European Journal of Pharmacology ∎ (∎∎∎∎) ∎∎∎–∎∎∎
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 Q1 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66
Contents lists available at ScienceDirect
European Journal of Pharmacology journal homepage: www.elsevier.com/locate/ejphar
Cardiovascular pharmacology
Influence of CYP2C19 polymorphisms on platelet reactivity and clinical outcomes in ischemic stroke patients treated with clopidogrel Li-Na Qiu a,1, Yan Sun b,1, Lin Wang c,d, Rui-Fa Han b, Xiao-Shuang Xia a, Jie Liu a, Xin Li a,n a
Department of Neurology, the Second Hospital of Tianjin Medical University, Tianjin 300211, China Tianjin Key Laboratory of Urology, Tianjin Institute of Urology Medical University, Tianjin 300211, China c Department of Geratology, the Second Hospital of Tianjin Medical University, Tianjin 300211, China d Tianjin Institute of Geratology, Tianjin 300211, China b
art ic l e i nf o
a b s t r a c t
Article history: Received 27 September 2014 Received in revised form 28 November 2014 Accepted 28 November 2014
CYP2C19 genetic polymorphisms influence clopidogrel response and clinical outcomes of cardiovascular disease. However, data on their relationship in stroke patients are scarce. We aimed to investigate the influence of CYP2C19 polymorphisms on platelet reactivity and clinical outcomes in ischemic stroke patients treated with clopidogrel. A total of 211 patients were enrolled. All patients were given clopidogrel treatment and underwent CYP2C19 genotyping and platelet function testing by flow cytometry including adenosine diphosphate-induced platelet aggregation (ADP-PAg) and platelet activation markers (PAC-1, CD62P and CD63). The modified Rankin Scale (mRS) was used and ischemic events were evaluated. A total of 129 (61.1%) of the 211 enrolled patients were carriers of CYP2C19 lossof-function (LOF) alleles (n2, n3). After clopidogrel therapy for 7 days, the levels of ADP-PAg, PAC-1, CD62P and CD63 were higher in carriers than noncarriers. CYP2C19 carriage was associated with more frequent high residual platelet reactivity. CYP2C19 polymorphisms alone could explain 12.9%, 4.3%, 8.9% and 5.5% of the inter-individual variability of ADP-PAg, PAC-1, CD62P and CD63 after clopidogrel treatment, respectively. At 6-month follow-up, 38 (19%) patients were scored poor prognosis and 15 (7.6%) ischemic events were observed. Carriers had poorer prognosis than noncarriers (P¼ 0.025). No significant association of CYP2C19 carriage with ischemic events was found. Multiple regression analysis showed that CYP2C19 carriage was an independent predictor of poor prognosis (odds ratio, 3.01; 95% confidence interval, 1.23–7.38; P ¼0.016). In conclusion, carriage of the CYP2C19 LOF allele has significant influence on clopidogrel response and prognosis in patients with ischemic stroke. & 2014 Published by Elsevier B.V.
Chemical compounds studied in this article: Clopidogrel (PubChem CID: 60606) Adenosine diphosphate (PubChem CID: 6022) Keywords: CYP2C19 Genetic polymorphism Clopidogrel Platelet function tests Flow cytometry Ischemic stroke
1. Introduction Platelet activation and aggregation play pivotal roles in the onset of ischemic stroke (Freynhofer et al., 2012; Tohgi et al., 1991). Elevated platelet activation and aggregation level lead to thrombin generation and vascular injury, which contribute substantially to the pathophysiology of ischemic stroke. Clopidogrel is a prodrug requiring biotransformation to form active metabolites, which act by irreversibly inhibiting the platelet P2Y12 receptor and thus
Abbreviations: ADP-PAg, adenosine diphosphate-induced platelet aggregation; LOF, loss-of-function; mRS, the modified Rankin Scale; RPR, residual platelet reactivity; HRPR, high residual platelet reactivity n Correspondence to: Department of Neurology, the Second Hospital of Tianjin Medical University, no. 23 Pingjiang Road, Hexi District, Tianjin 300211, China. Tel.: þ 86 22 8832 8514; fax: þ 86 22 2830 9286. E-mail address: [email protected] (X. Li). 1 These two authors contributed equally to this article.
platelet aggregation. It is commonly used to regulate the activated platelet for secondary prevention of ischemic stroke (Jauch et al., 2013) and to prevent early recurrence (Kennedy et al., 2007). Clopidogrel was superior to aspirin with a relative risk reduction of 7.3% in ischemic stroke patients especially in high-risk subgroups in CAPPIE study (Committee, 1996). Although the efficacy of clopidogrel is clear, observational studies have demonstrated that there exists considerable variability in clopidogrel response and suboptimal response to clopidogrel can result in recurrent ischemic events in cardiovascular disease (Gurbel et al., 2007) and stroke (Bennett and Yan, 2013). Numerous factors including clinical, cellular and genetic aspects contribute to poor response of clopidogrel (Campo et al., 2011b). Currently, the genetic polymorphism has been considered as the most important determinant (Campo et al., 2010b). The cytochrome P450 (CYP) 2C19 enzyme system, which contributes both of two oxidative metabolic steps of clopidogrel activation, had been reported to be a certain factor of the wide inter-individual variability
http://dx.doi.org/10.1016/j.ejphar.2014.11.037 0014-2999/& 2014 Published by Elsevier B.V.
Please cite this article as: Qiu, L.-N., et al., Influence of CYP2C19 polymorphisms on platelet reactivity and clinical outcomes in ischemic stroke patients treated with clopidogrel. Eur J Pharmacol (2014), http://dx.doi.org/10.1016/j.ejphar.2014.11.037i
67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90
L.-N. Qiu et al. / European Journal of Pharmacology ∎ (∎∎∎∎) ∎∎∎–∎∎∎
2
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66
of clopidogrel response (Mega et al., 2009). Carriers of at least one loss-of-function (LOF) allele of CYP2C19 (known as CYP2C19 n2 and CYP2C19 n3 alleles) have been reported to have lower levels of the active clopidogrel metabolism, reduced platelet inhibition and a higher prevalence of clinical outcomes (Brandt et al., 2007; Frere et al., 2008; Simon et al., 2009). While most studies were conducted in patients with cardiovascular disease, data on their relationship in stroke patients are scarce. In consideration of the poor clinical prognosis of stroke and high stroke recurrence (Antithrombotic Trialists' Collaboration, 2002), it merits considerable attention to clopidogrel response in stroke patients. In the present study, we aimed to evaluate the association of CYP2C19 polymorphisms with clopidogrel response and clinical sequelae in clopidogrel-treated Chinese patients who suffered ischemic stroke.
2. Materials and methods 2.1. Study population Between April 2012 and December 2013, a total of 211 patients were recruited from the Second Hospital of Tianjin Medical University, if they were admitted to hospital within a week after the symptom onset and were diagnosed as acute ischemic stroke by a neurologist. Exclusion criteria included: (1) treatment of anticoagulants, thrombolytic agents and other antiplatelet drugs within 2 weeks; (2) the presence of cranial bleeding or active hemorrhage; trauma, surgery; deep vein or arterial thrombosis within the preceding 3 months; (3) severe hepatic or renal dysfunction; malignant diseases; chronic inflammatory diseases or infectious conditions at study entry. All patients enrolled were given clopidogrel (75 mg once daily) therapy, underwent CYP2C19 genotyping once and platelet function testing before and 7 days after the clopidogrel regimen. The genotyping and platelet function testing were performed by two independent groups, who were unaware of patient individual information and testing results of each other. The study was approved by the Ethics Committee of the Second Hospital of Tianjin Medical University and written informed consent was obtained from every patient before enrollment. 2.2. Platelet function testing Blood samples for platelet function tests with flow cytometry were drawn using a standardized technique as previously described (Schmitz et al., 1998). Blood was anticoagulated with 3.8% sodium citrate (0.129 M/L in dilution 1:10). Platelets aggregation was assessed on a flow cytometer (FCL500MPL, Beckman Coulter, America) by stimulating with 20 μmol/L ADP (Sigma Biosciences, America). The peridinin chlorophyll protein (PerCP)conjugated anti-CD61 (Becton Dickinson Biosciences, America) was applied as a general platelet marker to identify the platelets. After platelets in whole blood were stimulated with ADP for 5 min, the blood mixture was compounded with saturating concentration of anti-CD61 PerCP and incubated at room temperature in the dark for 15 min. Then the sample was fixed with 1% paraformaldehyde in phosphate buffered saline for 30 min. After immunolabeling and fixation, the samples were analyzed on the flow cytometer. Side light scatter and expression of CD61 were used to discriminate platelets from other blood cells. Then 5000 platelets were gated. The result was expressed as the percentage of aggregates in the CD61-identified platelets. Platelet activation markers were determined using antibodies including fluorescein isothiocyanate (FITC)-labeled PAC-1 (activated glycoprotein IIb/IIIa receptors), phycoethrin (PE)-labeled
antibodies agonist CD62P (P-selectin) or CD63 (lysosome-associated membrane protein) (Becton-Dickinson Biosciences, America). Arg-Gly-Asp-Ser (RGDS) tetrapeptide (Sigma Biosciences, America) and mouse-IgG1 (Becton-Dickinson Biosciences, America) were used as isotype-negative controls to define nonspecific binding. The anti-CD61 PerCP was used to identify the platelets. The process of incubation and fixation was as described above. Then the samples were analyzed on FCL500MPL. Platelets were discriminate from other blood cells by using side light scatter and expression of CD61. Results were expressed as percentage of antibody-positive platelets which exceeded 99% of the control platelets. 2.3. CYP2C19 genotyping Genomic DNA was isolated using a commercially available DNA extractor kit (Cwbiotech, Beijing, China) according to the manufacturer's instructions. Polymerase chain reaction–restriction fragment length polymorphisms (PCR–RELPs) for the CYP2C19 LOF alleles on 2 polymorphic positions, n2 (681G4A, rs4244285) and n3 (636G4A, rs4986893), were performed as previously described (de Morais et al., 1994). The primers of CYP2C19 n2 and n3 were designed and synthesized by BGI Beijing Corporation (Table 1). Fast-digest restriction enzyme SmaI and BamHI (Takara biotechnology, Dalian, China) for CYP2C19 n2 and n3 was used. Random sampling from each genotype of CYP2C19 for direct DNA sequencing confirmed the genotyping results, and the results were 100% concordant. The CYP2C19 genotypes were classified into three subgroups: an extensive metabolizer (EM) carrying normal function alleles (CYP2C19 n1/n1), an intermediate metabolizer (IM) carrying one LOF allele (n1/n2 or n1/n3) and a poor metabolizer (PM) carrying two LOF alleles (n2/n2, n2/n3 or n3/n3). 2.4. Data collection and follow-up Data collection and follow-up were completed by another independent group and were unaware of the genotypic and platelet function information. Clinical data were collected from all patients including demographic characteristics and stroke risk factors on admission. Follow-up was performed at the end of 6 months after enrollment by clinical visits or telephone interviews. The primary end point was defined as a composite of recurrence of ischemic stroke, nonfatal myocardial infarction and death. Ischemic stroke recurrence was diagnosed as a focal neurological deficit lasting more than 24 h with ischemic cerebral lesions confirmed by computed tomography (CT) or magnetic resonance imaging (MRI). Myocardial infarction was diagnosed when two of the following three criteria were met: typical symptoms; increased cardiac-enzyme levels; and diagnostic electrocardiographic changes. Cardiovascular death was defined as deaths occurring within 24 h after symptoms onset without other causes evidence (Lonn et al., 2006). Clinical follow-up was censored when any above endpoint was defined or 6-month follow-up finished. At the same time, the modified Rankin Scale (mRS) was used. A good prognosis was defined as mRS r 2, while mRS 4 2 was considered as poor prognosis. Table 1 The primer sequences of CYP2C19 n2 (G681A) and n3 (G636A). Alleles
Primer sequences
CYP2C19 G681A
F 50 -ACCAGAGCTTGGCATATTGTATCT-30 R 50 -GATTCTTGGTGTTCTTTTACTTTCT-30
CYP2C19 G636A
F 50 -TTTCATCCTGGGCTGTGCTC-30 R 50 -TGTACTTCAGGGCTTGGTCAAT-30
Abbreviations: F, forward; R, reverse.
Please cite this article as: Qiu, L.-N., et al., Influence of CYP2C19 polymorphisms on platelet reactivity and clinical outcomes in ischemic stroke patients treated with clopidogrel. Eur J Pharmacol (2014), http://dx.doi.org/10.1016/j.ejphar.2014.11.037i
67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100 101 102 103 104 105 106 107 108 109 110 111 112 113 114 115 116 117 118 119 120 121 122 123 124 125 126 127 128 129 130 131 132
L.-N. Qiu et al. / European Journal of Pharmacology ∎ (∎∎∎∎) ∎∎∎–∎∎∎
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66
2.5. Statistical methods The statistical analysis was performed by IBM SPSS 19.0 software. Two-sided P-value o0.05 was considered to be significant. Continuous data were expressed as mean 7S.D. and categorical data were presented as counts (percentages). Allele frequencies were tested for Hardy Weinberg Equilibrium, using the chi-square test. Baseline characteristics and platelet function tests between carriers and noncarriers were compared with independent-sample t tests for continuous variables and chi-square tests or Fisher's exact tests for categorical variables. Platelet function parameters before and after clopidogrel treatment were compared with pairwise t tests. The correlation between platelet activation markers and ADP-PAg was evaluated with the Pearson correlation coefficient (r) and the Student's t-test. Univariate and multivariate linear regression analysis with the coefficient of determination (R2) calculation was used to identify the independent contribution of CYP2C19 LOF allele carriage and other variables to the interindividual variability in platelet reactivity. Significant independent predictors of clinical prognosis were detected by using a multivariate binary logistic analysis.
3. Results 3.1. Patient characteristics and the frequencies of CYP2C19 genotypes
Table 2 Baseline characteristics of the study population. Characteristics
CYP2C19 genotypes
t/χ2
P
3.998 0.687 0.211 1.237 2.407 0.019 1.789 1.259 0.348 0.811 1.573 0.209 1.466 1.096 0.015 0.637 0.398 0.418 1.273 2.656
0.688 0.407 0.833 0.266 0.121 0.891 0.181 0.262 0.555 0.368 0.210 0.648 0.688 0.344 0.967 0.982 0.189 0.757 0.348 0.868
Noncarriers (n¼ 82) Carriers (n ¼129) Age (years) Female sex BMI (kg/m2) Current smoking Hypertension Coronary artery disease Diabetes mellitus Hyperlipidemiaa Previous stroke/TIA Usage of statins Usage of PPI Usage of CCB Neutrophil (%) Hemoglobin (g/dl) Platelet ( 109/L) Fibrinogen (g/L) Cholesterol (mmol/L) Triglyceride (mmol/L) LDL-C (mmol/L) HDL-C (mmol/L)
67.4 7 13.6 34 (41.5%) 24.27 1.8 25 (30.5%) 48 (58.5%) 30 (36.6%) 22 (26.8%) 38 (46.3%) 26 (31.7%) 51 (62.2%) 29 (35.4%) 25 (30.5%) 71.5 711.4 137.3 7 15.8 220.1 761.2 3.45 7 0.99 5.03 7 0.86 1.737 0.98 3.45 7 0.88 0.98 7 0.21
66.7 7 11.5 61 (47.3%) 24.17 2.3 49 (38.0%) 89 (69.0%) 46 (35.7%) 46 (35.7%) 70 (54.3%) 46 (35.7%) 71 (55.9%) 56 (44.1%) 35 (27.6%) 70.67 9.6 139.9 7 21.2 219.8 7 61.5 3.45 71.06 4.88 70.84 1.7770.86 3.31 71.16 0.977 0.25
Data are presented as mean 7 S.D. or n (%). Abbreviations: BMI, body mass index; TIA, transient ischemic attack; PPI, proton pump inhibitor; CCB, calcium-channel blocker; LDL-C, low-density lipoprotein cholesterol; HDL-C, high-density lipoprotein cholesterol. a
During the study period, a total of 211 patients were enrolled. The distribution of CYP2C19 polymorphisms were 82 (38.9%), 80 (37.9%), 17 (8.0%), 21 (10.0%), 8 (3.8%) and 3 (1.4%) for n1/n1, n1/n2, n1/n3, n2/n2, n2/n3, and n3/n3, respectively. Therefore, 129 (61.1%) patients were carriers of at least one CYP2C19 LOF allele [IM: 97 (46.0%), PM: 32 (15.1%)] with 82 (38.9%) patients were noncarriers. The frequencies of CYP2C19 n2 and n3 in the cohort were 30.8% and 7.3%, respectively. The proportions of genotypes for each allele in the population were consistent with those predicted by the Hardy–Weinberg equilibrium for polymorphisms (P 40.05). Clinical characteristics of carriers and noncarriers are listed in Table 2. Variables known to be associated with platelet reactivity were well balanced between carriers and noncarriers.
3
Hypercholesterolemia or hyperglyceridemia.
3.2. Platelet function tests in carriers and noncarriers As shown in Fig. 1A, both ADP-PAg and platelet activation markers (PAC-1, CD62P and CD63) at baseline (before clopidogrel treatment) were consistent between carriers and noncarriers (all P values 4 0.05). After clopidogrel treatment for 7 days, the four platelet function parameters including ADP-PAg, PAC-1, CD62P and CD63 were significantly diminished (all P values o 0.001). The residual platelet reactivity (RPR) was significantly higher in carriers compared with noncarriers (all P values o 0.05, Fig. 1B). The multivariate liner regression model demonstrated that carriage of the CYP2C19 LOF allele was significantly associated with RPR including ADP-PAg, PAC-1, CD62P and CD63. CYP2C19 polymorphisms explained 12.9%, 4.3%, 8.9% and 5.5% of the interindividual variability of ADP-PAg, PAC-1, CD62P and CD63 after clopidogrel treatment, respectively, compared with 37.5%, 24.7%, 34.4% and 18.5% when clinical variables were included (Table 3). The clinical variables including age, hypertension, acute coronary disease, diabetes mellitus and basal platelet reactivity were independently associated with inter-individual variability in RPR. High residual platelet reactivity (HRPR) was defined as Z75th percentiles of ADP-PAg, PAC-1, CD62P and CD63 in this study. The corresponding cutoff values were 28.54%, 42.92%, 24.32% and 27.33%, respectively. HRPR with ADP-PAg Z28.54% was detected in 43 patients (33.3%) in carriers compared with 10 patients
Fig. 1. Association of CYP2C19 loss-of-function allele carriage with platelet reactivity including ADP-PAg, PAC-1, CD62P and CD63 at baseline (A) and after clopidogrel treatment (B). Data are shown as mean þstandard error (error bars). ADP-PAg, adenosine diphosphate-induced platelet aggregation. nP o0.05.
(12.2%) patients in noncarriers (P ¼0.001). HRPR with PAC1 Z42.92% was detected in 42 patients (25.1%) in carriers compared with 11 patients (13.4%) in noncarriers (P ¼0.002). HRPR with CD62P Z24.32% was detected in 41 patients (31.8%) in carriers compared with 12 patients (14.6%) in noncarriers
Please cite this article as: Qiu, L.-N., et al., Influence of CYP2C19 polymorphisms on platelet reactivity and clinical outcomes in ischemic stroke patients treated with clopidogrel. Eur J Pharmacol (2014), http://dx.doi.org/10.1016/j.ejphar.2014.11.037i
67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100 101 102 103 104 105 106 107 108 109 110 111 112 113 114 115 116 117 118 119 120 121 122 123 124 125 126 127 128 129 130 131 132
L.-N. Qiu et al. / European Journal of Pharmacology ∎ (∎∎∎∎) ∎∎∎–∎∎∎
4
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66
Table 3 Linear regression analysis for determining association of platelet reactivity and other variables. ADP-PAg
CYP2C19 LOF allele Clinical variablesa CYP2C19 LOF allele þ clinical variables P for changeb
PAC-1
CD62P
CD63
R2
P
R2
P
R2
P
R2
P
12.9 28.9 37.5
o0.001 o0.001 o0.001 o0.001
4.3 21.6 24.7
0.002 o 0.001 o 0.001 o 0.001
8.9 28.9 34.4
o 0.001 o 0.001 o 0.001 o 0.001
5.5 13.6 18.5
0.001 o 0.001 o 0.001 o 0.001
Percentage of variability in platelet reactivity explained was defined as coefficient determination (R2), 100%. Abbreviations: LOF, loss-of-function; ADP-PAg, adenosine diphosphate-induced platelet aggregation. a Age, hypertension, coronary artery disease, diabetes mellitus and basal platelet reactivity were significantly associated with residual platelet reactivity in univariate analysis (P o 0.1). b P value for the change in R2 when adding clinical variables to CYP2C19 LOF allele carriage.
Fig. 2. Correlation between different platelet parameters including ADP-PAg, PAC-1, CD62P and CD63 by flow cytometry after clopidogrel treatment. Cut-off values at 75th percentiles are indicated by dotted lines (Z 28.54% for ADP-PAg, Z42.92% for PAC-1, Z24.32% for CD62P and Z 27.33% for CD63). ADP-PAg, adenosine diphosphate-induced platelet aggregation.
(P ¼0.005). HRPR with CD63 Z27.33% was detected in 40 patients (31.0%) compared with 12 patients (14.6%) in noncarriers (P ¼0.007). A significant correlation between ADP-PAg and three platelet activation markers was observed: r ¼ 0.207 (P ¼0.002) for ADP-PAg and PAC-1; r ¼0.279 (Po 0.001) for ADP-PAg and CD62P; r¼ 0.202 (P ¼0.003) for ADP-PAg and CD63 (Fig. 2).
3.3. CYP2C19 genotypes and clinical prognosis Clinical follow-up was completed for 94.6% (198/211) of the enrolled patients. At 6 months, poor prognosis was observed in 30 patients in carriers of the CYP2C19 LOF allele compared with 8 patients in noncarriers (24% vs 11.0%, P ¼0.025). The multivariate binary regression model showed that carriage of the CYP2C19 LOF
Please cite this article as: Qiu, L.-N., et al., Influence of CYP2C19 polymorphisms on platelet reactivity and clinical outcomes in ischemic stroke patients treated with clopidogrel. Eur J Pharmacol (2014), http://dx.doi.org/10.1016/j.ejphar.2014.11.037i
67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100 101 102 103 104 105 106 107 108 109 110 111 112 113 114 115 116 117 118 119 120 121 122 123 124 125 126 127 128 129 130 131 132
L.-N. Qiu et al. / European Journal of Pharmacology ∎ (∎∎∎∎) ∎∎∎–∎∎∎
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66
allele was a significant independent predictor of poor prognosis (OR 3.01, 95% CI 1.23–7.38, P¼ 0.016) (Table 4). Meanwhile, complicating coronary artery disease was also an independent risk factor of poor prognosis in ischemic stroke patients on clopidogrel treatment (OR 2.38, 95% CI 1.11–5.06, P¼ 0.025).There were 15 (7.6%) primary end points during 6-month follow-up. Of which, 10 (5.1%) were ischemic stroke, 2 (1.0%) were myocardial infarctions and 3 (1.5%) were cardiovascular deaths. The characteristic of each primary event is listed in Table 5. About a half of patients had stopped clopidogrel therapy when they suffered an event. The accumulated primary end points were occurred in 12 patients in carriers compared with 3 patients in noncarriers (9.6% vs 4.1%, P¼ 0.159).
4. Discussion In the present study, platelet function tests and clinical prognosis in ischemic stroke patients on clopidogrel treatment were assessed to evaluate the influence of CYP2C19 genetic polymorphisms on clopidogrel response. Previously, conducted in different population and using various methods, multiple studies have reported that carriers with at least one CYP2C19 LOF alleles mainly n2 n3 had significantly diminished platelet inhibition and a higher risk of clinical outcomes than noncarriers in patients with acute coronary syndrome (Collet et al., 2009; Mega et al., 2009, 2010; Shuldiner et al., 2009). In the present study, among patients with Table 4 Multivariate logical regression analysis for predictors of poor prognosis. Variables
OR (95% CI)
P
Age Female sex Current smoking Hypertension Coronary artery disease Diabetes mellitus Hyperlipidemia Previous stroke/TIA CYP2C19 LOF allele carriage
1.02 1.03 0.78 1.37 2.38 0.85 0.61 1.06 3.01
0.234 0.942 0.574 0.451 0.025 0.711 0.205 0.882 0.016
(0.99–1.05) (0.46–2.31) (0.33–1.84) (0.60–3.14) (1.11–5.06) (0.37–1.97) (0.27–1.32) (0.49–2.32) (1.23–7.38)
The abbreviations are as in Tables 1 and 2.
5
ischemic stroke on clopidogrel treatment, we demonstrated that the CYP2C19 LOF allele carriage was significantly associated with RPR, including ADP-PAg and platelet activation (PAC-1, CD62P and CD63). These platelet function parameters have been showed to be well inhibited by clopidogrel. What is more, three platelet activation markers significantly correlated with ADP-PAg, which is a widely used platelet function parameter. It suggested that they might be valuable for assessing clopidogrel response. Yang et al. (2013) found ADP-PAg after clopidogrel treatment for 7 days was significantly higher in carriers of one or two CYP2C19 LOF allele than in noncarriers among ischemic stroke patients, and concluded that CYP2C19 n2 or n3 LOF alleles were independently associated ADP-PAg measurements. Jia et al. (2013) reported the significant association between CYP2C19 LOF allele carriage and high prevalence of clopidogrel resistance, which was defined as a reduction of ADP-PAg before and after clopidogrel treatment. We defined clopidogrel resistance as HRPR with RPR in the fourth quartile, because platelet reactivity is continuous data and there is wide inter-individual variability. Regarding of the inter-individual variability of ADP-PAg, baseline low platelet reactivity may cause overestimated risk of poor responders at the determination of reduction of ADP-PAg, while HRPR after clopidogrel treatment remained with underestimated risk in good responders (Bonello et al., 2010). Meanwhile, baseline platelet reactivity was not associated with CYP2C19 genotypes in our study, as well as described in the previous study (Nakata et al., 2013). What is more, based on RPR after clopidogrel treatment, studies also have demonstrated that clopidogrel poor response was significantly associated with worse clinical outcomes. But it was insufficiently powered to detect stroke occurrence (Campo et al., 2010a). A meta-analysis including 14 studies with 11,959 patients showed that HRPR in patients with coronary artery disease undergoing percutaneous coronary intervention was a risk factor for stroke (Taglieri et al., 2014). Therefore, the RPR appeared to be a better assessment of ischemic risk than reduction of ADP-PAg. We found carriers of at least one CYP2C19 LOF allele had higher prevalence of HRPR, when platelet inhibition was assessed by either ADP-PAg or platelet activation (PAC-1, CD62P and CD63). The present study also found that carriers of CYP2C19 LOF alleles had 3.01-fold increased risk of poor prognosis in 6-month follow-up compared with noncarriers among ischemic stroke patients treated
Table 5 Characteristics of patients with primary events. Patients (gender, age [yrs])
ADP-PAg (HRPR)
PAC-1 (HRPR)
CD62P (HRPR)
Recurrent ischemic #1 (Male, 66) #2 (Female, 71) #3 (Male, 76) #4 (Male, 76) #5 (Female, 89) #6 (Male, 86) #7 (Female, 76) #8 (Male,71) #9 (Female, 73) #10 (Male, 73)
stroke 43.64 ( þ) 65.00 ( þ) 27.03 ( ) 51.88 ( þ) 32.94 ( þ) 27.06 ( ) 21.56 ( ) 12.91 ( ) 39.50 ( þ) 42.62 ( þ)
58.04 20.41 43.76 28.96 59.55 23.70 35.77 32.08 48.77 49.65
33.66 37.80 12.88 25.15 22.34 22.20 34.50 41.90 12.14 29.65
(þ ) () (þ ) () (þ ) () () () (þ ) (þ )
(þ) (þ) () (þ) () () (þ) (þ) () (þ)
CD63 (HRPR)
25.44 31.13 22.16 18.51 35.02 33.91 39.66 17.08 28.41 30.65
() (þ) () () (þ) (þ) (þ) () (þ) (þ)
Pre-treatment Interval (h)
Test to events (days)
Clopidogrel therapy duration (weeks)
Clopidgrel therapy mRS status score
15 24 31 22 29 22 48 23 52 24
35 59 44 98 138 55 61 78 107 166
4 8 4 12 12 7 8 8 8 12
Off On Off Off Off On On Off Off Off
3 3 3 2 3 2 1 1 4 4
Myocardial infarction #11 (Female,77) 15.93 ( ) #12 (Female, 55) 45.09 ( þ)
54.27 (þ ) 35.00 ( þ ) 63.16 (þ ) 41.96 ( þ )
52.99 ( þ ) 36 26.71 ( ) 17
79 29
11 4
On On
0 2
Vascular death #13 (Female,74) #14 (Male, 52) #15 (Male,86)
55.76 (þ ) 45.54 ( þ ) 51.85 (þ ) 35.30 ( þ ) 43.96 (þ ) 38.49 ( þ )
33.76 ( þ ) 20 54.67 ( þ ) 28 15.15 ( ) 16
7 16 2
1 2 0
On On On
6 6 6
50.28 ( þ) 20.08 ( ) 48.60 ( þ)
Platelet reactivity listed in the table including ADP-PAg, PAC-1, CD62P and CD63 are all residual platelet reactivity after clopidogrel treatment.þ or in the braces means yes or no for HRPR. Abbreviations: ADP-PAg, adenosine diphosphate-induced platelet aggregation; HRPR, high residual platelet reactivity; mRS, the modified Rankin Scale.
Please cite this article as: Qiu, L.-N., et al., Influence of CYP2C19 polymorphisms on platelet reactivity and clinical outcomes in ischemic stroke patients treated with clopidogrel. Eur J Pharmacol (2014), http://dx.doi.org/10.1016/j.ejphar.2014.11.037i
67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100 101 102 103 104 105 106 107 108 109 110 111 112 113 114 115 116 117 118 119 120 121 122 123 124 125 126 127 128 129 130 131 132
L.-N. Qiu et al. / European Journal of Pharmacology ∎ (∎∎∎∎) ∎∎∎–∎∎∎
6
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66
with clopidogrel. This finding was in consistent with that of Jia et al. (2013). Indeed, carriage of the CYP2C19 LOF allele was a major determinant of poor prognosis in Chinese stroke patients in the present study. While, the absence of an effect of the CYP2C19 LOF allele carriage on primary events was in contrast to findings of previous studies on acute coronary syndromes. Possible explanation for the divergence between our finding on ischemic stroke patients and previous studies on patients with acute coronary syndromes is the heterogeneous cause of stroke (Adams et al., 1993) compared with acute coronary syndrome or unmeasured factors including CYP2C19 n17 gain-of-function allele (Frere et al., 2009) potentially resulting in reduced inter-individual variability. The significant association between CYP2C19 LOF allele variants and diminished clopidogrel response including ADP-PAg, PAC-1, CD62P and CD63 in this study indicated that the whole-blood flow-cytometric method is capable to predict the gene mediated inter-individual variability of clopidogrel response. Light transmittance aggregometry (LTA) is most commonly used for measurement of platelet function. However, certain defects of LTA, such as time consuming, poor specificity and poor reproducibility, limit its use. Without centrifugation and using platelet identified monoclonal antibody, determination of platelet aggregation by whole blood flow cytometry could overcome the defects in LTA and has been proved to be a reliable method in measuring platelet function in our study. The present study found that the frequency of the CYP2C19 LOF allele in Chinese stroke patients was 38.1%, and carriers of at least one LOF allele of CYP2C19 constituted 61.1% of the cohort. In the previous study (Mega et al., 2009), the prevalence of carriage of CYP2C19 LOF allele was 30% in the overall cohort in Westerners. Simultaneously, in patients with CYP2C19 LOF alleles and suffering acute coronary syndrome, risk of adverse clinical events appeared to be higher in the Asian population compared to Westerner population (Jang et al., 2012). Similarly, 1-year cumulative recurrence rate of ischemic stroke was reported to be higher in Chinese (19.9%) (Wang et al., 2013), when compared to that in Westerners (9.4%) (Allen et al., 2010), Although the clinical outcomes of ischemic stroke in Chinese population were irrespective of CYP2C19 carriage in our study, the effect of CYP2C19 polymorphisms on prognosis of ischemic stroke patients was definite. Therefore, it is noteworthy to underscore that the relevant genotyping may guide a more individualized and thus optimizing therapeutic regimens which are essential for improvement of stroke prognosis and the possible prevention of stroke recurrence in Chinese patients. There are several potential limitations in our study. First, other genetic variants except CYP2C19 n2 n3 LOF alleles, such as CYP2C19 n17 gain-of-function allele and PON-1 gene polymorphism in our study, might also contribute to platelet reactivity, clopidogrel response and the likelihood of clinical sequelae. At present, the role of PON-1 in patients with cardiovascular disease was debated or marginal (Campo et al., 2011a), whether it affected the on-clopidogrel platelet reactivity in stroke patients needs further studies. Second, our investigation did not measure the platelet reactivity during the follow-up period, although the time-dependent relationship between the CYP2C19 variants and the antiplatelet effect of clopidogrel was reported in some studies (Wallentin et al., 2010). Third, whether the optional regimens such as altering the dosage or drugs would override the baleful effect of CYP2C19 polymorphisms on stroke patients merits further investigations.
5. Conclusion Taken together, the present study demonstrated that carriage of the CYP2C19 LOF allele had significant influence on antiplatelet effect of clopidogrel and clinical prognosis of Chinese patients with
ischemic stroke. Our findings provide certain evidence on genetic effect of CYP2C19 on clopidogrel responsiveness in stroke patients, but whether the prognosis data associated with CYP2C19 genotypes can be used to guide individualized and thus optimizing the therapeutic regimens for the secondary prevention of stroke needs further studies.
Acknowledgments This work was supported by Major Social Science Program of Tianjin Municipal Education Commission (2011ZD016). The work was also supported by Key Technology Research and Development Program of Science & Technology of Tianjin (12ZCZDSY03100) and Tianjin Municipal Natural Science Foundation (09JCYBJC11400). We are grateful to all patients participating in this study, and Wei Huang and Xiaodong Zhou of Tianjin Institute of Urology for their technical support.
References Adams Jr., H.P., Bendixen, B.H., Kappelle, L.J., Biller, J., Love, B.B., Gordon, D.L., Marsh 3rd, E.E., 1993. Classification of subtype of acute ischemic stroke. Definitions for use in a multicenter clinical trial. TOAST. Trial of Org 10172 in Acute Stroke Treatment. Stroke 24, 35–41. Allen, N.B., Holford, T.R., Bracken, M.B., Goldstein, L.B., Howard, G., Wang, Y., Lichtman, J.H., 2010. Geographic variation in one-year recurrent ischemic stroke rates for elderly medicare beneficiaries in the USA. Neuroepidemiology 34, 123–129. Antithrombotic Trialists' Collaboration, 2002. Collaborative meta-analysis of randomised trials of antiplatelet therapy for prevention of death, myocardial infarction, and stroke in high risk patients. BMJ 324, 71–86. Bennett, D., Yan, B., 2013. Suboptimal response to clopidogrel: a genetic risk factor for recurrent ischaemic stroke. J. Clin. Neurosci. 20, 767–770. Bonello, L., Tantry, U.S., Marcucci, R., Blindt, R., Angiolillo, D.J., Becker, R., Bhatt, D.L., Cattaneo, M., Collet, J.P., Cuisset, T., Gachet, C., Montalescot, G., Jennings, L.K., Kereiakes, D., Sibbing, D., Trenk, D., Van Werkum, J.W., Paganelli, F., Price, M.J., Waksman, R., Gurbel, P.A., Working Group on High On-Treatment Platelet Reactivity, 2010. Consensus and future directions on the definition of high ontreatment platelet reactivity to adenosine diphosphate. J. Am. Coll. Cardiol. 56, 919–933. Brandt, J.T., Close, S.L., Iturria, S.J., Payne, C.D., Farid, N.A., Ernest 2nd, C.S., Lachno, D.R., Salazar, D., Winters, K.J., 2007. Common polymorphisms of CYP2C19 and CYP2C9 affect the pharmacokinetic and pharmacodynamic response to clopidogrel but not prasugrel. J. Thromb. Haemost. 5, 2429–2436. Campo, G., Ferraresi, P., Marchesini, J., Bernardi, F., Valgimigli, M., 2011a. Relationship between paraoxonase Q192R gene polymorphism and on-clopidogrel platelet reactivity over time in patients treated with percutaneous coronary intervention. J. Thromb. Haemost. 9, 2106–2108. Campo, G., Fileti, L., de Cesare, N., Meliga, E., Furgieri, A., Russo, F., Colangelo, S., Brugaletta, S., Ferrari, R., Valgimigli, M., 3T/2R Investigators, 2010. Long-term clinical outcome based on aspirin and clopidogrel responsiveness status after elective percutaneous coronary intervention: a 3T/2R (tailoring treatment with tirofiban in patients showing resistance to aspirin and/or resistance to clopidogrel) trial substudy. J. Am. Coll. Cardiol. 56, 1447–1455. Campo, G., Fileti, L., Valgimigli, M., Tebaldi, M., Cangiano, E., Cavazza, C., Marchesini, J., Ferrari, R., 2010b. Poor response to clopidogrel: current and future options for its management. J. Thromb. Thrombolysis 30, 319–331. Campo, G., Miccoli, M., Tebaldi, M., Marchesini, J., Fileti, L., Monti, M., Valgimigli, M., Ferrari, R., 2011b. Genetic determinants of on-clopidogrel high platelet reactivity. Platelets 22, 399–407. Collet, J.P., Hulot, J.S., Pena, A., Villard, E., Esteve, J.B., Silvain, J., Payot, L., Brugier, D., Cayla, G., Beygui, F., Bensimon, G., Funck-Brentano, C., Montalescot, G., 2009. Cytochrome P450 2C19 polymorphism in young patients treated with clopidogrel after myocardial infarction: a cohort study. Lancet 373, 309–317. CAPRIE Steering Committee, 1996. A randomised, blinded, trial of clopidogrel versus aspirin in patients at risk of ischaemic events (CAPRIE). CAPRIE Steering Committee. Lancet 348, 1329–1339. de Morais, S.M., Wilkinson, G.R., Blaisdell, J., Nakamura, K., Meyer, U.A., Goldstein, J.A., 1994. The major genetic defect responsible for the polymorphism of Smephenytoin metabolism in humans. J. Biol. Chem. 269, 15419–15422. Frere, C., Cuisset, T., Gaborit, B., Alessi, M.C., Hulot, J.S., 2009. The CYP2C19n17 allele is associated with better platelet response to clopidogrel in patients admitted for non-ST acute coronary syndrome. J. Thromb. Haemost. 7, 1409–1411. Frere, C., Cuisset, T., Morange, P.E., Quilici, J., Camoin-Jau, L., Saut, N., Faille, D., Lambert, M., Juhan-Vague, I., Bonnet, J.L., Alessi, M., 2008. Effect of cytochrome p450 polymorphisms on platelet reactivity after treatment with clopidogrel in acute coronary syndrome. Am. J. Cardiol. 101, 1088–1093.
Please cite this article as: Qiu, L.-N., et al., Influence of CYP2C19 polymorphisms on platelet reactivity and clinical outcomes in ischemic stroke patients treated with clopidogrel. Eur J Pharmacol (2014), http://dx.doi.org/10.1016/j.ejphar.2014.11.037i
67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100 101 102 103 104 105 106 107 108 109 110 111 112 113 114 115 116 117 118 119 120 121 122 123 124 125 126 127 128 129 130 131 132
L.-N. Qiu et al. / European Journal of Pharmacology ∎ (∎∎∎∎) ∎∎∎–∎∎∎
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30
Freynhofer, M.K., Bruno, V., Wojta, J., Huber, K., 2012. The role of platelets in atherothrombotic events. Curr. Pharm. Des. 18, 5197–5214. Gurbel, P.A., Becker, R.C., Mann, K.G., Steinhubl, S.R., Michelson, A.D., 2007. Platelet function monitoring in patients with coronary artery disease. J. Am. Coll. Cardiol. 50, 1822–1834. Jang, J.S., Cho, K.I., Jin, H.Y., Seo, J.S., Yang, T.H., Kim, D.K., Kim, D.S., Seol, S.H., Kim, D.I., Kim, B.H., Park, Y.H., Je, H.G., Jeong, Y.H., Lee, S.W., 2012. Meta-analysis of cytochrome P450 2C19 polymorphism and risk of adverse clinical outcomes among coronary artery disease patients of different ethnic groups treated with clopidogrel. Am. J. Cardiol. 110, 502–508. Jauch, E.C., Saver, J.L., Adams Jr., H.P., Bruno, A., Connors, J.J., Demaerschalk, B.M., Khatri, P., McMullan Jr., P.W., Qureshi, A.I., Rosenfield, K., Scott, P.A., Summers, D.R., Wang, D.Z., Wintermark, M., Yonas, H., American Heart Association Stroke Council Council on Cardiovascular Nursing Council on Peripheral Vascular Disease Council on Clinical Cardiology, 2013. Guidelines for the early management of patients with acute ischemic stroke: a guideline for healthcare professionals from the American Heart Association/American Stroke Association. Stroke 44, 870–947. Jia, D.M., Chen, Z.B., Zhang, M.J., Yang, W.J., Jin, J.L., Xia, Y.Q., Zhang, C.L., Shao, Y., Chen, C., Xu, Y., 2013. CYP2C19 polymorphisms and antiplatelet effects of clopidogrel in acute ischemic stroke in China. Stroke 44, 1717–1719. Kennedy, J., Hill, M.D., Ryckborst, K.J., Eliasziw, M., Demchuk, A.M., Buchan, A.M., FASTER Investigators, 2007. Fast assessment of stroke and transient ischaemic attack to prevent early recurrence (FASTER): a randomised controlled pilot trial. Lancet Neurol. 6, 961–969. Lonn, E., Yusuf, S., Arnold, M.J., Sheridan, P., Pogue, J., Micks, M., McQueen, M.J., Probstfield, J., Fodor, G., Held, C., Genest Jr., J., Heart Outcomes Prevention Evaluation Investigators, 2006. Homocysteine lowering with folic acid and B vitamins in vascular disease. N. Engl. J. Med. 354, 1567–1577. Mega, J.L., Close, S.L., Wiviott, S.D., Shen, L., Hockett, R.D., Brandt, J.T., Walker, J.R., Antman, E.M., Macias, W., Braunwald, E., Sabatine, M.S., 2009. Cytochrome p-450 polymorphisms and response to clopidogrel. N. Engl. J. Med. 360, 354–362. Mega, J.L., Simon, T., Collet, J.P., Anderson, J.L., Antman, E.M., Bliden, K., Cannon, C.P., Danchin, N., Giusti, B., Gurbel, P., Horne, B.D., Hulot, J.S., Kastrati, A., Montalescot, G., Neumann, F.J., Shen, L., Sibbing, D., Steg, P.G., Trenk, D., Wiviott, S.D., Sabatine, M.S., 2010. Reduced-function CYP2C19 genotype and risk of adverse clinical outcomes among patients treated with clopidogrel predominantly for PCI: a meta-analysis. JAMA 304, 1821–1830.
7
Nakata, T., Miyahara, M., Nakatani, K., Wada, H., Tanigawa, T., Komada, F., Hoshino, K., Aoki, T., Nishimura, Y., Tamaru, S., Ito, M., Nishikawa, M., 2013. McLordd groupRelationship between CYP2C19 loss-of-function polymorphism and platelet reactivities with clopidogrel treatment in Japanese patients undergoing coronary stent implantation. Circ. J. 77, 1436–1444. Schmitz, G., Rothe, G., Ruf, A., Barlage, S., Tschope, D., Clemetson, K.J., Goodall, A.H., Michelson, A.D., Nurden, A.T., Shankey, T.V., 1998. European Working Group on Clinical Cell Analysis: consensus protocol for the flow cytometric characterisation of platelet function. Thromb. Haemost. 79, 885–896. Shuldiner, A.R., O’Connell, J.R., Bliden, K.P., Gandhi, A., Ryan, K., Horenstein, R.B., Damcott, C.M., Pakyz, R., Tantry, U.S., Gibson, Q., Pollin, T.I., Post, W., Parsa, A., Mitchell, B.D., Faraday, N., Herzog, W., Gurbel, P.A., 2009. Association of cytochrome P450 2C19 genotype with the antiplatelet effect and clinical efficacy of clopidogrel therapy. JAMA 302, 849–857. Simon, T., Verstuyft, C., Mary-Krause, M., Quteineh, L., Drouet, E., Meneveau, N., Steg, P.G., Ferrieres, J., Danchin, N., Becquemont, L., French Registry of Acute S.T. E. Non-S.T.E.M.I.I.Genetic determinants of response to clopidogrel and cardiovascular events. N. Engl. J. Med. 360, 363–375. Taglieri, N., Bacchi Reggiani, M.L., Palmerini, T., Ghetti, G., Saia, F., Gallo, P., Moretti, C., Dall’Ara, G., Marrozzini, C., Marzocchi, A., Rapezzi, C., 2014. Risk of stroke in patients with high on-clopidogrel platelet reactivity to adenosine diphosphate after percutaneous coronary intervention. Am. J. Cardiol. 113, 1807–1814. Tohgi, H., Suzuki, H., Tamura, K., Kimura, B., 1991. Platelet volume, aggregation, and adenosine triphosphate release in cerebral thrombosis. Stroke 22, 17–21. Wallentin, L., James, S., Storey, R.F., Armstrong, M., Barratt, B.J., Horrow, J., Husted, S., Katus, H., Steg, P.G., Shah, S.H., Becker, R.C., PLATO investigators, 2010. Effect of CYP2C19 and ABCB1 single nucleotide polymorphisms on outcomes of treatment with ticagrelor versus clopidogrel for acute coronary syndromes: a genetic substudy of the PLATO trial. Lancet 376, 1320–1328. Wang, Z., Li, J., Wang, C., Yao, X., Zhao, X., Wang, Y., Li, H., Liu, G., Wang, A., Wang, Y., 2013. Gender differences in 1-year clinical characteristics and outcomes after stroke: results from the China National Stroke Registry. PloS One 8, e56459. Yang, J., Zhao, H.D., Tan, J., Ding, Y.L., Gu, Z.Q., Zou, J.J., 2013. CYP2C19 polymorphism and antiplatelet effects of clopidogrel in Chinese stroke patients. Pharmazie 68, 183–186.
Please cite this article as: Qiu, L.-N., et al., Influence of CYP2C19 polymorphisms on platelet reactivity and clinical outcomes in ischemic stroke patients treated with clopidogrel. Eur J Pharmacol (2014), http://dx.doi.org/10.1016/j.ejphar.2014.11.037i
31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 Q1 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66
Contents lists available at ScienceDirect
European Journal of Pharmacology journal homepage: www.elsevier.com/locate/ejphar
Cardiovascular pharmacology
Influence of CYP2C19 polymorphisms on platelet reactivity and clinical outcomes in ischemic stroke patients treated with clopidogrel Li-Na Qiu a,1, Yan Sun b,1, Lin Wang c,d, Rui-Fa Han b, Xiao-Shuang Xia a, Jie Liu a, Xin Li a,n a
Department of Neurology, the Second Hospital of Tianjin Medical University, Tianjin 300211, China Tianjin Key Laboratory of Urology, Tianjin Institute of Urology Medical University, Tianjin 300211, China c Department of Geratology, the Second Hospital of Tianjin Medical University, Tianjin 300211, China d Tianjin Institute of Geratology, Tianjin 300211, China b
art ic l e i nf o
a b s t r a c t
Article history: Received 27 September 2014 Received in revised form 28 November 2014 Accepted 28 November 2014
CYP2C19 genetic polymorphisms influence clopidogrel response and clinical outcomes of cardiovascular disease. However, data on their relationship in stroke patients are scarce. We aimed to investigate the influence of CYP2C19 polymorphisms on platelet reactivity and clinical outcomes in ischemic stroke patients treated with clopidogrel. A total of 211 patients were enrolled. All patients were given clopidogrel treatment and underwent CYP2C19 genotyping and platelet function testing by flow cytometry including adenosine diphosphate-induced platelet aggregation (ADP-PAg) and platelet activation markers (PAC-1, CD62P and CD63). The modified Rankin Scale (mRS) was used and ischemic events were evaluated. A total of 129 (61.1%) of the 211 enrolled patients were carriers of CYP2C19 lossof-function (LOF) alleles (n2, n3). After clopidogrel therapy for 7 days, the levels of ADP-PAg, PAC-1, CD62P and CD63 were higher in carriers than noncarriers. CYP2C19 carriage was associated with more frequent high residual platelet reactivity. CYP2C19 polymorphisms alone could explain 12.9%, 4.3%, 8.9% and 5.5% of the inter-individual variability of ADP-PAg, PAC-1, CD62P and CD63 after clopidogrel treatment, respectively. At 6-month follow-up, 38 (19%) patients were scored poor prognosis and 15 (7.6%) ischemic events were observed. Carriers had poorer prognosis than noncarriers (P¼ 0.025). No significant association of CYP2C19 carriage with ischemic events was found. Multiple regression analysis showed that CYP2C19 carriage was an independent predictor of poor prognosis (odds ratio, 3.01; 95% confidence interval, 1.23–7.38; P ¼0.016). In conclusion, carriage of the CYP2C19 LOF allele has significant influence on clopidogrel response and prognosis in patients with ischemic stroke. & 2014 Published by Elsevier B.V.
Chemical compounds studied in this article: Clopidogrel (PubChem CID: 60606) Adenosine diphosphate (PubChem CID: 6022) Keywords: CYP2C19 Genetic polymorphism Clopidogrel Platelet function tests Flow cytometry Ischemic stroke
1. Introduction Platelet activation and aggregation play pivotal roles in the onset of ischemic stroke (Freynhofer et al., 2012; Tohgi et al., 1991). Elevated platelet activation and aggregation level lead to thrombin generation and vascular injury, which contribute substantially to the pathophysiology of ischemic stroke. Clopidogrel is a prodrug requiring biotransformation to form active metabolites, which act by irreversibly inhibiting the platelet P2Y12 receptor and thus
Abbreviations: ADP-PAg, adenosine diphosphate-induced platelet aggregation; LOF, loss-of-function; mRS, the modified Rankin Scale; RPR, residual platelet reactivity; HRPR, high residual platelet reactivity n Correspondence to: Department of Neurology, the Second Hospital of Tianjin Medical University, no. 23 Pingjiang Road, Hexi District, Tianjin 300211, China. Tel.: þ 86 22 8832 8514; fax: þ 86 22 2830 9286. E-mail address: [email protected] (X. Li). 1 These two authors contributed equally to this article.
platelet aggregation. It is commonly used to regulate the activated platelet for secondary prevention of ischemic stroke (Jauch et al., 2013) and to prevent early recurrence (Kennedy et al., 2007). Clopidogrel was superior to aspirin with a relative risk reduction of 7.3% in ischemic stroke patients especially in high-risk subgroups in CAPPIE study (Committee, 1996). Although the efficacy of clopidogrel is clear, observational studies have demonstrated that there exists considerable variability in clopidogrel response and suboptimal response to clopidogrel can result in recurrent ischemic events in cardiovascular disease (Gurbel et al., 2007) and stroke (Bennett and Yan, 2013). Numerous factors including clinical, cellular and genetic aspects contribute to poor response of clopidogrel (Campo et al., 2011b). Currently, the genetic polymorphism has been considered as the most important determinant (Campo et al., 2010b). The cytochrome P450 (CYP) 2C19 enzyme system, which contributes both of two oxidative metabolic steps of clopidogrel activation, had been reported to be a certain factor of the wide inter-individual variability
http://dx.doi.org/10.1016/j.ejphar.2014.11.037 0014-2999/& 2014 Published by Elsevier B.V.
Please cite this article as: Qiu, L.-N., et al., Influence of CYP2C19 polymorphisms on platelet reactivity and clinical outcomes in ischemic stroke patients treated with clopidogrel. Eur J Pharmacol (2014), http://dx.doi.org/10.1016/j.ejphar.2014.11.037i
67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90
L.-N. Qiu et al. / European Journal of Pharmacology ∎ (∎∎∎∎) ∎∎∎–∎∎∎
2
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66
of clopidogrel response (Mega et al., 2009). Carriers of at least one loss-of-function (LOF) allele of CYP2C19 (known as CYP2C19 n2 and CYP2C19 n3 alleles) have been reported to have lower levels of the active clopidogrel metabolism, reduced platelet inhibition and a higher prevalence of clinical outcomes (Brandt et al., 2007; Frere et al., 2008; Simon et al., 2009). While most studies were conducted in patients with cardiovascular disease, data on their relationship in stroke patients are scarce. In consideration of the poor clinical prognosis of stroke and high stroke recurrence (Antithrombotic Trialists' Collaboration, 2002), it merits considerable attention to clopidogrel response in stroke patients. In the present study, we aimed to evaluate the association of CYP2C19 polymorphisms with clopidogrel response and clinical sequelae in clopidogrel-treated Chinese patients who suffered ischemic stroke.
2. Materials and methods 2.1. Study population Between April 2012 and December 2013, a total of 211 patients were recruited from the Second Hospital of Tianjin Medical University, if they were admitted to hospital within a week after the symptom onset and were diagnosed as acute ischemic stroke by a neurologist. Exclusion criteria included: (1) treatment of anticoagulants, thrombolytic agents and other antiplatelet drugs within 2 weeks; (2) the presence of cranial bleeding or active hemorrhage; trauma, surgery; deep vein or arterial thrombosis within the preceding 3 months; (3) severe hepatic or renal dysfunction; malignant diseases; chronic inflammatory diseases or infectious conditions at study entry. All patients enrolled were given clopidogrel (75 mg once daily) therapy, underwent CYP2C19 genotyping once and platelet function testing before and 7 days after the clopidogrel regimen. The genotyping and platelet function testing were performed by two independent groups, who were unaware of patient individual information and testing results of each other. The study was approved by the Ethics Committee of the Second Hospital of Tianjin Medical University and written informed consent was obtained from every patient before enrollment. 2.2. Platelet function testing Blood samples for platelet function tests with flow cytometry were drawn using a standardized technique as previously described (Schmitz et al., 1998). Blood was anticoagulated with 3.8% sodium citrate (0.129 M/L in dilution 1:10). Platelets aggregation was assessed on a flow cytometer (FCL500MPL, Beckman Coulter, America) by stimulating with 20 μmol/L ADP (Sigma Biosciences, America). The peridinin chlorophyll protein (PerCP)conjugated anti-CD61 (Becton Dickinson Biosciences, America) was applied as a general platelet marker to identify the platelets. After platelets in whole blood were stimulated with ADP for 5 min, the blood mixture was compounded with saturating concentration of anti-CD61 PerCP and incubated at room temperature in the dark for 15 min. Then the sample was fixed with 1% paraformaldehyde in phosphate buffered saline for 30 min. After immunolabeling and fixation, the samples were analyzed on the flow cytometer. Side light scatter and expression of CD61 were used to discriminate platelets from other blood cells. Then 5000 platelets were gated. The result was expressed as the percentage of aggregates in the CD61-identified platelets. Platelet activation markers were determined using antibodies including fluorescein isothiocyanate (FITC)-labeled PAC-1 (activated glycoprotein IIb/IIIa receptors), phycoethrin (PE)-labeled
antibodies agonist CD62P (P-selectin) or CD63 (lysosome-associated membrane protein) (Becton-Dickinson Biosciences, America). Arg-Gly-Asp-Ser (RGDS) tetrapeptide (Sigma Biosciences, America) and mouse-IgG1 (Becton-Dickinson Biosciences, America) were used as isotype-negative controls to define nonspecific binding. The anti-CD61 PerCP was used to identify the platelets. The process of incubation and fixation was as described above. Then the samples were analyzed on FCL500MPL. Platelets were discriminate from other blood cells by using side light scatter and expression of CD61. Results were expressed as percentage of antibody-positive platelets which exceeded 99% of the control platelets. 2.3. CYP2C19 genotyping Genomic DNA was isolated using a commercially available DNA extractor kit (Cwbiotech, Beijing, China) according to the manufacturer's instructions. Polymerase chain reaction–restriction fragment length polymorphisms (PCR–RELPs) for the CYP2C19 LOF alleles on 2 polymorphic positions, n2 (681G4A, rs4244285) and n3 (636G4A, rs4986893), were performed as previously described (de Morais et al., 1994). The primers of CYP2C19 n2 and n3 were designed and synthesized by BGI Beijing Corporation (Table 1). Fast-digest restriction enzyme SmaI and BamHI (Takara biotechnology, Dalian, China) for CYP2C19 n2 and n3 was used. Random sampling from each genotype of CYP2C19 for direct DNA sequencing confirmed the genotyping results, and the results were 100% concordant. The CYP2C19 genotypes were classified into three subgroups: an extensive metabolizer (EM) carrying normal function alleles (CYP2C19 n1/n1), an intermediate metabolizer (IM) carrying one LOF allele (n1/n2 or n1/n3) and a poor metabolizer (PM) carrying two LOF alleles (n2/n2, n2/n3 or n3/n3). 2.4. Data collection and follow-up Data collection and follow-up were completed by another independent group and were unaware of the genotypic and platelet function information. Clinical data were collected from all patients including demographic characteristics and stroke risk factors on admission. Follow-up was performed at the end of 6 months after enrollment by clinical visits or telephone interviews. The primary end point was defined as a composite of recurrence of ischemic stroke, nonfatal myocardial infarction and death. Ischemic stroke recurrence was diagnosed as a focal neurological deficit lasting more than 24 h with ischemic cerebral lesions confirmed by computed tomography (CT) or magnetic resonance imaging (MRI). Myocardial infarction was diagnosed when two of the following three criteria were met: typical symptoms; increased cardiac-enzyme levels; and diagnostic electrocardiographic changes. Cardiovascular death was defined as deaths occurring within 24 h after symptoms onset without other causes evidence (Lonn et al., 2006). Clinical follow-up was censored when any above endpoint was defined or 6-month follow-up finished. At the same time, the modified Rankin Scale (mRS) was used. A good prognosis was defined as mRS r 2, while mRS 4 2 was considered as poor prognosis. Table 1 The primer sequences of CYP2C19 n2 (G681A) and n3 (G636A). Alleles
Primer sequences
CYP2C19 G681A
F 50 -ACCAGAGCTTGGCATATTGTATCT-30 R 50 -GATTCTTGGTGTTCTTTTACTTTCT-30
CYP2C19 G636A
F 50 -TTTCATCCTGGGCTGTGCTC-30 R 50 -TGTACTTCAGGGCTTGGTCAAT-30
Abbreviations: F, forward; R, reverse.
Please cite this article as: Qiu, L.-N., et al., Influence of CYP2C19 polymorphisms on platelet reactivity and clinical outcomes in ischemic stroke patients treated with clopidogrel. Eur J Pharmacol (2014), http://dx.doi.org/10.1016/j.ejphar.2014.11.037i
67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100 101 102 103 104 105 106 107 108 109 110 111 112 113 114 115 116 117 118 119 120 121 122 123 124 125 126 127 128 129 130 131 132
L.-N. Qiu et al. / European Journal of Pharmacology ∎ (∎∎∎∎) ∎∎∎–∎∎∎
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66
2.5. Statistical methods The statistical analysis was performed by IBM SPSS 19.0 software. Two-sided P-value o0.05 was considered to be significant. Continuous data were expressed as mean 7S.D. and categorical data were presented as counts (percentages). Allele frequencies were tested for Hardy Weinberg Equilibrium, using the chi-square test. Baseline characteristics and platelet function tests between carriers and noncarriers were compared with independent-sample t tests for continuous variables and chi-square tests or Fisher's exact tests for categorical variables. Platelet function parameters before and after clopidogrel treatment were compared with pairwise t tests. The correlation between platelet activation markers and ADP-PAg was evaluated with the Pearson correlation coefficient (r) and the Student's t-test. Univariate and multivariate linear regression analysis with the coefficient of determination (R2) calculation was used to identify the independent contribution of CYP2C19 LOF allele carriage and other variables to the interindividual variability in platelet reactivity. Significant independent predictors of clinical prognosis were detected by using a multivariate binary logistic analysis.
3. Results 3.1. Patient characteristics and the frequencies of CYP2C19 genotypes
Table 2 Baseline characteristics of the study population. Characteristics
CYP2C19 genotypes
t/χ2
P
3.998 0.687 0.211 1.237 2.407 0.019 1.789 1.259 0.348 0.811 1.573 0.209 1.466 1.096 0.015 0.637 0.398 0.418 1.273 2.656
0.688 0.407 0.833 0.266 0.121 0.891 0.181 0.262 0.555 0.368 0.210 0.648 0.688 0.344 0.967 0.982 0.189 0.757 0.348 0.868
Noncarriers (n¼ 82) Carriers (n ¼129) Age (years) Female sex BMI (kg/m2) Current smoking Hypertension Coronary artery disease Diabetes mellitus Hyperlipidemiaa Previous stroke/TIA Usage of statins Usage of PPI Usage of CCB Neutrophil (%) Hemoglobin (g/dl) Platelet ( 109/L) Fibrinogen (g/L) Cholesterol (mmol/L) Triglyceride (mmol/L) LDL-C (mmol/L) HDL-C (mmol/L)
67.4 7 13.6 34 (41.5%) 24.27 1.8 25 (30.5%) 48 (58.5%) 30 (36.6%) 22 (26.8%) 38 (46.3%) 26 (31.7%) 51 (62.2%) 29 (35.4%) 25 (30.5%) 71.5 711.4 137.3 7 15.8 220.1 761.2 3.45 7 0.99 5.03 7 0.86 1.737 0.98 3.45 7 0.88 0.98 7 0.21
66.7 7 11.5 61 (47.3%) 24.17 2.3 49 (38.0%) 89 (69.0%) 46 (35.7%) 46 (35.7%) 70 (54.3%) 46 (35.7%) 71 (55.9%) 56 (44.1%) 35 (27.6%) 70.67 9.6 139.9 7 21.2 219.8 7 61.5 3.45 71.06 4.88 70.84 1.7770.86 3.31 71.16 0.977 0.25
Data are presented as mean 7 S.D. or n (%). Abbreviations: BMI, body mass index; TIA, transient ischemic attack; PPI, proton pump inhibitor; CCB, calcium-channel blocker; LDL-C, low-density lipoprotein cholesterol; HDL-C, high-density lipoprotein cholesterol. a
During the study period, a total of 211 patients were enrolled. The distribution of CYP2C19 polymorphisms were 82 (38.9%), 80 (37.9%), 17 (8.0%), 21 (10.0%), 8 (3.8%) and 3 (1.4%) for n1/n1, n1/n2, n1/n3, n2/n2, n2/n3, and n3/n3, respectively. Therefore, 129 (61.1%) patients were carriers of at least one CYP2C19 LOF allele [IM: 97 (46.0%), PM: 32 (15.1%)] with 82 (38.9%) patients were noncarriers. The frequencies of CYP2C19 n2 and n3 in the cohort were 30.8% and 7.3%, respectively. The proportions of genotypes for each allele in the population were consistent with those predicted by the Hardy–Weinberg equilibrium for polymorphisms (P 40.05). Clinical characteristics of carriers and noncarriers are listed in Table 2. Variables known to be associated with platelet reactivity were well balanced between carriers and noncarriers.
3
Hypercholesterolemia or hyperglyceridemia.
3.2. Platelet function tests in carriers and noncarriers As shown in Fig. 1A, both ADP-PAg and platelet activation markers (PAC-1, CD62P and CD63) at baseline (before clopidogrel treatment) were consistent between carriers and noncarriers (all P values 4 0.05). After clopidogrel treatment for 7 days, the four platelet function parameters including ADP-PAg, PAC-1, CD62P and CD63 were significantly diminished (all P values o 0.001). The residual platelet reactivity (RPR) was significantly higher in carriers compared with noncarriers (all P values o 0.05, Fig. 1B). The multivariate liner regression model demonstrated that carriage of the CYP2C19 LOF allele was significantly associated with RPR including ADP-PAg, PAC-1, CD62P and CD63. CYP2C19 polymorphisms explained 12.9%, 4.3%, 8.9% and 5.5% of the interindividual variability of ADP-PAg, PAC-1, CD62P and CD63 after clopidogrel treatment, respectively, compared with 37.5%, 24.7%, 34.4% and 18.5% when clinical variables were included (Table 3). The clinical variables including age, hypertension, acute coronary disease, diabetes mellitus and basal platelet reactivity were independently associated with inter-individual variability in RPR. High residual platelet reactivity (HRPR) was defined as Z75th percentiles of ADP-PAg, PAC-1, CD62P and CD63 in this study. The corresponding cutoff values were 28.54%, 42.92%, 24.32% and 27.33%, respectively. HRPR with ADP-PAg Z28.54% was detected in 43 patients (33.3%) in carriers compared with 10 patients
Fig. 1. Association of CYP2C19 loss-of-function allele carriage with platelet reactivity including ADP-PAg, PAC-1, CD62P and CD63 at baseline (A) and after clopidogrel treatment (B). Data are shown as mean þstandard error (error bars). ADP-PAg, adenosine diphosphate-induced platelet aggregation. nP o0.05.
(12.2%) patients in noncarriers (P ¼0.001). HRPR with PAC1 Z42.92% was detected in 42 patients (25.1%) in carriers compared with 11 patients (13.4%) in noncarriers (P ¼0.002). HRPR with CD62P Z24.32% was detected in 41 patients (31.8%) in carriers compared with 12 patients (14.6%) in noncarriers
Please cite this article as: Qiu, L.-N., et al., Influence of CYP2C19 polymorphisms on platelet reactivity and clinical outcomes in ischemic stroke patients treated with clopidogrel. Eur J Pharmacol (2014), http://dx.doi.org/10.1016/j.ejphar.2014.11.037i
67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100 101 102 103 104 105 106 107 108 109 110 111 112 113 114 115 116 117 118 119 120 121 122 123 124 125 126 127 128 129 130 131 132
L.-N. Qiu et al. / European Journal of Pharmacology ∎ (∎∎∎∎) ∎∎∎–∎∎∎
4
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66
Table 3 Linear regression analysis for determining association of platelet reactivity and other variables. ADP-PAg
CYP2C19 LOF allele Clinical variablesa CYP2C19 LOF allele þ clinical variables P for changeb
PAC-1
CD62P
CD63
R2
P
R2
P
R2
P
R2
P
12.9 28.9 37.5
o0.001 o0.001 o0.001 o0.001
4.3 21.6 24.7
0.002 o 0.001 o 0.001 o 0.001
8.9 28.9 34.4
o 0.001 o 0.001 o 0.001 o 0.001
5.5 13.6 18.5
0.001 o 0.001 o 0.001 o 0.001
Percentage of variability in platelet reactivity explained was defined as coefficient determination (R2), 100%. Abbreviations: LOF, loss-of-function; ADP-PAg, adenosine diphosphate-induced platelet aggregation. a Age, hypertension, coronary artery disease, diabetes mellitus and basal platelet reactivity were significantly associated with residual platelet reactivity in univariate analysis (P o 0.1). b P value for the change in R2 when adding clinical variables to CYP2C19 LOF allele carriage.
Fig. 2. Correlation between different platelet parameters including ADP-PAg, PAC-1, CD62P and CD63 by flow cytometry after clopidogrel treatment. Cut-off values at 75th percentiles are indicated by dotted lines (Z 28.54% for ADP-PAg, Z42.92% for PAC-1, Z24.32% for CD62P and Z 27.33% for CD63). ADP-PAg, adenosine diphosphate-induced platelet aggregation.
(P ¼0.005). HRPR with CD63 Z27.33% was detected in 40 patients (31.0%) compared with 12 patients (14.6%) in noncarriers (P ¼0.007). A significant correlation between ADP-PAg and three platelet activation markers was observed: r ¼ 0.207 (P ¼0.002) for ADP-PAg and PAC-1; r ¼0.279 (Po 0.001) for ADP-PAg and CD62P; r¼ 0.202 (P ¼0.003) for ADP-PAg and CD63 (Fig. 2).
3.3. CYP2C19 genotypes and clinical prognosis Clinical follow-up was completed for 94.6% (198/211) of the enrolled patients. At 6 months, poor prognosis was observed in 30 patients in carriers of the CYP2C19 LOF allele compared with 8 patients in noncarriers (24% vs 11.0%, P ¼0.025). The multivariate binary regression model showed that carriage of the CYP2C19 LOF
Please cite this article as: Qiu, L.-N., et al., Influence of CYP2C19 polymorphisms on platelet reactivity and clinical outcomes in ischemic stroke patients treated with clopidogrel. Eur J Pharmacol (2014), http://dx.doi.org/10.1016/j.ejphar.2014.11.037i
67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100 101 102 103 104 105 106 107 108 109 110 111 112 113 114 115 116 117 118 119 120 121 122 123 124 125 126 127 128 129 130 131 132
L.-N. Qiu et al. / European Journal of Pharmacology ∎ (∎∎∎∎) ∎∎∎–∎∎∎
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66
allele was a significant independent predictor of poor prognosis (OR 3.01, 95% CI 1.23–7.38, P¼ 0.016) (Table 4). Meanwhile, complicating coronary artery disease was also an independent risk factor of poor prognosis in ischemic stroke patients on clopidogrel treatment (OR 2.38, 95% CI 1.11–5.06, P¼ 0.025).There were 15 (7.6%) primary end points during 6-month follow-up. Of which, 10 (5.1%) were ischemic stroke, 2 (1.0%) were myocardial infarctions and 3 (1.5%) were cardiovascular deaths. The characteristic of each primary event is listed in Table 5. About a half of patients had stopped clopidogrel therapy when they suffered an event. The accumulated primary end points were occurred in 12 patients in carriers compared with 3 patients in noncarriers (9.6% vs 4.1%, P¼ 0.159).
4. Discussion In the present study, platelet function tests and clinical prognosis in ischemic stroke patients on clopidogrel treatment were assessed to evaluate the influence of CYP2C19 genetic polymorphisms on clopidogrel response. Previously, conducted in different population and using various methods, multiple studies have reported that carriers with at least one CYP2C19 LOF alleles mainly n2 n3 had significantly diminished platelet inhibition and a higher risk of clinical outcomes than noncarriers in patients with acute coronary syndrome (Collet et al., 2009; Mega et al., 2009, 2010; Shuldiner et al., 2009). In the present study, among patients with Table 4 Multivariate logical regression analysis for predictors of poor prognosis. Variables
OR (95% CI)
P
Age Female sex Current smoking Hypertension Coronary artery disease Diabetes mellitus Hyperlipidemia Previous stroke/TIA CYP2C19 LOF allele carriage
1.02 1.03 0.78 1.37 2.38 0.85 0.61 1.06 3.01
0.234 0.942 0.574 0.451 0.025 0.711 0.205 0.882 0.016
(0.99–1.05) (0.46–2.31) (0.33–1.84) (0.60–3.14) (1.11–5.06) (0.37–1.97) (0.27–1.32) (0.49–2.32) (1.23–7.38)
The abbreviations are as in Tables 1 and 2.
5
ischemic stroke on clopidogrel treatment, we demonstrated that the CYP2C19 LOF allele carriage was significantly associated with RPR, including ADP-PAg and platelet activation (PAC-1, CD62P and CD63). These platelet function parameters have been showed to be well inhibited by clopidogrel. What is more, three platelet activation markers significantly correlated with ADP-PAg, which is a widely used platelet function parameter. It suggested that they might be valuable for assessing clopidogrel response. Yang et al. (2013) found ADP-PAg after clopidogrel treatment for 7 days was significantly higher in carriers of one or two CYP2C19 LOF allele than in noncarriers among ischemic stroke patients, and concluded that CYP2C19 n2 or n3 LOF alleles were independently associated ADP-PAg measurements. Jia et al. (2013) reported the significant association between CYP2C19 LOF allele carriage and high prevalence of clopidogrel resistance, which was defined as a reduction of ADP-PAg before and after clopidogrel treatment. We defined clopidogrel resistance as HRPR with RPR in the fourth quartile, because platelet reactivity is continuous data and there is wide inter-individual variability. Regarding of the inter-individual variability of ADP-PAg, baseline low platelet reactivity may cause overestimated risk of poor responders at the determination of reduction of ADP-PAg, while HRPR after clopidogrel treatment remained with underestimated risk in good responders (Bonello et al., 2010). Meanwhile, baseline platelet reactivity was not associated with CYP2C19 genotypes in our study, as well as described in the previous study (Nakata et al., 2013). What is more, based on RPR after clopidogrel treatment, studies also have demonstrated that clopidogrel poor response was significantly associated with worse clinical outcomes. But it was insufficiently powered to detect stroke occurrence (Campo et al., 2010a). A meta-analysis including 14 studies with 11,959 patients showed that HRPR in patients with coronary artery disease undergoing percutaneous coronary intervention was a risk factor for stroke (Taglieri et al., 2014). Therefore, the RPR appeared to be a better assessment of ischemic risk than reduction of ADP-PAg. We found carriers of at least one CYP2C19 LOF allele had higher prevalence of HRPR, when platelet inhibition was assessed by either ADP-PAg or platelet activation (PAC-1, CD62P and CD63). The present study also found that carriers of CYP2C19 LOF alleles had 3.01-fold increased risk of poor prognosis in 6-month follow-up compared with noncarriers among ischemic stroke patients treated
Table 5 Characteristics of patients with primary events. Patients (gender, age [yrs])
ADP-PAg (HRPR)
PAC-1 (HRPR)
CD62P (HRPR)
Recurrent ischemic #1 (Male, 66) #2 (Female, 71) #3 (Male, 76) #4 (Male, 76) #5 (Female, 89) #6 (Male, 86) #7 (Female, 76) #8 (Male,71) #9 (Female, 73) #10 (Male, 73)
stroke 43.64 ( þ) 65.00 ( þ) 27.03 ( ) 51.88 ( þ) 32.94 ( þ) 27.06 ( ) 21.56 ( ) 12.91 ( ) 39.50 ( þ) 42.62 ( þ)
58.04 20.41 43.76 28.96 59.55 23.70 35.77 32.08 48.77 49.65
33.66 37.80 12.88 25.15 22.34 22.20 34.50 41.90 12.14 29.65
(þ ) () (þ ) () (þ ) () () () (þ ) (þ )
(þ) (þ) () (þ) () () (þ) (þ) () (þ)
CD63 (HRPR)
25.44 31.13 22.16 18.51 35.02 33.91 39.66 17.08 28.41 30.65
() (þ) () () (þ) (þ) (þ) () (þ) (þ)
Pre-treatment Interval (h)
Test to events (days)
Clopidogrel therapy duration (weeks)
Clopidgrel therapy mRS status score
15 24 31 22 29 22 48 23 52 24
35 59 44 98 138 55 61 78 107 166
4 8 4 12 12 7 8 8 8 12
Off On Off Off Off On On Off Off Off
3 3 3 2 3 2 1 1 4 4
Myocardial infarction #11 (Female,77) 15.93 ( ) #12 (Female, 55) 45.09 ( þ)
54.27 (þ ) 35.00 ( þ ) 63.16 (þ ) 41.96 ( þ )
52.99 ( þ ) 36 26.71 ( ) 17
79 29
11 4
On On
0 2
Vascular death #13 (Female,74) #14 (Male, 52) #15 (Male,86)
55.76 (þ ) 45.54 ( þ ) 51.85 (þ ) 35.30 ( þ ) 43.96 (þ ) 38.49 ( þ )
33.76 ( þ ) 20 54.67 ( þ ) 28 15.15 ( ) 16
7 16 2
1 2 0
On On On
6 6 6
50.28 ( þ) 20.08 ( ) 48.60 ( þ)
Platelet reactivity listed in the table including ADP-PAg, PAC-1, CD62P and CD63 are all residual platelet reactivity after clopidogrel treatment.þ or in the braces means yes or no for HRPR. Abbreviations: ADP-PAg, adenosine diphosphate-induced platelet aggregation; HRPR, high residual platelet reactivity; mRS, the modified Rankin Scale.
Please cite this article as: Qiu, L.-N., et al., Influence of CYP2C19 polymorphisms on platelet reactivity and clinical outcomes in ischemic stroke patients treated with clopidogrel. Eur J Pharmacol (2014), http://dx.doi.org/10.1016/j.ejphar.2014.11.037i
67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100 101 102 103 104 105 106 107 108 109 110 111 112 113 114 115 116 117 118 119 120 121 122 123 124 125 126 127 128 129 130 131 132
L.-N. Qiu et al. / European Journal of Pharmacology ∎ (∎∎∎∎) ∎∎∎–∎∎∎
6
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66
with clopidogrel. This finding was in consistent with that of Jia et al. (2013). Indeed, carriage of the CYP2C19 LOF allele was a major determinant of poor prognosis in Chinese stroke patients in the present study. While, the absence of an effect of the CYP2C19 LOF allele carriage on primary events was in contrast to findings of previous studies on acute coronary syndromes. Possible explanation for the divergence between our finding on ischemic stroke patients and previous studies on patients with acute coronary syndromes is the heterogeneous cause of stroke (Adams et al., 1993) compared with acute coronary syndrome or unmeasured factors including CYP2C19 n17 gain-of-function allele (Frere et al., 2009) potentially resulting in reduced inter-individual variability. The significant association between CYP2C19 LOF allele variants and diminished clopidogrel response including ADP-PAg, PAC-1, CD62P and CD63 in this study indicated that the whole-blood flow-cytometric method is capable to predict the gene mediated inter-individual variability of clopidogrel response. Light transmittance aggregometry (LTA) is most commonly used for measurement of platelet function. However, certain defects of LTA, such as time consuming, poor specificity and poor reproducibility, limit its use. Without centrifugation and using platelet identified monoclonal antibody, determination of platelet aggregation by whole blood flow cytometry could overcome the defects in LTA and has been proved to be a reliable method in measuring platelet function in our study. The present study found that the frequency of the CYP2C19 LOF allele in Chinese stroke patients was 38.1%, and carriers of at least one LOF allele of CYP2C19 constituted 61.1% of the cohort. In the previous study (Mega et al., 2009), the prevalence of carriage of CYP2C19 LOF allele was 30% in the overall cohort in Westerners. Simultaneously, in patients with CYP2C19 LOF alleles and suffering acute coronary syndrome, risk of adverse clinical events appeared to be higher in the Asian population compared to Westerner population (Jang et al., 2012). Similarly, 1-year cumulative recurrence rate of ischemic stroke was reported to be higher in Chinese (19.9%) (Wang et al., 2013), when compared to that in Westerners (9.4%) (Allen et al., 2010), Although the clinical outcomes of ischemic stroke in Chinese population were irrespective of CYP2C19 carriage in our study, the effect of CYP2C19 polymorphisms on prognosis of ischemic stroke patients was definite. Therefore, it is noteworthy to underscore that the relevant genotyping may guide a more individualized and thus optimizing therapeutic regimens which are essential for improvement of stroke prognosis and the possible prevention of stroke recurrence in Chinese patients. There are several potential limitations in our study. First, other genetic variants except CYP2C19 n2 n3 LOF alleles, such as CYP2C19 n17 gain-of-function allele and PON-1 gene polymorphism in our study, might also contribute to platelet reactivity, clopidogrel response and the likelihood of clinical sequelae. At present, the role of PON-1 in patients with cardiovascular disease was debated or marginal (Campo et al., 2011a), whether it affected the on-clopidogrel platelet reactivity in stroke patients needs further studies. Second, our investigation did not measure the platelet reactivity during the follow-up period, although the time-dependent relationship between the CYP2C19 variants and the antiplatelet effect of clopidogrel was reported in some studies (Wallentin et al., 2010). Third, whether the optional regimens such as altering the dosage or drugs would override the baleful effect of CYP2C19 polymorphisms on stroke patients merits further investigations.
5. Conclusion Taken together, the present study demonstrated that carriage of the CYP2C19 LOF allele had significant influence on antiplatelet effect of clopidogrel and clinical prognosis of Chinese patients with
ischemic stroke. Our findings provide certain evidence on genetic effect of CYP2C19 on clopidogrel responsiveness in stroke patients, but whether the prognosis data associated with CYP2C19 genotypes can be used to guide individualized and thus optimizing the therapeutic regimens for the secondary prevention of stroke needs further studies.
Acknowledgments This work was supported by Major Social Science Program of Tianjin Municipal Education Commission (2011ZD016). The work was also supported by Key Technology Research and Development Program of Science & Technology of Tianjin (12ZCZDSY03100) and Tianjin Municipal Natural Science Foundation (09JCYBJC11400). We are grateful to all patients participating in this study, and Wei Huang and Xiaodong Zhou of Tianjin Institute of Urology for their technical support.
References Adams Jr., H.P., Bendixen, B.H., Kappelle, L.J., Biller, J., Love, B.B., Gordon, D.L., Marsh 3rd, E.E., 1993. Classification of subtype of acute ischemic stroke. Definitions for use in a multicenter clinical trial. TOAST. Trial of Org 10172 in Acute Stroke Treatment. Stroke 24, 35–41. Allen, N.B., Holford, T.R., Bracken, M.B., Goldstein, L.B., Howard, G., Wang, Y., Lichtman, J.H., 2010. Geographic variation in one-year recurrent ischemic stroke rates for elderly medicare beneficiaries in the USA. Neuroepidemiology 34, 123–129. Antithrombotic Trialists' Collaboration, 2002. Collaborative meta-analysis of randomised trials of antiplatelet therapy for prevention of death, myocardial infarction, and stroke in high risk patients. BMJ 324, 71–86. Bennett, D., Yan, B., 2013. Suboptimal response to clopidogrel: a genetic risk factor for recurrent ischaemic stroke. J. Clin. Neurosci. 20, 767–770. Bonello, L., Tantry, U.S., Marcucci, R., Blindt, R., Angiolillo, D.J., Becker, R., Bhatt, D.L., Cattaneo, M., Collet, J.P., Cuisset, T., Gachet, C., Montalescot, G., Jennings, L.K., Kereiakes, D., Sibbing, D., Trenk, D., Van Werkum, J.W., Paganelli, F., Price, M.J., Waksman, R., Gurbel, P.A., Working Group on High On-Treatment Platelet Reactivity, 2010. Consensus and future directions on the definition of high ontreatment platelet reactivity to adenosine diphosphate. J. Am. Coll. Cardiol. 56, 919–933. Brandt, J.T., Close, S.L., Iturria, S.J., Payne, C.D., Farid, N.A., Ernest 2nd, C.S., Lachno, D.R., Salazar, D., Winters, K.J., 2007. Common polymorphisms of CYP2C19 and CYP2C9 affect the pharmacokinetic and pharmacodynamic response to clopidogrel but not prasugrel. J. Thromb. Haemost. 5, 2429–2436. Campo, G., Ferraresi, P., Marchesini, J., Bernardi, F., Valgimigli, M., 2011a. Relationship between paraoxonase Q192R gene polymorphism and on-clopidogrel platelet reactivity over time in patients treated with percutaneous coronary intervention. J. Thromb. Haemost. 9, 2106–2108. Campo, G., Fileti, L., de Cesare, N., Meliga, E., Furgieri, A., Russo, F., Colangelo, S., Brugaletta, S., Ferrari, R., Valgimigli, M., 3T/2R Investigators, 2010. Long-term clinical outcome based on aspirin and clopidogrel responsiveness status after elective percutaneous coronary intervention: a 3T/2R (tailoring treatment with tirofiban in patients showing resistance to aspirin and/or resistance to clopidogrel) trial substudy. J. Am. Coll. Cardiol. 56, 1447–1455. Campo, G., Fileti, L., Valgimigli, M., Tebaldi, M., Cangiano, E., Cavazza, C., Marchesini, J., Ferrari, R., 2010b. Poor response to clopidogrel: current and future options for its management. J. Thromb. Thrombolysis 30, 319–331. Campo, G., Miccoli, M., Tebaldi, M., Marchesini, J., Fileti, L., Monti, M., Valgimigli, M., Ferrari, R., 2011b. Genetic determinants of on-clopidogrel high platelet reactivity. Platelets 22, 399–407. Collet, J.P., Hulot, J.S., Pena, A., Villard, E., Esteve, J.B., Silvain, J., Payot, L., Brugier, D., Cayla, G., Beygui, F., Bensimon, G., Funck-Brentano, C., Montalescot, G., 2009. Cytochrome P450 2C19 polymorphism in young patients treated with clopidogrel after myocardial infarction: a cohort study. Lancet 373, 309–317. CAPRIE Steering Committee, 1996. A randomised, blinded, trial of clopidogrel versus aspirin in patients at risk of ischaemic events (CAPRIE). CAPRIE Steering Committee. Lancet 348, 1329–1339. de Morais, S.M., Wilkinson, G.R., Blaisdell, J., Nakamura, K., Meyer, U.A., Goldstein, J.A., 1994. The major genetic defect responsible for the polymorphism of Smephenytoin metabolism in humans. J. Biol. Chem. 269, 15419–15422. Frere, C., Cuisset, T., Gaborit, B., Alessi, M.C., Hulot, J.S., 2009. The CYP2C19n17 allele is associated with better platelet response to clopidogrel in patients admitted for non-ST acute coronary syndrome. J. Thromb. Haemost. 7, 1409–1411. Frere, C., Cuisset, T., Morange, P.E., Quilici, J., Camoin-Jau, L., Saut, N., Faille, D., Lambert, M., Juhan-Vague, I., Bonnet, J.L., Alessi, M., 2008. Effect of cytochrome p450 polymorphisms on platelet reactivity after treatment with clopidogrel in acute coronary syndrome. Am. J. Cardiol. 101, 1088–1093.
Please cite this article as: Qiu, L.-N., et al., Influence of CYP2C19 polymorphisms on platelet reactivity and clinical outcomes in ischemic stroke patients treated with clopidogrel. Eur J Pharmacol (2014), http://dx.doi.org/10.1016/j.ejphar.2014.11.037i
67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100 101 102 103 104 105 106 107 108 109 110 111 112 113 114 115 116 117 118 119 120 121 122 123 124 125 126 127 128 129 130 131 132
L.-N. Qiu et al. / European Journal of Pharmacology ∎ (∎∎∎∎) ∎∎∎–∎∎∎
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30
Freynhofer, M.K., Bruno, V., Wojta, J., Huber, K., 2012. The role of platelets in atherothrombotic events. Curr. Pharm. Des. 18, 5197–5214. Gurbel, P.A., Becker, R.C., Mann, K.G., Steinhubl, S.R., Michelson, A.D., 2007. Platelet function monitoring in patients with coronary artery disease. J. Am. Coll. Cardiol. 50, 1822–1834. Jang, J.S., Cho, K.I., Jin, H.Y., Seo, J.S., Yang, T.H., Kim, D.K., Kim, D.S., Seol, S.H., Kim, D.I., Kim, B.H., Park, Y.H., Je, H.G., Jeong, Y.H., Lee, S.W., 2012. Meta-analysis of cytochrome P450 2C19 polymorphism and risk of adverse clinical outcomes among coronary artery disease patients of different ethnic groups treated with clopidogrel. Am. J. Cardiol. 110, 502–508. Jauch, E.C., Saver, J.L., Adams Jr., H.P., Bruno, A., Connors, J.J., Demaerschalk, B.M., Khatri, P., McMullan Jr., P.W., Qureshi, A.I., Rosenfield, K., Scott, P.A., Summers, D.R., Wang, D.Z., Wintermark, M., Yonas, H., American Heart Association Stroke Council Council on Cardiovascular Nursing Council on Peripheral Vascular Disease Council on Clinical Cardiology, 2013. Guidelines for the early management of patients with acute ischemic stroke: a guideline for healthcare professionals from the American Heart Association/American Stroke Association. Stroke 44, 870–947. Jia, D.M., Chen, Z.B., Zhang, M.J., Yang, W.J., Jin, J.L., Xia, Y.Q., Zhang, C.L., Shao, Y., Chen, C., Xu, Y., 2013. CYP2C19 polymorphisms and antiplatelet effects of clopidogrel in acute ischemic stroke in China. Stroke 44, 1717–1719. Kennedy, J., Hill, M.D., Ryckborst, K.J., Eliasziw, M., Demchuk, A.M., Buchan, A.M., FASTER Investigators, 2007. Fast assessment of stroke and transient ischaemic attack to prevent early recurrence (FASTER): a randomised controlled pilot trial. Lancet Neurol. 6, 961–969. Lonn, E., Yusuf, S., Arnold, M.J., Sheridan, P., Pogue, J., Micks, M., McQueen, M.J., Probstfield, J., Fodor, G., Held, C., Genest Jr., J., Heart Outcomes Prevention Evaluation Investigators, 2006. Homocysteine lowering with folic acid and B vitamins in vascular disease. N. Engl. J. Med. 354, 1567–1577. Mega, J.L., Close, S.L., Wiviott, S.D., Shen, L., Hockett, R.D., Brandt, J.T., Walker, J.R., Antman, E.M., Macias, W., Braunwald, E., Sabatine, M.S., 2009. Cytochrome p-450 polymorphisms and response to clopidogrel. N. Engl. J. Med. 360, 354–362. Mega, J.L., Simon, T., Collet, J.P., Anderson, J.L., Antman, E.M., Bliden, K., Cannon, C.P., Danchin, N., Giusti, B., Gurbel, P., Horne, B.D., Hulot, J.S., Kastrati, A., Montalescot, G., Neumann, F.J., Shen, L., Sibbing, D., Steg, P.G., Trenk, D., Wiviott, S.D., Sabatine, M.S., 2010. Reduced-function CYP2C19 genotype and risk of adverse clinical outcomes among patients treated with clopidogrel predominantly for PCI: a meta-analysis. JAMA 304, 1821–1830.
7
Nakata, T., Miyahara, M., Nakatani, K., Wada, H., Tanigawa, T., Komada, F., Hoshino, K., Aoki, T., Nishimura, Y., Tamaru, S., Ito, M., Nishikawa, M., 2013. McLordd groupRelationship between CYP2C19 loss-of-function polymorphism and platelet reactivities with clopidogrel treatment in Japanese patients undergoing coronary stent implantation. Circ. J. 77, 1436–1444. Schmitz, G., Rothe, G., Ruf, A., Barlage, S., Tschope, D., Clemetson, K.J., Goodall, A.H., Michelson, A.D., Nurden, A.T., Shankey, T.V., 1998. European Working Group on Clinical Cell Analysis: consensus protocol for the flow cytometric characterisation of platelet function. Thromb. Haemost. 79, 885–896. Shuldiner, A.R., O’Connell, J.R., Bliden, K.P., Gandhi, A., Ryan, K., Horenstein, R.B., Damcott, C.M., Pakyz, R., Tantry, U.S., Gibson, Q., Pollin, T.I., Post, W., Parsa, A., Mitchell, B.D., Faraday, N., Herzog, W., Gurbel, P.A., 2009. Association of cytochrome P450 2C19 genotype with the antiplatelet effect and clinical efficacy of clopidogrel therapy. JAMA 302, 849–857. Simon, T., Verstuyft, C., Mary-Krause, M., Quteineh, L., Drouet, E., Meneveau, N., Steg, P.G., Ferrieres, J., Danchin, N., Becquemont, L., French Registry of Acute S.T. E. Non-S.T.E.M.I.I.Genetic determinants of response to clopidogrel and cardiovascular events. N. Engl. J. Med. 360, 363–375. Taglieri, N., Bacchi Reggiani, M.L., Palmerini, T., Ghetti, G., Saia, F., Gallo, P., Moretti, C., Dall’Ara, G., Marrozzini, C., Marzocchi, A., Rapezzi, C., 2014. Risk of stroke in patients with high on-clopidogrel platelet reactivity to adenosine diphosphate after percutaneous coronary intervention. Am. J. Cardiol. 113, 1807–1814. Tohgi, H., Suzuki, H., Tamura, K., Kimura, B., 1991. Platelet volume, aggregation, and adenosine triphosphate release in cerebral thrombosis. Stroke 22, 17–21. Wallentin, L., James, S., Storey, R.F., Armstrong, M., Barratt, B.J., Horrow, J., Husted, S., Katus, H., Steg, P.G., Shah, S.H., Becker, R.C., PLATO investigators, 2010. Effect of CYP2C19 and ABCB1 single nucleotide polymorphisms on outcomes of treatment with ticagrelor versus clopidogrel for acute coronary syndromes: a genetic substudy of the PLATO trial. Lancet 376, 1320–1328. Wang, Z., Li, J., Wang, C., Yao, X., Zhao, X., Wang, Y., Li, H., Liu, G., Wang, A., Wang, Y., 2013. Gender differences in 1-year clinical characteristics and outcomes after stroke: results from the China National Stroke Registry. PloS One 8, e56459. Yang, J., Zhao, H.D., Tan, J., Ding, Y.L., Gu, Z.Q., Zou, J.J., 2013. CYP2C19 polymorphism and antiplatelet effects of clopidogrel in Chinese stroke patients. Pharmazie 68, 183–186.
Please cite this article as: Qiu, L.-N., et al., Influence of CYP2C19 polymorphisms on platelet reactivity and clinical outcomes in ischemic stroke patients treated with clopidogrel. Eur J Pharmacol (2014), http://dx.doi.org/10.1016/j.ejphar.2014.11.037i
31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59
