Involvement of CYP2B6 in N-demethylation of ketamine in human liver microsomes

Ketamine is metabolized by cytochrome P450 (CYP) leading to production of p harmacologically active products and contributing to drug excretion. We ide ntified the CYP enzymes involved in the N-demethylation of ketamine enantio mers using pooled human liver microsomes and microsomes from human B-lympho blastoid cells that expressed CYP enzymes. The kinetic data in human liver microsomes for the (R)- and (S)-ketamine N-demethylase activities could be analyzed as two-enzyme systems. The K-m values were 31 and 496 muM for (R)- ketamine, and 24 and 444 muM for (S)-ketamine. Among the 12 cDNA-expressed CYP enzymes examined, CYP2B6, CYP2C9, and CYP3A4 showed high activities for the N-demethylation of both enantiomers at the substrate concentration of 1 mM. CYP2B6 had the lowest K-m value for the N-demethylation of (R)- and ( S)-ketamine (74 and 44 muM, respectively). Also, the intrinsic clearance (C Lint: V-max/K-m) of CYP2B6 for the N-demethylation of both enantiomers were 7 to 13 times higher than those of CYP2C9 and CYP3A4. Orphenadrine (CYP2B6 inhibitor, 500 muM) and sulfaphenazole (CYP2C9 inhibitor, 100 muM) inhibit ed the N-demethylase activities for both enantiomers (5 muM) in human liver microsomes by 60 to 70%, whereas cyclosporin A (CYP3A4 inhibitor, 100 muM) failed to inhibit these activities. In addition, the anti-CYP2B6 antibody inhibited these activities in human liver microsomes by 80%, whereas anti-C YP2C antibody and anti-CYP3A4 antibody failed to inhibit these activities. These results suggest that the high affinity/low capacity enzyme in human l iver microsomes is mediated by CYP2B6, and the low affinity/high capacity e nzyme is mediated by CYP2C9 and CYP3A4, CYP2B6 mainly mediates the N-demeth ylation of (R)- and (S)-ketamine in human liver microsomes at therapeutic c oncentrations (5 muM).