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Cytochrome P450 3A-Dependent Metabolism of a Potent and Selective γ-Aminobutyric Acid Aα2/3 Receptor Agonist in Vitro: Involvement of Cytochrome P450 3A5 Displaying Biphasic Kinetics

Drug Metabolism and Disposition, ISSN: 0090-9556, Vol: 35, Issue: 8, Page: 1301-1307
2007
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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-2 H -1,2,4-triazol-3-ylmethoxy)-3-(2-fluorophenyl)-1,2,4-triazolo[4,3 b ]pyridazine (TPA023), a selective agonist of human γ-aminobutyric acid A receptor α2 and α3 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 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 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, and CYP3A5 may also contribute at higher concentrations of the compound.

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