Short Communication HUMAN CYTOCHROME P450 INDUCTION AND INHIBITION POTENTIAL OF CLEVIDIPINE AND ITS PRIMARY METABOLITE

نویسنده

  • P. BLANCHARD ROBERT J. CLARK
چکیده

Clevidipine is a short-acting dihydropyridine calcium channel antagonist under development for treatment of perioperative hypertension. Patients treated with clevidipine are likely to be comedicated. Therefore, the potential for clevidipine and its major metabolite H152/81 to elicit drug interactions by induction or inhibition of cytochrome P450 was investigated. Induction of CYP1A2, CYP2C9, and CYP3A4 was examined in primary human hepatocytes treated with clevidipine at 1, 10, and 100 M. Clevidipine was found to be an inducer of CYP3A4, but not of CYP1A2 or CYP2C9, at the 10 M and 100 M concentrations of clevidipine tested. Induction response for CYP3A4 to 100 M clevidipine was approximately 20% of that of the positive control inducer rifampicin. The response of H152/81 was similar. Using cDNA-expressed enzymes, clevidipine inhibited CYP2C9, CYP2C19, and CYP3A4 activities with IC50 values below 10 M, whereas CYP1A2, CYP2D6, and CYP2E1 activities were not substantially inhibited (IC50 values >70 M). The Ki values for CYP2C9 and CYP2C19 were 1.7 and 3.3 M, respectively, and those for CYP3A4 were 8.3 and 2.9 M, using two substrates, testosterone and midazolam, respectively. These values are at least 10 times higher than the highest clevidipine concentration typically seen in the clinic. Little or no inhibition by H152/81 was found for the enzyme activities mentioned above (IC50 values > 69 M). The present study demonstrates that it is highly unlikely for clevidipine or its major metabolite to cause cytochrome P450-related drug interactions when used in the dose range required to manage hypertension in humans. Clevidipine (CLE), butyroxymethyl methyl 4-(2 , 3 -dichlorophenyl)-2,6-dimethyl-1,4-dihydropyridine-3,5-dicarboxylate (Fig. 1), is in phase III clinical trials as a rapid acting calcium channel antagonist for intravenous control of blood pressure (Nordlander et al., 2004). Clevidipine is rapidly hydrolyzed and inactivated by esterases in blood and extravascular tissues to its primary metabolite H152/81 (Ericsson et al., 1999, 2000). CLE may have advantages over other antihypertensives. For example, CLE effectively controlled blood pressure in hypertensive patients after undergoing elective coronary bypass grafting, and hemodynamic changes were less pronounced with CLE, compared with sodium nitroprusside (Powroznyk et al., 2003). CLE successfully decreased systemic vascular resistance and mean arterial pressure without changing heart rate, cardiac index, or cardiac filling pressures (Bailey et al., 2002). Clevidipine is a member of the dihydropyridine class of compounds, which have been shown to be inducers and/or inhibitors of cytochromes P450. For example, nifedipine, nicardipine, and isradipine are potent inducers for CYP3A4, CYP2B, and CYP2C in human hepatocyte cultures (Drocourt et al., 2001), and nifedipine induces CYP2C in coronary artery segment endothelial cells (Fisslthaler et al., 2000). In addition, nicardipine, benidipine, manifipine, and barnidipine are potent inhibitors of many P450 isoforms (Katoh et al., 2000). In this study, we have investigated whether CLE and/or H152/81 have the potential to cause P450 induction or inhibition, known causes of drug-drug interactions in humans (Lin and Lu, 2001). Materials and Methods Hepatocytes, Enzymes, and Chemicals. Primary cultured human hepatocytes were obtained from BD Biosciences Discovery Labware (Woburn, MA) (donors 2, 3, and 4) or CellzDirect Inc. (Tucson, AZ) (donors 1 and 5). Donor number, age, sex, and race were as follows: 1/ 51/F/His; 2/41/M/Cau; 3/57/ M/Cau; 4/6/F/AA; 5/61/F/Cau. Medical history was unremarkable, although two donors (1 and 3) had unspecified medication for hypertension. The cDNA-expressed P450 enzymes (Supersomes or lymphoblast cell-derived), hepatocyte culture medium, NADPH regenerating system (NADP , glucose 6-phosphate, glucose-6-phosphate dehydrogenase), and epidermal growth factor were from BD Biosciences. Glutamine and Fungizone (amphotericin B) were obtained from Invitrogen (Carlsbad, CA). 4-[C]-(S)-Mephenytoin was purchased from Amersham Biosciences (Piscataway, NJ). Other chemicals were of high purity grade and purchased from Sigma-Aldrich (St. Louis, MO) or J.T. Baker (Phillipsburg, NJ). Hepatocyte Culture, Treatment, and Induction Assays. Human hepatocytes plated in collagen I-coated 24-well plates were maintained in culture at least 48 h before treatment in hepatocyte culture medium supplemented with 10 g/l epidermal growth factor, 50 g/ml gentamycin, 2 mM L-glutamine, and 0.75 g/ml Fungizone. Cells were treated in triplicate with 0.08% dimethyl sulfoxide vehicle, 20 M BNF, 20 M RIF, CLE, or H152/81 at concentrations of 1, 10, and 100 M for 72 h, with medium change and replenishment every 24 h. After treatment, hepatocytes were washed with medium and then incubated with P450 isoform-specific probe substrates. Cells were incubated at 37°C for 60 min with 100 M phenacetin (CYP1A2) or 100 M diclofenac (CYP2C9), or 30 min with 200 M testosterone (CYP3A4) in volumes of 200 to 400 l. The reaction was stopped by mixing a 175l aliquot of incubation medium with 21.9 l of 70% perchloric acid (phenacetin), a 300l aliquot of incubation medium with 90 l of acetonitrile/acetic acid, 94:6 v/v (diclofenac), or a 300l aliquot of incubation medium with 150 l of acetonitrile (testosterone). Catalytic activity was determined by quantifying probe substrate metabolites in cell culture medium using high-perforArticle, publication date, and citation information can be found at http://dmd.aspetjournals.org. doi:10.1124/dmd.105.006569. ABBREVIATIONS: CLE, clevidipine; BNF, -naphthoflavone; RIF, rifampicin; P450, cytochrome P450; Cau, Caucasian; His, Hispanic; AA, African American. 0090-9556/06/3405-734–737$20.00 DRUG METABOLISM AND DISPOSITION Vol. 34, No. 5 Copyright © 2006 by The American Society for Pharmacology and Experimental Therapeutics 6569/3112709 DMD 34:734–737, 2006 Printed in U.S.A. 734 at A PE T Jornals on Jne 9, 2017 dm d.aspurnals.org D ow nladed from mance liquid chromatography with absorbance detection. Acetamidophenol, 4 -hydroxydiclofenac, and 6 -hydroxytestosterone metabolites were measured as described previously (Stresser et al., 2004). Enzyme Inhibition Studies. Enzyme inhibition analysis was carried out using cDNA-expressed P450 enzymes as described previously (Stresser et al., 2004), with total protein concentration standardized to 0.4 mg/ml. CLE or H152/81 at 10 concentrations ranging from 0.01 to 300 M was tested in duplicate. For Ki determination, three substrate concentrations were used and were incubated with or without three linearly spaced concentrations of CLE, chosen based on the results from the IC50 determinations. The apparent Ki was determined by nonlinear curve-fitting using SigmaPlot (v. 8) with Enzyme Kinetics Module 1.1 (SPSS Inc., Chicago, IL). Comparisons among competitive, noncompetitive, and mixed inhibition models and choice of best fit were conducted using Akaike’s information criterion and inspection of the residuals and Dixon plots. Statistical Analysis. Statistically significant differences between groups were determined by analysis of variance (Minitab Statistical software, release 13.31; Minitab Inc., State College, PA). When nonhomogeneity in the withintreatment variances was indicated, data were log-transformed (to stabilize the variances). Significant differences (p 0.05) between groups treated with test substance and vehicle-only treated groups were determined using Dunnett’s post hoc test.

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تاریخ انتشار 2006