Co-Existence of CYP2C19*1/*2 and ABCB1c.3435 CT Genotype has a Potential Impact on Clinical Outcome in CAD Patients Treated with Clopidogrel

Cardiovascular diseases are a leading cause of death worldwide. Coronary artery disease (CAD) is the most common type of cardiovascular disease, and for those patients, Percutaneous Coronary Intervention (PCI) with stenting is the standard of care. However, in many cases post-operative patients develop major cardiovascular events (MACE) as cardiac death, myocardial infarction, stroke, and stent thrombosis which are serious concerns (1). According to guidelines, antiplatelet therapy is the first-line option in primary and secondary prevention of most cardiovascular diseases, followed by a thrombotic event, but despite successful treatment, the possibilities for recurrent ischemic events still exist (2, 3). Therefore, the use of drug-eluting stents in combination with dual antiplatelet therapy of aspirin and clopidogrel significantly reduces the incidence of ischemic events and stent thrombosis in patients with CAD (4).

Clopidogrel (adenosine diphosphate receptor P2Y₁₂ blocker) is a prodrug that needs to be converted into an active drug by several hepatic cytochrome P450 (CYP) enzymes. For this reason, the activity of these enzymes is assumed as the primary determinant for therapeutic response to this drug. CYP2C19 plays a key role in the metabolic transformation. The CYP2C19 gene is highly polymorphic, and therefore the association between CYP2C19 gene variants and clopidogrel efficacy appears to be clinically actionable. The presence of certain CYP2C19 polymorphisms can lead to variations in the level of functional proteins that influence the degree of clopidogrel metabolites and promote different inter-individual clopidogrel responses (5, 6). Specifically, the presence of any dysfunctional CYP2C19 allele (*2,*3,*4,*5) is associated with adverse cardiovascular events, whereas the presence of CYP2C19 allele (*17) is associated with increased risk of bleeding (7, 8). Guidelines recommend the testing of CYP2C19*2 *3 and *17 in order to avoid adverse outcomes in patients with CAD treated with clopidogrel (9). According to the presence of reduced-function alleles, patients are classified into two clinically significant categories: intermediate metabolizers and poor metabolizers. Due to the insufficient activity of the enzyme these patients are suggested to consider alternative P2Y12 inhibitors (ticagrelor and ticlopidine) (10).

The intestinal absorption of clopidogrel is mediated by ATP-dependent drug efflux pump, and P-glycoprotein, transporting a high variety of molecules across the extra-and intra-cellular membranes. Although it is expressed mostly on the intestinal epithelial cells, an increased expression can alter the bioavailability of clopidogrel. The P-glycoprotein is encoded by the ABCB1 gene located on chromosome 7 (11). Among several single nucleotide polymorphisms (SNPs) that were examined within this gene, the ABCB1 C3435T has been shown to have an effect on absorption of clopidogrel (12). Namely, individuals carrying the loss of function allele variant were associated with lower levels of the active drug metabolite and were considered to have a high rate of adverse clinical outcomes (13, 14). However, recent studies have presented conflicting results on the association of the ABCB1 C3435T and adverse events in patients treated with clopidogrel.

It is known that the pharmacodynamic response of clopidogrel can vary among individuals. Nearly 25% of patients treated with standard doses of clopidogrel experience low ex vivo inhibition of ADP-induced thrombocyte aggregation. The precise mechanism of resistance of clopidogrel is still unclear, although additional factors including epigenetics, demographics, complications and drug-drug interactions may also be involved in the response heterogeneity (15).

The aim of this study was to evaluate the association between the presence of the ABCB1 C3435T and CYP2C19*2 polymorphisms and the clinical cardiovascular outcome in post-operative patients with coronary artery disease treated with clopidogrel.

By comparison of the results obtained from the genotyping and clinical presentation of the disease we observed that a negative clinical outcome is more frequent in the subgroup of patients treated with clopidogrel that carry the CYP2C19 *1/*2 genotype together with the ABCB1 CT genotype. These results indicate that those patients are at an increased risk of adverse cardiovascular events vs patients who are homozygotes for the normal allele (22.73% vs 9.62%; OR 3.455; 95% CI= [0.936–12.743], p=0.05722 (Table 2). Consequently, allelic distribution of the reduced-function allele, in the CYP2C19 and ABCB1 genes, was higher in patients with worse cardiovascular outcome (36.36% vs 21.15%) (Table 3, Figure 1). Additionally, in the subgroup of patients presented with the CYP2C19*1/*1 genotype and co-existence of the ABCB1 CC or CT genotype, we noted a trend towards higher risk of MACE occurrence compared to patients with the TT genotype (OR=1.316 TT→CT; OR=3.056 TT→CC).

Figure 1.

Based on the results from the CYP2C19/ABCB1 genotyping, patents were divided into three groups according to combined genotype/phenotype: extensive metabolizers (EM), intermediate metabolizers (IM) and poor metabolizers (PM) (Table 4). Patients carrying normal function alleles were classified as extensive metabolizers, patients carrying at least one or two loss-of-function allele were classified as intermediate metabolizers, whereas the patients carrying two loss-of-function alleles were classified as poor metabolizers. In the subgroup of patients with negative outcome, the presence of intermediate metabolizers was more frequent compared to the subgroup of patients with positive outcome (0.7272 vs 0.5192; p=0.02805). The frequency of patients referred to as poor metabolizers was higher in the subgroup with a positive outcome (0.2272 vs 0.3077; p=0.22842), but without statistical significance (Table 4). Therefore, the patients classified as intermediate metabolizers, carrying at least one loss-of-function allele were assumed to have higher risk of adverse cardiovascular events or MACE.

The main goal of optimal antiplatelet therapy for patients with acute coronary disease and/or undergoing percutaneous coronary intervention is to reduce the incidence of cardiovascular events. The clinical outcome of clopidogrel, as a well-established antiplatelet therapy, has high inter-individual variability within patients

Although recent studies present contradictory data, our results support the association between the presence of the CYP2C19 *2, alone or in combination with the ABCB1 C and an increased risk for adverse cardiovascular events compared to the carriers of the alternative alleles. The concomitant inheritance of the genotype CYP2C19*1/*2 and ABCB1 CT have shown a significant trend toward increased risk for MACE. These finding are line with data presented in the studies conducted on a larger sample size. (16, 17, 18).

According to another study ABCB1 C3435T and CYP2C19 *2 polymorphisms are recognized as significant, independent predictors for the primary endpoint of cardiovascular death, myocardial infarction or stroke. The presented results indicate that the risk for MACE is higher in patients, either as carriers of a CYP2C19 or ABCB1 reduced-function allele, or both (19). In our study it was also demonstrated that patients with normal CYP2C19 genetic status and presence of the ABCB1 CT/CC genotype have a higher incidence of MACE, indicating that the presence of the ABCB1 C allele could be a potential negative predictor for disease outcome in patients treated with clopidogrel.

In a large cohort, conducted on 2208 patients with an acute myocardial infarction receiving clopidogrel therapy, Simon et al., presented no significant association between the ABCB1 and CYP2C19 polymorphisms and the clinical outcome, but the presence of two CYP2C19-deficient alleles and either one or two ABCB1 variant alleles was associated with rate of events five times higher when compared to patients with the wild-type (20). Another study also supports the evidence that patients who are poor metabolizers are at greater risk of thrombotic events when treated with clopidogrel (17, 21).

Several studies including the TRITON TIMI study evaluated the contribution of ABCB1 variants in patient carrying the risk allele CYP2C19*2 and confirmed that clopidogrel response depends on the complex mechanisms of action, including hepatic activation and also that variable response can occur due to other factors such as polymorphisms in other genes involved in clopidogrel pharmacokinetics and pharmacodynamics.

Since this study was conducted on a small sample size, limitations should be considered when interpreting the results. This was the first conducted study in Macedonia to evaluate the concomitant influence of the ABCB13435 CT and CYP2C19*1/*2 genotypes on clinical cardiovascular outcomes in coronary artery disease patients on clopidogrel treatment. The evaluation was based on occurred major adverse cardiovascular events and plasma concentrations of clopidogrel, and its active metabolites were not monitored during the follow up. Moreover, we cannot completely exclude the probability that other risk factors, such as BMI, smoking status, lifestyle, diet etc., influence the clinical cardiovascular outcome. However, the main limitation of this study was the small number of patients included, leading to restricted statistical significance for some of the results. Additional, larger studies will be of great importance to further evaluate the relation between concomitant inheritance of the ABCB1 C3435T and CYP2C19*2 polymorphisms and clopidogrel treatment outcomes.

In summary, our results support the previous data on the association between the presence of the CYP2C19 *2 alone, or in combination with the ABCB1 C, and the increased risk of MACE compared to the carriers of the wild type alleles. Namely, a higher incidence of major adverse cardiovascular events was detected in patients with concomitant inheritance of the CYP2C19*1/*2 and ABCB1 CT genotype and in patients with normal CYP2C19 genetic status and presence of the ABCB1 3435C allele. The results also indicate that the presence of ABCB1 C343T polymorphism might be potentially considered as an independent predictor of adverse cardiovascular events in patients treated with clopidogrel. However, this was an initial study conducted on a subset of the Macedonian population, and the results should be confirmed with a larger cohort.