Abstract

Isoforms of cytochrome P-450 (CYP) involved in the metabolism of gallopamil enantiomers were identified by measuring the disappearance rate of parent drug from an incubation mixture with human liver microsomes and recombinant human CYPs. Mean (± S.D.) intrinsic clearances (CL_int) of R(+)- andS(−)-gallopamil in human liver microsomes were 0.320 ± 0.165 and 0.205 ± 0.107 ml/min/mg protein, respectively. These values were highly correlated with the 6β-hydroxylation activity of testosterone, a marker substrate of CYP3A4 (r = 0.977 and 0.900 forR(+)- and S(−)-gallopamil, respectively,p < .001). Ketoconazole and troleandomycin, selective inhibitors of CYP3A4, and polyclonal antibodies raised against CYP3A4/5 markedly reduced the CL_int of gallopamil enantiomers in human liver microsomes. Among the 10 recombinant human CYP isoforms, CYP3A4 exhibited the highest CL_int of gallopamil enantiomers, and CYP2C8 and CYP2D6 also exhibited appreciable activity. When the contribution of CYP3A4 to the total metabolic clearance of gallopamil enantiomers in human liver microsomes was estimated by relative activity factor, the mean (± S.D.) contributions were 92 ± 18 and 68 ± 19% forR(+)- and S(−)-gallopamil, respectively. These values were comparable to the rates of immunoinhibition by antibodies raised against CYP3A4/5 observed in human liver microsomes. The present study suggests that CYP3A4 is a major isoform involved in the overall metabolic clearance of gallopamil enantiomers in the human liver, and that the present approach based on disappearance rate may be applicable to identify major isoforms of CYP involved in the metabolism of a drug in human liver microsomes.