The long-term goal of this project is to assess the toxicological and pharmacological significance of large interindividual differences in P450 enzyme levels in human liver. We propose to determine the effects of freezing and storing human liver ont eh activity of P450 enzymes, to prepare inhibitory antibodies for in vitro studies of xenobiotic metabolism by human P450 enzymes, and to evaluate whether in vitro studies of xenobiotic metabolism with human-derived material accurately reflect the metabolic fate of xenobiotics in humans. The overall hypothesis is that in vitro studies of xenobiotic metabolism with human-derived material can be used to predict the metabolic fate of xenobiotics in humans. One of our specific aims is to test the hypothesis that, in humans, the O- demethylation and N-demethylation of dextromethorphan are catalyzed by CYP2D6 and CYP3A enzymes, respectively, and to evaluate dextromethorphan (cough syrup) as an in vivo probe of CYP2D6 and CYP3A activity in human volunteers. The goal of this study is to develop a non-invasive and inexpensive method to measure CYP3A activity in human volunteers. The goal of this study is to develop a non-invasive and inexpensive method to measure CYP3A activity in vivo, which varies more than 30 fold among human subjects and which determines the metabolic fate of numerous drugs and chemicals of environmental concern. Two other specific aims focus on cases where in vitro data appear to conflict with clinical observations of drug metabolism. The first case concerns diazepam and omeprazole. Poor metabolizers of S-mephenytoin are also poor metabolizers of diazepam and omeprazole, which suggests that the same CYP2C enzyme metabolizes all three drugs. However, our preliminary data suggest that neither diazepam nor omeprazole is a substrate for CYP2Cmp, which catalyzes the 4'-hydroxylation of S.mephenytoin. We propose to identify which human P450 enzymes are responsible for metabolizing diazepam and omeprazole as our initial approach to reconciling the conflicting data from in vivo and in vitro studies. The second case concerns the antipsychotic drug, haloperidol. The major urinary metabolites of haloperidol are formed by N- dealkylation.Theoretically, this reaction cannot be catalyzed by CYP2D6 (based on current models of its active site). Nevertheless, individuals lacking CYP2D6 are poor metabolizers of haloperidol. A clue to this paradox is that haloperidol is rapidly reduced in blood and liver, hence, we hypothesize that the rate-limiting step in the elimination of haloperidol is the re-oxidation of reduced haloperidol to haloperidol by CYP2D6. We also postulate that the enzyme responsible for N-dealkylating haloperidol is the same enzyme that generates a neurotoxic pyridinium metabolite, HPP+. The conflict between in vitro and in vivo observations must be resolved before in vitro approaches to reaction phenotyping can be used to accurately predict the metabolic fate of xenobiotics in humans. If such approaches can be validated, it will be possible to predict (1) which chemicals will likely inhibit the metabolism of a xenobiotic, (2) which chemicals will likely induce the metabolism of a xenobiotic, and (3) which ethnic groups will have a high incidence of poor and extensive metabolizers. These studies are significant because reaction phenotyping in vitro with a potentially toxic chemical, coupled with in vivo phenotyping with a relatively harmless chemical (or genotyping), will improve risk assessments and enable exposure levels to be determined on an individual basis.
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