Abstract

Ketamine is metabolized by cytochrome P450 (CYP) leading to production of pharmacologically active products and contributing to drug excretion. We identified the CYP enzymes involved in theN-demethylation of ketamine enantiomers using pooled human liver microsomes and microsomes from human B-lymphoblastoid cells that expressed CYP enzymes. The kinetic data in human liver microsomes for the (R)- and (S)-ketamineN-demethylase activities could be analyzed as two-enzyme systems. The K _m values were 31 and 496 μM for (R)-ketamine, and 24 and 444 μM for (S)-ketamine. Among the 12 cDNA-expressed CYP enzymes examined, CYP2B6, CYP2C9, and CYP3A4 showed high activities for theN-demethylation of both enantiomers at the substrate concentration of 1 mM. CYP2B6 had the lowestK _m value for theN-demethylation of (R)- and (S)-ketamine (74 and 44 μM, respectively). Also, the intrinsic clearance (CL_int: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 μM) and sulfaphenazole (CYP2C9 inhibitor, 100 μM) inhibited the N-demethylase activities for both enantiomers (5 μM) in human liver microsomes by 60 to 70%, whereas cyclosporin A (CYP3A4 inhibitor, 100 μM) failed to inhibit these activities. In addition, the anti-CYP2B6 antibody inhibited these activities in human liver microsomes by 80%, whereas anti-CYP2C antibody and anti-CYP3A4 antibody failed to inhibit these activities. These results suggest that the high affinity/low capacity enzyme in human liver microsomes is mediated by CYP2B6, and the low affinity/high capacity enzyme is mediated by CYP2C9 and CYP3A4. CYP2B6 mainly mediates the N-demethylation of (R)- and (S)-ketamine in human liver microsomes at therapeutic concentrations (5 μM).