Vol. 27, Issue 11, 1254-1259, November 1999

Identification of Human Cytochrome P-450 Isoforms Involved in Metabolism of R(+)- and S()-Gallopamil: Utility of In Vitro Disappearance Rate

Akio Suzuki, Izumi Iida, Fumie Tanaka, Masayuki Akimoto, Kiyomi Fukushima, Masayoshi Tani, Takashi Ishizaki, and Kan Chiba

Pharmaceutical Research Laboratories, Taisho Pharmaceutical Co., Ltd. (A.S., I.I., F.T., M.A., K.F.), Saitama; Department of Clinical Pharmacology, Research Institute (A.S., I.I., T.I.) and Department of General Surgery (M.T.), International Medical Center of Japan, Tokyo; and Laboratory of Biochemical Pharmacology and Toxicology, Faculty of Pharmaceutical Sciences, Chiba University (K.C.), Chiba, Japan

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(+)- and S()-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 6-hydroxylation activity of testosterone, a marker substrate of CYP3A4 (r = 0.977 and 0.900 for R(+)- 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% for R(+)- 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.