Background: Metabolism and clearance of racemic methadone are stereoselective and highly variable, yet the mechanism remains largely unknown. Initial in vitro studies attributed methadone metabolism to cytochrome P4503A4 (CYP3A4). CYP3A4 was also assumed responsible for methadone clearance in vivo. Nevertheless, recent clinical data do not support a primary role for CYP3A4 and suggest that CYP2B6 may mediate methadone clearance. Expressed CYP2B6 and also CYP2C19 N-demethylate methadone in vitro. This investigation tested the hypothesis that CYPs 2B6, 3A4, and/or 2C19 are responsible for stereoselective methadone metabolism in human liver microsomes and in vivo.
Methods: N-demethylation of racemic methadone and individual enantiomers by expressed CYPs 2B6, 2C19, and 3A4 was evaluated. Stereoselective microsomal methadone metabolism was quantified, compared with CYP 2B6 and 3A4 content, and probed using CYP isoform-selective inhibitors. A crossover clinical investigation (control, CYP2B6 and CYP3A4 induction by rifampin, CYP3A inhibition by troleandomycin and grapefruit juice) evaluated stereoselective methadone disposition.
Results: At clinical concentrations, methadone enantiomer N-demethylation by recombinant CYPs 2B6, 3A4, and 2C19 was S > R, S = R, and S << R. Greater stereoselective metabolism (S > R) occurred in livers expressing high levels of CYP2B6 compared with CYP3A4. Clopidogrel, troleandomycin, and (+)-N-3-benzyl-nirvanol, selective inhibitors of CYPs 2B6, 3A4, and 2C19, respectively, inhibited microsomal methadone metabolism by 50-60%, 20-30%, and less than 10%. Only inhibition by clopidogrel was stereoselective. Clinically, rifampin diminished both R- and S-methadone plasma concentrations, but troleandomycin and grapefruit juice altered neither R- nor S-methadone concentrations. Plasma R/S-methadone ratios were increased by rifampin but unchanged by CYP3A inhibition.
Conclusions: These results suggest a significant role for CYP2B6, but not CYP3A, in stereoselective human methadone metabolism and disposition.