Catalytic role of cytochrome P4502B6 in the N-demethylation of S-mephenytoin.
In vitro methods were used to identify the cytochrome P450 (CYP) enzyme(s) involved in S-mephenytoin N-demethylation. S-Mephenytoin (200 microM) was incubated with human liver microsomes, and nirvanol formation was quantitated by reversed-phase HPLC. S-Mephenytoin N-demethylase activity in a panel of human liver microsomes ranged 35-fold from 9 to 319 pmol/min/mg protein and correlated strongly with microsomal CYP2B6 activity (r = 0.91). Additional correlations were found with microsomal CYP2A6 and CYP3A4 activity (r = 0.88 and 0.74, respectively). Microsomes prepared from human beta-lymphoblastoid cells transformed with individual P450 cDNAs were assayed for S-mephenytoin N-demethylase activity. Of 11 P450 isoforms (P450s 1A1, 1A2, 2A6, 2B6, 2E1, 2D6, 2C8, 2C9, 2C19, 3A4, and 3A5) tested, only CYP2B6 catalyzed the N-demethylation of S-mephenytoin with an apparent K(m) of 564 microM. Experiments with P450 form-selective chemical inhibitors, competitive substrates, and anti-P450 antibodies were also performed. Troleandomycin, a mechanism-based CYP3A selective inhibitor, and coumarin, a substrate for CYP2A6 and therefore a potential competitive inhibitor, failed to inhibit human liver microsomal S-mephenytoin N-demethylation. In contrast, orphenadrine, an inhibitor of CYP2B forms, produced a 51 +/- 4% decrease in S-mephenytoin N-demethylase activity in human liver microsomes and a 45% decrease in recombinant microsomes expressing CYP2B6. Also, both CYP2B6-marker 7-ethoxytrifluoromethylcoumarin O-deethylase and S-mephenytoin N-demethylase activities were inhibited by approximately 65% by 5 mg anti-CYP2B1 IgG/mg microsomal protein. Finally, polyclonal antibody inhibitory to CYP3A1 failed to inhibit S-mephenytoin N-demethylase activity. Taken together, these studies indicate that the N-demethylation of S-mephenytoin by human liver microsomes is catalyzed primarily by CYP2B6.
Heyn
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Department of Drug Metabolism and Pharmacokinetics, Rhône-Poulenc Rorer Research and Development, Collegeville, PA 19426-0107, USA.
White
R B
RB
Stevens
J C
JC
D000911:Antibodies, Monoclonal / Q000494:pharmacology; D000927:Anticonvulsants / Q000378:metabolism*; D001189:Aryl Hydrocarbon Hydroxylases; D003374:Coumarins / Q000494:pharmacology; D065637:Cytochrome P-450 CYP2A6; D065702:Cytochrome P-450 CYP2B6; D051544:Cytochrome P-450 CYP3A; D065607:Cytochrome P-450 Enzyme Inhibitors; D003577:Cytochrome P-450 Enzyme System / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D007527:Isoenzymes / Q000235:genetics / Q000378:metabolism*; D007700:Kinetics; D008617:Mephenytoin / Q000031:analogs & derivatives / Q000378:metabolism*; D008862:Microsomes, Liver / Q000187:drug effects / Q000201:enzymology*; D006899:Mixed Function Oxygenases / Q000235:genetics / Q000378:metabolism; D010089:Oxidoreductases, N-Demethylating / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D014217:Troleandomycin / Q000494:pharmacology
D000911:Antibodies, Monoclonal; D000927:Anticonvulsants; D003374:Coumarins; D065607:Cytochrome P-450 Enzyme Inhibitors; D007527:Isoenzymes; D011994:Recombinant Proteins; C014544:ethylphenylhydantoin; D003577:Cytochrome P-450 Enzyme System; C030123:coumarin; D014217:Troleandomycin; D006899:Mixed Function Oxygenases; D001189:Aryl Hydrocarbon Hydroxylases; C585710:CYP2A6 protein, human; C585599:CYP2B6 protein, human; C104464:CYP3A protein, human; D065637:Cytochrome P-450 CYP2A6; D065702:Cytochrome P-450 CYP2B6; D051544:Cytochrome P-450 CYP3A; C510163:CYP3A4 protein, human; D010089:Oxidoreductases, N-Demethylating; D008617:Mephenytoin