HUMAN N-DEMETHYLATION OF (S)-MEPHENYTOIN BY CYTOCHROME P450S 2C9 AND 2B6

We tested the ability of human liver microsomes (HLMs) and recombinant human cytochrome P450 (CYP or P450) isoforms to catalyze the N-demeth ylation of nirvanol-free (S)-mephenytoin [(S)-MP] in vitro. In mixed H LMs, the kinetics of (S)-MP N-demethylation suggested two contributing activities. A high-affinity/low-capacity component exhibited a K-M of 174.1 mu M and a V-max of 170.5 pmol/mg protein/min, whereas a low-af finity/high-capacity component exhibited a K-M of 1911 mu M and a V-ma x of 3984 pmol/mg protein/min. The activity of the high-affinity compo nent was completely abolished by sulfaphenazole, with little effect on the low-affinity component. Of the recombinant P450 isoforms tested, only CYP2B6 and CYP2C9 formed nirvanol from (S)-MP. The K-M value (150 +/- 42 mu M) derived for recombinant CYP2C9 was close to that obtaine d for the high-affinity/low-capacity component in mixed HLMs (K-M = 17 4.1 mu M). The predicted contribution of this activity at concentratio ns (1-25 mu M) achieved after a single 100-mg dose of racemic MP is ap proximately 30% of the rate of nirvanol formation, At concentrations o f >1000 mu M, we estimate that >90% of the rate can be explained by th e low-affinity activity (CYP2B6). Therefore, the N-demethylation of (S )-MP to nirvanol may be a useful means of probing the activity of CYP2 B6 in vitro when concentrations of >1000 mu M are used, but it is unli kely to be a suitable phenotyping tool for this isoform in vivo, where concentrations of >1000 mu M are rarely encountered.