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Keywords:

  • aspirin;
  • clopidogrel;
  • dipyridamole;
  • GPIIb/IIIa antagonists;
  • non-steroidal anti-inflammatory drugs;
  • TP-antagonists

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Pharmacokinetic variability
  5. Pharmacodynamic variability
  6. ‘Resistance’ to aspirin and clopidogrel
  7. Implications for the practicing physician
  8. Implications for the clinical investigator
  9. Acknowledgements
  10. References

Summary.  The aim of this review article is to discuss the main determinants of the interindividual variability in response to antiplatelet agents. The main sources of pharmacokinetic and pharmacodynamic variability are reviewed, with particular emphasis on aspirin and clopidogrel. The term ‘resistance’ is uninformative of the mechanism(s) underlying interindividual variability in response to these antiplatelet agents, and is potentially misleading. Increased awareness of the distinct factors potentially interfering with the desired antiplatelet effects of aspirin or clopidogrel, particularly avoidable drug interactions, may ultimately result in better patient management than requesting unnecessary costly tests of platelet function. Similarly, new studies addressing the interindividual variability in response to these antiplatelet agents should rely upon mechanism-based biochemical end-points rather than platelet aggregation measurements. As with any drug used to prevent atherothrombosis, treatment ‘failure’ can occur with aspirin or clopidogrel perhaps not surprisingly, given the multifactorial nature of atherothrombosis. There is no scientific basis for changing antiplatelet therapy in the face of a treatment ‘failure’, as we cannot be sure whether a second vascular event occurring in the same patient will reflect the same pathophysiological event that led to the first. Moreover, we have no controlled evidence that changing therapy is a more effective strategy than maintaining an evidence-based therapy.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Pharmacokinetic variability
  5. Pharmacodynamic variability
  6. ‘Resistance’ to aspirin and clopidogrel
  7. Implications for the practicing physician
  8. Implications for the clinical investigator
  9. Acknowledgements
  10. References

Practicing physicians have long recognized that individual patients show quite substantial variability in response to the same drug treatment [1]. Important sources of variability have been characterized, as outlined in Fig. 1.

image

Figure 1. Variables that determine the complex relationship between prescribed drug dosage and drug effects on clinical outcome.

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The aim of this review article is to discuss the main determinants of the interindividual variability in response to antiplatelet agents. A case will be made against using the term ‘resistance’ to indicate such a variable antiplatelet response. Finally, both the practical and theoretical implications of this conceptual framework will be outlined.

Pharmacokinetic variability

  1. Top of page
  2. Abstract
  3. Introduction
  4. Pharmacokinetic variability
  5. Pharmacodynamic variability
  6. ‘Resistance’ to aspirin and clopidogrel
  7. Implications for the practicing physician
  8. Implications for the clinical investigator
  9. Acknowledgements
  10. References

For any given drug, there may be wide variation in its pharmacokinetic properties among individuals. This will result in quite variable plasma concentrations of the drug and/or its active metabolite(s) in different individuals receiving the same dose of the drug. In principle, one would expect that a threshold drug concentration is required in order to achieve a critical level of inhibition of a platelet enzyme or receptor. A standard therapeutic dose of the antiplatelet drug may or not achieve such a threshold concentration in the individual patient because of the factors outlined in Fig. 1, even in the presence of ideal compliance with the prescribed regimen.

The following examples illustrate the potential contribution of some of these factors. Dipyridamole is a pyrimidopyrimidine derivative with vasodilator and antiplatelet properties [2]. The mechanism of action of dipyridamole as an antiplatelet agent has been a subject of controversy. Both inhibition of cyclic nucleotide phosphodiesterase [the enzyme that degrades cyclic adenosine monophosphate (AMP) to 5′-AMP, resulting in the intraplatelet accumulation of cyclic AMP, a platelet inhibitor] and blockade of the uptake of adenosine (which acts at A2 receptors for adenosine to stimulate platelet adenylyl cyclase and thus increase cyclic AMP) have been suggested. Moreover, direct stimulation of prostacyclin (PGI2) synthesis and protection against its degradation have been reported, although the dipyridamole concentrations required to produce these effects far exceed the low micromolar plasma levels achieved after oral administration of conventional doses (100–400 mg daily) [2]. The absorption of dipyridamole from conventional formulations is quite variable and may result in low systemic bioavailability of the drug. A modified-release formulation of dipyridamole with improved bioavailability has been developed in association with low-dose aspirin [3]. Dipyridamole is eliminated primarily by biliary excretion as a glucuronide conjugate and is subject to enterohepatic recirculation. A terminal half-life of 10 h has been reported. This is consistent with the twice-a-day regimen used in recent clinical studies. Although the clinical efficacy of dipyridamole (75 mg tid), alone or in combination with aspirin, has been questioned on the basis of earlier randomized trials [4], the whole issue has been reopened by the reformulation of the drug to improve bioavailability and the results of the ESPS-2 study of the new preparation in 6602 patients with prior stroke or transient ischemic attack (TIA) [5]. Whether the favorable results obtained in ESPS-2 reflect the higher dose (400 vs. 225 mg daily) and improved systemic bioavailability of modified-release dipyridamole compared with conventional formulations, or the substantially larger sample size and statistical power of the study as compared with previous trials, remains to be established. The combination of modified release dipyridamole and low-dose aspirin has been approved by the Food and Drug Administration (FDA) based on the results of ESPS-2, and the same regimen is currently being compared with clopidogrel in the PROFESS trial.

A second example of pharmacokinetic variability is provided by the poor oral bioavailability of GPIIb/IIIa antagonists that combined with the target of approximately 50% inhibition of platelet aggregation resulted in poor antiplatelet activity in many patients [2].

Ticlopidine and clopidogrel are structurally related thienopyridines with platelet inhibitory properties. Both drugs selectively inhibit adenosine diphosphate (ADP)-induced platelet aggregation with no direct effects on arachidonic acid metabolism [2]. Neither ticlopidine nor clopidogrel affects ADP-induced platelet aggregation when added in vitro, up to 500 μM, thus suggesting that in vivo hepatic transformation to an active metabolite(s) is necessary for their antiplatelet effects. A short-lived metabolite of clopidogrel has been characterized. Clopidogrel and, probably, ticlopidine induce irreversible alterations of the platelet receptor P2Y12 mediating inhibition of stimulated adenylyl cyclase activity by ADP [6]. Permanent modification of a platelet ADP receptor by thienopyridines is consistent with time-dependent, cumulative inhibition of ADP-induced platelet aggregation upon repeated daily dosing with ticlopidine or clopidogrel and with slow recovery of platelet function after drug withdrawal [2].

The pharmacokinetics of clopidogrel are somewhat different from those of ticlopidine. Thus, after administration of single oral doses (up to 200 mg) or repeated doses (up to 100 mg daily), unchanged clopidogrel was not detectable in peripheral venous plasma. Concentrations of 1–2 ng mL−1 were measured in the plasma of patients who received 150 mg d−1 of clopidogrel for 16 days. The main systemic metabolite of clopidogrel is the carboxylic acid derivative, SR 26334. Based on measurements of circulating levels of SR 26334, it has been inferred that clopidogrel is rapidly absorbed and extensively metabolized. The plasma elimination half-life of SR 26334 is approximately 8 h. As noted above, clopidogrel, inactive in vitro, is metabolically transformed by the liver into a short-lived active platelet inhibitor. However, the interindividual variability in this metabolic activation is still being assessed, and there are no published data on whether liver impairment decreases the ability of clopidogrel to inhibit platelet function. As the cytochrome P450 isozymes CYP3A4 and 3A5 metabolize clopidogrel faster than other human P450 isozymes and are the most abundant P450s in human liver, they are predicted to be predominantly responsible for the activation of clopidogrel in vivo [7]. When clopidogrel and atorvastatin, a CYP3A4 substrate, are present at equimolar concentrations in vitro, clopidogrel metabolism is inhibited by  >90% [7].

Clopidogrel treatment exhibited marked interindividual variability in inhibiting platelet function in three different studies of patients undergoing elective percutaneous coronary intervention (PCI) and stenting [8–10]. A variable proportion of these patients was considered to be clopidogrel ‘non-responders’ or to have clopidogrel ‘resistance’ based on ADP-induced platelet aggregation. Three separate studies [8,11,12] suggest that concurrent treatment with lipophilic statins that are substrates of CYP3A4 (e.g. atorvastatin and simvastatin) may interfere with the inhibitory effects of clopidogrel on platelet function. In the study of Lau et al. [11] atorvastatin, but not pravastatin, attenuated the antiplatelet effect of clopidogrel in a dose-dependent manner. Because many drugs are metabolized by CYP3A4, it is likely that other drugs (e.g. cyclosporine and erythromycin) may modify the systemic bioavailability of the active metabolite of clopidogrel and affect its clinical efficacy. Moreover, variable metabolic activity of CYP3A4 may contribute to the interindividual variability in the platelet inhibitory effects of clopidogrel [13]. In fact, percent platelet aggregation after clopidogrel inversely correlated with CYP3A4 activity [13].

Although ex vivo measurements of ADP-induced platelet aggregation have suggested a pharmacokinetic interaction between a CYP3A4-metabolized statin and clopidogrel, post hoc analyses of placebo-controlled studies of clopidogrel have failed to detect a statistically significant clinical interaction between the two [14,15]. However, it should be emphasized that retrospective post hoc analyses have limitations that preclude definitive conclusions. Moreover, the lack of information on statin daily doses used in these trials notably restricts our ability to assess the dose-dependence of potential drug interactions.

Pharmacodynamic variability

  1. Top of page
  2. Abstract
  3. Introduction
  4. Pharmacokinetic variability
  5. Pharmacodynamic variability
  6. ‘Resistance’ to aspirin and clopidogrel
  7. Implications for the practicing physician
  8. Implications for the clinical investigator
  9. Acknowledgements
  10. References

Considerable interindividual variation in the response to drugs remains after the concentration of the drug in plasma has been adjusted to a target value. For some drugs, this pharmacodynamic variability accounts for much of the total variation in response among individuals [1].

Genetic variability of the enzyme or receptor targeted by antiplatelet drugs may alter the expected pharmacological response. Thus, an increased number of ADP receptors associated with the H2 haplotype of the P2Y12 gene may diminish the antiplatelet effect of thienopyridines that only provide partial P2Y12 blockade [16]. Although several polymorphisms of cyclooxygenase (COX)-1 [17] and -2 [18] have been described, none of these has been associated with diminished COX inhibition by aspirin. On the other hand, several lines of evidence suggest that the PlA1/A2 polymorphism of platelet GPIIb/IIIa may explain some of the variability in the response to GPIIb/IIIa antagonists [19–21]. In a substudy of the OPUS-TIMI-16 (orbofiban in patients with unstable coronary syndromes – thrombolysis in myocardial infarction 16) trial, there was a significant interaction between treatment (placebo and orbofiban) and the PlA polymorphism for bleeding [19]. Thus, while orbofiban increased bleeding in non-carriers in a dose-dependent fashion, it did not increase bleeding events in PlA2 carriers [19]. There was no interaction between treatment and the PlA polymorphism for the primary efficacy end-point. However, in the patients receiving orbofiban, there was a higher risk of a primary event and myocardial infarction in PlA2 carriers compared with non-carriers [19]. Whether the influence of the PlA genotype in modifying the clinical response to an oral GPIIb/IIIa antagonist applies also to short-term i.v. administration of this class of antiplatelet drugs is as yet unknown, though ex vivo and in vitro studies demonstrate reduced inhibition by abciximab in platelets with PlA2 polymorphism [20].

A pharmacodynamic interaction potentially affecting the antiplatelet effect of aspirin is related to the two-step mechanism of COX-1 inactivation by the drug [2]. Thus, concomitant administration of non-steroidal anti-inflammatory drugs (NSAIDs), such as ibuprofen [22] and naproxen [23], may interfere with the irreversible inactivation of platelet COX-1 by low-dose aspirin. This is due to competition between NSAIDs and aspirin for a common docking site within the COX-1 channel (Arg120), which aspirin binds to with weak affinity prior to irreversible acetylation of Ser 529 [2]. This pharmacodynamic interaction does not occur with NSAIDs endowed with some degree of COX-2 selectivity, including diclofenac [22].

Oral GPIIb/IIIa antagonists can activate platelets, at least in some individuals, acting as partial agonists, usually at concentrations below the therapeutic range [24–26]. GPIIb/IIIa is not a passive receptor, rather like all integrins it responds to ligand binding by activating the cell. Thus, fibrinogen binding leads to signals that further activate platelets and are essential for platelet aggregation. Several studies suggest that ligands designed to bind to the receptor and prevent platelet aggregation may paradoxically activate the receptor so that it can bind ligand or perhaps directly trigger some activating signals [24–26].

The same phenomenon may occur with thromboxane-receptor (TP) antagonists. Whether agonistic activity of these compounds is differentially expressed as a function of variable concentration of the endogenous TP agonists remains to be established.

An additional factor contributing to phamacodynamic variability is related to the extent and duration of platelet receptor blockade. In fact, a relatively constant level of high-grade inhibition throughout the dosing interval appears to represent an essential requirement for translating the antiplatelet effect into reduced risk of vascular events. This is ensured by: (i) the covalent nature of drug–receptor interaction, resulting in permanent inactivation of a platelet protein that cannot be resynthesized during 24 h, as in the case of aspirin and clopidogrel; (ii) the constant plasma levels being maintained through continuous infusion of the antagonist, as in the case of abciximab, epifibatide and tirofiban, or through an extended release formulation, as in the case of dipyridamole [2].

At variance with the requirements for antithrombotic efficacy, those associated with bleeding complications (most often from pre-existing upper gastrointestinal lesions) most likely involve transient, high-grade inhibition of the same receptors that may occur occasionally in a small percentage of the exposed individuals. This may explain the apparent paradox of two distinct classes of drugs endowed with same antiplatelet activity causing excess bleeding complications in the absence of any antithrombotic efficacy, i.e. traditional NSAIDs and oral GPIIb/IIIa antagonists.

Finally, a potential role for alternative sources of the endogenous platelet agonist that is targeted by the antiplatelet drug is exemplified by aspirin-insensitive sources of TXA2 biosynthesis [27]. Thus, aspirin-insensitive TXA2 biosynthesis has been described in patients with unstable angina [28–30] as well as in patients with poststroke dementia [31]. Both COX-2 expression in inflammatory cells endowed with TX-synthase [27] and in newly formed platelets [32] could account for TXA2 biosynthesis in these settings. Cipollone et al. [33] have recently reported that acute myocardial infarction occurring in aspirin-treated patients is associated with increased COX-2 expression in platelets and monocytes trapped in aspirated coronary thrombi, a potential mechanism of aspirin treatment failure.

‘Resistance’ to aspirin and clopidogrel

  1. Top of page
  2. Abstract
  3. Introduction
  4. Pharmacokinetic variability
  5. Pharmacodynamic variability
  6. ‘Resistance’ to aspirin and clopidogrel
  7. Implications for the practicing physician
  8. Implications for the clinical investigator
  9. Acknowledgements
  10. References

The term ‘aspirin resistance’ has been used to describe a number of different phenomena, including the inability of aspirin to: (i) protect individuals from thrombotic complications; (ii) cause a prolongation of the bleeding time; (iii) inhibit TXA2 biosynthesis; or (iv) produce an anticipated effect on one or more in vitro tests of platelet function [34]. The fact that some patients may experience recurrent vascular events despite long-term aspirin therapy should be properly labeled as ‘treatment failure’. This is a common phenomenon occurring with any drug (e.g. lipid-lowering or antihypertensive drugs). Given the multifactorial nature of atherothrombosis, it is not surprising that only a fraction (usually one-quarter to one-third) of all vascular complications can be prevented by any single preventive strategy.

Similarly, the term ‘clopidogrel resistance’ has been used to denote non-responsiveness of ADP-induced platelet aggregation following standard clopidogrel therapy [35].

The term ‘resistance’ is uninformative of the mechanism(s) underlying interindividual variability in response to aspirin or clopidogrel, and is potentially misleading. Thus, it implies that something can be measured that has direct bearing to clinical efficacy and, depending on its results, may lead to a change in antiplatelet treatment. In fact, the relevance of the various functional indexes of platelet capacity that can be measured ex vivo to the actual occurrence of platelet activation and inhibition in vivo is largely unknown [34,35]. Thus, we suggest that the term ‘resistance’ should be abandoned in order to advance our understanding of the distinct factors contributing to the interindividual variability in response to aspirin or clopidogrel (Table 1).

Table 1.  Main determinants of the interindividual variability in the antiplatelet effects of aspirin and clopidogrel
DeterminantAspirinClopidogrel
Dependence on systemic bioavailabilityLargely independentCompletely dependent
Influence of liver metabolismPartially inactivates active moietyConverts prodrug to active moiety
Influence of pharmacokinetic interactionsNot relevantLipophilic statins and other drugs may reduce formation of the active metabolite
Influence of pharmacodynamic interactionsSome NSAID prevent irreversible inactivation of the targetUnknown
Extraplatelet sources of the platelet agonistMonocyte and platelet COX-2 can produce TXA2Not relevant
Ratio of recommended dose to minimum effective dose for full pharmacodynamic effect2–3≤1?

Implications for the practicing physician

  1. Top of page
  2. Abstract
  3. Introduction
  4. Pharmacokinetic variability
  5. Pharmacodynamic variability
  6. ‘Resistance’ to aspirin and clopidogrel
  7. Implications for the practicing physician
  8. Implications for the clinical investigator
  9. Acknowledgements
  10. References

A test of platelet function should not be performed simply because an assay is available. In fact, no test of platelet function is currently recommended to assess the antiplatelet effects of aspirin or clopidogrel in the individual patient [2,36]. At present, ‘resistance’ to aspirin or clopidogrel should not be looked for in the clinical setting, because there is no convincing evidence for an association with clinical events conditioning cost-effective changes in antiplatelet therapy [35].

Increased awareness of the distinct factors potentially interfering with the desired antiplatelet effects of aspirin or clopidogrel, particularly avoidable drug interactions, may ultimately result in better patient management than requesting unnecessary, costly tests of platelet function.

As with any drug (antithrombotic, lipid-lowering or antihypertensive) used to prevent atherothrombosis, treatment ‘failure’ can occur with aspirin or clopidogrel perhaps not surprisingly, given the multifactorial nature of atherothrombosis. There is no scientific basis to change antiplatelet therapy in the face of a treatment ‘failure’, as we cannot be sure whether a second vascular event occurring in the same patient will reflect the same pathophysiological event that led to the first. Moreover, we have no controlled evidence that changing therapy is a more effective strategy than maintaining an evidence-based therapy.

Implications for the clinical investigator

  1. Top of page
  2. Abstract
  3. Introduction
  4. Pharmacokinetic variability
  5. Pharmacodynamic variability
  6. ‘Resistance’ to aspirin and clopidogrel
  7. Implications for the practicing physician
  8. Implications for the clinical investigator
  9. Acknowledgements
  10. References

We have a pretty detailed molecular understanding of how aspirin and clopidogrel work in preventing arterial thrombosis [2]. Thus, new studies addressing the interindividual variability in response to these antiplatelet agents should rely upon mechanism-based biochemical end-points rather than platelet aggregation measurements [34,35].

Serum TXB2 and urinary 11-dehydro-TXB2 provide reliable information on the maximal biosynthetic capacity of circulating platelets ex vivo and on the actual rate of TXA2 biosynthesis in vivo, respectively [37]. These measurements have been used extensively to characterize the clinical pharmacology of aspirin as an antiplatelet drug [38]. In patients treated with low-dose aspirin, serum TXB2 levels reflect the adequacy of platelet COX-1 inhibition and its duration, while urinary TX-metabolite excretion provides a non-invasive, time-integrated index of aspirin-insensitive sources of TXA2 biosynthesis [2,27].

Similarly, because the extent of residual P2Y1-dependent platelet aggregation induced by ADP varies considerably among patients with congenital P2Y12 deficiency [39] or healthy subjects in whom P2Y12 function has been completely blocked in vitro by saturating concentrations of specific antagonists [35], ADP-induced platelet aggregation is not the most appropriate test to evaluate the interindividual variability in response to clopidogrel [35]. Thus, measurement of ADP-induced inhibition of adenylyl cyclase, which is uniquely mediated by P2Y12, would provide a mechanism-based biochemical end-point for assessing the adequacy of clopidogrel's pharmacodynamics [35].

Reliable assessment of the adequacy and persistence of the expected pharmacodynamic effect of aspirin on platelet COX-1 would require a long-term, controlled study comparing aspirin to another antiplatelet drug in a sizeable group of stable patients requiring antiplatelet therapy. Clopidogrel would be an ideal comparator, because of remarkable similarities in the mechanism of action (permanent inactivation of a platelet protein), pharmacokinetics (short half-life of the active moiety) and dosing regimen (once daily). It should be noted, however, that while aspirin is currently used at doses that represent a 2.5- to 10-fold excess over the dose of 30 mg necessary and sufficient to fully inactivate platelet COX-1 activity upon repeated daily dosing [40], clopidogrel is used routinely at or near the threshold dose that appears to cause a ceiling effect on ADP-induced platelet aggregation upon repeated daily dosing [2].

Finally, given the size of the relative risk reduction (typically, 25% to 30% in high-risk patients) associated with long-term antiplatelet prophylaxis, novel studies aiming to detect an attenuation or loss of this protective effect, as a function of specific causes of interindividual variability in response to aspirin or clopidogrel, should have both the sensitivity and specificity necessary to detect such a small ‘signal’. None of the studies published so far meets these requirements, and estimates of relative risk of recurrent atherothrombotic events associated with aspirin [41] or clopidogrel [42] ‘resistance’ are simply unrealistic.

Similarly, because of limitations with post hoc analyses and observational studies of drug interactions (e.g. between ibuprofen and aspirin or between atorvastatin and clopidogrel), well-designed clinical trials are needed to specifically evaluate the clinical read-outs of these interactions.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Pharmacokinetic variability
  5. Pharmacodynamic variability
  6. ‘Resistance’ to aspirin and clopidogrel
  7. Implications for the practicing physician
  8. Implications for the clinical investigator
  9. Acknowledgements
  10. References

Supported by grants from the Italian Ministry of University and Research and the European Union (contract no. 005033 - EICOSANOX) to the Center of Excellence on Aging of the ‘G. d'Annunzio’ University Foundation. The expert editorial assistance of Daniela Basilico is gratefully acknowledged.

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  2. Abstract
  3. Introduction
  4. Pharmacokinetic variability
  5. Pharmacodynamic variability
  6. ‘Resistance’ to aspirin and clopidogrel
  7. Implications for the practicing physician
  8. Implications for the clinical investigator
  9. Acknowledgements
  10. References
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