CATALYTIC ROLE OF CYTOCHROME P4502B6 IN THE N-DEMETHYLATION OF S-MEPHENYTOIN

In vitro methods were used to identify the cytochrome P450 (CYP) enzym e(s) involved in S-mephenytoin N-demethylation. S-Mephenytoin (200 mu M) was incubated with human liver microsomes, and nirvanol formation w as quantitated by reversed-phase HPLC. S-Mephenytoin N-demethylase act ivity in a panel of human liver microsomes ranged 35-fold from 9 to 31 9 pmol/min/mg protein and correlated strongly with microsomal CYP2B6 a ctivity (r = 0.91), Additional correlations were found with microsomal CYP2A6 and CYP3A4 activity (r = 0.88 and 0.74, respectively), Microso mes prepared from human beta-lymphoblastoid cells transformed with ind ividual P450 cDNAs were assayed for S-mephenytoin N-demethylase activi ty. 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 mu M. Experiments with P 450 form-selective chemical inhibitors, competitive substrates, and an ti-P450 antibodies were also performed. Troleandomycin, a mechanism-ba sed CYP3A selective inhibitor, and coumarin, a substrate for CYP2A6 an d therefore a potential competitive inhibitor, failed to inhibit human liver microsomal S-mephenytoin N-demethylation, In contrast, orphenad rine, 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 CYP2 B6-marker 7-ethoxytrifluoromethylcoumarin O-deethylase and S-mephenyto in N-demethylase activities were inhibited by similar to 65% by 5 mg a nti-CYP2B1 IgG/mg microsomal protein. Finally, polyclonal antibody inh ibitory to CYP3A1 failed to inhibit S-mephenytoin N-demethylase activi ty. Taken together, these studies indicate that the N-demethylation of S-mephenytoin by human liver microsomes is catalyzed primarily by CYP 2B6.