Metabolically-based drug-drug interactions are a major cause of morbidity and mortality in the therapeutic treatment of disease. Although significant advances have been made in our understanding of the etiology of such adverse drug reactions, quantitative prediction of these events at early stages of drug development and clinical use remains elusive. This Program Project Grant focuses on understanding the in vitro to in vivo relationships for drug-drug interactions involving the human cytochrome P450 family of enzymes and drug transporters. In the current proposal, we will investigate several emerging factors that complicate our ability to accurately predict changes in drug metabolizing enzyme and drug transporter activity that result from the co-administration of multiple interacting species; (i) non-hyperbolic kinetics and cooperative ligand binding to human P450s, (ii) sequential oxidative metabolism and the formation of metabolic-intermediate (MI) complexes, (iii) the variable contribution made by Type II binding to inhibitor Ki, (iv) the role(s) of drug transporters such as the human OCTs, MDRs and MRPs, and (v) the interplay of drug-metabolizing enzymes and drug transporters in drug interactions associated with induction mechanisms. In Project 1 we will synthesize novel high affinity inhibitors for human CYP2C enzymes and combine this with site-directed mutagenesis studies to enable construction of discriminating computational and structural models for the prediction of inhibitor Ki. In Project 2, we will explore the mechanistic basis for 'allosteric' kinetic behavior with CYP3A4, towards a variety of ligands including caffeine, acetaminophen, pyrene and hypericin using fluorescence and NMR-based approaches. In Project 3, we will explore reasons for the discrepancy between in vivo and in vitro Kis for the test compounds, itraconazole and fluvoxamine and develop new kinetic models for sequential metabolism and MI complex formation with macrolides and antidepressants. In Project 4 we will determine the molecular and genetic factors that influence, both in vivo and in vitro, the magnitude and spectrum of human P450 enzymes and drug transporters induced by the anti-HIV protease inhibitors, nelfinavir and ritonavir. Collectively, these studies should provide new conceptual and practical tools to achieve quantitative predictions of drug-drug interactions.
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