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Amlodipine Inhibits Rat Microsomal Cytochrome P450-Mediated Drug Biotransformation
Amlodipine Inhibits Rat Microsomal Cytochrome P450-Mediated Drug Biotransformation ROBERT K. DROBITCH*‡, ROMAN A. MCLELLAN*§,
AND
KENNETH W. RENTON*X
Received May 9, 1997, from the *Department of Pharmacology, Dalhousie University, Halifax, Nova Scotia, Canada B3H 4H7. Accepted for publication August 18, 1997X. Present addresses: ‡Department of Pharmaceutical Sciences, Wayne State University, Detroit, MI 48202, and §Division of Molecular Toxicology, Institute of Environmental Medicine, Karolinska Institute, Stockholm, Sweden. Abstract 0 Calcium channel antagonists have been shown to inhibit cytochrome P-450-mediated metabolism both in vitro and in vivo. The purpose of the present study was to examine the effect of amlodipine on a suite of rat hepatic microsomal cytochrome P-450 activities to determine the potential for drug interactions. In this study, amlodipine (0.05 and 0.5 mM) decreased CYP1A-mediated ethoxyresorufin O-deethylase activity in microsomes prepared from noninduced (56 and 73% inhibition) and pyridine-induced (30 and 51% inhibition) rats. Amlodipine reduced pentoxyresorufin O-deethylase activity (a marker for CYP2B) to 15% of control in incubations utilizing microsomes from phenobarbital-treated rats, but had no effect on this enzyme reaction in noninduced microsomes. The para-nitrophenol hydroxylase, erythromycin N-demethylase, and lauric acid ω and ω −1 hydroxylase activities were significantly inhibited by 1 mM amlodipine in both noninduced and induced microsomes. These results suggest that amlodipine inhibits a number of different P450 forms and therefore has the potential to inhibit the metabolism of a large number of drugs.
The calcium channel antagonists are an important group of agents used in the treatment of angina pectoris, hypertension, and other cardiovascular disorders. Amlodipine is one of a group of “second generation” dihydropyridine-based calcium channel antagonists developed to increase vasculatory selectivity with less negative inotropy and much longer halflife than the prototype of this group, nifedipine. Nifedipine, diltiazem, and verapamil have been shown to inhibit the cytochrome P450-mediated in vitro metabolism of a number of compounds, including propranolol, aminopyrine, and cyclosporine (CyA).1-4 Verapamil and diltiazem have also been reported to decrease the clearance of drugs in vivo,5-7 but nifedipine appears to have little or no effect on the in vivo elimination of drugs.7-9 As far as amlodipine is concerned, the disappearance rate of propranolol in isolated rat hepatocytes was reduced by 30% when amlodipine is present at equimolar concentrations to that of propranolol.4 However, the degree of inhibition by amlodipine was much less than for other calcium channel antagonists, such as diltiazem (46% inhibition) and nifedipine (50% inhibition), at similiar concentrations.4 In controlled human studies, amlodipine had no effect on the metabolism of CyA, benazepril, or digoxin.10-12 To date, no drug interactions have been reported that can be attributed to cytochrome P-450 inhibition by amlodipine, but this drug has not been in widespread clinical use for an extended time. Therefore, to determine if inhibition of cytochrome P450 forms by amlodipine has any potential for drug/drug interactions, we measured the effect of amlodipine on a suite of rat hepatic microsomal cytochrome P-450 activities including ethoxyresorufin and pentoxyresorufin O-deethylase (EROD and PROD, respectively), para-nitrophenol hydroxylase (PNPH), erythromycin N-demethylase (EMND), and lauric acid ω and ω -1 X
Abstract published in Advance ACS Abstracts, November 1, 1997.
© 1997, American Chemical Society and American Pharmaceutical Association
hydroxylase (LAH) activities, which have high specificity for the CYP1A, CYP2B, CYP2E, CYP3A, and CYP4A P450 isozymes, respectively.
Materials and Methods Drugs and ChemicalssPyridine, phenobarbital, and dexamethasone were purchased from Fisher Scientific Company (Fair Lawn, NJ), BDH Inc. (Toronto, ONT, Canada), and Organon Teknika (Toronto, ONT, Canada), respectively. All other drugs and chemicals were purchased from Sigma Chemical Company (St. Louis, MO). Animals and TreatmentssMale Sprague-Dawley rats, obtained from Charles River Breeding Laboratories (Montreal, QUE, Canada), were housed on clay chips and fed Purina rat chow and water ad libitum and acclimatized to our facilities for 7 days prior to treatment. To induce high levels of the CYP1A and CYP2E1 isozymes, rats (n ) 3) were pretreated with a single dose of pyridine [200 mg/kg in saline, by intraperitoneal (ip) injection]. To induce high levels of CYP2B, rats (n ) 3) received phenobarbital (80 mg/kg in saline, ip) daily for 3 days. For the induction of CYP3A2, rats (n ) 3) received a single dose of dexamethasone (20 mg/kg in saline, by oral gavage). Corresponding control animals (n ) 3) for these studies received saline vehicle. For the induction of CYP4A1, animals (n ) 3) received clofibrate (250 mg/kg in olive oil, ip) daily for 4 days, and control animals (n ) 3) received olive oil vehicle. Animals were sacrificed 24 h after receiving the last dose of inducer or corresponding vehicle, the livers were removed, and hepatic microsomes were prepared by differential cetrifugation according to the method of el Defrawry el Masry et al.13 Microsomes were suspended in glycerol buffer (50 mM potassium phosphate, 0.4% potassium chloride, 20% glycerol) and stored at -80 °C. Microsomal Enzyme ActivitysTotal microsomal protein was determined by the method of Lowry et al.14 For each enzyme form, in vitro activity was determined in microsomes obtained from a group of control rats and a group of animals pretreated with the appropritate inducer. In vitro activities were measured in the absence and presence of amlodipine. In each case, the concentration of amlodipine utilized was ∼10-fold the substrate concentration to ensure that a purely competitive type interaction would be observed. EROD and PROD activities (CYP1A and CYP2B, respectively) were determined in incubations containing NADPH (0.1 mM), and ethoxyor pentoxyresorufin (0.5 mM) in the absence or presence of amlodipine (50 or 500 mM) according to the methods of Burke et al.15,16 PNPH activity (CYP2E1) was measured according to the method of Koop.17 Incubations contained para-nitrophenol (0.25 mM) and NADPH (1.0 mM) in the absence or presence of amlodipine (2.5 mM). EMND activity (CYP3A2) was determined in incubation mixtures containing NADPH (2.0 mM) and erythromycin (0.1 mM) in the absence or presence of amlodipine (1.0 mM) according to Arlotto et al.18 LAH activity (CYP4A1) was determined with an incubation mixture containing lauric acid (0.1 mM) and NADPH (0.8 mM) in the absence or presence of amlodipine (1.0 mM), according to Orton and Parker.19 Statistical AnalysissAll data are expressed as percentage of control activity ((SD) and are the mean of three separate animals. For each microsomal preparation, isozyme-specific enzyme activities measured in the absence and presence of amlodipine were compared by paired t tests, with a value of p < 0.05 considered as significant.
Results and Discussion The purpose of this study was to examine the effect of amlodipine on rat hepatic microsomal activities that are
S0022-3549(97)00188-3 CCC: $14.00
Journal of Pharmaceutical Sciences / 1501 Vol. 86, No. 12, December 1997
Figure 1sEffect of amlodipine on in vitro CYP1A, CYP2B activity in rat hepatic microsomes. Ethoxyresorufin O-dealkylase (CYP1A) and pentoxyresorufin Odealkylase (CYP2B) activities in rat liver microsomes were determined in the presence of amlodipine (0.05 or 0.50 mM) as described in the Methods. Microsomes were prepared from animals pretreated with specific inducers and from corresponding noninduced animals. Actual values for noninduced (open bar) and induced (filled bar) preparations, respectively: ethoxyresorufin O-dealkylase ) 32.6 and 447.8 pmol resorufin formed/mg protein/min; pentoxyresorufin O-dealkylase ) 5.7 and 22.6 pmol resorufin formed/mg protein/min. Activities are expressed as a percentage of the activity determined in the absence of amlodipine (±SD) for preparations from noninduced (n ) 3) or induced (n ) 3) animal groups. Key: * significantly different from corresponding activity in absence of amlodipine (p < 0.05).
relatively specific for the CYP1A, CYP2B, CYP2E, CYP3A, and CYP4A P450 isozymes to determine the potential for drug interactions between amlodipine and concurrently administered medications. Activities were measured in microsomes obtained from control rats as well as from rats pretreated with inducers to increase the proportional content of specific isozymes. Amlodipine (0.05 and 0.5 mM) decreased CYP1A-mediated EROD activity in microsomes prepared from noninduced and pyridine-induced rats (Figure 1). Amlodipine significantly reduced PROD activity, a marker for CYP2B, in incubations utilizing microsomes from phenobarbital-treated rats, but had no effect on this enzyme reaction in noninduced microsomes (Figure 1). The PNPH, EMND, and LAH activities were significantly inhibited by 1 mM amlodipine in both noninduced and induced microsomes (Figure 2). These results suggest that amlodipine inhibits a number of different P450 forms and therefore has the potential to inhibit the metabolism of a large number of drugs. Treatment of hypertension and angina pectoris with amlodipine or other calcium channel antagonists often involves the use of concommitent medications; thus, this potential for amlodipine to alter the metabolism of concurrently administered drugs requires consideration. Amlodipine is 99% protein bound and thus low free drug concentrations are present in plasma. Because the inhibition of the P450 isozyme-specific activities occurred at amlodipine concentrations significantly higher than the plasma concentrations achieved in vivo, it may be argued that a potential drug interaction is unlikely. However, such an argument may not always hold for calcium channel blocking drugs. For example, Vercruysse et al.4 reported that the metabolism of propranolol by isolated rat hepatocytes was reduced by 50% when nifedipine was added to a concentration of 30 mM. Although this concentration greatly exceeds plasma concentrations observed in humans 1502 / Journal of Pharmaceutical Sciences Vol. 86, No. 12, December 1997
Figure 2sEffect of amlodipine on para-nitrophenol hydroxylase (CYP2E1), erythromycin N-demethylase (CYP3A2), and lauric acid hydroxylase (CYP4A1) activities in rat hepatic microsomes. Activities were determined in the presence of amlodipine as described in the Methods. Microsomes were prepared from animals pretreated with specific inducers and from corresponding noninduced animals. Actual values for noninduced (open bar) and induced (filled bar) preparations, respectively: para-nitrophenol hydroxylase ) 0.55 and 0.83 nmol 4-nitrocatechol formed/mg protein/min; erythromycin N-demethylase ) 0.17 and 0.37 nmol formaldehyde formed/mg protein/min; lauric acid hydroxylase ) 0.48 and 4.7 nmol lauric acid metabolized/mg protein/min. Activities are expressed as a percentage of the activity determined in the absence of amlodipine (±SD) for preparations from noninduced (n ) 3) or induced (n ) 3) animal groups. Key: * significantly different from corresponding activity in absence of amlodipine (p < 0.05).
or rats, nifedipine causes significant inhibition of propranolol metabolism in vivo in rats.20 Pichard et al.3 reported a Ki for diltiazem of 51-75 mM for inhibition of CyA oxidase activity (a CYP3A-mediated activity) in primary cultures of human hepatocytes and microsomes. This value exceeds plasma concentrations of 0.1 to 0.7 mM21; however, diltiazem has been reported to reduce CyA metabolism in humans.22 These examples point out the difficulty in assessing potential in vivo drug interactions based solely on in vitro conditions. Unknown factors involved in the extrapolation of in vitro concentrations to the intact animal include the partitioning of amlodipine into the lipid environment of the cytochrome P450, which may reduce actual concentrations of amlodipine at the site of biotransformation in vitro, and the uncertainty as to the actual concentrations achieved at the site of biotransformation in vivo. In the case of amlodipine, this study points out the possibility of an interaction and alerts users of the drug of such a possibility. Only clinical observations will be able to determine if interactions occur in clinical practice. Although a recent report by Toupance et al.12 indicates that amlodipine does not significantly alter the metabolism of CyA in an adult renal transplant population, the potential for an interaction between amlodipine and CyA in other patient groups, such as pediatric renal transplant patients or cases of renal failure, cannot be ruled out. For example, although nifedipine appears to have little effect on the elimination of CyA in adult renal transplant patients,8,9 it has been reported to inhibit CYA metabolism in pediatric renal transplant patients.23 The results of the present study, in addition to the inhibition of propranolol metabolism (CYP2D-mediated) by amlodipine reported by Vercruysse et al.4 indicate a significant potential for clinically relevant drug interactions in patients receiving amlodipine concurrently with other medications. Abbreviations: CyA, cyclosporin A; CYP, cytochrome P450; EMND, erythromycin N-demethylase; EROD, ethoxyresorufin O-deethylase; ip, intraperitoneal; LAH, lauric acid ω and
ω -1 hydroxylase; PNPH, para-nitrophenol hydroxylase; PROD, pentoxyresorufin O-deethylase.
References and Notes 1. Renton, K. W. Biochem. Pharmacol. 1985, 34, 2549-2553. 2. Tjia, J. F.; Back, D. J.; Breckenbridge, A. M. Br. J. Clin. Pharmacol. 1989, 28, 362-365. 3. Pichard, L.; Fabre, I.; Fabre, G.; Domergue, J.; Saint Aubert, B.; Mourad, G,; Maurel, P. Drug Metab. Dispos. 1990, 18, 595606. 4. Vercruysse, I.; Vermeulen, A. M.; Belpaire, F. M.; Massart, D. L.; Dupont, A. G. Fundam. Clin. Pharmacol. 1994, 8, 373-378. 5. Abernethy, D. R.; Egan, J. M.; Dickinson, Twazzu.H.; Carrum, G. J. Pharmacol. Exp. Ther. 1988, 244, 994-999. 6. Robson, R. A.; Graenkel, M.; Barrett, L. J.; Birkett, D. J. Br. J. Clin. Pharmacol. 1988, 25, 402-403. 7. Sketris, I. S.; Methot, M. E.; Nicol, D.; Belitsky, P.; Knox M. G. Ann. Pharmacother. 1994, 28, 1227-1231. 8. McNally, P.; Mistry, N.; Idle, J.; Walls, J.; Feehally, J. Transplantation 1989, 48, 1071. 9. Tortorice, K. L.; Heim-Duthoy, K. L.; Awny, W. M.; Rao, K. V.; Kasiske, B. L. Ther. Drug Monit. 1990, 12, 321-328. 10. Schwartz, J. B. J. Cardiovasc. Pharmacol. 1988, 12, 1-5. 11. Sun, J. X.; Cipriano, A.; Chan, K.; John, V. A. Eur. J. Clin. Pharmacol. 1994, 47, 285-289. 12. Toupance, O.; Lavaud, S.; Canivet, E.; Bernaud, C.; Hotton, J. M.; Chanard, J. Hypertension 1994, 24, 297-300. 13. el Defrawry el Masry, S.; Cohen, G. M.; Mannering, G. J. Drug Metab. Dispos. 1974, 2, 267-278.
14. Lowry, O. H.; Rosenbrough, N. J.; Farr, A. L.; Randall R. J. J. Biol. Chem. 1951, 193, 265-275. 15. Burke, M. D.; Prough, R. A.; Mayer, R. T. Drug Metab. Dispos. 1977, 5, 1-8. 16. Burke, M. D.; Thompson, S.; Elcombe, C. R.; Halpert, J.; Haaparanta, T.; Mayer, R. T. Biochem. Pharmacol. 1985, 34, 3337-3345. 17. Koop, D. R. Mol. Pharmacol. 1986, 29, 399-404. 18. Arlotto, M. A.; Sonderfan, A. J.; Klaassen, C. D.; Parkinson, A. Biochem. Pharmacol. 1987, 36, 3859-3866. 19. Orton, T. C.; Parker, G. Drug Metab. Dispos. 1982, 10, 110115. 20. Vercruysse, I.; Schoors, D. F.; Massart, D. L.; Dupont A. Cardiovasc. Drugs Ther. 1993, 7, 721-726. 21. Piepho, R. W.; Bloedow, D. C.; Lacz, J. P.; Runser D. J.; Dimmit, D. C.; Browne, R. K. Am. J. Cardiol. 1982, 49, 525-528. 22. Kohlhaw, K.; Wonigeit, K.; Frei, U.; Oldhafer, K.; Neumann, K.; Pichlmayr, R. Transplant. Proc. 1988, 20 (Suppl 2), 572574. 23. Crocker, J. F. S.; Renton, K. W.; LeVatte, T. L.; McLellan, D. Pediatr. Nephrol. 1994, 8, 408-411.
Acknowledgments K.W.R. is supported by a grant from the Medical Research Council of Canada and R.K.D. is a recipient of a fellowship from the Dalhousie Medical Research Foundation, Halifax.
JS970188T
Journal of Pharmaceutical Sciences / 1503 Vol. 86, No. 12, December 1997
AND
KENNETH W. RENTON*X
Received May 9, 1997, from the *Department of Pharmacology, Dalhousie University, Halifax, Nova Scotia, Canada B3H 4H7. Accepted for publication August 18, 1997X. Present addresses: ‡Department of Pharmaceutical Sciences, Wayne State University, Detroit, MI 48202, and §Division of Molecular Toxicology, Institute of Environmental Medicine, Karolinska Institute, Stockholm, Sweden. Abstract 0 Calcium channel antagonists have been shown to inhibit cytochrome P-450-mediated metabolism both in vitro and in vivo. The purpose of the present study was to examine the effect of amlodipine on a suite of rat hepatic microsomal cytochrome P-450 activities to determine the potential for drug interactions. In this study, amlodipine (0.05 and 0.5 mM) decreased CYP1A-mediated ethoxyresorufin O-deethylase activity in microsomes prepared from noninduced (56 and 73% inhibition) and pyridine-induced (30 and 51% inhibition) rats. Amlodipine reduced pentoxyresorufin O-deethylase activity (a marker for CYP2B) to 15% of control in incubations utilizing microsomes from phenobarbital-treated rats, but had no effect on this enzyme reaction in noninduced microsomes. The para-nitrophenol hydroxylase, erythromycin N-demethylase, and lauric acid ω and ω −1 hydroxylase activities were significantly inhibited by 1 mM amlodipine in both noninduced and induced microsomes. These results suggest that amlodipine inhibits a number of different P450 forms and therefore has the potential to inhibit the metabolism of a large number of drugs.
The calcium channel antagonists are an important group of agents used in the treatment of angina pectoris, hypertension, and other cardiovascular disorders. Amlodipine is one of a group of “second generation” dihydropyridine-based calcium channel antagonists developed to increase vasculatory selectivity with less negative inotropy and much longer halflife than the prototype of this group, nifedipine. Nifedipine, diltiazem, and verapamil have been shown to inhibit the cytochrome P450-mediated in vitro metabolism of a number of compounds, including propranolol, aminopyrine, and cyclosporine (CyA).1-4 Verapamil and diltiazem have also been reported to decrease the clearance of drugs in vivo,5-7 but nifedipine appears to have little or no effect on the in vivo elimination of drugs.7-9 As far as amlodipine is concerned, the disappearance rate of propranolol in isolated rat hepatocytes was reduced by 30% when amlodipine is present at equimolar concentrations to that of propranolol.4 However, the degree of inhibition by amlodipine was much less than for other calcium channel antagonists, such as diltiazem (46% inhibition) and nifedipine (50% inhibition), at similiar concentrations.4 In controlled human studies, amlodipine had no effect on the metabolism of CyA, benazepril, or digoxin.10-12 To date, no drug interactions have been reported that can be attributed to cytochrome P-450 inhibition by amlodipine, but this drug has not been in widespread clinical use for an extended time. Therefore, to determine if inhibition of cytochrome P450 forms by amlodipine has any potential for drug/drug interactions, we measured the effect of amlodipine on a suite of rat hepatic microsomal cytochrome P-450 activities including ethoxyresorufin and pentoxyresorufin O-deethylase (EROD and PROD, respectively), para-nitrophenol hydroxylase (PNPH), erythromycin N-demethylase (EMND), and lauric acid ω and ω -1 X
Abstract published in Advance ACS Abstracts, November 1, 1997.
© 1997, American Chemical Society and American Pharmaceutical Association
hydroxylase (LAH) activities, which have high specificity for the CYP1A, CYP2B, CYP2E, CYP3A, and CYP4A P450 isozymes, respectively.
Materials and Methods Drugs and ChemicalssPyridine, phenobarbital, and dexamethasone were purchased from Fisher Scientific Company (Fair Lawn, NJ), BDH Inc. (Toronto, ONT, Canada), and Organon Teknika (Toronto, ONT, Canada), respectively. All other drugs and chemicals were purchased from Sigma Chemical Company (St. Louis, MO). Animals and TreatmentssMale Sprague-Dawley rats, obtained from Charles River Breeding Laboratories (Montreal, QUE, Canada), were housed on clay chips and fed Purina rat chow and water ad libitum and acclimatized to our facilities for 7 days prior to treatment. To induce high levels of the CYP1A and CYP2E1 isozymes, rats (n ) 3) were pretreated with a single dose of pyridine [200 mg/kg in saline, by intraperitoneal (ip) injection]. To induce high levels of CYP2B, rats (n ) 3) received phenobarbital (80 mg/kg in saline, ip) daily for 3 days. For the induction of CYP3A2, rats (n ) 3) received a single dose of dexamethasone (20 mg/kg in saline, by oral gavage). Corresponding control animals (n ) 3) for these studies received saline vehicle. For the induction of CYP4A1, animals (n ) 3) received clofibrate (250 mg/kg in olive oil, ip) daily for 4 days, and control animals (n ) 3) received olive oil vehicle. Animals were sacrificed 24 h after receiving the last dose of inducer or corresponding vehicle, the livers were removed, and hepatic microsomes were prepared by differential cetrifugation according to the method of el Defrawry el Masry et al.13 Microsomes were suspended in glycerol buffer (50 mM potassium phosphate, 0.4% potassium chloride, 20% glycerol) and stored at -80 °C. Microsomal Enzyme ActivitysTotal microsomal protein was determined by the method of Lowry et al.14 For each enzyme form, in vitro activity was determined in microsomes obtained from a group of control rats and a group of animals pretreated with the appropritate inducer. In vitro activities were measured in the absence and presence of amlodipine. In each case, the concentration of amlodipine utilized was ∼10-fold the substrate concentration to ensure that a purely competitive type interaction would be observed. EROD and PROD activities (CYP1A and CYP2B, respectively) were determined in incubations containing NADPH (0.1 mM), and ethoxyor pentoxyresorufin (0.5 mM) in the absence or presence of amlodipine (50 or 500 mM) according to the methods of Burke et al.15,16 PNPH activity (CYP2E1) was measured according to the method of Koop.17 Incubations contained para-nitrophenol (0.25 mM) and NADPH (1.0 mM) in the absence or presence of amlodipine (2.5 mM). EMND activity (CYP3A2) was determined in incubation mixtures containing NADPH (2.0 mM) and erythromycin (0.1 mM) in the absence or presence of amlodipine (1.0 mM) according to Arlotto et al.18 LAH activity (CYP4A1) was determined with an incubation mixture containing lauric acid (0.1 mM) and NADPH (0.8 mM) in the absence or presence of amlodipine (1.0 mM), according to Orton and Parker.19 Statistical AnalysissAll data are expressed as percentage of control activity ((SD) and are the mean of three separate animals. For each microsomal preparation, isozyme-specific enzyme activities measured in the absence and presence of amlodipine were compared by paired t tests, with a value of p < 0.05 considered as significant.
Results and Discussion The purpose of this study was to examine the effect of amlodipine on rat hepatic microsomal activities that are
S0022-3549(97)00188-3 CCC: $14.00
Journal of Pharmaceutical Sciences / 1501 Vol. 86, No. 12, December 1997
Figure 1sEffect of amlodipine on in vitro CYP1A, CYP2B activity in rat hepatic microsomes. Ethoxyresorufin O-dealkylase (CYP1A) and pentoxyresorufin Odealkylase (CYP2B) activities in rat liver microsomes were determined in the presence of amlodipine (0.05 or 0.50 mM) as described in the Methods. Microsomes were prepared from animals pretreated with specific inducers and from corresponding noninduced animals. Actual values for noninduced (open bar) and induced (filled bar) preparations, respectively: ethoxyresorufin O-dealkylase ) 32.6 and 447.8 pmol resorufin formed/mg protein/min; pentoxyresorufin O-dealkylase ) 5.7 and 22.6 pmol resorufin formed/mg protein/min. Activities are expressed as a percentage of the activity determined in the absence of amlodipine (±SD) for preparations from noninduced (n ) 3) or induced (n ) 3) animal groups. Key: * significantly different from corresponding activity in absence of amlodipine (p < 0.05).
relatively specific for the CYP1A, CYP2B, CYP2E, CYP3A, and CYP4A P450 isozymes to determine the potential for drug interactions between amlodipine and concurrently administered medications. Activities were measured in microsomes obtained from control rats as well as from rats pretreated with inducers to increase the proportional content of specific isozymes. Amlodipine (0.05 and 0.5 mM) decreased CYP1A-mediated EROD activity in microsomes prepared from noninduced and pyridine-induced rats (Figure 1). Amlodipine significantly reduced PROD activity, a marker for CYP2B, in incubations utilizing microsomes from phenobarbital-treated rats, but had no effect on this enzyme reaction in noninduced microsomes (Figure 1). The PNPH, EMND, and LAH activities were significantly inhibited by 1 mM amlodipine in both noninduced and induced microsomes (Figure 2). These results suggest that amlodipine inhibits a number of different P450 forms and therefore has the potential to inhibit the metabolism of a large number of drugs. Treatment of hypertension and angina pectoris with amlodipine or other calcium channel antagonists often involves the use of concommitent medications; thus, this potential for amlodipine to alter the metabolism of concurrently administered drugs requires consideration. Amlodipine is 99% protein bound and thus low free drug concentrations are present in plasma. Because the inhibition of the P450 isozyme-specific activities occurred at amlodipine concentrations significantly higher than the plasma concentrations achieved in vivo, it may be argued that a potential drug interaction is unlikely. However, such an argument may not always hold for calcium channel blocking drugs. For example, Vercruysse et al.4 reported that the metabolism of propranolol by isolated rat hepatocytes was reduced by 50% when nifedipine was added to a concentration of 30 mM. Although this concentration greatly exceeds plasma concentrations observed in humans 1502 / Journal of Pharmaceutical Sciences Vol. 86, No. 12, December 1997
Figure 2sEffect of amlodipine on para-nitrophenol hydroxylase (CYP2E1), erythromycin N-demethylase (CYP3A2), and lauric acid hydroxylase (CYP4A1) activities in rat hepatic microsomes. Activities were determined in the presence of amlodipine as described in the Methods. Microsomes were prepared from animals pretreated with specific inducers and from corresponding noninduced animals. Actual values for noninduced (open bar) and induced (filled bar) preparations, respectively: para-nitrophenol hydroxylase ) 0.55 and 0.83 nmol 4-nitrocatechol formed/mg protein/min; erythromycin N-demethylase ) 0.17 and 0.37 nmol formaldehyde formed/mg protein/min; lauric acid hydroxylase ) 0.48 and 4.7 nmol lauric acid metabolized/mg protein/min. Activities are expressed as a percentage of the activity determined in the absence of amlodipine (±SD) for preparations from noninduced (n ) 3) or induced (n ) 3) animal groups. Key: * significantly different from corresponding activity in absence of amlodipine (p < 0.05).
or rats, nifedipine causes significant inhibition of propranolol metabolism in vivo in rats.20 Pichard et al.3 reported a Ki for diltiazem of 51-75 mM for inhibition of CyA oxidase activity (a CYP3A-mediated activity) in primary cultures of human hepatocytes and microsomes. This value exceeds plasma concentrations of 0.1 to 0.7 mM21; however, diltiazem has been reported to reduce CyA metabolism in humans.22 These examples point out the difficulty in assessing potential in vivo drug interactions based solely on in vitro conditions. Unknown factors involved in the extrapolation of in vitro concentrations to the intact animal include the partitioning of amlodipine into the lipid environment of the cytochrome P450, which may reduce actual concentrations of amlodipine at the site of biotransformation in vitro, and the uncertainty as to the actual concentrations achieved at the site of biotransformation in vivo. In the case of amlodipine, this study points out the possibility of an interaction and alerts users of the drug of such a possibility. Only clinical observations will be able to determine if interactions occur in clinical practice. Although a recent report by Toupance et al.12 indicates that amlodipine does not significantly alter the metabolism of CyA in an adult renal transplant population, the potential for an interaction between amlodipine and CyA in other patient groups, such as pediatric renal transplant patients or cases of renal failure, cannot be ruled out. For example, although nifedipine appears to have little effect on the elimination of CyA in adult renal transplant patients,8,9 it has been reported to inhibit CYA metabolism in pediatric renal transplant patients.23 The results of the present study, in addition to the inhibition of propranolol metabolism (CYP2D-mediated) by amlodipine reported by Vercruysse et al.4 indicate a significant potential for clinically relevant drug interactions in patients receiving amlodipine concurrently with other medications. Abbreviations: CyA, cyclosporin A; CYP, cytochrome P450; EMND, erythromycin N-demethylase; EROD, ethoxyresorufin O-deethylase; ip, intraperitoneal; LAH, lauric acid ω and
ω -1 hydroxylase; PNPH, para-nitrophenol hydroxylase; PROD, pentoxyresorufin O-deethylase.
References and Notes 1. Renton, K. W. Biochem. Pharmacol. 1985, 34, 2549-2553. 2. Tjia, J. F.; Back, D. J.; Breckenbridge, A. M. Br. J. Clin. Pharmacol. 1989, 28, 362-365. 3. Pichard, L.; Fabre, I.; Fabre, G.; Domergue, J.; Saint Aubert, B.; Mourad, G,; Maurel, P. Drug Metab. Dispos. 1990, 18, 595606. 4. Vercruysse, I.; Vermeulen, A. M.; Belpaire, F. M.; Massart, D. L.; Dupont, A. G. Fundam. Clin. Pharmacol. 1994, 8, 373-378. 5. Abernethy, D. R.; Egan, J. M.; Dickinson, Twazzu.H.; Carrum, G. J. Pharmacol. Exp. Ther. 1988, 244, 994-999. 6. Robson, R. A.; Graenkel, M.; Barrett, L. J.; Birkett, D. J. Br. J. Clin. Pharmacol. 1988, 25, 402-403. 7. Sketris, I. S.; Methot, M. E.; Nicol, D.; Belitsky, P.; Knox M. G. Ann. Pharmacother. 1994, 28, 1227-1231. 8. McNally, P.; Mistry, N.; Idle, J.; Walls, J.; Feehally, J. Transplantation 1989, 48, 1071. 9. Tortorice, K. L.; Heim-Duthoy, K. L.; Awny, W. M.; Rao, K. V.; Kasiske, B. L. Ther. Drug Monit. 1990, 12, 321-328. 10. Schwartz, J. B. J. Cardiovasc. Pharmacol. 1988, 12, 1-5. 11. Sun, J. X.; Cipriano, A.; Chan, K.; John, V. A. Eur. J. Clin. Pharmacol. 1994, 47, 285-289. 12. Toupance, O.; Lavaud, S.; Canivet, E.; Bernaud, C.; Hotton, J. M.; Chanard, J. Hypertension 1994, 24, 297-300. 13. el Defrawry el Masry, S.; Cohen, G. M.; Mannering, G. J. Drug Metab. Dispos. 1974, 2, 267-278.
14. Lowry, O. H.; Rosenbrough, N. J.; Farr, A. L.; Randall R. J. J. Biol. Chem. 1951, 193, 265-275. 15. Burke, M. D.; Prough, R. A.; Mayer, R. T. Drug Metab. Dispos. 1977, 5, 1-8. 16. Burke, M. D.; Thompson, S.; Elcombe, C. R.; Halpert, J.; Haaparanta, T.; Mayer, R. T. Biochem. Pharmacol. 1985, 34, 3337-3345. 17. Koop, D. R. Mol. Pharmacol. 1986, 29, 399-404. 18. Arlotto, M. A.; Sonderfan, A. J.; Klaassen, C. D.; Parkinson, A. Biochem. Pharmacol. 1987, 36, 3859-3866. 19. Orton, T. C.; Parker, G. Drug Metab. Dispos. 1982, 10, 110115. 20. Vercruysse, I.; Schoors, D. F.; Massart, D. L.; Dupont A. Cardiovasc. Drugs Ther. 1993, 7, 721-726. 21. Piepho, R. W.; Bloedow, D. C.; Lacz, J. P.; Runser D. J.; Dimmit, D. C.; Browne, R. K. Am. J. Cardiol. 1982, 49, 525-528. 22. Kohlhaw, K.; Wonigeit, K.; Frei, U.; Oldhafer, K.; Neumann, K.; Pichlmayr, R. Transplant. Proc. 1988, 20 (Suppl 2), 572574. 23. Crocker, J. F. S.; Renton, K. W.; LeVatte, T. L.; McLellan, D. Pediatr. Nephrol. 1994, 8, 408-411.
Acknowledgments K.W.R. is supported by a grant from the Medical Research Council of Canada and R.K.D. is a recipient of a fellowship from the Dalhousie Medical Research Foundation, Halifax.
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Journal of Pharmaceutical Sciences / 1503 Vol. 86, No. 12, December 1997