Functional characterization of 40 CYP2B6 allelic variants by assessing efavirenz 8-hydroxylation

Genetic variations within cytochrome P450 2B6 (CYP2B6) contribute to inter-individual variation in the metabolism of clinically important drugs, including cyclophosphamide, bupropion, methadone and efavirenz (EFZ). In this study, we performed an in vitro analysis of 40 CYP2B6 allelic variant proteins including seven novel variants identified in 1070 Japanese individuals. Wild-type and 39 variant proteins were heterologously expressed in 293FT cells to estimate the kinetic parameters (Km, Vmax, and CLint) of EFZ 8-hydroxylation and 7-ethoxy-4-trifluoromethylcoumarin (7-ETC) O-deethylation activities. The concentrations of CYP2B6 variant holo-enzymes were measured by using carbon monoxide (CO)-reduced difference spectroscopy, and the wild-type and 28 variants showed a peak at 450 nm. The kinetic parameters were measured for the wild-type and 24 variant proteins. The values for the remaining 15 variants could not be determined because the enzymatic activity was not detected at the highest substrate concentration used. Compared to wild-type, six variants showed significantly decreased EFZ 8-hydroxylation CLint values, while these values were significantly increased in another six variants, including CYP2B6.6. Although 7-ETC O-deethylation CLint values of CYP2B6 variants did not differ significantly from that of CYP2B6.1, the CLint ratios obtained for 7-ETC O-deethylation were highly correlated with EFZ 8-hydroxylation. Furthermore, three-dimensional structural modeling analysis was performed to elucidate the mechanism of changes in the kinetics of CYP2B6 variants. Our findings could provide evidence of the specific metabolic activities of the CYP2B6 proteins encoded by these variant alleles.