Selectivity and Mechanisms of Human Cytochrome P450 Inhibition by Chlormethiazole

Abstract

The cytochrome P450 (CYP) superfamily of enzymes are heme containing enzymes that primarily perform oxidative reactions to a variety of substrates, including small molecule drugs and other xenobiotics. Formation of polar metabolites by metabolism of small molecule drugs by CYP enzymes is often a major determinant of the clearance of those drugs, and changes in the activity of the enzymes involved in the metabolism of a particular xenobiotic can lead to alteration of pharmacokinetics for that xenobiotic. The most frequent cause for change in CYP activity is enzyme inhibition, which can result in accumulations of a xenobiotic, and those accumulations have the potential to result in overdose toxicity. However, CYP inhibition can be beneficial if the formation of toxic metabolites can be blocked, or a desired increase in drug exposure is desirable. CYP mediated drug-drug interactions have been the focus of intensive research since they are common and potentially harmful, and can have dramatic implications for drug development. This dissertation explores the selectivity of CYP inhibition by the GABAA agonist chlormethiazole (CMZ) and the mechanisms of inhibition of cytochrome P450 2E1 (CYP2E1), 2B6 (CYP2B6), and 3A4/5 (CYP3A4/5) by CMZ. CMZ elicits a clinically significant drug-drug interaction (DDI) with chlorzoxazone due to potent CYP2E1 inhibition in vivo. It has been reported that chlormethiazole is a time-dependent inhibitor of CYP2E1 and CYP2B6, but the mechanisms were not investigated. Herein, we identified CYP3A4/5 as being susceptible to time-dependent inhibition by CMZ, and we were able to identify a potential role for an N-oxide metabolite in the mechanisms of time-dependent inhibition of CYP2E1 and CYP3A4/5. We also determined that time- dependent inhibition of CYP2B6, 2E1, and CYP3A4/5 by CMZ was NADPH-dependent and irreversible by dialysis. The formation of CMZ N-oxide by CYP2E1 and CYP3A4/5 appears to play a major role in the inactivation of these enzymes. Thiazole N-oxides are quasi-stable carbenes, which should be capable of forming metabolic intermediate (MI) complexes and possibly N-alkylated porphyrins, as other carbenes do. We found that CYP3A4 makes a MI complex when incubated with CMZ, and that the addition of dithionite and CMZ N-oxide to purified CYP3A4 replicates the observed complex. In the case of CYP2E1, we were able to identify an unstable alkylated porphyrin species that consisted of a mono-oxygenated CMZ metabolite attached to protoporphyrin IX, and we were able to generate a MI complex with the addition of CMZ N-oxide and dithionite to purified CYP2E1. Therefore, we propose that both enzymes generate MI complexes with CMZ N-oxide, but the MI complex of CYP2E1 can rearrange to an unstable N-alkylated porphyrin species. CYP2B6 mechanistic data was inconclusive, but does not refute the idea that the N-oxide metabolite is playing a role in the time-dependent inhibition. No distinct spectral changes or adducts were detected in recombinant CYP2B6 incubations. However, CMZ N-oxide is produced by CYP2B6, so it is possible that the same unstable alkylated porphyrin found for CYP2E1 could be formed by CYP2B6 at a rate that is too low to be detected. In summary, we have investigated the selectivity of inhibition for the major liver CYP isoforms by CMZ and the mechanisms of time-dependent inhibition for CYP2E1, CYP2B6, and CYP3A4 by CMZ. The N-oxide metabolite of CMZ appears to play a major role in the time-dependent inhibition of CYP2E1 and CYP3A4, and that effect is likely mediated by the formation of its carbene tautomer. The mechanism of time-dependent inhibition of CYP2B6 by CMZ is not clear since we do not have direct evidence of the molecular target of inhibition.