Metabolically-based, inhibitory drug-drug interactions are a major cause of morbidity and mortality in the therapeutic treatment of diseases. Although significant advances have been made in our understanding of the etiology of such adverse interactions, quantitative prediction of these events at early stages of drug development and clinical use remains elusive. Understanding the in vitro to in vivo relationships for interactions involving the major family of drug metabolizing enzymes, the cytochromes P450 (CYP), has been the focus of this Program Project since its inception. In the current proposal, we will examine four factors that we believe may underlie our inability to predict accurately changes in drug metabolism during polytherapy: (1) the relationship between inhibitor concentration in circulating blood and at the site of drug biotransformation (i.e., at the enzyme active site); (2) the relationship between substrate and inhibitor structure and binding affinity for the active site; (3) the dynamic interplay between simultaneous enzyme induction and inhibition from a single interacting agent; and (4) time-dependent and mechanism- based changes in intestinal first-pass metabolism following oral drug administration. In Project 1, we will determine for a series of CYP2C and 1A2 inhibitors, how inhibitor lipophilicity and plasma protein binding affect the concentration of the inhibitor at the enzyme active site and ultimately its in vivo potency. In Project 2, we will combine computational modeling with site-directed mutagenesis to identify the structural determinants that make a drug either a good inhibitor or good substrate of CYP2C9 and CYP2C19. In Project 3, we will determine how certain orally administered inhibitors of CYP3A4 preferentially alter intestinal, rather than hepatic, first-pass metabolism. Finally,, for Project 4, we will determine whether the paradoxical inhibitory and inductive effects of small, polar drug molecules on CYP2E1- and CYP2A6- dependent drug metabolism is mediated, in part, by their ability to interfere with the interaction between the enzyme and its important co- enzyme, cytochrome b/5. Overall, the studies proposed in this Program Project grant should provide major conceptual and methodological advances for the field of drug metabolism and improve our ability to develop safe, new therapeutic agents and manage existing drug therapies.