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Journal of Thrombosis and Haemostasis, 11: 1627–1639

DOI: 10.1111/jth.12318

REVIEW ARTICLE

Pharmacogenomics in cardiovascular disease: focus on aspirin and ADP receptor antagonists € T Z , * M . L O R D K I P A N I D Z E† and E . L . G R O V E * M . W UR *Department of Cardiology, Aarhus University Hospital, Aarhus, Denmark; and †Centre for Cardiovascular Sciences, Institute of Biomedical Research, College of Medical and Dental Sciences, University of Birmingham, Birmingham, UK

To cite this article: W€ urtz M, Lordkipanidze M, Grove EL. Pharmacogenomics in cardiovascular disease: focus on aspirin and ADP receptor antagonists. J Thromb Haemost 2013; 11: 1627–39.

Summary. Antiplatelet agents like aspirin and adenosine diphosphate receptor antagonists are effective in reducing recurrent ischemic events. Considerable inter-individual variability in the platelet inhibition obtained with these drugs has initiated a search for explanatory mechanisms and ways to improve treatment. In recent years, numerous genetic polymorphisms have been linked with reduced platelet inhibition and lack of clinical efficacy of antiplatelet drugs, particularly clopidogrel and aspirin. Consequently, attempts to adjust antiplatelet treatment according to genotype have been made, but the clinical benefit has been modest in studies performed so far. The progress in genome science over the last decade and the declining cost of sequencing technologies hold the promise of enabling genetically tailored antiplatelet therapy. However, more evidence is needed to clarify which polymorphisms may serve as targets to improve treatment. The present review outlines the panel of polymorphisms affecting the benefit of aspirin and adenosine diphosphate receptor antagonists, including novel and ongoing studies evaluating whether genotyping may be beneficial in tailoring antiplatelet therapy. Keywords: ADP receptor antagonists; aspirin; cardiovascular disease; clopidogrel; pharmacogenomics.

Introduction Antiplatelet agents like aspirin and adenosine diphosphate (ADP) receptor antagonists markedly reduce cardiovascuCorrespondence: Erik L. Grove, Department of Cardiology, Aarhus University Hospital, Skejby, Brendstrupgaardsvej 100, 8200 Aarhus, Denmark. Tel.: +45 7845 2029; fax: +45 7845 2260. E-mail: [email protected] Received 21 March 2013 Manuscript handled by: M. Cattaneo Final decision: F. R. Rosendaal, 6 June 2013 © 2013 International Society on Thrombosis and Haemostasis

lar morbidity and mortality in patients with preexisting cardiovascular disease. In particular, antiplatelet drugs are widely used by interventional cardiologists because they remain essential in maintaining stent patency and prevent thrombus formation. The burden of these drugs on worldwide healthcare expenses is indisputable: aspirin is the most sold drug worldwide and clopidogrel remains the second most prescribed drug globally although it is being increasingly substituted with the newer and more potent ADP receptor antagonists, prasugrel or ticagrelor. Despite their proven clinical effect, a considerable number of patients respond inadequately to aspirin, ADP receptor antagonists or both. The efficacy of antiplatelet drugs can be defined by platelet function testing or the ability of the drugs to prevent cardiovascular events [1]. Numerous platelet function tests are available, but their clinical use is hampered by considerable inter-assay variability and the lack of standardized cut-off values [2]. Treatment failure can be explained by various mechanisms, which may be broadly categorized into biological, clinical, pharmacodynamic and genetic mechanisms [3]. With regard to genetic mechanisms, an estimated 30% of the variability in platelet aggregation seen in the general population is ascribed to heritable factors [4]. Genetic variation has long been determined by a targeted approach using well-known variants in candidate genes hypothesized to influence platelet response to antiplatelet agents. In recent years, pharmacogenetics (i.e. the study of single genes and their effect) has gradually evolved into pharmacogenomics (i.e. the study of all of a person’s genes, including their effects and interactions) [5]. This enables unbiased screening for common variants across the entire genome with genome-wide association studies. The large amounts of genetic information derived from contemporary genomics hold the promise of improved treatment options, but at the same time they constitute a translational barrier that needs to be surmounted before personal genomics and personalized medicine can be integrated into the management of cardiovascular disease. The present review outlines the pharmacogenomics of aspirin and ADP receptor antagonists and discusses present and future challenges in the field.

1628 M. W€ urtz et al Table 1 Pharmacology of aspirin and ADP receptor antagonists Agent

Mechanism of action

Metabolism

Reversibility

Onset of effect

Dosing

Aspirin

Cyclooxygenase-1 inhibition

Prodrug

Irreversible

30–60 min

Clopidogrel

Thienopyridine, ADP receptor antagonist

Prodrug

Irreversible

2–4 h

Prasugrel

Thienopyridine, ADP receptor antagonist

Prodrug

Irreversible

30 min

Ticagrelor

Non-thienopyridine, allosteric ADP receptor antagonist

Active drug

Reversible

30 min

LD: 300–500 mg MD: 75–100 mg day LD: 300–600 mg MD: 75 mg day 1 LD: 60 mg MD: 10 mg day 1* LD: 180 mg MD: 90 mg b.i.d.

1

ADP, adenosine diphosphate; b.i.d., bis in die (twice daily); LD, loading dose; MD, maintenance dose. *Dose reduction to 5 mg day 1 in patients with low body weight (< 60 kg) and high age (> 75 years).

Aspirin: pharmacology and clinical use Aspirin irreversibly acetylates platelet cyclooxygenase (COX)-1, thereby inhibiting the conversion of arachidonic acid into thromboxane (TX) A2, a potent vasoconstrictor and platelet activator (Table 1). Platelets are particularly sensitive to the effect of aspirin, being anucleate and thus incapable of regenerating significant levels of active COX1 once it has been inactivated. This enables aspirin to provide potent platelet inhibition at daily doses as low as 30 mg [6]. Aspirin significantly reduces the risk of recurrent arterial thrombosis and a dose of 75–81 mg daily is recommended across the entire spectrum of cardiovascular diseases, from stable coronary artery disease (CAD) to acute coronary syndromes (ACS). Higher doses are still being used, but they confer an increased bleeding risk while providing no incremental protection from cardiovascular events [6]. ADP receptor antagonists: pharmacology and clinical use Four different ADP receptor antagonists are currently available for clinical use: ticlopidine, clopidogrel, prasugrel and ticagrelor. Clopidogrel, now available in its generic form, is a second-generation thienopyridine and is the most thoroughly investigated ADP receptor antagonist in terms of clinical efficacy and safety, but also in terms of response variability and genetic reasons for this. It has almost completely replaced ticlopidine, because it can be given once daily and has a much better safety profile. Clopidogrel is a prodrug and requires hepatic bioactivation (Table 1). Approximately 85% is hydrolyzed into inactive metabolites, leaving only 15% of administered clopidogrel to form the active clopidogrel thiol metabolite [7]. This is mediated by a two-step oxidative process regulated by the cytochrome P450 (CYP) system (Fig. 1). The conversion of clopidogrel to 2-oxo-clopidogrel is mediated by CYP1A2, CYP2B6 and CYP2C19, which contributes most significantly to the oxidative process. The subsequent conversion of 2-oxo-clopidogrel to the active thiol metabolite is mediated by CYP2B6, CYP2C9, CYP2C19 and, most importantly, CYP3A4 (Fig. 1). Ultimately,

only 2% ends up occupying the purinergic P2Y12 receptor, thereby inhibiting ADP-mediated platelet aggregation [8]. Like clopidogrel, prasugrel is a prodrug belonging to the thienopyridine family with its inherent need for bioactivation. Prasugrel is activated in a one-step oxidative process and, unlike clopidogrel, none of the drug is shunted to an inactive pathway. Unlike the thienopyridines, the ATP analogue ticagrelor inhibits the P2Y12 receptor reversibly and does not require hepatic bioactivation (Table 1). Prasugrel and ticagrelor are more potent platelet function inhibitors than clopidogrel and are being widely used in combination with aspirin in the setting of ACS. Pharmacogenomics of aspirin Numerous candidate genes have been investigated to establish potential associations with the antiplatelet effect of aspirin. Only a few of these are related to the COX-1 dependent pathway of platelet activation. The panel of investigated genes includes those encoding platelet surface receptors (the fibrinogen receptor, collagen receptors, ADP receptors, etc.) and key enzymes in the hemostatic system (COX enzymes, coagulation factors, etc.) (Table 2) [9]. In particular, the influence of the IIIa subunit of the dimeric glycoprotein (GP) IIb/IIIa fibrinogen receptor has been amply documented. Attention has centered on the human platelet antigen system-1a isoform of GP IIIa, often referred to as the Platelet Antigen (PlA). Initially, the T1565?C polymorphism (PlA1/A2; rs5918) was linked with the occurrence of MI [10,11], and subsequent in vitro studies pointed towards a reduced aspirin response in PlA2 carriers, both healthy volunteers [12] and patients with cardiovascular disease [13]. Although these findings have been contrasted [14], a systematic review confirmed that the PlA2 allele does indeed impart a less responsive phenotype, which seems most pronounced in healthy individuals [15]. Given that COX-1 is the specific target of aspirin, it is plausible that genetic variation in the PTGS1 gene encoding this enzyme may affect aspirin efficacy. Halushka et al. [16] demonstrated in aspirin-treated healthy individuals © 2013 International Society on Thrombosis and Haemostasis

Pharmacogenomics in cardiovascular disease 1629

Intestinal cell

P-GP

Clopidogrel Prasugrel Ticagrelor

Ticagrelor

Liver Clopidogrel 2C19 2B6 1A2

2-oxoclopidogrel 2C19 2C9 2B6 3A4/5

Prasugrel 2C19 2C9 2B6 3A4/5

Active metabolite

Active metabolite

P2Y12 Platelet

Fig. 1. A schematic presentation of the absorption, metabolism and anti-aggregatory effect of clopidogrel, prasugrel and ticagrelor. Clopidogrel is activated by a two-step oxidative process in the liver, whereas only one oxidative step is needed for the activation of prasugrel. The most important cytochrome P450 enzymes mediating the hepatic bioactivation of clopidogrel and prasugrel are depicted. Ticagrelor does not require hepatic bioactivation. P-GP, P-glycoprotein (multidrug resistance protein-1).

that two polymorphisms, A-842?G (rs10306114) and C50?T (rs3842787), are in complete linkage disequilibrium and that arachidonic acid-induced platelet aggregation varies according to A-842?G/C50?T haplotype. This may be true in cardiovascular patients as well [17], but contradictory studies exist and evidence is sparse. Although low-dose aspirin inhibits the COX-2 pathway only marginally, studies have also investigated the influence of the G-765?C polymorphism (rs20417) in the COX-2 gene (PTGS2), yielding inconsistent results [18,19]. TXA2 production is highly dependent on COX-1 activity, which in turn is reduced by aspirin. Polymorphisms in the TX receptor (TP) gene (TBXA2R) have been shown to influence platelet response to TXA2 [20]. Interestingly, certain genetic variants may also reduce the ability of aspirin to inhibit platelet function. In a healthy Asian population, individuals homozygous for 924T of © 2013 International Society on Thrombosis and Haemostasis

the TXA2 receptor C924?T polymorphism (rs4523) showed reduced sensitivity to aspirin [21], as did those homozygous for the minor allele of the C795?T polymorphism (rs1131882) in Caucasian healthy volunteers [22] and in patients with diabetes and CAD [23]. P2Y1 and P2Y12 are purinergic receptors mediating platelet activation upon stimulation by ADP. Addition of aspirin to ADP receptor antagonists synergistically inhibits platelet aggregation, and a crosstalk between ADPand TX-dependent platelet activation exists. Accordingly, two polymorphisms in the P2Y1 gene (P2RY1), C893?T (rs1065776) [24] and A1622?G (rs701265) [14], have been proposed to influence aspirin efficacy in platelet aggregation studies. Moreover, various polymorphisms in the P2Y12 gene (P2RY12) have been described, some of which determine two haplotypes (H1/H2; constituted by rs10935838, rs2046934, rs5853517, rs6809699) of particular interest. The H2 haplotype may determine clopidogrel efficacy but does not seem to influence aspirin efficacy [25]. Collagen is a key platelet activator, although less specific than arachidonic acid for the action of aspirin. Collagen functions through specific collagen receptors, namely GP Ia/IIa and GP VI. The T13254?C polymorphism (rs1613662) in the GP VI gene (GP6) was associated with increased platelet aggregation in one study [26] but not in a more recent one including aspirin-treated cardiovascular patients [27]. In contrast, when investigating the C807?T polymorphism (rs1126643) in the GP Ia gene (ITGA2), Su et al. [28] reported the T allele to almost quadruple the risk of increased platelet aggregation during aspirin treatment in 200 atherosclerotic patients. The patients were Chinese and the results have not been replicated in Caucasian cohorts. A trimeric complex assembled from GP Ib, IX and V forms the receptor through which von Willebrand factor exerts its prothrombotic effects. The a-subunit of GP Ib contains the binding site for von Willebrand factor and is highly polymorphic. The C-5?T polymorphism (Kozak sequence, rs2243093) was linked with high on-aspirin platelet reactivity in some studies [29,30] but current evidence is inconclusive. In addition to the aforementioned polymorphisms, genetic variants of other hemostatic proteins have been investigated, including platelet-activating factor acetylhydrolase, platelet endothelial aggregation receptor-1 (PEAR1, rs12041331; [31]) and coagulation factor (F) XIII. The G994?T polymorphism (rs1051931) of the latter was recently shown in Caucasian diabetics to be significantly associated with a prolonged closure time as assessed by the PFA-100 assay [23]. Finally, there are data suggesting that aspirin not only influences platelet activation and aggregation, but also influences coagulation. This is due in part to the fact that aspirin acetylates not only COX but also other blood proteins, most importantly fibrinogen, although higher aspirin doses are

1630 M. W€ urtz et al Table 2 Polymorphisms reported to interact with the antiplatelet effect of aspirin

Gene

Gene product

Nucleotide change or alternative name*

ITGB3 PTGS1 PTGS1 PTGS2 TBXA2R TBXA2R P2RY1 P2RY1 P2RY12

GP IIIa COX-1 COX-1 COX-2 TXA2 receptor TXA2 receptor P2Y1 P2Y1 P2Y12

PlA1/A2 (T1565?C) A-842?G/C50?T C22?T G-765?C C924?T C795?T C893?T A1622?G H2 haplotype

GP6 ITGA2 GP1BA PLA2G7 F13A1 PEAR1

GP VI GP Ia GP Iba Platelet-activating factor acetylhydrolase Coagulation factor XIII Platelet endothelial aggregation receptor-1

T13254?C C807?T C-5?T Kozak G994?T G34?T A?G

Ref SNP ID†

Key studies

rs5918 rs10306114/rs3842787 rs1236913 rs20417 rs4523 rs1131882 rs1065776 rs701265 H1/H2; constituted by rs10935838, rs2046934, rs5853517, rs6809699 rs1613662 rs1126643 rs2243093 rs1051931 rs5985 rs12041331

[12,13,15] [16,17] [18,19] [21] [22,23] [24] [14,24,25] [25]

[26,27] [28] [13,29,30] [21,23] [35,36] [31]

Summary of evidence: Accumulating evidence supports a genetic basis, particularly the PlA1/A2 polymorphism in ITGB3, for a reduced antiplatelet effect of aspirin. Importantly, the results are inconsistent and largely derived from pharmacodynamics studies. Some polymorphisms have only been sparsely explored, including those in PLA2G7 and PEAR1, and for most polymorphisms there is a lack of studies with clinical endpoints. Moreover, a reduced aspirin response is a complex phenotypic trait, and the lack of well-established cut-off values and low concordance between different platelet function tests makes it difficult to directly compare results across studies. At present, genotyping is not a recommendable clinical strategy to assess the antiplatelet effect of aspirin. *As most often termed in the existing literature. †According to NCBI’s SNP database, dbSNP: http://www.ncbi.nlm.nih.gov/projects/SNP/ (accessed 17 April 2013). COX, cyclooxygenase; GP, glycoprotein; SNP, single nucleotide polymorphism; TXA2, thromboxane A2; UDP, uridine diphosphate; UGT, uridine diphosphateglucuronosyltransferase.

required for this [32,33]. As a consequence, the fibrin clots formed in the presence of aspirin have a looser structure, which facilitates the fibrinolytic process [34]. Among the pharmacogenetic variants of importance in this setting, the G34?T polymorphism (rs5985) in the coagulation FXIII A-subunit has been reported to affect cardiovascular protection by aspirin [35], and a recent pharmacodynamic study indicated that a reduced antiplatelet effect of aspirin was more frequent in T allele carriers [36]. Overall, the inconsistent results of numerous pharmacogenetic studies conducted through the last decades allow for only very weak conclusions to be made. A systematic review concluded that among a multitude of polymorphisms, only the PlA1/A2 polymorphism was associated with aspirin efficacy [15], and this association was less pronounced in cardiovascular patients than in healthy individuals. Therefore, in the context of aspirin, the current level of evidence does not justify the use of genotyping for clinical purposes.

Absorption

Intestinal absorption of clopidogrel depends on the membrane-associated drug efflux transporter P-glycoprotein (multidrug resistance protein-1). This key protein limits transport of clopidogrel across the intestinal wall and is encoded by ABCB1. A reduced peak concentration of clopidogrel and its active metabolite was found in 3435T homozygotes (rs1045642) compared with carriers of the wild-type C allele [37]. This may translate into lower platelet inhibition [38] and increased cardiovascular risk [38,39], although evidence is conflicting [40–43]. In particular, results of the large TRITON-TIMI 38 [38] and PLATO [41] genetic sub-studies are directly opposite and suggest that TT homozygosity (TRITON-TIMI 38) and CC homozygosity (PLATO) carry the increased cardiovascular risk. In light of these results, ABCB1 genotyping should not be performed in routine clinical practice. Metabolism

Pharmacogenomics of clopidogrel It is well established that platelet inhibition by clopidogrel depends to a large extent on the amount of active metabolite. Therefore, recent efforts to understand the pharmacogenomics of clopidogrel have focused on the panel of genes involved in its absorption and metabolism (Table 3).

The cytochrome P450 family A variety of CYP enzymes are involved in clopidogrel metabolism, including CYP2C19, CYP2C9, CYP3A4, CYP3A5, CYP1A2, CYP2B6, etc. The CYP2C19 enzyme, which is involved in both oxidative steps of clopidogrel metabolism, has attracted most attention and yielded the most consistent results. Different allelic variants of CYP2C19 have been © 2013 International Society on Thrombosis and Haemostasis

Pharmacogenomics in cardiovascular disease 1631 Table 3 Polymorphisms reported to interact with the antiplatelet effect of clopidogrel Key studies

Gene

Gene product

Nucleotide change or alternative name*

ABCB1 CYP2C19‡ CYP2C19 PON1 CES1 P2RY12

P-glycoprotein Cytochrome P450 2C19 Cytochrome P450 2C19 Paraoxonase-1 Carboxylesterase-1 P2Y12

C3435?T *2 *17 Q192?R G143?E H2 haplotype

ITGA2 ITGB3

GP Ia GP IIIa

C807?T T1565?C

Ref SNP ID† rs1045642 rs4244285 rs12248560 rs662 rs71647871 H1/H2; constituted by rs10935838, rs2046934, rs5853517, rs6809699 rs1126643 rs5918

Pharmacokinetic/ pharmacodynamic

Clinical

[37,38,40,43] [48–52,54,55] [67–69] [42,43,71–75] [77,78] [51,79–81]

[38–42] [39,49,54,57–63] [66,69,70] [42,72,73,75,76] [77] [39]

[82,83] [84]

[39] [39]

Summary of evidence: Consistent data associate the antiplatelet effect of clopidogrel with loss-of-function polymorphisms, namely in genes involved in clopidogrel metabolism. The *2 variant of CYP2C19 has been shown to reduce clopidogrel response in numerous pharmacokinetic and pharmacodynamic studies, thereby possibly increasing the risk of adverse events, particularly stent thrombosis. Studies suggest that increasing clopidogrel dose or switching to different antiplatelet drugs can increase platelet inhibition in *2 carriers, but any clinical benefit of doing so remains to be proven. The *17 gain-of-function variant of CYP2C19 may increase bleeding risk, but results are inconsistent and have not been replicated in prospective clinical studies. Routine genotyping to identify patients with inadequate clopidogrel response is currently not advisable, but ongoing clinical trials powered for clinical endpoints will determine if genotyping may be justified in selected clinical settings. *As most often termed in the existing literature. †According to NCBI’s SNP database, dbSNP: http://www.ncbi.nlm.nih.gov/projects/SNP/ (accessed 17 April 2013). ‡Various CYP2C19 variants may be associated with the pharmacodynamic and clinical effect of clopidogrel. Only the *2 and *17 variants are detailed herein, while others are briefly mentioned in the main text. For further details, see Table S3 in Verschuren et al. [46]. ADP, adenosine diphosphate; GP, glycoprotein.

investigated, including *1 (wild-type allele), *4 and *17, but the *2 and *3 loss-of-function alleles have been particularly scrutinized [44]. In essence, a number of allelic CYP variants are associated with a reduced response to clopidogrel (loss-of-function), including CYP2C19*2 (rs4244285), CYP2C19*3 (rs4986893), CYP2C19*4 (rs28399504), CYP2C19*5 (rs56337013), CYP2C9*2 (rs1799853), CYP2C9*3 (rs1057910), CYP2B6*6 (rs3745274), CYP2B6*9 (G516? T), CYP3A4*1B (rs2740574), CYP3A4*3 (rs4986910) and CYP3A5*3 (rs776746). Another group of variants is associated with a normal response, including CYP2C19*1 (wild-type, hence no NCBI dbSNP identifier), CYP2C9*1 (wild-type) and CYP3A5*1 (wild-type). Finally, one allelic variant is associated with an enhanced response (gain-offunction): CYP2C19*17 (rs1248560) [45,46]. Genetic variants of other enzymes involved in clopidogrel metabolism, paraoxonase-1 (PON-1) and carboxylesterase 1 (CES-1), may be linked with clopidogrel efficacy. In the following, the CYP2C19*2 loss-of-function and CYP2C19*17 gainof-function alleles as well as PON1 and CES1 polymorphisms are emphasized. CYP2C19*2: pharmacokinetics and pharmacodynamics The CYP2C19*2 allele is carried by almost one-third of Western Europeans and half of East Asians. This common genetic variant was recently identified as the most prominent genetic contributor to clopidogrel response variability [40,44]. The abundance of pharmacodynamic studies supporting this finding may reflect that plasma exposure to the active clopidogrel metabolite is reduced © 2013 International Society on Thrombosis and Haemostasis

in *2 carriers as suggested in pharmacokinetic studies [47–49]. The first platelet aggregation study to document an association included 28 healthy volunteers, of whom 20 were wild-type homozygotes (*1/*1) and eight were carriers of the loss-of-function allele (*1/*2). ADPinduced platelet aggregation following clopidogrel 75 mg treatment, but not at baseline, was increased in *2 carriers [50]. These findings were replicated in larger in vitro studies on healthy volunteers [48,49] and at-risk cardiovascular patients [51–54]. Brandt et al. [48] showed in healthy volunteers receiving clopidogrel 300 mg that *2 carriage was significantly associated with reduced pharmacokinetic and pharmacodynamic responses. Furthermore, in 60 patients undergoing coronary percutaneous coronary intervention (PCI), Gladding et al. [51] confirmed the reduced effect in *2 carriers and suggested that doubling loading and maintenance doses may yield particular benefit in these patients compared with wild-type homozygotes. However, in a recent prospective study including unstable patients undergoing PCI, doubling the maintenance dose was able to overcome a reduced clopidogrel response in only 44% of those initially categorized as non-responders [55]. CYP2C19*2: clinical outcome In March 2010, the US Food and Drug Administration issued a boxed warning on the label for clopidogrel (Plavixâ, Bristol-Myers Squibb/Sanofi Pharmaceuticals Partnership, Bridgewater, NJ, USA) highlighting that patients with loss-of-function CYP2C19 genotypes do not derive optimal platelet inhibition from clopidogrel and may be at increased risk of

1632 M. W€ urtz et al

thrombotic events [56]. This warning was based on large meta-analyses unequivocally confirming that the CYP2C19 genotype is independently associated with adverse cardiovascular events [57,58]. The warning was accompanied by the approval of the INFINITIâ (Autogenomics, Inc., Vista, CA, USA) CYP2C19 genotyping assay intended for the detection of specific loss-of-function CYP2C19 variants (*2, *3 and *17). Various studies have evaluated clinical outcome in patients at low (stable CAD) and high (ACS) cardiovascular risk. Sibbing et al. [59] genotyped 2485 consecutive patients undergoing PCI (one-third with ACS) after clopidogrel loading and showed an increased 30-day risk of stent thrombosis in *2 carriers, particularly high in *2 homozygotes. The association between *2 carriage and stent thrombosis has been documented in other studies [49,60,61], with the tripled risk observed in the TRITONTIMI 38 genetic sub-study serving as the most striking evidence [49]. Another study even reported the *2 allelic variant to carry a 6-fold 1-year risk of stent thrombosis and a 3.7-fold risk of the combined primary endpoint of death, myocardial infarction and urgent revascularization [60]. Data from a French registry including 2208 patients with acute myocardial infarction confirmed that *2 homozygosity is linked with a reduced clinical effect of clopidogrel. In contrast, heterozygosity (*1/*2) did not reduce clopidogrel efficacy compared with homozygosity for the wild-type allele (*1/*1) [39]. This suggests a gene dosage effect (i.e. a relationship between phenotype and the number of variant alleles), which corresponds with recent data showing that reduced clopidogrel response in *2 heterozygotes, but not *2 homozygotes, can be compensated for by increasing clopidogrel doses [54]. In this pharmacodynamic study by Mega et al. [54], stable CAD *2 carriers were subjected to four 14-day periods of clopidogrel treatment with a 75 mg increase between each period, reaching a maximum of 300 mg daily. Only in *2 homozygotes was residual platelet reactivity sustained despite dose increments. However, the proposed gene dosage effect contrasts with some previous reports. Even more contrasting are the results of a recent study including data from two randomized trials comparing clopidogrel with placebo: the Clopidogrel in Unstable Angina to Prevent Recurrent Events (CURE) trial (ACS patients) and the Atrial Fibrillation Clopidogrel Trial with Irbesartan for Prevention of Vascular Events (ACTIVE) A trial (atrial fibrillation patients) [62]. In both cohorts, clopidogrel sustained its clinical benefit irrespective of CYP2C19*2 carrier status. Similarly, a genetic sub-study of the Clopidogrel for High Atherothrombotic Risk and Ischemic Stabilization, Management, and Avoidance (CHARISMA) trial showed no difference in event rates across different CYP2C19 genotypes. Again, this was a study confined to stable patients managed non-invasively [63]. Taken together, there is strong evidence that carriage of one or two loss-of-function CYP2C19*2 alleles confers

an increased risk of adverse events in patients treated with clopidogrel. The association is most pronounced for the distinct outcome of stent thrombosis, which is analogous to the more pronounced risk reduction seen with ADP receptor antagonists for this specific outcome compared with broader cardiovascular outcomes. Two meta-analyses merging most of the currently available data support this, although acknowledging that validity is limited by the very low prevalence of stent thrombosis (approximately 1.5%) [57,58]. Importantly, these meta-analyses did not include data from the aforementioned CURE and ACTIVE A studies or the CHARISMA study, in which placebo was used as the comparator as opposed to most other studies comparing different clopidogrel doses or newer ADP receptor antagonists. Other recent meta-analyses reported a less substantial influence of the CYP2C19 genotype on the clinical effect of clopidogrel [64–66]. CYP2C19*17 The CYP2C19*17 polymorphism (rs12248560) yields the most prominent gain of clopidogrel efficacy described so far. The CYP2C19*17 allele causes high enzyme activity and rapid metabolization via the CYP2C19 route, whether present in duplicate (*17/ *17) or coupled with the wild-type allele (*1/*17) [67]. In a retrospective study including 598 ACS patients, Frere et al. [68] reported that *17 carriage was associated with lower platelet reactivity following a 600 mg clopidogrel loading dose. Sibbing et al. [69] corroborated these findings in a study showing that the *17 allele conferred lower platelet aggregation and increased bleeding risk in 1524 patients undergoing PCI. While increasing the risk of bleeding, the *17 allele did not protect against ischemic events. These findings have been extended to patients treated with a 75 mg maintenance dose [70]. It is important to notice that not all studies have confirmed these associations, and in particular it remains controversial whether or not the CYP2C19*17 allele is protective against ischemic events. A recent meta-analysis reported a protective effect of CYP2C19*17 [66], but so far genotyping for this polymorphism should be reserved for research purposes. Paraoxonase-1 The PON-1 enzyme was recently suggested as a key mediator of clopidogrel bioactivation, particularly involved in the second oxidative step transforming the intermediate metabolite 2-oxo-clopidogrel into the active metabolite. In patients with CAD, homozygosity for the Q allele of the common Q192?R polymorphism (rs662) of PON1 was associated with increased risk of stent thrombosis as well as a reduction in PON-1 enzyme activity, active metabolite levels and platelet inhibition [42]. Another study suggested an association with high platelet reactivity in clopidogrel-treated Chinese ACS patients [71], whereas many other studies found no impact of PON1 genotype on clopidogrel response in terms of pharmacokinetics and dynamics [43,72–75] or © 2013 International Society on Thrombosis and Haemostasis

Pharmacogenomics in cardiovascular disease 1633

clinical outcome [72,73,75,76]. Genotyping for PON1 should not be undertaken in clinical settings. Carboxylesterase-1 Carboxylesterase-1 (CES1) is a widely expressed serine esterase that hydrolyses clopidogrel into its inactive metabolites [77]. A recently identified single nucleotide variation G143?E (rs71647871) induces a substantially impaired catalytic activity of CES1 [78]. Lewis et al. [77] examined the contribution of the G143? E polymorphism in the PAPI Study. Carriers of the 143E allele had significantly higher levels of clopidogrel active metabolite and lower platelet aggregation levels compared with 143G allele homozygotes [77]. In a validation cohort of cardiovascular patients on clopidogrel therapy, carriers of the 143E allele had a significantly higher clopidogrel response compared with 143G allele homozygotes [77]. Although the study was not powered for clinical outcomes, 13.7% of patients who were homozygous for the 143G allele experienced a cardiovascular event at 12 months, and 0% of 143E allele carriers (P = 0.44); this requires validation for clinical outcomes in larger cohorts. Platelet surface receptors

ADP receptor antagonists inhibit platelet function by blocking the P2Y12 receptor. The H2 haplotype of the P2Y12 gene (P2RY12), although rare, may be associated with increased ADP-induced platelet aggregation during clopidogrel treatment [79–81]. GP Ia (collagen receptor) and GP IIIa (fibrinogen receptor) are some of the most prominent glycoproteins covering the platelet surface. Polymorphisms in GP Ia (C807?T, rs1126643) [82,83] and GP IIIa (T1565?C, rs5918) [84] may influence platelet function, but hardly affect clopidogrel response clinically [39]. Pharmacogenomics of new ADP receptor antagonists: prasugrel and ticagrelor Prasugrel

From a theoretical point of view, the efficacy of prasugrel is less susceptible to genetic variation than that of clopidogrel; prasugrel is primarily metabolized by CYP3A4, CYP3A5 and CYP2B6, rendering it less dependent on CYP2C19 [85], which accounts for most of the genetic variability seen with clopidogrel. Supporting this is the fact that prasugrel, unlike clopidogrel, seems unaffected by co-treatment with CYP inhibitors such as proton pump inhibitors [86]. Two head-to-head comparisons of clopidogrel and prasugrel showed no differences in terms of pharmacokinetic or pharmacodynamic effects of prasugrel between patients with and without common genetic polymorphisms [47,48]. Mega et al. [87] extended these findings by reporting equal event rates irrespective of genotype in the 1466 ACS patients allocated to prasugrel © 2013 International Society on Thrombosis and Haemostasis

in the TRITON-TIMI 38 trial. In contrast, a very recent pharmacodynamic study showed higher platelet reactivity in *2 carriers and lower platelet reactivity in *17 carriers compared with non-carriers by a highly P2Y12 receptorspecific test (the vasodilator-stimulated phosphoprotein phosphorylation assay), but not the more commonly used platelet aggregation assay. In this study, all patients underwent PCI and were treated with prasugrel 10 mg daily. Platelet reactivity was assessed 1 month after PCI [88]. Not only was *2 and *17 carrier status associated with platelet reactivity in opposite directions, it was also associated with bleeding complications in this cohort of stable CAD patients. Ticagrelor

Ticagrelor is an ATP analogue that does not require metabolism in the liver. At present, the genetic substudy of PLATO is the only study investigating the impact of genetic variability on the antiplatelet effect of ticagrelor [41]. Overall, the study showed that none of the following CYP2C19 alleles influenced the risk of thrombosis or bleeding with ticagrelor in ACS patients: *2, *3, *4, *5, *6, *7, *8 and *17. The study also investigated the effects of the ABCB1 polymorphism. The ABCB1 C3435?T (rs1045642) genotype may affect the expression of the P-glycoprotein efflux transporter, which might be important as ticagrelor is a putative P-glycoprotein substrate. In the PLATO sub-study, the ABCB1 polymorphism was not associated with ischemic or bleeding events in patients treated with ticagrelor. Genetically tailored therapy as an approach to improve antiplatelet therapy In view of the compelling association between some genetic traits and intensity of platelet inhibition by antiplatelet drugs, the possibility of tailoring antiplatelet therapy seems intellectually appealing. The evidence base for this strategy, however, is minute, and many inferences are made on surrogate endpoints. The best studied association is that between CYP2C19 polymorphisms and clopidogrel efficacy. CYP2C19*2-based strategies

Several studies have investigated the possible benefit of changing antiplatelet therapy in carriers of CYP2C19 loss-of-function alleles. Bonello et al. [89] showed that up to four consecutive clopidogrel loading doses were able to overcome residual platelet reactivity in 90% of patients carrying the CYP2C19*2 allele. In the randomized crossover study CLOVIS-2, high platelet reactivity on clopidogrel could be overcome by increasing the loading dose of clopidogrel in heterozygous CYP2C19*2 carriers but not in homozygous carriers [90]. This study, along with a

1634 M. W€ urtz et al

comparable study by Mega et al., [38,41,87], suggests that high doses of clopidogrel provide adequate platelet inhibition in the vast majority of patients, because the proportion of *2 homozygous patients is small, at least in Caucasians. However, the GIFT study randomized 741 patients to a standard or high maintenance dose of clopidogrel based on platelet function testing after PCI and followed patients for 6 months [91]. In this study, carriers of at least one reduced-function CYP2C19 allele had a significant increase in the risk of high platelet reactivity on clopidogrel, regardless of the clopidogrel dosing strategy employed [91]. Switching from clopidogrel to a stronger ADP receptor antagonist thus may be a better treatment strategy in *2 carriers. In RESET GENE, patients with reduced clopidogrel response were randomized to either high-dose clopidogrel or prasugrel for 15 days [92]. Interestingly, high-dose clopidogrel and prasugrel were equally effective in inhibiting platelet aggregation in CYP2C19*2 non-carriers, but highdose clopidogrel was significantly less effective in CYP2C19*2 carriers, whereas prasugrel effectively abolished high platelet reactivity in this small study. In RAPID GENE, patients undergoing PCI were randomized to either standard therapy or rapid genotyping for CYP2C19*2 [93]. Carriers of the *2 allele were given prasugrel 10 mg daily, whereas non-carriers and patients randomized to standard therapy received clopidogrel 75 mg daily. The mean platelet reactivity of CYP2C19*2 carriers at 1 week in the genotyping group treated with prasugrel was significantly lower than in *2 carriers given standard treatment. Addition of cilostazol, a phosphodiesterase inhibitor, on top of standard dual antiplatelet therapy, was tested in a recent study including 126 patients with myocardial infarction. Adjunctive cilostazol significantly reduced the rate of high residual platelet reactivity in *2/*3 carriers, but not in wild-type homozygotes [94]. Taken together, these studies suggest that intensified antiplatelet therapy may be beneficial in carriers of the CYP2C19*2 allele. The recently published Clinical Pharmacogenetics Implementation Consortium Guidelines for Cytochrome P450-2C19 (CYP2C19) Genotype and Clopidogrel Therapy suggest switching therapy based on CYP2C19 genotype [44]. The authors suggest that carriers of CYP2C19*2 are treated with prasugrel or other alternative therapies, whereas CYP2C19*1 homozygotes or CYP2C19*17 carriers should be given standard clopidogrel therapy. Prospective randomized studies showing a benefit of genotype-based adjustment of antiplatelet therapy for clinical outcomes are yet to be completed, but a number of trials are underway. The RAPID STEMI study will randomize patients to genetic testing and intensified therapy with prasugrel in CYP2C19*2 carriers vs. standard treatment; participants will be followed-up for clinical outcomes at 30 days (NCT01452139). In the GIANT study, all participants will be genotyped for CYP2C19 status, and treatment will

be adjusted in *2 carriers to either an increase of the clopidogrel dosage or switch to prasugrel for 12 months. Clinical outcomes at 1 year will be reported (NCT01134380). In the TAILOR-PCI study, patients who undergo PCI will be randomized to a conventional therapy arm (i.e. to receive clopidogrel without prospective genotyping guidance) vs. a prospective CYP2C19 genotype-based approach (ticagrelor in *2 or *3 carriers, clopidogrel 75 mg once daily in non-carriers). All patients will be followed-up for 1 year to assess the frequency of major adverse cardiovascular events (NCT01742117). These studies will inform clinical practice on the applicability of genotype-derived antiplatelet strategies. Novel approaches to pharmacogenomics of clopidogrel

Despite strong associations seen in candidate gene studies, the CYP2C19 polymorphisms only explain up to 12% of the variability in platelet response to clopidogrel [40]. The vastly unexplained portion of platelet response variability suggests that other important as yet unidentified genetic determinants may be involved. While most studies have concentrated on a candidate gene approach to test pharmacogenetic associations with clopidogrel response, Price et al. [95] have carried out a whole exome analysis in 392 subjects taking part in the GRAVITAS trial. The GIFT EXOME study results confirmed the association between clopidogrel response and the CYP2C18 and CYP2C19 cluster, and revealed two new genes, ATP2B2 (a member of the P-type primary ion transport ATPases that play a critical role in intracellular calcium homeostasis) and TIAM2 (a guanine nucleotide exchange factor that activates Rac1, which plays a central role in secretion-dependent platelet aggregation), previously unknown to play a role in platelet response to clopidogrel [96]. While this study with an expected sample size of 1000 clopidogrel-treated participants is still ongoing, and the new loci need to be confirmed independently, the whole exome sequencing approach appears to have some promise in elucidating the complex pharmacogenomics of platelet response to clopidogrel. Gene–gene interactions and gene–environment interactions Modern high-throughput analyses of thousands of individuals for a vast number of gene variants allow for the identification of subtle gene–gene interactions. In fact, from a biological point of view, this strategy appears more promising than candidate gene studies given that one polymorphism alone is unlikely to explain a reduced effect of antiplatelet drugs. Hopefully, genome-wide association studies will provide a better insight into reduced antiplatelet drug responses resulting from multiple inherited gene variants with prothrombotic effects, although the effect size of these variants may be insignificant when considered individually. © 2013 International Society on Thrombosis and Haemostasis

Pharmacogenomics in cardiovascular disease 1635

Our knowledge about gene–environment interactions and their importance for antiplatelet drug responses remains limited. The interesting finding that clopidogrel reduces cardiovascular events and morbidity only in smokers may represent such an interaction [97]. Moreover, there are studies suggesting that the pharmacogenetic interaction between CYP2C19 and clopidogrel is partly determined by the patients’ clinical status (e.g. whether or not they have undergone PCI) [98]. Environmental factors influence pharmacogenetic interactions, but further studies are warranted. Conclusions and perspectives Oral antiplatelet therapy is the therapy of choice to reduce the risk of atherothrombotic events in patients with ACS and in patients undergoing PCI. Importantly, a considerable number of patients still experience stent thrombosis and other platelet-dependent cardiovascular events despite antiplatelet treatment. Current practice guidelines advocate a ‘one size fits all’ approach, but the responsiveness to antiplatelet drugs is subject to considerable inter-individual variability, and reduced platelet inhibition has been linked to atherothrombotic events [3]. Ongoing research therefore aims to develop better strategies to improve antiplatelet treatment. This is a difficult task in view of the already high efficacy of antiplatelet agents in patients at risk of cardiovascular events. Genetic polymorphisms should be considered as one of several factors contributing to residual platelet aggregation [3]. What is really of interest for a clinician is whether or not the patient derives adequate platelet inhibition from the antiplatelet drug being administered. In that sense, platelet aggregation is a more rational endpoint than any of the parameters affecting platelet aggregation. Essentially, genotyping provides very limited incremental information in patients identified by platelet function testing to have high residual platelet aggregation during antiplatelet therapy; however, according to clinical guidelines, genetic testing may be considered in carefully selected high-risk patients when clopidogrel is used (e.g. patients undergoing complex PCI procedures) [99,100]. In these patients, changing treatment by dose adjustments or use of alternative drugs is intriguing, although the evidence to support this change of practice outside clinical trials is limited. In the context of bleeding, evidence is largely confined to the *17 allele of CYP2C19, which seems to be associated with low platelet reactivity and increased bleeding risk. However, for the prediction of bleeding in patients treated with antiplatelet agents, platelet function testing seems the most obvious strategy, as emphasized by recent guidelines [101,102]. This gap in knowledge is recognized by the National Heart, Lung and Blood Institute Working Group [98], which encourages research into the full spectrum of genetic variants associated with platelet biology and function including: gene–gene and gene–environment interactions © 2013 International Society on Thrombosis and Haemostasis

governing response to antiplatelet therapy; bringing genetic testing into clinical practice; and determining the utility of platelet function and genetic testing in clinically applicable algorithms. The ongoing studies on genetically-tailored antiplatelet therapy are highly awaited, as they may provide support for incorporating a patient’s genotype with demographic factors, co-medications and lifestyle choices into clinical algorithms, and may thus lead to improvements in clinical practice. Disclosure of Conflicts of Interest E. L. Grove has received speaker honoraria from AstraZeneca, Bayer, Boehringer Ingelheim and Pfizer and serves on advisory boards for AstraZeneca, Bayer and Bristol-Myers Squibb. M. Lordkipanidze has received speaker honoraria from Eli Lilly and has served as a consultant for Roche. M. W€ urtz has no conflicts of interest to declare.

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