Identification of Human Cytochrome P-450 Isoforms Involved in Metabolism of R(1)- and S(2)-gallopamil: Utility of in Vitro Disappearance Rate

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 (6 S.D.) intrinsic clearances (CLint) of R(1)and S(2)-gallopamil in human liver microsomes were 0.320 6 0.165 and 0.205 6 0.107 ml/min/mg protein, respectively. These values were highly correlated with the 6b-hydroxylation activity of testosterone, a marker substrate of CYP3A4 (r 5 0.977 and 0.900 for R(1)and S(2)gallopamil, respectively, p < .001). Ketoconazole and troleandomycin, selective inhibitors of CYP3A4, and polyclonal antibodies raised against CYP3A4/5 markedly reduced the CLint of gallopamil enantiomers in human liver microsomes. Among the 10 recombinant human CYP isoforms, CYP3A4 exhibited the highest CLint 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 (6 S.D.) contributions were 92 6 18 and 68 6 19% for R(1)and S(2)-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. Gallopamil, a methoxy derivative of verapamil, is a calcium-channel antagonist used for the treatment of coronary artery diseases (e.g., vasospastic or variant angina pectoris). As is the case with verapamil, gallopamil is a chiral compound administered as a racemic mixture of R(1)and S(2)-enantiomers. It is well established that the pharmacokinetics of racemic verapamil is stereoselective; the total systemic clearance of S(2)-verapamil is greater than that of R(1)-enantiomer after i.v. administration, and the preferential first-pass metabolism of S(2)-verapamil occurs after oral administration (Eichelbaum et al., 1984; Vogelgesang et al., 1984). Unlike verapamil, the first-pass metabolism of gallopamil is not stereoselective, despite its similarity in the structure and disposition to verapamil (Gross et al., 1997). The pharmacokinetics of gallopamil is characterized by a metabolismand flow-dependent clearance with its distinct first-pass metabolism (Stieren et al., 1983). Major metabolic processes of gallopamil are N-dealkylation and O-demethylation in humans in vivo (Stieren et al., 1983; Weymann et al., 1989) and in rat and human liver microsomes in vitro (Mutlib and Nelson, 1990a,b) (Fig. 1). The formation of N-dealkylated and N-demethylated metabolites of verapamil has been demonstrated to be catalyzed mainly by an isoform of cytochrome P-450 (CYP), CYP3A4 (Kroemer et al., 1993). However, the candidate human CYP isoform(s) involved in the metabolism of gallopamil has not been identified. Identification of CYP isoform(s) has generally been performed by measuring the metabolite(s) production rate with a specific pathway of metabolism, although the metabolic process of a new drug is usually not known in the early stage of drug development. In contrast, the disappearance rate of a drug from incubation medium may be equally useful for this purpose, and in certain circumstances this approach appears to be more appropriate than an approach based on use of a specific metabolic pathway (Houston, 1994). However, little information is available whether the disappearance rate is applicable to the identification of CYP isoform(s) involved in the metabolism of new drugs. The aim of the present study was to identify the major CYP isoform(s) involved in metabolism of R(1)and S(2)-gallopamil by measuring the disappearance rate of parent compound from an incubation mixture with human liver microsomes and recombinant human CYP isoforms. Send reprint requests to: Kan Chiba, Ph.D., Laboratory of Biochemical Pharmacology and Toxicology, Faculty of Pharmaceutical Sciences, Chiba University, 1–33 Yayoi-cho, Inage-ku, Chiba-shi, Chiba 263-8522, Japan. E-mail: [email protected] 1 Abbreviations used are: CYP, cytochrome P-450; TAO, troleandomycin; CLint, intrinsic clearance; RAF, relative activity factor. 0090-9556/99/2711-1254–1259$02.00/0 DRUG METABOLISM AND DISPOSITION Vol. 27, No. 11 Copyright © 1999 by The American Society for Pharmacology and Experimental Therapeutics Printed in U.S.A. 1254 at A PE T Jornals on O cber 9, 2017 dm d.aspurnals.org D ow nladed from Materials and Methods Chemicals and Reagents. R(1)and S(2)-gallopamil HCl, and Lu46973 (internal standard) were obtained from Knoll AG (Ludwigshafen, Germany). Testosterone and troleandomycin (TAO) were purchased from Sigma Chemical Co. (St. Louis, MO). 6b-Hydroxytestosterone and ketoconazole were obtained from Sumika Chemical Analysis Service (Osaka, Japan) and BIOMOL Research Laboratories, Inc. (Plymouth Meeting, PA), respectively. Acetonitrile, methanol, and other reagents of analytical grade were purchased from Wako Pure Chemical Industries, Ltd. (Osaka, Japan). Microsomal preparations from 10 different recombinant human CYP isoforms (i.e., CYP1A2, CYP2A6, CYP2B6, CYP2C8, CYP2C9-Arg, CYP2C9-Cys, CYP2C19, CYP2D6, CYP2E1, and CYP3A4) expressed in the human B lymphoblastoid cell line (AHH-1) were purchased from Gentest Corp. (Woburn, MA). Inhibitory antibody to human CYP3A4/5 was purchased from Amersham International (Tokyo, Japan). Human Liver Microsomes. The microsomal fraction was prepared from human livers by differential centrifugation as described previously (Chiba et al., 1993a). Liver samples (HL-31, HL-32, HL-33, HL-34, HL-35, HL-37, HL-38, HL-39, and HL-40) were obtained, as excess material removed during surgery on liver, from nine Japanese patients undergoing partial hepatectomy at the Department of General Surgery, International Medical Center of Japan (Tokyo, Japan). After determination of protein concentration (Lowry et al., 1951), the individual microsomal samples were aliquoted, frozen, and stored at 280°C until used. Use of human liver samples for the study was approved by the Institutional Ethics Committee, International Medical Center of Japan. Incubation Conditions with Human Liver Microsomes and Recombinant Human CYP Isoforms. The basic incubation medium contained 0.1 mg/ml human liver microsomes, 15 mM MgCl2, 1.5 mM NADP, 10 mM glucose 6-phosphate, 1.5 I.U./ml glucose 6-phosphate dehydrogenase, 100 mM potassium phosphate buffer (pH 7.4), 0.1 mM EDTA, and 0.5 mM R(1)and S(2)-gallopamil HCl, in a final volume of 200 ml. The mixtures were incubated at 37°C in a shaking water bath for 0, 5, 10, 15, and 20 min except for the inhibition study in which incubations were carried out for 0, 10, 20, 30, and 40 min. The reaction was terminated by addition of 10 ml of perchloric acid and 50 ml of a methanolic solution of the internal standard (0.2 mg Lu 46973/ml in methanol). After termination of the incubation, the mixtures were centrifuged at 10,000 rpm for 1 min, and the supernatants were injected onto an HPLC apparatus as described below. Throughout the study, we used 0.5 mM as the substrate concentration to determine the disappearance rates of gallopamil enantiomers. This concentration was determined by a preliminary study that showed the disappearance rates of gallopamil enantiomers at 0.25 mM did not differ from those at 0.5 mM in human liver microsomes, suggesting that metabolism of gallopamil enantiomers is not saturated at these substrate concentrations (i.e., 0.5 mM is estimated to be in the range of gallopamil concentrations in which gallopamil shows the first order disappearance from the incubation mixture). The incubation conditions used for the 10 different recombinant human CYP isoforms obtained from genetically engineered B lymphoblastoid cells were essentially the same as those used for human liver microsomes, except for incubation time (120 min) and the concentration of microsomes used (1 mg/ml). HPLC Assay. The determination of R(1)and S(2)-gallopamil in the incubation mixture was performed by an HPLC-fluorescence detection method. The HPLC system consisted of a model LC-6A pump (Shimadzu, Kyoto, Japan), model SIL-6A autosampler (Shimadzu), model CTO-6A column oven (Shimadzu), model RF-535 fluorescence detector (Shimadzu), model C-R3A integrator (Shimadzu), and J’sphere ODS-L80 column (150 3 2.0 mm internal diameter, YMC Co., Ltd., Kyoto, Japan). The mobile phase consisted of 0.12% perchloric acid and acetonitrile (68:32 v/v) delivered at 0.3 ml/min. Column temperature was maintained at 40°C. The excitation and emission wavelengths of the fluorescence detector were set at 235 and 315 nm, respectively. Analytes were quantified by comparison with the standard curve generated from peak area ratios of gallopamil against the internal standard. Intraand interassay coefficients of variation were ,5%. Intrinsic Clearance of R(1)and S(2)-Gallopamil. The intrinsic clearances (CLint) of gallopamil enantiomers in nine different human liver microsomes were estimated from the volume of medium (V) and the half-life of substrate disappearance (T1/2) with the following equation: CLint 5 V 3 0.693/T1/2 (Chenery et al., 1987). The half-life of R(1)and S(2)-gallopamil in the incubation medium was calculated by the regression analysis of semi-