CLOPIDOGREL RESPONSE IN ACUtE CORONARY SYNDROME PAtIENtS WItH CYP 2 Y 12 * 2 AND CYP 2 Y 12 * 3 POLYMORPHISMS

Ischemic heart disease is the leading cause of death and disability in the world with a fairly high portion coming from low and middle-income countries (LMICs). Deaths due to ischemic heart disease and acute coronary syndrome (ACS) appear at a younger age in LMICs countries compared with high-income countries, often occurring during the productive years [1]. Data from the Jakarta Cardiovascular Study in 2008 at the Heart Hospital Harapan Kita shows the prevalence of myocardial infarction at 5,29%-4,12% and 7,6% in women and men, respectively. Over the past seven years, the prevalence of myocardial infarction has increased by 4,09% or an average of 0,6% per year [2]. Clopidogrel is an irreversible inhibitor of the platelet P2Y12 adenosine diphosphate receptor, which is the standard drug therapy in the management of ACSs and in patients who will be undergoing percutaneous coronary intervention (PCI) [3-5]. Clopidogrel combined with CLOPIDOGREL RESPONSE IN ACUtE CORONARY SYNDROME PAtIENtS WItH CYP2Y12*2 AND CYP2Y12*3 POLYMORPHISMS

dose (LD) and action PCI.Blood samples were taken at 48 hours after the loading dose.Blood samples were centrifuged to obtain blood plasma.Plasma is stored at a temperature of minus 20° C until the sample size is met.Genomic DNA extractions from whole blood for PCR were carried out simultaneously after all samples collected.Polymorphism data obtained from PCR were performed in Biomedical Laboratory Faculty of Medicine, Andalas University.
Data on PRU values were obtained from examinations conducted at the central laboratory of Dr. M. Djamil hospital, Padang.Examination of platelet reactivity was performed within four hours from blood sampling.Examination of clopidogrel inhibitory activity against P2Y12 receptor (P2Y12 reactivity unit) was measured by using VerifyNow ® software and was performed at the cardiovascular ward, Dr. M. Djamil hospital.
Examination of genetic polymorphisms.Genomic DNA was extracted from frozen anti-coagulated whole blood samples from each of the 30 subjects using a GF-1 Blood DNA Extraction Kit (Vivantis, catalog number GF-BD-050) according to the manufacturer's instructions.Genomic DNA concentration and purity was quantified in duplicate using NanoDrop spectrophotometer.DNA samples were stored at -20 o C.
CYP2C19*2 PCR reactions were performed in a 25 μl total volume that contained 400 nM primer 2C19Ex5U and 400 nM primer 2C19Ex5L (synthesized by Integrated DNA Technologies, Singapore), 1x GoTaq Green Master mix (Promega, catalog number M7122), and  [3,4].CYP2C19 is the major enzyme in the biotransformation of clopidogrel.Genetic variations in this enzyme may affect clopidogrel activation and slow down the barriers of platelets during clopidogrel therapy [3].Currently, there is no data about CYP2C19 polymorphism in Indonesian populations, especially ethnic Minang.This study aimed to obtain data regarding enzyme CYP2C19 polymorphisms in ethnic Minang.These were assessed by studying the P2Y12 platelet activity through assessing P2Y12 Reactivity Unit (PRU) in the blood of sampled individuals who had received clopidogrel.Knowledge of CYP2C19 polymorphisms in particular populations should be helpful guides for antiplatelet therapy in patients with the ACS.

Subjects.
Our sample population was 30 patients with diagnosed ACS who received clopidogrel for the prevention of ischemic stroke in September 2016.This research is an observational study conducted prospectively in patients with ACSs who received clopidogrel therapy.This research was conducted in ward cardiology, cardiovascular care unit (CVCU), Biomedical Laboratory and Department of Pharmacology and Therapeutics, Faculty of Medicine, Andalas University, from August to November 2016.
The inclusion criteria of the subject are patients with a diagnosis of ACSs, male or female, age 18-65 years.Exclusion criteria are pregnancy, liver dysfunction, creatinine clearance <30 ml/min.Informed consent was obtained from each patient before study enrollment, and the study protocol was approved by the committee on ethics of Faculty of Medicine, Andalas University in Padang, Indonesia.
Data collection technique.Data were collected from medical records of all outpatient and inpatient Section Cardiovascular RSMD receiving clopidogrel therapy.The patient record data taken included characteristics/ demographic data (name, age, sex, weight, date of entry into care, medical record numbers), the results of anamnesis, physical examination, the results of initial laboratory examination, diagnosis and treatment of patients.The above data were used for the selection of research subjects.
Examination of genetic polymorphisms and PRU done using blood samples from patients.Blood samples were taken in patients who have been using clopidogrel loading washed using 3M sodium acetate and chilled with absolute ethanol.
The purified PCR products were sent for sequencing using a Sanger DNA sequencing method to Macrogen, Inc., South Korea.Then the DNA sequences were analyzed using Geneious 7.0.6 bioinformatics software.The results were classified as the extensive, intermediate, and poor metabolizer.The extensive metabolizer was defined as a patient who had a homozygous wild-type genotype (CYP2C19*1/*1).The intermediate metabolizer was defined as a patient who had a heterozygous genotype with at least one CYP2C19*1 allele (CYP2C19*1/*2 or *1/*3), and a poor metabolizer was classified as a patient who had a homozygous (CYP2C19*2/*2 or *3/*3) or heterozygous (CYP2C19*2/*3) genotype with a mutant allele [7].
Statistical analysis.Basic characteristic data were presented with descriptive statistics.The Shapiro-Wilk test was used to test for normal distribution.The relationship between CYP2C19 metabolizers with PRU values was assessed using the Kruskal-Wallis One-Way ANOVA on ranks.Results are considered significant when p<0,05.Quantitative data collected was processed and analyzed using SPSS version 17.0.

Results
We enrolled 30 patients with ACS who were treated at the Dr. M. Djamil hospital who met the inclusion criteria and had signed an informed consent.The age range was 38-75 years, with a mean age of 57,30 years.A total of 80% of the subjects were male, and 20% female.Almost all patients had risk factors for coronary artery disease (CAD) with only one patient without any risk factors.Smoking and hypertension were the biggest risk factors of CAD (56,67%) for the subject of the study, followed by diabetes mellitus, dyslipidemia and menopause (Tab.1).
Results from the PRU testing indicated 63,3% of the study subjects with PRU values <208 were classified as responsive patients, and 36,7% of the study subjects with PRU values >208 patients were classified as resistant (Fig. 1).Results from the sequencing of alleles CYP2C19*2 and CYP2C19*3 showed that the proportion of wild-type patients is still high.Although some patients were homozygous mutant.

Discussion
Among of the research subjects only one was aged under 40 years.There are various risk factors that facilitate the occurrence of ischemic heart disease.Most patients with ACS have an acute manifestation of coronary artery plaque, atheromatous tear, or rupture.This situation is followed by a process of platelet aggregation and activation of the coagulation pathway, forming a platelet-rich thrombus.This thrombus can clog a coronary artery, either totally or partially.Some extrinsic factors such as fever, anemia, thyrotoxicosis, hypotension, tachycardia can trigger the occurrence of ACS in patients who already have plaque atherosclerosis [8].In accordance with the literature, patients with acute coronary syndromes were primarily male.This study also found almost all subjects had other risk factors such as diabetes, hypertension, dyslipidemia and menopause.Clopidogrel resistance is a condition in which clopidogrel is less effective in certain patients [9].This study found 37% of subjects had clopidogrel resistance (Fig. 1).Patients with clopidogrel resistance are at risk of serious complications, which are sometimes fatal.These patients may experience a heart attack or stroke caused by abnormal blood clot formation.Stents can cause blood clots (thrombosis) that can block blood flow.This study found that 63% of the subjects were still responsive to clopidogrel, based on PRU value.This result was in line with the results from sequencing the CYP2C19*2 and CYP2C19*3 exons, which showed that the number of wild-type alleles was much higher than that the mutant alleles (homozygous and heterozygous) in the subject population (Tab.2).This showed the genetic variants that modulate the absorption and metabolism of clopidogrel could also impact on clopidogrel response [9,10].A point mutation in CYP2C19 gene could lead to a defective or nonfunctional protein, or inactive enzyme, resulting in the lack of ability to metabolize clopidogrel into an active metabolite [7].
About 46,7% patients were extensive metabolizers (Tab.3).Similar to the results seen by Gonzalez, et al. 45% of carotic artery stenting patients were extensive metabolizers [4].Baseline PRU was significantly higher among intermediate-poor metabolizers compared with ultra-rapid or extensive metabolizers.In our patients, PRUs among intermediate-poor metabolizers were higher than extensive metabolizers but not significant (Tab.4).Subraja et al. found a higher proportion of extensive metabolizers (62,3%) among cerebrovascular patients [11].Lee, et al. found that extensive metabolizers had significantly lower PRU than intermediate-poor metabolizers [12].Poor metabolizers should not be treated with clopidogrel [13].Determination of CYP2C19 polymorphism could increase the effectiveness of antithrombotic therapy.
Our study has some important limitations.It is a single-center study with a small number of patients.It is advisable in future studies to involve a greater number of subjects so that it is more representative of the population.The need remains for examination of plasma levels of clopidogrel to determine the effects of polymorphisms of genes encoding the metabolism and absorption is the reduction in plasma levels of clopidogrel so that correlations between genetic polymorphisms with drug levels in the blood can be assessed.

Conclusion
ACS patients with functional loss of CYP2C19 activity have lower platelet activity when treated with clopidogrel.Routine CYP2C19 and PRU testing may be warranted.Further research is needed with a larger number of patients to investigate the pharmacogenetic profile of CYP2C19 and the cause of clopidogrel resistance in Minang patients.

Conflict of Interest
Authors declare that there is no conflict of interest.