A randomized controlled trial to assess the efficacy and safety of doubling dose clopidogrel versus ticagrelor for the treatment of acute coronary syndrome in patients with CYP2C19*2 homozygotes

Abstract

Background: Compared with non-reversible, indirect P2Y12 inhibitor clopidogrel, ticagrelor is a reversible, direct acting inhibitor. The CYP2C19*2 allele is a common genetic variant in individuals that need given higher clopidogrel in acute coronary syndrome patients. Objective: We aimed to assess a pharmacogenetic approach of doubling dose clopidogrel compare with standard dose of ticagrelor among carriers with the CYP2C19*2 homozygotes. Materials and methods: We compared ticagrelor (180 mg loading dose, 90 mg twice daily thereafter) with clopidogrel (600 mg loading dose, 150 mg daily thereafter) for the prevention of cardiovascular events in CYP2C19*2 homozygotes patients admitted to the hospital with an acute coronary syndrome, with or without ST-segment elevation. Results: After genetic test to identify carriers of the CYP2C19*2 allele from 2295 patients, 224 cases with CYP2C19*2 homozygotes were enrolled into our prospective, randomized trial. Patients were random assignment with colpidogrel group (n = 112) and ticagrelor group (n = 112). The two groups were similar in terms of baseline characteristics. After the first 600 mg loading dose of clopidogrel, patients carrying two CYP2C19*2 allele had weaker PRU inhibition (39.8±37.4 vs 27.9±12.4; P = 0.001) and more bleeding adverse events (20.5% vs. 7.1%; hazard ratio = 2.88; 95% [CI], 1.34-6.15; P = 0.001) compared to those taking standard dose of ticagrelor. Conclusions: In CYP2C19*2 carriers with ACS, ticagrlor is as effective as high clopidogrel in reducing platelet reactivity, particularly in first days. This study suggests that ticagrelor may be much better than doubling dose clopidogrel in patients with CYP2C19*2 in according to platelet reactivity monitoring. Use of ticagrelor instead of clopidogrel may eliminate the need for genetic testing and lead to less mild bleeding adverse.

Introduction

Platelets play a major pathogenic role in thrombus formation. Clopidogrel treatment in combination with aspirin can prevent recurrent ischaemic events after an acute coronary syndrome (ACS) [1,2]. However, many ACS patients on current standard dual antiplatelet therapy still have thrombotic events because clinical responses to clopidogrel are highly variable between patients. Those patients are clinically designated as clopidogrel resistant or non-responders [3,4]. Clopidogrel is a prodrug that has to be converted into an active metabolite by the hepatic cytochrome P450 (CYP) system [5,6]. The polymorphisms of CYP2C19 might be one of the factors influencing the individual susceptibility. Moreover, CYP2C19 is a key enzyme in this activation process, and the presence of the loss-of-function CYP2C19 681G>A polymorphism (CYP2C19*2) is associated with reduced clopidogrel responsiveness in healthy people [7] and in patients with ACS [8] or percutaneous coronary intervention (PCI) [9]. It has been proved that the CYP2C19*2 variant was associated with a significant increase in the rate of cardiovascular events among patients who under clopidogrel treatment [10]. Then, a strategy of raising the dose of clopidogrel or changing for more potent antiplatelet drugs (such as ticagrelor) may help overcome poor response and improve clinical outcome.

In contrast, CYP2C19 genotype was shown no effect on pharmacodynamics in patients during ticagrelor therapy [11]. And ticagrelor have shown more effective that clopidogrel in the treatment of acute coronary syndrome, it can significantly reduce the rate of death as compared with clopidogrel [12]. However, ticagrelor as a new antiplatelet drug is more expensive than clopidogrel. It needs take twice daily for its pharmacokinetic characteristics and will conduct to less compliance in patients. Recently, it suggested that people carries the risk of increased thrombotic events if patients miss a ticagrelor dose [13,14].

The United States FDA has issued a black box warning about carriers of CYP2C19*2 alleles (http://products.sanofi.us/plavix/plavix.html) and they suggested that these carriers should receive a higher dose of clopidogrel or another drug. Evidence suggests that carriers of at least one CYP2C19*2 allele had a relative reduction of 32.4% in plasma exposure to the active metabolite of clopidogrel, as compared with noncarriers [15]. What’s more, patients with two copies of CYP2C19*2 allele had a higher risk of cardiovascular system event than patients with one or none allele [16]. Similar findings have been noted for ACS patients with the 2-flod maintenance dose of clopidogrel, which has possible better prognostic implications [17,18]. Current guidelines offer a choice among clopidogrel or ticagrelor on top of aspirin for dual antiplatelet ACS therapy. Thus, with the availability of ticagrelor, doctors are challenged to determine the right treatment strategies for individual with CYP2C19*2 allele while minimizing the risk of bleeding and maximizing the efficacy. So we assessed the effect of the twice dosage of clopidogrel or ticagrelor in two cohort of patients who were identified with two copies of CYP2C19*2 loss-of-function polymorphism.

Methods

Study design

It was a single center, randomized trial. A computerized random-number generator was used to draw up an allocation schedule by the first. This prospective study enrolling consecutive patients undergoing non-emergent PCI was performed. An independent data and safety monitoring board monitored the trial and had access to all data. The sponsor coordinated the data management. This study protocol was approved by the local ethic committee in China. All patients that gave informed consent for genetic testing were prospectively included.

Inclusion/exclusion criteria

The study randomized 224 patients who carries CYP2C19*2 homozygotes within 24 hours of an acute coronary syndrome (either ST-elevation or non-ST-elevation) to ticagrelor (n = 112) or clopidogrel (n = 112). Patients were excluded if they had periprocedural P2Y12 inhibitors administration, a history of stroke/transient ischemic attack, bleeding diathesis, chronic oral anticoagulation treatment, PCI, hemodynamic instability, platelet count less than 100 g/L, hematocrit 30%, creatinine clearance 30 ml/min, severe hepatic dysfunction, history of bleeding diathesis, and concurrent severe illness with expected survival of 1 month.

Therapeutic strategy

Ticagrelor (AstraZeneca) was administered as a loading dose of 180 mg followed by 90 mg twice daily, and clopidogrel (Bristol-Myers Squibb and Sanofi) was given as a 600 mg loading dose followed by 150 mg daily of maintenance therapy (for patients who had not been previously taking clopidogrel). In contrast, clopidogrel (300 mg) was administered in the first day and followed by 75 mg daily. Usually, platelet inhibition is dose-related up to a single dose of 400 mg of clopidogrel, then a dose of 75 mg per day produces the same level of inhibition after one week [19]. All patients received background aspirin therapy at a dose of 75 mg per day.

Laboratory test

Blood sample collection

Whole blood for clinical laboratory test and genotyping were obtained from the arterial sheath of all patients directly after diagnostic angiography. DNA was extracted from peripheral blood lymphocytes by standard procedures. All tests were done by nurses and the staff of clinical examination.

CYP2C19*2 genotyping

Genotyping for known variants *2 of the CYP2C19 gene was performed by PCR amplification using primer oligonucleotides designed with Primer3 software tool. Amplification was done with two oligonucleotide primers: Forward, 5’-TGCAATAATTTTCCCACTATCATTG-3’; Reverse, 5’-CCAAAATATCACTTTCCATAAAAGCA-3’.

Polymorphism screening was undertaken with the ABI PRISM dye terminator method. DNA sequencing gave identical genotypes by cycle sequencing kit procedure and loaded on to a capillary automatic sequencer ABI3130xl (Applied Biosystems, USA).

PRU analysis

The initial blood drawn was discarded to avoid measuring platelet activation induced by needle puncture; blood was collected into a Vacutainer containing 3.8% trisodium citrate and filled to 8 ml. The Vacutainer was inverted 3 to 5 times for gentle mixing and sent immediately to the hemostasis laboratory. For the VerifyNow P2Y12 assay, blood sample was performed as described by the manufacturer (Accumetrics, USA). Reported measures included P2Y12 Reaction Units (PRU) and P2Y12 device-reported percent inhibition.

Statistical analysis

Continuous data are expressed as mean ± SD. Categorical data are expressed as frequencies. Means of age, body weight and platelet reactivity unit are compared with Student t tests at a two-tailed 5% level. Frequencies of dyslipidemia, hypertension, diabetes mellitus, smoking, history of myocardial infarction, proportion of ST elevation myocardial infarction and creatinine clearance < 60 ml/min are compared using chi-square test at a two-tailed 5% level. All statistical analyses were performed using SPSS 19.0 software.

Results

We recruited 224 patients from October 2012 through September 2013 by the flow chart (Figure 1). Of 2295 patients with genotype assessment, 224 (9.8%) were identified to have CYP2C19*2 homozygote variant (Table 1) and were all randomized to two group. 1093 (47.6%) were wild-type homozygotes (*1/*1), 978 (42.6%) were heterozygotes (*1/*2), and 224 (9.8%) were homozygous (*2/*2) with respect to the *2 allelic variant. The allelic frequency of CYP2C19*2 was 31.1% (Table 1). We noted no deviation from the expected proportions of genotypes in the selected population predicted by the Hardy-Weinberg equilibrium for polymorphisms.

Figure 1.

Study Flow Chart.

Table 1.

CYP2C19*2 genotype frequencies in patients

SNPs	Allele frequencies		Genotype frequencies			Total
CYP2C19 mutation	*1	*2	*1/*1	*1/*2	*2/*2
Number	2071	1202	1093	978	224	2295
Frequencies (%)	68.9	31.1	47.6	42.6	9.8

Baseline characteristics, such as comorbidities, previous myocardial infarction, drugs, and procedural characteristics, were similar in both groups. In short, Baseline characteristics were well balanced between the two groups defined by CYP2C19*2 genotype (Table 2). Until day 30, there was no obvious side effects leading to study drug discontinuation, every person completed this study. 224 patients had complete follow-up: 112 patients in the doubling clopidogrel group and 112 were given standard ticagrelor treatment (Figure 1).

Table 2.

Baseline characteristics of the patients, according to treatment group

Baseline Characteristic	Ticagrelor Group (N = 112)	Clopidogrel Group (N = 112)	Two-sided p-value
Race	100% (Han Chinese)	100% (Han Chinese)	1.00
Male	70% (86)	71% (87)	0.98
Age, yrs	66±8	67±9	0.36
Body weight, kg ± SD	71±18	70±19	0.67
Dyslipidemia	71% (87)	72% (88)	0.98
Hypertension	30% (36)	32% (39)	0.78
Diabetes mellitus	22% (27)	23% (28)	0.98
Smoking	55% (67)	50% (61)	0.98
Previous myocardial infarction	27% (33)	29% (35)	0.89
ST elevation myocardial infarction	41% (50)	39% (47)	0.79
CrCl < 60 mL/min	12% (15)	10% (12)	0.68

Individual platelet reactivity units (PRU) according to treatment and baseline platelet reactivity values are depicted in Figure 2. Present study showed that the baseline of platelet function was 283.2±12 PRU on aspirin + clopidogrel therapy versus 279.5±16.2 PRU on aspirin + ticagrelor therapy) in CYP2C19*2 homozygotes. After that, the PRU was significantly lower for ticagrelor (34.5±21.0 PRU) compared with clopidogrel (76.6±55.2 PRU) at day 15 (P = 0.000, and their difference is 42.1 (95% confidence interval [CI], 31.1 to 53.1). However, individual PRU at the end day of treatment period are depicted (Clopidogrel PRU 39.8±37.4, 95% [CI]: 32.8 to 46.8 vs. ticagrelor PRU 27.9±12.4, 95% [CI]: 25.6 to 30.2, P = 0.001) in Figure 3.

Figure 2.

Platelet reactivity units (PRU) after treatment sequence.

Figure 3.

Individual platelet reactivity unit values at day 30 after treatment.

No patient exhibited a major adverse cardiovascular event or a major bleeding event at either treatment group. There was no difference in major bleeding. But there were some differences between ticagrelor and clopidogrel in minor bleeding. 23 patients under clopidogrel during the first period reported minimal bleeding events. On the contrary, only 8 patients under ticagrelor reported a mild bleeding event. Thus, patients who treated by clopidogrel were at significantly higher risk for the safety outcome of minimal bleeding events (20.5% vs. 7.1%; hazard ratio for ticagrelor, 2.88; 95% confidence interval [CI], 1.34 to 6.15; P = 0.001).

Discussions

The present study first aimed to compare ticagreor with clopidogrel in ACS patients with two copies of CYP2C19*12 mutation. We hypothesized that persistence of high dose clopidogrel on treatment platelet reactivity could block the P2Y12 receptor as same as ticagrelor. In the present study no significance difference at major events was observed between clopidogrel and ticagrelor group during treatment period. We have demonstrated that clopidgrel provides a little stronger than ticagrelor platelet inhibition and more minor bleeding adverse at last. The results support that people with CYP2C19*2 alleles need rising the dose of clopidogrel to induce an optimal platelet reactivity inhibition. However, the only significant difference between two groups was platelet reactivity inhibition after the first loading dose of clopidogrel at day 15. It means ticagrelor was a faster onset of action in the platelet inhibition and an approximately 2-fold higher potency than clopidogrel. These findings would suggest that ticagrelor is a more rapid and efficacious treatment for ACS than is clopidogrel, irrespective of CYP2C19*2 polymorphisms.

We have further demonstrated that using this kind of doubling clopidogrel loading dose in patients with high on treatment platelet reactivity was associated with higher bleeding adverse. However, previous studies have shown that compared with 300 mg loading dose of clopidogrel, treatment with a 600 mg loading dose can reduced nearly 4 times of non-responsiveness [20] and significance lower rate of cardiovascular death or non-fatal myocardial infarction [21]. It implied that simple doubling dose was not a good choice for all patients with *2 alleles. There may be other CYPs mutations should be considered. CYP2C19*2 polymorphism is not the sole determinant of the response to clopidogrel: implications for its monitoring [22]. The variant allele CYP2C9*3 and *17 play an important role in the response to clopidogrel in patients on dual antiplatelet therapy undergoing coronary stenting [23,24].

First originality is that we have used the VerifyNow as a specific platelet assay to assess platelet reactivity after doubling clopidogrel loading dose (600 mg). In addition, we selected the subjects with two copies of CYP2C19 for the clinical trial at the first time. Only patients who were identified CYP2C19*2 homozygotes were assessed in this comparative study. The CYP2C19 gene polymorphisms are differently distributed according to ethnicity in world population: African (20.2%); Caucasian (16.9%); Mulatto (16.5%); and Amerindian (10.4%) individuals, respectively [25]. In fact, it has been demonstrated that Asian populations had a much higher proportion (> 50%) than caucasian or others [26]. In conclusion, the CYP2C19*2 induced clopidogrel resistance can be managed in some patients by increasing the given dose. But the twice dose of clopidogrel is not responsible for all persons according to platelet reactivity. The comparative risks of long-term cardiovascular events during clopidogrel treatment are not clear. Long-term prognosis should be considered during clopidogrel or ticagrelor exposure. Ticagrelor may be useful in these patients to improve platelet reactivity inhibition. Therefore, for CYP2C19*2 cases, physician use the ticagrelor instead of clopidogrel is a more effective and safe solution for ACS patients.

Disclosure of conflict of interest

None.