Enzyme kinetics of clausenamide enantiomers in rat liver microsomes

AimTo investigate the enzyme kinetics of (-)-3S,4r,5r,6S-clausenamide［(-)-Clau］ and (+)-3r,4S,5S,6r-clausenamide［(+)-Clau］ catalyzed by rat liver microsomes and compare their stereoselective differences. MethodsAn In Vitro metabolic system was built by using rat liver microsomes and NADPH-generating system. Clau and its metabolites were determined simultaneously by a reversed-phase high performance liquid chromatography. The kinetic parameters, K_m, V_max, and metabolic rate, V_max/K_m, were calculated by Eadie-Hofstee plot. ResultsIn the metabolic system, (-)-Clau was found to be mainly metabolized to 7-hydroxy-, 5-hydoxy- and 4-hydroxy-Clau, and 7-hydroxylation was a preferential pathway which exhibited higher V_max/K_m value (0.135 μＬ·min^-1·mg^-1) than those of 5-　and 4-hydroxylation (0.063 and 0.068　μＬ·min^-1·mg^-1, respectively). For (+)-Clau, it was mainly metabolized to 4-hydroxy-Clau, whereas 7-hydroxy- and 4-hydroxy-Clau were so small that they could not be detected systematically. 4-Hydroxylation of (+)-Clau showed highest V_max/K_m value (0.547　μＬ·min^-1·mg^-1) among all the metabolites tested, which was 8.0 times　higher than that of 4-hydroxylation of its antipode. ConclusionThe data indicated that there were obvious substrate stereoselective differences in the hydroxylation metabolism of (+)- and (-)-Clau, which provided an explanation of the difference of pharmacokinetic characteristics of Clau enantiomers in rats.