Selective Inhibition of Dog Hepatic Cytochromes P450 2b11 and 3a12

In the present study, N-(α-methylbenzyl-)-1-aminobenzotriazole (MBA) and ketoconazole (KET) were identified as the inhibitors with selectivity towards 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 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 CYP CO-binding spectrum. These data suggest that the inactivation involved covalent modification of CYP 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 CYP reaction phenotype studies with DLM using MBA and KET as selective inhibitors of CYP2B11 and CYP3A12, respectively. JPET #77651 4 Dog is an important and commonly-used species for the preclinical assessment of metabolism, pharmacokinetics, safety and efficacy in drug discovery and preclinical development. Cytochromes P450 (CYPs) in dog play a pivotal role in drug biotransformation and clearance. To date, nine dog cytochrome P450 genes have been been expressed individually as functional enzymes in baculovirus-insect cells, which have enabled the evaluation of their substrate selectivity and the identification of dog CYP isoform marker substrates (Shou et al., 2003). The advantage of such isoform markers is that they help screen new chemical entities as inhibitors of specific CYPs in pooled dog liver microsomes (DLM) and provide useful probes for selecting isoform-selective inhibitors that can be used to assess the contribution of a CYP, or subclass of CYP, to the metabolism of a drug or drug candidate. Many chemical inhibitors have been known to selectively inhibit human and animal CYPs. In the absence of CYP isoform selective substrates, however, their selectivity towards …