CYP2C19 polymorphisms and effects of polymorphic CYP2C19 on clopidogrel response in Vietnamese stroke patients

To research CYP2C19 polymorphism and effects of CYP2C19 polymorphisms on clopidogrel response in ischemic stroke patients. Subjects and methods: A prospective and cross-sectional analysis was implemented among stroke patients in a 103 Military Hospital, from the beginning of 2017 to August 2018.

Journal of military pharmaco-medicine no6-2019 CYP2C19 POLYMORPHISMS AND EFFECTS OF POLYMORPHIC CYP2C19 ON CLOPIDOGREL RESPONSE IN VIETNAMESE STROKE PATIENTS Do Duc Thuan1; Pham Dinh Dai1; Tran Van Khoa2 SUMMARY Objectives: To research CYP2C19 polymorphism and effects of CYP2C19 polymorphisms on clopidogrel response in ischemic stroke patients Subjects and methods: A prospective and cross-sectional analysis was implemented among stroke patients in a 103 Military Hospital, from the beginning of 2017 to August 2018 144 patients with cerebral infarction were given clopidogrel at 75 mg daily and platelet aggregation was measured using light transmittance aggregometry with adenosine diphosphate µg/L after days Clopidogrel resistance was controlled until it got more than 50%, and identification of polymorphisms by Sanger sequencing Results: Frequency of patients carrying at least one allele CYP2C19*1 was 59.72%, at least one allele CYP2C19*2 was 92.36% and at least one allele CYP2C19*3 was 43.75% Frequency of poor metabolizer of clopidogrel was 40.29% Clopidogrel resistance in the CYP2C19*1/*1, CYP2C19*1/*2 plus CYP2C19*1/*3 genotypes was 45.83% In the same manner, the frequency of CYP2C19*2/*3 genotype was 54.17% Conclusion: Ischemic stroke patients with the CYP2C19*2,*3 alleles had a higher incidence and were also a contributing factor in increasing the resistance to clopidogrel * Keywords: Stroke; CYP2C19 gene; Polymorphism; Clopidogrel INTRODUCTION Clopidogrel (methyl [+]-[S]-a-[2chlorophenyl]-6.7-dihydrothieno [3,2-c] pyridine-5 [4H]-acetate hydrogen sulfate) is an oral thienopyridine derivation Clopidogrel, a kind of pro-drug, after absorption in gastrointestinal tract, is metabolized in liver by CYP2C19 enzyme This is an isoenzyme of cytochrome P450 [5] Benefits of clopidogrel have been widely recognized in treatment and prevention of ischemic stroke but some patients still face recurrent ischemic stroke although they are being treated with clopidogrel [10] One reason is polymorphism of the CYP2C19 gene [5] In Asia, the CYP2C19* 2,*3 allele genotypes account for a high proportion and these genotypes not support metabolism of clopidogrel, resulting in poor efficacy of clopidogrel in treatment and prophylaxis happens within ischemic stroke patients when there are thrombotic events [7] 103 Military Hospital Vietnam Military Medical University Corresponding author: Do Duc Thuan (dothuanvien103@gmail.com) Date received: 19/06/2019 Date accepted: 05/08/2019 140 Journal of military pharmaco-medicine no6-2019 In Vietnam, clopidogrel has been widely prescribed in treatment of ischemic stroke [1, 3] In order to improve the clinical efficacy of clopidogrel, this research takes a Look on CYP2C19 polymorphism and its impacts on clopidogrel response in patients with cerebral infarction SUBJECTS AND METHODS Subjects 144 consecutive patients were treated at Stroke Department, 103 Militayr Hospital from Jun 2017 to March 2018 * Inclusion criteria: Patients were diagnosed by definition stroke of WHO in 1989 [11] and confirmed by 1.5 Tesla brain magnetic resonance imaging or layers computed tomography All patients were treated with clopidogrel 75 mg once daily (Sanofi Company Ltd) for at least days The treatment regimen was unchanged during the study in each patient Protective neuron medication was the same in the all patients: Cerebrolysin 20 mL IV, piracetam g IV, choline alfoscerate g IM * Exclusion criteria: Allergy to clopidogrel; using anticoagulation or other antiplatelet drugs in the preceding weeks or during the study period; patients with acute ischemic stroke were treated with IV alteplase; patients with carotid endarterectomy or carotid angioplasty and stenting; patients who were severe water and electrolyte disorder or decreased level of consciousness, infarction > 1/3 MCA territory on MRI or CT-scan, hemorrhagic image on MRI or CT-scan, hemoglobin < 80 g/L or > 160 g/L, platelet count < 100,000/L or > 450,000/L; patients who had the following diseases: myocardial infarction, atrial fibrillation, heart failure level or (New York Heart Association), infective endocarditis, heart valve surgery, congenial heart disease, hepatitis, cirrhosis, liver cancer, estimated glomerular filtration rate (eGFR) < 30 (mL/min/1.73 m2) or they were being on a kidney machine and other severe diseases and patients refused to participate in the study were also uninvolved in this study Methods - Methods: A prospective, cross-sectional analysis at the Department of Stroke, 103 Military Hospital from June 2017 to March 2018 - Platelet aggression assay: Using light transmission aggregometry (LTA) with adenosine5′-diphosphate (ADP) µg/L Patients abstained ≥ hours before blood samples were collected Blood samples were drawn into vacutainer tubes containing 0.5 mL of 3.2% sodium citrate (BectonDickinson,SanJose, CA), preserved at 18 - 250C and processed in 60 mins by a Chrono-Log 530 of American at Hematology Department, 103 Military Hospital Clopidogrel resistance definition: Clopidogrel resistance was defined as platelet aggregation > 50% ADP5 µg/Linduced platelet aggression assay was performed by LTA at one point: treated with clopidogrel 75 mg once daily for at least days [6] - CYP2C19 polymorphism assay: DNA was extracted from peripheral blood (anticoagulated EDTA) by DNA blood mini kit (Qiagen, Germany) Amplification PCR on Mastercycle ProS (Eppendorf, Germany) using specific primers from IDT-American 141 Journal of military pharmaco-medicine no6-2019 Company Temperature cycle: 95oC x [94oC x min, 57oC x (*2) or 56oC x (*3), 72 x min] x 35 cycles, 72oC x 20 mins, stop at 4oC electrophoresis testing PCR products Good product with electrophoresis is light, clear and only one 168 bp band for (*2), 270 bp band for (*3) CYP2C19 polymorphisms (CYP2C19*2 and CYP2C19*3) were determined by Sanger sequencing using Big Dye Terminator v3.1 cycle sequencing kit All analyses were performed by using BioEdit software at Department of Biology and Medical Genetics, Military Medical University RESULTS AND DISCUSSION Patient population P2C19 gene assay was performed in 150 patients, but patients were excluded because of lack of research criteria Table 1: Demographic, baseline clinical and procedural characteristics of the patients *1/*1 (n = 6) *1/*2, *1/*3 (n = 80) *2/*3 (n = 58) Total (n = 144) p value 71.33 ± 7.20 67.35 ± 11.17 68.10 ± 10.96 67.82 ± 10.92 > 0.05 Male (%, n) 71.43 (5) 56.25 (45) 53.45 (31) 55.86 (81) > 0.05 Diabetes mellitus (%, n) 33.33 (2) 26.25 (21) 32.76 (19) 29.17 (42) > 0.05 Hypertension (%, n) 100.00 (6) 85.00 (68) 84.50 (49) 85.42 (123) > 0.05 Current smoking (%, n) 33.33 (2) 18.75 (15) 18.97 (11) 19.44 (28) > 0.05 0.0 (0) 7.50 (6) 6.90 (4) 6.94 (10) > 0.05 83.33 (5) 67.50 (54) 75.86 (44) 71.53 (103) > 0.05 Genotype Age (yrs) (X ± SD) Alcoholism (%, n) Hyperlipidemia (%, n) Uric acid serum (mmol/L) 345.08 ± 93.60 360.36 ± 102.01 364.55 ± 109.04 361.30 ± 103.91 > 0.05 243.53 ± 24.76 245.52 ± 60.93 245.94 ± 56.35 245.60 ± 57.78 > 0.05 Calcium-channel blocker (%, n) 16.67 (1) 67.50 (54) 57.17 (32) 60.42 (87) > 0.05 Angiotensin-converting enzyme inhibitor (%, n) 50.00 (3) 33.75 (27) 32.76 (19) 34.03 (49) > 0.05 Statins (%, n) 50.00 (3) 38.75 (31) 27.59 (16) 34.72 (50) > 0.05 Platelet count × 10 /L (Allele CYP2C19*1: *1; CYP2C19*2: *2; CYP2C19*3: *3) There were no significant differences in the ratio of sex, diabetes mellitus, hypertension, smoking habit, alcoholism, hyperlipidemia, calcium-channel blocker, angiotensin-converting enzyme inhibitor and statin usage between the three groups with p > 0.05 Age, uric acid serum, platelet count in peripheral blood were also no significant differences each other with p > 0.05 142 Journal of military pharmaco-medicine no6-2019 CYP2C19 polymorphisms in ischemic stroke patients Figure 1: Frequencies of CYP2C19 alleles in ischemic stroke patients Frequencies of patients with CYP2C19*1 and CYP2C19*3 were 59.72% and 43.75%, respectively and frequency of patients with CYP2C19*2 showed the highest frequency of 92.36% Patients carrying at least one CYP2C19*1 allele accounted for 59.72% (normal function allele) Frequency of CYP2C19*1 allele was equivalent to that in the Asian population of study by Yamazaki H (61.5%) [15], but it was lower than that in the study by Yang et al with stroke patients used clopidogrel, who carried at least one CYP2C19*1 allele accounted for 92.90% [16] Frequency of patients with at least one loss-of-function allele (CYP2C19*2,*3) was higher than that in other studies In 2009, Veiga et al studied Vietnamese people, the results showed that frequency of people carrying at least one CYP2C19*2 allele was 31%, at least one CYP2C19*3 allele was 6% [13] In 2007, Nguyen Thuy Mau et al studied in the myocardial infarction patients, the result showed that the frequency of people carrying at least one CYP2C19*2 allele was 54.7%, at least one CYP2C19*3 allele was 7.8% [2] There were several reasons for these differences such as subjects, population and the time of study, etc Table 2: Distributed CYP2C19 genotype and phenotype in ischemic stroke patients Phenotype Extensive metabolizer (EM) Intermediate metabolizer (IM) Poor metabolizer (PM) Genotype Frequency % (n) *1/*1 4.17 (6) *1/*2, *1/*3 55.56 (80) *2/*3 40.29 (58) 143 Journal of military pharmaco-medicine no6-2019 Frequencies of EM phenotype (CYP2C19*1/*1), IM phenotype (CYP2C19*1/*2 or *1/*3) and PM phenotype (CYP2C19 *2/*3) were 4.17%, 55.56% and 40.29%, respectively Scott S.A et al reported that among patients with EM was 35 - 50%, IM was 18 45% and PM was - 15% [12] In 2012, Yang J et al studied in Chinese stroked patients with EM frequency of 47.54%, IM frequency of 45.36% and 7.1% as for PM [16] In our study, IM frequency was lower, but PM frequency was higher than that in other studies This is a warning to stroke patients in Vietnam Effects of polymorphic CYP2C19 on clopidogrel response Table 3: Effects of CYP2C19 polymorphism on platelet aggression Genotype (n) Platelet aggression (X ± SD) *1/*1 (7) 30.00 ± 14.97 *1/*2 (75) 39.20 ± 18.02 *1/*3 (5) 40.60 ± 10.71 *2/*3 (58) 47.02 ± 18.40 Total (145) p < 0.05 42.01 ± 18.29 Platelet aggression mean was the smallest value among the patients with CYP2C19*1/*1 genotype (30.00 ± 14.97%) and it had the highest number among the patients carried two loss-of-function allele (CYP2C19*2/*3) genotype (47.02 ± 18.40%) Platelet aggression median was significantly different between the three genotype groups with p < 0.05 Our observation, regarding to CYP2C19*2, CYP2C19*3 alleles in Vietnamese stroked patients is supported by the results of previous studies (Yang J.C et al [16], Y.H Jeong et al (2011) [8], Guillaume Paré et al (2010) [17]) reported that carriage of the CYP2C19*2, CYP2C19*3 allele to its active metabolite clopidogrel may be reduced, resulting in decreased inhibition of platelets Table 4: Effects of CYP2C19 polymorphism on clopidogrel resistance Genotype Clopidogrel resistance Clopidogrel sensitive n = 48 n = 96 *1/*1, *1/*2, *1/*3 45.83% (22) 66.67% (64 ) *2/*3 54.17%(26) 33.33% (32 ) Total 33.10% (48) 66.90% (96) p < 0.05 Frequency of clopidogrel resistance in patients with CYP2C19*1/*1 genotype and only one loss-of-function allele was 45.83% Patients carried two loss-of-function allele had the highest frequency of clopidogrel resistance (54.17%) Frequency of clopidogrel resistance was significantly different between the three genotype groups with p < 0.05 144 Journal of military pharmaco-medicine no6-2019 Many previous studies had found that, people carried loss-of-function alleles (CYP2C19*2, CYP2C19*3) [9, 14] showed clopidogrel resistance Clopidogrel is a pro-drug, which requires metabolism by the hepatic cytochrome P450 (CYP) The main enzyme responsible for activating clopidogrel is CYP2C19 [4, 5] People carried loss-of-function alleles will have loss-of-function enzyme which not metabolize the pro-drug into activating form [5, 14] So December 3, 2010, The U.S Food and Drug Administration (FDA) has added a boxed warning on clopidogrel for use in patients carried loss-of-function CYP2C19 allele who not effectively metabolize the drug and therefore may not receive the full beneficial platelet aggression of the drug [7] CONCLUSIONS The study was conducted on 144 Vietnamese patients with ischemic stroke, we had found that: Allelic frequency of CYP2C19*1 was 59.72% in which CYP2C19*1/*1 homozygous frequency was 4.17% CYP2C19*2 allele showed the highest frequency of 92.36% without any CYP2C19*2/*2 homozygous phenotype Allelic frequency of CYP2C19*3 only found in the form of heterozygous, accounted for 43.75% Frequency of poor metabolized phenotype was 40.29% Patients carried CYP2C19*2, CYP2C19*3 showed the increased platelet aggression median and frequency of clopidogrel resistance after treatment Platelet aggression mean in patients with CYP2C19*1/*2, CYP2C19*1/*3 and CYP2C19*2/*3 genotypes were 39.20 ± 18.02%, 40.60 ± 10.71% and 47.02 ± 18.40%, respectively Frequencies of clopidogrel resistance in patients with CYP2C19*1/*1, CYP2C19*1/*2 plus CYP2C19*1/*3 and CYP2C19*2/*3 genotypes were 45.83% and 54.17%, respectively LIMITATIONS OF RESEARCH In addition to the allele CYP2C19*1,*2,*3, there are some other alleles CYP2C19 (CYP2C19*4, *5, *6…, *17…) and some variants of certain genes that regulate the absorption, metabolism and coding for receptors of clopidogrel This aspect was not included in the present study Additionally, our study have not flow-up recurrent ischemic stroke patients who carry loss-of-function allele using clopidogrel, the number of subjects who were recruited from only center was limited as well A multicenter study involving a larger sample size, another genes and follow-up study are needed to confirm our findings REFERENCE Nguyễn Văn Chương Nhồi máu não Thực hành lâm sàng Thần kinh học, Tập Nhà xuất Y học Hà Nội 2005, tr.43-73 Nguyễn Thị Thúy Mậu, Vũ Ngọc Trung, Nguyễn Hữu Hiếu CS Bước đầu đánh giá mối liên quan đa hình gen CYP2C19 độ ngưng tập tiểu cầu BN nhồi máu tim cấp số bệnh viện Hà Nội Tạp chí Khoa học ĐHQGHN 2017, tập 33, số 2, tr.68-74 Nguyễn Văn Thơng Xử trí cấp cứu BN đột quỵ não cấp Bệnh học Thần kinh Nhà xuất Y học Viện Nghiên cứu Khoa học Y Dược lâm sàng 108 Hà Nội 2013, tr.31-41 145 Journal of military pharmaco-medicine no6-2019 Ancrenaz V, Daali Y, Fontana P et al Impact of genetic polymorphisms and drugdrug interactions on clopidogrel and prasugrel response variability Curr Drug Metab 2010, 11 (8), pp.667-677 New Eng Land Journal of Medicine 2008, 359, pp.1238-1251 Dahabreh I.J, Moorthy D, Lamont J.L et al Testing of CYP2C19 variants and platelet reactivity for guiding antiplatelet treatment AHRQ Publication Rockville (MD) 2013 12 Scott S.A, Sangkuhl K, Gardner E.E, Stein C.M, Hulot J.S, Johnson J.A, Roden D.M, Klein T.E, Shuldiner A.R Clinical pharmacogenetics implementation consortium guidelines for cytochrome P450-2C19 (CYP2C19) genotype and clopidogrel therapy Clin Pharmacol Ther 2011, 90 (2), pp.328-332 Gurbel P.A, Tantry U.S Clopidogrel resistance? Thrombosis Research 2007, 120, pp.311-321 Holmes D.R, Jr Dehmer G.J, Kaul S et al ACCF/AHA clopidogrel clinical alert: Approaches to the FDA "boxed warning": A report of the American College of Cardiology Foundation Task Force on clinical expert consensus documents and the American Heart Association endorsed by the Society for Cardiovascular Angiography and Interventions and the Society of Thoracic Surgeons J Am Coll Cardiol 2010, 56 (4), pp.321-341 11 Sacco R.L, Kasner S.E, Broderick J.P et al An updated definition of stroke for the 21st century Stroke 2013, 44, pp.2064-2089 13 Veiga M.I, Asimus S, Ferreira P.E et al Pharmacogenomics of CYP2A6, CYP2B6, CYP2C19, CYP2D6, CYP3A4, CYP3A5 and MDR1 in Vietnam Eur J Clin Pharmacol 2009, 65, pp.355-363 14 VlachoJannis G.J, Dimitropoulos G, Alexopoulos D Clopidogrel resistance: Current aspects and future directions Hellenic Journal of Cardiology 2011, 52, pp.236-245 Jeong Y.H, Tantry U.S, Kim I S et al Effect of CYP2C19*2 and *3 loss-of-function alleles on platelet reactivity and adverse clinical events in East Asian acute myocardial infarction survivors treated with clopidogrel and aspirin Circ Cardiovasc Interv 2011, (6), pp.585-594 15 Yamazaki Hiroshi Fifty years of cytochrome P450 research Springer Japan 2014 Mega J.L, Close S.L, Wiviott S.D et al Cytochrome P-450 polymorphisms and response to clopidogrel N Engl J Med 2009, 360, pp.354-362 17 Paré Guillaume, R Mehta Shamir, Yusuf Salim, Anand Sonia, J Connolly Stuart, Hirsh Jack, Simonsen Katy, Bhatt Deepak, Fox Keith, Eikelboom John Effects of CYP2C19 genotype on outcomes of clopidogrel treatment 2010, Vol 363, pp.1704-1714 10 Sacco R.L, Diener H.C, Yusuf S et al Aspirin and extended-release dipyridamole versus clopidogrel for recurrent stroke The 146 16 Yang J, Zhao H D, Tan J et al CYP2C19 polymorphism and antiplatelet effects of clopidogrel in Chinese stroke patients Pharmazie 2012, 68 (3), pp.183-186  ... that in other studies This is a warning to stroke patients in Vietnam Effects of polymorphic CYP2C19 on clopidogrel response Table 3: Effects of CYP2C19 polymorphism on platelet aggression Genotype... Journal of military pharmaco-medicine no6-2019 CYP2C19 polymorphisms in ischemic stroke patients Figure 1: Frequencies of CYP2C19 alleles in ischemic stroke patients Frequencies of patients with CYP2C19* 1... takes a Look on CYP2C19 polymorphism and its impacts on clopidogrel response in patients with cerebral infarction SUBJECTS AND METHODS Subjects 144 consecutive patients were treated at Stroke Department,