Therapy with a P2Y12 receptor blocker added to aspirin remains the cornerstone of pharmacological therapy in patients with arterial diseases to prevent recurrent ischemic events [1]. Continuous downstream intracellular signaling from the P2Y12 receptor is essential for sustained platelet activation and aggregation, as well as for subsequent stable platelet-rich thrombus generation at the site of vascular injury causing thrombotic event occurrences [2]. Antithrombotic efficacy observed in multiple randomized clinical trials has provided strong rationale for adding a P2Y12 receptor blocker to aspirin therapy [1]. Despite the availability of more potent P2Y12 receptor blockers such as prasugrel and ticagrelor, clopidogrel remains the dominant P2Y12 inhibitor used in patients with arterial diseases.

Clopidogrel is an oral, selective platelet P2Y12 receptor blocker. It is a second-generation thienopyridine prodrug. Following intestinal administration, 85% of clopidogrel is hydrolyzed predominantly by hepatic carboxylesterase I to an inactive carboxylic acid metabolite, i.e. SR26334, and the remaining 15% is rapidly metabolized in a two-step process by hepatic CYP enzymes to an unstable active thiolactone metabolite. CYP2C19 plays a major role in clopidogrel metabolism in both steps [3]. In modeling studies, the active thiolactone metabolite is predicted to destabilize interactions of the G-coupled oligomeric P2Y12 receptor with adenosine diphosphate (ADP) by binding permanently to the free thiol of Cys97 and thereby preventing ADP-induced platelet activa- tion and aggregation for the lifetime of the platelet [2, 4].

A pivotal study conducted in patients undergoing elective stenting in 2003 demonstrated a wide variation in pharmacodynamic response to clopidogrel. Most importantly, a limited or no inhibition of ADP-induced platelet aggregation was demonstrated in nearly one in three patients. This phenomenon was at first described as clopidogrel “non-responsiveness” or clopidogrel “resis­tance” [5]. Later, it was suggested that high post-treatment platelet reactivity (HPR) to ADP is a more appropriate way to describe this phenomenon and more useful in correlating with recurrent ischemic events [6]. Multiple translational and observational research studies have linked HPR to recurrent ischemic events such as myocardial infarction (MI) and stent thrombosis in patients treated with coronary stenting. Several lines of evidence strongly suggest that variable and insufficient active metabolite generation are the primary explanations for clopidogrel response variability and nonresponsiveness, respectively [7]. Functional variability in hepatic cytochromes, particularly CYP2C19, has been reported as a major cause of clopidogrel response variability. CYP2C19 isoenzyme activity is strongly influenced by the presence of single nucleotide polymorphisms (SNPs). In addition, epigenetic factors such as demographic variables, comorbidities, and drug-drug interaction at the CYP level further influence platelet reactivity in patients treated with clopidogrel, independent of SNP carriage [8].

A genome-wide association study (GWAS) conducted in healthy volunteers demonstrated that loss-of-function (LoF) polymorphisms of CYP2C19 (*2 and *3) were associated with decreased clopidogrel active metabolite exposure and less platelet inhibition [9]. In a replication study with clopidogrel-treated patients who underwent percutaneous coronary intervention (PCI), carriers of the LoF CYP2C19*2 allele had ~2.4× higher cardiovascular (CV) event occurrence at 12 months compared to non-carriers [9]. The influence of CYP2C19 LoF carrier status on clopidogrel-induced platelet inhibition has been shown to be more significant among poor metabolizers who carry two LoF alleles than in patients carrying one LoF allele [10]. Similarly, an increased risk of the composite endpoint of CV death, MI, or stroke among carriers of one LoF allele (1.6×) and carriers of two LoF alleles (1.8×), compared to non-carriers, was demonstrated in a collaborative meta-analysis of trials primarily involving PCI-treated patients (11). The same meta-analysis demonstrated a dose-dependent association of LoF carriage with 2.67-times increased risk of stent thrombosis among carriers of one LoF allele, and a 3.97-times increased risk of stent thrombosis among carriers of two LoF alleles compared to non-carriers [11]. The influence of the gain-of-function allele (GoF) (CYP2C19*17) on ADP-induced platelet function and post-PCI clinical events is smaller compared to that of CYP2C19 LoF alleles.

The prevalence of LoF and GoF alleles differ significantly in different ethnicities. LoF allele carriage is more frequent in Asians, particularly East Asians, whereas GoF allele carriage occurs more frequently among Europeans and Africans [12, 13]. The genotype-based CYP2C19 metabolizer status indicating CYP2C19 enzyme activity and their prevalence in different ethnicities are set out in Table 1. However, despite the higher prevalence of the LoF allele carriage, East Asian patients demonstrate a lower risk for post-PCI ischemic event occurrences along with an elevated risk for bleeding. This phenomenon has been called the “East Asian Paradox” [14].

The pharmacodynamic effects of ticagrelor and prasugrel are not significantly influenced by CYP2C19 genetic polymorphisms. These agents are associated with less response variability, greater platelet inhibition and low rates of HPR. In genetic sub-studies of major randomized clinical trials, treatment with prasugrel and ticagrelor was associated with significantly lower major adverse cardiovascular events (MACE) rates compared to clopidogrel in patients carrying a LoF allele [15, 16].

The International Clopidogrel Pharmacogenomics Consortium (ICPC) developed a pharmacogenomic polygenic response score (PgxRS) to study the impact of 31 candidate gene polymorphisms on platelet reactivity in coronary artery disease (CAD) patients treated with clopidogrel [17]. Earlier it had been suggested that antithrombotic efficacy could be improved by treating patients with HPR or with LoF presence with prasugrel or ticagrelor. It was also postulated that the reservation of potent P2Y12 inhibitors for the latter patients as compared to uniform treatment of all patients with prasugrel or ticagrelor would be associated with greater safety [18]. Multiple subsequent studies have demonstrated that the anti-ischemic effects of clopidogrel are comparable to those of potent P2Y12 receptor inhibitors in LoF non-carriers. These observations have provided a strong rationale for selective treatment of patients with CYP2C19 LoF carriage, as determined by laboratory testing with prasugrel or ticagrelor [18].

Multiple randomized clinical trials have explored the utility of CYP2C19 genetic testing to personalize P2Y12 receptor-based dual antiplatelet therapy (DAPT) [19–21]. The TROPICAL ACS (Testing Responsiveness to Platelet Inhibition on Chronic Antiplatelet Treatment for Acute Coronary Syndromes) trial primarily demonstrated the utility of the de-escalation of prasugrel to clopidogrel therapy based on platelet function testing in ACS patients undergoing PCI. In a genotyping sub-study of TROPICAL ACS, significant correlations between CYP2C19*2 and CYP2C19*17 carrier status with platelet reactivity in patients treated with clopidogrel were demonstrated. However, regarding the prediction of ischemic and bleeding risk, there was no added benefit of genotyping with the phenotype-guided de-escalation approach [22].

In the POPular Genetics trial, patients undergoing PCI for ST-segment elevation myocardial infarction (n = 2488) who received genotype-guided P2Y12 inhibitor treatment had lower Platelet Inhibition and Patient Outcomes (PLATO) major bleeding events (9.8% vs. 12.5%; HR, 0.78; P = 0.04) and similar rates of net adverse clinical events (defined as death from any cause, MI, definite stent thrombosis, stroke, or PLATO major bleeding at 12 months (5.1% vs. 5.9%; P <0.001 for noninferiority) compared to patients who received uniform ticagrelor or prasugrel therapy [19]. In the TAILOR PCI (Tailored Antiplatelet Therapy Following PCI) trial, a randomized investigation of 5302 CAD patients undergoing PCI, CYP2C19 LoF carriers treated with ticagrelor or prasugrel had lower MACE (CV death, MI, stroke, stent thrombosis, and severe myocardial ischemia) rates compared to patients randomized to conventional clopidogrel therapy (4.0% vs. 5.9%; HR, 0.66; P = 0.06) [20]. Interestingly, in a prespecified analysis of this trial that assessed multiple events per patient, the genotype-guided strategy was associated with a significantly lower rate of ischemic events (HR, 0.60; P = 0.01), with no significant difference in major/minor bleeding (HR, 1.36; P = 0.39) [23]. A collaborative study of 3342 patients undergoing PCI in a real-world setting demonstrated a lower rate of major thrombotic events in LoF carriers treated with prasugrel or ticagrelor compared to patients treated with clopidogrel (adjusted HR, 0.56), but a similar rate of thrombotic events in CYP2C19 LoF non-carriers (adjusted HR, 1.07) and a similar rate of bleeding with alternative therapy versus clopidogrel in both groups [24]. It was also demonstrated that there was no significant influence of CYP2C19*17 genotype on post-PCI prescribing decisions or clinical outcomes [25].

Combining CYP2C19 genotype testing, platelet reactivity, and clinical risk factors in a pharmacogenetic-driven algorithm (POPular Risk Score [PRiS]) showed usefulness in tailoring DAPT in patients undergoing drug-eluting stenting; in patients with PRiS ≥2, ticagrelor has been demonstrated to reduce MACE without an increase in bleeding risk compared to the uniform use of clopidogrel; in patients with PRiS <2, clopidogrel therapy was associated with bleeding risk with a comparable incidence of MACE [26]. This study highlighted the utility of combining genetic testing with platelet function testing and demographic variables to improve patient outcomes.

In addition to CAD patients, the utility of CYP2C19 genotype testing has been explored in large-scale trials of patients with stroke. In the randomized Clopidogrel in High-Risk Patients with Acute Nondisabling Cerebrovascular Events (CHANCE-2 trial), Chinese patients with CYP2C19 LoF presence and with minor ischemic stroke or transient ischemic attack (TIA) had a reduced risk of stroke at 90 days (6.0% vs. 7.6%; HR, 0.77; P = 0.008) when treated with ticagrelor versus clopidogrel and a similar risk of moderate/severe bleeding (0.3% vs. 0.3%) [27] (Table 2).

Total bleeding events were more common in the ticagrelor group, and the clinical outcomes were consistent among the intermediate and poor CYP2C19 metabolizers [28]. Among different subgroups, the benefit of ticagrelor over clopidogrel has been seen in patients with normal renal function, single small subcortical infarction (SSSI), non-elevated remnant cholesterol (RC) levels, and without intracranial artery stenosis (ICAS) [29–32]. It is worth mentioning that time-wise assessment in the CHANCE-2 trial showed a benefit associated with ticagrelor therapy mainly in the first week, with only a small additional benefit in the next 2 weeks, and the overall superiority of ticagrelor over clopidogrel persisted at the 12 month follow-up [33, 34]. In a recent RCT, personalized antiplatelet therapy in acute stroke/TIA patients based on the CYP2C19 genotype testing and urinary 11-dihydroxy thromboxane (Tx)B2 (a marker of aspirin response) levels was associated with favorable neurological function and reduced bleeding risk compared to standard treatment [35]. A similar benefit was also shown in another RCT where antiplatelet therapy guided by pharmacogenomics and clinical characteristics was associated with decreased vascular event risk in patients with stroke/TIA [36]. These trials signify the higher recurrent risk of stroke in CYP2C19 LoF carriers treated with clopidogrel [37].

GUIDELINES ADDRESSING CYP2C19 GENOTYPE TESTING

In 2010, the FDA issued a “boxed warning” stating that healthcare professionals should consider other antiplatelet medications or alternative dosing in patients who are poor metabolizers of clopidogrel (CYP2C19 LoF carriers) [38]. Despite the demonstration of a graded effect of CYP2C19 LoF carriage [11], the American Heart Association and American College of Cardiology guidelines for CYP2C19 genotyping have not changed since 2011, when they recommended against routine genotyping — a class III (i.e., no benefit) recommendation [39]. The 2020 European Society of Cardiology guidelines for the management of ACS recommend CYP2C19-guided antiplatelet therapy as an alternative to 12 months of DAPT with prasugrel or ticagrelor [40]. The latter was based on the results from the POPular Genetics trial [19]. The 2022 Clinical Pharmacogenetics Implementation Consortium (CPIC) guidelines recommended the use of alternative P2Y12 inhibitors in CYP2C19 poor metabolizers, in patients with acute coronary syndromes, and/or those undergoing PCI (strong recommendation), and for other CV/neurovascular indications (moderate recommendation) [15]. Finally, the 2023 European Society of Cardiology guidelines for the management of acute coronary syndrome briefly mention CYP2C19 genetic testing for P2Y12 therapy de-escalation [41].

The main reason for not giving a strong recommendation for genotyping is that the guidelines require high-quality evidence from more than one large RCT, meta-analyses of high-quality RCTs, or one or more RCTs corroborated by high-quality registry studies for a class I/level A recommendation (i.e., that the test should be performed) [39]. It is difficult to achieve such evidence at this time because it is prohibitively costly to conduct a large-scale trial of the size required to reach adequate power. Moreover, trials of personalized therapies are not of interest to most pharmaceutical companies due to concerns about market share loss. Finally, it is unethical, given the body of evidence currently available, to randomize CYP2C19 LoF carriers to clopidogrel, since they suffer harm. In this scenario, a non-inferiority trial like the POPular Genetics trial, and the integration of evidence across multiple studies in meta-analyses may be the only option, but would be unlikely to provide findings conclusive enough to reach a class I recommendation in the minds of guideline writers [19].

FUTURE PERSPECTIVES

Despite the FDA’s “boxed warning”, robust data implicating a poorer clinical outcome in LoF carriers treated with clopidogrel, and the cost-effectiveness of personalizing antiplatelet therapy by genotyping, including genotype testing in routine clinical practice has been less robust. Furthermore, uniform use of more potent P2Y12 inhibitors is associated with greater bleeding and higher cost and lower adherence, whereas clopidogrel is effective in the majority of CYP2C19 LoF non-carrier patients. Therefore, at this time, the totality of evidence strongly suggests that physicians should perform genetic testing to identify patients with high-risk arterial disease to determine whether they are suitable for clopidogrel therapy. However, current physician preference for P2Y12 inhibitors is not genotype-guided [42–44]. The current American Heart Association statement supporting genotype-guided antiplatelet therapy may provide the impetus to change present practice [45]. As an aside, the seemingly absolute requirements for large RCT results stand against the requirements of guideline writers in other specialties (e.g., oncology) who have been willing to consider the entire hierarchy of evidence.

Real-time PCR (TaqMan system, laboratory-based testing) using genomic DNA from whole blood samples is time-consuming and requires certified personnel and a laboratory to perform CYP2C19 genotyping for clinical use. Point-of-care (POC) genotype testing may have its greatest impact when implemented during the hospital stay when the physician can decide on the genetically predicted optimal choice of P2Y12 receptor inhibitor for loading and maintenance. Development of advanced point-of-care genetic testing technology with rapid turnaround times has opened the door for personalized antiplatelet therapy strategies. Currently, a POC CYP2C19 genotyping assay using buccal swabs (Cube, Genomadix, Kanata, ON, Canada, earlier SPARTAN) is available. There is a 99.1% concordance between the SPARTAN assay and TaqMan genotyping assay results [20]. Genedrive, another POC testing method, is also available [46].

POC genotyping assay is simple, user-friendly, and can be performed even in an outpatient clinic for quick antiplatelet therapy personalization. In a recent study, the feasibility of routine bedside CYP2C19 genetic testing for guiding antiplatelet therapy in a community setting was explored using the SPARTAN system [47]. CYP2C19 genetic testing was performed in the catheterization laboratory before PCI and the results were available within one hour for decision-making by the interventional cardiologist. Adherence to the best practice advisory (BPA) by clinicians was noted in 93% of patients undergoing PCI; 87% of CYP2C19 LoF allele carriers were prescribed prasugrel or ticagrelor, and 95% of non-carriers were prescribed clopidogrel [47]. These findings from a community hospital study reinforce the feasibility of routine CYP2C19 genotyping in large academic centers. In 2024, POC genotyping can be used to rapidly personalize antiplatelet therapy, reserving clopidogrel for the wild type, and prasugrel or ticagrelor for the LoF carriers.

Article information

Conflict of interest: PAG received consulting fees and/or honoraria from Bayer, Vectura/Otitopic, Janssen, Cleveland Clinic Foundation, Wolters Kluwer Pharma, Web MD Medscape, Baron and Budd, Premier Health Care Resource, Baim Institute, and Medforce; institutional research grants from Accriva Diagnostics, AstraZeneca, Bayer, Cronos, Janssen Pharmaceuticals Inc., Haemonetics, Hikari Dx, Idorisa, Labcorp Drug Development, Novartis, Prolocor, Recor Medical, Vectura Limited, Zoll Medical Corporation; in addition, PAG has two patents i.e. ‘Detection of restenosis risk in patients issued’ and “Assessment of cardiac health and thrombotic risk in a patient”. He was an expert witness in a lawsuit associated with Plavix. Other authors report no conflict of interest.

Funding: This study was funded by the Sinai Center for Thrombosis Research and Drug Development.

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