Abstract

Methadone is a long-acting opioid with considerable unexplained interindividual variability in clearance. Cytochrome P450 2B6 (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 coexpression system containing P450 reductase. Additionally, the influence of coexpressing cytochrome b₅, whose role in metabolism can be inhibitory or stimulatory depending on the P450 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. Coexpression 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, whereas 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 of pharmacogenetic variability in clinical methadone metabolism and clearance.