Planning
Committee and Session Chairs:
Magang Shou,
Merck & Co., Inc.
Albert P. Li,
Advanced Pharmaceutical Sciences, Inc.
Wednesday, June 15, 2005
Session 1: New Technologies
and Approaches for the Evaluation of Drug-Drug Interaction (DDI)
Chair: Albert Li
8:00 AM
9:00 AM REGISTRATION
8:00 AM 5:00 PM EXHIBITS
8:50 AM 9:00 AM Exhibitor Presentation Qualyst, Inc.
9:00 AM 9:05 AM
Welcoming Remarks
(Albert
P. Li; The ADMET Group, LLC; Rockville, MD)
9:05 AM 9:45 AM
Overview: Present Status, Challenges, and New Approaches to Assess
Drug-Drug Interaction Potential
(Albert
P. Li; The ADMET Group, LLC.; Rockville, MD)
The present status of the
application of in vitro experimental systems in drug-drug interactions
evaluation will be discussed, with emphasis on challenges and new approaches.
The use of in vitro assays to evaluate cytotoxic drug-drug interactions to aid
the understanding of drug toxicity and to identify possible risk factors will be
introduced.
9:45 AM
10:30 AM
Early Prediction of Drug-Drug Interactions in Pharmaceutical R&D:
Use of Reactive Oxygen Species to Assess CYP450 3A4 Inhibition (Kenneth R. Brouwer, Qualyst, Inc.;
Research Triangle Park, NC) Cytochrome P450 3A4 metabolizes more drugs than any other CYP enzyme,
and 3A4 inhibition may significantly impact the disposition of co-administered
therapeutics. Existing fluorescent 3A4
inhibition assays require use of fluorogenic probe substrates. Qualyst has developed ACCURATE, a
high-throughput assay measuring the 3A4 inhibition of compounds by using a
reactive oxygen species (ROS)-sensitive fluorescent probe. This assay permits the use of a
pharmaceutically-relevant substrate (e.g. testosterone) to assess inhibitory
potential of a compound, and provides improved predictability of LCMS
results.
10:30 AM 10:45 AM BREAK
10:45 AM
11:30 AM
Confounding Factors in the Identification of DDI in CYP2C9 (Dan Rock; Pfizer, Inc.;
St. Louis, MO)Fluorescent DDI screening aids in the
early identification of compounds that can influence CYP metabolism and act to
minimize the number of compounds that transition into secondary assays. DDI can be influenced by the presence of
multiple binding sites. Furthermore, multiple substrate binding conformations
can lead to multiple metabolites, both of which can complicate the
identification and interpretation of DDI.
CYP2C9 has been shown to bind multiple substrates. Fluorescent-based DDI is described in context
of incubation conditions and probe selection as factors that influence DDI
identification.
11:30 AM
12:15 PM
A Combined
Model Approach for Studying CYP450 Enzyme Induction and Inhibition (Odette A. Fahmi, Pfizer Inc.;
Groton, CT) It has been a common practice to study P450 induction
using primary hepatocyte cultures, while inhibition and time-dependent
inactivation studies are investigated using human liver microsomes. We
investigated the use of a novel human hepatocyte clone (Fa2N-4) as an
alternative reagent for identifying both
P450 inducers and inhibitors. Enzyme
activity and induction levels from total RNA extracts from Fa2N-4 cells treated
with a panel of known inducers and known time-dependent inactivators are
measured simultaneously. This presentation reviews the current status of
predicting CYP450 net outcome effect.
12:15 PM 1:30 PM LUNCH
1:30 PM 1:40 PM Exhibitor Presentation XenoTech, LLC
1:40 PM 2:25 PM
In Vitro Cell Model to Assess CYP Induction and
Mechanism-Based Inhibition Using Cryopreserved Human Hepatocytes (Jose Silva, Johnson & Johnson, PRD; Raritan, NJ) Primary cultured human hepatocytes are commonly used to assess the
potential for CYP induction. However,
the limited availability of fresh human hepatocytes is a major drawback. One of the promising developments that may
circumvent this limitation is the recent success in the cryopreservation of
human hepatocytes. This presentation
will focus on a 96-well plate cryopreserved human hepatocyte culture model
being used at Johnson & Johnson, PRD, to assess both CYP induction and
mechanism-based CYP inhibition by drug candidates.
2:25 PM
3:10 PM
Gene Expression Profiling in Preclinical Pharmaceutical
Research (Greg Slatter PhD,
Rosetta Inpharmatics LLC (A subsidiary of Merck & Co., Inc.; Seattle, WA) Gene expression profiling using DNA
microarrays has the potential to improve how the pharmaceutical industry
selects and develops drug candidates. Compound-specific transcriptional
fingerprints are identified that can indicate diverse biological
phenotypes. One experiment can transcend
chemical template-specific pharmacology to afford clues about potential adverse
events and effects on drug metabolism and transport. As gene annotations
improve, and genomic, metabonomic and proteomic technologies are integrated and
become mainstream, the biological sequelae of chemical exposure will be
elucidated earlier in development and in greater detail than ever before.
3:10 PM 3:25 PM BREAK
3:25 PM 4:10 PM
Standardizing CYP Inhibition Studies Through Optimum Use of Technology (Brian Ogilvie, XenoTech, LLC; Lenexa, KS) Several
in vitro approaches have been developed to assess the potential for a drug
(or other xenobiotic) to inhibit the human CYP enzymes involved in drug
metabolism. In recent years, advances in
automation and analytical techniques (and the availability of deuterated
metabolites) have allowed for more robust experimental design. The development of highly sensitive,
validated LC/MS/MS methods allows for the use of uniformly low microsomal
protein concentrations (≤0.1 mg/mL) and uniformly short incubation times
with marker substrate (5 minutes). In
addition, a simple combination of manual and automated liquid handling for
sample preparation can allow for high precision as well as higher
throughput. This presentation will
describe the preferred test system and experimental methods, and the sensible
application of technology to achieving these goals as well as the consequences
of poor experimental design.
4:10 PM 4:55 PM
Rationalization of Source Errors in Predicting of Inhibition-Based
Metabolic Drug-Drug Interactions from In Vitro Data
(Larry C Wienkers, Amgen, Inc.; Seattle WA)
The prediction of a drug's inhibitory potential are projected
based on the systemic concentration of the inhibitor [I] and the kinetic
parameters derived from in vitro experiments (Ki). Unfortunately,
misidentification of the drug's inhibitory potency can lead to inaccurate
predictions of in vivo drug-drug interactions potential. The current
presentation will describe the principles underlying the generation of in vitro
inhibition information and highlight sources of bias and error encountered in
generating these in vitro determinations.
4:55 PM 5:30
PM PANEL DISCUSSION: Session I
END OF DAY
Thursday, June 16, 2005
Session 2: Mechanisms of
Drug-Drug Interaction
Chair: James Halpert
8:00 AM 9:00 AM REGISTRATION
8:00 AM 5:00 PM EXHIBITS
8:50 AM 9:00 AM Exhibitor Presentation APSciences, Inc.
9:00 AM
9:45 AM
Structure-Function of Cytochromes P450: Implications
for Prediction of Drug Metabolism and Drug Interactions (James R. Halpert, University of
Texas Medical
Branch; Galveston
Texas) Progress in prediction
of cytochrome P450-mediated drug interactions requires knowledge of the
dynamics of enzyme-ligand interactions, including conformational changes that
accompany ligand binding. Our x-ray
crystal structures of ligand-free and ligand-bound P450 2B4 demonstrate a
remarkable conformational change from an open to a closed form upon ligand
binding. Studies of P450 3A4 suggest
that simultaneous binding of multiple ligands as well as conformational changes
contribute to cooperativity and partial inhibition, consistent with recent
x-ray crystal structures from other laboratories.
9:45 AM 10:30 AM
Time-Dependent CYP Inhibition: A Practical Industry Perspective (Tristan S. Maurer,
Pfizer Global Research and Development; Groton, CT) Drug-drug interaction mechanisms
arising from time-dependent alterations of functional enzyme concentrations are
particularly challenging to predict in a quantitative manner due to confounding
processes that regulate endogenous enzyme synthesis and degradation. Of
particular concern is mechanism-based CYP 3A4 inactivation, which substantially
increases clinical inhibitory potency beyond that expected from traditional
preclinical competitive interaction studies. Prediction approaches that account
for the additional complexity of this process do improve predictive accuracy
and are increasingly being incorporated into new strategies to reduce attrition
10:30 AM 10:45 AM BREAK
10:45 AM
11:30 AM
Differential Behavior of CYP3A4 and CYP3A5 in Drug-Drug Interactions (Kenneth
E. Thummel, University of Washington; Seattle, WA) Many metabolically-based drug-drug
interactions occur as the direct result of perturbations in the expression
and/or function of hepatic and intestinal CYP3A enzymes. Although most of the pharmacokinetic effects
of CYP3A modulators can be attributed to changes in CYP3A4 catalytic activity,
the polymorphic enzyme CYP3A5 may also be involved. Moreover, this factor may contribute to
inter-individual differences in the magnitude of an interaction observed in vivo, because CYP3A4 and CYP3A5 do
not behave identically with regard to substrate and inhibitor binding affinity
and the generation of reactive inhibitory metabolites. Recent in
vitro and in vivo from our lab
and others suggests that CYP3A5 is generally less sensitive to inhibition than
CYP3A4 by both reversible and irreversible processes. This presentation will explore the
mechanistic basis for these differences and potential clinical consequences. Supported
in part by NIH GM63666 and GM32165.
11:30 AM 12:15 PM
Complex Ligand Binding and Cytochrome P450 3A4 (William M. Atkins,
University of
Washington;
Seattle, WA) Cytochrome P450 3A4 is the major enzymatic
determinant of drug metabolism, and it exhibits complex ligand binding and
catalytic behavior, including positive and negative allosterism. These complexities hamper prediction of drug
clearance and drug-drug interactions. In
order to understand the molecular mechanisms underlying these allosteric
effects, surface plasmon resonance, 1H-15N-HSQC NMR, and
fluorescence methods have been used to monitor multiple ligand binding,
multiple ligand orientation, and protein conformational changes that contribute
to allosteric effect and drug interactions.
These methods demonstrate the importance of ligand dynamics, protein-protein
interactions, and protein dynamics in CYP3A4-dependent processes.
12:15
PM 1:30 PM LUNCH BREAK
1:30 PM
2:15 PM
Effect of Polymorphisms in P450s on PK in the Sprague-Dawley Rat (Tom Rushmore, Merck Research Labs, West
Point, PA) When
clearance of a drug is mediated by a single P450, significant variability in
exposure may be observed in humans and in animals. During basic discovery studies with Compound
X, a large variation in systemic exposure was observed in Sprague-Dawley rats
following oral dosing. The large
variability in exposure meant that no clear relationship between dose and
exposure could be established using the routine SD rat population(s). Genetic analysis of the CYP2D gene locus in
the SD rat revealed several polymorphisms whose presence was predictive for
systemic exposure of Compound X. Data
describing the identification, analysis and functional consequences (activity)
for several novel polymorphisms in several rat CYP2D genes will be presented.
2:15 PM
3:00 PM
Mechanism-based Inhibition of P450s: Drug Discovery Perspective
(Weichao Chen, Merck & Co., Inc.
San Diego,
CA) Many drugs
(such as erythromycin, fluvoxamine and ethynyl estradiol) cause drug
interactions through mechanism-based P450 inhibition. It is critical to select
candidates with minimum potential of mechanism-based inhibition of P450s in
early drug discovery. However, characterization of mechanism-based inhibitors
usually requires detailed enzyme kinetics studies to measure kinetic constants
(such as KI and Kinact). These studies typically
have limited throughput to support drug discovery programs. Specific examples
will be presented to illustrate how to utilize simplified assays to establish
structure-metabolism relationship and to reduce or remove the potential
liability of mechanism-based inhibition.
3:00 PM 3:30
PM PANEL DISCUSSION Session II
3:30 PM 3:45
PM BREAK
Session 3:
Pharmacokinetic Drug-Drug
Interaction and its Prediction from In Vitro ADME Data
Chair: Rene Levy
3:45 PM 4:30
PM
Quantitative Prediction of Clinical Induction Liability from In Vitro
Data (Bernard Murray, Abbott Laboratories, Chicago, IL)
Despite an increasing
understanding of the mechanisms governing induction of enzymes and transporters
there is still no means by which to reliably predict the quantitative liability
of drug candidates as inducers in man from in vitro data. We have found a
simple PD model to show promise in this regard but plasma protein binding and
active tissue uptake of inducers can still confound the plasma
concentration-response relationship.
4:30 PM 5:15
PM
Predicting Clinical DDI Potential Using Preclinical Parameters
(Raj Nagaraja,
Millenium Pharmaceuticals, Inc; Cambridge, MA)
Poly-pharmacy in patients is routine either to increase the effectiveness
of treating one disease, or to treat more than one disease. Pharmacokinetic
interaction between two concurrently administered drugs could potentially alter
the disposition properties of individual drugs and thus compromising the
treatment goal or leading to exaggerated toxicity. A large majority of compounds
and drug candidates are substrates for cytochrome P450 (CYP450) enzyme mediated
biotransformation in the liver. Qualitative nature and wide range of in vitro
parameters have compounded the DDI potential prediction of CYP inhibitors. In
vitro and in vivo pre-clinical and clinical parameters of known inhibitors of
CYP3A4 were used to reconstruct their pharmacokinetic profiles, and the
magnitude of interaction with midazolam, a probe substrate for CYP3A4, through
hybrid PBPK model. Use of liver distribution of these inhibitors in rats and Ki
values determined using midazolam as substrate generally improved the accuracy
of drug-drug interaction (DDI) predictions. This approach makes it possible to
use the model in drug discovery and development areas to predict the magnitude
of interaction of NCEs and guide the clinical groups to better design the
interaction studies.
END OF DAY
Friday, June 17, 2005
Session 3: Continued
Pharmacokinetic
Drug-Drug Interaction and its Prediction from In Vitro ADME Data
Chair: Rene Levy
8:00 AM 9:00
AM REGISTRATION
8:00 AM
12:00 PM EXHIBITS
9:00 AM 9:45 AM
Breast Cancer Resistance
Protein (BCRP) Mediated Drug-Drug Interactions in Drug Development
(Cindy Xia,
Millennium Pharmaceuticals, Inc., Cambridge, MA)
Breast cancer resistance protein (BCRP), also known as
ABCG2 or ABCP or MXR, is a member of the ATP-binding cassette (ABC) transporter
G family. BCRP functions as a biological barrier which extrudes xenobiotics out
of cells. Recent studies have demonstrated that BCRP plays an important role in
drug absorption, disposition, and elimination. In vitro and in vivo
strategies to evaluate hepatic and intestinal BCRP-mediated drug transports and
drug-drug interactions will be discussed.
9:45 AM - 10:30 AM
- PANEL
DISCUSSION Session III
10:30
AM 10:45 AM BREAK
Session 4:
Current Status of
Clinical Drug-Drug Interaction
Chair: Evan Kharasch
10:45 AM - 11:30 AM
Clinical Interactions with Long-Acting Opioids and HIV/AIDS Drugs
(Evan D. Kharasch, University of Washington; Seattle, WA)
Long-acting opioids are the primary therapy for treating opiate addiction and
chronic pain. HIV/AIDS drugs notoriously cause desired and undesirable
drug interactions, and HIV/AIDS-drug abuse co-treatment is common. While
in vitro metabolic drug interactions data abounds, there is less information on
the mechanism of clinical interactions. Recent clinical investigations, using in
vivo probes, have evaluated the mechanism of pharmacokinetic and pharmacodynamic
interactions between long acting opioids and HIV/AIDS drugs, and evaluated the
effects of HIV/AIDS drugs on the activities of intestinal and hepatic CYP3A and
transporters. These studies have shown some surprising differences from in vitro
results, requiring revision of long-accepted mechanisms and demonstrating the
necessity of clinical studies to confirm in vitro models.
11:30 AM -
12:15 PM
In Vivo Behavior of Sensitive CYP3A Substrates
(Isabelle Ragueneau-Majlessi,
University of
Washington;
Seattle,
WA)
CYP3A is a major determinant of first-pass metabolism of oral
drugs. Using the Metabolism & Transport Drug Interaction Database and DRUGS@FDA,
we obtained a comprehensive list of major CYP3A substrates. We studied the most
sensitive of these substrates (i.e. inhibited substrates with plasma AUC
values increased 5-fold or more) and evaluated: i) the contribution of
intestinal metabolism, ii) the possible role(s) of transporters and iii) the
practical consequences of such marked interactions.
12:15 PM -
1:00 PM
Differential Exposure
based on Subject Phenotype
(Carol
Collins, University of Washington, Seattle, WA)
The recent
concept paper on drug interaction studies (www.fda.gov/ohrms/dockets/ac/04/briefing/2004-4079B1_04_Topic2-TabA.pdf
) by the FDA indicates that minor pathways may become significant in subjects
lacking a particular enzyme (poor metabolizers). Consequently, the evaluation of
potential drug interactions via the minor pathway may be appropriate in these
subjects. This presentation will review currently available information in both
published literature and labeling monographs on increased drug exposure
resulting from inhibition of minor pathways in poor metabolizers.
1:00 PM -
1:30 PM - PANEL DISCUSSION: Session IV
END OF CONFERENCE
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