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-demethylation of nirvanol-free (S)-mephenytoin [(S)-MP] in vitro. In mixed HLMs, the kinetics of (S)-MP N-demethylation suggested two contributing activities. A high-affinity/low-capacity component exhibited a KM of 174.1 microM and a Vmax of 170.5 pmol/mg protein/min, whereas a low-affinity/high-capacity component exhibited a KM of 1911 microM and a Vmax of 3984 pmol/mg protein/min. The activity of the high-affinity component 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 KM value (150 +/- 42 microM) derived for recombinant CYP2C9 was close to that obtained for the high-affinity/low-capacity component in mixed HLMs (KM = 174.1 microM). The predicted contribution of this activity at concentrations (1-25 microM) achieved after a single 100-mg dose of racemic MP is approximately 30% of the rate of nirvanol formation. At concentrations of >1000 microM, we estimate that >90% of the rate can be explained by the low-affinity activity (CYP2B6). Therefore, the N-demethylation of (S)-MP to nirvanol may be a useful means of probing the activity of CYP2B6 in vitro when concentrations of >1000 microM are used, but it is unlikely to be a suitable phenotyping tool for this isoform in vivo, where concentrations of >1000 microM are rarely encountered.
Ko
J W
JW
Division of Clinical Pharmacology, Department of Medicine, Georgetown University Medical Center, Washington, DC 20007, USA.
Desta
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Flockhart
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D000927:Anticonvulsants / Q000378:metabolism / Q000493:pharmacokinetics*; D001189:Aryl Hydrocarbon Hydroxylases; D065702:Cytochrome P-450 CYP2B6; D065729:Cytochrome P-450 CYP2C9; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism*; D006801:Humans; D007527:Isoenzymes / Q000378:metabolism; D007700:Kinetics; D008617:Mephenytoin / Q000031:analogs & derivatives / Q000378:metabolism / Q000493:pharmacokinetics*; D008745:Methylation; D008862:Microsomes, Liver / Q000378:metabolism; D010089:Oxidoreductases, N-Demethylating / Q000378:metabolism*; D010641:Phenotype; D013237:Stereoisomerism; D039181:Steroid 16-alpha-Hydroxylase; D013250:Steroid Hydroxylases / Q000378:metabolism*; D013379:Substrate Specificity
D000927:Anticonvulsants; D007527:Isoenzymes; C014544:ethylphenylhydantoin; D003577:Cytochrome P-450 Enzyme System; D013250:Steroid Hydroxylases; C450260:CYP2C9 protein, human; D065729:Cytochrome P-450 CYP2C9; D001189:Aryl Hydrocarbon Hydroxylases; C585599:CYP2B6 protein, human; D065702:Cytochrome P-450 CYP2B6; D039181:Steroid 16-alpha-Hydroxylase; D010089:Oxidoreductases, N-Demethylating; D008617:Mephenytoin