Toxicological consequences may occur when drug interactions involve drugs with narrow therapeutic indices such as the cytocbrome P450 2C9 (CYP2C9) substrates warfarin and phenytoin. To minimize risk to humans, accurate predictions of in vivo drug metabolism, pharmacokinetics and drug interactions from in vitro data are needed. Recently, careful examination of drug metabolism kinetic data has demonstrated that atypical kinetics of cytochrome P450 mediated reactions may occur with greater frequency than previously believed. Atypical kinetic profiles such as autoactivation, activation and substrate inhibition have been observed for several P450 isoforms including CYP2C9. These atypical kinetic processes may be either homotropic (involving only one substrate) or heterotropic (involving a substrate and another effector/substrate). Since these kinetic patterns are observed in vitro and investigators use these in vitro data to predict in vivo pharmacokinetics, it is imperative that the mechanisms for these interactions be studied to form a better foundation for in vitro-in vivo predictions of human drug metabolism and drug interactions. We hypothesize that critical amino acid changes in the CYP2C9 enzyme can alter the degree of cooperativity observed, that key molecule structural characteristics are required for heterotropic cooperativity, that these cooperativity interactions can be modeled and cooperativity can be observed in vivo. Using expressed CYP2C9 allelic variants and site-directed mutagenesis, metabolism of CYP2C9 substrates, which exhibit various kinetic profiles, and their interactions with a known effector (and a series of structural analogs) will be studied and modeled. Additionally, in vivo studies of a CYP2C9 substrate and a known (in vitro) effector will be conducted. Taken together, these results will improve CYP2C9 in vitro-in vivo predictive capabilities.
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