Nefiracetam metabolism by human liver microsomes: role of cytochrome P450 3A4 and cytochrome P450 1A2 in 5-hydroxynefiracetam formation

An in-vitro study was conducted to investigate the metabolism of nefiraceta m in human liver microsomes and to identify the enzymes responsible for the metabolism. Nefiracetam was hydroxylated by human liver microsomes to 5-hy droxynefiracetam (5-OHN). Eadie-Hofstee plots for the formation of 5-OHN su ggested substrate activation. The kinetic parameters, apparent K-m, V-max, and Hill coefficient, for the formation of 5-OHN by pooled human liver micr osomes were 4012 muM, 2.66 nmol min(-1) (mg protein)(-1), and 1.65, respect ively. The formation of 5-OHN was significantly correlated with cytochrome P450 (CYP)3A4-mediated testosterone 6 beta -hydroxylase activity and dextro methorphan N-demethylase activity. The 5-OHN formation was inhibited (94 %) by antibody to human CYP3A4/5. The 5-OHN formation was also inhibited by t he CYP3A4 inhibitors ketoconazole and troleandomycin, but not significantly inhibited by several other P450 inhibitors. The microsomes containing cDNA -expressed CYP3A4 formed 5-OHN with sigmoidal kinetics. CYP3A5-containing m icrosomes did not form 5-OHN. These results indicated that CYP3A, most like ly CYP3A4, was the major isozyme responsible for the formation of 5-OHN in human liver microsomes. CYP1A2 and CYP2C19 microsomes were also capable of forming 5-OHN. However, the contribution of CYP1A2 was considered to be rel atively minor compared with that of CYP3A4, and the contribution of CYP2C19 was assumed to be negligible, based on the result of the immunoinhibition study and taking into account both the turnover rate by each isozyme and th e relative abundance of each isozyme in human liver. We conclude that on av erage the formation of 5-OHN, the major metabolite of nefiracetam, is princ ipally mediated by CYP3A4 with a relatively minor contribution by CYP1A2.