GENETIC POLYMORPHISMS OF ABCB1, CYP2C19, CYP3A5, CYP4F2 AND PLATELET REACTIVITY IN THE EARLY PHASES OF ACUTE CORONARY SYNDROMES

Abstract

Aims

The aim was to study 7 polymorphic markers of genes encoding proteins involved in the absorbtion, metabolism and pharmacokinetics of clopidogrel among patients with an acute coronary syndrome (ACS).

Materials and Methods

81 ACS and PCI patients older than 18 years treated with dual antiplatelet therapy. Platelet function testing and ABCB1, CYP2C19, CYP3A5, CYP4F2 genotyping were performed. Predictive role of categorical variables such as genotypes (carriers and non-carriers of polymorphism) on platelet reactivity (PRU, PI) was assessed by logistic regression (for categorical outcomes) and linear regression (for continuous outcomes) analysis. A p-value < 0.05 was considered significant. Allele frequencies were estimated by gene counting, and Hardy-Weinberg equilibrium was tested using the chi-square test.

Results

Regarding clopidogrel response, 62 patients (76,5 %) were clopidogrel responders and 19 were non-responders (23,5 %). Mean PRU value and the percentage of platelet inhibition were 170,0 ± 50,9 PRU and 28,6 ± 19,9 %, respectively. The effects of CYP2C19*2 polymorphisms on PRU (166,0 ± 50,8 vs. 190,7 ± 48,2, p < 0.038) and PI (30,6 ± 20,0 vs. 18,1 ± 16,3, p < 0.013) was observed and the rates of HPR were lower in CYP2C19*1/*1 than that in CYP2C19*1/*2 + CYP2C19*2/*2 (16,2% vs. 53,8%  p < 0.0067). Whereas there was no significant difference in PRU value and PI at < 5 days between the rest of polymorphisms (p > 0,05).  By the logistic regression analysis, CYP2C19*2 (OR: 4,365, CI: 1,25 – 17,67, p = 0,022) was independent predictor of HPR at < 5 days, as were stent diameter (OR: 0,219, CI: 0,002 – 0,229, p = 0,049). The rest of polymorphisms had no influence. The linear regression was used to calculate the odds ratio of changes in on-clopidogrel platelet reactivity (PRU and PI) between carriers and non-carriers of the minor allelic variant: for PRU – CYPC19*2 – 190,7 ± 48,2 vs. 166,0 ± 50,8  (OR=0,27; р=0.026) and CYPC19*17 – 153,8 ± 62,3 vs. 176,8 ± 44,1 (OR=0,3; р=0,01); for PI – 18,1 ± 16,3 vs. 30,6 ± 20,8  (OR=0,33; р=0,005) and CYPC19*17 – 34,9 ± 25,0 vs. 25,9 ± 16,9 (OR=0,32; р=0,006), the rest of polymorphisms had no influence.

Conclusions

We found that on-clopidogrel platelets reactivity in the early phase of ACS is influenced primarily by CYP2C19 polymorphisms. We believe that the findings of the present study could supply additional evidence regarding the clinical appropriateness of the CYP2C19 genetic testing for tailoring antiplatelet therapy in the early phase of ACS.

Keywords: clopidogrel, ticagrelor,  prasugrel, acute coronary syndrome, pharmacogenetics, cytochrome P450, polymorphism, percutaneous coronary intervention

Introduction

As a standard of care for patients with acute coronary syndrome undergoing percutaneous coronary intervention, dual antiplatelet therapy is routinely used [1]. The P2Y12 inhibitor clopidogrel is a prodrug, therefore it needs to be metabolized in the liver, which makes its pharmacokinetics dependent on the activity of the enzymes involved. For clopidogrel, these enzymes being CYP2C19, CYP2B6, CYP1A2 for transformation of the prodrug into 2-oxo-clopidogrel as stage one, and CYP2C19, CYP2B6, CYP3A4, CYP3A5, CYP2C9 to transform that intermediate product into an active metabolite as stage two [2]. Not only does metabolism of clopidogrel involve a two-stage transformation, but also tiny transporter P-glycoproteins to facilitate prodrug absorbtion at the apical membranes of the intestinal cells. This P-glycoprotein is encoded by the ABCB1 gene. As a result, there are numerous sites for a possible breakdown which lead to altered response to clopidogrel. These causes for altered clopidogrel response could be divided into three groups: pharmacokinetic markers like intestinal absorbtion, metabolic activation in the liver and pharmacodynamic markers. These causes have a clear link to variants of the genes coding the enzymes involved, so called polymorphisms. These polymorphisms which are either suspected or proven causes are mentioned in this article as follows: ABCB1 C3435T, ABCB1 C>T, rs4148738 for P-glycoprotein; CYP2C19*17 C-806T, CYP2C19*2 681G>A, CYP2C19*3 636G>A, CYP3A5*3 A6986G for genes CYP2C19 and CYP3A5 of the cytochrome P450 enzymes family; CYP4F2 C(Val433Met)T as a pharmacodynamic marker for CYP4F2 enzyme which is also responsible for epoxyeicosatrienoic acids metabolism [3].

This altered response to clopidogrel poses a challenge to physician and could potentionally lead towards such complications as stent thrombosis or bleeding [4,5]. Although, studies suggest that the presence of polymorphisms does affect laboratory clopidogrel resistance, the clinical implications for routine genetic testing are not stated according to the current guidelines [6]. This is so because the “critical mass” of the studies showing that altered response to clopidogrel might seriously jeopardize the safety of percutaneous coronary intervention and cause stent thrombosis or bleeding has not been achieved yet.

One of the way to assess the response to clopidogrel is to measure platelet reactivity with the VerifyNow P2Y12 Assay. The therapeutic range is over 85 but less than 208 platelet reactivity units (PRUs) [7]. While PRU level under 85 may be a predictor of a bleeding event, PRU level over 208 is a sign that the higher clopidogrel reactivity is present and a physician might consider taking action. This is the time for a personalized approach. There were some trials which assessed doubling and tripling the dose of clopidogrel for low responders [8,9]. However, the current strategy for low responders is switching to another P2Y12 inhibitor like ticagrelor or prasugrel [10].

Studies have shown that polymorphisms are more frequent in Asians than in Caucasians [11] Therefore, a physician might consider measuring platelet reactivity for such a patient or switch to another P2Y12 inhibitor.

Materials and methods

Study population

The study protocol was approved by the Ethics Committee of Russian Medical Academy of Continuous Professional Education, Ministry of Health of the Russian Federation, Moscow, and all patients gave written informed consent for participation. It was conducted in accordance with the Declaration of Helsinki and consistent with applicable guidelines for good clinical practice. Patients included in this study were recruited from  October 2014 to September 2015 in Moscow city clinical hospital №1, Moscow, Russian Federation. Those enrolled 81 ACS and PCI patients older than 18 years were treated with dual antiplatelet therapy, 100 mg of aspirin daily and either a 300 or 600 mg loading dose following 75 mg maintenance dose daily of clopidogrel. Postprocedural therapy consisted of 100 mg aspirin once daily and 75 mg clopidogrel once daily. The major exclusion criteria included high risk of bleeding, thrombocytopenia, contraindications to aspirin or clopidogrel, malignancies, pregnancy, and cerebrovascular events within the past 3 months. These factors included age, sex, smoking, diabetes mellitus, hypertension (BP ≥ 140/90 mmHg), hyperlipidemia, bodymass index, family history of coronary artery disease, previous myocardial infarction, and coronary stents were obtained from patient’s files or interview.

Blood sampling

A total of two blood samples from all 81 patients were collected on the 3-5^th day after the last dose of clopidogrel taken for genotyping and platelet reactivity. Blood (2 ml each) for DNA analysis was sampled from the peripheral vein using ethylene diamine tetra acetate (EDTA) tubes (VACUETTE® (Greiner Bio-One, Austria) and stored at −80 °C. Blood samples (2 ml each) were taken in tubes with 3.2% sodium citrate for the measurement of platelet activity in response to ADP at 4 h after the last dose of clopidogrel taken [12].

Extraction of peripheral blood DNA

Genomic DNA was extracted from peripheral blood by using “DNA ‑ EKSTRAN-1” (ZAO Syntol, Russia) according to the manufacturer’s protocol. The quantitative concentration of DNA was measured by the Nanodrop Spectrophotometer (ND-2000, USA).

ABCB1, CYP2C19, CYP3A5 and CYP4F2 genotyping

A panel of 7 SNP of ABCB1 (C3435T, rs1045642), ABCB1 (C>T, rs4148738), CYP2C19*2 (681G>A, rs4244285), CYP2C19*3 (636G>A, rs4986893), CYP2C19*17 (C-806T, rs1224856), CYP3A5*3 (A6986G, rs776746), CYP4F2 (C>T, Val433Met, rs2108622) was selected based on previous investigations [12-14]. Base numbering and allele definitions follow the nomenclature of the Human Cytochrome P450 (CYP) Allele Nomenclature Committee (www.cypalleles.ki.se). The genotypes were determined with a TaqMan® Single Nucleotide Polymorphism Genotyping Assay kit and a TaqMan Universal PCR Master Mix (Applied Biosystems, Foster City, CA, USA), according to the manufacturer’s instructions, using an ABI PRISM® Sequence Detector 7000 (Applied Biosystems). All the PCR reactions were carried out in a 10-μL reaction volume containing genomic DNA 15 ng, oligonucleotide primers 0.5 pM, 1 μL 10 PCR buffer, deoxynucleotides (dNTPs) 250 μM, magnesium chloride 3 mM, and DNA polymerase 0.25 U. The cycling program involved preliminary denaturation at 95°C for 10 min, followed by 30 cycles of denaturation at 95°C for 30 s, annealing at 60°C for 60 s, and elongation at 72°C for 60 s, followed by a final elongation step at 72°C for 7 min [15].

Platelet function assay

Platelet function tests were performed within 1 h of sample acquisition utilizing VerifyNow P2Y12 assay (Accumetrics, San Diego, CA, USA) within 5 days after clopidogrel loading (early phase). The glycoprotein IIb/IIIa inhibitors and intravenous anticoagulants other than unfractionated heparin were not approved for use in patients with ACS in this study. The level of platelet aggregation is expressed in P2Y12 reactivity unit (PRU) and in platelet percentage inhibition (PI). The assessment was performed within 1 hour after venous blood collection. The VerifyNow system is a rapid point-of-care platelet function test system. The VerifyNow P2Y12 test measures ADP-induced platelet aggregation and reports results as P2Y12 Reaction Units (PRU). A second channel in the test device activates platelets through the thrombin receptor pathway, which is P2Y12-receptor-independent and provides a simultaneous estimate of baseline total platelet function, which is reported as “Base PRU”.  The percent inhibition of ADP-induced platelet aggregation is calculated from the PRU and Base PRU values. A cutoff value for higher platelet reactivity (HPR) was defined as PRU > 208 in the present study, despite the lack of consensus on the cutoff value associated with clinical outcomes [16]. The reference values were PRU values < 208 (responders to clopidogrel) or PRU values > 208 (high OTR to clopidogrel or clopidogrel non-responders). Moreover, a cutoff for lower platelet reactivity (LPR) was defined as PRU < 85.

Statistical analysis

Qualitative variables were expressed as relative and absolute frequencies and quantitative variables as the mean (M) ± standard deviation (SD). The Kolmogorov Smirnov test was used to assess normality. Continuous variable comparisons were performed using the Student T test and analysis of variance. The chi-square test and analysis of variance (ANOVA) were used to compare variables between the subgroups, and the Fisher exact test was used when any expected cell count was <5 for a contingency table.  Differences in data between subgroups were tested by either ANOVA when values were normally distributed and the non-parametric Mann‑Whitney U-test or Kruskal‑Wallis test when the distribution normality test failed. Predictive role of categorical variables such as genotypes (carriers and non-carriers of polymorphism) on platelet reactivity (PRU, PI) was assessed by logistic regression (for categorical outcomes) and linear regression (for continuous outcomes) analysis.  A p-value < 0.05 was considered significant. Allele frequencies were estimated by gene counting, and Hardy-Weinberg equilibrium was tested using the chi-square test. Statistical analysis was performed using Statistical Package for Social Sciences (SPSS) 20.0 (IBM Corporation, USA).

Results

Characteristics of the study population and genotyping

A total of 81 Caucasian patients were included in the study. In 81 ASC patients, 77 received PCI, and 4 received standard medical treatment without PCI. There were no significant differences in the demographic, clinical, and laboratory findings between the study subgroups according to platelet reactivity levels (Table 1).

Table 1. Baseline demographics and clinical characteristics of the study cohort with resistant to clopidogrel (PRU > 208) and a normal response to clopidogrel (PRU < 208)