The present study was performed to compare the influence of cytochrome P459 2C19 (CYP2C19) *2 and *17 genetic variants on the platelet response to clopidogrel and prasugrel maintenance therapy and to assess the relation between platelet reactivity and bleeding complications. A total of 730 patients were included (517 patients treated with clopidogrel 150 mg/day and 213 discharged with prasugrel 10 mg). Platelet reactivity was assessed at 1 month with the platelet reactivity index vasodilator-stimulated phosphoprotein (PRI VASP). High on-treatment platelet reactivity was defined as PRI VASP >50% and low on-treatment platelet reactivity (LTPR) as PRI VASP <20%. The patients were classified according to their genotypes as poor metabolizers (*2/non *17), intermediate metabolizers (*2/*17 or non *2/non *17) and ultrametabolizers (non *2/*17). At 1 month, the prasugrel response was significantly better than the clopidogrel response in all groups of patients, with a lower incidence of high on-treatment platelet reactivity but a greater incidence of LTPR, regardless of the genetic variants. The genetic distribution had a significant effect on the mean PRI VASP values, the incidence of high on-treatment platelet reactivity, and LTPR with both clopidogrel and prasugrel (p <0.05 for all). LTPR identified a group of patients at a greater risk of bleeding (odds ratio 4.8, 95% confidence interval 2.7 to 8.3; p <0.0001). In conclusion, the present study showed that both clopidogrel and prasugrel have genetic modulation by CYP2C19 *2 and *17 alleles and that prasugrel provides greater platelet inhibition, regardless of the genotypes. In addition, LTPR was associated with a greater risk of bleeding.
Aspirin and clopidogrel has been the reference standard therapy for patients undergoing percutaneous coronary intervention and/or after acute coronary syndrome. A large variability in the clopidogrel response has been described, with clinical consequences. The cytochrome P459 2C19 (CYP2C19)*2 loss-of-function allele is associated with a marked decrease in the platelet response to clopidogrel and an impaired prognosis in clopidogrel treated patients. In contrast, the gain of the function allele CYP2C19*17 has been linked with a better response to clopidogrel and, accordingly, a greater risk of bleeding. Prasugrel provides faster, more potent, and more predictable platelet inhibition compared to clopidogrel, resulting in a significant reduction in ischemic events and an increase in bleeding complications in the overall TRial to Assess Improvement in Therapeutic Outcomes by Optimizing Platelet InhibitioN with Prasugrel–Thrombolysis In Myocardial Infarction (TRITON TIMI 38) study. Early studies did not find any significant influence of CYP genetics variants on the pharmacokinetic and pharmacodynamic response to prasugrel or on the clinical outcome in prasugrel-treated patients, although a recent analysis from our group found a significant effect of these genetic variants on prasugrel response with a specific assay for measuring P2Y12 receptor blockade. We performed the present analysis to compare the influence of CYP2C19*2 and *17 allele on the chronic response to clopidogrel and prasugrel and to compare the platelet inhibition induced with both drugs according to the genetic background. We also analyzed the relation between platelet reactivity and bleeding complications with both drugs.
Methods
Consecutive patients admitted for non–ST-segment elevation acute coronary syndrome or ST-segment elevation myocardial infarction in our institution were eligible for the present prospective study if they had undergone successful percutaneous coronary intervention and were treated at discharge with clopidogrel 150 mg or prasugrel 10 mg. The exclusion criteria were a history of bleeding diathesis, previous stroke or transient ischemic attack, contraindications to antiplatelet therapy, platelet count <100 g/L, and creatinine clearance <25 ml/min. The response to clopidogrel and prasugrel was assessed at the 1-month clinical follow-up visit using the platelet reactivity index vasodilator-stimulated phosphoprotein (PRI VASP). Adherence was systematically assessed during this consultation using a specifically designed questionnaire. The ethics committee of our institution approved the study protocol, and all patients gave written informed consent for participation. Blood samples for testing the clopidogrel response were drawn at the 1-month follow-up visit and sent immediately to the hemostasis laboratory to determine the VASP phosphorylation state of whole blood. We used a standardized flow cytometric assay (platelet VASP, Diagnostica Stago/BioCytex, Asnières, France), which is an adaptation of the method previously described by Schwarz et al. PRI VASP was calculated using the formula: PRI VASP = [MFI (PGE1) − MFI (PGE1+ADP) /MFI PGE1 ] × 100, where MFI is the median fluorescence intensity, PGE1 is prostaglandin E 1 , and ADP is adenosine diphosphate. High on-treatment platelet reactivity (HTPR) was defined as PRI VASP >50%, as recently proposed, and low on-treatment platelet reactivity (LTPR) as PRI VASP <20%, as previously proposed. Genomic DNA was extracted from peripheral blood leukocytes using the salting-out method. CYP2C19*2 and CYP2C19*17 genotyping was done using the amplification refractory mutation system-polymerase chain reaction in duplex reaction. The reactions were made in a final volume of 12.5 μl, using 200 nM of primer (except for *2), primer (300 nM), 0.15 U of Taq DNA polymerase (Qbiotaq, MP Biomedicals, Santa Ana, California) in its accompanying buffer, 200 μM of accompanying deoxynucleotide triphosphates, and 25 ng of genomic DNA. The cycling conditions were as follows: a first denaturing step, for 1 minute, 30 seconds at 94°C, then 35 cycles of 30 seconds at 95°C, 30 seconds at 60°C, and 50 seconds at 72°C, a final extension step for 3 minutes at 72°C, with cooling to 15°C. The polymerase chain reaction products were run on 2% agarose gels, and the genotypes were assigned according the polymerase chain reaction product sizes observed. CYP2C19*2 and *17 is defined by rs4244285 and rs12248560, respectively. Patients were classified according to their genotypes as poor metabolizers (*2 “loss-of-function” allele carriers/*17 “gain-of-function” allele noncarriers), intermediate metabolizers (*2 “loss-of-function” allele carriers/*17 “gain-of-function” allele carriers and *2 “loss-of-function” allele noncarriers/*17 “gain-of-function” allele noncarriers), and ultrametabolizers (*2 “loss-of-function” allele noncarriers/*17 “gain-of-function” allele carriers). The Hardy-Weinberg equilibrium was tested for each polymorphism to detect potential stratification bias or genotyping errors. Clinical follow-up was planned for all patients at 1 month. The clinical end point was the occurrence of nonaccess site-related bleeding events according to the Bleeding Academic Research Consortium (BARC) definitions of type 1, 2, 3, or 5. The primary objectives of the present study were (1) to compare the prasugrel and clopidogrel response in different groups of patients classified according to their genotypes, (2) to determine the effect of the CYP2C19*2 and *17 alleles on the clopidogrel and prasugrel response, as assessed by PRI VASP, and the incidence of HTPR (defined as PRI VASP >50%) and LTPR (defined as PRI VASP <20%), and (3) to determine the occurrence of bleeding complications and their relation to the PRI VASP in the whole population and with each drug. Statistical analysis was performed using the GraphPad Prism software, version 4.00 (GraphPad Software, La Jolla, California). Continuous variables were analyzed for a normal distribution using the Shapiro-Wilk test and are expressed as the mean ± SD. Categorical variables are expressed as frequencies and percentages. Comparisons between groups were done using the chi-square or Fisher exact test for categorical variables and the t test for continuous variables. One-way analysis of variance was used for comparisons between the different groups. p Values <0.05 were considered statistically significant. Assuming that carriers of the *2 and *17 alleles represent 1/3 of the entire population, and expecting in these patients a 5% absolute difference in PRI VASP compared with “noncarrier” patients, a total of ≥205 patients were needed in both groups to detect the expected difference, with an estimated power of 80% at a 2-sided α of 0.05.
Results
A total of 730 patients were included in the present analysis (517 clopidogrel treated and 213 prasugrel treated). The patients treated with prasugrel were significantly younger, more likely to be men, had a lower body weight, and more likely to be treated after ST-segment elevation myocardial infarction ( Table 1 ). In the whole population, the rate of patients with HTPR was 27% (n = 198). The genotype frequencies for each allele considered separately were consistent with the Hardy-Weinberg predictions. At 1 month, the mean PRI VASP in the clopidogrel-treated patients was significantly greater than that in the prasugrel-treated patients (41 ± 18% vs 29 ± 14%, p <0.0001; Figure 1 ). The rate of HTPR was significantly greater for the clopidogrel-treated patients (35% vs 7%, odds ratio [OR] 7, 95% confidence interval [CI] 4.2 to 12.6; p <0.0001), and the rate of LTPR (PRI VASP <20%) was significantly greater for the prasugrel-treated patients (31% vs 10%, OR 4, 95% CI 2.7 to 6.1; p <0.0001). The superiority of prasugrel over clopidogrel was observed in all subgroups of patients defined according to their genetic profile: poor metabolizers, PRI VASP 46 ± 20% with clopidogrel versus 35 ± 15% with prasugrel (p = 0.005); intermediate metabolizers, PRI VASP 40 ± 18% with clopidogrel versus 30 ± 14% with prasugrel (p <0.0001); and ultrametabolizers, PRI VASP 39 ± 18% with clopidogrel versus 25 ± 12% with prasugrel (p <0.0001; Figure 1 ). In the clopidogrel-treated patients, this classification was associated with the mean PRI VASP values and with the incidence of HTPR and LTPR with clopidogrel 150 mg ( Figure 2 ). The rate of patients with HTPR was 50% in the poor metabolizer group (n = 58 of 116), 33% in the intermediate metabolizer group (n = 81 of 249), and 29% in the ultrametabolizer group (n = 44 of 152). The rate of patients with LTPR was 6% in the poor metabolizer group (n = 7 of 116), 10% in the intermediate metabolizer group (n = 26 of 249), and 13% in the ultrametabolizer group (n = 20 of 152 (p <0.01; Figure 2 ). In the prasugrel-treated patients, this classification was associated with the mean PRI VASP values and with the incidence of HTPR and LTPR with prasugrel 10 mg ( Figure 2 ). The rate of patients with HTPR was 19% in the poor metabolizer group (n = 8 of 42), 6% in the intermediate metabolizer group (n = 6 of 107), and 1% in the ultrametabolizer group (n = 1 of 64). The rate of patients with LTPR was 14% in the poor metabolizer group (n = 7 of 116), 24% in the intermediate metabolizer group (n = 26 of 249), and 36% in the ultrametabolizer group (n = 20 of 152; p <0.05; Figure 2 ). In the overall population, the incidence of bleeding complications at 1 month was 8% (n = 60), including 32 with BARC 1, 22 with BARC 2, and 6 with BARC 3 bleeding events. In the whole population, patients developing bleeding complications according to the BARC definitions had a significantly lower PRI VASP than did the patients without complications: 26 ± 15% versus 30 ± 14% (p <0.0001; Figure 3 ). In the subgroups of clopidogrel-treated (n = 517) and prasugrel-treated (n = 213) patients, those with bleeding complications had a significantly lower PRI VASP than did patients without complications (28 ± 17% vs 41 ± 18% [p <0.0001] and 24 ± 14% vs 30 ± 14% [p = 0.03], respectively; Figure 3 ). In the whole population, pooling the clopidogrel and prasugrel treatments, the patients with LTPR (PRI VASP <20%) had a significantly greater rate of bleeding complications than did the patients without LTPR (22% [n = 26 of 120] vs 6% [n = 34 of 610]; OR 4.8, 95% CI 2.7 to 8.3; p <0.0001; Figure 4 ). This association remained significant on multivariate analysis (OR 3.5, 95% CI 2.2 to 9.6; p <0.01). In the subgroups of clopidogrel-treated (n = 517) and prasugrel-treated (n = 213) patients, patients with LTPR (PRI VASP <20%) had a significantly greater rate of bleeding complications than did the patients without LTPR: 19% (n = 10 of 53) versus 4% (n = 18 of 464; OR 6, 95% CI 2.6 to 13.8; p <0.0001) and 24% (n = 16 of 67) versus 11% (n = 16 of 146; OR 2.5, 95% CI 1.2 to 5.5; p = 0.02; Figure 4 ).
| Variable | Clopidogrel (n = 517) | Prasugrel (n = 213) | p Value |
|---|---|---|---|
| Age (yrs) | 64 ± 12 | 58 ± 10 | <0.01 |
| Women | 92 (18%) | 23 (11%) | 0.02 |
| Non–ST-segment elevation acute coronary syndrome | 367 (71%) | 117 (55%) | <0.01 |
| ST-segment elevation myocardial infarction | 150 (29%) | 96 (45%) | <0.01 |
| Weight (kg) | 78 ± 15 | 82 ± 15 | 0.02 |
| Hypertension | 305 (59%) | 83 (39%) | <0.01 |
| Diabetes mellitus | 161 (31%) | 64 (30%) | 0.79 |
| Smoker | 195 (38%) | 106 (50%) | 0.003 |
| Dyslipidemia ∗ | 287 (55%) | 104 (49%) | 0.14 |
| Familial history of coronary artery disease | 181 (35%) | 36 (17%) | <0.01 |
| Left ventricular ejection fraction (%) | 55 ± 8 | 55 ± 7 | 0.90 |
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