Cytochrome P450 3A-dependent metabolism of a potent and selective γ-aminobutyric AcidAα2/3 receptor agonist in vitro:: Involvement of cytochrome P450 3A5 displaying biphasic kinetics

In vitro metabolism studies were conducted to determine the human cytochrome P450 enzyme(s) involved in the biotransformation of 7-(1,1-dimethylethyl)-6-(2-ethyl-2H-1,2,4-triazol-3-ylmethoxy)-3( 2-fluorophenyl)-1,2,4-triazolo[4,3b] pyridazine (TPA023), a selective agonist of human gamma-aminobutyric acid(A) receptor alpha 2 and alpha 3 subunits. Incubation of TPA023 with NADPH-fortified human liver microsomes resulted in the formation of t-butyl hydroxy TPA023, N-desethyl TPA023, and three minor metabolites. Both t-butyl hydroxylation and N-deethylation reactions were greatly inhibited (>85%) in the presence of CYP3A-selective inhibitory antibodies and chemical inhibitors, indicating that members of the CYP3A subfamily play an important role in TPA023 metabolism. Eadie-Hofstee plots of t-butyl hydroxylation and N-deethylation in pooled CYP3A5-rich human liver microsomes revealed a low K-m (3.4 and 4.5 mu M, respectively) and a high K-m (12.7 and 40.0 mu M, respectively) component. For both metabolites, the high Km component was not observed with a pool of microsomal preparations containing minimal levels of CYP3A5. Preincubation of liver microsomes with mifepristone (selectivity for CYP3A4 > CYP3A5) greatly inhibited both t-butyl hydroxylation and N-deethylation (> 75%); however, the residual activities were significantly higher in the pooled CYP3A5-rich liver microsomes (p < 0.0005). In addition, elevated levels of residual t-butyl hydroxylase and N-deethylase activities were observed in the presence of both CYP3A5-rich and CYP3A5-deficient preparations when the substrate concentration increased from 4 to 40 mu M. In agreement, metabolite formation catalyzed by recombinant CYP3A5 was described by a biphasic model. It is concluded that CYP3A4 plays a major role in TPA023 metabolism, and CYP3A5 may also contribute at higher concentrations of the compound.