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ABSORPTION, DISTRIBUTION, METABOLISM, AND EXCRETION

Selective Inhibition of Dog Hepatic CYP2B11 and CYP3A12

Department of Drug Metabolism, Merck Research Laboratories, West Point, Pennsylvania

In the present study, N-(-methylbenzyl-)-1-aminobenzotriazole (MBA) and ketoconazole (KET) were identified as the inhibitors with selectivity toward dog CYP2B11 and CYP3A12, respectively. Their selectivity was evaluated using phenacetin O-deethylation (CYP1A), diazepam (DZ) N1-demethylation (CYP2B11), diclofenac 4'-hydrxylation (CYP2C21), bufuralol 1'-hydroxylation (CYP2D11), and DZ C3-hydroxylation (CYP3A12) activities in dog liver microsomes (DLM). MBA exhibited potent mechanism-based inhibition of DZ N1-demethylase activity catalyzed by both baculovirus-expressed CYP2B11 and DLM. In both cases, inhibition was characterized by a low K_I (0.35 and 0.46 µM, respectively) and high k_inact (1.5 and 0.56 min^–1, respectively). Despite complete loss of DZ N1-demethylase activity in the presence of MBA, there was no significant loss of cytochrome P450 (P450) CO-binding spectrum. These data suggest that the inactivation involved covalent modification of P450 apoprotein, instead of the prosthetic heme moiety. A homology model of CYP2B11 was constructed, based on the crystal structure of rabbit CYP2C5, for docking the substrate (DZ) and the inhibitor (MBA), respectively. The model, within the limits of our approximations, helped explain the substrate specificity and inhibitor selectivity of CYP2B11. In contrast to MBA, KET was identified as a potent and selective reversible (competitive) inhibitor of CYP3A12 (K_I = 0.13–0.33 µM). In fact, complete inhibition of CYP3A12-dependent DZ C3-hydroxylation was possible at a low KET concentration (1 µM). Therefore, it is concluded that one can attempt to conduct P450 reaction phenotype studies with DLM using MBA and KET as selective inhibitors of CYP2B11 and CYP3A12, respectively.

Address correspondence to: Dr. Magang Shou, Department of Drug Metabolism, WP75A-203, Merck Research Laboratories, West Point, PA 19486. E-mail: magang_shou{at}merck.com

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