a
a
*When appropriate
13
Substrate Decision Tree
TheFDAandEMArecommendevaluationofallNCEsassubstratesofat
leasttwotransporters:
All NCEs
Determine whether
NME is a P-gp
and/or BCRP
substrate in vitro
Hepatic or biliary
secretion major?
e.g., ≥25% of total
clearance or unknown?
Renal active secretion
major? e.g., ≥25%
of total clearance
or unknown?
Determine whether
NME is an OATP1B1
and/or OATP1B3
substrate in vitro
Determine whether
NME is an OAT1,
OAT3 and/or OCT2
substrate in vitro
Othertransporters(e.g.MRP2)mayneedtobestudiedbasedonknowledgeof
otherdrugsinthesametherapeuticclass.
Inhibitor Decision Tree
TheFDAandEMArecommendevaluationofallNCEsforinhibitionofatleast
fourtransporters:
All NCEs
P-gp and
BCRP
inhibitor
evaluation
OATP1B1
and OATP1B3
inhibitor
evaluation
OAT1, OAT3,
and OCT2
inhibitor
evaluation
Inhibitor evaluation
for other transporters
(i.e. BSEP, OCT1, MATEs)
when appropriate
Adapted from: FDA Draft Guidance on Drug Interaction Studies. February 2012.
EMA Guidlines on the Investigation of Drug Interactions. June 2012.
14
©2012AbsorptionSystems.Allrightsreserved.
Which Transporter and When?
Lead
Generation
Lead
Optimization
Candidate
Selection
Toxicology
& Safety
FTIH to
Phase II
P-gp (ABCB1)
OAT1 (SLC22A6)
BCRP (ABCG2)
OAT3 (SLC22A8
OATP1B1 (SLCO1B1)
OCT1 (SLC22A1)
OATP1B3 (SLCO1B3)
OCT2 (SLC22A2)
Phase III
& Final
Launch
BSEP
Adapted from presentation by Joseph Polli and Caroline Lee
General considerations for which transporters to evaluate and when
should include: therapeutic area (potential co-medications), product profile,
development plan, clearance route (hepatic vs. renal), chemical structure,
and physicochemical properties.
Because it is recommended to evaluate all NCEs as inhibitors of P-gp, BCRP,
OATP1B1, OATP1B3, OAT1, OAT3, and OCT2, and as substrates of P-gp and
BCRP, it makes sense to investigate these transporters relatively early in drug
development. Knowing if your compound is a potential substrate or inhibitor of
one of these transporters can help you plan clinical studies down the road.
If there is evidence of significant hepatic/biliary clearance, then substrate
assessment of OATPs should be performed. Similarly, if renal clearance is
important, then OAT and OCT substrate interaction should be assessed.
Depending on the cholestatic potential of the compound, it may be important
to evaluate BSEP.
If your compound is found to interact with one or more transporters, there
may be implications for drug labeling and additional studies (clinical studies
or non-clinical mechanistic studies) may be required.
Based on drug-specific properties (solubility, permeability, metabolism), it
may be possible to waive clinical DDI studies, even if your compound is a
transporter substrate. Find more information on biowaivers in the intestinal
transporters section.
15
Tools for Investigating Transporters
Methodsforelucidatingtransporterinteractionsinclude:
Clinical Methods
Non Clinical Methods
•ComparativePK
•ATPaseActivity
•PolymorphicVariants
•Cell-basedAssays
•KnockoutAnimals
•Vesicles
AshighlightedbytheFDA’sCriticalPathInitiative,in vitrostudiesarepartof
anintegratedapproachtoreduceunnecessaryclinicalstudies.
“Inmanycases,negativefindingsfromearlyin vitro
andearlyclinicalstudiescaneliminatetheneedforlater
clinicalinvestigationsofdrug-druginteractionpotential.”
FDA Draft Guidance, Feb. 2012
Whilenegativein vitroresultscanprecludetheneedforclinicaldrug-drug
interaction(DDI)studies,insomecasesitisalsopossibletowaiveclinical
studiesforcompoundsthatdointeractwithtransporters(findmoreinformation
onbiowaiversintheintestinaltransporterssection).IfclinicalDDIstudies
arerequired,thenin vitroresultsmayserveasthebasisfordesignofthose
studies.Similarly,theresultsofclinicalDDIstudieshelptoexplainandrefine
predictivein vitromodelsortools.
In Vitro
Model/Tools
Explain
In Vivo
DDI Studies
Predict
Adapted from “Transporter-Mediated Drug-Drug Interactions
(DDIs)”, Lei Zhang, Ph.D., CDER, FDA, Clinical Pharmacology
Advisory Committee Meeting, Atlanta, GA, March 17, 2010.
16
For efflux transporter interaction assessments, Absorption Systems uses both
unidirectional (basolateral-to-apical) and bidirectional assay formats specified
by the FDA, as illustrated below. Each assay plate typically contains 12 or 24
dual assay chambers. Cells are cultured on polycarbonate filters until they
reach confluence and differentiate to form a confluent monolayer. At that time,
a sample of the monolayers is tested for barrier properties and transporter
activity as a quality control measure for the batch.
Apical Chamber
Basolateral Chamber
Filter
Cell Monolayer
In addition to Caco-2, other polarized epithelial cell lines, such as knockdown
Caco-2 lines, MDR1-transfected MDCK, BCRP-transfected MDCK, and MDCK,
are used in a similar assay format. All of Absorption Systems’ cell lines are
extensively characterized and validated to ensure rugged, reproducible results.
17
During efflux transporter substrate assessments, the test compound is
added to the buffer solution of the apical chamber for apical-to-basolateral
(A¨B) transport assessment (the physiological absorptive direction) or to the
basolateral chamber for basolateral-to-apical (BÄ) transport assessment
(the efflux or secretory direction).
The rate of transport or flux is divided by the drug concentration in the dosing
chamber to derive an apparent permeability (Papp) value. If a compound is
passively transported, the Papp values measured in either direction should be
approximately equal and the efflux ratio (the BÄ Papp divided by the A¨B
Papp) should be close to 1.
•Efflux ratios > 2 suggest the
cells are actively secreting the
drug into the apical chamber.
Papp =
dCr /dt x vr
A x Co
•Efflux ratios < 0.5 suggest
active absorption of the drug
from the apical chamber.
ER =
Papp (BÄ)
Papp (A¨B)
•When two cell lines are
evaluated in parallel (such
as wild-type and transporter
knockdown cells), the relative
efflux ratio (RER) is calculated.
RER =
RWT
RKD
For inhibition evaluation with efflux transporters, the unidirectional (basolateralto-apical) or bidirectional permeability of a probe substrate is evaluated in
the presence and absence of a test compound. The following equations are
applicable when the bidirectional format is used:
Efflux Ratio (ER) =
Papp (BÄ)
Papp (A¨B)
Papp (BÄ)
Corrected Efflux
=
-1
Ratio (CER )
Papp (A¨B)
18
If the corrected efflux ratio of the probe substrate (CER) is reduced by more
than 50% in the presence of the highest concentration of the test compound
(CER/TC), then the compound is likely an inhibitor, and an IC50 evaluation should
proceed. For example, for P-gp inhibition assessment, the corrected efflux
ratio of digoxin (CERD) is evaluated:
(CERD - CERD/TC )
x 100 ≥ 50%
CERD
For uptake transporter interaction assessments, cellular uptake is evaluated in
24-well plates. For substrate evaluation, buffer containing the test compound
is added to the well and incubated for various durations. At a given time point,
dosing solution is aspirated, the cells are washed, and lysis buffer is used to
collect the cells from the well. The influx rate (IR) of the test compound into the
cells is evaluated. Influx rate is the concentration of test compound normalized
to protein content and incubation time. The influx rate ratio (IRR) is then
calculated by comparing uptake in the transfected and vector control cell lines:
Influx Rate Ratio (IRR) =
IRTF
IRVC
• IRR values > 2.0 indicate that the compound is being actively taken up
into the cells by the uptake transporter of interest
• Substrate classification is further confirmed by a challenge with an
inhibitor at a single time point. Inhibition >50% supports a positive
substrate classification
IR: Influx rate (concentration of probe substrate normalized to protein content)
TF: Transfected Cells
VC: Vector Control Cells
19
For inhibitor evaluation, the format is similar to that used for substrate
assessment. In inhibition assessments, however, a single duration is used
that has been optimized for each probe substrate. The IR of a probe substrate
is evaluated in the presence and absence of a test compound. Net influx rate
(NIR) and percent inhibition are calculated:
Net Influx Rate (NIR) = IRTF - IR vc
Percent Inhibition = 1-
NIRTC
NIRw/outTC
x 100
• Inhibition >50% suggests that the test compound is an inhibitor of the
uptake transporter
IR: Influx rate (concentration of probe substrate normalized to protein content)
TF: Transfected Cells
VC: Vector Control Cells
TC: Test Compound
20
CellPort Technologies:
Innovative Solution for a More Definitive Assay System
We selected a human cell line (Caco-2, C2BBE1 clone) expressing the
efflux transporters P-gp, BCRP, and MRP2, and have knocked down the
expression of one transporter at a time. This was achieved using short
hairpin RNA (shRNA) in a lentiviral vector, an approach that is capable
of producing stable phenotypes.
Lentiviral Particle
Nucleus
mRNA
Dicer RNase III
mRNA Cleavage
Suppression
siRNA
RISC
siRNA
Targeting
Our cell lines for evaluation of hepatic and renal uptake transporters were
created by stable transfection of transporter genes into HEK293 cells. For
initial set-up, stable transfections are more laborious and challenging than
transient transfections. However, they result in permanent insertion into the
genome, allowing us to fully characterize the phenotype of our cells. You can
benefit from Absorption Systems’ well-characterized, stable cell lines, which
are ready to go.
All of Absorption Systems’ cell lines have been
extensively characterized and validated:
Molecular Biology for Phenotypic Expression
• Quantitative PCR for mRNA expression
• Western blot for protein expression
• Immunofluorescence for protein localization
Assay Window and Phenotype Stability
• Growth rate
• Passage number
• Days in culture
• Assay window
Appropriate and Robust Functional Expression
• Bidirectional transport and/or uptake
• Polarization and barrier properties
21
Our CellPort Analytics™ database captures up to ten years of data for our
cell lines. This tool helps us ensure our cell lines are behaving optimally,
according to stringent criteria we’ve established based on extensive
knowledge and experience.
Knockdown cell lines can be evaluated in different ways. For example, as a
standalone model the P-gp knockdown is a cleaner BCRP assay system. Or,
when used in combination with control cells, it can identify P-gp substrates.
P-gp
WT
BCRP
P-gp-KD
MRP2
BCRP-KD
VS
VS
VS
22
MRP2-KD
Absorption Systems Has the Tools You Need.
Absorption Systems offers validated assays for investigating all of the
transporters recommended by the FDA and the EMA. We also offer an
array of other transporters, such as PepT1, OATP2B1, MCT, NTCP,
and amino acid transporters.
Drug Safety
Absorption Systems
Portfolio
P-gp
BCRP
MRP2
BSEP
OATP1B1
OATP1B3
OCT1
OCT2
OAT1
OAT3
MATE1
MATE2K
a
a
a
a
a
a
a
a
a
a
a
a
Drug Delivery
PepT1
Amino Acid
OATP1A2
OATP2B1
a
a
a
a
Radiolabel and fluorescent probe substrates are available
23
Intestinal
Transporters
Overview
Intestinal Transporters
P-gp
BCRP
MRP3
MRP2
ASBT
PepT1
OATP
MCT1
OST
Blood
OCT1
Intestine
ASC/LAT
Due to ubiquitous expression of P-gp (MDR1, ABCB1) and BCRP (ABCG2),
the current FDA and EMA guidelines recommend evaluation of interaction
(both substrate and inhibitor assessment) with these efflux transporters.
25
Test Systems for Intestinal Efflux Transporters
For evaluation of intestinal efflux transporters, CellPort Technologies is an
optimal test system. CellPort is an innovative solution that provides a more
definitive assay system. Using shRNA-mediated interference in a lentiviral
vector, efflux transporters BCRP, P-gp, and MRP2 were individually and
stably knocked down in human Caco-2 cells.
CPT-B1 = CellPort BCRP knockdown
CPT-P1 = CellPort P-gp knockdown
CPT-M1 = CellPort MRP2 knockdown
Transporter Knockdown is Selective and Effective
Based on quantitative PCR, knockdown of targeted transporter is specific
and significant, while the expression of other transporters is maintained at
normal levels.
mRNA Expression Relative
to Parental Caco-2
1.2
0.8
0.6
0.4
0.2
0.0
26
■ CPT-M1
■ CPT-B1
■ CPT-P1
1.0
MRP2
P-gp
BCRP
Fluorescent (green) antibody is specific for the cell-surface protein, BCRP.
Nuclei are stained blue with DAPI. BCRP is clearly knocked down in CPT-B1.
Fluorescence
in parental Caco-2
Fluorescence
in vector control
No green fluorescence
in CPT-B1
Similar immunofluorescence images demonstrate knockdown of P-gp in
CPT-P1 cells.
Parental Caco-2
CPT-P1
% of Efflux Ratio vs. Caco-2
Bidirectional permeability of probe substrates demonstrates appropriate
knockdown of transporter function.
140
130
120
110
100
90
80
70
60
50
40
30
20
10
0
■ CPT-M1
■ CPT-B1
■ CPT-P1
E3S
Digoxin
27
Functional data verify comparable BCRP function, P-gp function, and barrier
properties between parental Caco-2 cells and vector control cells.
Transport Properties
Vector Control
Parental Caco-2
Estrone-3-Sulfate (E3S) Efflux
E3S A¨B Papp
0.74
0.63
E3S BÄ Papp
10.36
10.69
14
17
Digoxin A¨B Papp
0.33
0.20
Digoxin BÄ Papp
8.24
3.69
25
19
E3S Efflux Ratio
Digoxin Efflux
Digoxin Efflux Ratio
Barrier properties
Atenolol A¨B Papp
0.39
0.35
Propranolol A¨B Papp
13.88
22.4
446
477
TEER
Characteristics of Knockdown Cells
Typical data for CPT-B1, CPT-P1, and vector control cells are shown below:
Control
CPT-B1
Digoxin Efflux Ratio
18.65
21.50
CPT-P1
4.99
Estrone-3-sulfate (E3S) Efflux Ratio
25.58
2.60
40.21
TEER
477
556
543
Atenolol A¨B Papp
0.13
0.41
0.33
Propranolol A¨B Papp
17.2
23.2
17.23
CPT-B1:
1. low E3S efflux (i.e., low BCRP function)
2. remaining E3S transport completely inhibited by the selective
BCRP inhibitor FTC
3. high digoxin efflux (i.e., normal P-gp function)
4. good barrier properties.
CPT-P1:
1. low digoxin efflux ratio (i.e., P-gp function)
2. high E3S efflux (i.e., normal BCRP function)
3. good barrier properties.
28
Caco-2 Validation and Ruggedness Testing
Based on our validation data, Caco-2 cell monolayers used between
days 21 and 28 post-seeding and seeded between passages 58 and 76
are suitable for testing.
Our validation of Caco-2 cells included an evaluation of digoxin efflux
ratios in the presence and absence of cyclosporine A (CsA) to assess P-gp
function. Digoxin is a known substrate of P-gp and CsA is a known inhibitor.
Monolayers were tested for digoxin efflux in bidirectional permeability assays
on multiple batches of cells at different passage numbers and days in culture.
Digoxin exhibited an efflux ratio >10, which was reduced to approximately 1 in
the presence of CsA. These results confirm the presence of P-gp-mediated
efflux activity in Caco-2 cells 21 to 28 days after seeding. The efflux function
of P-gp was consistently observed throughout the tested range of passages.
Prior to an assay, each batch of cells undergoes quality control testing.
The permeabilities of control compounds digoxin, E3S, atenolol (low
permeability marker), propranolol (high permeability marker), and lucifer
yellow are measured. Each monolayer used in a transport experiment is
verified by a pre-experiment TEER measurement and by calculating the Papp
of a monolayer integrity marker compound after the experiment.
Caco-2 Cell Batch Certification Criteria
Atenolol Papp
≤ 0.5
Propranolol Papp
10 – 30
Lucifer Yellow Papp
≤ 0.4
Digoxin Ratio: Papp (BÄ) / Papp (A¨B)
> 10
E3S Ratio: Papp (BÄ) / Papp (A¨B)
≥ 25
Pre-Experiment TEER Value
Post-Experiment Lucifer Yellow Papp
450 – 650
≤ 0.8
29
MDR1-MDCK Validation and Ruggedness Testing
Expression of human and canine P-gp in MDR1-MDCK cells and parental
MDCK cells as a function of cell passage number (from 10 to 50) was
characterized and verified by Western blot, RT-PCR, and sequence analysis.
The expression levels of human and canine P-gp were similar between
passages 10 to 50 in MDR1-MDCK cells as was the expression of canine
P-gp in parental MDCK cells.
P15 P15’ P25 P25’ P35 P35’ P45 P45’ (-) (+)
Fig.1 Effect of cell passage (P) on expression of human P-gp in MDR1-MDCK with colchicine and
without colchicine (’). (-) negative control MDCKII cells, and (+) positive control purified human
P-gp protein control. An immunoblot analysis of human P-gp protein expression (20 μg protein/
lane) was done by using a species-specific monoclonal antibody for human P-gp.
Canine P-gp was detected in both parental MDCK and MDR1-MDCK
cells. There was no apparent difference in canine P-gp mRNA expression
levels between the two cell lines. As a test for efflux pump function, the
permeabilities of rhodamine123 (Rh123) and digoxin were similar in cells
from different passages and at different days after seeding.
Protein and mRNA levels for human P-gp in MDR1-MDCK cells were similar
across different passages. Results using known P-gp inhibitors correlated
with the rank order of their P-gp affinities. The data demonstrate that the
MDR1-MDCK cell line is a rugged in vitro model for P-gp interaction assays.
30
Cells between 7 and 11 days of growth are used for transport assays. The
quality of every batch of monolayers is certified using control compounds
prior to release for use. Each monolayer used in a transport experiment is
verified by a pre-experiment TEER measurement and by calculating the
Papp of a monolayer integrity marker compound after the experiment.
MDR1-MDCK Cell Batch Certification Criteria
Atenolol Papp
< 0.5
Propranolol Papp
10-30
Lucifer Yellow Papp
< 0.4
Digoxin Papp A¨B
< 0.1
>100
Pre-Experiment TEER Value ≥1400
≤ 0.8
MDCK Cell Batch Certification Criteria
Atenolol Papp
< 0.5
Propranolol Papp
10-30
Lucifer Yellow Papp
< 0.4
10 to 50
Pre-Experiment TEER Value
≥1400
≤ 0.8
IC50 Evaluation in Validated Test Systems
A series of known P-gp inhibitors with varying potencies was tested using
several validated cell-based test systems and IC50 values were determined.
The commonly used P-gp inhibitors CsA, ketoconazole, GF120918, and
verapamil were evaluated in Caco-2, MDR1-MDCK, and MDCK cell
monolayers with digoxin as the probe substrate. IC50 values were calculated
according to six different methods to identify the least variable approach,
used to establish our internal protocol. While the sensitivities of the cell lines
varied due to P-gp expression levels, the rank order of inhibitor potency was
maintained and was reproducible.
31
Current Challenges
A multitude of challenges exist with current in vitro test systems. When cells
express multiple efflux transporters, identifying which transporter mediates
drug efflux is difficult. Interpreting results from non-human cells in which a
human transporter is over-expressed is complicated by the background of a
non-human version of the same transporter.
Chemical Inhibitors Lack Specificity
Almost all known chemical inhibitors of drug transporters interact with
multiple transporters. Non-specific pharmacologic probes can only confirm
the involvement of a transporter but cannot identify which one is responsible
for efflux of your test compound.
The Best Solution to Overcome Today’s Challenges
CellPort Technologies has an innovative solution for efflux with a more
definitive assay system. CellPort is an innovative technology funded by FDA
grants to provide more definitive transporter data. By selective knockdown of
specific efflux transporters, CellPort eliminates dependence on non-selective
chemical inhibitors.
Current Challenges
Collaborator
Test systems in which transporters are
over-expressed can lead to artifacts
Chemical inhibitors lack specificity and
require optimization
Transporter phenotyping: Human cell lines
express multiple efflux transporters
Promiscuous probe substrates make it
difficult to assess inhibition potential
32
The Absorption Systems Advantage
Not all in vitro cell systems are ideal for in vitro drug investigations.
Absorption Systems offers a variety of different cell models to best mimic
the target in vivo site. Over-expressing cell lines may exaggerate drug-drug
transporter interactions. However, if the drug targets a tissue type that
over-expresses certain transporters, these cell lines may be ideal. This
can also provide valuable information regarding SAR. By having the ability
to run several well-characterized in vitro systems in parallel, Absorption
Systems is better equipped to predict in vivo outcomes.
Exaggerated
Response
80
Relative Efflux Ratio (RER)
40
Caco-2 vs CPT-P1
MDR1-MDCK vs MDCK
20
Exaggerated
Response
15
10
Positive
Classification
5
0
Negative
Classification
Digoxin
Atenolol
False
Negative
Fexofenadine Loperamide
Labetalol
33
Selecting the appropriate in vitro tool is critical for predicting and/or explaining
clinical drug-drug interactions. AstraZeneca’s thrombin inhibitor, ximelagatran,
is a pro-drug that is metabolized to the active drug, melagatran, in the liver.
Both pro-drugs and drugs are actively secreted into bile. After the drug was
approved, a clinical drug interaction with erythromycin became evident, which
was unanticipated based on in vitro CYP metabolism studies.
To investigate this phenomenon, bidirectional permeability of ximelagatran
was assessed across Caco-2, CPT-P1 (P-gp knockdown), and CPT-M1
(MRP2 knockdown) cell monolayers. Intracellular accumulation of
ximelagatran and its metabolites was also measured.
Effects of P-gp and MRP2 Knockdown
on Intracellular Accumulation of Melagatran
4000
Melagatran (pmol)
3000
n AgB
n B gA
2000
1000
0
Vector Control
Ximel
OH-mel
Et-mel
Melagatran
Digoxin
P-gp Knockdown
Caco-2
47
7
NA
97
36
MRP2 Knockdown
Efflux Ratio
CPT-P1
CPT-M1
5
42
1
10
NA
NA
6
96
7
39
As highlighted above, the efflux ratio of ximelagatran and its metabolites were
all substantially reduced in P-gp knockdown cells but not in MRP2 knockdown
cells, indicating that ximelagatran and melagatran are substrates of P-gp.
This fact explains the observed clinical interaction with erythromycin, a known
inhibitor of P-gp.
With multiple transporters present and the ability to generate metabolites inside
the cell, CellPort provided an ideal platform to investigate this phenomenon.
34
Waiving Clinical Studies
Class II
Class I
Ketoprofen
Naproxen
Carbamazepine
Class IV
Propranolol
Verapamil
Metoprolol
Class III
Furosemide
Hydrochlorothiazide
Low
Per meab il it y
High
Translating Cellular Science into Human Outcomes
Absorption Systems’ Caco-2 cells are uniquely validated for both transporter
interaction assessment and BCS classification. This test system may allow
you to waive clinical DDI studies and/or human BA/BE studies based on
certain drug-specific properties (high solubility, high permeability, high
metabolism). Only Absorption Systems’ validated Caco-2 system provides
the most accurate threshold for permeability classification.
Ranitidine
Cimetidine
Atenolol
Vancomycin
Solubility
Low
High
35
We Can Make In Vivo Interaction Studies Unnecessary.
According to the 2000 FDA BCS Guidance, compounds that are classified
as Class I (highly soluble, highly permeable) are eligible for BCS biowaivers.
For such compounds, the rate and extent of drug absorption is unlikely
to be affected by drug dissolution and/or GI residence time, and in vivo
bioequivalence studies (for new formulations, etc.) may be waived based on
in vitro permeability and solubility data.
Furthermore, for BCS Class I compounds, it is unlikely that absorption will be
limited by efflux transporters. Thus, it may be possible to waive clinical DDI
studies as well:
“If in vitro experiments demonstrate that an NME is a
P-gp substrate, additional drug-specific factors may
be considered before determining whether an in vivo
drug interaction study is warranted. For example, the
bioavailability of the BCS Class I or BDDCS Class I
NMEs that are highly soluble, highly permeable, and
highly metabolized may not be significantly affected by
a co-administered drug that is a P-gp inhibitor, and thus,
an in vivo interaction study may not be needed.”
L. Zhang et al., “Predicting Drug-Drug Interactions: An FDA Perspective” (2009)
The AAPS Journal
Absorption Systems has conducted over 125 in vitro BCS studies for
classification and biowaivers over the last 10 years.
Absorption Systems BCS Studies by Year
2011
60
55
50
45
40
2010
2008
2009
2007
2006
2005
2004
2002
36
2003
35
30
25
20
15
10
5
0
Asdemonstratedbyourvalidationdata,Caco-2accuratelypredictshuman
intestinalabsorption.Caco-2Pappvaluescorrelatewithhumanfraction
absorbeddatafromtheliterature(seebelow).Forthisreason,in vitroCaco-2
testingisareliablesurrogateforin vivobioequivalencestudies.Infact,more
than90%ofthenon-clinicaldatasubmittedandacceptedforbiowaivers
isbasedonCaco-2.Caco-2testingisasprevalentashumanPKdatafor
biowaiversubmissions.
Human Fab vs. Caco-2 AgB P for 61 compounds
% fraction absorbed orally in humans (literature data)
app
100
90
80
Highly Absorbed
70
60
50
40
30
20
10
I
0I
I
I
0.1
1
10
100
Caco-2 AgB Papp (x10-6cm/s, log scale)
37
Three Steps to BCS
Our validated 3-step study design allows
for early identification of BCS biowaiver
candidates and optimization of protocol
elements. These steps include:
1.Pre-qualification and determination of
the eligibility of test compound for BCS
biowaiver. Your go/no-go decision point.
2.Protocol optimization and conduct of
FDA-required experiments to establish
protocol for pivotal studies.
3.GLP BCS classification of permeability
and/or solubility.
Our Uniquely Validated Caco-2 Cells May Allow
You to Waive Clinical P-gp/BCRP Studies and/or
Human BA/BE Studies.
Redefining the threshold for permeability classification
The threshold defined by Absorption Systems’ High Permeability Internal
Standard (HPIS) is the lowest available. This novel internal standard has a
Papp value about 7-fold lower than metoprolol and fraction absorbed > 95% in
humans. Multiple biowaivers have been accepted using this novel HPIS.
Caco-2 Papp (x 10-6 cm/s)
100
10
Antipyrine:
Metoprolol:
Pindolol:
Papp ~63
Papp ~28
Papp ~17
Minoxidil:
Papp ~4
1
Our High Permeability
Internal Standard
(HPIS) identifies high
permeability at a
lower threshold.
0.1
0.01
High
38
Low
Translating Cellular Science into Human Outcomes
Here’s an example of how and which clinical studies you could potentially
waive by using Absorption Systems’ dually validated Caco-2 test system:
P-gp Interaction
Study
BCRP Interaction
Study
Substrate Assessment
• Caco-2 vs. CPT-P1
• Caco-2 vs. CPT-B1
Inhibition Assessment
• Caco-2
• Probe Substrate: Digoxin
• CPT-P1
• Probe Substrate: Cladribine
BCS Classification Study
• Caco-2
BCS Classification Study
• Caco-2 Cells
Possibly Waive
• Clinical P-gp Study
• Human BA/BE
Possibly Waive
• Clinical BCRP Study
• Human BA/BE
39
A Decade of BCS History
Our extensive experience continues to facilitate innovative solutions and
enable in vitro classification for a broader range of drugs. This, combined with
our 10-day turnaround for pre-qualification and our cost-effective designs,
saves you time and money. Just a few reasons why Absorption Systems
averages more than 30 BCS studies each year.
January 2011
May 2010
2009
December 2008
July 2008
• Present educational seminar to FDA new drug reviewers
• FDA recommends biowaivers for cytotoxic drugs
and highly variable drugs
• Present educational seminar to FDA generic drug reviewers
• FDA inspection results in ANDA pre-qualification
2007
• FDA audits again to confirm choice of HPIS
2006
• Addition of pH 6.5 option and introduction of new HPIS
2003 - 2005
Summer 2002
Fall 2001
Spring 2001
August 2000
40
• FDA audits again
• Cross-validate assay based on customer and FDA feedback
• Our first BCS customer receives biowaiver approval
• FDA audits first BCS study conducted at Absorption Systems
• Conduct first customer-sponsored BCS study
• Validate Caco-2 for BCS
• FDA issues BCS Guidance
Test Systems for Intestinal Uptake Transporters
Transporters for peptides, amino acids, monocarboxylic acids, and vitamins
are often exploited for targeted drug delivery. The most well-studied intestinal
uptake transporter, PepT1, regulates transport of protein digestion products
(di- and tri-peptides), as well as peptidomimetic drugs (β-lactam antibiotics,
renin inhibitors, and nonpeptide pro-drugs). Induced PepT1 expression is
observed in patients with ulcerative colitis, Crohn’s disease, and short-bowel
syndrome. PepT1 expression is also modified as a result of nutritional and
metabolic alterations.
Another important class of intestinal uptake transporters is the amino acid
uptake transporters. In addition to naturally occurring amino acids, these
transporters are known to transport drugs such as L-DOPA, melphalan,
triiodothyronine, thyroxine, gapabentin, and valacyclovir. They are classified
on the basis of their functional differences:
Amino Acid Uptake Transporter
Characteristics
Substrate
Na+-dependent ASC, System A
Ala-preferring, small neutral
amino acids
Alanine
Na+-dependent ASC, System B0,+
Neutral and cationic amino
acids preferring
Arginine
Na+-independent LAT
Large neutral amino acids
preferring
Phenylalanine
The broad substrate specificity of these uptake transporters provides great
potential for targeted drug delivery of amino acid-based drugs and pro-drugs.
For example, valacylovir is a pro-drug formed by esterifying the 3-hydroxyl
group of acyclovir with L-valine. Valacyclovir increases the oral bioavailability
of acyclovir (an antiviral nucleoside) 3-5 fold. Its uptake is mediated by the
PepT1 and amino acid B0,+ transporters.
Caco-2 cells can be used to evaluate these transporters as, upon
differentiation, these cells are morphologically and functionally similar to the
absorptive epithelial cells of the small intestine. Absorption Systems has
optimized the induction of PepT1 via GlySar teatment. We’ve shown this
induction to increase uptake of PepT1 substrates like cephalexin by ~90%.
Ref: Katragadda S et al., International Journal of Pharmaceutics 362 (2008) 93-101
Bhardwaj R et al., JPET 314 (2005) 1093-1100. Adibi S, AJPGI 285 (2003) G779-G788
41
Transporter Reference Card: P-gp
Transporter: P-gp (P-glycoprotein, MDR1, ABCB1)
Key Facts: Most-studied transporter; suggested for evaluation by FDA
and EMA; ABC efflux transporter
Localization: Ubiquitous (intestine, kidney, liver, etc.)
CellPort Advantage:
• Stable P-gp knockdown eliminates dependence on promiscuous
• Transfected MDCK is a highly sensitive test system.
• Provides the potential to waive clinical DDI studies based on drugspecific factors. Only Absorption Systems’ validated Caco-2 system
provides the most accurate threshold for permeability class
(see section on waiving clinical studies).
Substrate Evaluation
Recommended for all NCEs
Caco-2 vs. CPT-P1 (P-gp knockdown)
Caco-2 +/- chemical inhibitors
Test Systems Available
MDR1-MDCK vs. MDCK
MDR1-MDCK +/- chemical inhibitors
CPT-B1 (BCRP knockdown) +/- chemical inhibitors
Assay Type
Bidirectional permeability of test compound at
multiple concentrations
Positive Control Substrate
Digoxin
Chemical Inhibitors
CsA or ketoconazole
Inhibitor Evaluation
CPT-B1 (BCRP knockdown)
Caco-2
MDR1-MDCK
42
Assay Type
Unidirectional or bidirectional permeability of probe
substrate +/- test compound; IC50 evaluation when
applicable
Probe Substrate
Digoxin or 3H-digoxin
Positive Control Inhibitors
CsA or ketoconazole
Transporter Reference Card: BCRP
Transporter: BCRP (Breast Cancer Resistance Protein, ABCG2)
Key Facts: Suggested for evaluation by FDA and EMA;
ABC efflux transporter
CellPort Advantage:
•Stable BCRP knockdown eliminates dependence on promiscuous
• Transfected MDCK is a highly sensitive test system. •Provides the potential to waive clinical DDI studies based on drugspecific factors. Only Absorption Systems’ validated Caco-2 system
provides the most accurate threshold for permeability class
(see section on waiving clinical studies).
Caco-2 vs. CPT-B1 (BCRP knockdown)
CPT-P1 (P-gp knockdown) +/- chemical inhibitors
BCRP-MDCK vs. MDCK
BCRP-MDCK +/- chemical inhibitors
Assay Type
LC-MS/MS: Cladribine
Fluorescent: Pheophorbide A
Chemical Inhibitors
Ko143 or FTC
CPT-P1 (P-gp knockdown)
Caco-2
BCRP-MDCK
Assay Type
Unidirectional or bidirectional permeability of probe
substrate +/- test compound; IC50 when applicable
Probe Substrate
LC-MS/MS: Cladribine
Fluorescent: Pheophorbide A
Ko143 or FTC
43
Transporter Reference Card: MRP2
Transporter: MRP2 (Multidrug Resistance Protein, ABC2)
Key Facts: ABC efflux transporter
CellPort Advantage:
•Stable MRP2 knockdown eliminates dependence on promiscuous
•Provides the potential to waive clinical DDI studies based on drugspecific factors. Only Absorption Systems’ validated Caco-2 system
provides the most accurate threshold for permeability class (see section
on waiving clinical studies).
Caco-2 vs. CPT-M1 (MRP2 knockdown)
CPT-P1 (P-gp knockdown) +/- chemical inhibitors
Assay Type
CDCFDA
Chemical Inhibitor
MK571
44
Caco-2
Assay Type
Unidirectional or bidirectional permeability of
probe substrate +/- test compound; IC50 evaluation
when applicable
Probe Substrate
CDCFDA
Positive Control Inhibitor
MK571
Transporter Reference Card: PepT1
Transporter: PepT1 (peptide transporter, SLC15A1)
Key Facts: Proton-dependent, oligopeptide solute carrier
Localization: Intestinal brush border
CellPort Advantage:
•Highly sensitive test system with induced PepT1 expression
•Caco-2 is a human cell line; no interference from animal transporters.
•Optimize absorption of peptide pro-drugs.
Recommended for amino acids,
peptides, and pro-drugs
Caco-2 cells induced with 48-hour GlySar treatment
Assay Type
Unidirectional (AgB) permeability assessment of
test compound +/- GlySar with pH gradient; optional
evaluation of concentration dependence, induced vs.
wild-type cells
Valacyclovir
Chemical Inhibitor
GlySar
Recommended for amino acids,
peptides, and pro-drugs
Caco-2 cells induced with 48-hour GlySar treatment Assay Type
Unidirectional (AgB) permeability assessment of probe
substrate +/- test compound with pH gradient
Probe Substrate
Valacyclovir Positive Control Inhibitor
GlySar
45
Transporter Reference Card: Amino Acid Uptake Transporters
Transporter: ASC, LAT
Key Facts: Na+-dependent ASC, System A; Ala-preferring,
small neutral amino acids
Na+-dependent ASC, System B0,+; neutral and
cationic amino acids preferring
Na+-independent LAT; large neutral amino acids preferring
Localization: Intestinal brush border, brain, placenta, kidney, heart
CellPort Advantage:
•Probes established for small cationic and large neutral amino acids.
•Optimize absorption of amino acid pro-drugs.
Recommended for amino acids and pro-drugs
Caco-2 cells
Assay Type
Uptake of probe substrate +/- test compound in
Caco-2 cells
Probe Substrate
3
H-Amino Acid (Alanine, Phenylalanine, or Arginine) Unlabeled amino acid (Alanine, Phenylalanine, or
Arginine)
Note: Mannitol is marker for non-specific entrapment
46
Transporter Reference Card: OATP1A2
Transporter: OATP1A2 (SLC21A3)
Key Facts: Solute-linked organic anion transporter;
broad substrate specificity
Localization: Intestine, kidney, liver and brain
CellPort Advantage:
•HEK293 is a human cell line; no interference from animal transporters.
•Use stable transfection rather than transient for consistent gene
expression in the same cell line over time, avoiding the caveats
associated with repetitive transient transfection.
Recommended when appropriate based on test
compound properties
OATP1A2-HEK293 cells vs. control
Assay Type
Uptake of test compound in OATP1A2-transfected cells
vs. control
Atorvastatin
Chemical Inhibitor
Ritonavir
compound properties
OATP1A2-HEK293 cells vs. control
Assay Type
OATP1A2-transfected cells vs. control; single
concentration or IC50
Probe Substrate
Atorvastatin
Ritonavir
47
Transporter Reference Card: OATP2B1
Transporter: OATP2B1 (SLCO2B1)
Key Facts: Solute-linked organic anion transporter;
narrow substrate specificity
Localization: Intestine, kidney, liver
CellPort Advantage:
compound properties
OATP2B1-HEK293 cells vs. control
Assay Type
Uptake of test compound in OATP2B1-transfected cells
vs. control Positive Control Substrate
Rosuvastatin
Chemical Inhibitors
Atorvastatin
48
compound properties
OATP2B1-HEK293 cells vs. control
Assay Type
OATP2B1-transfected cells vs. control; single
concentration or IC50
Probe Substrate
Rosuvastatin
Atorvastatin
Hepatic
Transporters
Overview
Hepatic Uptake Transporters
MRP3
P-g
p
BSEP
P
Bile
TE1
MA
MR
P2
MRP6
OST
BCR
MRP4
NTCP
OATP2B1
OATP1B3
OATP1B1
OAT7
OAT2
Blood
OCT1
The FDA and EMA both recommend OATP1B1 (SLCO1B1) and OATP1B3
(SLCO1B3) inhibitor assessment for all NCEs. In addition to OATP1B1 and
OATP1B3, the EMA recommends assessment of BSEP. Both agencies agree
that other transporters should be evaluated for inhibition when appropriate.
If hepatic clearance is significant, then evaluation of an NCE as a substrate
for OAT1B1 and OAT1B3 is additionally recommended.
49
Test Systems for Hepatic Uptake Transporters
We have cell lines that were created by over-expressing hepatic OATP1B1,
OATP1B3, or OCT1 in HEK293 human embryonic kidney cells. For OATP1B1
and OATP1B3, we have both wild-type and mutant variants. We use these
models for uptake assays, to evaluate substrate and inhibitor potential.
OATP1B1
Recombinant Over-expression
OATP1B1*1A
Wild-type variant
OATP1B1*1B
A388G mutation
OATP1B3
Recombinant Over-expression
OATP1B3*1
Wild-type variant
OATP1B3*2
T334G and G669A mutations
Vector Control
Control Plasmid
Transfection
Identical plasmid without
coding sequence
Nuclei are stained blue with DAPI. OATP1B1 on the surface of the cells is
stained green with a fluorescent antibody. There is no green fluorescence
in the vector control cells, whereas the transfected cells show robust
OATP1B1 expression.
OATP1B1-transfected HEK Cells
DAPI
OATP1B1
Mock-transfected
DAPI
50
OATP1B1
DAPI + OATP1B1
Transporter Transfections Are Stable
Our cell lines are stably, not transiently, transfected. All cells lines exhibit
robust expression, both molecularly and functionally, over several passages,
ensuring consistency and reproducibility between data sets. Additionally,
HEK293 parental cell lines show little to no expression of other uptake
transporters; therefore, the uptake due to transporter interactions is a result
of the specific transporter being expressed.
Uptake of Fluorescent Probes in Transfected Cell Lines
Uptake (µM)
0.20
0.15
OATP1B1
14-fold increase in FMTX
uptake compared to
HEK293 control cells
0.10
0.05
0.00
HEK293
OATP1B1
0.25
Uptake (µM)
0.20
OATP1B3
16-fold increase in
FMTX uptake compared
to HEK293 control cells
0.15
0.10
0.05
0.00
HEK293
OATP1B3
1.8
1.6
Uptake (µM)
1.4
OCT1
16-fold increase
in ASP uptake
compared to HEK293
control cells
1.2
1.0
0.8
0.6
0.4
0.2
0.0
HEK293
OCT1
51
All of our HEK hepatic transporter systems have been subjected to a rigorous
panel of experiments to confirm their suitability for substrate and inhibitor
assessments. The Km values determined with our systems for known and
confirmed substrates are comparable to values reported in the literature.
Net Influx Rate (pmol/mg/min)
Determination of Km and Vmax
20
15
OATP1B1
10
Km = 0.304 µM
Vmax = 18.9 pmol/mg/min
5
0
0
1
2
3
4
5
6
Atorvastatin Concentration (µM)
10
8
OATP1B3
6
Km = 2.01 µM
4
4
0.0
0
1
2
3
4
5
5
6
Atorvastatin Concentration (µM)
4000
3000
OCT1
Km = 56.6 µM
Vmax = 4616 pmol/mg/min
2000
1000
0
0
50
100
150
200
250
MPP+ Concentration (µM)
Not all compounds interact in the same way with uptake transporters.
We determine the optimal concentration and time for all related substrate
assessment studies using a 3 time point/3 concentration matrix (9 conditions
total). This initial study helps us to determine the optimal assay conditions
for any test compound.
52
Using a panel of known inhibitors, we demonstrated that our hepatic transporter
systems exhibit inhibition of substrate uptake. Additionally, we further
characterized these inhibitors and established IC50 data for strong and moderate
inhibitors (based on our initial inhibitor panels).
Determination of IC50
% Activity Remaining
120
100
80
OATP1B1
60
l Rifamycin SV IC50=0.407
40
n Rifampicin IC50=5.28
20
0
-10
-9
-8
-7
-6
-5
-4
Log Concentration (M)
120
100
80
OATP1B3
60
l Rifamycin SV IC50=0.244
40
n Rifampicin IC50=3.56
20
0
-10
-9
-8
-7
-6
-5
-4
120
100
80
OCT1
60
l Repaglinide IC50=116
40
n Quinine IC50=109
20
0
-10
-9
-8
-7
-6
-5
-4
-3
53
Solute-linked carrier organic anion transporter
Localization: Sinusoidal (basolateral) membrane of liver
CellPort Advantage:
• HEK293 is a human cell line; no interference from animal transporters
• Prevalent OATP1B1*1B variant available.
•Clinically relevant probe substrate.
•Provides definitive inhibitor assessment required for all NCEs.
Recommended if hepatic or biliary excretion is
major (≥25% total clearance)
OATP1B1-HEK293 vs. control
Assay Type
Uptake of test compound in OATP-transfected cells
vs. control
LC-MS/MS: Atorvastatin
Radioactive: 3H-atorvastatin
Fluorescent: FMTX
Chemical Inhibitor
Rifamycin SV
Assay Type
Uptake of probe substrate +/- test compound in OATPtransfected cells vs. control; single concentration or IC50
Probe Substrate
Fluorescent: FMTX
Rifamycin SV
54
Solute-linked carrier organic anion transporter
Localization: Sinusoidal (basolateral) membrane of liver
CellPort Advantage:
•Prevalent OATP1B3*2 variant available.
•Clinically relevant probe substrate.
•Provides definitive inhibitor assessment required for all NCEs.
Recommended if hepatic or biliary excretion is
major (≥25% total clearance)
Assay Type
Uptake of test compound in OATP-transfected cells
vs. control
Fluorescent: FMTX
Chemical Inhibitor
Rifamycin SV
Assay Type
Uptake of probe substrate +/- test compound in OATPtransfected cells vs. control; single concentration or IC50
Probe Substrate
Fluorescent: FMTX
Rifamycin SV
55
Transporter Reference Card: OCT1
Transporter: OCT1 (SLC22A1)
Key Facts: Suggested for evalutation by EMA when appropriate;
organic cation uptake transporters; pump substrates
from blood to liver for hepatic extraction
Localization: Liver, intestines, kidney
CellPort Advantage:
Recommended when appropriate
OCT1-HEK293 vs. control
Assay Type
Uptake of test compound in OCT1-transfected cells
vs. control
LC-MS/MS: MPP+ (1 methyl-4-phenylpyridinium iodide)
Positive Control Substrate Radioactive: 3H-metformin
Fluorescent: ASP
Chemical Inhibitor
Repaglinide
56
Assay Type
Uptake of probe substrate +/- test compound in OCT1transfected cells vs. control; single concentration or IC50
Probe Substrate
Radioactive: 3H-metformin
Fluorescent: ASP
Repaglinide
Transporter Reference Card: BSEP
Transporter: BSEP (ABCB11)
Key Facts: Suggested for evaluation by EMA, and also
by FDA when appropriate; ABC efflux transporter
Localization: Canalicular (apical) membrane of liver
CellPort Advantage:
• Robust expression of the transporter in vesicles from
over-expressing cells
• Inside-out vesicles increase sensitivity of the test system
through direct access to the transporter
• Validated using clinically relevant inhibitors
hBSEP-expressing vesicles
Assay Type
Uptake of test compound in hBSEP-expressing
vesicles in presence of ATP vs. AMP
3
Chemical Inhibitors
CsA
H-taurocholic acid
Recommended for all NCEs (EMA) or when
appropriate (FDA)
hBSEP-expressing vesicles
Assay Type
hBSEP-expressing vesicles in presence of ATP
vs. AMP; single concentration or IC50
Probe Substrate
3
CsA
H-taurocholic acid
57
Renal
Transporters
Overview
Renal Transporters
According to the FDA and EMA guidelines, all NCEs should be evaluated
for inhibition of OAT1 (SLC22A6), OAT3 (SLC22A8), and OCT2 (SLC22A2).
Additional transporters, such as MATE1 and MATE2, should also be
assessed when appropriate. Depending on the extent of renal clearance,
substrate evaluation should also be considered.
Blood
Urine
OAT4
OATP4C1
URATI
OCT2
PepT1, PepT2
OAT1
MRP2, MRP4
OAT2
OAT3
MATE1, MATE2
P-gp
OCTN1, OCTN2
59
Test Systems for Renal Uptake Inhibitors
We have created cell lines which over-express renal OAT1, OAT3, or OCT2
in HEK293 human embryonic kidney cells. We use these models to predict
substrate and inhibitor potential with regard to specific uptake transporters.
Nuclei are stained blue with DAPI. OCT2 on the surface of the cells is
stained green with a fluorescent antibody. There is no green fluorescence
in the HEK293 control cells, whereas the transfected cells show robust
OCT2 expression.
OCT2-transfected HEK Cells
DAPI
HEK-OCT2
DAPI+HEK-OCT2
HEK293
DAPI+HEK293
Mock-transfected
DAPI
60
Transporter Transfection Is Stable
These cells were transfected stably, rather than transiently, and show robust
expression, both molecularly and functionally, over several passages, ensuring
consistency and reproducibility between data sets. Additionally, HEK293
parental cell lines show little to no expression of other uptake transporters;
therefore, the uptake due to transporter interactions is a result of the specific
transporter being expressed.
Uptake of Fluorescent Probes in Transfected Cell Lines
1.5
Uptake (µM)
1.2
OCT2
14-fold increase in ASP
uptake compared to
0.9
0.6
0.3
0.0
HEK293
OCT2
.80
Uptake (µM)
.70
.60
OAT1
263-fold increase in 6CF
uptake compared to
.50
.40
.30
.20
.10
.00
HEK293
OAT1
.12
Uptake (µM)
.10
OAT3
.08
29-fold increase in 5CF
uptake compared to
.06
.04
.02
0.0
HEK293
OAT3
61
All HEK transporter systems have been subjected to a rigorous panel of
experiments to confirm their suitability for substrate and inhibitor assessments.
Using known and confirmed substrates, we were able to determine Km values
in our systems that are comparable to values reported in the literature.
Determination of Km and Vmax
4000
OCT2
3000
Km = 27.1 µM
2000
1000
0
0
50
100
150
200
250
MPP+ Concentration (µM)
200
150
OAT1
Km = 23.73 µM
100
50
0
0
20
40
80
6
60
PAH Concentration (µM)
750
500
OAT3
250
Km = 22 µM
0
0
25
50
75
100
125
Furosemide Concentration (µM)
Due to the fact that not all compounds interact in the same way with uptake
transporters, we determine the optimal concentration and time for all related
substrate assessment studies using a 3 time point/3 concentration matrix
(9 conditions total). These more rigorous initial studies help us to determine
the time and concentration to use for subsequent studies.
62
Inhibitor Assessment
We used a panel of known inhibitors for each transporter to demonstrate
inhibition. Additionally, we further characterized these inhibitors and
determined IC50 values for strong and moderate inhibitors (based on the
data from our inhibitor panels).
Determination of IC50
120
100
80
60
OCT2
40
l Imipramine IC50=51.8
20
n Quinidine IC50=276
0
-10
-9
-8
-7
-6
-5
-4
-3
-2
150
OAT1
100
l Probenecid IC50= 4.14
50
n Bumetanide IC50=6.08
20
-50
-4
-2
0
2
4
Log Concentration (µM)
125
100
OAT3
75
l Probenecid IC50=9.3
50
n Rosuvastatin IC50=21.7
25
0
-1
0
1
2
3
Log Concentration (µM)
63
Transporter Reference Card: OAT1
Transporter: OAT1 (SLC22A6)
solute-linked carrier organic anion transporter
Localization: Basolateral membrane of kidney proximal tubule cells;
also found in brain, placenta, eyes, and smooth muscle
CellPort Advantage:
Recommended if renal secretion is major
(≥25% total clearance)
OAT1-HEK293 vs. control
Assay Type
Uptake of test compound in OAT1-transfected cells
vs. control
Positive Control
Substrate
LC-MS/MS: para-Aminohippurate (PAH)
Fluorescent: 6-Carboxyfluorescein
Chemical Inhibitor
Probenecid
64
Assay Type
Uptake of probe substrate +/- test compound in OAT1transfected cells vs. control; single concentration or IC50
Probe Substrate
LC-MS/MS: para-Aminohippurate (PAH)
Radioactive: 3H-PAH
Probenecid
Transporter Reference Card: OAT3
Transporter: OAT3 (SLC22A8)
solute-linked carrier organic anion transporter
Localization: Basolateral membrane of kidney proximal tubule cells
CellPort Advantage:
Assay Type
Uptake of test compound in OAT3-transfected cells vs.
control
Positive Control
Substrate
LC-MS/MS: Furosemide
Radioactive: 3H-E3S
Chemical Inhibitor
Probenecid
Assay Type
Uptake of probe substrate +/- test compound in OAT3transfected cells vs. control; single concentration or IC50
Probe Substrate
LC-MS/MS: Furosemide
Radioactive: 3H-E3S
Probenecid
65
Transporter Reference Card: OCT2
Transporter: OCT2 (SLC22A2)
solute-linked carrier cation transporter
Localization: Primarily in kidney, also in liver, brain, lungs, etc.
CellPort Advantage:
Assay Type
Uptake of test compound in OCT2-transfected cells
vs. control
Positive Control
Substrate
Fluorescent: ASP
Chemical Inhibitor
Imipramine
66
Assay Type
Uptake of probe substrate +/- test compound in OCT2transfected cells vs. control; single concentration or IC50
Probe Substrate
Fluorescent: ASP
Imipramine
Transporter Reference Card: MATE1/2K
Transporter: MATE1/2K (SLC47A1/SLC47A2)
Key Facts: Suggested for evaluation by FDA and EMA,
when appropriate; SLC transporter
Localization: Kidney; Liver (MATE1)
CellPort Advantage:
• HEK293 is a human cell line; no interference from animal transporters.
• Clinically relevant probe substrate.
• Provides definitive inhibitor assessment required for all NCEs.
MATE1-HEK293 vs. control; MATE2K-HEK293
vs. control
Assay Type
Uptake of test compound in MATE-transfected cells
vs. control
Positive Control
Substrate
Metformin
Chemical Inhibitor
Cimetidine
MATE1-HEK293 vs. control; MATE2K-HEK293
vs. control
Assay Type
Uptake of probe substrate +/- test compound in MATEtransfected cells vs. control; single concentration or IC50
Probe Substrate
Metformin
Cimetidine
67
Concentration
Selection
Overview
Concentration Selection for Transporter Evaluations
Important Considerations for In Vitro Transporter Assays:
• The choice of concentration(s)
• Sampling time points (linear range, appropriate signal to noise ratio)
• Calculation methods suitable for the test system
• Choice of probe substrates or inhibitors that allow for extrapolation
to the clinical situation
Concentration Selection for Substrate Assessment
Concentrations selected for substrate assessment should be based around
the plasma Cmax of the test compound to ensure that results are clinically
relevant. If three concentrations are evaluated, then typically 0.5, 2 and 10 µM
would be chosen.
Concentration Selection for Inhibitor Assessment
Concentrations selected for inhibitor assessment are based on the
physiological site of interaction.
FDA
EMA
Intestinal Efflux
0.1x [I]2
0.1x [I]2
Hepatic Uptake
10x total Cmax
25x [I]u,inlet,max (administered orally) or
50x unbound Cmax (administered i.v.)
Renal Uptake
10x unbound Cmax
50x unbound Cmax
Hepatic Efflux
10x total Cmax
50x unbound Cmax
[I]2 : the concentration of drug in the intestinal lumen, defined as the highest dose divided by 250 mL
[I] u,inlet,max : the unbound hepatic inlet concentration of drug
Other factors to consider in selecting concentrations:
• Sensitivity of analytical method
• Solubility of the test compound
• Tolerability of the test compound in the test system
If solubility is a limitation for testing relevant concentrations of your test
compound, consider the use of protein or excipients. Absorption Systems
has evaluated the impact of bovine serum albumen (BSA) on known
probe substrates and optimized experimental conditions to ensure proper
classification during inhibition assays. We have also confirmed that the
following excipients have no influence on monolayer integrity, P-gp function,
or BCRP function at specific concentrations:
• PEG-400
• Tween 80
• DMSO
• HP-β-CD
• NMP
• Methanol
A regulatory viewpoint on transporter-based drug interactions L. Zhang, Y. (D.)
Zhang, J. M. Strong, K. S. Reynolds, S.-M. Huang Xenobiotica, 2008, Vol. 38, No. 7-8, 709-724
1
69
FAQs
Overview
Frequently Asked Questions (FAQs)
Q–– When will the FDA draft guidance on drug interactions be finalized?
A–– A “new draft guidance,” was published in February 2012; it is unclear when a
finalized guidance will be issued.
Q–– How does the FDA draft guidance compare to the EMA transporter
guidelines?
A–– There are some different expectations regarding the transporters to be
evaluated, as highlighted below:
FDA Guidance
2012
EMA Guideline
2012
a
a
a
a*
a
a
a
a*
a
a
a
a*
P-gp
BCRP
MRP
BSEP
OATP1B1
OATP1B3
a
a
a*
a*
a
a
OCT1
OCT2
OAT1
OAT3
MATE1/2
a
a
*When appropriate
Q–– When CLH and CLR are not available, can I use preclinical data to
determine them?
A–– You can, but you should certainly be cautious of species differences.
Human results would be best.
Q–– If CLR is <1% of total clearance, do I still need to perform an inhibition
assessment with renal transporters?
A–– Yes. Although the compound may not be a substrate of OATs or OCTs,
it could be an inhibitor and thereby interfere with the renal clearance of
other drugs.
71
Q–– [I]2 doesn’t take into account solubility. How do we overcome solubility
problems?
A–– Protein (BSA) or appropriate excipients can be used to enhance solubility
so that a broader range of concentrations may be tested. Absorption Systems
has evaluated a number of excipients that are ideal for in vitro drug transporter
studies: they do not alter monolayer integrity or affect transporter function.
We can check if these excipients improve solubility of your test compound(s).
Q–– If in vitro experiments for determination of P-gp/BCRP substrate interactions
result in an efflux ratio ≥ 2, do I need to always perform an in vivo DDI study?
A–– No, this may not be necessary if is determined that efflux is not rate limiting
because the compound has favorable drug specific-factors, such as high
permeability and high solubility. You can determine if your compound has
good intestinal absorption using a validated in vitro test system such as
Absorption Systems’ Caco-2 monolayer system. See section on waiving
clinical studies (under intestinal transporters).
Q–– Why should I run these studies at Absorption Systems?
A–– Absorption Systems has:
•
•
•
•
Innovative, proprietary technology funded by the FDA
Extensively validated test systems
Ability to waive clinical DDI and/or BA/BE studies
Experience and expertise in drug permeability and transporters
Q–– Is it possible to evaluate “sticky” compounds with low recovery using an
in vitro test system?
A–– Yes, various techniques may be implemented to alleviate non-specific
binding (NSB). As a diagnostic measure, the NSB of the test compound
to the experimental apparatus is assessed prior to any permeability
experiment. If the recovery is low, then a pre-incubation step is included
to saturate binding sites. For vectorial assays, 1% BSA is included in the
receiver compartment to improve recovery. At the end of a permeability
experiment, donor and receiver compartments may be rinsed with organic
solvent to collect residual test compound. Cellular accumulation may
also be measured.
Q–– What are the timelines and prices for these transporter assays?
A–– For more information about our study design, prices, and timelines visit
us online at www.absorption.com/assays or call us at 610-280-7300.
72
Drug Transporter Scientific Publications
The Role of a Basolateral Transporter in Rosuvastatin Transport and its
Interplay with Apical BCRP in Polarized Cell Monolayer Systems
Jibin Li, Ying Wang, Wei Zhang, Yuehua Huang, Kristin Hein, and Ismael J Hidalgo
Drug Metab. Dispos. published ahead of print August 1, 2012, dmd.112.045666
Models to Predict Unbound Intracellular Drug Concentrations in the
Presence of Transporters
Ken R. Korzekwa1, Swati Nagar1, Jalia Tucker1, Erica A. Weiskircher 2, Sid Bhoopathy2,
and Ismael J. Hidalgo2
1
Department of Pharmaceutical Sciences, Temple University School of Pharmacy,
Philadelphia, Pennsylvania
2
Absorption Systems, Exton, Pennsylvania
Drug Metab. Dispos. 2012; 40 865-876
Use of Transporter Knockdown Caco-2 Cells to Investigate the In Vitro
Efflux of Statins
Jibin Li1, Donna A. Volpe2, Ying Wang1, Wei Zhang1, Chris Bode1, Albert Owen1,
and Ismael J. Hidalgo1
1
2
Food and Drug Administration, Laboratory of Clinical Pharmacology,
Silver Spring, Maryland
Drug Metab. Dispos. 2011; 39 1196-1202
Silencing the Breast Cancer Resistance Protein Expression and Function
in Caco-2 Cells Using Lentiviral Vector-Based Short Hairpin RNA
Zhang W, Li J, Allen SM, Weiskircher EA, Huang Y, George RA, Fong RG,
Owen A and Hidalgo IJ.
Drug Metab. Dispos. 2009 Apr; 37(4):737-44.
Investigation of the Involvement of P-glycoprotein and Multidrug ResistanceAssociated Protein 2 in the Efflux of Ximelagatran and Its Metabolites by Using
Short Hairpin RNA Knockdown in Caco-2 Cells
Darnell M1, Karlsson JE1, Owen A 2, Hidalgo IJ2, Li J2, Zhang W2, Andersson TB2
1
Clinical Pharmacology and DMPK, AstraZeneca R&D, Mölndal, Sweden.
2
Drug Metab. Dispos. 2010 Mar;38(3):491-7. Epub 2009 Dec 18.
Stably Transfected HEK293 Cell Assays for Assessing Drug Interaction with
the Human Hepatic Transporters OATP1B1 and OATP1B3
Jibin Li, Samantha Allen, Ying Wang, Abby Herberger, Yuehua Huang, Alison Rush,
Albert Owen, Per Artursson, Ismael J. Hidalgo
AAPS Drug Transporters Workshop Mar. 2011, Poster W3011
Development of Cell-based Assays for Assessing Drug Interactions with
Human Renal Transporters
Jibin Li, Ying Wang, Abby Herberger, Samantha Allen, Libin Li, Yuehua Huang,
Alison Rush, Wei Zhang, Albert Owen, Ismael J. Hidalgo
73
Establishment and Characterization of a HEK293 Cell Line with Stable
Expression of Human Organic Cation Transporter
Wei Zhang, Erica A. Weiskircher, Rebecca A. George, Samantha M. Allen,
Yuehua Huang, Jibin Li, Albert Owen, Ismael J. Hidalgo
AAPS Drug Transporters Workshop Mar. 2011, Poster T2024
Applications of CellPort CPT-P1 Cells in Identifying P-gp Substrates In Vitro
Ying Wang, Abby Herberger, Jibin Li, Wei Zhang, Paula Kardos, Alison Rush,
Albert Owen, Ismael J. Hidalgo
Development of a Human Cell-based BCRP Inhibition Assay using
CellPort CPT-P1 Cell Monolayers and Cladribine as the Probe Substrate
Libin Li, Justin Wright, Amanda Budow, Sid Bhoopathy, Albert Owen, Ismael J. Hidalgo
Development of Transfected HEK293 Cell Lines Stably Expressing Individual
Human Organic Anion Transporters (OATs)
Wei Zhang, Samantha M. Allen, Erica A. Weiskircher, Abby L. Herberger,
Yuehua Huang, Rebecca A. George, Jibin Li, Albert Owen, Ismael J. Hidalgo
AAPS Drug Transporters Workshop Mar. 2011, Poster T2025
Mass Action Kinetic Model for P-gp Mediated Transport across Confluent Cell
Monolayers: A Sensitive Tool to Facilitate Mechanistic Understanding of DDIs
Annie Albin Lumen1,3 Libin Li2, Jibin Li2, Albert Owen2, Ismael J. Hidalgo2,
Harma Ellens3, Joe Bentz1
1
Drexel University, Philadelphia Pennsylvania,
2
3
GlaxoSmithKline, King of Prussia, Pennsylvania
AAPS Drug Transporters Workshop Mar. 2011 Poster M1013
Use of Caco-2 Knockdown Cells to Investigate Transporter-Mediated Efflux
of Statin Drugs
Donna Volpe1, Jibin Li2, Ying Wang2, Wei Zhang2, Chris Bode2, Albert Owen2,
Ismael J. Hidalgo2
1
Food and Drug Administration, Silver Spring, Maryland
2
AAPS/PSWC Nov. 2010, Poster T3371
Induction of Breast Cancer Resistance Protein (BCRP) and Enhanced Transport
of Estrone-3-sulfate and Pheophorbide A Across Caco-2 Cell Monolayers
Wei Zhang, Samantha M. Allen, Yuehua Huang, Erica A. Weiskircher,
Rebecca A. George, Jibin Li, Albert Owen, Ismael J. Hidalgo
Determination of Substrate Specificity and Relative Activity of Uptake and
Efflux Transporters in a New Cell Line: OATP1B1-Transfected MDR1-MDCK Cells
Wei Zhang, Erica A. Weiskircher, Samantha M. Allen, Yuehua Huang,
Rebecca A. George, Jibin Li, Albert Owen, Ismael J. Hidalgo
74
Determination of BCRP Activity and Evaluation of Potential BCRP-Mediated
Drug-Drug Interactions in Caco-2 Cells Using the Fluorescent Substrate
Pheophorbide A
Ying Wang, Abby Herberger, Jibin Li, Albert Owen, Ismael J. Hidalgo
Fluorescence Assay for Evaluation of MRP2 Based Drug Interactions
Fany B. Guerra, Jibin Li, Albert Owen, Ismael J. Hidalgo
ISSX Sept. 2010
In Vitro Permeability Systems to Assess P-gp Substrate Potential
Qing Wang, Yuehua Huang, Blair DeFulvio, Allison McLaughlin, Paula Kardos,
Susan Rawa, Ismael J. Hidalgo
AAPS Annual Meeting November 2011 Poster M1296
Cell-based Radioactive Assays for Assessing Drug Interactions with Human
Renal Uptake Transporters
Jibin Li, Wei Zhang, Ying Wang, Samantha M. Allen, Abby L. Herberger, Albert J.
Owen, Ismael J. Hidalgo
Drug Transporters: ‘Not as Easy as ABC’ A case study demonstrating benefits
and limitations of test systems and experimental designs when elucidating
complex transporter interactions
Libin Li, Ying Wang, Erica A. Weiskircher, Samantha M. Allen, Yuehua Huang,
Vatsala Naageshwaran, Sid Bhoopathy, Albert J. Owen, and Ismael J. Hidalgo
Rapid Screening of Human OATP1B1- and OATP1B3-Mediated Drug Interactions
in Stably Transfected Human Embryonic Kidney HEK293 Cell Lines Using Flow
Cytometry and Fluorescence Microplate Methods
Steve Wright1, Ying Wang2, Jibin Li2, Albert Owen2, Ismael J. Hidalgo2
1
Department of Biology, Carson-Newman College, Jefferson City, Tennessee
2
ISSX 17th North American Meeting November 2011 Poster P347
Using Lentiviral Vector-Based shRNA to Silence the Expression and Function
of ABC Transporter(s) in Caco-2 Cells
Chris Bode, Sid Bhoopathy, Ismael Hidalgo
Development of a Rapid Cell-based Assay using Flow Cytometry for Assessing
Drug Interactions with Human Sodium-dependent Taurocholate Cotransporting
Polypeptide (NTCP)
Steve Wright1, Matt Wilkerson1, Jibin Li2, Ying Wang2, Albert Owen2, Ismael J. Hidalgo2
1
Department of Biology, Carson-Newman College, Jefferson City, Tennessee
2Absorption Systems, Exton, Pennsylvania
75
Identification of OCT1-mediated interaction of antimicrobial drugs with
metformin using stably transfected OCT1 HEK293 cells
Wei Zhang, Samantha M. Allen, Jibin Li, Erica A. Weiskircher, Yuehua Huang,
Ying Wang, Albert Owen and Ismael J. Hidalgo
AAPS Annual Meeting October 2012 Poster M1322
In Vitro Systems to Assess the Potency of Selected Uptake Transporter Inhibitors
Blair Meziewieski, Joseph Rager, Libin Li, Jessica Helwig, Sid Bhoopathy,
Ismael J. Hidalgo, and Qing Wang
Ex Vivo Evaluation of Regional Differences in the Expression and Function of
Active Drug Transporters and Metabolic Enzymes in Rat and Human Intestine
Xuexuan Wang, Fany Guerra, Jerline Joseph, Yuehua Huang, Jennifer Winans,
Sid Bhoopathy and Ismael J. Hidalgo
AAPS Annual Meeting October 2012 Poster T2298
In Vitro Assays for Assessing Drug Interactions with Human Bile Salt Export Pump
Jibin Li, Steve Wright, Wei Zhang, Ying Wang, Yuehua Huang, Ismael J. Hidalgo
Determination of Efflux Transporter Membrane Location using Caco-2
Knockdown Cells
Erica Weiskircher1, Allison McLaughlin1, Jessica Helwig1, Sid Bhoopathy1,
Swati Nagar 2, Ken Korzekwa2, and Ismael Hidalgo1
1
Absorption Systems, Exton, Pennsylvania 2
Temple University, Philadelphia, Pennsylvania
DVDMDG The Rozman Symposium June 2012
76
Glossary
Overview
Glossary of Terms
ABC: ATP-binding cassette, a term referring to a common feature of one of
the two superfamilies (the other is the SLC superfamily) of drug transporters.
Members of the ABC superfamily, including P-gp, BCRP and the MRP family,
are responsible for active efflux of drugs, other xenobiotics, and endogenous
substances from cells throughout the body. The extrusion of substrates is
driven by hydrolysis of ATP by the transporter itself.
Absorption: Before a drug can exert a pharmacological effect in tissues, it
generally has to reach the systemic circulation—often via a mucous surface
such as that in the digestive tract (intestinal absorption). Factors such as poor
solubility, chemical instability in the stomach, and inability to permeate the
intestinal wall can all limit the extent to which a drug is absorbed after oral
administration. Absorption critically determines a drug’s bioavailability.
ADME: An acronym for Absorption, Distribution, Metabolism, and Excretion
(or Elimination).
Apparent Permeability: The apparent permeability coefficient (also known as
Papp) is a parameter that is determined in an in vitro or ex vivo transport assay
system. It is referred to as apparent permeability because its value represents
the composite effects of traversing all permeation pathways across the test
system and does not represent the permeability across any single barrier, such
as the unstirred water layer, the cell membrane or the tight junctions between
epithelial cells. It is equal to (dQr/dt)/(A x C0), where dQr/dt is the cumulative
amount of test compound appearing in the receiver compartment of the assay
system vs. time, A is the area of absorption (cell monolayer or tissue), and C0
is the initial concentration of the test compound in the donor compartment
of the assay system. Papp can be determined in either or both directions
across an assay system. For cell monolayer assay systems the directions are
referred to as apical to basolateral (A¨B) and vice versa. For ex vivo tissue
systems the directions are typically referred to as mucosal to serosal and
vice versa. A significant directional dependence in Papp values suggests that
active absorption or efflux mechanisms mediate compound transport across
the assay system. When determined in an appropriate assay system, such
as Caco-2 cell monolayers or human intestinal tissue strips, the rank order
of a compound’s Papp value as compared to control reference compounds of
known human absorption potential has been demonstrated to be predictive of
the in vivo absorption classification of the same compound in humans.
BCRP: An acronym for breast cancer resistance protein, product of the
human ABCG2 gene and a member of the ABC (ATP-binding cassette)
transporter superfamily. BCRP is expressed in the apical membrane of
epithelial and endothelial cells in many tissues of the body, including intestine,
liver, kidney, central nervous system (blood-brain barrier) and placenta.
77
It was originally characterized as one of the gene products mediating
resistance to cancer chemotherapeutic drugs; its involvement in drug
disposition and drug-drug interactions was recognized more recently.
BCS: See Biopharmaceutics Classification System
Bioavailability: (also known as %F) The fraction of administered drug
that reaches the systemic circulation after enteral administration. Absolute
bioavailability is the ratio of the area under the curve (AUC) of the blood
(or plasma) concentration versus time curve after enteral administration
divided by the AUC after intravenous administration, appropriately adjusted
for differences in dose. Relative bioavailability is the AUC ratio after dosing
by two different enteral dose routes or two different dosing formulations,
etc. Bioavailability is affected by various barriers to enteral drug absorption,
including solubility, stability, permeability, active efflux and metabolism.
Bioavailability may be expressed as a fraction or as a percent of the
administered dose.
Biopharmaceutics: The study of the effect of a drug’s formulation and
physicochemical properties on its absorption and disposition.
Biopharmaceutics Classification System: The Biopharmaceutical
Classification System (BCS) was developed in the 1990s by a collaboration
of academic, industrial and US FDA scientists in order to provide guidelines
for the development of oral dosage forms. It was formalized in the US in
2000, when the FDA published the Guidance for Industry, Waiver of In Vivo
Bioavailablity and Bioequivalence Studies for Immediate-Release Solid
Oral Dosage Forms Based on a Biopharmaceutics Classification System.
The European Medicines Agency (EMA) and the World Health Organization
(WHO) have since adopted the BCS in whole or in part. The basic principle
behind the classification system is that the absorption of immediate-release,
orally administered drugs is driven by two properties, the solubility of the
drug in the gastrointestinal tract and the permeability of the drug across
the intestinal epithelial cell barrier. Proper in vitro surrogates for these two
properties should allow prediction of a drug’s absorption in vivo from in
vitro assays. A drug’s solubility classification in the BCS is a function of the
intended human dose. Drugs whose solubility under appropriate conditions
exceeds the highest dose strength dissolved in 250 mL of aqueous medium
are classified as highly soluble, i.e., Class I or III according to the BCS
scheme. Drugs not meeting these criteria are classified as Class II or IV.
Class I and Class II drugs have high permeability in an appropriate
permeability assay system that has been validated with compounds of known
in vivo human fractional absorption after oral administration. Drugs not
meeting these criteria are class III, if they have high solubility, or class IV,
if their solubility is low.
78
Biotransformation: See Metabolism
Biowaiver: An exemption granted by the US FDA from conducting human
bioequivalence studies when the active pharmaceutical ingredient(s) meet
certain solubility and permeability criteria in vitro and when the dissolution
profile of the dose form meets the requirements for an “immediate” release
dose form. Biowaivers are based on the Biopharmaceutics Classification
System (BCS) category of the active ingredient. Currently BCS class I and
some class III compounds are eligible for biowaivers.
BSEP: An acronym for bile salt export pump, product of the human
ABCB11 gene and a member of the ABC (ATP-binding cassette) transporter
superfamily. BSEP is expressed in the canalicular membrane of hepatocytes
in the liver, and is involved in the excretion of bile acids into the bile. When
inhibited, it induces cholestasis as a result of transcellular accumulation of
these bile salts.
Buffer: A compound or mixture of compounds which, added to a solution,
helps maintain a certain pH. A buffered solution resists changes in pH if either
acids or bases are added to the solution or if the solution is diluted.
Caco-2: Human colonic carcinoma cells. The Caco-2 cell line is widely used
in in vitro assays to predict the absorption of candidate drug compounds
across the intestinal epithelial cell barrier. The assay requires that the
absorption rate (apparent permeability, or Papp) of a compound be determined
approximately 21 days after Caco-2 cell seeding to allow the formation of a
confluent monolayer and cell differentiation (appropriate localization of active
transporters to the apical or basolateral plasma membrane).
Calcein AM: A non-fluorescent compound that is cleaved to a fluorescent
compound by non-specific intracellular esterases. Calcein AM (the
acetoxymethyl ester of the fluorescent dye, calcein) is a substrate of the drug
efflux transporter P-glycoprotein (P-gp).
CellPort CPT-B1: the first product in the CellPort Technologies® product
line from Absorption Systems. It was created by using RNA interference to
achieve stable knockdown of the expression of BCRP in human Caco-2 cells.
CellPort Technologies®: Absorption Systems product line, consisting of all
products and services related to drug transporters.
CL: See Clearance
Clearance: Drug elimination from the body, regardless of the route or
mechanism. The units are volume per time (L/hr). Therefore, clearance (CL)
is defined as the volume of fluid cleared of drug per unit time.
CPT-B1: see CellPort CPT-B1
79
CYP: Shorthand for cytochrome P450, a family of membrane-bound,
heme-containing oxygenases that are active in a range of metabolic and
biosynthetic pathways. They are involved in the metabolism (breakdown) of
both endogenous and exogenous (xenobiotic) substrates, including drugs and
environmental chemicals, and in the synthesis of metabolome components
such as leukotrienes, prostacyclins, HETEs, retinoic acids, steroids and bile
acids. They are divided into families of structurally related proteins based on
their gene sequence. Families are designated by a number, their subfamilies
by a letter and their species-specific subfamily by a second number. Thus,
CYP 3A4 is a human CYP that is a member of the CYP 3 family and CYP 3A
subfamily. The study of CYPs is complicated by the fact that the metabolic
products and pathways of individual CYPs overlap and their relative levels
of expression differ among species, making interspecies extrapolation of
xenobiotic metabolism extremely problematic. In addition, the expression
of some CYPs varies greatly among individual human subjects, and some
are inducible (expression increased) by drugs, dietary constituents, or
environmental chemicals. Within a species, some CYPs are expressed
exclusively, or nearly so, in particular tissues, while others are expressed in
multiple tissues but at very different levels. In general, hepatic CYPs are the
most important drug-metabolizing enzymes, but other (non-CYP) enzymes
are important for many drugs, and CYPs in other organs can be significant
depending on the route of administration (e.g., intestinal CYPs for oral
drugs, dermal CYPs for topical drugs, nasal CYPs for intranasal drugs, etc.).
Numerous clinical drug-drug interactions have been reported involving CYPs
(e.g., one drug as a substrate, another co-administered drug as an inhibitor)
and can result in therapeutic failure or death.
CYP450: See CYP
Cytochrome P450: See CYP
DDI: See Drug-drug Interaction
Distribution: The reversible transfer of xenobiotics from one compartment in
the body to another compartment, e.g., across the blood-brain barrier from the
plasma to the extracellular fluid of the brain.
DM: See Metabolism
Drug: A chemical substance that can alter or influence the responsiveness
of a living organism. A therapeutic chemical substance that is used for the
diagnosis, prevention, cure or amelioration of an unwanted health condition
(FDA definition). Drugs are usually xenobiotics (i.e., substances chemically
foreign to the organism).
80
Drug-drug Interaction: A pharmacokinetic drug-drug interaction (DDI)
involves two (or more) co-administered drugs, often referred to as the
perpetrator and the victim: when co-administered with the perpetrator, the
systemic exposure (Cmax or AUC), tissue-specific exposure, or side effects
(toxicity) of the victim drug are affected to an extent that is clinically significant.
This type of DDI typically occurs when the victim drug is a substrate of a
drug-metabolizing enzyme or transporter and the perpetrator drug is a
substrate or inhibitor of the same enzyme or transporter. Qualitatively, the
pharmacokinetic consequences of a metabolism-mediated DDI are fairly
straightforward: enzyme inhibition can increase the exposure to a drug that
is administered as the active species and can decrease the exposure to
the active moiety of a pro-drug. Enzyme induction has the opposite effect.
The consequences of transporter-mediated DDIs are much more difficult
to predict; in fact, the clinical impact of a transporter-mediated DDI on a
particular tissue or organ is often much more significant than the effect on
systemic exposure. Whereas the risk of many clinical DDIs can be predicted
qualitatively based on in vitro testing, reliable quantitative prediction of the
magnitude of a clinical DDI is not yet possible in most cases.
Drug Metabolism: See Metabolism
Efflux: The process by which molecules (typically, drugs or other xenobiotics)
are pumped out of cells. This process is monitored in laboratory assays using
polarized epithelial cell lines such as Caco-2, which express several efflux
pumps (e.g., P-gp, BCRP and MRP2) in the apical membrane and others
in the basolateral membrane. Efflux is catalyzed by such efflux pumps for
compounds that are substrates. The cell-based laboratory test systems are
models of various cells in the body, which express many of the same efflux
transporters as well as others.
Efflux Pumps: See Membrane Transport Proteins
Efflux Ratio: In in vitro and ex vivo assays, the efflux ratio is defined as the
ratio of secretory to absorptive apparent permeability (Papp): (BÄ Papp) /
(A¨B Papp). The magnitude of this ratio is used as an indicator of active vs.
passive transport through cells. At Absorption Systems, efflux is classified as
significant when the efflux ratio is at least 3.0 and the BÄ Papp is at least
1.0 x 10-6 cm/s.
HEK293: Continuous cell line derived from human embryonic kidney. HEK293
cells are frequently transfected with a vector containing one or more genes in
order to create an in vitro model in which a protein of interest is over-expressed.
Hit: A molecule with confirmed pharmacologic activity in a screen for efficacy.
81
Investigational New Drug (also known as IND): The purpose of an IND
application is to provide data showing that it is reasonable to begin tests of a
new drug in humans. IND regulations are covered in 21 CFR 312.
There are two categories of IND: commercial and research (non-commercial).
An IND must contain information in three broad areas: animal pharmacology
and toxicology studies; manufacturing information; and clinical protocols and
investigator information.
Once an IND is submitted, the sponsor must wait thirty calendar days before
initiating any clinical trials. During this time, the FDA has an opportunity
to review the IND for safety to ensure that research subjects will not be
subjected to unreasonable risk. There are four types of IND:
• Company- (or institution-) sponsored IND: This is the traditional type of IND.
• Investigator IND: Submitted by a physician who both initiates and conducts
an investigation, and under whose immediate direction the investigational
drug is administered or dispensed. A physician might submit such an IND
to propose studying an unapproved drug, or an approved product for a new
indication or in a new patient population.
• Emergency Use IND: Allows the FDA to authorize use of an experimental
drug in an emergency situation that does not allow time for submission of an
IND in accordance with the normal process. It is also used for patients who
do not meet the criteria of an existing study protocol, or if an approved study
protocol does not exist.
• Treatment IND: Used to facilitate access by patients to promising new
drugs for serious or immediately life-threatening conditions while late-stage
clinical trials (in which enrollment may be closed) are conducted and NDA
review takes place.
ITC White Paper: An authoritative review on drug transporters by the
International Transporter Consortium (ITC), a group of experts in the
transporter field from the pharmaceutical industry, academia and the FDA.
The paper, published in Nature Reviews Drug Discovery in March 2010
(Giacomini KM, et al., Membrane transporters in drug development. Nature
Rev Drug Disc 2010 March;9(3):215-36), was immediately recognized as the
most relevant reference on the topic to date.
In Vitro: Literally, “in glass” (Latin). The term is used to describe a process or
reaction that is carried out in an artificial environment such as a test tube, petri
dish or 96-well plate. The reaction mixture can include a biologically derived
material, such as a tissue extract, cultured cells, or a purified protein. Other in
vitro assays, such as physicochemical measurements for solubility or pKa, do
not contain any biological matrix.
82
LC-MS/MS: Liquid chromatography – tandem mass spectrometry. Mass
spectrometry (MS) is an analytical technique to measure the mass-to-charge
ratio (m/z) of ions. It is most generally used to find the composition of a
physical sample by generating a mass spectrum representing the masses of
the components of a sample. In addition to a number of broader applications,
in drug development LC-MS/MS is generally used to quantify the amount of a
compound in a sample using carefully designed methods (mass spectrometry
is not inherently quantitative) and to determine the structure of a compound
by analyzing its fragmentation pattern. In LC-MS/MS, molecules are partially
resolved by rapid LC, followed by detection via tandem mass spectrometry
(MS/MS). This mode of detection is exquisitely sensitive due to the operator’s
ability to tune the instrument to a particular m/z value to the exclusion of other
molecular species that may be present.
Lead Optimization: A portion of the drug discovery continuum, lead
optimization is the complex process of refining the chemical structure of a “hit”
to improve its drug characteristics, with the goal of producing a pre-clinical
drug candidate. Typically, by the lead optimization stage many compounds
have been screened and many “hits” discarded for various reasons. At this
stage, analogues of a biologically active compound or a series of structurally
related compounds are synthesized, the goal being to find a molecule with the
most favorable combination of physicochemical (e.g. solubility), pharmacologic
(i.e. therapeutic efficacy, potency, and specificity), pharmacokinetic (e.g.
metabolic stability), and toxicologic (i.e. safety) properties.
Liquid Chromatography: (LC) is the science of separating molecules,
applied in a liquid mobile phase, based on differences in affinity for a solid
stationary phase relative to the mobile phase, often while varying the
composition of the mobile phase.
Mass Spectrometer: is a device used for mass spectrometry, and produces
a mass spectrum of a sample to find its composition. This is normally
achieved by ionizing the sample and separating ions of differing masses
and recording their relative abundance by measuring intensities of ion flux.
A typical mass spectrometer comprises three parts: an ion source, a mass
analyzer, and a detector.
MATE: An acronym for Multidrug And Toxin Extrusion transporter, product
of the human SLC47 gene and a member of the SLC (solute carriers)
transporter superfamily. MATE1 is primarily expressed in the kidney and
also in liver. MATE2 and MATE2-K are expressed only in the kidney, and
are localized to the brush-border membranes (BBM) of the renal proximal
tubules. The MATE family is proposed to play an important role in protecting
the kidney from cationic toxins. So far, transport characteristics of the original
hMATE2 remain unclear. MATE2-K is currently the only functional isoform in
the MATE2 subfamily.
83
MDR Protein: Multi-drug resistance protein 1 is the protein product of the
human MDR1 gene and is a member of the ABC (ATP-binding cassette)
superfamily of plasma membrane transporter proteins that pump drugs (such
as some anti-cancer drugs) and other xenobiotics out of the cytoplasm of
cells. This particular family of transporters is expressed by mammalian cells,
but analogous proteins are expressed by bacteria, where they contribute to
antibiotic resistance. As the name suggests, the gene encoding the MDR1
protein was discovered in drug-resistant tumors. MDR1 is synonymous with
human P-glycoprotein (Pgp), a name that was actually first applied to the
bacterial version of the protein. In the context of drug discovery in general, the
MDR1 protein can have a major impact on the pharmacokinetic properties of
drugs by reducing absorption from the gastrointestinal tract, limiting distribution
from the blood into the brain, and/or contributing to biliary excretion from
hepatocytes. MDR1 protein can also be a locus of drug-drug interactions in
patients taking an inhibitor and a substrate of the transporter at the same time.
Because MDR1 protein is expressed in so many locations in the human body
that play key roles in pharmacokinetics, the effects of modulation of its activity
(induction or inhibition) are complex and difficult to predict.
MDR1-MDCK: Madin-Darby Canine Kidney cells transfected with the human
multi-drug resistance gene. Confluent monolayers made from these cells can
be used to assess a test compound’s potential as a P-gp substrate.
Membrane Transport Proteins: Membrane proteins whose primary function
is to facilitate the transport of molecules across a biological membrane.
Included in this broad category are proteins involved in active transport,
facilitated transport and ion channels. They play key roles in drug absorption,
distribution and excretion, and are sometimes involved in drug toxicity and
drug-drug interactions. They may be indirectly involved in drug metabolism as
well, due to their involvement in drug uptake into hepatocytes.
Metabolism: Metabolism, one mechanism of clearance of drugs and other
xenobiotics, is the irreversible biochemical transformation of a compound to
another chemical (metabolite). The metabolite is usually more polar (watersoluble) and, therefore, more readily excreted, than the parent compound;
thus, metabolism facilitates drug excretion. Conceptually, metabolism is often
divided into Phase I (oxidation or reduction) and Phase II (conjugation with
negatively charged functional groups), which are closely coupled in intact cells
such that Phase II metabolites are often present at much higher concentrations
than Phase I metabolites. In the context of toxicology, metabolism is often
referred to as activation; the same enzymes that facilitate excretion of
compounds via metabolism can also produce highly reactive metabolites or
metabolic intermediates, which bind to cellular proteins or DNA, sometimes
84
leading to cell death or cancer. Many drug-drug interactions take place at
the level of drug metabolism, via inhibition or induction of drug-metabolizing
enzymes, potentially resulting in toxicity or therapeutic failure, respectively.
MRP2: Multidrug resistance-associated protein 2, a member of the MRP
family of drug transporters and the product of the human ABCC2 gene.
Previously known at CMOAT, MRP2 is expressed in the apical membrane
of epithelial and endothelial cells in the liver, kidney, placenta, intestine,
blood-brain barrier and many other tissues. It is also expressed in the apical
membrane of Caco-2 cells.
NCE: New chemical entity, a compound not previously described in the
scientific literature.
New Chemical Entity: See NCE
Nonclinical: See Preclinical. The terms are often used interchangeably, but
sometimes the term “nonclinical” is preferred because some nonclinical studies
are performed in parallel with, not literally prior to, clinical trials of a drug.
OATP: An acronym for organic anion transporting polypeptide, a family of
solute-linked carrier (SLC) drug transporters. The major human OATPs are
OATP1B1 (formerly known as OATP-C or OATP2; product of the SLCO1B1
gene in humans), OATP1B3 (formerly known as OATP8; product of the
SLCO1B3 gene) and OATP2B1 (formerly known as OATP-B; product of the
SLCO2B1 gene). OATP1B1 and OATP1B3 are expressed in the sinusoidal
(basolateral) membrane of hepatocytes in the liver. A number of clinically
significant, even fatal, increases in exposure to drugs have been reported
involving the hepatic OATPs due to inhibition by co-administered drugs or
pharmacogenetics (reduced expression or expression of a less active variant).
As a result, drug developers and drug regulatory agencies now pay close
attention to new drugs that are substrates of OATP1B1 and/or OATP1B3.
Because some of the highly prescribed statin drugs are substrates of
OATP1B1 and/or OATP1B3, drug developers and drug regulators also pay
close attention to new drugs that are inhibitors of these transporters. The
expression of OATP1A2 and OATP2B1 is more diverse, including intestine,
kidney and brain in addition to liver.
OCT: An acronym for organic cation transporter, a family of solute-linked
carrier (SLC) drug transporters including OCT1 (product of the human
SLC22A1 gene), OCT2 (SLC22A2) and OCT3 (SLC22A3). Expression of
OCT1 is limited to the liver, OCT2 is expressed primarily in kidney (also brain,
lungs, testes, etc.), and OCT3 is expressed in many tissues. The involvement
of OCT2 in renal drug clearance and the fact that it has been implicated in
more than one clinical drug-drug interaction has drawn the attention of drug
developers and global drug regulatory authorities.
85
Papp: See Apparent Permeability
P-glycoprotein: See P-gp
P-gp: P-gp is short for P-glycoprotein, a 170-kDa integral membrane
transport protein present in a variety of types of mammalian cells. P-gp was
first identified as a primary cause of multidrug resistance in tumors. Using
ATP hydrolysis as an energy source, it transports its substrates, typically
sterols or hydrophobic amines, into the gut, out of the brain, into urine, into
bile, out of the gonads, or out of other organs. An understanding of P-gp and
related transport proteins is key to designing strategies for the improvement of
therapeutic efficacy of drugs that are their substrates and to anticipating their
role in mediating drug-drug and drug-diet interactions.
P-gp Inhibitor: A compound that binds to P-gp and inhibits its transport
activity. P-gp inhibitors are useful research tools; their potential clinical
applications include reversal of resistance to cancer chemotherapy.
Pharmacokinetics: Operationally, the term describes the time course of the
variation of a drug’s concentration in various body tissues, as well as blood,
blood plasma and urine, following dosing. It is the study of ADME, i.e., the
absorption, distribution from plasma into tissues, metabolism and excretion
(elimination) of drugs. These factors, combined with dosage, determine
the concentration of a drug at its site(s) of action as a function of time.
Pharmacokinetics frequently utilizes mathematical equations to describe and
model the concentration vs. time profile of a drug in sampled body fluids such
as plasma. Compartmental models are widely used to calculate fundamental
parameters from preclinical or clinical experimental data.
Phase 0: See Preclinical
PK: see Pharmacokinetics
Preclinical: Literally, “before the clinic”. In drug discovery and development,
the term refers to in vitro or in vivo (animal) studies that are performed prior to
or concurrent with human clinical trials of a compound.
Recovery: The extraction efficiency of an analytical process, reported as a
percentage of the known amount of an analyte carried through the sample
extraction and processing steps of the method. Recovery is most valid when
an internal standard is carried through the process along with the analyte
of interest. In some applications, low recovery is not a problem because the
observed amount of analyte can simply be normalized to the recovery of the
internal standard. In other cases, such as a cell-based permeability study,
low recovery is indicative of a high degree of non-specific binding and can
confound the interpretation of the observed results.
86
Reproducibility: The between-run, or inter-batch, precision of an
assay. It typically refers to sets of results obtained with the same test
material on different days, by different operators, using different analytical
instruments, in different laboratories, etc. It can be viewed as the variation
in measurements obtained when two or more people perform the same
assay on identical test articles.
Ruggedness: The reproducibility of an assay under a variety of normal, but
variable, test conditions. Variable conditions might include different instruments,
operators, reagent lots, etc. Ruggedness provides an estimate of experimental
reproducibility with unavoidable error. In the process of developing an assay,
it is necessary to consider the effect of environmental factors on the output
of the assay. If these effects are not considered, the results of the assay in
actual use may not be as accurate as expected. The purpose of a ruggedness
test is to identify the variables (experimental factors) that strongly influence
the measurements provided by the assay, and to determine how closely these
variables need to be controlled. Ruggedness tests do NOT determine the
optimal conditions for the assay.
SLC: Solute-linked carrier, a descriptor of one of the two superfamilies
(the other is the ABC superfamily) of drug transporters. Members of the
SLC superfamily, including the OATP, OAT and OCT families, are responsible
for uptake of drugs, other xenobiotics, and endogenous substances by cells
throughout the body. The mechanism of transport by these transporters is
not well understood; uptake of a drug is coupled to export of another molecule,
whose identity is not known. Theoretically, SLC transporters can operate as
either uptake or efflux transporters, but physiologically they always mediate
uptake of drugs.
Substrate: In biochemistry, a substrate is a molecule undergoing a reaction,
for which the presence of an enzyme greatly increases the rate by lowering
the activation energy when the substrate binds to the enzyme’s active site.
In ADME assays, a substrate is a test compound.
Transporters: see Membrane Transport Proteins
Uptake Pumps: see Membrane Transport Proteins
Validated Cell Line: In the context of ADME assays, a validated cell line
is a cell line that has been demonstrated to predict the clinical behavior of
compounds, e.g., in vivo potency, efficacy or bioavailability. Examples include
the rank order of in vitro permeability across Caco-2 cell monolayers vs. human
fractional absorption and CYP induction in hepatocytes vs. effect on clinical
pharmacokinetics when co-dosed with a sensitive substrate of the same CYP.
The rank order responses must be reproducible and rugged with respect to
typical changes in assay conditions for the cell line to be considered validated.
87
Validation: Method validation is the process of establishing the performance
characteristics and limitations of a method and the identification of the
influences which may change these characteristics and to what extent. It
includes accuracy, precision, specificity, sensitivity, reproducibility, stability,
predetermined limits for acceptance of data based on QC standards, and
extensive documentation. A related process is verification, the confirmation by
examination and provision of objective evidence that specified requirements
have been fulfilled. Assay parameter optimization is a separate process that
precedes assay validation and/or may be iterative with it. For example, assay
validation may identify parameters for which the assay should have been
optimized; after further optimization, the assay would need to be re-validated.
Xenobiotic: A biologically (pharmacologically, endocrinologically, and/
or toxicologically) active substance that is not produced endogenously and
is, therefore, foreign to a living organism. Examples include drugs, dietary
components, carcinogens, and chemicals that have been introduced into the
environment by artificial means.
88

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