Methadone pharmacogenetics in vitro and in vivo: Metabolism by CYP2B6 polymorphic variants and genetic variability in paediatric disposition

Abstract

Aims: Methadone metabolism and clearance are determined principally by polymorphic cytochrome P4502B6 (CYP2B6). Some CYP2B6 allelic variants affect methadone metabolism in vitro and disposition in vivo. We assessed methadone metabolism by CYP2B6 minor variants in vitro. We also assessed the influence of CYP2B6 variants, and P450 oxidoreductase (POR) and CYP2C19 variants, on methadone clearance in surgical patients in vivo.

Methods: CYP2B6 and P450 oxidoreductase variants were coexpressed with cytochrome b₅ . The metabolism of methadone racemate and enantiomers was measured at therapeutic concentrations and intrinsic clearances were determined. Adolescents receiving methadone for surgery were genotyped for CYP2B6, CYP2C19 and POR, and methadone clearance and metabolite formation clearance were determined.

Results: In vitro, CYP2B6.4 was more active than wild-type CYP2B6.1. CYPs 2B6.5, 2B6.6, 2B6.7, 2B6.9, 2B6.17, 2B6.19 and 2B6.26 were less active. CYPs 2B6.16 and 2B6.18 were inactive. CYP2B6.1 expressed with POR variants POR.28, POR.5 and P228L had lower rates of methadone metabolism than wild-type reductase. In vivo, methadone clinical clearance decreased linearly with the number of CYP2B6 slow metabolizer alleles, but was not different in CYP2C19 slow or rapid metabolizer phenotypes, or in carriers of the POR*28 allele.

Conclusions: Several CYP2B6 and POR variants were slow metabolizers of methadone in vitro. Polymorphisms in CYP2B6, but not CYP2C19 or P450 reductase, affected methadone clearance in vivo. CYP2B6 polymorphisms 516G>T and 983T>C code for canonical loss of function variants and should be assessed when considering genetic influences on clinical methadone disposition. These complementary translational in vitro and in vivo results inform on pharmacogenetic variability affecting methadone disposition in patients.

Keywords: CYP2B6; CYP2B6 polymorphisms; cytochrome P4502B6; methadone.

Figure 1.

Metabolism of racemic methadone by CYP2B6 variants at therapeutic concentrations of methadone enantiomers. Results are the mean ± SD of triplicate determinations. *P<0.05 vs CYP2B6.1

Figure 2.

Metabolism of racemic methadone to EDDP by CYP2B6 variants. Symbols are R-EDDP (circles) and S-EDDP (triangles) formation from racemic methadone. Lines are predicted concentrations based on kinetic parameters obtained by nonlinear regression analysis of measured concentrations.

Figure 3.

Metabolism of single enantiomers R- or S-methadone and racemate RS-methadone to EDDP catalyzed by CYP2B6.1 (left) and CYP2B6.4 (right). Symbols are R-EDDP (circles) and S-EDDP (triangles) formation from single enantiomers (blue) or racemate (black). Lines are predicted concentrations based on kinetic parameters obtained by nonlinear regression analysis of measured concentrations.

Figure 4.

Metabolism of racemic methadone catalyzed by POR variants at therapeutic concentrations of methadone enantiomers. Results are the mean ± SDof triplicate determinations. *P<0.05 vs POR.1

Figure 5.

Metabolism of racemic methadone to EDDP catalyzed by POR polymorphic variants. Symbols are R-EDDP (circles) and S-EDDP (triangles) formation from racemic methadone. Lines are predicted concentrations based on kinetic parameters obtained by nonlinear regression analysis of measured concentrations.

Figure 6.

Relationship between CYP2B6 allelic variants and clearance of R- and S-methadone clearance and EDDP formation clearance. CYP2B19 phenotypes were based on diplotypes, as described in Methods. CYP2B6 slow metabolizer alleles in the stdy population included *6,*7,*9,*18.

Figure 7.

Relationship between CYP2C19 and P450 oxidoreductase allelic variants and clearance of R- and S-methadone clearance and EDDP formation clearance. CYP2C19 phenotypes were based on diplotypes, as described in Methods.