Received for publication January 10, 2007.
Revised April 13, 2007.
Accepted for publication April 23, 2007.

CYTOCHROME P450 3A-DEPENDENT METABOLISM OF A POTENT AND SELECTIVE GABA-A_2/3 RECEPTOR AGONIST IN VITRO: INVOLVEMENT OF CYTOCHROME P450 3A5 DISPLAYING BIPHASIC KINETICS

Abstract

In vitro metabolism studies were conducted to determine the human cytochrome P450 enzyme(s) involved in the biotransformation of TPA023 [7-(1,1-dimethylethyl)-6-(2-ethyl-2H-1,2,4-triazol-3-ylmethoxy)-3-(2-fluorophenyl)-1,2,4-triazolo[4,3b]pyridazine], a selective agonist of human -aminobutyric acid_A receptor 2 and 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 µM, respectively) and a high K_m (12.7 and 40.0 µM, respectively) component. For both metabolites, the high K_m 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 activites 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 µM. 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, while CYP3A5 may also contribute at higher concentrations of the compound.

Key words: CYP3A, kinetics