Aspirin is perhaps the most cost-effective therapy in medicine.

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1 Review Translational Success Stories highlight how basic discoveries have led to clinical advances (such as the use of new drugs or diagnostic modalities in patients). This initiative reflects the renewed emphasis of our Journal on translational research. It is hoped that these articles will stimulate efforts to translate basic insights into clinical practice. Historical Lessons in Translational Medicine Cyclooxygenase Inhibition and P2Y12 Antagonism Desmond J. Fitzgerald, Garret A. FitzGerald Abstract: The development of drugs that inhibit platelets has been driven by a combination of clinical insights, fundamental science, and sheer luck. The process has evolved as the days of stumbling on therapeutic gems, such as aspirin, have long passed and have been replaced by an arduous process in which a drug is designed to target a specific protein implicated in a well-characterized pathophysiological process, or so we would like to believe. The development of antiplatelet therapy illustrates the importance of understanding the mechanisms of disease and the pharmacology of the compounds we develop, coupled with careful clinical experimentation and observation and, yes, still, a fair bit of luck. (Circ Res. 2013;112: ) Key Words: aspirin platelet prostaglandin Aspirin is perhaps the most cost-effective therapy in medicine. Its evolution as a mainstay of cardioprotection illustrates interlocking contributions of industry and academia, the importance of astute clinical observation, and the pivotal integration of basic and clinical pharmacology in drug repurposing, prompting hypotheses then addressed by large, placebo-controlled, randomized trials. The analgesic potential of salicylate variously had been appreciated by the ancient Egyptians, Hippocrates, Pliny the Elder, Jesuits in South America, and a Protestant pastor in Chipping Norton. These interlocking trails of discovery eventually led to a dye manufacturer in Leverkusen in the late 19th century. This story, together with the personalities and politics surrounding the synthesis of acetyl salicylic acid at Bayer by Felix Hoffmann and its marketing in mid 1899 as aspirin, is nicely told by Mueller and Scheidt. 1 We pick up the story with the uncontrolled though astute observations of a private practitioner, L. L. Craven, in the late 1940s, that the gingival bleeding of his tonsillectomy patients treated with aspirin suggested an aspect of drug action that could be exploited for cardioprotection. 2 His uncontrolled observations, suggesting protection from myocardial infarction (MI) by aspirin, were buried in obscurity until the development of light-transmission platelet aggregometry and standardized approaches to measuring the bleeding time by Quick, Weiss, O Brien, and Born in the 1960s focused attention on hemostasis and the platelet in particular as a target of aspirin action. 3 6 In the mean time, Vane, Moncada, Ferrara, Gryglewski, and their colleagues at the Royal College of Surgeons and the Wellcome Laboratories in Beckenham had been using an ingenious technique, superfusion bioassay (dripping blood over a mesmerizing sequence of animal tissues), to elucidate, by their distinct effects, the contrasting biology of a diverse family of lipid mediators, 7 the prostaglandins (PGs), first described as products of ram seminal vesicle PG G/H synthase by von Euler. 8 PG synthase possesses both cyclooxygenase (COX) and peroxidase activity to generate sequentially the endoperoxide intermediates, PGG 2 and PGH 2, that are then acted on by specific PG synthases that are expressed in a relatively cell-specific fashion so that most cells make 1 or 2 dominant products (Figure 1). Colloquially, the PG G/H synthase enzyme is referred to as COX. Vane s group showed that nonsteroidal anti-inflammatory drugs (NSAIDs), including aspirin, acted to prevent PG synthesis by blocking COX 9 and discovered a platelet inhibitory vasodilator PG product of endothelial COX, 10 confirmed through a collaboration with scientists at Upjohn as prostacyclin, also known as PGI Elegant structural elucidation using mass Original received October 10, 2012; revision received November 28, 2012; accepted December 6, In November 2012, the average time from submission to first decision for all original research papers submitted to Circulation Research was 15.8 days. UCD Conway Institute and Java Clinical Research, University College Dublin, Ireland (D.J.F.); and the Institute for Translational Medicine and Therapeutics, University of Pennsylvania, Philadelphia, PA (G.A.F.). Correspondence to G.A. FitzGerald, Institute for Translational Medicine and Therapeutics, Translational Research Center, 10th Floor, Room 116, 3400 Civic Center Blvd, Bldg 421, Philadelphia, PA garret@upenn.edu 2013 American Heart Association, Inc. Circulation Research is available at DOI: /CIRCRESAHA

2 Fitzgerald and FitzGerald Translational Success Story 175 Non-standard Abbreviations and Acronyms COX GI MI NSAID PCI PG Tx TxM VASP cyclooxygenase gastrointesitinal myocardial infarction nonsteroidal anti-inflammatory drug percutaneous coronary interventions prostaglandin thromboxane thromboxane metabolite; 2,3-dinor TxB2 vasodilator-stimulated phosphoprotein spectrometry of the major product of platelet COX as thromboxane (Tx) A 2 a vasoconstrictor and platelet agonist was achieved by Samuelsson et al at Karolinska. 12,13 Together with lipoxygenase and epoxygenase products of the 20 carbon unsaturated fatty acid and arachidonic acid (AA), these compounds are termed eicosanoids, with εικωσι being Greek for 20. J. R. Vane, B. Samuelsson, and M. Hamberg shared the Nobel Prize for their discoveries in Low-Dose Aspirin In St. Louis, Roth and Majerus 14 made a discovery that the platelet was a special target of aspirin. Using (acetyl- 3 H) aspirin, they showed that a platelet protein was acetylated by aspirin and speculated that this was platelet COX (Figure 2). Platelets prepared from aspirin-treated donors did not incorporate any aspirin-derived radioactivity into the particulate protein for 2 days after drug treatment, which, they suggested, reflected megakaryocyte acetylation. Full pretreatment uptake of radioactivity was not obtained until 12 days thereafter, a course of increasing incorporation of radioactivity that paralleled that of platelet turnover. The integration of these observations with the production of TxA 2 by platelet COX and elucidation of the clinical pharmacology of aspirin required the development of biomarkers of TxA 2 formation (Figure 3). A problem was that TxA 2 rapidly dehydrated to an inactive product, TxB 2, with a half-life of approximately 30 seconds; 15 this was then metabolized to chemically stable, but biologically inactive, products that were detectable in plasma and urine. 16 Two approaches to the challenge were deployed. Patrignani et al 17 used immunoreactive measurement of serum TxB 2 to reflect indirectly the capacity of platelets to form TxA 2 and found that, as expected, repeated administration of low doses of aspirin that suppressed this index only partially ex vivo when administered acutely, cumulatively suppressed platelet Tx production completely on chronic dosing. This contrasted with their measurement of the inactive hydrolysis product of PGI 2, 6-keto-PGF 1α, in urine, which was not altered by chronic administration of this low dose (0.45 mg/kg per day; 32 mg/d for a 70-kg man) of aspirin. Using mass spectrometric based analysis of major urinary metabolites 2,3-dinor 6-keto-PGF 1α (prostaglandin I [prostacyclin] metabolite) and 15-keto-13,14-dihydro-2,3-dinor-6- keto-pgf 1α we were able reliably to estimate actual in vivo production rates of a PG, in this case of PGI 2, in humans for the first time. 18 Extrapolation from metabolite excretion in response to infusion of a range of concentrations of exogenous PGI 2 allowed us to conclude that the eicosanoid functioned as a local autacoid and not as a circulating hormone as had been inferred based on estimates of its concentration in plasma using superfusion bioassay and immunoassays. We subsequently reached a similar conclusion by measuring stable metabolites Figure 1. Biosynthetic pathway of prostanoids. Arachidonic acid, a constituent of cell membrane lipid domains, is released by phospholipases, especially a cytosolic phospholipase (PL) A 2, is mobilized by physical and chemical stimuli, and is subject to metabolism by prostaglandin (PG) G/H synthases to form sequentially PG endoperoxides, PGG 2 and PGH 2. The latter is then subject to further metabolism by synthases and isomerases to form thromboxane (Tx) A 2 and PGs, each of which activates G-protein-coupled receptors termed T,E,D, I of F prostanoid (P) receptors. Cells typically make 1 or 2 dominant PGs. Besides DP1, PGD 2 also activates at low concentrations a member of the N-formylmethionyl-leucyl-phenylalinine (fmlp) receptor superfamily, termed CRTH2 or, colloquially, DP2. VSMCs, vascular smooth muscle cells. Reproduced with permission from Ricciotti E, FitzGerald GA. Arterioscler Thromb Vasc Biol. 2011;31:

3 176 Circulation Research January 4, 2013 COOH OCOCH3 COOH OH Serv 52 OH9 PGH-synthase 1 Aspirin Ser 529 OCOCH3 Salicylic Acid PGH-synthase 1 (inactive) COOH o COOH COOH Figure 2. Aspirin inhibits platelet cyclooxygenase (COX)-1. Aspirin irreversibly acetylates Ser529 in the human COX-1, converting to salicylic acid, a weak reversible COX inhibitor. Covalent modification of platelet COX-1 disrupts biotransformation of arachidonic acid to prostaglandin G 2 (PGG 2 ), and subsequently to TxA 2, for the lifetime of that platelet. o Arachidonic Acid OOH PGG2 Arachidonic Acid in urine and plasma (2,3-dinor TxB 2 [TxM] and 11-dehydro TxB 2 ) with regard to TxA 2 ; 19 indeed, in this case, aside from quantitative inaccuracies attributable to the analytic approach, estimates of Tx biosynthesis based on measurements of plasma TxB 2 were further confounded by ex vivo platelet activation. 20 These studies, affording maximal estimates of PGI 2 and TxA 2 in the circulation of 1 to 2 pg/ml, confounded a substantial literature suggesting that both circulated at concentrations orders of magnitude higher than we reported. 21 Using urinary metabolites, we could show that aspirin did cumulatively suppress systemic Tx synthesis in vivo, 22 and using the unique pattern of recovery from inhibition of a platelet source (10 12 days), as opposed to from nucleated cells (hours), conclude that under physiological conditions both Figure 3. Indices of biosynthesis and the capacity to form prostacyclin (PGI 2 ) and thromboxane (TxA 2 ). The primary bioactive products, TxA 2 and PGI 2, rapidly degrade into the inactive hydrolysis products, TxB 2 and 6-keto-PGF 1α. Measurements of these latter products are poor indices of the biosynthesis of prostaglandins because they are confounded by ex vivo formation of the parent compounds in blood and other biological samples. They are, however, useful markers of the capacity of a tissue to generate the parent compound, for example, measurements of serum TxB 2 as an index of cyclooxygenase activity in platelets. These products are enzymatically metabolized further in a tissue-specific manner to products that are excreted in urine. Measurements of these chemically stable products reflect in vivo formation of the parent compounds.

4 Fitzgerald and FitzGerald Translational Success Story 177 Table. Explanation of Clinical Trial Acronyms COMMIT ISAR-REACT CURE CREDO ESPRIT TARGET PCI-CURE PLATO PRONTO TRITON-TIMI 38 Clinical Trials Clopidogrel and Metoprolol in Myocardial Infarction Trial Intracoronary Stenting and Antithrombotic Regimen: Rapid Early Action for Coronary Treatment Clopidogrel in Unstable angina to prevent Recurrent ischemic Events Clopidogrel for Reduction of Events During Observation Enhanced Suppression of the Platelet IIb/IIIa Receptor with Integrilin Therapy Tirofiban and Reopro Give Similar Efficacy Outcome Trial Percutaneous Coronary Intervention-Clopidogrel in Unstable angina to prevent Recurrent ischemic Events Platelet Inhibition and Patient Outcomes Comparison of Ticagrelor (AZD6140) and Clopidogrel in Patients with Acute Coronary Syndrome Plavix Reduction Of New Thrombus Occurrence Trial to Assess Improvement in Therapeutic Outcomes by Optimizing Platelet Inhibition with Prasugrel-Thrombolysis in Myocardial Infarction the dinor and 11-dehydro metabolites of Tx were derived predominantly from platelets in healthy volunteers. 23 However, the discrepancy between the capacity of platelets to form Tx compared with actual biosynthetic rates of formation meant that activation of Tx formation in another cellular source in vivo for example, macrophages could dilute the relative contribution of platelets to metabolite measurements in urine. Although our estimates of the dose-dependent impact of aspirin on Tx formation aligned with those of Patrignani and Patrono, we found that even relatively low doses of 80 to 160 mg/d had a measurable suppressive effect on prostaglandin I (prostacyclin) metabolite excretion. Subsequent studies explained this discrepancy, whereas urinary prostaglandin I (prostacyclin) metabolite reflected predominantly extrarenal systemic sources of PGI 2 biosynthesis and urinary 6-keto- PGF 1α was derived predominantly from the kidney. 24 Thus, low doses of aspirin in the range of 70 to 100 mg/d would be expected to suppress completely platelet Tx despite having a minor suppressive effect on extrarenal sources of PGI 2, such as the systemic vasculature, that is, a relative dosedependent biochemical selectivity by aspirin for inhibition of platelet TxA 2. Cardioprotection From Low-dose Aspirin: Mechanistic Insights The original observations that aspirin inhibited platelet aggregation led to a fervid resuscitation of the original proposal by Craven that aspirin might afford cardioprotection. However, during the ensuing decade a series of contradictory results were reported as investigators tried to detect a reduction in heart attacks and strokes by aspirin. In retrospect, the confusion seems to have derived from a failure to select the most susceptible populations to evaluate this aspect of aspirin action and to size appropriately clinical trials based on a reasonable expectation of its impact. The development of methods to study Tx biosynthesis afforded an important biomarker. Thus, we reported that Tx formation was markedly elevated in patients presenting with MI, but in most cases levels rapidly declined toward normal within 1 to 2 days. Thus, clinical trials in which the index infarct necessary for entry to the trial may have occurred days, weeks, or months before randomization to aspirin or placebo were likely to be seriously underpowered for detection of drug effect. Rather, patients with unstable angina the acute coronary syndrome (ACS) had phasic increases in urinary TxM that corresponded to their ischemic episodes and thus constituted a population enriched for evaluation of the cardioprotective effects of aspirin. 25 This was born out by the results of 3 randomized, placebo-controlled trials of aspirin, respectively, at 75 mg/d, 324 mg/d, and 1420 mg/d, with each showing a similar cardioprotective impact approximately a 50% decrease in the incidence of MI and of vascular deaths Quite an impressive impact of an inexpensive therapy! Although these trials did not involve a direct comparison across aspirin doses, they suggested that a low dose was probably at least as effective as higher doses of aspirin, laying to rest an argument by some that if low-dose aspirin was good, then more would be better. 29 The value of a biomarker of drug effect was further emphasized by a chance discovery. When collecting serial samples from patients admitted with acute MI, we noticed remarkable interindividual variation in urinary TxM. At the time, streptokinase only was administered to patients with MI who presented within a few hours of the onset of chest pain, and by comparing patients on either side of this time window who otherwise had infarcts of comparable severity, we realized that administration of the thrombolytic was associated with incremental elevation of TxM. 30 Subsequently, we showed that a similar increment in TxM caused by tissue plasminogen activator could be abrogated by pretreatment with a single dose of aspirin 325 mg orally. 31 In studies of experimental MI in dogs, we could show that this reflected platelet activation secondary to clot disruption and that the extent and complications of the infarct could be limited by pharmacological suppression of TxA 2 or blockade of its action. 32,33 At the time, it was thought that thrombolytic drugs were actually platelet-inhibitory, as they are if used at sufficient concentrations in vitro. However, here were data consistent with drug efficacy being compromised by concomitant platelet activation in vivo. Cardioprotection From Low-Dose Aspirin: Clinical Trials Evidence that this was the case emerged with the completion of the International Study of Infarct Survival-2 clinical trial by Collins et al. 34 Here, the efficacy of streptokinase (a 1-hour intravenous infusion of 1.5 MU) or aspirin (1 month

5 178 Circulation Research January 4, 2013 of 160 mg/d of the enteric-coated drug) alone was compared with the combination of these and with placebo in > patients within a median of 5 hours from the onset of MI. Aspirin and streptokinase each significantly reduced vascular deaths but, importantly, their effects were additive, consistent with our hypothesis; mortality was 8% among patients allocated to both active agents vs 13.2% in the placebo group. Subsequently, many randomized trials of aspirin have been completed to address the question of its efficacy in cardioprotection. Perhaps, the best summary of this work has been provided by the overview analyses performed by the Antithrombotic Trialists Collaboration led by the Oxford Clinical Trials Services Unit. In summary, this work has confirmed that aspirin reduces the secondary incidence of MI, stroke, and important vascular events, and secondary analyses have supported the contention that this benefit holds true irrespective of the presence of comorbidities, such as hypertension or diabetes mellitus, and is uninfluenced by gender or age. 35,36 The magnitude of benefit and pretreatment of cardiovascular risk are directly related, although the risk of a serious gastrointestinal (GI) bleed from low-dose aspirin, approximately a doubling of placebo rates, is unrelated to cardiovascular risk. 37 Thus, the benfit risk ratio favors the use of aspirin in secondary prevention, although there has been some dispute as to how long such benefit may be sustained. 38 By contrast, the use of aspirin in primary prevention of cardiovascular disease remains much more debatable. The absolute benefit declines, but GI risk is unchanged. Thus, although aspirin prevents the primary incidence of MI, there is approximate numeric equivalence between infarcts prevented and serious GI bleeds caused and, unlike the setting of secondary prevention, an overall impact on vascular deaths was not observed. 37 However, it is likely that a much larger trial than those presently reported would be necessary to detect a reasonable estimate of the segregation of benefit from risk. Meantime, a systematic review of observational studies by Garcia Rodriguez suggests that the incidence of serious GI bleeds caused by aspirin increases quite steeply with age in those >60 years. 39 The outcome of an ongoing prospective trial of primary prevention by aspirin in the elderly 40 will be of interest. Additionally, there is evidence from overview analyses of the multiple, albeit rather small, placebo-controlled trials of aspirin suggesting efficacy in the prevention of deep venous thrombosis and pulmonary embolism in postoperative patients. 41 An impact of platelet inhibition would be consistent with the evidence from a model of flow-restricted initiation of venous thrombosis in mice in which monocyte adhesion to venous endothelium is followed by platelet-facilitated neutrophil recruitment with consequent tissue factor release and fibrin deposition. 42 Aspirin also has been shown to reduce the incidence of recurrent thromboembolism in patients who have discontinued anticoagulant therapy. 43,44 Overview analysis of these 2 small trials (402 and 822 patients, respectively) suggests that the overall incidences of both thromboembolism and important vascular events are each reduced by approximately one-third in aspirin-treated patients. 45 Although this is a more complex and inflammatory form of vascular occlusive disease than arterial thrombosis, platelet activation is integral to its pathogenesis. At-Swim-2 Birds: A Tale of 2 COXs The COX enzyme was cloned initially from sheep seminal vesicles in 1988 independently by Smith and Needleman, 46,47 and subsequent mutational analysis, particularly by the groups of Smith and Marnett, 48 turned out to have predicted with remarkable accuracy the structural topology of the enzyme when its crystallization was accomplished by Garavito et al. 49 A striking feature of the enzyme was a hydrophobic channel through which the lipid substrate gained access to the COX active site, buried deep within the protein. The target serine residue was positioned so that acetylation by aspirin prevented access of the substrate to the active site (Figure 4). NSAIDs differ from aspirin in that they are reversible, competitive inhibitors that occupy the active site, and this provided a basis for a drug drug interaction described. Two groups led by Simmons 50 and Herschman 51 simultaneously reported in 1992 an immediate early gene with PG synthetic capability, an enzyme now known colloquially as COX-2. Despite exhibiting remarkable conservation of sequence with COX-1, COX-2 differed in that it is highly transcriptionally regulated by inflammatory cytokines and mitogens, and thus it was assumed to be the dominant source of PGs relevant to the mediation of pain and inflammation and relevant to the role of PGs in the promotion of cancer. COX-1, by contrast, was attributed the role of making PGs that subserved housekeeping functions, such as the preservation of GI epithelial integrity and hemostasis. The GI bleeds that complicated administration of many NSAIDs, such as ibuprofen and naproxen, were attributed to inhibition of platelet COX-1 dependent Tx formation and of gastroduodenal COX-1 dependent PGE 2 and PGI 2, which afforded local cytoprotection. This prompted immediate interest in developing drugs that might target COX-2 specifically, thereby conserving efficacy while minimizing GI intolerance. Traditional screening approaches rapidly yielded molecules that preferentially inhibited COX-2. These could be categorized ex vivo by comparing suppression of serum TxB 2 with that of an assay of lipopolysaccharide-induced COX-2-dependent PGE 2 formation developed by Patrignani and Patrono. 52 Structural elucidation of COX-2 by Browner et al 53 revealed a striking similarity to COX-1 (Figure 5), but one difference was a side pocket in the hydrophobic channel that seemed to afford a basis for compounds that favored inhibition of COX- 2. Although the distinction between a constitutively expressed COX-1 and a highly regulated COX-2 was not as clear-cut as originally proposed, 2 NSAIDs purposefully developed for selective inhibition of COX-2, rofecoxib and lumiracoxib, did result in significantly less serious GI adverse events than older nonselective comparators in randomized trials. Although this is consistent with the original COX-2 hypothesis, the explanation is not that straightforward. COX-2 plays an important role in GI ulcer healing, which is delayed by its inhibition or deletion. 54,55 Moreover, deletion of COX-1 alone does not result in ulceration in mice; coincident inhibition of 1 form of COX with deletion of the other is necessary to result in GI lesions, 56 at least in mice. The rush to accept the GI safety benefits of COX-2 selective NSAIDs, driven aggressively (still, in the case of celecoxib) by direct-to-consumer advertising, became uncoupled from the growing evidence of a mechanism-based cardiovascular

6 Fitzgerald and FitzGerald Translational Success Story 179 Figure 4. Nonsteroidal anti-inflammatory drug (NSAID) interaction to undermine the antiplatelet effect of aspirin. Cyclooxygenase (COX) enzymes exist as dimers and the substrate arachidonic acid gains access to the catalytic site of 1 monomer via a hydrophobic channel, whereas the second monomer stabilizes the protein to facilitate the interaction. Acetylation of its target serine residue obstructs access of arachidonic acid (AA) to the catalytic site, blocking thromboxane A 2 (TxA 2 ) formation irreversibly in the case of platelet COX-1. Nonspecific traditional NSAIDs, such as ibuprofen, block COX-1 reversibly as competitive active site inhibitors and, if in previous occupancy of the active site, block access of aspirin to its target serine. Functionally, this would replace sustained maximal suppression of platelet TxA 2 throughout a typical dosing interval with transient maximal inhibition sufficient for platelet inhibition for a fraction of the dosing interval. Reproduced with permission from Catella-Lawson et al. 67 hazard. It has been estimated that rofecoxib and celecoxib caused > acute MIs and strokes and deaths in the first 5 years of their release in the United States market. 57 Furthermore, it seems that the incidence of such events may have been underreported. 58 This is the equivalent to the impact of smoking on cardiovascular risk. Such drugs disrupt the cardioprotective properties of COX-2 dependent PGI 2 formation by the heart and vasculature, leading to an increase in cardiovascular events, such as MI, stroke, and heart failure. This hazard has been documented in 8 placebo-controlled trials of 3 structurally distinct, purposefully developed selective inhibitors rofecoxib, celecoxib, and valdecoxib. 59,60 Less conclusive evidence from observational studies implicates some of the older drugs (Figure 6), such as diclofenac, which is selective for inhibition of COX-2 as celecoxib. 61 Aspirin Resistance The estimated incidence of this condition varies widely. However, this is not surprising, given its loose definition. Often based on a single measurement of the failure of aspirin to inhibit agonist-induced platelet activation ex vivo, the condition is reported to affect approximately one-third of patients using aspirin. This gave rise to an industry based on devices capable of single point-of-care estimates of platelet aggregability. 62 An alternative approach was the measurement of immunoreactive urinary 11-dehydro TxB 2. Post hoc analysis of this metabolite in samples collected during a clinical trial related the interquartile levels to the likelihood of clinical cardiovascular events on aspirin. 63 This has led to Food and Drug Administration (FDA) approval of an immunoassay for this purpose. However, serious concerns were raised as to how validly these biomarkers of aspirin response might predict the failure of clinical cardioprotection from aspirin. 64 As with all drugs, treatment failures occur in patients using low-dose aspirin. This may reflect a failure to ingest the drug frequently enough to sustain maximal inhibition of platelet Tx or low bioavailability from enteric-coated preparations. 65 We have shown that the relationship between suppression of Tx and Tx-dependent platelet activation is nonlinear; suppression of serum TxB 2 of >95% is necessary to inhibit platelet

7 180 Circulation Research January 4, 2013 Figure 5. Structural similarity of cyclooxygenase (COX)-1 with COX-2. Overlay of tertiary structures reflects a considerable conservation of structure despite marked differences in transcriptional regulation. *Cyclooxygenase catalytic site, deep within the protein. Peroxidase (POX) site designates that associated with the prostaglandin H synthase peroxidase reaction. Reproduced with permission from FitzGerald and Loll. 174 function, so that below a critical dose, aspirin may fail to prevent platelet activation in vivo. 66 Another possible form of resistance is the interaction of aspirin with the commonly coadministered NSAIDs, such as ibuprofen. Here, we showed that previous administration of single doses of the NSAID blocked access of low-dose aspirin to its target serine, undermining the constant inhibition of platelet function by aspirin during the typical dosing interval and replacing it with the transient functionally relevant inhibition of platelet COX-1 by ibuprofen (Figure 4). More impressively, when ibuprofen was administered 400 mg 3 times per day and aspirin 81 mg was administered chronically before the morning dose of ibuprofen, the interaction was maintained. 67 Thus, under chronic dosing conditions it was insufficient to change the order of drug administration to avoid such resistance. The development of direct assays of COX-1 acetylation in platelets will permit us to address this mechanism and its clinical implications directly. Aside from undermining the cardioprotection of aspirin, combined therapy also confers an interactive risk of GI bleeding, also a feature of the combination of aspirin with other antiplatelet drugs. We failed to observe an interaction between the platelet inhibitory effects of aspirin with the COX-2 selective NSAID, rofecoxib, reflecting the absence of COX-2 in mature human platelets. Patrignani showed an interaction similar to that with ibuprofen using a different nonselective NSAID, naproxen. 68 We recently have attempted to estimate the incidence of true pharmacological resistance to aspirin, such as might result from variants in genes in the COX-1/TxA 2 synthesis response pathway or in genes relevant to the uptake and disposition of aspirin. We sought a phenotype of resistance that was biochemically internally consistent, reflecting a failure of aspirin-dependent inhibition of serum TxB 2, urinary 11-dehydro TxB 2, and agonistinduced platelet aggregation, that was stable over time, and that was specific to aspirin and not shared with another antiplatelet drug (in this case, clopidogrel). Using this approach, we failed to find a single case of true resistance to aspirin in 400 volunteers. 69 Rather, we found that although apparent resistance was common at a single point in time, it was not stable over time and reflected the delayed and variable absorption of enteric-coated but not immediate-release aspirin. Furthermore, although we could clearly define volunteers displaying resistance using either platelet aggregation or serum TxB 2, highly accurate measurements of urinary 11-dehydro TxB 2 by mass spectrometry showing incomplete suppression by aspirin in the population failed to discriminate responders from nonresponders at the individual level. These observations question 3 aspects of common practice: (1) the use of acute point-of-care assessment of the platelet aggregation response to aspirin as a guide to clinical utility; (2) the use of the FDA-approved urinary 11-dehydro TxB 2 immunoassay for diagnosis of aspirin resistance; and (3) the use of enteric-coated forms of aspirin (which never have been shown to have improved efficacy of GI tolerance compared with cheaper immediate-release aspirin) for cardioprotection. Alternative Approaches to Disrupting the TxA 2 Synthesis/Response Pathway Downstream of COX-1, platelet PGH 2 is converted to TxA 2 by Tx synthase. Several companies developed Tx synthase inhibitors with a view to accumulation of its PGH 2 endoperoxide substrate that can be used by endothelial PGI 2 synthase to augment formation of PGI We obtained evidence consistent with PG endoperoxide rediversion in humans 71,72 and animal models of thrombosis and, in the latter, evidence that this contributed to platelet inhibition. 73 Unfortunately, PGH 2 substitutes for TxA 2 as an agonist of the Tx receptor (T prostanoid receptor [TP]) undermining the clinical use of this strategy, 73 and the approach was abandoned after several inhibitors reached human trials. Specific TP antagonists were developed at a time when the efficacy of low-dose aspirin for cardioprotection was beginning to be widely accepted. This unfortunate timing a more expensive way of making low-dose aspirin and the choice of challenging clinical end points (restenosis after angioplasty)

8 Fitzgerald and FitzGerald Translational Success Story 181 Figure 6. Characteristics of representative traditional nonsteroidal anti-inflammatory drugs (NSAIDs) and those drugs purposefully developed to inhibit selectively cyclooxygenase (COX)-2 [(pd)nsaids]. Reproduced with permission from Grosser T, Smyth E, FitzGerald, GA. Analgesic-anti-pyretic and anti-inflammatory agents and drugs employed in the treatment of gout. In: Brunton L, Chabner B, Knollman B, eds. Goodman & Gilman s The Pharmacological Basis of Therapeutics. McGraw-Hill; 2009: for proof of principle largely led to their abandonment. An exception was a recently completed large-scale phase 3 trial of a TP antagonist that failed to detect superiority over lowdose aspirin in the secondary prevention of stroke. 74 From a mechanistic point of view, it is difficult to see how this could have been successful. Superiority of this approach would have been dependent on (1) the biological importance of avoidance of the small reduction in PGI 2 biosynthesis observed with 100 mg/d aspirin, and (2) the incremental importance of blocking TP activation not only by Tx but also by unconventional ligands, such as the isoprostane products of lipid peroxidation. 75 However, although isoprostanes and other indices of lipid peroxidation are markedly elevated by reperfusion, they are minimally and transiently altered, if at all, in patients who entered into these studies, as shown, for example, in a study of stroke. 76 Furthermore, detection of the cardiovascular consequences of suppressing PGI 2 formation 59 by an average of 15% to 20% 22 would be a considerable challenge. Parenthetically, the similar GI bleeding rates attributable to the 2 treatments in this trial 74 imply that such events with low-dose aspirin are attributable largely, if not entirely, to platelet inhibition and that suppressing cytoprotective PGI 2 and PGE 2 is relevant to bleeding resulting only from higher doses of aspirin. One elegant approach prompted by these studies was the combination of Tx synthase inhibitors with TP antagonism to block Tx and PGH 2 -dependent receptor activation despite enhancing PGI 2 formation. Studies of animal models revealed the benefit of augmenting endogenous PGI 2 formation while suppressing Tx in preventing thrombosis by acute combinations of such drugs or by single agents possessing both activities. 73 Although some combination drugs have been evaluated in humans, they have not sustained both aspects of drug action comparatively through the dosing interval. The clinical utility of this approach, which remains theoretically attractive, has yet to be tested with appropriate drugs in humans, despite the convincing preclinical proof of principle. Aspirin and Cancer Gabriel Kune, a surgeon from Melbourne, was the first to notice in an observational study published in 1988 that aspirin use was underrepresented in cases of colon cancer compared with their matched controls after adjustment for multiple potentially confounding variables, including the presence of arthritis and cardiovascular disease. He declined to speculate on the mechanism, but he suggested that the prospect of chemoprevention should spur further investigation. 77 His report was followed by a series of confirmatory observational studies and the report of overexpression of COX-2 in colonic and other cancers, including a report that related the intensity of its expression to prognostic outcome. 78 Proof of principle seemed to be afforded when it was reported that crossing mice deficient in COX-2 into those

9 182 Circulation Research January 4, 2013 bearing the Apc Δ 716 mutation restrained the development of intestinal polyps, an impact replicated by treatment with a COX-2 selective inhibitor. 79 A complication in this story was that subsequently it was shown that crossing mice deficient in COX-1 into the same model had the same effect on polyposis. 80 However, products of COX-2 in tumors may have functional relevance, and we know that this enzyme is asymmetrically upregulated by mitogens. We have reported that deletion of epithelial COX-2 delays breast cancer tumor progression in mice by augmenting type 1 immune responses. 81 Despite these findings and the observational data of the impact of aspirin at low doses (which would mostly inhibit just COX-1) on colon cancer, placebo-controlled chemoprevention studies of COX-2 inhibitors proceeded mostly without an aspirin (or nonselective NSAID) arm in patients with the precancerous syndrome of familial polyposis coli. These studies established that both rofecoxib and celecoxib delayed polyp formation when compared with placebo. Ironically, it was precisely these studies that revealed an unambiguous signal of cardiovascular hazard from COX-2 selective NSAIDs. The results of the Adenomatous Polyp Prevention on Vioxx trial led to the withdrawal of rofecoxib, although celecoxib remains on the market despite similar evidence of a cardiovascular hazard. 59 However, its approval for use by patients with familial polyposis coli has now been withdrawn by the FDA. More recently, Rothwell et al have provided supportive evidence for the role of COX inhibition in cancer prevention. In their overview analyses of data, including long-term follow up from placebo-controlled trials of aspirin for cardioprevention, it appeared that aspirin use was associated with a reduced incidence and metastatic spread of and death from colon cancer and a variety of other cancers. 82,83 Some interesting findings of the impact of dose can be gleaned from these large datasets. Although there is a remarkable paucity of randomized comparisons of different doses of aspirin in cardioprotection, indirect comparisons are consistent with an inverse dose response relation. 35,36 This could be attributed to progressive inhibition of COX-2 dependent PGI 2 formation 22 with increasing doses of aspirin along with complete inhibition of platelet COX-1 derived TxA 2 at all doses. The same analysis shows no apparent dose dependency for the putative effect of aspirin on tumor prevention. One could go farther and attribute the apparent chemopreventative effect of aspirin to its inhibition of platelet function. Together with Pedersen, we reported in 1984 that aspirin inhibited platelet COX-1 in the presystemic circulation and that as one descended the dose response curve, the platelet inhibitory effects of aspirin were progressively restricted to that site of action. 84 Working with Charman at Sterling Research Laboratories, we calculated the dose and rate of delivery of aspirin that would optimally confine its action to the presystemic circulation by delivering aspirin continuously by GI intubation. 85 From these experimental data we developed a slowrelease matrix formulation that satisfied those requirements. 86 When compared with a conventional formulation of the same 75-mg oral dose, the matrix formulation not only suppressed platelet Tx completely but also left unaltered an index of stimulated systemic vascular PGI 2 formation, that is, the increment in urinary PGIM evoked by intravenous bradykinin in healthy volunteers. 87 This form of aspirin was used by Meade in the Thrombosis Prevention Study (TPT), a primary prevention study of aspirin in patients with demographic and biochemical features suggestive of elevated cardiovascular risk. 88 The matrix formulation of aspirin reduced the incidence of nonfatal ischemic heart disease compared with placebo in TPT, but unfortunately the trial lacked a conventional formulation of the same dose of aspirin as a comparator. Nevertheless, TPT contributed perhaps the largest amount of data of all the trials surveyed by Rothwell for cancer prevention, 82,83 and there seemed to be no additional impact in trials using even higher doses of conventional formulations. How might platelet inhibition influence cancer prognosis? There is considerable literature implicating metastatic spread via platelets, 89 and their role in the promotion of inflammation 90 and angiogenesis 91 also may be relevant. Based on these findings, it is evident that suppression by aspirin of any products of platelet activation, not just eicosanoids, may be relevant to cancer promotion. However, Halushka 92 has reported overexpression of TxS and the β isoform of TP in human bladder cancer along with elevated urinary TxB 2. Moreover, Honn 93 has implicated the platelet 12-lipoxygenase product, 12-hydroxy eicosatetraenoic acid, in carcinogenesis, although it is not suppressed by aspirin. Finally, it is possible that upregulation of tumor COX-2 may be regulated, in part, by the products of COX-1, perhaps explaining the similar impact of deleting or inhibiting either enzyme in Apc mice. Although it has long been known that eicosanoids can regulate expression of COX-2 in vitro, the importance of this mechanism in vivo has emerged with cell-selective deletions of the enzyme. Deletion of COX-2 in one cell type has been associated with upregulation of COX-2 in another, 94,95 suggesting removal of an endogenous restraint. Perhaps, products of COX-1 in platelets regulate, in part, the expression of COX-2 in tumors. There are considerable challenges to performing prospective, placebo-controlled, randomized trials of low-dose aspirin to assess its singular impact on tumor survival. What kind of evidence of benefit from cancer prevention will be sufficient to tip the benefit risk ratio in patients who have never had a cardiovascular event in favor of their chronically ingesting aspirin? Such calculations will depend on refinement of the estimates of the magnitude of the benefit in cancer prevention by further analyses of aspirin trials, strengthening of the evidence for plausibility, and perhaps obtaining evidence of adjunctive benefit in prospective trials of rare forms of accelerated cancer. In summary, we have a structural basis for the mechanism of aspirin action and that of other NSAIDs with validated, but indirect, quantitative assays of their inhibitory effect ex vivo and in vivo. The structural features of COX-1 and COX-2 are remarkably similar. Further, Smith has established that whereas both COX-1 and COX-2 exist as dimers of apparently identical monomers in the resting enzymes, they differ during catalysis when a nonfunctioning subunit provides structural support enabling its partner monomer to catalyze the COX reaction. 96 Furthermore, binding of 1 monomer with a nonsubstrate fatty acid can regulate the

10 Fitzgerald and FitzGerald Translational Success Story 183 catalytic reactivity of its partner and its response to pharmacological inhibitors. 97 A clear net cardioprotective benefit for aspirin has been established, and this outweighs the risk of serious GI adverse effects sufficiently to justify its use in the secondary prevention of MI, stroke, and serious vascular events. Although there are several potential causes of clinical failure of a treatment that blocks only one pathway of platelet activation, the incidence of true pharmacological resistance to aspirin, if it exists, is extremely low. Patients using aspirin for secondary prevention may derive an additional benefit from tumor delay or prevention. By contrast, the net primary cardioprotective value of aspirin remains arguable approximately 1 serious nonfatal vascular event prevented for every serious GI bleed caused. An incremental tumor-preventative effect might tip the balance in favor of chronic aspirin treatment in such patients. However, to make such calculations, we need more precise estimates of the magnitude of this putative benefit and the extent to which this might favor particular subgroups of patients and perhaps greater evidence of mechanistic plausibility. Unlike the case of cardioprotection, the clinical evidence for a cancer benefit is likely to fall short of prospective, placebo-controlled clinical trials. From Bench to Bedside and Back to the Bench Emergence of P2Y12 Adenosine Diphosphate Receptor Antagonists The experience with clopidogrel (Plavix), which for many years was the world s second-best selling medicine, with global sales peaking at $9.6 billion in 2010, contradicts current paradigms of drug development. The molecular target of clopidogrel was not identified until after the drug was marketed. Dosing was determined based on clinical experience and changing therapeutic strategies. There were marked variances in drug response, with the emergence of nonresponders as a driver in the development of dosing strategies and successor drugs. A key issue was its safety and effectiveness when combined with other antithrombotics, such as aspirin, with the clinical outcomes of trials encouraging more intensive platelet inhibition. The favorable clinical experience motivated a more fundamental pharmacological analysis, which identified the target and the metabolic disposition of clopidogrel, allowing a logical refinement of its usage. The story of clopidogrel exemplifies the reality of translational medicine: observations from the clinical use of clopidogrel triggered a reevaluation of the relevant biology that spurred further basic research, clinical Figure 7. Signaling pathways in platelets for adenosine diphosphate (ADP) and site of action of ADP antagonists. ADP activates several distinct ADP receptors and signaling. Also shown is the impact on vasodilator-stimulated phosphoprotein (VASP) mediated through P2Y12 activation. The arrows indicate key signaling events.

11 184 Circulation Research January 4, 2013 Oxidation CYP2C19 Clopidogrel Active metabolite Carboxyl Esterase Inactive metabolites P2Y12 P2Y12 P2Y12 P2Y12 Figure 8. Schematic of the absorption and metabolism of clopidogrel. Oral clopidogrel is predominantly metabolized by liver carboxylesterases to inactive metabolites. Lesser amounts are metabolized by liver cytochrome P450 enzymes, including CYP2C19, which forms the active metabolite. investigation, and the evolution of routine medical practice in an iterative cycle a case of bedside to bench to bedside. Here, we discuss some of the fundamental science and findings from experimental studies in humans that have translated into better clinical use of clopidogrel. These include basic pharmacology, analysis of the genetic variation in some of the proteins involved in its metabolism and action, functional testing of responses, and both small and large clinical trials. Strikingly, these findings influenced therapeutic practice more rapidly than is usually the case and the outcomes of these translational studies have fostered the development of alternatives to clopidogrel. Clopidogrel Clopidogrel inhibits the part of the platelet response induced by adenosine diphosphate (ADP), which is released from the dense granules of activated platelets. Released ADP provides a critical feedback loop within a circuit that amplifies the platelet response to weak agonists, such as epinephrine. ADP is also derived from other cells, including erythrocytes and endothelial cells at the site of vascular injury. The overall effect is to promote the development and stability of nascent thrombus. Preclinical Development: the Complexity of Clopidogrel Clopidogrel (then designated PCR 4099) was first reported in 1987 to be an inhibitor of ADP-induced platelet activation. 98 Notably, the molecular target for clopidogrel (and ticlopidine, collectively referred to as thienopyridines) was unknown at the time and could be inferred only from its pharmacological activity. There are 3 receptors for ADP on platelets: a P2X-type ion channel linked receptor and 2 P2Y-type G-protein-coupled receptors P2Y1 and P2Y12 (Figure 7). Because these receptors had not been cloned, they were characterized on the basis of their downstream coupling to activation of phospholipase C and inhibition of adenylyl cyclase as the P2T PLC and P2T AC receptors and were deduced to couple to G αq and G αi, respectively. Other potent platelet agonists, such as thrombin and TxA 2, activate G-protein-coupled receptors and downstream signaling pathways that are distinct from the ADP pathways, an important point that provides a rationale for combination therapy. 99 The 2 thienopyridines had similar platelet effects, although ticlopidine had a worse adverse effect profile (severe bone marrow toxicity and skin rashes) and was replaced by clopidogrel (despite its efficacy in the secondary prevention of stroke) in clinical development. Clopidogrel blocked the adenylate cyclase linked ADP response, which was sufficient to limit ADP-induced platelet aggregation and fibrinogen binding. However, other ADP effects on platelets, such as shape change, were not inhibited. Platelet activation by other agonists was affected slightly, 100 explained by the inhibition of the secondary wave of platelet aggregation in response to ADP released on platelet activation. Clopidogrel bound to platelet ADP receptors irreversibly, 101 which was to have important clinical consequences. Another important discovery during preclinical development was that clopidogrel is a prodrug, with hepatic metabolism required to form the highly reactive and short-lived compound responsible for its activity (Figure 8). 102 The requirement for cytochrome P450 family of enzymes for metabolism was established in Pharmacokinetic characterization showed that clopidogrel was rapidly metabolized, with little parent compound detectable 2 hours after dosing. Radiolabeled compound is excreted equally in urine and feces, with a small proportion (2%) covalently attached to platelets that declined over time, with a half-life of 7 days. 103 Clinical Development One of the most influential clinical trials with clopidogrel was the Clopidogrel Versus Aspirin in Patients at Risk of Ischemic Events (CAPRIE) study (Table), reported in This study of almost patients compared daily dosing of 75 mg clopidogrel with 325 mg/d aspirin for the secondary prevention of cardiovascular events. This dose was chosen based on small dose-finding pharmacodynamic studies showing that clopidogrel resulted in an effect equivalent to that of ticlopidine. 104 CAPRIE demonstrated a relative risk reduction of 8.7% for clopidogrel compared with aspirin, a small effect that nevertheless was statistically significant, and a favorable adverse effect profile. Although the benefit over aspirin was small, the FDA gave a qualified approval for clopidogrel for the secondary prevention of cardiovascular disease. 103 Evolution of the Clinical Use of Clopidogrel After its initial licensing for secondary prevention of cardiovascular events, subsequent clinical trials explored, in an iterative way, different dosing strategies and treatment indications for clopidogrel. The application of clopidogrel was guided by experimental studies demonstrating a critical role for platelets