title: Selective inhibition of dog hepatic CYP2B11 and CYP3A12.
abstract: In the present study, N-(alpha-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 microM, 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 microM). In fact, complete inhibition of CYP3A12-dependent DZ C3-hydroxylation was possible at a low KET concentration (1 microM). 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.
authors.affiliation: Department of Drug Metabolism, Merck Research Laboratories, West Point, PA 19486, USA.
authors.forename: Ping
authors.initials: P
authors.lastname: Lu
authors.forename: Suresh B
authors.initials: SB
authors.lastname: Singh
authors.forename: Brian A
authors.initials: BA
authors.lastname: Carr
authors.forename: Yulin
authors.initials: Y
authors.lastname: Fang
authors.forename: Cathie Dong
authors.initials: CD
authors.lastname: Xiang
authors.forename: Thomas H
authors.initials: TH
authors.lastname: Rushmore
authors.forename: A David
authors.initials: AD
authors.lastname: Rodrigues
authors.forename: Magang
authors.initials: M
authors.lastname: Shou
mesh_terms: D000818:Animals; D001189:Aryl Hydrocarbon Hydroxylases / Q000037:antagonists & inhibitors* / Q000378:metabolism; D001665:Binding Sites / Q000187:drug effects / Q000502:physiology; D065607:Cytochrome P-450 Enzyme Inhibitors; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism; D000072467:Cytochrome P450 Family 2; D004285:Dogs; D004305:Dose-Response Relationship, Drug; D004791:Enzyme Inhibitors / Q000494:pharmacology; D007654:Ketoconazole / Q000494:pharmacology; D008099:Liver / Q000187:drug effects / Q000201:enzymology*; D013250:Steroid Hydroxylases / Q000037:antagonists & inhibitors* / Q000378:metabolism
chemical_list: D065607:Cytochrome P-450 Enzyme Inhibitors; D004791:Enzyme Inhibitors; D003577:Cytochrome P-450 Enzyme System; D013250:Steroid Hydroxylases; D001189:Aryl Hydrocarbon Hydroxylases; D000072467:Cytochrome P450 Family 2; C090278:cytochrome P-450 CYP2B11 (dog); C475779:cytochrome P-450 CYP3A12 (canine); D007654:Ketoconazole