Abstract
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)-MPN-demethylation suggested two contributing activities. A high-affinity/low-capacity component exhibited aKM of 174.1 μM and aVmax of 170.5 pmol/mg protein/min, whereas a low-affinity/high-capacity component exhibited aKM of 1911 μM and aVmax 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 μM) derived for recombinant CYP2C9 was close to that obtained for the high-affinity/low-capacity component in mixed HLMs (KM = 174.1 μM). The predicted contribution of this activity at concentrations (1–25 μM) achieved after a single 100-mg dose of racemic MP is approximately 30% of the rate of nirvanol formation. At concentrations of >1000 μM, 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 μM are used, but it is unlikely to be a suitable phenotyping tool for this isoform in vivo, where concentrations of >1000 μM are rarely encountered.
Footnotes
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Send reprint requests to: David A. Flockhart, M.D., Ph.D., Division of Clinical Pharmacology, Georgetown University Medical Center, 3900 Reservoir Road, NW, Washington DC, 20007.
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This work was supported in part by United States Public Health Service Grant T32-GM08386 from the National Institute of General Medical Sciences and by a fellowship award to J.-W.K. from the World Health Organization (WPRO 0630/95).
- Abbreviations used are::
- MP
- mephenytoin
- CYP or P450
- cytochrome P450
- HL
- human liver
- HLMs
- human liver microsomes
- Received August 13, 1997.
- Accepted April 17, 1998.
- The American Society for Pharmacology and Experimental Therapeutics
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