The antiplatelet agent clopidogrel has proved to be an important agent for the management of patients who are at risk of cardiovascular events. The CAPRIE.
COMMENTARY title
Clopidogrel Resistance: Pharmacokinetic or Pharmacogenetic? Neville F. Ford, MD, PhD, FCP
Clopidogrel is important for the management of acute coronary syndromes and, along with aspirin, is recommended in the American College of Cardiology/American Heart Association guideline. It is also used along with aspirin, during the placement of coronary artery stents. Clopidogrel resistance was recognized in such procedures, as several patients did not have the anticipated platelet aggregation response to an ex vivo adenosine diphosphate challenge. From the EXCELSIOR study, which investigated the phenomenon, it was appreciated that it was present prior to treatment with clopidogrel and was therefore an intrinsic property of the patient’s platelets. From other studies, it was appreciated that the patients who had clopidogrel resistance had a defective allele *2/ in the CYP2C19 gene. Furthermore, there was a dose response evident in that the homozygotes CYP2C19*2/*2 had platelets that responded even less well to
clopidogrel than the heterozygotes CYP2C19*2 that responded less well than the wild-type homozygote. The involvement of the phenomenon with CYP2C19 led some to believe that it was a pharmacokinetic issue. However, the major oxidative metabolic pathway for clopidogrel by which the reactive intermediate is formed is CYP3A4. It is suggested that there is a linkage between a polymorphism of the platelet receptor P2Y12 and the polymorphism of CYP2C19.
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ischemia. In this study in 12 562 patients with ACS, clopidogrel with aspirin was compared to aspirin alone. Clopidogrel reduced the risk of ischemic events, with the benefits being evident within 24 hours of initiating therapy. Clopidogrel plus aspirin has become the standard of care for patients with ACS based on these and related findings.3
he antiplatelet agent clopidogrel has proved to be an important agent for the management of patients who are at risk of cardiovascular events. The CAPRIE study1 involved 19 185 patients with acute coronary syndromes (ACS) who were randomized in a blinded manner to clopidogrel (75 mg once daily) or aspirin (325 mg once daily). The combined endpoint was vascular death, myocardial infarction, or ischemic stroke. There was a mean follow-up of 1.9 years. Clopidogrel was shown to be more effective than aspirin in reducing the number of such cardiovascular events (a relative risk reduction of 8.7%; P = .043). Another study (CURE)2 used the same primary endpoint but had as a second primary endpoint refractory
From Woodfield Clinical Consulting LLC, Green Valley, Arizona. Submitted for publication November 11, 2008; revised version accepted January 11, 2009. Address for correspondence: Neville F. Ford, MD, PhD, FCP, Woodfield Clinical Consulting LLC, 5481 South Acacia Creek Drive, Green Valley, AZ 85622; e-mail: neville@ woodfieldclinical.com. DOI: 10.1177/0091270009332433
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Keywords: Cardiovascular; clinical pharmacology; chemistry; clinical trials; disease management; pharmacogenetics; pharmacokinetics and drug metabolism Journal of Clinical Pharmacology, 2009;49:506-512 © 2009 the American College of Clinical Pharmacology
CLOPIDOGREL RESISTANCE Clopidogrel has also developed an important role in the management of patients during and following placement of a coronary artery stent.4 The study by Gurbel et al4 identified a condition that has come to be called clopidogrel resistance. There was considerable intersubject variability in the platelet inhibitory response in patients undergoing coronary artery stenting. This clopidogrel resistance was not something that developed over time; it was a phenomenon that was evident within the first 24 hours. A lot has been written about the phenomenon. A study by Müller et al5 had findings that provided a useful
CLOPIDOGREL RESISTANCE insight. A cohort of patients (105) undergoing elective coronary stent placement and receiving regular aspirin therapy (100 mg daily) was treated with a bolus of clopidogrel (600 mg). Blood samples were drawn at 4 and 24 hours postdosing. The effect of clopidogrel on ex vivo platelet function was investigated using 5 and 20 µM/L adenosine diphosphate (ADP) as the challenges. Inhibition of platelet aggregation was calculated as a percentage of the absolute reduction in maximum aggregation relative to predosing values. Platelet inhibition of >30% was considered to be a responder, and values 500 µM) were not as
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s ubstantial.” It should have been more clearly stated that effects at such concentrations are not likely to be clinically relevant. These rather limited data have been reported and even extended by others; for example, Farid et al15 have stated that “clopidogrel’s activation involves two sequential steps by CYP3A, CYP1A2, CYP2C9, CYP2C18 and/or CYP2B6.” Based on the in vivo data described above, this does not appear to be the case. PHARMACOKINETIC EXPLANATIONS OF CLOPIDOGREL RESISTANCE There have been several efforts to make an attribution for clopidogrel resistance to pharmacokinetic effects. There is general agreement that the major oxidative metabolic pathway for clopidogrel is CYP3A4.18 In an in vitro model, atorvastatin lactone, which is also a substrate for CYP3A4, was shown to successfully compete with clopidogrel.18 These findings were supported by an in vivo study that evaluated platelet aggregation in patients undergoing elective coronary artery stent placement, some of whom who were taking pravastatin, atorvastatin, or no statins at all.19 The effects of clopidogrel on platelet aggregation were assessed predosing and at 24 hours postdosing. Pravastatin, which is not a CYP3A4 substrate, had no effect, but atorvastatin showed a dose-response relationship, decreasing the clopidogrel effect on platelet aggregation with increasing dose. This study by Lau et al,19 though, involved small sample sizes (atorvastatin 10 mg [7 patients], 20 mg [7 patients], and 40 mg [5 patients]). In a subsequent study by Hochholzer et al,20 involving 1001 patients, no significant effect of CYP3A4 metabolizable statins on clopidogrel-induced platelet aggregation was observed. In a commentary by Bates et al21 on the Hochholzer paper, the authors recapped the basis for the view that CYP3A4 is the major oxidative metabolic pathway for clopidogrel. They failed to make a sound case that atorvastatin inhibited clopidogrel metabolism by competition for CYP3A4. There have been no other reports of such CYP3A4 inhibitory activity by atorvastatin with other CYP3A4 substrates.22 A post hoc analysis of the Clopidogrel for the Reduction of Events During Observation (CREDO) study also found no evidence for CPY3A4 metabolizable statins affecting the performance of clopidogrel.23 A study looked at whether CYP3A5 polymorphism could account for clopidogrel resistance, as clopido grel was also a substrate for CYP3A5. This study by
CLOPIDOGREL RESISTANCE Suh et al24 showed that when CYP3A4 metabolism was inhibited by itraconazole, some participants could still convert clopidogrel to its active metabolite, presumably by a CYP3A5 pathway, but participants who had the CYP3A5*3/*3 genotype were unable to affect it. It was an interesting hypothesis, but it was difficult to see how it would account for the magni tude of the effect that is seen. A prospective study showed that the CYP3A5 genotype affected neither the pharmacokinetics nor the effects of clopidogrel on platelet aggregation.25 Another interesting approach to a better under standing of the clopidogrel resistance phenomenon was the pharmacogenetic study by Hulot et al.26 This study was conducted in 28 healthy young white men whose platelets had a normal aggregation response (>50%) to 5- and 10-µM ADP challenges. They were genotyped for single-nucleotide polymorphisms (SNPs) in the genes that Kurihara et al17 had conclu ded were involved in the metabolism of clopidogrel— namely, CYP2C19, CYP2B6, CYP1A2, and CYP3A4/5. They did consider that clopidogrel resistance might be associated with genetic variants in the P2Y12 receptor on the platelet but concluded that such “genetic variants are unlikely to have a marked effect on the response to clopidogrel.” The study showed that participants with a defective allele in CYP2C19 (ie, CYP2C19*2) had an impaired responsiveness to a 10-µM ADP challenge after a 7-day course of clopidogrel at 75 mg per day. An important difference in these findings and in subsequent work is that this effect was of more gradual onset. The authors offered a pharmacokinetic explanation for the result, suggesting that the findings reflected lower exposure to the reactive metabolite, although they did not measure drug or metabolite concentrations. If this were true, then CYP2C19 must be playing a major role in the metabolism of clopidogrel. A CYP2C19 inhibitor would have a similar clinical effect. A preliminary report by Gilard et al27 suggested that this was the case. Using VASP methodology (BiodisStago, Asnières, France) as a measure of platelet reactivity on 105 consecutive angioplasty patients who were taking aspirin and clopidogrel, Gilard et al27 showed that patients taking proton pump inhibitors (PPIs) had significantly higher VASP values than those who did not. As most PPIs are CYP2C19 inhibitors, the conclusion was that inhibition of the CYP2C19 pathway reduced the formation of the active metabolite from clopidogrel. This clinical observation led to a prospective, double-blind, placebo-controlled, randomized trial, the Omeprazole Clopidogrel Aspirin (OCLA) study.28
commentary
All consecutive patients undergoing elective coronary artery stent placement were considered for inclusion. The volunteers were all on aspirin. They received a loading dose of clopidogrel (300 mg) and then 75 mg daily for 7 days. They were randomized to omeprazole (20 mg once daily) or placebo. VASP methodology was used to determine the treatment effect on platelet function. The Biodis-Stago commercial kit was based on the method of Schwarz et al.29 VASP measure ments were made predosing on day 1 and on day 7 following the final dosing. It was concluded that omeprazole significantly decreased the antiplatelet effect of clopidogrel, as determined by VASP, and that PPIs should not be added to clopidogrel therapy based on these findings. The editorial comment by Gurbel et al,30 which was associated with this paper, pointed out several design problems with the study. Their conclusions were limited by the fact that the patient’s status was not determined prospectively. How many of them were poor responders, and how were they distributed between the 2 treatment groups? All patients with CYP2C19 polymorphism should have been excluded, as it was known that CYP2C19*2 patients respond less well to clopidogrel. Gurbel et al30 subscribe to the view that clopidogrel resistance is a pharmacokinetic issue, but they point out, “CYP2C19 does not appear to be a major hepatic pathway for clopidogrel metabolism.” In regard to concomitant administration of omeprazole to aspirin and clopidogrel-treated patients, it was believed that the findings of the Gilard et al27 study did not warrant a proscription against the use of omeprazole in these patients. Two abstracts appeared at the recent European Society of Cardiology meeting by Sibbing et al31 and Zairis et al32 that described drug interaction studies in groups of patients (256 and 312, respectively) who were undergoing coronary stenting and subsequent treatment with clopidogrel and PPIs that were CYP2C19 inhibitors. There was no evidence in either study for a reduction of antip latelet efficacy with clopidogrel, as would have been expected if CYP2C19 played a clinically significant role in the metabolism of clopidogrel. PHARMACOGENETIC EXPLANATION OF CLOPIDOGREL RESISTANCE The phenomenon of clopidogrel resistance was also evident in the results from the EXCELSIOR study (Impact of Extent of Clopidogrel Induced Platelet Inhibition During Elective Stent Implanta tion on Clinical Event Rate).33 The ex vivo platelet response to 5 µM/L of ADP in 802 consecutive
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FORD patients undergoing elective coronary artery stent placement could be divided into 4 groups: 32%. Major adverse cardiac events (MACE)—that is, death, myocardial infarction, and target lesion revascularization in the 30 days after the procedure—correlated well with the level of platelet aggregation prior to administration of the 600-mg loading dose of clopidogrel. It is therefore less likely that clopidogrel resistance is a consequence of clopidogrel exposure levels and more likely that it is an intrinsic property of the patient’s platelets. However, in the editorial review of the study by Alfonso and Angiolillo,34 it was noted that ischemic events occurred in 10% of patients at 30 days and 20% at 6 months. Ninety percent of these patients had maximal ADP-induced platelet aggregation values >50%. There was a plea for better standardization of platelet aggregation tests so that better between-study comparisons could be made. Alfonso and Angiolillo,34 though, considered EXCELSIOR “a landmark study.” The Framingham Heart Study35 provides an intere sting insight into a potential mechanism for inter subject variability in platelet aggregation. A cohort (2413; 1363 women) was studied using epinephrine, collagen, and ADP as the ex vivo stimuli. It was concluded that heritable factors play a major role in determining platelet aggregation. However, both the GP IIIa and the fibrinogen Hind III β-148 genotypes make only a small contribution. It was suggested that some other proteins play a role. A protein that has genetic variability and that could be associated with clopidogrel is P-glycoprotein. Clopidogrel is a CYP3A4 substrate, and such subs trates are commonly associated with the efflux transporter P-glycoprotein (or MDR1). Taubert et al36 have described a study that explains the doseresponse relationship that has been empirically developed for clopidogrel. Using Caco-2 cells and inhibitors of P-glycoprotein (P-gp), researchers have subsequently shown clinically that P-gp is an intestinal efflux transporter of clopidogrel. Efflux of clopidogrel from the intestinal tract limits the magnitude of the rising dose effect; 900 mg does not have an incremental effect over 600 mg.37 The study also measured both clopidogrel and active metabolite in these participants. The values showed considerable intersubject variability, but within subject, there was a consistent relationship of Cmax of clopidogrel to the active metabolite, as had previously been reported.14 This supports the view that CYP3A4 oxidation of clopidogrel is not the rate-limiting factor in deter mining the high interindividual variability in active
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metabolite concentrations. However, attempts to link genetic polymorphisms in P-gp to drug disposition yielded conflicting results. Giusti et al,38 in a study with 1419 patients with acute coronary syndrome on aspirin and clopidogrel therapy, showed that there was a gene-effect relation ship for CYP2C19. In total, 974 patients were wild type *1/*1, 406 were heterozygotes *1/*2, and 40 were homozygotes *2/*2, a distribution that is con sistent with the polymorphisms being in HardyWeinberg equilibrium. Carriers of the *2 allele had significantly higher platelet aggregation values than the noncarriers for an ADP challenge, and the homozygotes *2/*2 were significantly higher than the heterozygotes. These differences were not evident with an arachidonic acid challenge. This distribution of responders, semiresponders, and nonresponders was similar to that reported in a smaller study by Müller et al.5 These findings provide an explanation for the results of the EXCELSIOR study,33 in which the clinical outcome could be directly related to the response of the patient’s platelets to ADP prior to clopidogrel. A study by Frere et al39 in 603 patients with non-ST elevation acute coronary syndromes used VASP methodology to assess platelet reactivity after dosing with clopidogrel. The patients were genotyped for CYP3A4*1B, CYP3A5*3, and CYP2C19*2. The CYP2C19 polymorphisms were significantly associ ated with ADP-induced platelet aggregation and the VASP index, but CYP3A4*1B and CYP3A5*3 polymorphisms were not. In these authors’ view, their data suggest that the CYP2C19*2 allele was what influences posttreatment platelet reactivity and the clopidogrel response. It is important to realize that such a genetic property means that patients with the CYP2C19*2 allele will have more reactive platelets pretreatment, and this property has nothing to do with clopidogrel. It means that patients with the CYP2C19*2 allele will have more reactive platelets after dosing with clopidogrel than patients without such an allele, but this will be true for other agents that work by blocking the P2Y12 receptor on the platelet, such as prasugrel.40 A recently published substudy of the EXCELSIOR study41 has supported this view. In the editorial comment on this substudy by Cairns and Eikelboom,42 there are obviously still some misunderstandings about the nature of clopidogrel resistance. The authors define it as “incomplete blockade of the platelet membrane P2Y12 receptor in a patient, who is compliant with clopidogrel therapy.” The problem is with the P2Y12 receptor, as the patient’s genetic profile suggests, not with clopidogrel. They also erroneously state that
CLOPIDOGREL RESISTANCE conversion of clopidogrel to its active metabolite involves CYP2C19. A recent publication from Daiichi Sankyo and Lilly based on an analysis of the 1477 patients in the TRITON-TIMI-38 study43 who took clopidogrel, along with data obtained from 162 healthy participants, showed that the patients with a reduced-function allele (ie, *2) had lower AUCs of the active metabolite of clopidogrel, diminished platelet inhibition, and a higher rate of cardiovascular events.44 However, it had been shown by Taubert et al14 that the AUC of the thiol acid active metabolite did not correlate with the degree of platelet inhibition. This metabolite is very unstable (t1/2, 42 ± 24 minutes), is highly variable between participants, and represents only 0.02% of the major metabolite in plasma. These authors also consider that “CYP2C19 contributes in both of the two sequential oxidation steps of clopidogrel activa tion.” This statement is at variance with the generally accepted activation mechanism by CYP3A4 and Figure 6 in Savi et al.9 Their data, however, do support well the previous views that patients with one or both *2 alleles in their CYP2C19 have platelets that are more sensitive to the aggregating effects of ADP. It would have been especially helpful if these authors had applied their analysis to the patients in TRITONTIMI-38 who had taken prasugrel. This might have enabled one to separate drug-related effects from the intrinsic properties of their platelets. CONCLUSION It is suggested that a polymorphism in the P2Y12 gene has a linkage with the CYP2C19*2 gene that has not been identified yet. However, gene sequence variations of the P2Y12 receptor are known to be associated with coronary artery disease.45 Investi gation in patients with ACS46 of a known polymorphism in P2Y12, T744C, which has been associated with enhanced platelet aggregation in healthy volun teers,47 did not provide any insights. It is likely, though, that a linkage will be found. It will then be clear that clopidogrel resistance is a misnomer and that it is a pharmacogenetic, not pharmacokinetic, phenomenon. The patient’s CYP2C19 genetic status then might be considered as a “biomarker” of how the patient’s platelets will react to ADP. However, from the clinical perspective, if the patient is known to have CYP2C19 *2 polymorphism, then the loading dose of clopidogrel should certainly be 600 mg, and because clopidogrel is well tolerated at this dose, it should be considered generally for all patients. Although only the 300-mg dose is FDA approved as
commentary
a loading dose for PCI, “a 600 mg dose is increasingly used in daily clinical practice.”34 Financial disclosure: Neville F. Ford, MD, PhD, FCP, has provided consulting services for the following companies, which have projects related to this area of research: Bristol-Myers Squibb, Cogentus Pharmaceuticals, Daiichi-Sankyo, and Sanofi-Aventis. None of these companies provided support for this manuscript.
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