Carboxylesterase 1-Mediated Drug–Drug Interactions between Clopidogrel and Simvastatin

Abstract

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.

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 metabolites^2,3) (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 CYP3A4^7,8) (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.

MATERIALS AND METHODS

Materials

S-(+)-Clopidogrel, 2-oxo-clopidogrel, 2-bromo-3′-methoxy acetophenone (MPB) derivatized clopidogrel active metabolite (cis-clopidogrel thiol metabolite, clopidogrel-AM), clopidogrel carboxylate, and the internal standards d₄-clopidogrel, 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 mM 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 mM). 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 CES1-expressing 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 mM 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 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 mM 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).

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.

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 with an NADPH generating system. Consistent with the CES1 cell s9 incubation study, simvastatin significantly inhibited the hydrolysis of clopidogrel, 2-oxo-clopidogrel, and clopidogrel-AM 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 clopidogrel-AM (Figs. 2A–C) whereas markedly decreasing the formation of all 3 carboxylate metabolites (Figs. 2D–F).

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<0.05, ** p<0.01, *** p<0.001 versus control.

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.

Fig. 3. CES1-Mediated Hydrolysis of Simvastatin and Enalapril

The concentrations of the hydrolytic metabolites simvastatin acid (right y-axis) and enalaprilat (left y-axis) were determined after incubation of the substrates with various concentrations of CES1 cell s9 fractions. Data are presented as mean±S.D. (n=3).

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).

Fig. 4. Inhibitory Effect of Clopidogrel on CES1-Mediated PNPA Hydrolysis

CES1 activity was assessed by determining the formation of the hydrolytic metabolite PNP. The relative CES1 activity in control group was defined as 100%. Each data point represents mean±S.D. of triplicated experiments.

Fig. 5. Influence of Clopidogrel on CES1-Catalyzed Simvastatin Activation

The formation of simvastatin acid was determined after simvastatin (20 µM) was incubated with various concentrations of clopidogrel (10 to 500 µM) in CES1 cell s9 fractions. Data are presented as mean±S.D. (n=3).

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 intermediate and active metabolites, and is also considered to be the requisite enzyme for simvastatin activation. Another major advantage of the present study is that we were able to determine the formation of both the intermediate metabolite 2-oxo-clopidogrel, and the final active metabolite clopidogrel-AM for the assessment of potential interactions between these two agents. Previously published studies addressing this issue have been focused only on the parent compound and failed to evaluate the formation of the intermediate and active metabolites.

The determination of clopidogrel and its five metabolites illustrated the details about the influence of simvastatin on clopidogrel metabolism and provided clues about the drug interaction mechanisms as well. As a CES1 inhibitor (Fig. 1), simvastatin significantly decreased hydrolytic metabolism of clopidogrel, 2-oxo-clopidogrel, and clopidogrel-AM. However, co-incubation of simvastatin significantly enhanced the formation of the intermediate metabolite 2-oxo-clopidogrel. Metabolic elimination of 2-oxo-clopidogrel is governed by 2 distinctive pathways, i.e., the formation of its hydrolytic metabolite catalyzed by CES1 and the biotransformation of the active metabolite catalyzed by CYP enzymes (CYP2B6, CYP2C9, CYP2C19 and CYP3A4).²⁾ Therefore, the increase of 2-oxo-clopidogrel is likely due to the collective effect of the inhibition of CES1 and CYP3A enzymes by simvastatin.

It has been speculated by several investigators that as a CYP3A inhibitor, simvastatin could decrease the formation of clopidogrel-AM leading to impaired antiplatelet activity of clopidogrel treatment.⁷⁾ However, a number of recently published clinical studies suggested that co-administration of simvastatin didn’t significantly affect the pharmacological activity of clopidogrel.^23,24) Consistent with those clinical observations, our in vitro study revealed no any significant changes of the production of the active metabolite following the co-incubation of clopidogrel and simvastatin. Clopidogrel and its intermediate and active metabolites undergo complex metabolism involving multiple enzymes. Clopidogrel-AM is formed from the metabolism of 2-oxo-clopidogrel catalyzed by CYP2B6, 2C9, 2C19 and 3A4, and then eliminated by CES1-catalyzed hydrolysis.^2,3) Therefore, both the CYP enzymes and CES1 regulate the production of clopidogrel-AM. It is a plausible interpretation that the inhibition of CES1 and the presence of higher concentrations of 2-oxo-clopidogrel have offset the effect of CYP3A inhibition of simvastatin on clopidogrel activation resulting in essentially no change in the net formation of clopidogrel-AM.

CES1 has been speculated to be the major esterase involved in simvastatin activation,⁴⁾ though experimental evidence is currently lacking. The in vitro incubation study indicated that CES1 is unlikely to play a significant role in simvastatin activation (Fig. 3). Additionally, the CES1 inhibitor clopidogrel did not alter the metabolism of simvastatin (Fig. 5), which provides further evidence supporting that CES1 is not the primary enzyme activating simvastatin. Further study is warranted to identify the enzyme(s) responsible for the activation of simvastatin.

In summary, the present study demonstrated that simvastatin significantly inhibited CES1-catalyzed hydrolysis of clopidogrel, 2-oxo-clopidogrel and clopidogrel-AM, and significantly enhanced the formation of 2-oxo-clopidogrel. However, co-incubation of simvastatin didn’t affect the production of clopidogrel-AM, which appears to be due to the inhibition of both the CYP3A4-mediated activation and the CES1-mediated hydrolytic deactivation pathways of clopidogrel-AM. These data may help to explain the negative findings of the assessments of clopidogrel and simvastatin interactions as observed in clinical studies. Furthermore, contrary to previous speculation, CES1 is not an efficient enzyme catalyzing simvastatin activation.

Conflict of Interest

The authors declare no conflict of interest.

Supplementary Materials

The online version of this article contains supplementary materials.

REFERENCES

• 1) Tang M, Mukundan M, Yang J, Charpentier N, LeCluyse EL, Black C, Yang D, Shi D, Yan B. Antiplatelet agents aspirin and clopidogrel are hydrolyzed by distinct carboxylesterases, and clopidogrel is transesterificated in the presence of ethyl alcohol. J. Pharmacol. Exp. Ther., 319, 1467–1476 (2006).
• 2) Kazui M, Nishiya Y, Ishizuka T, Hagihara K, Farid NA, Okazaki O, Ikeda T, Kurihara A. Identification of the human cytochrome P450 enzymes involved in the two oxidative steps in the bioactivation of clopidogrel to its pharmacologically active metabolite. Drug Metab. Dispos., 38, 92–99 (2010).
• 3) Zhu HJ, Wang X, Gawronski BE, Brinda BJ, Angiolillo DJ, Markowitz JS. Carboxylesterase 1 as a determinant of clopidogrel metabolism and activation. J. Pharmacol. Exp. Ther., 344, 665–672 (2013).
• 4) Laizure SC, Herring V, Hu Z, Witbrodt K, Parker RB. The role of human carboxylesterases in drug metabolism: have we overlooked their importance? Pharmacotherapy, 33, 210–222 (2013).
• 5) Prueksaritanont T, Gorham LM, Ma B, Liu L, Yu X, Zhao JJ, Slaughter DE, Arison BH, Vyas KP. In vitro metabolism of simvastatin in humans [SBT]identification of metabolizing enzymes and effect of the drug on hepatic P450s. Drug Metab. Dispos., 25, 1191–1199 (1997).
• 6) Müller I, Besta F, Schulz C, Li Z, Massberg S, Gawaz M. Effects of statins on platelet inhibition by a high loading dose of clopidogrel. Circulation, 108, 2195–2197 (2003).
• 7) Zahno A, Brecht K, Bodmer M, Bur D, Tsakiris DA, Krahenbuhl S. Effects of drug interactions on biotransformation and antiplatelet effect of clopidogrel in vitro. Br. J. Pharmacol., 161, 393–404 (2010).
• 8) Mach F, Senouf D, Fontana P, Boehlen F, Reber G, Daali Y, de Moerloose P, Sigwart U. Not all statins interfere with clopidogrel during antiplatelet therapy. Eur. J. Clin. Invest., 35, 476–481 (2005).
• 9) Saw J, Brennan DM, Steinhubl SR, Bhatt DL, Mak KH, Fox K, Topol EJ. CHARISMA Investigators. Lack of evidence of a clopidogrel–statin interaction in the CHARISMA trial. J. Am. Coll. Cardiol., 50, 291–295 (2007).
• 10) Serrano Júnior CV, Soeiro Ade M, Araújo LF, Jabot B, Rached F, Orii NM, Nicolau JC, Duarte AJ, Ramires JA. Lack of clopidogrel–statin interaction in patients undergoing coronary stent implantation. Arq. Bras. Cardiol., 95, 321–327 (2010).
• 11) Lewis JP, Horenstein RB, Ryan K, O’Connell JR, Gibson Q, Mitchell BD, Tanner K, Chai S, Bliden KP, Tantry US, Peer CJ, Figg WD, Spencer SD, Pacanowski MA, Gurbel PA, Shuldiner AR. The functional G143E variant of carboxylesterase 1 is associated with increased clopidogrel active metabolite levels and greater clopidogrel response. Pharmacogenet. Genomics, 23, 1–8 (2013).
• 12) Fukami T, Takahashi S, Nakagawa N, Maruichi T, Nakajima M, Yokoi T. In vitro evaluation of inhibitory effects of antidiabetic and antihyperlipidemic drugs on human carboxylesterase activities. Drug Metab. Dispos., 38, 2173–2178 (2010).
• 13) Zhu HJ, Patrick KS, Yuan HJ, Wang JS, Donovan JL, DeVane CL, Malcolm R, Johnson JA, Youngblood GL, Sweet DH, Langaee TY, Markowitz JS. Two CES1 gene mutations lead to dysfunctional carboxylesterase 1 activity in man: clinical significance and molecular basis. Am. J. Hum. Genet., 82, 1241–1248 (2008).
• 14) Takahashi M, Pang H, Kawabata K, Farid NA, Kurihara A. Quantitative determination of clopidogrel active metabolite in human plasma by LC-MS/MS. J. Pharm. Biomed. Anal., 48, 1219–1224 (2008).
• 15) Tuffal G, Roy S, Lavisse M, Brasseur D, Schofield J, Delesque Touchard N, Savi P, Bremond N, Rouchon MC, Hurbin F, Sultan E. An improved method for specific and quantitative determination of the clopidogrel active metabolite isomers in human plasma. Thromb. Haemost., 105, 696–705 (2011).
• 16) Gu Q, Chen X, Zhong D, Wang Y. Simultaneous determination of enalapril and enalaprilat in human plasma by liquid chromatography-tandem mass spectrometry. J. Chromatogr. B Analyt. Technol. Biomed. Life Sci., 813, 337–342 (2004).
• 17) Zhu HJ, Appel DI, Peterson YK, Wang Z, Markowitz JS. Identification of selected therapeutic agents as inhibitors of carboxylesterase 1: Potential sources of metabolic drug interactions. Toxicology, 270, 59–65 (2010).
• 18) Yang AY, Sun L, Musson DG, Zhao JJ. Application of a novel ultra-low elution volume 96-well solid-phase extraction method to the LC/MS/MS determination of simvastatin and simvastatin acid in human plasma. J. Pharm. Biomed. Anal., 38, 521–527 (2005).
• 19) Kim MJ, Jeong ES, Park JS, Lee SJ, Ghim JL, Choi CS, Shin JG, Kim DH. Multiple cytochromes P450s are involved in the generation of a pharmacologically active thiol metabolite, whereas paraoxonase 1 and carboxylesterase 1 catalyze the formation of a thiol metabolite isomer from ticlopidine. Drug Metab. Dispos., 42, 141–152 (2014).
• 20) Bonello L, Tantry US, Marcucci R, Blindt R, Angiolillo DJ, Becker R, Bhatt DL, Cattaneo M, Collet JP, Cuisset T, Gachet C, Montalescot G, Jennings LK, Kereiakes D, Sibbing D, Trenk D, Van Werkum JW, Paganelli F, Price MJ, Waksman R, Gurbel PA, Working Group on High On-Treatment Platelet Reactivity. Consensus and future directions on the definition of high on-treatment platelet reactivity to adenosine diphosphate. J. Am. Coll. Cardiol., 56, 919–933 (2010).
• 21) Parodi G, Marcucci R, Valenti R, Gori AM, Migliorini A, Giusti B, Buonamici P, Gensini GF, Abbate R, Antoniucci D. High residual platelet reactivity after clopidogrel loading and long-term cardiovascular events among patients with acute coronary syndromes undergoing PCI. JAMA, 306, 1215–1223 (2011).
• 22) Hamm CW, Bassand JP, Agewall S, Bax J, Boersma E, Bueno H, Caso P, Dudek D, Gielen S, Huber K, Ohman M, Petrie MC, Sonntag F, Uva MS, Storey RF, Wijns W, Zahger D, Bax JJ, Auricchio A, Baumgartner H, Ceconi C, Dean V, Deaton C, Fagard R, Funck-Brentano C, Hasdai D, Hoes A, Knuuti J, Kolh P, McDonagh T, Moulin C, Poldermans D, Popescu BA, Reiner Z, Sechtem U, Sirnes PA, Torbicki A, Vahanian A, Windecker S, Windecker S, Achenbach S, Badimon L, Bertrand M, Botker HE, Collet J-P, Crea F, Danchin N, Falk E, Goudevenos J, Gulba D, Hambrecht R, Herrmann J, Kastrati A, Kjeldsen K, Kristensen SD, Lancellotti P, Mehilli J, Merkely B, Montalescot G, Neumann F-J, Neyses L, Perk J, Roffi M, Romeo F, Ruda M, Swahn E, Valgimigli M, Vrints CJ, Widimsky P, ESC Committee for Practice Guidelines. ESC Guidelines for the management of acute coronary syndromes in patients presenting without persistent ST-segment elevation: The Task Force for the management of acute coronary syndromes (ACS) in patients presenting without persistent ST-segment elevation of the European Society of Cardiology (ESC). Eur. Heart J., 32, 2999–3054 (2011).
• 23) Gorchakova O, von Beckerath N, Gawaz M, Mocz A, Joost A, Schomig A, Kastrati A. Antiplatelet effects of a 600 mg loading dose of clopidogrel are not attenuated in patients receiving atorvastatin or simvastatin for at least 4 weeks prior to coronary artery stenting. Eur. Heart J., 25, 1898–1902 (2004).
• 24) Malmström RE, Ostergren J, Jorgensen L, Hjemdahl P, CASTOR investigators. Influence of statin treatment on platelet inhibition by clopidogrel—a randomized comparison of rosuvastatin, atorvastatin and simvastatin co-treatment. J. Intern. Med., 266, 457–466 (2009).