MECHANISMS OF DRUG INTERACTIONS AND REACTIVE METABOLITES

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The long-term objectives of the research described in this application are to understand, at the molecular level, the mechanisms of interaction between drugs that are biotransformed to toxic metabolites and other drugs which modulate the toxicity. Knowledge of factors involved in the generation and disposition of reactive metabolites is important to the safe use of drugs, and can provide insights into biochemical and chemical pathways that form toxic metabolites. Specifically in this investigation, mechanisms of activation and induction of cytochrome P450s that catalyze the oxidation of the widely used analgesic/antipyretic, acetaminophen, will be investigated. The rationale for these studies is that acetaminophen can cause life-threatening hepatic necrosis which is primary mediated by N-acetyl-p-benzoquinone imine, a P450 oxidative metabolite of acetaminophen, and other drugs used with acetaminophen may increase the formation rate of this toxic metabolite. First, the mechanism of activation of CYP3A2-mediated acetaminophen oxidation by caffeine will be examined. Antibodies prepared to P450 reductase and cytochrome b5, and site-directed mutants to CYP3A2 will be used to determine which amino acid residues are involved in the activation process. Sites for mutation will be chosen based on the finding that CYP3A1, which is ~90% homologous to CYP3A2, is not activated significantly by caffeine. Secondly, the mechanism of induction of CYP2E1 by the antituberculosis drug, isoniazid, will be investigated by spectral and kinetic studies of the stability of apoprotein, holoprotein, and mRNA. The time-course of drug interactions of isoniazid with acetaminophen and the more specific CYP2E1 substrate, chlorzoxazone, will be examined in detail in both humans and rats, to develop a model for interactions of drug that may stabilize cytochromes P450 against degradation. Third, the hypothesis that the new gastric acid antisecretory drug, omeprazole, increases the formation clearance of N-acetyl-p-benzoquinone imine from acetaminophen in humans by inducing CYP1A2, will be tested. The effects of omeprazole treatment and pretreatment on the metabolism of acetaminophen and on the 3-demethylation of caffeine, a CYP1A2 probe reaction, will be determined in human volunteers. Finally, the major human isoforms that catalyze acetaminophen oxidation to the toxic quinone imine and the non-toxic catechol metabolite, 3- hydroxyacetaminophen, will be identified. NMR paramagnetic relaxation methods will then be used to determine if formation rates of the two products correlate with the orientation of acetaminophen at the active site of the P450 isoforms identified.