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Carboxylesterase 1-Mediated Drug–Drug Interactions between Clopidogrel and Simvastatin Xinwen Wang,a,b Hao-Jie Zhu,*,a and John Seth Markowitzc a
Department of Clinical, Social, and Administrative Sciences, University of Michigan; Ann Arbor, MI 48109–1065, U.S.A.: b The Key Laboratory of Drug Metabolism and Pharmacokinetics, China Pharmaceutical University; Nanjing 210009, China: and c Department of Pharmacotherapy and Translational Research, and Center for Pharmacogenomics, University of Florida; Gainesville, FL 32610, U.S.A. Received September 23, 2014; accepted November 10, 2014 Patients with coronary artery disease often receive concurrent treatment with clopidogrel and a hydroxymethylglutaryl (HMG)-CoA reductase inhibitor medication. Accordingly, potential drug–drug interactions associated with the concomitant use of these agents present an area of concern. Both CYP enzymes and carboxylesterase 1 (CES1) are involved in the metabolism of clopidogrel, while CES1 is believed to be the enzyme responsible for the activation of simvastatin. Some in vitro studies have suggested that simvastatin could attenuate clopidogrel activation via inhibiting CYP3A activity. However, these findings have not found support in several recently published clinical investigations. The present study addresses these inconsistencies by exploring the potential role of CES1 in the metabolism of clopidogrel and simvastatin. Our in vitro human liver s9 fraction incubation study demonstrated that simvastatin significantly enhanced the formation of the intermediate metabolite 2-oxo-clopidogrel, and inhibited the CES1-mediated hydrolysis of clopidogrel, 2-oxo-clopidogrel, and the active metabolite. However, the production of the active metabolite remained unchanged. Conversely, clopidogrel was not found to influence the CES1 mediated hydrolysis (activation) of simvastatin. Moreover, we provided evidence that CES1 is not an efficient enzyme for catalyzing simvastatin activation. In summary, the inhibitory effect of simvastatin on the hydrolysis of clopidogrel and its principal metabolites may have offset the influence of simvastatin-mediated inhibition of CYP3A, and permitted the unaltered formation of the clopidogrel active metabolite. These data help explain the conflicting accounts in previous reports regarding clopidogrel and simvastatin interactions by taking into consideration CES1; they suggest that the interactions are unlikely to significantly influence clinical outcomes. Key words
carboxylesterase 1; drug–drug interaction; clopidogrel; simvastatin; prodrug activation
Clopidogrel is one of the most commonly used oral antiplatelet agents. As a prodrug, clopidogrel requires a two-step conversion to its active metabolite in order to exert its antiplatelet activity. The majority of the absorbed clopidogrel dose never enters the bioactivation cascade since ca. 85% of the parent compound is hydrolyzed by human carboxylesterase 1 (CES1) to its major, albeit inactive carboxylic acid metabolite, clopidogrel carboxylic acid,1) whereas the remainder (ca. 15%) is oxidized to the intermediate metabolite 2-oxo-clopidogrel by the hepatic CYP isoenzymes CYP2C19, CYP1A2, and CYP2B6. 2-Oxo-clopidogrel is further metabolized by CYP2B6, CYP2C9, CYP2C19 and CYP3A4 to form the final active 5-thiol metabolite (clopidogrel-AM).2) Additionally, 2-oxo-clopidogrel and clopidogrel-AM are further subject to CES1-mediated hydrolysis, forming their respective inactive carboxylate metabolites2,3) (Fig. S1A). Simvastatin, among the most widely prescribed of the “statin” family, is indicated for the treatment of hypercholesterolemia. Simvastatin is a prodrug requiring activation to its hydroxyl acid form, simvastatin acid, via cleavage of the lactone ring of the parent compound before exerting its potent inhibitory effect on HMG-CoA reductase, the rate limiting enzyme in cholesterol biosynthesis. This requisite activation has been assumed to be a process catalyzed by CES1,4) though definitive evidence is currently lacking. In addition to hydrolysis, simvastatin also undergoes extensive oxidative metabolism in the liver, which is primarily catalyzed by CYP3A4, with CYP3A5 contributing to a lesser extent (Fig. S1B). Some of
the hydrolytic products of those oxidative metabolites exhibit HMG-CoA reductase inhibition properties as well. Moreover, the active metabolite simvastatin acid is further metabolized by CYP3A4, with less contribution from CYP3A5, CYP2C8 and uridine 5′-diphosphate (UDP) glucuronosyltransferase.5) It has been suspected that the activation and antiplatelet effect of clopidogrel could be attenuated under conditions of co-administration with certain statins, such as simvastatin, since CYP3A4 is involved in the metabolism of both compounds and simvastatin is a potent CYP3A4 inhibitor.5,6) Some in vitro and ex vivo studies showed that simvastatin significantly impaired the biotransformation and antiplatelet activity of clopidogrel via inhibition of CYP3A47,8) (Fig. S1). However, recently published clinical reports have suggested that the interaction between clopidogrel and simvastatin is unlikely to occur.9,10) To date, hepatic CYP enzymes have been the primary focus of drug–drug interaction studies involving clopidogrel and statins, whereas CES1 has not been studied for its contribution to potential drug–drug interactions between clopidogrel and statins. We and other investigators recently demonstrated that decreased CES1 activity resulting from inhibition or genetic polymorphisms can lead to increased activation and improved therapeutic outcomes of clopidogrel pharmacotherapy,3,11) and simvastatin serves as both a substrate and inhibitor of CES1.4,12) In the present study, we conducted an in vitro study to elucidate the potential role of CES1 in the interaction between clopidogrel and simvastatin.
* To whom correspondence should be addressed. e-mail: [email protected] © 2015 The Pharmaceutical Society of Japan
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MATERIALS AND METHODS Materials S-(+)-Clopidogrel, 2-oxo-clopidogrel, 2-bromo3′-methoxy acetophenone (MPB) derivatized clopidogrel active metabolite (cis-clopidogrel thiol metabolite, clopidogrelAM), clopidogrel carboxylate, and the internal standards d4clopidogrel, d6 -simvastatin were all purchased from Toronto Research Chemicals Inc. (Toronto, Canada). Simvastatin was the product from Abcam (Cambridge, MA, U.S.A.) and TCI America Inc. (Portland, OR, U.S.A.), respectively. p-Nitrophenyl acetate (PNPA), p-nitrophenol (PNP), the derivatizing agent MPB, and the CES1 inhibitor bis(4-nitrophenyl) phosphate (BNPP) were purchased from Sigma-Aldrich (St. Louis, MO, U.S.A.). Pooled human liver s9 fractions were obtained from XenoTech LLC (Lenexa, KS, U.S.A.). The hydrolytic metabolites of 2-oxo-clopidogrel and clopidogrel-AM were obtained via incubation of the respective parent compounds (100 µM) with the cell s9 fractions (1 mg protein/mL) prepared from the transfected cells stably expressing wild type human CES1.3) The hydrolytic reactions were completed in 90 min at 37°C. The completion of the bioconversion was confirmed by analyzing the remaining parent compounds via high performance liquid chromatography-tandem mass spectrometry (LC-MS/MS) assay. All other chemicals and reagents were of the highest analytical grade commercially available. Effect of Simvastatin on CES1-Mediated Clopidogrel Hydrolysis An in vitro study was conducted to assess the inhibitory effect of simvastatin on CES1-mediated clopidogrel hydrolysis.3,13) The development of the stable cell lines transfected with wild type CES1 and vector, and the preparation of s9 fractions from those cells were described in details in our previous publication.13) The s9 fractions, clopidogrel, and various concentrations of simvastatin were freshly prepared in the assay buffer (0.1 m M phosphate buffer) on the day of experiment. The s9 fraction and simvastatin were pre-incubated at 37°C for 10 min, followed by the addition of clopidogrel to initiate the reaction. The final concentrations of the s9 proteins and clopidogrel were 25 µg/mL and 20 µM, respectively, while the concentrations of simvastatin were 0, 2, 5, 10, 25, 50 µM. After incubation at 37°C for 20 min, the reaction was terminated by adding a 2-fold volume of acetonitrile containing the internal standard d4-clopidogrel (50 ng/mL). The samples were centrifuged at 16000×g at 4°C for 20 min to remove proteins. The resulting supernatant was then collected for LC-MS/MS analysis. The formation of clopidogrel carboxylate from clopidogrel was determined by a validated LC-MS/MS assay described previously.3) A preliminary experiment confirmed that the formation of the metabolite was linear with the concentrations of s9 proteins and the incubation times adopted in the study. CES1-catalyzed clopidogrel hydrolysis was calculated by subtracting the value of the hydrolytic metabolite found in the vector group. Influence of Simvastatin on Clopidogrel Metabolism Pooled human liver s9 fractions were pre-incubated with a reduced nicotinamide adenine dinucleotide phosphate (NADPH) generating system (0.1 mg/mL yeast glucose-6-phosphate dehydrogenase, 3 mg/mL NADP+, and 0.07 M glucose-6-phosphate) in the absence and presence of simvastatin at 37°C for 10 min. The reaction was initiated by adding clopidogrel. The final concentrations of the liver s9 fractions and clopidogrel in the reaction system were 4 mg/mL and 20 µM, respectively,
while the concentrations of simvastatin were 5 µM, 20 µM, and 50 µM. The final volume of the reaction system was 320 µL. The known CES1 inhibitor BNPP (10 µM) was included as a positive control. Samples (70 µL) were collected at 0 and 1.5 h after initiation of the reaction. The incubation time 1.5 h was chosen based on our previous study that determined that clopidogrel-AM was maximally formed approximately1.5 h from the initiation of the reaction.3) The reaction was terminated by adding a 2-fold volume of acetonitrile containing the internal standard d4-clopidogrel (50 ng/mL) and the derivatizing reagent MPB (5 m M). MPB was utilized to form the stable MPB derivative of unstable clopidogrel-AM to facilitate analysis. The mixtures were left standing at room temperature for 10 min to allow the derivatization reaction to proceed to completion.14,15) Samples were centrifuged at 16000 ×g at 4°C for 20 min to remove precipitated proteins. The resulting supernatant was then collected for LC-MS/MS analysis, and the results were calibrated by deducting the initial concentrations of each analyte at the 0 time point. Effect of Clopidogrel on Simvastatin Activation via CES1 A preliminary experiment was conducted to evaluate CES1-catalyzed simvastatin activation using the CES1expressing cells with vector-transfected cells as the negative control. In brief, simvastatin (40 µM) was incubated with various concentrations of the cell s9 fractions (0.1, 0.2, 0.5, 1 mg/mL) at 37°C for 20 min. The reaction was terminated by adding 200 µL of acetonitrile containing 500 ng/mL internal standard d6 -simvastatin. Precipitated proteins were then removed by centrifugation (16000×g for 20 min at 4°C). The supernatant (100 µL) was transferred to autosampler vials for the measurement of the simvastatin hydrolytic active metabolite, simvastatin acid, using the LC-MS/MS method described below. Additionally, the known CES1 substrate enalapril was included as the positive control in the study. The concentrations of the hydrolytic product enalaprilat were determined based on an established LC-MS/MS assay16) after enalapril (200 µM) was incubated with CES1 cell s9 fractions (0.1, 0.2, 0.5, 1 mg/mL) at 37°C for 10 min. To assess potential effect of clopidogrel on CES1-mediated activation of simvastatin, we performed an in vitro drug–drug interaction study using the CES1-expressing cell line. Similar to the clopidogrel hydrolysis methodology described above, the cell s9 fractions, simvastatin, and a series of concentrations of clopidogrel were prepared in the reaction buffer (0.1 m M phosphate buffer). The CES1 and vector s9 fractions were pre-incubated with clopidogrel at 37°C for 10 min. The reaction was initiated by adding the substrate simvastatin. The final concentrations of s9 fraction and simvastatin were 0.2 mg/mL and 20 µM, respectively, while the concentrations of clopidogrel were 10, 20, 100, 200, 500 µM. After incubation at 37°C for 20 min, the reaction was terminated, and the samples were prepared for the measurement of the concentrations of the formed active metabolite simvastatin acid using the method described above. The model CES1 substrate PNPA was included in the study as a positive control to assess inhibitory effect of clopidogrel on CES1 utilizing previously described methods.17) LC-MS/MS Assays The concentrations of clopidogrel and its five metabolites including 2-oxo-clopidogrel, clopidogrel-AM, clopidogrel carboxylate, 2-oxo-clopidogrel carboxylate, and clopidogrel-AM carboxylate were determined by an
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LC-MS/MS assay detailed elsewhere3) Quantification of simvastatin and simvastatin acid were performed on a Shimadzu HPLC system coupled with an AB Sciex API 3000 triple quadrupole mass spectrometer (MS) based on a published method with some minor modifications.18) The analytes were separated on a reverse phase column (Phenomenex, 125 Å Aqua C18, 2.0×50 mm, 5 micron). The mobile phase consisted of 70% acetonitrile containing 2 m M ammonium acetate and 0.2% formic acid, and was delivered at 0.20 mL/min. The MS detection was performed by electrospray ionization starting from negative mode (0–4.3 min), followed by positive mode (4.3–8.5 min). The following transitions were monitored in a multiple reaction monitoring (MRM) mode: simvastatin (m/z 419.1 [M+H]+→199.3), simvastatin acid (m/z 435.3 [M−H]−→319.0), and d6 -simvastatin acid (m/z 425.1 [M+H]+→199.3). Data were acquired and analyzed utilizing AB Sciex Analyst software, Version 1.4.2 (AB Sciex, Toronto, Canada). Statistical Analysis All data are presented as the mean±S.D. In the in vitro inhibition study, the IC50 values were estimated by fitting the inhibition data to sigmoidal dose–response equation using GraphPad Prism 6.0 software (GraphPad Software, San Diego, CA, U.S.A.). The IC50 is the concentration of inhibitor at which 50% of enzymatic activity is inhibited. The differences between treated and control groups were compared using one-way ANOVA, and were considered statistically significant when p-value was less 0.05.
RESULTS Simvastatin Inhibited CES1-Mediated Clopidogrel Hydrolysis We first determined the inhibitory effect of simvastatin on the CES1-mediated hydrolysis of clopidogrel using the s9 fractions prepared from the cells stably expressing CES1. The in vitro incubation study demonstrated that simvastatin inhibited CES1-mediated clopidogrel hydrolysis in a concentration dependent manner with an IC50 value of 18.3 µM (Fig. 1). The Influence of Simvastatin on Clopidogrel Activation To determine whether simvastatin-mediated CES1 inhibition could affect the bioactivation of clopidogrel, we conducted a further in vitro incubation study in human liver s9 fractions
Fig. 1. Inhibition of CES1-Mediated Clopidogrel Hydrolysis by Simvastatin The data are the means from 3 independent experiments with error bars representing S.D.
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with an NADPH generating system. Consistent with the CES1 cell s9 incubation study, simvastatin significantly inhibited the hydrolysis of clopidogrel, 2-oxo-clopidogrel, and clopidogrelAM as evidenced by the decreases of the formation of all 3 hydrolytic metabolites (Figs. 2D–F). Additionally, co-incubation of simvastatin significantly enhanced the concentrations of 2-oxo-clopidogrel (Fig. 2B) in the reaction system in a concentration dependent manner. However, simvastatin did not significantly affect the formation of the active-metabolite even at a high concentration (50 µM) (Fig. 2C), which is in agreement with the recent clinical observations.9,10) BNPP, a potent CES1 inhibitor, was included in the study as a positive control. As anticipated, BNPP significantly increased the concentrations of clopidogrel, 2-oxo-clopidogrel, and clopidogrelAM (Figs. 2A–C) whereas markedly decreasing the formation of all 3 carboxylate metabolites (Figs. 2D–F). Clopidogrel Did Not Affect CES1-Catalyzed Simvastatin Hydrolysis (Activation) CES1-mediated simvastatin activation was determined after incubation of simvastatin with various concentrations of CES1 cell s9 fractions. The data showed that CES1 activity on simvastatin activation was very low, and the production of simvastatin active metabolite was not significantly enhanced with the increase of the concentrations of the s9 fractions (Fig. 3). As a comparison, enalapril was efficiently converted to its hydrolytic metabolite enalaprilat following incubation with the CES1 cell s9 fractions (Fig. 3). The formation of enalaprilat was increased in proportion to the concentrations of the s9 fractions. Thus, CES1 appears not to be the primary enzyme responsible for the activation of simvastatin. Inhibitory effect of clopidogrel on CES1 was evaluated using the model substrate PNPA. Consistent with previous report,19) clopidogrel was found to be an inhibitor of CES1 with an IC50 value of 24.7 µM on PNPA hydrolysis (Fig. 4). However, clopidogrel did not exhibit any effect on the CES1-catalyzed hydrolysis (activation) of simvastatin under the present experimental conditions, even when clopidogrel concentration (500 µM) was 25-time higher than the substrate simvastatin (Fig. 5).
DISCUSSION Clopidogrel has become a mainstay in the management of patients following PCI and stent placement.20,21) In addition, statins are recommended in the current therapeutic guidelines for the treatment of ACS, and are commonly used in combination with clopidogrel.22) Several drug metabolizing enzymes including CYP3A4 and CES1 are involved in the biotransformation of both clopidogrel and simvastatin. Therefore, there has been considerable speculation and concern that potentially significant drug–drug interactions may occur when these two medications are co-administered.2–5) A number of in vitro and in vivo studies have been conducted to determine the potential for the interactions with the primary focus upon the effect of simvastatin-mediated CYP3A inhibition on the activation of clopidogrel.7,9,10) However, these studies have often led to inconsistent conclusions. The present investigation differs from previous investigations by taking the substantial contribution of CES1-catalyzed hydrolysis in clopidogrel metabolism into account given that this major hydrolase is the primary enzyme responsible for the deactivation of clopidogrel and its interme-
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Fig. 2.
Effect of Simvastatin on the Metabolism of Clopidogrel in Human Liver s9 Fractions
The concentrations of clopidogrel (A) and its five metabolites including 2-oxo-clopidogrel (B), clopidogrel-AM (C), clopidogrel carboxylate (D), 2-oxo-clopidogrel carboxylate (E), and clopidogrel-AM carboxylate (F), were determined after incubation of clopidogrel (20 µM) with pooled human liver s9 fractions (4 mg/mL) in the absence or presence of different concentrations of simvastatin (5, 20, and 50 µM). The known CES1 inhibitor BNPP (10 µM) was included as a positive control. Data are the averages of three independent experiments with error bars representing S.D. * p
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Carboxylesterase 1-Mediated Drug–Drug Interactions between Clopidogrel and Simvastatin Xinwen Wang,a,b Hao-Jie Zhu,*,a and John Seth Markowitzc a
Department of Clinical, Social, and Administrative Sciences, University of Michigan; Ann Arbor, MI 48109–1065, U.S.A.: b The Key Laboratory of Drug Metabolism and Pharmacokinetics, China Pharmaceutical University; Nanjing 210009, China: and c Department of Pharmacotherapy and Translational Research, and Center for Pharmacogenomics, University of Florida; Gainesville, FL 32610, U.S.A. Received September 23, 2014; accepted November 10, 2014 Patients with coronary artery disease often receive concurrent treatment with clopidogrel and a hydroxymethylglutaryl (HMG)-CoA reductase inhibitor medication. Accordingly, potential drug–drug interactions associated with the concomitant use of these agents present an area of concern. Both CYP enzymes and carboxylesterase 1 (CES1) are involved in the metabolism of clopidogrel, while CES1 is believed to be the enzyme responsible for the activation of simvastatin. Some in vitro studies have suggested that simvastatin could attenuate clopidogrel activation via inhibiting CYP3A activity. However, these findings have not found support in several recently published clinical investigations. The present study addresses these inconsistencies by exploring the potential role of CES1 in the metabolism of clopidogrel and simvastatin. Our in vitro human liver s9 fraction incubation study demonstrated that simvastatin significantly enhanced the formation of the intermediate metabolite 2-oxo-clopidogrel, and inhibited the CES1-mediated hydrolysis of clopidogrel, 2-oxo-clopidogrel, and the active metabolite. However, the production of the active metabolite remained unchanged. Conversely, clopidogrel was not found to influence the CES1 mediated hydrolysis (activation) of simvastatin. Moreover, we provided evidence that CES1 is not an efficient enzyme for catalyzing simvastatin activation. In summary, the inhibitory effect of simvastatin on the hydrolysis of clopidogrel and its principal metabolites may have offset the influence of simvastatin-mediated inhibition of CYP3A, and permitted the unaltered formation of the clopidogrel active metabolite. These data help explain the conflicting accounts in previous reports regarding clopidogrel and simvastatin interactions by taking into consideration CES1; they suggest that the interactions are unlikely to significantly influence clinical outcomes. Key words
carboxylesterase 1; drug–drug interaction; clopidogrel; simvastatin; prodrug activation
Clopidogrel is one of the most commonly used oral antiplatelet agents. As a prodrug, clopidogrel requires a two-step conversion to its active metabolite in order to exert its antiplatelet activity. The majority of the absorbed clopidogrel dose never enters the bioactivation cascade since ca. 85% of the parent compound is hydrolyzed by human carboxylesterase 1 (CES1) to its major, albeit inactive carboxylic acid metabolite, clopidogrel carboxylic acid,1) whereas the remainder (ca. 15%) is oxidized to the intermediate metabolite 2-oxo-clopidogrel by the hepatic CYP isoenzymes CYP2C19, CYP1A2, and CYP2B6. 2-Oxo-clopidogrel is further metabolized by CYP2B6, CYP2C9, CYP2C19 and CYP3A4 to form the final active 5-thiol metabolite (clopidogrel-AM).2) Additionally, 2-oxo-clopidogrel and clopidogrel-AM are further subject to CES1-mediated hydrolysis, forming their respective inactive carboxylate metabolites2,3) (Fig. S1A). Simvastatin, among the most widely prescribed of the “statin” family, is indicated for the treatment of hypercholesterolemia. Simvastatin is a prodrug requiring activation to its hydroxyl acid form, simvastatin acid, via cleavage of the lactone ring of the parent compound before exerting its potent inhibitory effect on HMG-CoA reductase, the rate limiting enzyme in cholesterol biosynthesis. This requisite activation has been assumed to be a process catalyzed by CES1,4) though definitive evidence is currently lacking. In addition to hydrolysis, simvastatin also undergoes extensive oxidative metabolism in the liver, which is primarily catalyzed by CYP3A4, with CYP3A5 contributing to a lesser extent (Fig. S1B). Some of
the hydrolytic products of those oxidative metabolites exhibit HMG-CoA reductase inhibition properties as well. Moreover, the active metabolite simvastatin acid is further metabolized by CYP3A4, with less contribution from CYP3A5, CYP2C8 and uridine 5′-diphosphate (UDP) glucuronosyltransferase.5) It has been suspected that the activation and antiplatelet effect of clopidogrel could be attenuated under conditions of co-administration with certain statins, such as simvastatin, since CYP3A4 is involved in the metabolism of both compounds and simvastatin is a potent CYP3A4 inhibitor.5,6) Some in vitro and ex vivo studies showed that simvastatin significantly impaired the biotransformation and antiplatelet activity of clopidogrel via inhibition of CYP3A47,8) (Fig. S1). However, recently published clinical reports have suggested that the interaction between clopidogrel and simvastatin is unlikely to occur.9,10) To date, hepatic CYP enzymes have been the primary focus of drug–drug interaction studies involving clopidogrel and statins, whereas CES1 has not been studied for its contribution to potential drug–drug interactions between clopidogrel and statins. We and other investigators recently demonstrated that decreased CES1 activity resulting from inhibition or genetic polymorphisms can lead to increased activation and improved therapeutic outcomes of clopidogrel pharmacotherapy,3,11) and simvastatin serves as both a substrate and inhibitor of CES1.4,12) In the present study, we conducted an in vitro study to elucidate the potential role of CES1 in the interaction between clopidogrel and simvastatin.
* To whom correspondence should be addressed. e-mail: [email protected] © 2015 The Pharmaceutical Society of Japan
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MATERIALS AND METHODS Materials S-(+)-Clopidogrel, 2-oxo-clopidogrel, 2-bromo3′-methoxy acetophenone (MPB) derivatized clopidogrel active metabolite (cis-clopidogrel thiol metabolite, clopidogrelAM), clopidogrel carboxylate, and the internal standards d4clopidogrel, d6 -simvastatin were all purchased from Toronto Research Chemicals Inc. (Toronto, Canada). Simvastatin was the product from Abcam (Cambridge, MA, U.S.A.) and TCI America Inc. (Portland, OR, U.S.A.), respectively. p-Nitrophenyl acetate (PNPA), p-nitrophenol (PNP), the derivatizing agent MPB, and the CES1 inhibitor bis(4-nitrophenyl) phosphate (BNPP) were purchased from Sigma-Aldrich (St. Louis, MO, U.S.A.). Pooled human liver s9 fractions were obtained from XenoTech LLC (Lenexa, KS, U.S.A.). The hydrolytic metabolites of 2-oxo-clopidogrel and clopidogrel-AM were obtained via incubation of the respective parent compounds (100 µM) with the cell s9 fractions (1 mg protein/mL) prepared from the transfected cells stably expressing wild type human CES1.3) The hydrolytic reactions were completed in 90 min at 37°C. The completion of the bioconversion was confirmed by analyzing the remaining parent compounds via high performance liquid chromatography-tandem mass spectrometry (LC-MS/MS) assay. All other chemicals and reagents were of the highest analytical grade commercially available. Effect of Simvastatin on CES1-Mediated Clopidogrel Hydrolysis An in vitro study was conducted to assess the inhibitory effect of simvastatin on CES1-mediated clopidogrel hydrolysis.3,13) The development of the stable cell lines transfected with wild type CES1 and vector, and the preparation of s9 fractions from those cells were described in details in our previous publication.13) The s9 fractions, clopidogrel, and various concentrations of simvastatin were freshly prepared in the assay buffer (0.1 m M phosphate buffer) on the day of experiment. The s9 fraction and simvastatin were pre-incubated at 37°C for 10 min, followed by the addition of clopidogrel to initiate the reaction. The final concentrations of the s9 proteins and clopidogrel were 25 µg/mL and 20 µM, respectively, while the concentrations of simvastatin were 0, 2, 5, 10, 25, 50 µM. After incubation at 37°C for 20 min, the reaction was terminated by adding a 2-fold volume of acetonitrile containing the internal standard d4-clopidogrel (50 ng/mL). The samples were centrifuged at 16000×g at 4°C for 20 min to remove proteins. The resulting supernatant was then collected for LC-MS/MS analysis. The formation of clopidogrel carboxylate from clopidogrel was determined by a validated LC-MS/MS assay described previously.3) A preliminary experiment confirmed that the formation of the metabolite was linear with the concentrations of s9 proteins and the incubation times adopted in the study. CES1-catalyzed clopidogrel hydrolysis was calculated by subtracting the value of the hydrolytic metabolite found in the vector group. Influence of Simvastatin on Clopidogrel Metabolism Pooled human liver s9 fractions were pre-incubated with a reduced nicotinamide adenine dinucleotide phosphate (NADPH) generating system (0.1 mg/mL yeast glucose-6-phosphate dehydrogenase, 3 mg/mL NADP+, and 0.07 M glucose-6-phosphate) in the absence and presence of simvastatin at 37°C for 10 min. The reaction was initiated by adding clopidogrel. The final concentrations of the liver s9 fractions and clopidogrel in the reaction system were 4 mg/mL and 20 µM, respectively,
while the concentrations of simvastatin were 5 µM, 20 µM, and 50 µM. The final volume of the reaction system was 320 µL. The known CES1 inhibitor BNPP (10 µM) was included as a positive control. Samples (70 µL) were collected at 0 and 1.5 h after initiation of the reaction. The incubation time 1.5 h was chosen based on our previous study that determined that clopidogrel-AM was maximally formed approximately1.5 h from the initiation of the reaction.3) The reaction was terminated by adding a 2-fold volume of acetonitrile containing the internal standard d4-clopidogrel (50 ng/mL) and the derivatizing reagent MPB (5 m M). MPB was utilized to form the stable MPB derivative of unstable clopidogrel-AM to facilitate analysis. The mixtures were left standing at room temperature for 10 min to allow the derivatization reaction to proceed to completion.14,15) Samples were centrifuged at 16000 ×g at 4°C for 20 min to remove precipitated proteins. The resulting supernatant was then collected for LC-MS/MS analysis, and the results were calibrated by deducting the initial concentrations of each analyte at the 0 time point. Effect of Clopidogrel on Simvastatin Activation via CES1 A preliminary experiment was conducted to evaluate CES1-catalyzed simvastatin activation using the CES1expressing cells with vector-transfected cells as the negative control. In brief, simvastatin (40 µM) was incubated with various concentrations of the cell s9 fractions (0.1, 0.2, 0.5, 1 mg/mL) at 37°C for 20 min. The reaction was terminated by adding 200 µL of acetonitrile containing 500 ng/mL internal standard d6 -simvastatin. Precipitated proteins were then removed by centrifugation (16000×g for 20 min at 4°C). The supernatant (100 µL) was transferred to autosampler vials for the measurement of the simvastatin hydrolytic active metabolite, simvastatin acid, using the LC-MS/MS method described below. Additionally, the known CES1 substrate enalapril was included as the positive control in the study. The concentrations of the hydrolytic product enalaprilat were determined based on an established LC-MS/MS assay16) after enalapril (200 µM) was incubated with CES1 cell s9 fractions (0.1, 0.2, 0.5, 1 mg/mL) at 37°C for 10 min. To assess potential effect of clopidogrel on CES1-mediated activation of simvastatin, we performed an in vitro drug–drug interaction study using the CES1-expressing cell line. Similar to the clopidogrel hydrolysis methodology described above, the cell s9 fractions, simvastatin, and a series of concentrations of clopidogrel were prepared in the reaction buffer (0.1 m M phosphate buffer). The CES1 and vector s9 fractions were pre-incubated with clopidogrel at 37°C for 10 min. The reaction was initiated by adding the substrate simvastatin. The final concentrations of s9 fraction and simvastatin were 0.2 mg/mL and 20 µM, respectively, while the concentrations of clopidogrel were 10, 20, 100, 200, 500 µM. After incubation at 37°C for 20 min, the reaction was terminated, and the samples were prepared for the measurement of the concentrations of the formed active metabolite simvastatin acid using the method described above. The model CES1 substrate PNPA was included in the study as a positive control to assess inhibitory effect of clopidogrel on CES1 utilizing previously described methods.17) LC-MS/MS Assays The concentrations of clopidogrel and its five metabolites including 2-oxo-clopidogrel, clopidogrel-AM, clopidogrel carboxylate, 2-oxo-clopidogrel carboxylate, and clopidogrel-AM carboxylate were determined by an
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LC-MS/MS assay detailed elsewhere3) Quantification of simvastatin and simvastatin acid were performed on a Shimadzu HPLC system coupled with an AB Sciex API 3000 triple quadrupole mass spectrometer (MS) based on a published method with some minor modifications.18) The analytes were separated on a reverse phase column (Phenomenex, 125 Å Aqua C18, 2.0×50 mm, 5 micron). The mobile phase consisted of 70% acetonitrile containing 2 m M ammonium acetate and 0.2% formic acid, and was delivered at 0.20 mL/min. The MS detection was performed by electrospray ionization starting from negative mode (0–4.3 min), followed by positive mode (4.3–8.5 min). The following transitions were monitored in a multiple reaction monitoring (MRM) mode: simvastatin (m/z 419.1 [M+H]+→199.3), simvastatin acid (m/z 435.3 [M−H]−→319.0), and d6 -simvastatin acid (m/z 425.1 [M+H]+→199.3). Data were acquired and analyzed utilizing AB Sciex Analyst software, Version 1.4.2 (AB Sciex, Toronto, Canada). Statistical Analysis All data are presented as the mean±S.D. In the in vitro inhibition study, the IC50 values were estimated by fitting the inhibition data to sigmoidal dose–response equation using GraphPad Prism 6.0 software (GraphPad Software, San Diego, CA, U.S.A.). The IC50 is the concentration of inhibitor at which 50% of enzymatic activity is inhibited. The differences between treated and control groups were compared using one-way ANOVA, and were considered statistically significant when p-value was less 0.05.
RESULTS Simvastatin Inhibited CES1-Mediated Clopidogrel Hydrolysis We first determined the inhibitory effect of simvastatin on the CES1-mediated hydrolysis of clopidogrel using the s9 fractions prepared from the cells stably expressing CES1. The in vitro incubation study demonstrated that simvastatin inhibited CES1-mediated clopidogrel hydrolysis in a concentration dependent manner with an IC50 value of 18.3 µM (Fig. 1). The Influence of Simvastatin on Clopidogrel Activation To determine whether simvastatin-mediated CES1 inhibition could affect the bioactivation of clopidogrel, we conducted a further in vitro incubation study in human liver s9 fractions
Fig. 1. Inhibition of CES1-Mediated Clopidogrel Hydrolysis by Simvastatin The data are the means from 3 independent experiments with error bars representing S.D.
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with an NADPH generating system. Consistent with the CES1 cell s9 incubation study, simvastatin significantly inhibited the hydrolysis of clopidogrel, 2-oxo-clopidogrel, and clopidogrelAM as evidenced by the decreases of the formation of all 3 hydrolytic metabolites (Figs. 2D–F). Additionally, co-incubation of simvastatin significantly enhanced the concentrations of 2-oxo-clopidogrel (Fig. 2B) in the reaction system in a concentration dependent manner. However, simvastatin did not significantly affect the formation of the active-metabolite even at a high concentration (50 µM) (Fig. 2C), which is in agreement with the recent clinical observations.9,10) BNPP, a potent CES1 inhibitor, was included in the study as a positive control. As anticipated, BNPP significantly increased the concentrations of clopidogrel, 2-oxo-clopidogrel, and clopidogrelAM (Figs. 2A–C) whereas markedly decreasing the formation of all 3 carboxylate metabolites (Figs. 2D–F). Clopidogrel Did Not Affect CES1-Catalyzed Simvastatin Hydrolysis (Activation) CES1-mediated simvastatin activation was determined after incubation of simvastatin with various concentrations of CES1 cell s9 fractions. The data showed that CES1 activity on simvastatin activation was very low, and the production of simvastatin active metabolite was not significantly enhanced with the increase of the concentrations of the s9 fractions (Fig. 3). As a comparison, enalapril was efficiently converted to its hydrolytic metabolite enalaprilat following incubation with the CES1 cell s9 fractions (Fig. 3). The formation of enalaprilat was increased in proportion to the concentrations of the s9 fractions. Thus, CES1 appears not to be the primary enzyme responsible for the activation of simvastatin. Inhibitory effect of clopidogrel on CES1 was evaluated using the model substrate PNPA. Consistent with previous report,19) clopidogrel was found to be an inhibitor of CES1 with an IC50 value of 24.7 µM on PNPA hydrolysis (Fig. 4). However, clopidogrel did not exhibit any effect on the CES1-catalyzed hydrolysis (activation) of simvastatin under the present experimental conditions, even when clopidogrel concentration (500 µM) was 25-time higher than the substrate simvastatin (Fig. 5).
DISCUSSION Clopidogrel has become a mainstay in the management of patients following PCI and stent placement.20,21) In addition, statins are recommended in the current therapeutic guidelines for the treatment of ACS, and are commonly used in combination with clopidogrel.22) Several drug metabolizing enzymes including CYP3A4 and CES1 are involved in the biotransformation of both clopidogrel and simvastatin. Therefore, there has been considerable speculation and concern that potentially significant drug–drug interactions may occur when these two medications are co-administered.2–5) A number of in vitro and in vivo studies have been conducted to determine the potential for the interactions with the primary focus upon the effect of simvastatin-mediated CYP3A inhibition on the activation of clopidogrel.7,9,10) However, these studies have often led to inconsistent conclusions. The present investigation differs from previous investigations by taking the substantial contribution of CES1-catalyzed hydrolysis in clopidogrel metabolism into account given that this major hydrolase is the primary enzyme responsible for the deactivation of clopidogrel and its interme-
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Fig. 2.
Effect of Simvastatin on the Metabolism of Clopidogrel in Human Liver s9 Fractions
The concentrations of clopidogrel (A) and its five metabolites including 2-oxo-clopidogrel (B), clopidogrel-AM (C), clopidogrel carboxylate (D), 2-oxo-clopidogrel carboxylate (E), and clopidogrel-AM carboxylate (F), were determined after incubation of clopidogrel (20 µM) with pooled human liver s9 fractions (4 mg/mL) in the absence or presence of different concentrations of simvastatin (5, 20, and 50 µM). The known CES1 inhibitor BNPP (10 µM) was included as a positive control. Data are the averages of three independent experiments with error bars representing S.D. * p
