In Vitro Identification of the Human Cytochrome P450 Enzymes Involved in the Metabolism of R(1)- and S(2)-carvedilol

Both the R(1) and the S(2) enantiomers of carvedilol were metabolized in human liver microsomes primarily to 4*(4OHC) and 5*(5OHC) hydroxyphenyl, 8-hydroxy carbazolyl (8OHC) and O-desmethyl (ODMC) derivatives. The S(2) enantiomer was metabolized faster than the R(1) enantiomer although the same P450 enzymes seemed to be involved in each case. A combination of multivariate correlation analysis, the use of selective inhibitors of P450, and microsomes from human lymphoblastoid cells expressing various human P450s enabled phenotyping of the enzymes involved in the oxidative metabolism of carvedilol. CYP2D6 was primarily responsible for 4OHC and 5OHC production, although considerable activity was observed in a CYP2D6 poor metabolizer liver and the variability of these activities across a human liver bank was not high. There was some evidence that CYP2E1, CYP2C9, and CYP3A4 were also involved in the production of these metabolites. CYP1A2 was primarily responsible for the 8OHC pathway with additional contributions from CYP3A4. The ODMC was clearly associated with CYP2C9 with some evidence for the partial involvement of CYP2D6, CYP1A2, and CYP2E1. With its complex P450 phenotype pattern and the known contribution of non-oxidative pathways of elimination, the activity (or lack of activity) of any particular P450 would have a limited influence on the disposition of carvedilol in an individual. Carvedilol (Coreg/Kredex, SmithKline Beecham Pharmaceuticals, Brentford, UK; Dilatrend/Eucardic, Boehringer Mannheim GmbH, Mannheim, Germany) is a b-adrenoceptor antagonist with vasodilating activity based on a1-blockade, available for the treatment of hypertension and congestive heart failure. This drug is used clinically as a racemic mixture of R(1)and S(2)-enantiomers, and maximum plasma concentrations after oral administration of 50 mg racemic carvedilol were reported to be 74 ng/ml (0.18 mM) (R(1)-carvedilol) and 29 ng/ml (0.07 mM) (S(2)-carvedilol) (1). Previous studies have shown that carvedilol is extensively metabolized in man giving products from both oxidation (fig. 1) and conjugation pathways (2). It was clear that P450 mono-oxygenases play an important role in the oxidative biotransformation of carvedilol, and the objective of this study was to identify the specific enzymes involved. The specific P450 enzyme(s) involved in the biotransformation of a drug can be the defining characteristic of its pharmacokinetic behavior, and drug interactions can occur when there is competition between two or more drugs for oxidation by the same P450 enzyme (3). Pharmacokinetic and clinical issues may also occur in individuals who are deficient in a specific P450 enzyme. For example, 5–10% of Caucasians are deficient in CYP2D6 and 3–5% of Caucasians and 20% of Far Eastern populations lack functional CYP2C19 (4). Thus, it is useful to delineate the involvement of particular P450 enzymes in metabolic pathways of new pharmaceuticals to explain and forecast interindividual pharmacokinetic variability and help predict drug-drug interactions. To identify which P450 enzymes are involved in the in vitro metabolism of carvedilol, a combination of selective inhibitors of human P450 enzymes, correlation analysis, and heterologous expression systems was used. Chemicals characterized as inhibitors of individual P450 enzymes include furafylline, sulfaphenazole, quinidine, and ketoconazole which are selective inhibitors of CYP1A2 (5), CYP2C9 (6), CYP2D6 (7, 8) and CYP3A (6, 9), respectively. Correlation analysis with known activities using specific substrates for the P450 enzymes in a human liver bank can help determine which P450 enzymes are responsible for the metabolism of a compound (10). Lymphoblastoid cells transfected with human P450 cDNAs can provide a source of individual enzymes which can demonstrate which P450 enzymes can contribute to the metabolism of a compound. Materials and Methods Chemicals. R(1)-carvedilol purity 98.10% and S(2)-carvedilol purity 99.87% and SK&F 108410 (BM 14.225) purity 99.54% (internal standard) were obtained from Chemical Development, SmithKline Beecham Pharmaceuticals. Metabolite standards 49-hydroxyphenyl carvedilol (4OHC), 59-hydroxyphenyl carvedilol (5OHC), 1-hydroxyphenyl carvedilol (1OHC), 8-hydroxy carbazolyl carvedilol (8OHC), and O-desmethyl carvedilol (ODMC) were supplied by Boehringer Mannheim GMBH, and were of purity greater than 94% (by HPLC). Ketoconazole and quinidine sulfate were obtained from Sigma Chemical Company (St Louis, MO). Sulfaphenazole and furafylline were obtained from Ultrafine Chemicals (Salford, UK). All other reagents were purchased from BDH (Poole, Dorset, UK), Fisher Scientific Equipment Ltd. (Loughborough, UK), May and Baker (Dagenham, Essex, UK), or Sigma Chemical Company and were of the purest grade available. Human Liver Tissue. Samples of human livers (N 5 27) were obtained from Vitron Inc. (Tucson, AZ) and the International Institute for the Advancement of Medicine (IIAM), Exton, PA. In all cases, the cause of death was not 1 Abbreviations used are: carvedilol, 1-(9H-carbazol-4-yloxy)-3[[2-(2-methoxy phenoxy)ethyl]amino]-2-propanol; P450, cytochrome P450; 4OHC, 49-hydroxyphenyl carvedilol; 5OHC, 59-hydroxyphenyl carvedilol; 1OHC, 1-hydroxyphenyl carvedilol; 8OHC, 8-hydroxycarbazolyl carvedilol; ODMC, O-desmethyl carvedilol. Send reprint requests to: Harriet G. Oldham, Ph.D., Drug Metabolism and Pharmacokinetics, SmithKline Beecham Pharmaceuticals, The Frythe, Welwyn, Hertfordshire AL6 9AR, England. 0090-9556/97/2508-0970–977$02.00/0 DRUG METABOLISM AND DISPOSITION Vol. 25, No. 8 Copyright © 1997 by The American Society for Pharmacology and Experimental Therapeutics Printed in U.S.A. 970 at A PE T Jornals on Sptem er 7, 2017 dm d.aspurnals.org D ow nladed from a result of any known biochemical deficiency in the liver, although several patients had taken or were administered drugs known to affect liver enzyme levels shortly before death. Microsomes derived from human B lymphoblastoid cells transfected with human cDNA for CYP1A1, CYP1A2, CYP2A6, CYP2B6, CYP2C9 co-expressed with P450 reductase, CYP2D6-Val, CYP3A4 co-expressed with P450 reductase, CYP2E1 co-expressed with P450 reductase, human P450 reductase, and control for native activity-containing vector were purchased from Gentest Corporation (Woburn, MA). Human liver microsomes were prepared by differential centrifugation. Incubation of Carvedilol with Human Liver Microsomes and Microsomes from lymphoblastoid cells transfected with human P450 cDNA. All incubations were carried out under similar conditions at 37°C in 50 mM potassium phosphate buffer (pH 7.4), using a NADPH generating system comprising NADP, glucose 6-phosphate, and glucose 6-phosphate dehydrogenase. Each incubation contained human liver microsomes at a final concentration of approximately 0.5 mg microsomal protein/ml or microsomes from lymphoblastoid cells transfected with human P450 cDNA at a final concentration of approximately 2 mg microsomal protein/ml except for CYP2D6 where 0.4 mg/ml was used. R(1)or S(2)-carvedilol (final concentration of 0.2–400 mM) was solubilized with acetonitrile, and in a typical microsomal incubation the final concentration of acetonitrile did not exceed 2% (w/v). After a 5-min pre-incubation, the reaction was initiated by the addition of a pre-warmed NADPH-generating system. The reaction was terminated after 10 min by adding 250 ml acetonitrile containing 3uM SK&F 108410 (internal standard) and 1 mg/ml ascorbic acid to prevent breakdown of 1-hydroxy carvedilol. After centrifugation, the supernatant was removed and analyzed by HPLC. HPLC of Carvedilol Incubations. Incubates were analyzed on a Hewlett Packard 1090A or a Merck-Hitachi (Poole, Dorset, UK) L6200 HPLC system. Detection was by fluorescence using either a Hewlett Packard (Cheadle Heath, Stockport, Cheshire, UK) 1046A or a Perkin Elmer (Beaconsfield, Bucks, UK) LC 240 fluorescence detector at either lex278 nm, lem320 nm or lex330 nm, lem380 nm. The HPLC method developed was based on that of Schaefer (11). Aliquots of each sample (50–100 ml) were injected onto a Supelco ABZ column (5 mm, 4.6 mm 3 15 cm) maintained at a temperature of approximately 40°C with a flow rate of 1.0 mlzmin. Elution conditions were a linear gradient of 75% solvent A (0.1 M ammonium acetate, pH 5.0):25% solvent B (acetonitrile: water (80:20 v:v) to either 60% A:40% B or 63.4% A: 36.6% B over 35 min, followed by a second linear gradient to 0% A:100% B at 37 min, followed by isocratic 100% B until 40 min and finally a linear gradient of 0% solvent A : 100% solvent B to 75% A:25% B until 45 min. The fluorescent peaks of interest on the chromatogram were integrated and expressed as the area under each peak. Rates of formation of carvedilol metabolites were evaluated from calibration lines for each metabolite constructed by determining the peak area ratios of known concentrations of authentic standards at a constant concentration of the internal standard. Inhibition of R(1)and S(2)-Carvedilol Metabolism. Inhibition of R(1)and S(2)-carvedilol metabolism was investigated by incubating R(1)or S(2)-carvedilol at a final concentration of 30 mM and approximately 0.5 mg protein/ml human liver microsomes in the absence and presence of each inhibitor. Quinidine (1 mM), selective for CYP2D6 (8), ketoconazole (1 mM), selective for CYP3A4 (6, 12), and sulfaphenazole (10 mM), selective for CYP2C9 (6, 13–16), were preincubated at approximately 37°C with the R(1)or S(2)-carvedilol, microsomes, and potassium phosphate buffer (pH 7.4) for 5 min, before adding a pre-warmed NADPH generating system to start the reaction. Furafylline (10 mM), selective for CYP1A2 (17), was preincubated at approximately 37°C with microsomes, NADPH generating system, and buffer for 10 min before prewarmed solutions of R(1)or S(2)-carvedilol were added to start the reaction. Data Analysis. All graphical analysis of data was performed using nonlinear regression with weighted data (1/y) with GraFit version 3 (R. J. Leatherbarrow: Erithacus Software Ltd, Staines, UK (1992)). Tests for statistical significance were performed using SAS/INSIGHT Version 6 (SAS Institute Inc, Cary, NC). A multiple linear regression analysis was used to select a model to identify the cytochrome P450 enzyme(s) responsible for the variability in the metabolism of R(1)and S(2)-carvedilol in 26 of the samples in the human liver microsomal bank. A multiple regression model was initially fitted using all known explanatory variables (cytochrome P450 enzyme activities). Type III For, equivalently, t-statistics were calculated for the exclusion of each explanatory variable from the model. The cytochrome P450 enzyme activity corresponding to the lowest value of the statistic was omitted if its p-value was greater than the 5% significance level and the change in the adjusted R value indicated that little explanatory power would be lost by its omission. The For t-statistics were then calculated for the new model and again the explanatory variable corresponding to the lowest value of the statistic was excluded if its p-value was greater than the 5% significance level and little explanatory power would be lost by its exclusion. These steps were repeated until no other cytochrome P450 enzyme activity could be omitted.