TY - JOUR T1 - Differences in Methadone Metabolism by CYP2B6 Variants JF - Drug Metabolism and Disposition JO - Drug Metab Dispos DO - 10.1124/dmd.115.064352 SP - dmd.115.064352 AU - Evan D. Kharasch AU - Christina Friedel AU - Sarah Gadel Y1 - 2015/01/01 UR - http://dmd.aspetjournals.org/content/early/2015/04/20/dmd.115.064352.abstract N2 - Methadone is a long-acting opioid with considerable unexplained interindividual variability in clearance. Cytochrome P4502B6 (CYP2B6) mediates clinical methadone clearance and metabolic inactivation via N-demethylation to 2-ethyl-1,5-dimethyl-3,3-diphenylpyrrolidine (EDDP). Retrospective studies suggest that individuals with the CYP2B6*6 allelic variant have higher methadone plasma concentrations. Catalytic activities of CYP2B6 variants are highly substrate- and expression-system dependent. This investigation evaluated methadone N-demethylation by expressed human CYP2B6 allelic variants in an insect cell co-expression system containing P450 reductase. Additionally, the influence of co-expressing cytochrome b5, whose role in metabolism can be inhibitory or stimulatory, and dependent on the CYP isoform and substrate, on methadone metabolism was evaluated. EDDP formation from therapeutic (0.25-1 μM) R- and S-methadone concentrations was CYP2B6.4 ≥ CYP2B6.1 ≥ CYP2B6.5 >> CYP2B6.9 ≈ CYP2B6.6, and undetectable from CYP2B6.18. Co-expression of b5 had small and variant-specific effects at therapeutic methadone concentrations, but at higher concentrations stimulated EDDP formation by CYP2B6.1, CYP2B6.4, CYP2B6.5, and CYP2B6.9 but not CYP2B6.6. In vitro intrinsic clearances were generally CYP2B6.4 ≥ CYP2B6.1 > CYP2B6.5 > CYP2B6.9 ≥ CYP2B6.6. Stereoselective methadone metabolism (S>R) was maintained with all CYP2B6 variants. These results show that methadone N-demethylation by CYP2B6.4 is greater compared with CYP2B6.1, while CYP2B6.9 and CYP2B6.6 (which both contain the 516G>T, Q172H polymorphism), are catalytically deficient. The presence or absence of b5 in expression systems may explain previously reported disparate catalytic activities of CYP2B6 variants for specific substrates. Differences in methadone metabolism by CYP2B6 allelic variants provide a mechanistic understanding for pharmacogenetic variability in clinical methadone metabolism and clearance. ER -