Clinical Therapeutics/Volume ], Number ], 2014

Role of Clinical Pharmacology in the Development of Antiplatelet Drugs Carlo Patrono, MD Department of Pharmacology, Catholic University School of Medicine, Rome, Italy ABSTRACT Purpose: This review discusses the role of clinical pharmacology in the development of low-dose aspirin and other antiplatelet agents during the past 30 years, emphasizing the main determinants of several success stories as well as of complete failures in the field. Methods: The author employs personal appraisal of the literature, with emphasis on personal contributions to the field. Findings: Low-dose aspirin provides an interesting paradigm of the independent development of a “new” antiplatelet agent by the medical/scientific community. Aspirin “resistance,” improved dosing regimens for personalized therapy, and chemoprevention of colorectal cancer are thoroughly discussed. The industrydriven development paradigm includes 12 mechanismbased antiplatelet agents. Of those completing Phase 3, only 6 have been approved for the acute treatment or secondary prevention of atherothrombosis. Inadequate Phase 2 studies were largely involved in Phase 3 failures. Implications: The design of mechanism-based pharmacodynamic biomarkers and sophisticated Phase 2 investigations appear as an important key to successful drug development in this field. Clinical pharmacology has an excellent track record in this endeavor, and its role needs to be expanded, as suggested by the case studies discussed in this review. Finally, the choice of appropriate platelet-dependent end points and homogeneous clinical settings for Phase 3 trials not only represent desirable objectives for an integrated scientific and regulatory discussion but also deserve proper ethical consideration by all stakeholders to avoid an unacceptable burden of drug toxicity and an unsustainable waste of financial resources. (Clin Ther. 2014;]:]]]–]]]) & 2014 Elsevier HS Journals, Inc. All rights reserved. Key words: aspirin, GPIIb/IIIa blockers, P2Y12 blockers, PAR-1 antagonists, TP antagonists.

HISTORICAL PERSPECTIVE The first randomized controlled trials (RCTs) of antiplatelet therapy were reported
40 years ago.1,2 They were aimed at the secondary prevention of myocardial infarction1 and stroke2 and yielded promising but inconclusive results because of relatively small sample sizes. The 3 main drugs used in those trials, that is, aspirin, dipyridamole, and sulfinpyrazone, had not been originally developed as antiplatelet agents but were found to produce antiplatelet effects years or decades after their initial marketing as analgesic/ antiinflammatory, vasodilatory, and uricosuric agents, respectively.3 Similarly, ticlopidine was synthesized in 1972 as a potential substitute for another thienopyridine, tinoridine, the antiinflammatory properties of which were published in 1970 by a team from Japan.3 Although the mechanism of action of aspirin as an antiplatelet agent was elucidated in the 1970s by the fundamental discoveries of Vane, Samuelsson, and Majerus (reviewed by Born and Patrono3), aspirin continued to be given at relatively high doses 3 or 4 times daily,4 based on alleged “non–cyclooxygenase (COX)-dependent” antithrombotic effects5 until the mid-1990s. In a review article published in 1995 in the New England Journal of Medicine, Barnett et al6 concluded that “the optimal dose of aspirin for stroke prevention has not been established, p. 246.” In the present article, I review the role of clinical pharmacology in the development of low-dose aspirin and other antiplatelet agents during the past 30 years, emphasizing the main determinants of several success stories as well as of complete fiascos in the field.

Accepted for publication October 20, 2014. http://dx.doi.org/10.1016/j.clinthera.2014.10.012 0149-2918/$ - see front matter & 2014 Elsevier HS Journals, Inc. All rights reserved.

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Clinical Therapeutics include (1) a thorough understanding of the mechanism of action at the molecular level (Figure 1); (2) the development of mechanism-based biomarkers for dose-finding studies; and (3) the design of adequately sized RCTs to test efficacy and safety in different clinical settings. The unique pharmacokinetic (PK) properties (ie, 40%–50% systemic bioavailability and short half-life) and pharmacodynamic (PD) features (ie, irreversible inactivation of the drug target) of aspirin are ideally suited for inhibiting anucleate platelets through a “hitand-run” mechanism of action. Until 1980, the measurement of agonist (eg, epinephrine, ADP) induced platelet aggregation represented the only method of investigating the antiplatelet effects of aspirin.3 Although apparently banal in their simplicity, turbidimetric measurements in platelet-rich plasma were instrumental in demonstrating platelet inhibition in vitro and ex vivo after oral dosing of the inhibitor.3 Optical aggregometry developed by Born in the early 1960s led to the discovery of the first aggregation inhibitors, that is, ATP and adenosine, and allowed for the early characterization of the inhibitory effects of aspirin on the second phase of aggregation in the late 1960s.3 However, optical aggregometry (and its modern bedside devices) is a less-than-ideal method of investigating the antiplatelet effect of aspirin.13 In fact,

INDEPENDENT DEVELOPMENT OF LOWDOSE ASPIRIN AS AN ANTIPLATELET AGENT The clinical development of low-dose aspirin as an antiplatelet agent was largely driven by the medical/ scientific community with public funding (reviewed by Patrono7). The positive aspects of this unusual paradigm of drug development are related to the impressive number of RCTs in many different clinical settings, which is probably unmatched by any commercially developed drug. Meta-analyses of these RCTs have been performed by the Antithrombotic Trialistsʼ Collaboration8,9 based on data from individual participants, and the results form the basis of current antithrombotic treatment guidelines in different areas of cardiovascular medicine.10 A potential drawback of industry-independent development is the lack of coordination in dose selection and clinical indications. This lack of coordination is most notably reflected in the designs of 9 primary prevention trials of low-dose aspirin (reviewed by Patrono11), with largely different inclusion/exclusion criteria, the results of which form a patchwork of inconclusive scientific and regulatory interpretation. Key factors in the success story of low-dose aspirin as an antiplatelet agent for the acute treatment and secondary prevention of coronary and cerebrovascular atherothrombosis (reviewed by Patrono et al12)

COOH

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Figure 1. Mechanism of action of aspirin as an antiplatelet agent. Aspirin acetylates the hydroxyl group of a serine residue at position 529 (Ser529) in the polypeptide chain of human platelet prostaglandin (PG) G/H synthase, resulting in the inactivation of cyclooxygenase catalytic activity. Aspirin-induced blockade of platelet prostaglandin G2 synthesis results in decreased biosynthesis of prostaglandin H2 and thromboxane A2.

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C. Patrono FitzGerald et al16 through measurements of the urinary excretion of major enzymatic metabolites of TXA2 (TXM) and prostaglandin (PG) I2 (PGI-M) in healthy volunteers treated with increasing doses of aspirin. Pedersen and FitzGerald19 made other important contributions to the clinical pharmacology of platelet inhibition, including the demonstration of the presystemic nature (ie, occurring in the portal blood) of platelet COX-1 inactivation by low-dose aspirin. Moreover, FitzGerald and colleagues20 developed a controlledrelease formulation of aspirin with negligible systemic bioavailability to maximize platelet COX-1 inactivation in the prehepatic circulation and to minimize endothelial COX-2 inhibition, a major source of PGI2 biosynthesis in humans,21 in the systemic vasculature. The consistent findings from 3 different groups of very rapid and virtually complete acetylation of platelet COX-1,14 suppression of platelet TXA2 production,15 and TXM excretion16 with the use of aspirin 160 mg PO led the Clinical Trial Service Unit of the University of Oxford (Oxford, United Kingdom) to design the first large-scale (n ¼ 17,187) randomized, placebo-controlled trial evaluating the efficacy and safety of this daily dose of aspirin in

the effect of aspirin is variably detected by functional assays, potentially leading to misclassification of responder as “resistant” phenotypes owing to poor reproducibility of functional measurements over time.13 Aspirin is often considered a “weak” platelet inhibitor because of its limited effects on aggregation induced by high concentrations of collagen or ADP.13 Although the initial evaluation of aspirin as an antithrombotic agent was based on functional assays that predicted the requirement of relatively high doses and 3- or 4-times-daily dosing regimens,2–6 further development of low-dose aspirin was largely based on biomarkers reflecting its mechanism of action.14–16 In 1980, the author reported a biochemical assay of platelet COX-1 activity based on radioimmunoassay measurements of thromboxane (TX) A2 production during whole blood clotting, as reflected by serum TXB2 concentrations (Figure 2).17 This allowed for the characterization of the time and dose dependence of the inhibitory effects of aspirin in healthy subjects,15,17,18 and for the demonstration of the cumulative nature of platelet COX-1 inactivation on repeated daily dosing, its saturability, and biochemical selectivity at low doses (Figure 3).15 Substantially similar data were obtained almost simultaneously by

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Figure 2. Whole blood thromboxane production as an index of platelet cyclooxygenase activity. Endogenously formed thrombin triggers a chain of enzymatic reactions that result in the time-dependent platelet production of thromboxane (TX) A2, which is measured as serum TXB2 after 1 hour at 371C. PG ¼ prostaglandin; PL = phospholipids; AA ¼ arachidonic acid. Reprinted with permission.17

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Clinical Therapeutics Aspirin 30 mg/d Urinary 6-keto-PGF1α

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Figure 3. Long-term effects of low-dose (0.45 mg/kg/d) aspirin on platelet thromboxane (TX) B2 and renal prostaglandin (PG) I2 synthesis. Serum TXB2 concentrations and urinary excretion of 6-keto-PGF1α were measured in 3 healthy subjects before, during, and after aspirin therapy. Mean (SEM) values are plotted as the percentages of pre-aspirin measurements. The arrows indicate the duration of daily aspirin therapy. Reprinted with permission.15

reducing 5-week vascular mortality in patients with a suspected acute myocardial infarction.22 The author and his team23 also demonstrated the extremely low rate of entry of TXA2 into and rapid clearance from the systemic circulation in humans and calculated its endogenous rate of biosynthesis under physiologic conditions. Having established the linearity of the fractional conversion of systemically infused TXB2 into urinary TXM, we and others went on to characterize episodic increases in TXA2 biosynthesis in acute coronary24,25 and cerebrovascular26,27 ischemic syndromes, as well as persistent TXA2dependent platelet activation in association with major cardiovascular risk factors28–33 and myeloproliferative disorders34 (Figure 4). These findings provided a rationale for exploring the efficacy and safety of lowdose aspirin in these settings (reviewed by Patrono et al12,34).

Effects of COX Inhibitors on the Platelet–Vascular Interface PGI2 is a potent inhibitor of platelet aggregation in response to a variety of agonists and is a vasodilator.35 The basal rate of entry of PGI2 into the systemic circulation is as low as that of TXA2,
0.1 ng/kg/min under physiologic circumstances, resulting in blood levels Z1 order of magnitude lower than the minimal concentration that inhibits platelet

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aggregation.36 However, the in vivo biosynthesis of PGI2, as reflected by PGI-M excretion, is markedly enhanced in patients with severe atherosclerosis and platelet activation,37 suggesting that it represents a homeostatic response of endothelial cells to accelerated platelet–vessel wall interactions.35 Because of a lack of specific inhibitors of PGI2 synthesis or action, this hypothesis was not adequately verified until the long-term RCTs of selective inhibitors of COX-238 (“coxibs”) and the genetic manipulation of the PGI2 receptor, IP.39 The latter directly demonstrated for the first time that PGI2 modulates platelet-vascular interactions in vivo and specifically limits the cardiovascular response to TXA2.40 The individual coxib trials and their meta-analyses41,42 unraveled the clinical consequences of profound suppression of vascular PGI2 biosynthesis by high-dose regimens of COX-2 inhibitors inadequately inhibiting platelet COX-1 activity, that is, a 40% increase in major cardiovascular events (MACEs) largely driven by a 2-fold increased risk for myocardial infarction.41,42 Although enhanced platelet activation in response to vascular injury might explain the early appearance of a cardiovascular hazard, additional effects of COX-2 inhibitors might contribute to late events and to the persistence of higher risk after drug discontinuation. The clinical pharmacology of selective COX-2 inhibition had, to some extent, predicted that there might be

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Figure 4. Calculated rate of thromboxane (TX) B2 production in healthy subjects, and urinary excretion rates of 11-dehydro-TXB2 in clinical settings characterized by high cardiovascular risk. A, the metabolic fate of TXA2 in vivo and the calculated rate of its production in healthy subjects on the basis of TXB2 infusions and the measurement of its major urinary metabolite.23 B, mean (SD) or median (interquartile range) urinary excretion rates of 11-dehydro-TXB2 in clinical settings characterized by high cardiovascular risk.25–33 CHD = coronary heart disease, PCI = percutaneous coronary intervention, T2DM = type 2 diabetes mellitus, TIA = transient ischemic attack. Reprinted with permission.34

cardiovascular consequences of an altered equilibrium of these homeostatic mechanisms,21 but no regulatory body at the time could have used this surrogate marker of cardiovascular risk as a basis for not approving the marketing of drugs with an improved gastrointestinal safety profile over conventional NSAIDs.

Aspirin “Resistance”: Fact or Fiction? The term resistance has been used to indicate an incomplete inhibition of platelet function with lowdose aspirin use, with widely variable estimates of the prevalence of resistance in different clinical settings and inconclusive data on its clinical significance.5,43 In fact, interest in optical aggregometry was resurrected by the first report of so-called “aspirin resistance” 20 years ago.44 Based on 4500 publications on the topic (reviewed by Patrono and Rocca43), it was proposed that aspirin resistance represents a true clinical diagnosis, dictating a change in antiplatelet therapy.45

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Because the vast majority of these studies were based on a single measurement of agonist-induced platelet aggregation at a variable interval after unwitnessed aspirin dosing by the patient, it was widely—and uncritically—assumed that a single determination of platelet function could determine whether aspirin had fully inhibited platelet COX-1 activity and could define a stable resistant phenotype based on largely arbitrary thresholds of functional response. The author and his colleagues13 designed a complex study to formally test this widely held assumption by comparing different assays of platelet biochemistry and function for their sensitivity and specificity in detecting the antiplatelet effect of aspirin 100 mg/d for 1 to 8 weeks in 48 healthy subjects. The study design included up to 8 repeated biochemical and functional measurements in the same subjects to assess the reproducibility of the different assays, as well as an analysis of the recovery after aspirin withdrawal.13 The latter provided biochemical and functional data

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Clinical Therapeutics throughout a wide range of inhibitory values of platelet COX-1 activity to fully describe the relationship between the inhibition of enzyme activity and the inhibition of TXA2-dependent platelet function.13 Among 6 functional and biochemical assays, serum TXB2 had the highest signal-to-noise ratio and the lowest interindividual and intraindividual variabilities. At variance with measurements of serum TXB2, for which every value among 4200 determinations during aspirin intake was suppressed by at the least 97% versus preaspirin values, measurements of different functional indexes categorized according to previously described thresholds identified 2% to 30% of “resistant” or “nonresponder” samples. However, analysis of prior and subsequent determinations performed in the same subject clearly identified the fluctuating nature of the apparent “nonresponder” phenotype, most likely reflecting the relatively poor intrasubject reproducibility of functional measurements.13 These findings clearly demonstrate that functional assays, particularly when performed only once, cannot predict which individuals have effective inhibition of platelet COX-1 activity in response to low-dose aspirin.13

Does One Aspirin Dosing Regimen Fit All? Although one daily dose of aspirin (ie, 75–100 mg) probably fits all in the capacity of full inactivation of platelet COX-1 activity, one dosing interval (ie, 24 hours) does not, because of marked interindividual variability in the rate of recovery of platelet TXA2 production during the 24-hour dosing interval.46–49 Approximately 10% to 12% of the circulating platelet pool is replaced each day in subjects with a normal platelet turnover. However, only a negligible recovery of platelet COX-1 activity is detectable in aspirintreated healthy subjects for 24 to 48 hours after dosing, most likely because aspirin also acetylates COX-1 of the bone marrow megakaryocytes.13–15 Reduced acetylation of platelet and megakaryocyte COX-1 by lowdose enteric-coated aspirin, as seen in association with high body mass index in both diabetic and nondiabetic subjects46,50,51 and in patients with the metabolic syndrome,52 as well as changes in platelet turnover, as seen in at least a fraction of the diabetic population46 and in the vast majority of patients with essential thrombocythemia,47 limit the duration of the antiplatelet effect of low-dose aspirin and may require twicedaily dosing for sustained COX-1 inhibition throughout

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the dosing interval (Figure 5).46,47,53,54 Whether incomplete inhibition of platelet TXA2 production during the second half of the dosing interval in about one third of the diabetic population treated with a conventional low-dose aspirin regimen may dilute its antithrombotic effect in recent primary prevention trials in diabetes, and contribute to their inconclusive results, is currently a matter of debate.11,55 This phenomenon of accelerated renewal of the drug target is dramatically amplified in the extreme clinical paradigm of essential thrombocythemia.47 Thus, a clear PD rationale and analytical tools are available for a personalized approach to antiplatelet therapy in this setting, and an improved regimen (ie, twice daily) of low-dose aspirin should be tested in an appropriately sized RCT.34

Low-Dose Aspirin and Chemoprevention of Colorectal Cancer: Is Platelet Inhibition a Biologically Plausible Mechanism of Action? The long-term use of NSAIDs, including aspirin, has been associated with reduced incidence and mortality of colorectal cancer (CRC) (reviewed by Patrono and colleagues56,57). In a meta-analysis of 17 observational studies, aspirin use was associated with a 38% reduction in CRC incidence.58 A large body of experimental evidence and a more limited amount of clinical data have suggested that COX-2 inhibition in different cellular targets was a most likely explanation for the apparent chemopreventive effect of NSAIDs.57 This evidence was considered sufficiently strong to convince both Merck & Co Inc and Pfizer Inc to embark on long-term, placebo-controlled RCTs of rofecoxib and celecoxib in patients with sporadic colorectal adenomas (reviewed by Thun et al59). Although those trials consistently demonstrated a 20% to 40% relative risk reduction in adenoma recurrence at 3 years, they also provided the clearest evidence for an increased risk for MACEs that appeared early during treatment (reviewed by Patrono and Baigent60). At about the same time, 4 independent placebo-controlled RCTs of low-dose (81–325 mg/d) aspirin were completed in a clinical setting similar to that of the coxib trials and demonstrated remarkably similar effects of low-dose aspirin in preventing sporadic adenoma recurrence,59 probably the earliest stage of colorectal carcinogenesis. Both the daily dose (as low as 81 mg) and dosing interval (24 hours) used in the aspirin trials would

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Figure 5. Altered pharmacodynamics of low-dose aspirin in a subgroup of patients with diabetes mellitus. Compared with healthy individuals, approximately one third of patients with diabetes, obesity, or both can have impaired acetylation of bone-marrow megakaryocyte (MK) prostaglandin G/H synthase 1 and 2 (also known as cyclooxygenase [COX] 1 and 2) in response to aspirin because of reduced systemic bioavailability of the drug, accelerated megakaryopoiesis, or both. Immunohistochemistry staining of bone-marrow megakaryocytes for prostaglandin G/H synthase 1 (A), and prostaglandin G/H synthase 2 (B). C, As a consequence, these patients also have a faster renewal of mature platelets with nonacetylated prostaglandin G/H synthase 1. D, Recovery of prostaglandin G/H synthase 1 activity leads to thromboxane A2 production and platelet aggregation. E, This phenomenon can be detected in the peripheral blood by measuring time-dependent changes in serum thromboxane B2 (the stable hydrolysis product of thromboxane A2), particularly during the 12–24-hour aspirin dosing interval. Faster recovery of platelet thromboxane production can be normalized by a regimen of twice-daily aspirin administration. Reprinted with permission.46

argue against the likelihood of significant COX-2 inhibition in intestinal epithelial and stromal cells as the mechanistic basis for this effect. Also, the chemoprevention of adenoma recurrence by aspirin did not display any apparent dose dependence within the 81to 325-mg dose range,59 another element arguing against a COX-2–dependent effect. Another important piece of evidence has been added to the mechanistic puzzle by the post hoc analyses of the vast majority of RCTs of aspirin for primary and secondary cardiovascular prevention.61–63 In 6 trials of daily low-dose (75–100 mg) aspirin in primary

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prevention (35,535 participants), the overall cancer incidence was reduced with aspirin use from 3 years onward (odds ratio, 0.76; 95% CI, 0.66–0.88; P = 0.0003), similarly in women and men.63 A metaanalysis of 51 RCTs reported a 12% risk reduction in non–cardiovascular-related death in both high-dose (4300 mg) and low-dose (o300 mg) aspirin trials, and in both primary and secondary prevention trials.63 The hallmarks of this chemopreventive effect of aspirin can be summarized as follows11,59: (1) evidence of chemoprevention is detectable at daily doses as low as 75 mg; (2) this effect is saturable at low

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Clinical Therapeutics doses; that is, much higher doses are not more effective61; (3) a reduced risk for CRC is associated with an alternate-day regimen of 100 mg, as recently reported in the long-term follow-up of the Womenʼs Health Study64; (4) this effect was also apparent in the Thrombosis Prevention Trial,61 a primary prevention trial using a controlled-release formulation of aspirin 75 mg developed for maximizing the presystemic inactivation of platelet COX-1 and for minimizing the systemic inhibition of vascular COX-2.20 These main features of the chemopreventive effect of aspirin remarkably mimic the “fingerprints” of its antithrombotic effect,11,59 suggesting a common mechanism of action.59,65 The author has argued that the inhibition of platelet COX-1 activity may represent a biologically plausible mechanism of action of the antithrombotic and chemopreventive effects of low-dose aspirin, by reducing the platelet participation in abnormal repair processes of vascular and mucosal injury that may progress to atherothrombotic occlusion and neoplastic

transformation, respectively.59,65 The release of inflammatory cytokines and growth factors from activated platelets at sites of intestinal mucosal lesions might in turn upregulate COX-2 expression in adjacent nucleated cells (eg, epithelial and stromal cells) and trigger the chain of molecular events leading to enhanced cellular proliferation, reduced apoptosis, and promotion of angiogenesis (Figure 6).59,65 A sequential involvement of platelet COX-1 and nucleated cell COX-2 in the early transformation of an apparently healthy intestinal epithelium into an adenomatous lesion would explain the similar chemopreventive effects of low-dose aspirin and coxibs against the recurrence of a sporadic colorectal adenoma over a 3-year time frame,59 as well as their longer-term benefits in reducing cancer incidence and mortality after 3 to 5 years.63 Experimental evidence supporting this hypothetical chain of events is beginning to emerge.66 The development of a massspectrometric assay to quantitate the acetylated form of COX-167 may allow for testing this mechanistic hypothesis by comparing the extent and duration of

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Figure 6. Platelet activation at sites of intestinal mucosal injury may trigger downstream signaling events leading to reduced apoptosis, enhanced cellular proliferation and angiogenesis. The figure illustrates the hypothesized mechanism by which the inhibition of cyclooxygenase (COX)-1 in platelets by low-dose aspirin may suppress the induction of COX-2 in adjacent nucleated cells of the intestinal mucosa in early stage neoplasia. The sequential involvement of COX-1 and COX-2 would explain the similar inhibitory effects of deletion of either gene on murine intestinal tumorigenesis, as well as the similar effects of low-dose aspirin and coxibs in preventing sporadic colorectal adenoma recurrence in man. IL ¼ interleukin; PDGF = Platelet-Derived Growth Factor; PG ¼ prostaglandin; TGF ¼ transforming growth factor; TX ¼ thromboxane. Reprinted with permission.59

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C. Patrono aspirin-induced modification of the enzyme in circulating platelets and putative extraplatelet sites of drug action.67 Eventually, prospective and adequately sized RCTs will be required for formal evaluation of the efficacy and safety of low-dose aspirin as a chemopreventive agent in high-risk individuals and/or in an adjuvant setting. One such trial, the Add-Aspirin Trial,68 recently started recruiting
10,000 patients with a primary cancer of the esophagus/stomach, receptor colon/rectum, breast, or prostate, who after initial therapy will be randomly assigned to receive aspirin 100 or 300 mg/d or placebo and followed up for 5 years. Biomarker substudies of this trial will be testing, among other things, whether the baseline rate of platelet activation in vivo predicts cancer recurrence and/or the response to low-dose aspirin.

INDUSTRY-DRIVEN DEVELOPMENT PARADIGM: SUCCESS VERSUS FAILURE In addition to aspirin, ticlopidine, clopidogrel, and dipyridamole, at least 12 mechanism-based antiplatelet agents have been developed during the past 20 years.69 These include 3 novel P2Y12 blockers, 7 glycoprotein (GP) IIb/IIIa blockers, 1 thromboxane/prostaglandin endoperoxide receptor (TP) antagonist, and 1 proteaseactivated receptor (PAR)-1 antagonist. Of those completing Phase 3, only 6 have been approved by the European Medicines Agency or the US Food and Drug Administration (FDA) for the acute treatment or secondary prevention of atherothrombosis.

P2Y12 Blockers The development of prasugrel and ticagrelor as P2Y12 blockers represents a success story of industryguided drug development aimed at improving over existing inhibitors of ADP-induced platelet aggregation. While ticlopidine and the structurally related thienopyridine clopidogrel were developed long before the molecular characterization of their mechanism of action,3 the chemical synthesis and pharmacologic characterization of prasugrel and ticagrelor benefited from the large body of evidence demonstrating substantial interindividual variability in the PK/PD relationship of clopidogrel.69,70 The improved PK features of prasugrel over clopidogrel, in terms of higher and less variable conversion of the prodrug into its active metabolite, were associated with more profound and consistent inhibition of ADP-induced

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platelet aggregation in Phase 2 studies.71,72 Similarly, the different mechanism of action of ticagrelor in reversibly blocking the ADP–P2Y12 interaction compared with permanent but incomplete inactivation of platelet P2Y12 by the active metabolite of clopidogrel led to faster, more complete, and less variable inhibition of ADP-induced platelet aggregation.73,74 Consistent with their PD superiority, both prasugrel and ticagrelor proved to be more effective than clopidogrel in reducing MACEs in patients with acute coronary syndromes (ACS) at the expense of an acceptable increase in the risk for major bleeding complications,75,76 as would be expected from more profound interference with an important mechanism of primary hemostasis. This safety issue prompted a group of European interventional cardiologists to initiate the Global Leaders study, 77 a clinical trial designed to compare 2 antiplatelet strategies after percutaneous coronary intervention (PCI) and stent implantation: (1) dual antiplatelet therapy (aspirin plus ticagrelor) for 1 month, followed by ticagrelor alone for another 23 months; and (2) dual antiplatelet therapy (aspirin plus ticagrelor [in patients with ACS] or plus clopidogrel [in stable patients]) for 12 months, followed by aspirin alone indefinitely. Apparently, the study rationale rests on an educated guess that profound P2Y12 blockade may make TXA2 inhibition redundant in terms of cardioprotection, while reducing bleeding risk during the first year of the study, and on the assumption that a potent P2Y12 blocker may be more effective than aspirin in preventing long-term ischemic complications. Unfortunately, no PD studies were conducted to probe these hypotheses before the launch of this large-scale clinical trial in 16,000 patients. Recent advances in the pharmacogenetics of the thienopyridines have opened the realistic prospect of a personalized choice of the most appropriate P2Y12 blocker and tailored dose adjustment for individual patients.69 Another P2Y12 blocker for intravenous administration, cangrelor, was not approved by the FDA because of substantially unclear risk–benefit assessment in Phase 3 trials. Despite adequate PK/PD features of this antiplatelet drug, issues with trial design as well as the nature of the benefit precluded its approval.

GPIIb/IIIa Blockers The successful development of abciximab was largely based on the understanding at the molecular

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Clinical Therapeutics level of a rare platelet disorder called Glanzman thrombasthenia, as well as on the technology for preparing monoclonal antibodies using murine hybridomas (reviewed by Born and Patrono3). One such antibody against GPIIb/IIIa (10E5) abolished the aggregation of normal platelets, blocked platelet– fibrinogen interaction, and inhibited clot retraction, thus reproducing a functional thrombasthenic phenotype.78 This antibody turned out to be an effective antithrombotic strategy when applied intravenously in the short term to reduce the risk for ischemic events in patients undergoing PCI.79 This represented an interesting example of academic translational research, whereby timely technology transfer from academia to industry was associated with the rapid clinical development of a novel antiplatelet agent.3 This development was followed by that of 2 additional intravenous GPIIb/IIIa blockers, eptifibatide and tirofiban.3 The efficacy and safety of short-term, high-grade platelet inhibition (ie, 480% inhibition of ADP-induced platelet aggregation) with 3 structurally unrelated intravenous GPIIb/IIIa blockers (reviewed by Patrono et al5) led several companies to develop oral agents that target the same receptor, with the expectation of extending the short-term benefit to the long-term management of ACS.3,69 After limited Phase 2 dosefinding studies targeting 25% to 50% inhibition of ADP-induced platelet aggregation with a low dose and 450% with a high dose, relatively large-scale Phase 3 trials of 4 oral GPIIb/IIIa blockers, xemilofiban, orbofiban, sibrafiban, and lotrafiban, were completed in patients with ACS.3,69 None of these agents was reported to have been more effective compared with placebo (when added to a regimen of aspirin) or aspirin. In fact, a meta-analysis of 4 RCTs in 33,326 patients with ACS found a consistent and statistically significant 37% increase in mortality, and a 74% increase in major bleeding.80 There was no statistically significant effect on myocardial infarction, and a 23% reduction in the need for urgent revascularization was the only detectable benefit.80 The low oral bioavailability of these agents and the target of
50% inhibition of platelet aggregation likely were associated with a relatively limited antiplatelet activity in many patients.3 The apparent dissociation of bleeding liability from antithrombotic efficacy may not be surprising, in light of a similar phenomenon noted with most reversible COX1 inhibitors.5 Thus, the transient, high-grade inhibition of

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platelet function in a relatively small percentage of treated individuals may be sufficient to explain the increase in hemorrhagic complications (most likely, from preexisting gastrointestinal lesions) while being inadequate to protect against coronary thrombosis.5

TP Antagonists A potential limitation of the antiplatelet effect of aspirin is related to the existence of extraplatelet sources (eg, monocytes/macrophages) of TXA2 biosynthesis that are less affected by a conventional low-dose regimen than platelet TXA2 production (Figure 7).69 Moreover, some F2-isoprostanes (eg, 8-iso-PGF2α) act as agonists of the platelet TP receptor, and their formation is not affected by aspirin because they are produced nonenzymatically through a process of oxygen radical–catalyzed lipid peroxidation (reviewed by Davì and Patrono81). A selective TP antagonist could effectively block the interaction of both aspirin-sensitive and -insensitive agonists with this platelet receptor and, at least theoretically, provide a superior antiplatelet strategy (Figure 7). Only 1 such compound, terutroban, has completed Phase 3 clinical development to test this hypothesis. Terutroban has undergone a rather unusual development program: (1) a number of small mechanistic studies in different clinical settings; (2) a Phase 2, dose-finding study in patients with peripheral arterial disease82; (3) a pilot study in patients with acute myocardial infarction; and (4) a large Phase 3 trial in patients with a recent noncardioembolic cerebral ischemic event.83 The PERFORM (Terutroban Versus Aspirin in Patients With Cerebral Ischaemic Events) study83 randomly assigned 419,000 patients to receive terutroban or low-dose aspirin and was stopped prematurely for futility. After a mean follow-up of 28 months, terutroban-treated and aspirin-treated patients had virtually identical rates of major ischemic events and bleeding complications.83 Perhaps the pathophysiologic role of aspirininsensitive agonists of the platelet TP receptor has been overemphasized,81 whereas the results of the PERFORM trial clearly suggest that platelet-derived TXA2 is the clinically relevant TP agonist in this type of patient. Also, little Phase 2 clinical investigation had been conducted in patients with cerebral ischemic events to characterize the actual biosynthesis of aspirin-insensitive TP agonists and their modulation by terutroban in this setting.

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Lipid peroxidation (nonenzymatic, aspirin insensitive)

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Inflammatory cell COX-2 (low-dose aspirin insensitive)

CH3

OH

OH COOH

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TP antagonist

TXA2

8-iso-PGF2α and other iso-eicosanoids

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α β γ

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Activation PLCβ

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Platelet aggregation, vasoconstriction, cell proliferation

Figure 7. Aspirin-insensitive agonists of the thromboxane receptor. The activation of platelets is induced by the interaction of several agonists with receptors expressed on the platelet membrane. The figure depicts outside-in signaling mediated by thromboxane (TX) A2 and isoprostanes. TXA2 is synthesized by activated platelets from arachidonic acid through the cyclooxygenase (COX) pathway. Once formed, TXA2 can diffuse across the membrane and activate other platelets. In platelets, there are 2 splice variants of the TXA2 receptor: thromboxane/prostaglandin endoperoxide receptor (TP) α and TPβ, which differ in their cytoplasmic tail. TPα and TPβ couple to the proteins Gq and G12 or G13, all of which activate phospholipase C (PLC). This enzyme degrades the membrane phosphoinositides (eg, phosphatidylinositol 4,5-bisphosphate [PIP2]), releasing the second messengers inositol triphosphate (IP3) and diacylglycerol (DAG). DAG activates intracellular protein kinase C (PKC), which causes protein phosphorylation. The release of IP3 increases cytosolic levels of Ca2þ, which is released from the endoplasmic reticulum. While platelet production of TXA2 is highly sensitive to inhibition by low-dose aspirin, the production of TXA2 by inflammatory cells expressing COX-2 is not. Similarly, the TP receptor can be activated by the F2-isoprostane, 8-iso-PGF2α, and other iso-eicosanoids that are produced nonenzymatically through a process of oxygen radical-catalyzed lipid peroxidation. The interaction of these aspirininsensitive agonists with the TP receptor can be blocked by the TP antagonist terutroban. From N Engl J Med Davı` G, Patrono C, Platelet activation and atherothrombosis, 357:2482–94, Copyright 2007 Massachusetts Medical Society. Reprinted with permission from Massachusetts Medical Society.81

PAR-1 Antagonists Despite recent improvements in dual antiplatelet therapy of ACS, the rate of MACEs at 1 year was still
10% in both prasugrel-75 and ticagrelor-treated patients.76 This finding raises the possibility that ] 2014

platelet agonists other than TXA2 and ADP play a role in this residual cardiovascular risk. Thrombin is a potent platelet agonist that interacts with 2 receptors, protease activated receptor (PAR)-1 and -4, that are activated through proteolytic cleavage.84 PAR-1 is

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Clinical Therapeutics considered the major human platelet thrombin receptor, and at least 2 receptor antagonists with PAR-1 selectivity have been developed.69 The rationale for adding a thrombin receptor antagonist to conventional antiplatelet therapy in high-risk settings was largely based on the hypothesis that PAR-1 antagonism might reduce atherothrombotic events without increasing the risk for bleeding.84 Based on a Phase 2 dose-finding study of vorapaxar (formerly, SCH530348) in 1030 patients undergoing nonurgent PCI, it was concluded that clinically significant TIMI major and minor bleedings were not increased with this PAR-1 antagonist, even when administered in addition to aspirin and clopidogrel.85 Typically, Phase 2 studies are not adequately sized to evaluate hard end points of efficacy or safety. Despite these obvious limitations, both industry and opinion leaders who act as its consultants have a tendency to overinterpret the clinical results of Phase 2 studies, particularly when they meet (often for the wrong reason) their expectations. Based on these promising results, 2 large-scale Phase 3 trials of vorapaxar versus placebo were launched: (1) the TRACER (Thrombin-Receptor Antagonist Vorapaxar in Acute Coronary Syndromes) study86 in 12,944 ACS patients; and (2) the TRA 2PTIMI 50 (Vorapaxar in the Secondary Prevention of Atherothrombotic Events) study87 in 26,449 patients who had a history of myocardial infarction, ischemic stroke, or peripheral arterial disease. In patients with ACS, the primary composite end point (an unusual mix of the traditional hard end points plus recurrent ischemia with rehospitalization, or urgent coronary revascularization) was not significantly reduced with the addition of vorapaxar to standard therapy, but the risk for major bleeding, including intracranial hemorrhage, was significantly increased.86 Despite triple antiplatelet therapy, the rate of myocardial infarction, stroke, or death from cardiovascular causes at 12 months was still
10%.86 This key secondary end point was significantly reduced, by 11%, with vorapaxar use, consistent with thrombin-induced platelet aggregation contributing to a small fraction of MACEs occurring despite treatment with aspirin and clopidogrel. The second Phase 3 trial, TRA 2P-TIMI 50,87 met its primary end point (the composite of myocardial infarction, stroke, or death from cardiovascular causes) in the overall study population, although with an

12

unclear benefit–risk balance. This reflected, at least in part, substantial heterogeneity in treatment effects in the 3 clinical presentations of atherothrombosis. Thus, although for patients with a history of myocardial infarction, inhibition of PAR-1 with vorapaxar was associated with a reduced risk for cardiovascular death or ischemic events with an acceptable increase in the risk for moderate or severe bleeding,88 no detectable benefit was apparent in patients with peripheral arterial disease89 and in patients with prior ischemic stroke.90 Such heterogeneity in treatment effects may not be surprising in light of similar findings from both CAPRIE (A Randomised, Blinded, Trial of Clopidogrel Versus Aspirin in Patients at Risk of Ischaemic Events)91 and CHARISMA (Clopidogrel and Aspirin Versus Aspirin Alone for the Prevention of Atherothrombotic Events)92 with similar designs, consistently suggesting that the contribution of the same pathway of platelet aggregation, and hence the clinical consequences of its inhibition, is substantially different in the coronary, cerebral, and peripheral presentations of atherothrombosis. Based on the results of the TRA 2P-TIMI 50 trial, the FDA approved the use of vorapaxar for the secondary prevention of ischemic events in patients with a previous myocardial infarction or peripheral arterial disease. Issues with the choice of the primary end point (TRACER) and with the study design (TRA 2P-TIMI 50) have limited the potential clinical indications of vorapaxar and made the participation of about half of the patients recruited in Phase 3 trials an unnecessary exercise with undue bleeding complications.

CONCLUSIONS Progress in antiplatelet therapy has occurred at an exponential rate during the past 20 years and has perhaps reached a plateau. Adding novel antiplatelet drugs one on top of the other(s) is probably not the best strategy for future drug development. On the other hand, the current trend for dropping aspirin from long-term therapy after ACS does not appear to be a rational solution to the safety concerns, in the absence of reliable mechanistic studies to support this approach and in light of new evidence on the additional benefits of long-term aspirin therapy. Rather, a fresh approach to understanding the pathophysiology of cardiovascular events occurring despite improved dual antiplatelet therapy would seem like a more promising avenue to identify novel drug targets.

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C. Patrono The design of mechanism-based PD markers and sophisticated Phase 2 investigations appear as important keys to successful drug development in this field. Clinical pharmacology has an excellent track record in this endeavor, and its role needs to be expanded, as suggested by the case studies discussed in this review. Finally, the choice of appropriate platelet-dependent end points and homogeneous clinical settings for Phase 3 trials not only represent desirable objectives for an integrated scientific and regulatory discussion but also deserve proper ethical consideration by all stakeholders to avoid an unacceptable burden of drug toxicity and an unsustainable waste of financial resources.

ACKNOWLEDGMENTS The authorʼs studies were supported by grants from the European Commission (FP6 EICOSANOX “Eicosanoids and Nitric Oxide: Mediators of Cardiovascular, Cerebral & Neoplastic Disease” and FP7 Innovative Medicines Initiative [IMI] SUMMIT Consortium) and from the Italian Ministry of University and Research (PRIN Project 2010-2011, protocol number 2010FHH32M). The expert editorial assistance of Patrizia Barbi and Daniela Basilico is gratefully acknowledged.

CONFLICTS OF INTEREST Dr. Patrono reports having received consultantʼs and speakerʼs fees from Bayer Pharmaceuticals Corporation, Eli Lilly and Co (the makers of prasugrel), and Merck & Co Inc (the makers of vorapaxar), as well as institutional grant support for investigator-initiated research from Bayer. The author has indicated that he has no other conflicts of interest with regard to the content of this article.

REFERENCES 1. Elwood PC, Cochrane AL, Burr ML, et al. A randomized controlled trial of acetyl salicylic acid in the secondary prevention of mortality from myocardial infarction. BMJ. 1974;1:436–440. 2. The Canadian Cooperative Study Group. A randomized trial of aspirin and sulfinpyrazone in threatened stroke. N Engl J Med. 1978;299:53–59. 3. Born G, Patrono C. Antiplatelet drugs. Br J Pharmacol. 2006;147(Suppl):S241–S251.

] 2014

4. Chesebro JH, Fuster V, Elveback LR, et al. Effect of dipyridamole and aspirin on late vein-graft patency after coronary bypass operations. N Engl J Med. 1984;310:209–214. 5. Patrono C, Coller B, FitzGerald GA, et al. Platelet-active drugs: the relationships among dose, effectiveness, and side effects: the Seventh ACCP Conference on Antithrombotic and Thrombolytic Therapy. Chest. 2004;126(Suppl): 234S–264S. 6. Barnett HJ, Eliasziw M, Meldrum HE. Drugs and surgery in the prevention of ischemic stroke. N Engl J Med. 1995;332:238–248. 7. Patrono C. Aspirin as an antiplatelet drug. N Engl J Med. 1994;330:1287–1294. 8. Antithrombotic Trialistsʼ Collaboration, Collaborative meta-analysis of randomised trials of antiplatelet therapy for prevention of death, myocardial infarction, and stroke in high risk patients. BMJ. 2002;324:71–86. 9. Antithrombotic Trialistsʼ Collaboration, Aspirin in the primary and secondary prevention of vascular disease: collaborative meta-analysis of individual participant data from randomised trials. Lancet. 2009;373:1849–1860. 10. Patrono C, Andreotti F, Arnesen H, et al. Antiplatelet agents for the treatment and prevention of atherothrombosis. Eur Heart J. 2011;32:2922–2932. 11. Patrono C. Low-dose aspirin in primary prevention: cardioprotection, chemoprevention, both, or neither? Eur Heart J. 2013;34:3403–3411. 12. Patrono C, García Rodríguez LA, Landolfi R, Baigent C. Low-dose aspirin for the prevention of atherothrombosis. N Engl J Med. 2005;353:2373–2383. 13. Santilli F, Rocca B, De Cristofaro R, et al. Platelet cyclooxygenase inhibition by low-dose aspirin is not reflected consistently by platelet function assays: implications for aspirin “resistance”. J Am Coll Cardiol. 2009;53:667–677. 14. Burch JW, Stanford N, Majerus PW. Inhibition of platelet prostaglandin synthetase by oral aspirin. J Clin Invest. 1978;612:314–319. 15. Patrignani P, Filabozzi P, Patrono C. Selective cumulative inhibition of platelet thromboxane production by low-dose aspirin in healthy subjects. J Clin Invest. 1982;69:1366–1372. 16. FitzGerald GA, Oates JA, Hawiger J, et al. Endogenous biosynthesis of prostacyclin and thromboxane and platelet function during chronic administration of aspirin in man. J Clin Invest. 1983;71:676–688. 17. Patrono C, Ciabattoni G, Pinca E, et al. Low dose aspirin and inhibition of thromboxane B2 production in healthy subjects. Thromb Res. 1980;17:317–327. 18. Patrono C, Ciabattoni G, Patrignani P, et al. Clinical pharmacology of platelet cyclooxygenase inhibition. Circulation. 1985;72:1177–1184. 19. Pedersen AK, FitzGerald GA. Dose-related kinetics of aspirin. Presystemic acetylation of platelet cyclooxygenase. N Engl J Med. 1984;311:1206–1211.

13

Clinical Therapeutics 20. Clarke RJ, Mayo G, Price P, FitzGerald GA. Suppression of thromboxane A2 but not of systemic prostacyclin by controlled-release aspirin. N Engl J Med. 1991;325:1137– 1141. 21. McAdam BF, Catella-Lawson F, Mardini IA, et al. Systemic biosynthesis of prostacyclin by cyclooxygenase (COX)-2: the human pharmacology of a selective inhibitor of COX2. Proc Natl Acad Sci USA. 1999;96: 272–277. 22. ISIS-2 (Second International Study of Infarct Survival) Collaborative Group. Randomised trial of intravenous streptokinase, oral aspirin, both, or neither among 17,187 cases of suspected acute myocardial infarction: ISIS-2. Lancet. 1988; 2:349–360. 23. Patrono C, Ciabattoni G, Pugliese F, et al. Estimated rate of thromboxane secretion into the circulation of normal humans. J Clin Invest. 1986;77:590–594. 24. Fitzgerald DJ, Roy L, Catella F, FitzGerald GA. Platelet activation in unstable coronary disease. N Engl J Med. 1986;315:983–989. 25. Vejar M, Fragasso G, Hackett D, et al. Dissociation of platelet activation and spontaneous myocardial ischemia in unstable angina. Thromb Haemost. 1990;63:163–168. 26. Koudstaal PJ, Ciabattoni G, van Gijn J, et al. Increased thromboxane biosynthesis in patients with acute cerebral ischemia. Stroke. 1993;24:219–223. 27. van Kooten F, Ciabattoni G, Patrono C, et al. Evidence for episodic platelet activation in acute ischemic stroke. Stroke. 1994;25:278–281. 28. Davì G, Catalano I, Averna M, et al. Thromboxane biosynthesis and platelet function in type II diabetes mellitus. N Engl J Med. 1990;322:1769–1774. 29. Davì G, Averna M, Catalano I, et al. Increased thromboxane biosynthesis in type IIa hypercholesterolemia. Circulation. 1992;85:1792–1798.

14

30. Di Minno G, Davì G, Margaglione M, et al. Abnormally high thromboxane biosynthesis in homozygous homocystinuria. Evidence for platelet involvement and probucolsensitive mechanism. J Clin Invest. 1993;92:1400–1406. 31. Davì G, Gresele P, Violi F, et al. Diabetes mellitus, hypercholesterolemia, and hypertension but not vascular disease per se are associated with persistent platelet activation in vivo. Evidence derived from the study of peripheral arterial disease. Circulation. 1997;96:69–75. 32. Davì G, Guagnano MT, Ciabattoni G, et al. Platelet activation in obese women: role of inflammation and oxidant stress. JAMA. 2002;288:2008–2014. 33. Minuz P, Patrignani P, Gaino S, et al. Increased oxidative stress and platelet activation in patients with hypertension and renovascular disease. Circulation. 2002;106:2800– 2805. 34. Patrono C, Rocca B, De Stefano V. Platelet activation and inhibition in polycythemia vera and essential thrombocythemia. Blood. 2013;121: 1701–1711. 35. Moncada S, Vane JR. Arachidonic acid metabolites and the interactions between platelets and bloodvessel walls. N Engl J Med. 1979; 300:1142–1147. 36. FitzGerald GA, Brash AR, Falardeau P, Oates JA. Estimated rate of prostacyclin secretion into the circulation of normal man. J Clin Invest. 1981;68:1272–1276. 37. FitzGerald GA, Smith B, Pedersen AK, Brash AR. Increased prostacyclin biosynthesis in patients with severe atherosclerosis and platelet activation. N Engl J Med. 1984;310: 1065–1068. 38. FitzGerald GA, Patrono C. The coxibs, selective inhibitors of cyclooxygenase-2. N Engl J Med. 2001; 345:433–442. 39. Murata T, Ushikubi F, Matsuoka T, et al. Altered pain perception

40.

41.

42.

43.

44.

45.

46.

47.

48.

and inflammatory response in mice lacking prostacyclin receptor. Nature. 1997;388:678–682. Cheng Y, Austin SC, Rocca B, et al. Role of prostacyclin in the cardiovascular response to thromboxane A2. Science. 2002;296:539–541. Kearney PM, Baigent C, Godwin J, et al. Do selective cyclo-oxygenase2 inhibitors and traditional nonsteroidal anti-inflammatory drugs increase the risk of atherothrombosis? Meta-analysis of randomised trials. BMJ. 2006;332:1302–1308. Coxib and traditional NSAID Trialistsʼ (CNT) Collaboration, Vascular and upper gastrointestinal effects of non-steroidal anti-inflammatory drugs: meta-analyses of individual participant data from randomised trials. Lancet. 2013;382:769–779. Patrono C, Rocca B. Drug insight: aspirin resistance—fact or fashion? Nat Clin Pract Cardiovasc Med. 2007;4:42–50. Helgason CM, Bolin KM, Hoff JA, et al. Development of aspirin resistance in persons with previous ischemic stroke. Stroke. 1994;25: 2331–2336. Gum PA, Kottke-Marchant K, Welsh PA, et al. A prospective, blinded determination of the natural history of aspirin resistance among stable patients with cardiovascular disease. J Am Coll Cardiol. 2003;41:961–965. Rocca B, Santilli F, Pitocco D, et al. The recovery of platelet cyclooxygenase activity explains interindividual variability in responsiveness to lowdose aspirin in patients with and without diabetes. J Thromb Haemost. 2012;10:1220–1230. Pascale S, Petrucci G, Dragani A, et al. Aspirin-insensitive thromboxane biosynthesis in essential thrombocythemia is explained by accelerated renewal of the drug target. Blood. 2012;119:3595–3603. Perneby C, Wallén NH, Rooney C, et al. Dose- and time-dependent

Volume ] Number ]

C. Patrono

49.

50.

51.

52.

53.

54.

55.

56.

antiplatelet effects of aspirin. Thromb Haemost. 2006;95:652– 658. Henry P, Vermillet A, Boval B, et al. 24-hour time-dependent aspirin efficacy in patients with stable coronary artery disease. Thromb Haemost. 2011;105:336–344. Maree AO, Curtin RJ, Dooley M, et al. Platelet response to low-dose enteric-coated aspirin in patients with stable cardiovascular disease. J Am Coll Cardiol. 2005;47:1258– 1263. Grosser T, Fries S, Lawson JA, et al. Drug resistance and pseudoresistance. An unintended consequence of enteric coating aspirin. Circulation. 2013;127:377–385. Smith JP, Haddad EV, Taylor MB, et al. Suboptimal inhibition of platelet cyclooxygenase-1 by aspirin in metabolic syndrome. Hypertension. 2012;59:719–725. Spectre G, Arnetz L, Östenson CG, et al. Twice daily dosing of aspirin improves platelet inhibition in whole blood in patients with type 2 diabetes mellitus and micro- or macrovascular complications. Thromb Haemost. 2011;106:491–499. Dillinger JG, Drissa A, Sideris G, et al. Biological efficacy of twice daily aspirin in type 2 diabetic patients with coronary artery disease. Am Heart J. 2012;164:600–606. Authors/Task Force Members, Rydén L, Grant PJ, et al. ESC Guidelines on diabetes, pre-diabetes, and cardiovascular diseases developed in collaboration with the EASD: the Task Force on Diabetes, Pre-Diabetes, and Cardiovascular Diseases of the European Society of Cardiology (ESC) and developed in collaboration with the European Association for the Study of Diabetes (EASD) [erratum in: Eur Heart J 2014 ;35:1824]. Eur Heart J 2013;34:3035-3087. Thun MJ, Henley SJ, Patrono C. Nonsteroidal anti-inflammatory drugs as anticancer agents:

] 2014

57.

58.

59.

60.

61.

62.

63.

64.

65.

mechanistic, pharmacologic, and clinical issues. J Natl Cancer Inst. 2002;94:252–266. Patrignani P, Patrono C. Cyclooxygenase inhibitors: from pharmacology to clinical read-outs. Biochim Biophys Acta. 2014. pii:S1388-1981 (14)00194-2. Algra AM, Rothwell PM. Effects of regular aspirin on long-term cancer incidence and metastasis: a systematic comparison of evidence from observational studies versus randomised trials. Lancet Oncol. 2012;13:518–527. Thun MJ, Jacobs EJ, Patrono C. The role of aspirin in cancer prevention. Nat Rev Clin Oncol. 2012; 9:259–267. Patrono C, Baigent C. Nonsteroidal anti-inflammatory drugs and the heart. Circulation. 2014;129:907–916. Rothwell PM, Wilson M, Elwin CE, et al. Long-term effect of aspirin on colorectal cancer incidence and mortality: 20 year follow-up of five randomised trials. Lancet. 2010;376: 1741–1750. Rothwell PM, Fowkes FG, Belch JF, et al. Effect of daily aspirin on long-term risk of death due to cancer: analysis of individual patient data from randomised trials. Lancet. 2011;377:31–41. Rothwell PM, Price JF, Fowkes FG, et al. Short-term effects of daily aspirin on cancer incidence, mortality, and non-vascular death: analysis of the time course of risks and benefits in 51 randomised controlled trials. Lancet. 2012;379:1602–1612. Cook NR, Lee IM, Zhang SM, et al. Alternate-day, low-dose aspirin and cancer risk: long-term observational follow-up of a randomized trial. Ann Intern Med. 2013;159:77–85. Patrono C, Patrignani P, García Rodríguez LA. Cyclooxygenaseselective inhibition of prostanoid formation: transducing biochemical selectivity into clinical read-outs. J Clin Invest. 2001;108:7–13.

66. Dovizio M, Maier TJ, Alberti S, et al. Pharmacological inhibition of platelet-tumor cell cross-talk prevents platelet-induced overexpression of cyclooxygenase-2 in HT29 human colon carcinoma cells. Mol Pharmacol. 2013;84:25–40. 67. Patrignani P, Tacconelli S, Piazuelo E, et al. Reappraisal of the clinical pharmacology of low-dose aspirin by comparing novel direct and traditional indirect biomarkers of drug action. J Thromb Haemost. 2014;12:1320–1330. 68. University College London, Medical Research Council, Clinical Trials Unit. Welcome to the AddAspirin website. www.addaspirin trial.org. Accessed [30 July 2014]. 69. Patrono C, Rocca B. The future of antiplatelet therapy in cardiovascular disease. Annu Rev Med. 2010; 61:49–61. 70. Simon T, Verstuyft C, Mary-Krause M, et al. French Registry of Acute ST-Elevation and Non-ST-Elevation Myocardial Infarction (FAST-MI) Investigators. Genetic determinants of response to clopidogrel and cardiovascular events. N Engl J Med. 2009;360:363–375. 71. Wiviott SD, Trenk D, Frelinger AL, et al. PRINCIPLE-TIMI 44 Investigators. Prasugrel compared with high loading- and maintenancedose clopidogrel in patients with planned percutaneous coronary intervention: the Prasugrel in Comparison to Clopidogrel for Inhibition of Platelet Activation and Aggregation-Thrombolysis in Myocardial Infarction 44 trial. Circulation. 2007;116:2923–2932. 72. Wallentin L, Varenhorst C, James S, et al. Prasugrel achieves greater and faster P2Y12 receptor-mediated platelet inhibition than clopidogrel due to more efficient generation of its active metabolite in aspirin-treated patients with coronary artery disease. Eur Heart J. 2008;29:21–30. 73. Husted S, Emanuelsson H, Heptinstall S, et al. Pharmacodynamics,

15

Clinical Therapeutics

74.

75.

76.

77.

78.

79.

80.

pharmacokinetics, and safety of the oral reversible P2Y12 antagonist AZD6140 with aspirin in patients with atherosclerosis: a double-blind comparison to clopidogrel with aspirin. Eur Heart J. 2006;27:1038–1047. Gurbel PA, Bliden KP, Butler K, et al. Randomized double-blind assessment of the ONSET and OFFSET of the antiplatelet effects of ticagrelor versus clopidogrel in patients with stable coronary artery disease: the ONSET/OFFSET study. Circulation. 2009;120:2577–2585. Wiviott SD, Braunwald E, McCabe CH, et al. TRITON-TIMI 38 Investigators. Prasugrel versus clopidogrel in patients with acute coronary syndromes. N Engl J Med. 2007; 357:2001–2015. Wallentin L, Becker RC, Budaj A, et al. PLATO Investigators. Ticagrelor versus clopidogrel in patients with acute coronary syndromes. N Engl J Med. 2009;361: 1045–1057. Comparative Effectiveness of 1 Month of Ticagrelor Plus Aspirin Followed by Ticagrelor Monotherapy Versus a Current-day Intensive Dual Antiplatelet Therapy in Allcomers Patients Undergoing Percutaneous Coronary Intervention With Bivalirudin and BioMatrix Family Drug-eluting Stent Use ClinicalTrials.gov. Identifier:NCT01813 435. Accessed July 30 2014. Coller BS, Peerschke EI, Scudder LE, Sullivan CA. A murine monoclonal antibody that completely blocks the binding of fibrinogen to platelets produces a thrombasthenic-like state in normal platelets and binds to glycoproteins IIb and/or IIIa. J Clin Invest. 1983; 72:325–338. Coller BS. Blockade of platelet GPIIb/IIIa receptors as an antithrombotic strategy. Circulation. 1995; 92:2373–2380. Chew DP, Bhatt DL, Sapp S, Topol EJ. Increased mortality with oral

16

81.

82.

83.

84.

85.

86.

platelet glycoprotein IIb/IIIa antagonists: a meta-analysis of phase III multicenter randomized trials. Circulation. 2001;103:201–206. Davì G, Patrono C. Platelet activation and atherothrombosis. N Engl J Med. 2007;357:2482–2494. Fiessinger JN, Bounameaux H, Cairols MA, et al. TAIPAD investigators. Thromboxane Antagonism with terutroban in Peripheral Arterial Disease: the TAIPAD study. J Thromb Haemost. 2010;8:2369–2376. Bousser MG, Amarenco P, Chamorro A, et al. PERFORM Study Investigators. Terutroban versus aspirin in patients with cerebral ischaemic events (PERFORM): a randomised, double-blind, parallelgroup trial. Lancet. 2011;377:2013– 2022. Coughlin SR. Protease-activated receptors in hemostasis, thrombosis and vascular biology. J Thromb Haemost. 2005;3:1800–1814. Becker RC, Moliterno DJ, Jennings LK, et al. TRA-PCI Investigators. Safety and tolerability of SCH 530348 in patients undergoing non-urgent percutaneous coronary intervention: a randomised, double-blind, placebo-controlled phase II study. Lancet. 2009;373: 919–928. Tricoci P, Huang Z, Held C, et al. TRACER Investigators. Thrombinreceptor antagonist vorapaxar in acute coronary syndromes. N Engl J Med. 2012;366:20–33.

87. Morrow DA, Braunwald E, Bonaca MP, et al. TRA 2P–TIMI 50 Steering Committee and Investigators. Vorapaxar in the secondary prevention of atherothrombotic events. N Engl J Med. 2012;366:1404–1413. 88. Scirica BM, Bonaca MP, Braunwald E, et al. TRA 21P-TIMI 50 Steering Committee and Investigators. Vorapaxar for secondary prevention of thrombotic events for patients with previous myocardial infarction: a prespecified subgroup analysis of the TRA 21P-TIMI 50 trial. Lancet. 2012;380:1317–1324. 89. Bonaca MP, Scirica BM, Creager MA, et al. Vorapaxar in patients with peripheral artery disease: results from TRA 2P-TIMI 50. Circulation. 2013;127:1522–1529. 90. Morrow DA, Alberts MJ, Mohr JP, et al. Thrombin Receptor Antagonist in Secondary Prevention of Atherothrombotic Ischemic Events–TIMI 50 Steering Committee and Investigators. Efficacy and safety of vorapaxar in patients with prior ischemic stroke. Stroke. 2013;44:691–698. 91. CAPRIE Steering Committee. A randomised, blinded, trial of clopidogrel versus aspirin in patients at risk of ischaemic events. Lancet. 1996;348:1329–1339. 92. Bhatt DL, Fox KA, Hacke W, et al. CHARISMA Investigators. Clopidogrel and aspirin versus aspirin alone for the prevention of atherothrombotic events. N Engl J Med. 2006;354:1706–1717.

Address correspondence to: Carlo Patrono, MD, Istituto di Farmacologia, Università Cattolica del S. Cuore, Largo Francesco Vito, 1, 00168 Rome, Italy. E-mail: [email protected]

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