MECHANISMS OF DRUG INTERACTIONS AND REACTIVE METABOLITES

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The long-term objectives of the research described in this project are to understand mechanisms by which the widely used analgesic/antipyretic, acetaminophen, interacts with other drugs, and to characterize at the molecular level enzymes involved in the interactions. This is important to avoid life-threatening hepatic necrosis initiated by N-acetyl-p- benzoquinone imine, a reactive metabolite of acetaminophen formed by several cytochrome P450s that metabolize many other drugs. Results of previous studies implicate CYP2E1 and CYP2A6 as major catalysts in the oxidation of acetaminophen to its toxic quinoneimine and non-toxic catechol metabolites, respectively, and inhibition/induction of CYP2E1 by other drugs, such as isoniazid and ethanol, can increase or decrease risk of hepatotoxicity caused by acetaminophen depending on the time of ingestion relative to consequences of ligand regulation of CYP2E1. We propose to continue our studies of P450s responsible for the oxidation of acetaminophen in the next grant period. The first Specific Aim of this proposal is to construct active site models of CYP2E1 and CYP2A6 that are consistent with the different product selectivities of these two P450 isoforms for the quinoneimine and catechol metabolites of acetaminophen. A multi-tiered approach will be used that incorporate the results of NMR paramagnetic relaxation studies, homology and CoMFA modelling, mutagenesis experiments, and studies with mechanism- based inactivators. The second Specific Aim is to assess the roles of CYP2E1, CYP3A4 and dysregulation of hepatocyte mitochondrial glutathione status in the market toxicity of acetaminophen in individuals who chronically consume ethanol. This interaction has been suggested to be the leading cause of acute liver failure in the U.S. The interactions is multifaceted and the proposed studies continue an evaluation of this interaction initiated during the last grant period. A rat model will be used to investigate the separate effects of changes in acetaminophen reactive metabolite formation clearance and hepatocyte mitochondrial GSH status on hepatocellular damage. A series of studies will be conducted in human volunteers to determine the contribution of CYP2E1 and CYP3A P450 isoforms to acetaminophen reactive metabolite formation in vivo at acetaminophen doses relevant to the interaction and the effects that moderate drinking of alcoholic beverages and starvation have on this clinical syndrome.