Clopidogrel: A multifaceted affair


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Review

Clopidogrel: A Multifaceted Affair

The Journal of Clinical Pharmacology XX(XX) 1–9 © 2014, The American College of Clinical Pharmacology DOI: 10.1002/jcph.413

Efrén Martínez-Quintana, MD, PhD1, and Antonio Tugores, PhD2

Abstract Clopidogrel has been the therapy of choice, combined with aspirin, against platelet aggregation in patients at risk of suffering a vascular thrombotic event. Not all patients respond equally to clopidogrel, an observation that has led to searching for a test that, in the clinical setting, could predict patients’ “resistance” to therapy. The evidence reveals a complex pharmacokinetic profile for clopidogrel, with multiple players involved, including cytochromes, characteristics of the target tissue, and accompanying clinical conditions. Despite FDA black box warnings recommending CYP2C19 genotyping before clopidogrel use, no robust evidence indicates that CYP2C19 function determines clinical response to the drug, either based on the presence of loss of function alleles or drug interactions with CYP2C19 inhibitors, like omeprazole. A tailored anti-aggregation treatment based on ex vivo platelet reactivity also seems unlikely due to the lack of robustness of most assays. The identification of clinical conditions that are at higher risk of new cardiovascular events, such as diabetes, obesity, coronary artery disease, or specific stenting procedures, seems to be a prudent approach to tailor anti-platelet therapy with more powerful drugs, accompanied by careful counseling to promote patient compliance.

Keywords clopidogrel, response, pharmacogenetics, clinical

Platelet anti-aggregation is the main therapeutic approach to prevent new cardiovascular complications in patients who have suffered a major acute cardiovascular event (MACE) and may have undergone a percutaneous coronary intervention (PCI).1 A well-established treatment regime has been the combination of aspirin, that blocks thromboxane synthesis, and clopidogrel, a prodrug that, upon biotransformation, irreversibly blocks the binding of adenosine diphosphate (ADP) to its platelet P2Y12 receptor (previously P2T), thus preventing the inactivation of adenylate cyclase (AC) and the subsequent activation of the glycoprotein GPIIb/IIIa complex, required for platelet aggregation.2 The clinical benefit of adding clopidogrel to aspirin therapy has been demonstrated by several large, multicenter, randomized, controlled trials. The CAPRIE trial (clopidogrel vs. aspirin in patients at risk of ischemic events) was the first to prove the benefit of clopidogrel over aspirin, showing a relative risk reduction of 8.7% for the secondary prevention of cardiovascular events in 19,185 patients with clinical manifestations of atherosclerosis.3 The CURE (clopidogrel in unstable angina to prevent recurrent events) study, performed in 12,562 patients with acute coronary syndrome (unstable angina or non-ST elevation myocardial infarction), demonstrated that a loading dose of 300 mg clopidogrel followed by a once daily treatment with 75 mg, in addition to aspirin (75– 325 mg/day) significantly reduced the risk of the combined endpoint of cardiovascular death, myocardial infarction, and stroke (9.3% of the patients in the clopidogrel group vs. 11.4% of the patients in the placebo group, relative risk

0.80, P < .001). Moreover, the percentages of patients with in-hospital refractory or severe ischemia, heart failure, and revascularization procedures were also significantly lower in patients receiving clopidogrel.4 The benefits of clopidogrel in the CURE trial were consistently demonstrated in low-, intermediate-, and high-risk patients with acute coronary syndromes, as stratified by TIMI risk score, thus supporting its use in all patients with documented non-ST elevation acute coronary syndromes.5 The downside of these trials was evidenced by the risk of major bleeding, that was higher in the clopidogrel group than in the placebo group (3.7% vs. 2.7%, relative risk 1.38, P ¼ .001), although without significant life-threatening episodes.4 The PCI-CURE study demonstrated that pretreatment with clopidogrel, in addition to aspirin, followed by longterm therapy after PCI resulted in a significant lower rate of cardiovascular death, myocardial infarction, or any revascularization (4.5% patients in the clopidogrel group vs. 6.4% in the placebo group; relative risk 0.70, P ¼ .03)

1

Cardiology Department, Complejo Hospitalario Universitario Insular Materno Infantil, Las Palmas de Gran Canaria, Spain 2 Research Unit, Complejo Hospitalario Universitario Insular Materno Infantil, Las Palmas de Gran Canaria, Spain Submitted for publication 2 October 2014; accepted 14 October 2014. Corresponding Author: Antonio Tugores, PhD, Unidad de Investigación, Complejo Hospitalario Universitario Insular Materno-Infantil, Avda Maritima del Sur, s/n. 35016, Las Palmas de Gran Canaria, Spain Email: [email protected]

2 without an increase in major bleeding.6 Similarly, others showed the benefit of long-term (12-month) treatment with clopidogrel after PCI, reducing by 26.9% the combined risk of death, myocardial infarction, or stroke as compared with placebo.7 Clopidogrel therapy has also been shown to improve the patency rate of the infarct-related artery and reduced ischemic complications in patients 75 years of age or younger who had myocardial infarction with ST-segment elevation and who received aspirin and a standard fibrinolytic regimen,8 further demonstrating to be a safe and effective therapy to reduce mortality and major vascular events during hospital admission.9 By contrast, addition of clopidogrel to aspirin did not show an incremental benefit in the reduction of adverse cardiovascular events in patients with either stable coronary disease or asymptomatic patients with multiple cardiovascular risk factors during a follow-up period of 28 months,10 whereas patients with an established history of prior stroke, myocardial infarction, or peripheral arterial disease had a significant reduction in the rate of cardiovascular death, myocardial infarction, or stroke.11 Therefore, the clinical approaches to both the stable and unstable phases of coronary artery disease are different. In the latter, a disrupted atherosclerotic plaque stimulates platelet aggregation and thrombus formation, and the antiplatelet therapy plays a fundamental role from the start, as rapid stabilization of the plaque is essential to reduce new and recurrent coronary events. That is why, in the setting of primary prevention, dual antiplatelet therapy does not produce a benefit, and conversely is associated with an increased bleeding risk, whereas in secondary prevention, an initial loading dose of a dual antiplatelet regimen, followed by a long treatment period of at least 1 year, should be administered in order to achieve maximal efficacy.7 Even when the correct patient group is targeted, a significant percentage of individuals treated with clopidogrel shows reduced response to the drug, as assessed by an increase in the risk of suffering recurrent ischemic events and both acute and subacute stent thrombosis.12–14 The concept of clopidogrel resistance arises then, in order to define the individual variability that leads to clopidogrel non-responsiveness, and the proposed hypotheses to explain it may be grouped into 3 main categories: (i) bioavailability of the drug, (ii) the target tissue, and (iii) other concurrent clinical conditions, including compliance.

Bioavailability Clopidogrel is administered orally as a prodrug, and is rapidly absorbed by the enterocyte against the efflux mediated by the ABCB1 transporter, also known as multidrug resistance 1 (MDR1) or P-glycoprotein.15

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Cytochrome P450 enzymes convert the prodrug to the active thiol metabolite through the formation of the intermediate 2-oxo clopidogrel, whereas esterases generate inactive acid metabolites. The final result is that less than 10%–15% of the original input becomes an active P2Y12 inhibitor.16 In addition, up to 99% of the parent compound and its metabolites are bound to plasma proteins.17 An early study observed that, upon administration of clopidogrel to healthy subjects, there was considerable inter-individual variability in the time of onset and in the prolonged bleeding time observed after the drug was discontinued, suggesting that the drug was metabolized at variable rates in different subjects.18 Based on this observation, it could be hypothesized that the bioavailability of the active metabolite would depend on the activity of factors that determine its absorption and/or metabolism, which may vary among individuals. As genetic variants may determine the function of many proteins, an initial feasible approach to detect functional variation is to screen for genetic variants in the genes of interest. The first gene to be screened for variants that could affect clopidogrel responsiveness was the one encoding for the ABCB1 transporter, leading to finding an influence of the variation C3435T (SNP ID: rs1045642) in clopidogrel absorption.15 This observation has been backed up by some clinical studies,19,20 although not by others.21–24 As the variant leads to a synonymous change in the ABCB1 protein (Ile1145Ile), it is difficult to evaluate the biochemical changes that such a variant could produce in ABCB1 function. Regarding the biotransformation process per se, the identification of the enzymes that are most important in the conversion of clopidogrel has been controversial. Although initial studies in liver microsomes revealed CYP3A isozymes as crucial in the biotransformation process,25 genetic analyses have emphasized the role of CYP2C19 as one fundamental enzyme.20,21,26–28 There are several loss of function alleles for CYP2C19, the most common being the one defined by the *2 haplotype, leading to a splicing defect, followed by the *3 allele, resulting in premature termination. Both alleles encode for an inactive protein product and are, therefore, considered null. The CYP2C19*17 haplotype defines a gain of function allele as a result of increased transcriptional activity at the CYP2C19 locus (http:// www.cypalleles.ki.se/cyp2c19.htm). Numerous studies, followed by large meta-analyses, have studied the influence of both CYP2C19 loss and gain of function genetic variants in the response to clopidogrel, providing a wealth of information that has been analyzed in recent reviews.29–31 As post hoc analysts point out, these studies and their subsequent meta-analyses have generated conflicting results due to several caveats associated with

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them. First, there is great heterogeneity in the study populations tested. Second, there are a variety of endpoints, ranging from ex vivo platelet function assays to several clinical endpoints. This is a controversial issue that will be discussed later under the target tissue section. Third, many of these studies largely ignore other concurrent situations that may affect the response to medication besides the genotype and, finally, combining large and smaller studies, while not accounting those with negative results that have not been published, may skew the final analysis. In conclusion, in the absence of a properly randomized clinical study, there is no robust evidence supporting that CYP2C19 genetic variants affect an individuals’ response to clopidogrel. Further evidence against CYP2C19 as an important genetic factor for clopidogrel comes from the study of drug interactions. Some observational studies have shown that some PPIs, and in particular omeprazole, a competitive substrate of CYP2C19, may decrease clopidogrel’s antiplatelet effectiveness as assessed by both ex vivo platelet function assays and by the evaluation of the risk to suffer a new acute coronary event.32,33 By contrast, other studies do not demonstrate a consistent interaction between clopidogrel and PPIs,34 and even identify PPI use as an independent risk factor for adverse cardiovascular events, regardless of clopidogrel use.35,36 Strong biochemical evidence supports CYP3A isozymes as the major players in clopidogrel bioactivation.25,37 The contribution of specific CYP3A genetic variants to the metabolism of clopidogrel has not been thoroughly explored. Alternatively, the concomitant use of drugs that interfere with CYP3A function has been associated with adverse outcomes. Drugs like calcium channel blockers, ketoconazole, erythromicin, and troleandomycin, all known to be CYP3A inhibitors, decrease clopidogrel biotransformation and diminish platelet aggregation ex vivo.38 However, clopidogrel can be used concomitantly with CYP3A inhibitors, such as statins and calcium-channel blockers, with no adverse clinical outcomes, as evidenced by a sub-study derived from the TRITON-TIMI 38 trial.39 Nonetheless, the importance of CYP3A is revealed by the administration of grapefruit juice, a potent enteric CYP3A inhibitor,40 to healthy subjects taking clopidogrel, causing almost a 90% reduction of both the peak concentration (Cmax) and the area under the concentration–time curve (AUC) of the active metabolite in plasma.41 Another enzyme proposed as necessary for clopidogrel conversion is serum paraxoxonase 1 (PON1). In particular, the common variant SNP ID rs662 (Q192R), leading to a decrease in enzymatic activity, was shown to negatively affect clopidogrel conversion.42 However, multiple clinical studies have failed to identify this variant as a determinant factor in the response to clopidogrel, both by ex vivo platelet functional assays

and by clinical outcome,43–45 an observation supported by biochemical evidence showing that PON1 only mediates the conversion of a minor thiol metabolite isomer and, therefore, is not necessary for the biotransformation process.46 Another factor affecting pharmacokinetic parameters is plasma protein binding, which is very high for clopidogrel and its metabolites. In this situation, alteration of the equilibrium between the active and the inactive metabolites, the latter in a large molar excess, may cause the release of active metabolite to the blood stream, resulting in enhanced platelet inhibition.17 Similarly, other drugs competing for plasma protein binding could cause this release, although this is a possibility that has not been tested.

The Target Bioconversion of clopidogrel produces an irreversible non-competitive inhibitor of the P2Y12 receptor that binds to the platelet surface during its entire lifespan (7–10 days). There are 2 related issues that are of interest to understand clopidogrel action on platelets: One is represented by the assays used to measure platelet function whereas the other is related to the biology of the platelet per se. Ex vivo platelet functional assays are commonly used to evaluate the action of clopidogrel. The gold standard should be to study receptor occupancy by measuring the binding of the agonist in the presence of the drug, but this assay is not practical in the clinical setting. So other surrogate assays have been developed based on the measurement of platelet aggregation in static solution or under a flow, platelet binding to fibrinogen, clot formation, or the detection of events downstream of the P2Y12 receptor. Light transmission aggregometry (LTA) and impedance aggregometry, together with the VASP phosphorylation assay, that measures a downstream event, have been the most widely used methods to (i) measure platelet function in response to clopidogrel and (ii) to correlate platelet function with clinical outcome.47 It is important to note that platelet functional assays ex vivo have not been properly validated before being used to determine efficacy in clinical studies. That is, not all platelet tests are equivalent: They do not measure the same effects and they may lead to different results. So, it is possible that one patient, at once, is considered “resistant” to clopidogrel by one method, but not by another. At the same time, a single individual might be considered “resistant” one day, but not the following, and vice versa.48–54 As a result of the adventurous exercise of using a tool before it is properly validated, there is a great concern about the competence of platelet function tests to measure clopidogrel efficacy and predict clinical outcome. Three large studies, TRILOGY ACS, ARCTIC and

4 GRAVITAS, could not find a correlation between the VerifyNow platelet function test and clinical outcome.55–57 A smaller study, the PEGASUS-PCI, suggested that measurement of platelet function by multiple electrode aggregometry (MEA) predicts stent thrombosis better than VASP phosphorylation, cone and platelet analyser, and the platelet function analyser (PFA-100) tests, an observation that was later reinforced in the MADONNA study.49,58 At the pharmacological level, the LTA assay correlates best with P2Y12 receptor occupancy—the gold standard—as compared to the VerifyNow P2Y12 assay, detection of platelet activation markers, VASP phosphorylation, or thromboelastography.48 Therefore, in view of the controversial evidence provided by these studies, it remains unclear as to which method and under what circumstances, platelet aggregation values are able to differentiate responders from non-responders. Beyond the tricky field of ex vivo platelet functional assays, the behavior of platelets shows, as it could be expected of a biological system, both inter- and intraindividual variability. Platelet number and size are variables that differ among individuals and have been proportionally related to cardiovascular risk.59 Larger platelets appear to have hyper-reactivity, a term that has also been used to define resistance to anti-aggregation therapy with clopidogrel.60 To better understand the role of hyper-reactivity in clopidogrel “resistance,” we should consider a situation where platelet reactivity is measured with an ex vivo assay, like the VASP assay, widely used in a great number of studies analyzing the response to clopidogrel. In this assay, platelet reactivity is defined by a platelet reactivity index (PRI) in response to the agonist, which decreases in response to clopidogrel. A specific threshold is set to determine response, ie, 50%, and every value above that threshold is defined as nonresponse. A problem arises when the levels of platelet reactivity are highly responsive to the agonist, with PRI steady-state levels well above the average. In such cases, a positive clopidogrel response might remain unnoticed if the resulting activity upon clopidogrel addition, in the presence of the agonist, is decreased but still above the pre-determined threshold. Likewise, hypo-reactive platelets may stay below the threshold, even when being nonresponsive to the drug.61 Therefore, platelets that are hyper-reactive before treatment are more likely to appear “resistant” to clopidogrel.62 This residual platelet aggregation (RPA) that persists after treatment has been associated with increased risk of atherothrombotic events during treatment.63–67 The hyper-reactive platelet phenotype has been described in certain situations such as patients with diabetes, obesity, or with elevated circulating levels of fibrinogen or inflammatory mediators, influencing both ex vivo platelet reactivity68–72 and clinical outcome.73–76 One of the biological mechanisms proposed to lead to

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platelet hyper-reactivity is downstream of the P2Y12 receptor. Binding of ADP to the receptor results in the inhibition of adenylate cyclase (AC) and decreased cyclic AMP (cAMP) production. Platelets isolated from both diabetics and obese subjects are hypo-responsive to the anti-aggregation effect of prostaglandin I 2 (PGI2), which activates AC, and nitric oxide (NO).77,78 Impaired response to cAMP is also found in patients with stable angina, another population with hyper-reactive platelets.79 As mentioned earlier, further complexity to this picture is added by the evidence that platelet reactivity is variable within an individual, as it has been evidenced by a recent study arising from the ELEVATE-TIMI 56 trial.54 One mechanism leading to intra-individual variability is circadian control, a situation that may explain why most of the cardiovascular acute events take place during the morning hours.80 Analysis of the TRITON-TIMI 38 trial reveals circadian variations in stent thrombosis,81 further stressing the need for several measurements of platelet reactivity in a single individual in order to fully appreciate the value of a specific ex vivo platelet function assay.

Other Clinical Factors As mentioned in the previous section, certain clinical conditions may determine a highly reactive platelet phenotype and, therefore, lead to clopidogrel “resistance.” Caution should be taken when the appreciation of the response to clopidogrel is based on platelet function assays, as some of the clinical factors that are influencing clopidogrel response do not show consistency between different assays, as some influence the assays per se.82,83 This adds a new confounding factor to the technical variability associated with the evaluation of platelet function ex vivo. Therefore, we believe that it is important to focus on clinical conditions that increase the risk of suffering new acute cardiovascular events, including stent thrombosis, instead of conditions that affect platelet reactivity ex vivo. Patients with type 2 diabetes mellitus are characterized by a pro-thrombotic status favored by resistance to antithrombotic physiological signals, shear-induced aggregation, non-enzymatic glycation of platelet glycoproteins, changes in the structure and conformation of platelets, increased oxidative stress, and increased platelet turnover,77 a situation that is more severe in patients with insulin-dependent diabetes.84 Diabetics are, therefore, a population of patients at high risk of recurrent thrombotic events even under clopidogrel therapy.24,73–75 Central obesity predisposes to thrombotic events,85 and overweight patients need a higher loading dose of clopidogrel and/or adjunt antithrombotic treatment to adequately inhibit platelet aggregation.86 Fortunately,

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platelet sensitivity to PGI2 and NO may be restored in these patients through weight loss.78 The presence of inflammatory markers in serum leads to bad prognosis in patients taking clopidogrel after stent implantation.87 Inflammation may favor thrombosis due to endothelial damage, leading to decreased vasodilation properties, an increase in procoagulant factors, inhibition of natural anticoagulant pathways, and reduced fibrinolytic activity.88 Certain acute coronary syndromes are, by themselves, negative factors among clopidogrel users.24 Angiographic evaluation reveals that bifurcation coronary lesions, instent restenosis, stent length, use of multiple stents, unplanned stenting, residual dissections, stent overlap, smaller final lumen diameter, combined use of different stent designs, incomplete apposition, or stent under expansion as seen in calcified arteries may favor acutely, subacutely, or late stent thrombosis irrespective of clopidogrel use.89 However, there is no convincing evidence showing differential risk of suffering stent thrombosis between bare metal stents (BMS) and drug-eluting stents (DES).90 Inappropriate dosing of clopidogrel may favor the recurrence of a new coronary event and stent thrombosis. A 600 mg loading dose of clopidogrel, which provides a faster and greater inhibition of platelet aggregation than the standard 300 mg loading dose and a higher transient maintenance dose during critical or high-risk periods (eg, after PCI) may reduce the risk of recurrent coronary events and early stent thrombosis.91 Nonetheless, despite the improvement in clopidogrel platelet inhibitory effect, a high loading dose followed by higher maintenance doses do not completely rescue responsiveness to clopidogrel.68 “Extreme underdosing” occurs when there is a lack of patient compliance. As expected, premature discontinuation of thienopyridines is associated with an increased risk of stent thrombosis.92 A large study identified that the level of persistence among clopidogrel users after 9 months treatment is above 75% in patients who have suffered an ACS and have undergone a PCI, but this level of compliance drops drastically to below 55% in those patients who have not undergone a PCI.93 This “lower risk” perception, added to price/socio-economical status, advanced age, female sex, and nuisance bleeding are issues leading to clopidogrel non-compliance.92,94,95

Clopidogrel Resistance: The Gordian Knot After reviewing the literature, a physician may ask: What is clopidogrel resistance? Is there a simple test to measure it before prescribing clopidogrel? If not, when should clopidogrel be prescribed? The answer to the first question has generated much debate.96 Treatment failure could be due to the complex

5 multifactorial nature of the atherothrombotic disease, where a second cardiovascular event might occur independent of treatment. Therefore, the definition of “resistance” seems to rather identify a population at risk of recurrent thrombotic events rather than its inability to respond to the drug. However, there is a strong support for the view that resistance to clopidogrel is a pharmacokinetic issue, so great efforts have been put to focus on the identification of surrogate markers that could predict clinical efficacy. Then, the second question arises: Is there a test to predict clopidogrel clinical efficacy? Platelet aggregation assays have been widely used to study this issue although, as we have seen, with inconsistent results due to both technical and biological issues. To date, it is unclear whether there is a sufficiently validated ex vivo platelet aggregation assay to predict clinical response to clopidogrel, although the PEGASUS and MADONNA studies strongly favor the MEA method.49,58 Nonetheless, besides technical considerations, an additional explanation for the lack of correlation between the measurement of platelet aggregation ex vivo and clinical endpoint efficacy is the possibility that some of the beneficial therapeutic effects of clopidogrel may go beyond platelet aggregation inhibition, reaching purinergic receptors in other relevant targets such as the vascular system, leading to increased nitric oxide availability and vasorelaxation.97,98 Platelet functional assays ex vivo have been used to evaluate genetic variants that could alter bioconversion of clopidogrel, especially the null CYP2C19 variants, and have emphasized the importance of this cytochrome in the conversion of clopidogrel. However, the lack of sufficient clinical evidence, and the fact that concomitant medication with omeprazole, a CYP2C19 substrate and inhibitor, has no clinical consequences, have questioned the utility of CYP2C19 genotyping in predicting response to clopidogrel. This lack of correlation is also evident at the pharmacokinetic level. Being carrier of a CYP2C19 null allele translates in roughly a 30% reduction of the active metabolite in serum, translating in an average 9% decreased platelet response.26 Likewise, concomitant administration of omeprazole with clopidogrel to healthy subjects may decrease the AUC for the active metabolite up to 47%, resulting in modest dose-independent effects: a 5.6% increase in maximal platelet aggregation induced by ATP, and even a 27% increase of the VASP index.99 Early clinical pharmacology in humans during Phase I trials for Plavix shows that transition from 100 to a 200 mg dose results in a 20% increase in platelet inhibition, whereas the ascension from 200 to 600 mg only attains an additional 10% increase (http://www. accessdata.fda.gov/drugsatfda_docs/nda/97/20839_Plavix_ clinphrmr_P1.pdf). Considering the clinical evidence, these observations question whether a 30% or 40%

6 decrease in the availability of a non-competitive compound could have a profound pharmacological effect as to determine responsiveness to the drug. By contrast, grapefruit juice, a strong inhibitor of enteric CYP3A isozymes, causes a dramatic effect on drug availability,41 supporting the view that clopidogrel is, primarily, an enteric CYP3A substrate. Even though a number of studies addressing the role of CYP3A inhibitors in the response to clopidogrel have failed to show relevant clinical significance,38,39 it seems prudent to avoid concomitant administration of clopidogrel with strong CYP3A inhibitors, especially grapefruit juice. Dosing regimes also show that the response to clopidogrel is beyond pharmacokinetics,67 and that other issues also affect this clinical response. Among these, high on treatment platelet reactivity seems to be the best predictor of adverse clinical outcomes. Thus, evaluating high platelet reactivity before treatment could be a wise option, although it does not seem practical in the clinical setting as urgent pharmacological intervention is crucial and anti-aggregation therapy is rapidly administered before a test can be performed. In summary, clopidogrel response variability, due to differential pharmacokinetics and dynamics, is multifactorial, so it is unlikely to be predicted by simple CYP2C19 genotyping and/or platelet aggregation tests.23,96 Thus, in the absence of a reliable predictive assay, the key issue is to identify patients at higher risk of suffering a new thrombotic event based on the available clinical evidence. Patients with diabetes, especially insulin-dependent, coronary disease conditions, and certain vascular lesions and stent-related issues may benefit from intensified antiplatelet therapy.63 In this context, the use of new and more potent P2Y12-receptor blockers, such as prasugrel and ticagrelor, might help. Prasugrel offers an alternative to clopidogrel with greater efficacy but with increased bleeding risk whereas treatment with ticagrelor, as compared with clopidogrel, significantly reduced the rate of death from vascular causes, myocardial infarction, or stroke without an increase in the rate of overall major bleeding.1,47 However, the benefit of using new P2Y12receptor antagonists may come at the additional cost of the increased risk of bleeding and therapy discontinuation, due to economic reasons. Special efforts should be made to educate the patients and, maybe, it should be considered to choose the cheaper generic clopidogrel alternative when socio-economical conditions are not favorable, a situation where the risk of non-compliance counterweighs the expected clinical benefit. As a final comment, it should be noted that prasugrel or ticagrelor has not been subjected to the same scrutiny as clopidogrel, so it remains to be determined whether specific conditions that determine “resistance” to clopidogrel also convey resistance to the other P2Y12 inhibitors. One example could be the case for platelet

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hyper-reactivity caused by biochemical events occurring downstream of the P2Y12 receptor, such as diminished response to PGI2. This is a defect that cannot, by principle, be rescued with another antagonist to the same receptor, defining drug class resistance rather than just clopidogrel resistance. Acknowledgments We would like to thank Dr. Gary Hardiman for helpful comments on the manuscript. We would like to apologize because, due to space constraints and the vast amount of published information available, we have been unable to cite all the work that deserves to be cited.

Declaration of Conflicting Interests The authors declare no conflicts of interest.

Funding Both authors have been funded by the Fundación Canaria de Investigación en Salud (FUNCIS) and by the Servicio Canario de Salud.

References 01. Michelson AD. Antiplatelet therapies for the treatment of cardiovascular disease. Nat Rev Drug Discov. 2010;9:154–169. 02. Schrör K. Clinical pharmacology of the adenosine diphosphate (ADP) receptor antagonist, clopidogrel. Vasc Med. 1998;3:247– 251. 03. Creager MA. Results of the CAPRIE trial: efficacy and safety of clopidogrel. Clopidogrel versus aspirin in patients at risk of ischaemic events. Vasc Med. 1998;3:257–260. 04. Yusuf S, Zhao F, Mehta SR, et al. Effects of clopidogrel in addition to aspirin in patients with acute coronary syndromes without STsegment elevation. N Engl J Med. 2001;345:494–502. 05. Budaj A, Yusuf S, Mehta SR, et al. Benefit of clopidogrel in patients with acute coronary syndromes without ST-segment elevation in various risk groups. Circulation. 2002;106:1622–1626. 06. Mehta SR, Yusuf S, Peters RJ, et al. Effects of pretreatment with clopidogrel and aspirin followed by long-term therapy in patients undergoing percutaneous coronary intervention: the PCI-CURE study. Lancet. 2001;358:527–533. 07. Steinhubl SR, Berger PB, Mann JT 3rd, et al. Clopidogrel for the reduction of events during observation. Early and sustained dual oral antiplatelet therapy following percutaneous coronary intervention: a randomized controlled trial. JAMA. 2002;288:2411–2420. 08. Sabatine MS, Cannon CP, Gibson CM, et al. Addition of clopidogrel to aspirin and fibrinolytic therapy for myocardial infarction with ST-segment elevation. N Engl J Med. 2005;352:1179–1189. 09. Chen ZM, Jiang LX, Chen YP, et al. Addition of clopidogrel to aspirin in 45,852 patients with acute myocardial infarction: randomised placebo-controlled trial. Lancet. 2005;366:1607–1621. 10. Bhatt DL, Fox KA, Hacke W, et al. Clopidogrel and aspirin versus aspirin alone for the prevention of atherothrombotic events. N Engl J Med. 2006;354:1706–1717. 11. Wang TH, Bhatt DL, Fox KA, et al. An analysis of mortality rates with dual-antiplatelet therapy in the primary prevention population of the CHARISMA trial. Eur Heart J. 2007;28:2200–2207. 12. Gurbel PA, Bliden KP, Hiatt BL, O’Connor CM. Clopidogrel for coronary stenting: response variability, drug resistance, and the effect of pretreatment platelet reactivity. Circulation. 2003;107:2908– 2913.

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Martínez-Quintana and Tugores 13. Matetzky S, Shenkman B, Guetta V, et al. Clopidogrel resistance is associated with increased risk of recurrent atherothrombotic events in patients with acute myocardial infarction. Circulation. 2004;109: 3171–3175. 14. Gurbel PA, Bliden KP, Samara W, et al. Clopidogrel effect on platelet reactivity in patients with stent thrombosis: results of the CREST study. J Am Coll Cardiol. 2005;46:1827–1832. 15. Taubert D, von Beckerath N, Grimberg G, et al. Impact of Pglycoprotein on clopidogrel absorption. Clin Pharmacol Ther. 2006;80:486–501. 16. Farid NA, Kurihara A, Wrighton SA. Metabolism and disposition of the thienopyridine antiplatelet drugs ticlopidine, clopidogrel, and prasugrel in humans. J Clin Pharmacol. 2010;50:126–142. 17. Ganesan S, Williams C, Maslen CL, Cherala G. Clopidogrel variability: role of plasma protein binding alterations. Br J Clin Pharmacol. 2013;75:1468–1477. 18. Mills DC, Puri R, Hu CJ, et al. Clopidogrel inhibits the binding of ADP analogues to the receptor mediating inhibition of platelet adenylate cyclase. Arterioscler Thromb. 1992;12:430–436. 19. Simon T, Verstuyft C, Mary-Krause M, et al. Genetic determinants of response to clopidogrel and cardiovascular events. N Engl J Med. 2009;360:363–375. 20. Mega JL, Close SL, Wiviott SD, et al. Genetic variants in ABCB1 and CYP2C19 and cardiovascular outcomes after treatment with clopidogrel and prasugrel in the TRITON-TIMI 38 trial: a pharmacogenetic analysis. Lancet. 2010;376:1312–1319. 21. Rideg O, Komócsi A, Magyarlaki T, et al. Impact of genetic variants on post-clopidogrel platelet reactivity in patients after elective percutaneous coronary intervention. Pharmacogenomics. 2011;12:1269–1280. 22. Jaitner J, Morath T, Byrne RA, et al. No association of ABCB1 C3435T genotype with clopidogrel response or risk of stent thrombosis in patients undergoing coronary stenting. Circ Cardiovasc Interv. 2012;5:82–88. 23. Frelinger AL 3rd, Bhatt DL, Lee RD, et al. Clopidogrel pharmacokinetics and pharmacodynamics vary widely despite exclusion or control of polymorphisms (CYP2C19, ABCB1, PON1), noncompliance, diet, smoking, co-medications (including proton pump inhibitors), and pre-existent variability in platelet function. J Am Coll Cardiol. 2013;61:872–879. 24. Siller-Matula JM, Lang IM, Neunteufl T, et al. Interplay between genetic and clinical variables affecting platelet reactivity and cardiac adverse events in patients undergoing percutaneous coronary intervention. PLoS One. 2014;9:e102701. 25. Clarke TA, Waskell LA. The metabolism of clopidogrel is catalyzed by human cytochrome P450 3A and is inhibited by atorvastatin. Drug Metab Dispos. 2003;31:53–59. 26. Mega JL, Close SL, Wiviott SD, et al. Cytochrome p-450 polymorphisms and response to clopidogrel. N Engl J Med. 2009;360: 354–362. 27. Shuldiner AR, O’Connell JR, Bliden KP, et al. Association of cytochrome P450 2C19 genotype with the antiplatelet effect and clinical efficacy of clopidogrel therapy. JAMA. 2009;302:849–857. 28. Sibbing D, Gebhard D, Koch W, et al. Isolated and interactive impact of common CYP2C19 genetic variants on the antiplatelet effect of chronic clopidogrel therapy. J Thromb Haemost. 2010; 8:1685–1693. 29. Sorich MJ, Polasek TM, Wiese MD. Challenges and limitations in the interpretation of systematic reviews: making sense of clopidogrel and CYP2C19 pharmacogenetics. Clin Pharmacol Ther. 2013;94:376–382. 30. Ford NF, Taubert D. Clopidogrel, CYP2C19, and a black box. J Clin Pharmacol. 2013;53:241–248. 31. Osnabrugge RL, Head SJ, Zijlstra F, et al. A systematic review and critical assessment of 11 discordant meta-analyses on reduced-

32.

33.

34.

35.

36.

37.

38.

39.

40.

41.

42.

43.

44.

45.

46.

47.

48.

function CYP2C19 genotype and risk of adverse clinical outcomes in clopidogrel users. Genet Med. 2014 [Epub ahead of print]. Sibbing D, Morath T, Stegherr J, et al. Impact of proton pump inhibitors on the antiplatelet effects of clopidogrel. Thromb Haemost. 2009;101:714–719. Ho PM, Maddox TM, Wang L, et al. Risk of adverse outcomes associated with concomitant use of clopidogrel and proton pump inhibitors following acute coronary syndrome. JAMA. 2009;301: 937–944. Dunn SP, Steinhubl SR, Bauer D, Charnigo RJ, Berger PB, Topol EJ. Impact of proton pump inhibitor therapy on the efficacy of clopidogrel in the CAPRIE and CREDO trials. J Am Heart Assoc. 2013;2:e004564. Bhatt DL, Cryer BL, Contant CF, et al. Clopidogrel with or without omeprazole in coronary artery disease. N Engl J Med. 2010;363: 1909–1917. Charlot M, Ahlehoff O, Norgaard ML, et al. Proton-pump inhibitors are associated with increased cardiovascular risk independent of clopidogrel use: a nationwide cohort study. Ann Intern Med. 2010;153:378–386. Kazui M, Nishiya Y, Ishizuka T, et al. 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. 2010;38:92–99. Siller-Matula JM, Trenk D, Krähenbühl S, Michelson AD, DelleKarth G. Clinical implications of drug–drug interactions with P2Y12 receptor inhibitors. J Thromb Haemost. 2014;12:2–13. Ojeifo O, Wiviott SD, Antman EM, et al. Concomitant administration of clopidogrel with statins or calcium-channel blockers: insights from the TRITON-TIMI 38 (trial to assess improvement in therapeutic outcomes by optimizing platelet inhibition with prasugrel-thrombolysis in myocardial infarction 38). JACC Cardiovasc Interv. 2013;6:1275–1281. Tassaneeyakul W, Vannaprasaht S, Yamazoe Y. Formation of omeprazole sulphone but not 5-hydroxyomeprazole is inhibited by grapefruit juice. Br J Clin Pharmacol. 2000;49:139–144. Holmberg MT, Tornio A, Neuvonen M, Neuvonen PJ, Backman JT, Niemi M. Grapefruit juice inhibits the metabolic activation of clopidogrel. Clin Pharmacol Ther. 2014;95:307–313. Bouman HJ, Schömig E, van Werkum JW, et al. Paraoxonase-1 is a major determinant of clopidogrel efficacy. Nat Med. 2011;17:110– 116. Lewis JP, Fisch AS, Ryan K, et al. Paraoxonase 1 (PON1) gene variants are not associated with clopidogrel response. Clin Pharmacol Ther. 2011;90:568–574. Sibbing D, Koch W, Massberg S, et al. No association of paraoxonase-1 Q192R genotypes with platelet response to clopidogrel and risk of stent thrombosis after coronary stenting. Eur Heart J. 2011;32:1605–1613. Trenk D, Hochholzer W, Fromm MF, et al. Paraoxonase-1 Q192R polymorphism and antiplatelet effects of clopidogrel in patients undergoing elective coronary stent placement. Circ Cardiovasc Genet. 2011;4:429–436. Dansette PM, Rosi J, Bertho G, Mansuy D. Cytochromes P450 catalyze both steps of the major pathway of clopidogrel bioactivation, whereas paraoxonase catalyzes the formation of a minor thiol metabolite isomer. Chem Res Toxicol. 2012;25:348– 356. Siller-Matula JM, Trenk D, Schrör K, et al. Response variability to P2Y12 receptor inhibitors: expectations and reality. JACC Cardiovasc Interv. 2013;6:1111–1128. Bal Dit Sollier C, Berge N, Boval B, Dubar M, Drouet L. Differential sensitivity and kinetics of response of different ex vivo tests monitoring functional variability of platelet response to clopidogrel. Thromb Haemost. 2010;104:571–581.

8 49. Siller-Matula JM, Delle-Karth G, Lang IM, et al. Phenotyping vs. genotyping for prediction of clopidogrel efficacy and safety: the PEGASUS-PCI study. J Thromb Haemost. 2012;10(4):529–542. 50. Bagoly Z, Sarkady F, Magyar T, et al. Comparison of a new P2Y12 receptor specific platelet aggregation test with other laboratory methods in stroke patients on clopidogrel monotherapy. PLoS One. 2013;8:e69417. 51. Lemesle G, Landel JB, Bauters A, et al. Poor agreement between light transmission aggregometry, Verify Now P2Y12 and vasodilatator-stimulated phosphoprotein for clopidogrel lowresponse assessment: a potential explanation of negative results of recent randomized trials. Platelets. 2013:31 [Epub ahead of print]. 52. Meen Ø, Brosstad F, Bjørnsen S, Pedersen TM, Erikssen G. Variability in aggregometry response before and after initiation of clopidogrel therapy. Scand J Clin Lab Invest. 2009;69:673–679. 53. Arméro S, Camoin Jau L, Omar Aït Mokhtar O, et al. Intraindividual variability in clopidogrel responsiveness in coronary artery disease patients under long term therapy. Platelets. 2010;21:503–507. 54. Hochholzer W, Ruff CT, Mesa RA, et al. Variability of individual platelet reactivity over time in patients treated with clopidogrel: insights from the ELEVATE-TIMI 56 trial. J Am Coll Cardiol. 2014;64:361–368. 55. Price MJ, Berger PB, Teirstein PS, et al. Standard- vs high-dose clopidogrel based on platelet function testing after percutaneous coronary intervention: the GRAVITAS randomized trial. JAMA. 2011;305:1097–1105. 56. Collet JP, Cuisset T, Rangé G, et al. Bedside monitoring to adjust antiplatelet therapy for coronary stenting. N Engl J Med. 2012;367: 2100–2109. 57. Gurbel PA, Erlinge D, Ohman EM, et al. Platelet function during extended prasugrel and clopidogrel therapy for patients with ACS treated without revascularization: the TRILOGY ACS platelet function substudy. JAMA. 2012;308:1785–1794. 58. Siller-Matula JM, Francesconi M, Dechant C, et al. Personalized antiplatelet treatment after percutaneous coronary intervention: the MADONNA study. Int J Cardiol. 2013;167:2018–2023. 59. de Gaetano G, Santimone I, Gianfagna F, Iacoviello L, Cerletti C. Variability of platelet indices and function: acquired and genetic factors. Handb Exp Pharmacol. 2012;210:395–434. 60. Michelson AD, Linden MD, Furman MI, et al. Evidence that preexistent variability in platelet response to ADP accounts for ‘clopidogrel resistance’. J Thromb Haemost. 2007;5:75–81. 61. van Werkum JW, Bouman HJ, Breet NJ, ten Berg JM, Hackeng CM. The Cone-and-Plate(let) analyzer is not suitable to monitor clopidogrel therapy: a comparison with the flowcytometric VASP assay and optical aggregometry. Thromb Res. 2010;126: 44–49. 62. Samara WM, Bliden KP, Tantry US, Gurbel PA. The difference between clopidogrel responsiveness and posttreatment platelet reactivity. Thromb Res. 2005;115:89–94. 63. Geisler T, Grass D, Bigalke B, et al. The residual platelet aggregation after deployment of intracoronary stent (PREDICT) score. J Thromb Haemost. 2008;6:54–61. 64. Patti G, Nusca A, Mangiacapra F, Gatto L, D’Ambrosio A, Di Sciascio G. Point-of-care measurement of clopidogrel responsiveness predicts clinical outcome in patients undergoing percutaneous coronary intervention results of the ARMYDA-PRO (antiplatelet therapy for reduction of myocardial damage during angioplastyplatelet reactivity predicts outcome) study. J Am Coll Cardiol. 2008;52:1128–1133. 65. Price MJ, Endemann S, Gollapudi RR, et al. Prognostic significance of post-clopidogrel platelet reactivity assessed by a point-of-care assay on thrombotic events after drug-eluting stent implantation. Eur Heart J. 2008;29:992–1000.

The Journal of Clinical Pharmacology / Vol XX No XX (2014) 66. Marcucci R, Gori AM, Paniccia R, et al. Cardiovascular death and nonfatal myocardial infarction in acute coronary syndrome patients receiving coronary stenting are predicted by residual platelet reactivity to ADP detected by a point-of-care assay: a 12-month follow-up. Circulation. 2009;119:237–242. 67. Parodi G, Marcucci R, Valenti R, et al. High residual platelet reactivity after clopidogrel loading and long-term cardiovascular events among patients with acute coronary syndromes undergoing PCI. JAMA. 2011;306:1215–1223. 68. Ang L, Palakodeti V, Khalid A, et al. Elevated plasma fibrinogen and diabetes mellitus are associated with lower inhibition of platelet reactivity with clopidogrel. J Am Coll Cardiol. 2008;52:1052– 1059. 69. Bonello-Palot N, Armero S, Paganelli F, et al. Relation of body mass index to high on-treatment platelet reactivity and of failed clopidogrel dose adjustment according to platelet reactivity monitoring in patients undergoing percutaneous coronary intervention. Am J Cardiol. 2009;104:1511–1515. 70. Cuisset T, Frere C, Quilici J, et al. Relationship between aspirin and clopidogrel responses in acute coronary syndrome and clinical predictors of non response. Thromb Res. 2009;123:597–603. 71. Li YG, Ni L, Brandt JT, et al. Inhibition of platelet aggregation with prasugrel and clopidogrel: an integrated analysis in 846 subjects. Platelets. 2009;20:316–327. 72. Bernlochner I, Steinhubl S, Braun S, et al. Association between inflammatory biomarkers and platelet aggregation in patients under chronic clopidogrel treatment. Thromb Haemost. 2010;104:1193– 1200. 73. Geisler T, Mueller K, Aichele S, et al. Impact of inflammatory state and metabolic control on responsiveness to dual antiplatelet therapy in type 2 diabetics after PCI: prognostic relevance of residual platelet aggregability in diabetics undergoing coronary interventions. Clin Res Cardiol. 2010;99:743–752. 74. Angiolillo DJ, Bernardo E, Sabaté M, et al. Impact of platelet reactivity on cardiovascular outcomes in patients with type 2 diabetes mellitus and coronary artery disease. J Am Coll Cardiol. 2007;50:1541–1547. 75. Andersson C, Lyngbæk S, Nguyen CD, et al. Association of clopidogrel treatment with risk of mortality and cardiovascular events following myocardial infarction in patients with, without diabetes. JAMA. 2012;308:882–889. 76. Dosh K, Berger PB, Marso S, et al. Relationship between baseline inflammatory markers, antiplatelet therapy, and adverse cardiac events after percutaneous coronary intervention: an analysis from the clopidogrel for the reduction of events during observation trial. Circ Cardiovasc Interv. 2009;2:503–512. 77. Ferroni P, Basili S, Falco A, Davi G. Platelet activation in type 2 diabetes mellitus. J Thromb Haemost. 2004;2:1282–1291. 78. Russo I, Traversa M, Bonomo K, et al. In central obesity, weight loss restores platelet sensitivity to nitric oxide and prostacyclin. Obesity (Silver Spring). 2010;18:788–797. 79. Chirkov YY, Chirkova LP, Sage RE, Horowitz JD. Impaired responsiveness of platelets from patients with stable angina pectoris to antiaggregating and cyclic AMP-elevating effects of prostaglandin E1. J Cardiovasc Pharmacol. 1995;25:961–966. 80. Scheer FA, Michelson AD, Frelinger AL 3rd, et al. The human endogenous circadian system causes greatest platelet activation during the biological morning independent of behaviors. PLoS One. 2011;6(9):e24549. 81. Mogabgab O, Wiviott SD, Cannon CP, et al. Circadian variation of stent thrombosis and the effect of more robust platelet inhibition: a post hoc analysis of the TRITON-TIMI 38 trial. J Cardiovasc Pharmacol Ther. 2013;18:555–559. 82. Elsenberg EH, van Werkum JW, van de Wal RM, et al. The influence of clinical characteristics, laboratory and inflammatory

Martínez-Quintana and Tugores

83.

84.

85.

86.

87.

88.

89.

90.

markers on “high on-treatment platelet reactivity” as measured with different platelet function tests. Thromb Haemost. 2009;102:719– 727. Gremmel T, Steiner S, Seidinger D, Koppensteiner R, Panzer S, Kopp CW. The influencing factors for clopidogrel-mediated platelet inhibition are assay-dependent. Thromb Res. 2011;128:352–357. Angiolillo DJ, Bernardo E, Ramírez C, et al. Insulin therapy is associated with platelet dysfunction in patients with type 2 diabetes mellitus on dual oral antiplatelet treatment. J Am Coll Cardiol. 2006;48:298–304. Morange PE, Alessi MC. Thrombosis in central obesity and metabolic syndrome: mechanisms and epidemiology. Thromb Haemost. 2013;110:669–680. Angiolillo DJ, Fernández-Ortiz A, Bernardo E, et al. Platelet aggregation according to body mass index in patients undergoing coronary stenting: should clopidogrel loading-dose be weight adjusted. J Invasive Cardiol. 2004;16:169–174. Dosh K, Berger PB, Marso S, et al. Relationship between baseline inflammatory markers, antiplatelet therapy, and adverse cardiac events after percutaneous coronary intervention: an analysis from the clopidogrel for the reduction of events during observation trial. Circ Cardiovasc Interv. 2009;2(6):503–512. Aksu K, Donmez A, Keser G. Inflammation-induced thrombosis: mechanisms, disease associations and management. Curr Pharm Des. 2012;18:1478–1493. Attizzani GF, Capodanno D, Ohno Y, Tamburino C. Mechanisms, pathophysiology, and clinical aspects of incomplete stent apposition. J Am Coll Cardiol. 2014;63(14):1355–1367. Pullara A, Longo G, Gonella A, et al. Incidence and very long- term outcomes of stent thrombosis after bare-metal or drug-eluting stent implantation: a retrospective analysis. Minerva Cardioangiol. 2013;61:1–9.

9 91. Cuisset T, Frere C, Quilici J, et al. Benefit of a 600-mg loading dose of clopidogrel on platelet reactivity and clinical outcomes in patients with non-ST-segment elevation acute coronary syndrome undergoing coronary stenting. J Am Coll Cardiol. 2006;48:1339– 1345. 92. Quadros AS, Welter DI, Camozzatto FO, et al. Identifying patients at risk for premature discontinuation of thienopyridine after coronary stent implantation. Am J Cardiol. 2011;107:685–689. 93. Sørensen R, Gislason GH, Fosbøl EL, et al. Initiation and persistence with clopidogrel treatment after acute myocardial infarction: a nationwide study. Br J Clin Pharmacol. 2008;66:875–884. 94. Roy P, Bonello L, Torguson R, et al. Impact of “nuisance” bleeding on clopidogrel compliance in patients undergoing intracoronary drug-eluting stent implantation. Am J Cardiol. 2008;102:1614–1617. 95. Serebruany V, Cherala G, Williams C, et al. Association of platelet responsiveness with clopidogrel metabolism: role of compliance in the assessment of resistance. Am Heart J. 2009;158:925–932. 96. Hurst NL, Nooney VB, Raman B, Chirkov YY, De Caterina R, Horowitz JD. Clopidogrel resistance: pre- vs post-receptor determinants. Vascul Pharmacol. 2013;59:152–161. 97. Heitzer T, Rudolph V, Schwedhelm E, et al. Clopidogrel improves systemic endothelial nitric oxide bioavailability in patients with coronary artery disease: evidence for antioxidant and antiinflammatory effects. Arterioscler Thromb Vasc Biol. 2006;26:1648–1652. 98. Bundhoo SS, Anderson RA, Sagan E, et al. Direct vasoactive properties of thienopyridine-derived nitrosothiols. J Cardiovasc Pharmacol. 2011;58:550–558. 99. Angiolillo DJ, Gibson CM, Cheng S, et al. Differential effects of omeprazole and pantoprazole on the pharmacodynamics and pharmacokinetics of clopidogrel in healthy subjects: randomized, placebo-controlled, crossover comparison studies. Clin Pharmacol Ther. 2011;89:65–74.