Conference Venue: San Diego Mission Valley Marriott

Featuring experts from the following institutions: Astex Technology, LLC; Bristol-Myers Squibb; Indiana University School of Medicine; Merck Research Labs; National Cancer Institute ; Pharmacia Corp.; Phase-1 Molecular Toxicology, Inc.; The Scripps Research Institute; University of Michigan; University of Pittsburgh; University of Texas Medical Branch; University of Tokyo; University of Washington; West Virginia University.

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Planning Committee Chairman:

Albert P. Li, PHASE-1 Molecular Toxicology, Inc.

Magang Shou, Merck Research Labs

Overview: Present and future technologies for the evaluation of drug-drug interaction potential (Albert P. Li; PHASE-1 Molecular Toxicology, Santa Fe, NM) In vitro human-based experimental systems: liver microsomes, hepatocytes, cDNA-expressed microsomes have been used extensively in the evaluation of drug-drug interactions potential. These techniques will be critically reviewed. The latest technologies, especially the application of expression genomics to evaluate enzyme induction and suppression, will be discussed.

Predicting Clinically Important Drug-drug Interactions (Stephen D. Hall, PhD Indiana University School of Medicine; Indianapolis, IN) Many clinically important drug-drug interaction arise from irreversible mechanisms of cytochrome P450 inhibition or other interactions that prolong the duration of inhibition. These non-reversible interactions require a modified approach to preclinical for drug interactions screening and mechanism specific prediction strategies. The common consequences of ignoring this class of drug interaction include the marketing of new drugs with unexpectedly high potency for cytochrome P450 inhibition and an unexpectedly prolonged duration of effect. Detection and prediction strategies for this class of drug interaction will be presented.

Predicting Metabolic Drug Interactions Using In Vitro Data: Are We There Yet? (A. David Rodrigues, Ph.D.; Merck Research Labs; West Point, PA) In recent years, it has become widely accepted that metabolic drug-drug interactions involving cytochromes P450 (CYP) can be forecast using in vitro data. However, numerous assumptions are made and many of the existing approaches, and models, are problematic and oversimplified. In reality, many compounds are inducers, do not exhibit simple competitive (reversible) inhibition, and are metabolized by non-CYP enzymes Therefore, quantitative predictions are difficult and many unforeseen drug-drug interactions occur despite weak inhibition of CYP enzymes in vitro.

Altering Cytochrome P450 Oxidation Reactions In Vitro: A Survey of Factors which Confound Successful In Vivo Extrapolations (Larry C. Wienkers; Pharmacia Corp.; Kalamazoo, MI) Atypical kinetics has been observed with a number of P450 enzymes, in particular CYP3A4. The unusual kinetic characteristics associated with CYP3A4 catalyzed reactions generally fall into one of four categories: activation of substrate oxidation by another compound (heterotropic activation); 2) autoactivation (homotropic activation); 3) partial inhibition and 4) substrate inhibition. Homotropic activation is most commonly characterized by a sigmoidal substrate velocity curve and has typically been rationalized by the binding of two substrate molecules within a single enzyme active site or by the existence of a separate allosteric. Although the multiple substrate-binding hypothesis may account for a large proportion of atypical kinetics associated with P450 reactions, it fails to consider the dynamic interplay between the P450 enzyme and its requisite co-enzyme(s). The presentation will highlight the role of protein-protein dynamics as well as other factors that may contribute to non-Michaelis-Menten kinetics observed with P450

Atypical Enzyme Kinetics: Their Effect on In Vitro-In Vivo Pharmacokinetic Predictions and Drug Interactions (Timothy S. Tracy; West Virginia University; Morgantown, WV) The observation of atypical enzyme kinetics, particularly involving cytochrome P450 enzymes, has become relatively common. This presentation will discuss various types of atypical enzyme kinetics observed and potential causes for their occurrence. Additionally, the effect of atypical kinetic profiles on in vitro-in vivo predictions of drug clearance and the potential for drug-drug interactions will be discussed.

Structure-function Analysis of Cytochromes P450 2B and 3A: Implications for Prediction of Substrate Specificity and Drug Interactions. (James R. Halpert, Ph.D.; The University of Texas Medical Branch
Galveston, TX) Homology models based on bacterial cytochrome P450 x-ray crystal structures or more recently P450 2C5 have been utilized to rationalize and predict inhibition and activation of cytochromes P450 from the 2B and 3A subfamilies. Site-directed mutagenesis has been invaluable in testing and validating the models. Recent efforts have focused on the role of substrate
access to the buried active site in determining rates and regioselectivity of substrate oxidation. X-ray crystallography of P450 2B enzymes is in progress.

Prediction of Inhibitory Drug Interactions using in vitro Inhibition Constants (Rene H. Levy; University of Washington; Seattle, WA) A number of approaches have been used to predict in vivo inhibitory drug-drug interactions based on in vitro data. These predictions are limited by two fundamental assumptions: (I) enzyme behavior in vitro is the same as in vivo, namely that the inhibition constant estimated by kinetic analysis of in vitro data using human liver microsomes and/or recombinant systems can be used as such; (ii) inhibitor concentration at the enzyme site can be estimated.using the free drug hypothesis or an alternative approach. We have conducted a number of studies with inhibitors such as fluconazole to demonstrate that the in vitro-in vivo gap in prediction can be quantified by the ratio Rki (in vitro Ki/in vivo Ki). We also used fluvoxamine as a model inhibitor because it inhibits CYP 1A2 and CYP2C19, and to a lesser extent CYP2C9, CYP2D6 and CYP3A4. It was found that the inhibition constants of fluvoxamine toward CYP1A2 and CYP2C19 are at least ten times greater in vitro than in vivo: specifically, the in vivo unbound Ki values of fluvoxamine for CYP1A2 and CYP2C19 were 3.6nM and 2nM, while the corresponding in vitro Ki values were 39nM and 76nM. Furthermore, the difference in Rki values for CYP1A2 and CYP2C19 was statistically different (10.8 vs. 38, p<0.05). Ongoing studies test several hypotheses (environmental effects, active metabolites, transport) to explain the in vitro-in vivo gap.

4:15 PM – 5:00 PM PANEL DISCUSSION: Present status and future approaches to accurately predict drug-drug interaction potential of pharmaceuticals

Prediction of Drug-Drug Interaction from Mechanism-Based Inhibition of Cytochrome P450 (Magang Shou, Ph.D., Department of Drug Metabolism, Merck Research Laboratories, West Point, PA ) It is now accepted widely that the cytochromes P450 (CYPs) represent the locus of most metabolic drug-drug interactions. The presentation will focus on the effects of mechanism-based CYP inhibitors on the pharmacokinetics (PK) of co-administered drugs. Both animal studies and in vitro studies will be described. In addition, a population PK model for enterohepatic recirculation (EHC) will be introduced to provide a reasonable approximation of individual subjects and to allow for an appropriate description of the PK properties of the given compounds in the presence of EHC.

Mechanism-Based Inactivation of Cytochrome P450s: Potential for Drug-Drug Interactions (Paul Hollenberg; University of Michigan; Ann Arbor, MI) Mechanism-based inactivation of the cytochromes P450 has resulted in a number of clinically significant drug interactions and is of considerable importance in many drug development programs. A large number of drugs undergo metabolic activation by P450s to very tight-binding or highly reactive intermediates that can then inactivate the P450 by binding to the heme or the apoprotein. Studies aimed at characterizing the kinetics and mechanism of inactivation, the site of adduct formation (heme or apoprotein), and the forms of P450 susceptible to inactivation by a candidate drug are extremely important in assessing the potential for drug-drug interactions in vivo. Functional groups shown to be problematic in this regard, approaches for identifying reactive intermediates and the sites of covalent adduct formation as well as the utilization of mechanism-based inactivators to gain information on the active site structures of these proteins will be discussed.

Role of Mechanism-based P450 Inhibition in Evaluation of P450 Drug-drug Interactions (Renke Dai; Bristol-Myers Squibb; Princeton, NJ) Clinically significant drug-drug interactions that derive from cytochrome P450 inhibition and induction pose potential liability concerns for pharmaceutical industry. In this presentation, in vitro mechanism-based P450 inhibition will be discussed in an attempt to predict the in vivo drug-drug interaction potential of drug candidates. This phenomenon becomes much more complicated when a drug candidate simultaneously inhibits and induces the same P450, where the pharmacokinetic drug-drug interaction is dependent on the balance between enzyme inhibition and induction.

How Important is the Role of P-Glycoprotein in Drug Interactions? (Jiunn Lin; Merck Research Labs; West Point, PA) Like CYP-mediated drug interactions, inhibition and induction of P-glycoprotein activity have been reported as causes of drug interactions. Because there is a striking overlapping substrate specificity between P-glycoprotein and CYP3A4, and these two proteins share similar inhibitors and inducers, many drug interactions may involve both P-glycoprotein and CYP3A4. Although attempts have been made to differentiate the relative contribution of P-glycoprotein and CYP3A4 to the overall drug interactions, there is still no simple way by which the relative contribution of these two systems can be quantified due to the complexity of the interplay involved between P-glycoprotein and CYP3A4. Unless the relative contribution can be quantitatively estimated, care should be taken in interpreting the role of P-glycoprotein in drug interactions.

3:15 PM – 4:00 PM – PANEL DISCUSSION: Clinical Significance of Mechanism-based and Transporter-based Drug-Drug Interactions

Crystal Structure of a Human Cytochrome P450 Enzyme (Harren Jhoti; Astex Technology Ltd.; Cambridge, UK)
Information on the 3-dimensional structure of the cytochrome P450 family of proteins has largely been derived from crystal structures of bacterial cytochrome P450 proteins. Despite many attempts, obtaining a crystal
structure of a human cytochrome P450 enzyme has, until now, proved intractable. Here we describe an approach that has resulted in the successful structure determination of a human cytochrome P450 enzyme. The crystal structure of the human cytochrome P450 enzyme will be described and
implications for rational design of drugs with improved metabolic profiles will be discussed.

Modeling Cytochrome P450 Interactions With Substrates and Inhibitors (Suresh B. Singh; Merck Research Laboratories, Rahway, NJ 07065) Models of cytochrome P450s and interactions with their substrates will be discussed. We will present quantitative/semi-quantitative models that allow us to predict binding orientations of CYP substrates. We developed a rapid semi-quantitative model for judging the relative susceptibility of different sites in drug molecules to metabolism by cytochrome P450 3A4. This model was used to calculate metabolically labile sites for 50 CYP3A4 substrates whose major sites of metabolism are known in literature. We will also discuss our quantitative models that are reasonably accurate in distinguishing inhibitors from non-inhibitors of CYPs.

Structural Studies of Substrate Complexes with Microsomal P450s (Eric F. Johnson; The Scripps Research Institute; La Jolla, CA) Structural studies of microsomal P450s can identify steric constraints and specific interactions that control the oxidation pathways in these enzymes. Our studies of 2C P450s indicate that adaptive conformational changes occur when substrates bind and that multiple modes of binding are event for some substrates. A combination of structural studies and functional assessment with panel of substrates will facilitate the prediction of drug-drug interactions related to P450mediatedmetabolism.

Exploring Potential Adverse Drug Reactions through Molecular Profiling (Roger Ulrich, Rosetta Inpharmatics-Merck Research Laboratories, Kirkland, WA) Examining the transcriptional output of the genome can provide valuable details on the biological effects of drugs. Currently, large-scale monitoring of gene expression is most effectively done using microarrays, which when appropriately analyzed can provide for the transcriptional evaluation of thousands of genes at one time. In addition to detecting downstream effects that result from interaction with the intended drug target, expression profiling illuminates effects on untoward targets including induction and repression of genes coding for drug metabolism and transport proteins, and genes that implicate cell dysfunction or death. Since microarrays have been constructed for the human genome as well as many animal toxicology models (mouse, rat, dog) it is possible to examine for potential species differences in responses to chemical exposure. Thus, gene expression profiling has enabled the toxicologist to more effectively identify compound hazards and, with a further understanding of the biological implications of altered gene expression, toxicogenomics may ultimately help better predict human health risks.

Selection and Application of Probe Substrates for Assessing Potential Drug-Drug Interactions In Vivo (Reginald Frye, University of Pittsburgh; Pittsburgh, PA) Determining the potential for drug-drug interactions early in drug development is critical. An attractive in vivo approach for screening for the potential for drug-drug interactions is the use of probe drugs selectively metabolized by an individual enzyme (e.g., cytochrome P450). The use of probe drugs, which can be administered individually or simultaneously (i.e., the “cocktail” approach), may provide valuable information on the selectivity, magnitude, and relevance of the in vivo effect(s) a NME may have on individual enzymes, including both inhibitory and/or inductive effects. This session will address the use of probe drugs and the cocktail approach for targeting clinical drug-drug interaction studies.

Glutathione S-transferase: Structure, Mechanism, and Structure-based Drug Design (Xinhua Ji; National Cancer Institute; Frederick, MD) Glutathione S-transferases (GST) catalyze the addition of glutathione to xenobiotic substrates that have an electrophilic functional group. Many tumors become drug resistant by over expressing the pi isoform of GST (GSTP). We are attempting to design agents that will overcome this drug resistance by generating NO, which is cytotoxic/cytostatic to tumor cells, selectively in the active site of GSTP. Structural information especially that for the transition state, has been playing a crucial role in this effort.

3:00 PM – 3:30 PM – PANEL DISCUSSION: In Silico Prediction of Drug-Drug Interactions

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