BMJ
Confidential: For Review Only Ticagrelor with Aspirin for Platelet Reactivity in Minor Stroke or Transient Ischemic Attack Patients: A Randomized, Open-label, Blinded-endpoint Trial
Journal: BMJ
Manuscript ID BMJ-2018-047607.R1
Article Type: Research
BMJ Journal: BMJ
Date Submitted by the 27-Feb-2019 Author:
Complete List of Authors: WANG, YILONG; Tiantan Comprehensive Stroke Center, Tiantan Hospital, Capital Medical University, Beijing, China, Chen, Weiqi; Beijing Tiantan Hospital, Capital Medical University Lin, Yi; Department of Neurology and Institute of Neurology, First Affiliated Hospital, Fujian Medical University Meng, Xia; Beijing Tiantan Hospital Capital Medical University, Department of Neurology Chen, Guohua; Department of Neurology, Wuhan No.1 Hospital Wang, zhimin; Department of Neurology, Taizhou First People's Hospital, Huangyan Hospital of Wenzhou Medical University Wu, Jialing; Department of Neurology, Tianjin Huanhu Hospital Wang, Dali; Department of Neurology, North China University of Science and Technology Affiliated Hospital, Li, Jianhua; Department of Neurology, the First Hospital of Fangshan District Cao, Yibin; Department of Neurology, Tangshan Gongren Hospital Xu, Yu-ming ; The First Affiliated Hospital of Zhengzhou University, Department of Neurology Zhang, Guohua; Department of Neurology, the Second Hospital of Hebei Medical University, Li, Xiaobo; Department of Neurology, Northern Jiangsu People's Hospital, Clinical Medical School, Yangzhou University PAN, YUESONG; Beijing Tiantan Hospital, Capital Medical University, Department of Neurology LI, HAO; Tiantan Comprehensive Stroke Center, Tiantan Hospital, Capital Medical University, Beijing, China, Zhao, Xingquan; Beijing Tiantan Hospital Capital Medical University, Department of Neurology Liu, Liping; Beijing Tiantan Hospital Capital Medical University, Department of Neurology Lin, Jinxi; Department of Neurology, Beijing Tiantan Hospital, Capital Medical University, Dong, Kehui; Department of Neurology, Beijing Tiantan Hospital, Capital Medical University, Jing, Jing; Department of Neurology, Beijing Tiantan Hospital, Capital Medical University,
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1 2 3 Johnston, S Claiborne; University of Texas, Austin, Neurology 4 Wang, David; INI Stroke Network, OSF Healthcare System, University of 5 Illinois College of Medicine, 6 Wang, Yongjun; Department of Neurology, Beijing Tiantan Hospital, 7 Capital Medical University, 8 9 Keywords: Platetlet Reactivity, Ticagrelor, Stroke, TIA, CYP2C19 10 11 Confidential: For Review Only 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 https://mc.manuscriptcentral.com/bmj BMJ Page 2 of 36
Wang Page 1 1 2 3 Ticagrelor with Aspirin for Platelet Reactivity in Minor Stroke or Transient 4 5 6 Ischemic Attack Patients: A Randomized, Open-label, Blinded-endpoint Trial 7 Yilong Wang, MD, PhD1,2,3,4*, Weiqi Chen, MD, PhD1,2,3,4*, Yi Lin, MD, PhD5, Xia Meng, MD, 8 PhD1,2,3,4, Guohua Chen, MD6, Zhimin Wang, MD7, Jialing Wu, MD8, Dali Wang, MD9, Jianhua 9 10 11 12 13 14 10 Li, MD , Yibin Cao , MD, Yuming Xu , MD, Guohua Zhang , MD, Xiaobo Li , MD, 1,2,3,4 1,2,3,4 1,2,3,4 11 YuesongConfidential: Pan, PhD , Hao Li, PhD ,For Xingquan Review Zhao, MD, PhD Only, Liping Liu, MD, 12 PhD1,2,3,4, Jinxi Lin, MD, PhD1,2,3,4, Kehui Dong, MD1,2,3,4, Jing Jing, MD, PhD1,2,3,4, S. 13 Claiborne Johnston, MD, PhD15, David Wang, DO, FAHA, FAAN16, Yongjun Wang, MD1,2,3,4; 14 On Behalf of the PRINCE Protocol Steering Group 15 16 17 Yilong Wang and Weiqi Chen contributed equally to the manuscript. 18 19 1 Department of Neurology, Beijing Tiantan Hospital, Capital Medical University, Beijing, China 20 2 China National Clinical Research Center for Neurological Diseases, Beijing, China 21 3 Center of Stroke, Beijing Institute for Brain Disorders, Beijing, China 22 4 Beijing Key Laboratory of Translational Medicine for Cerebrovascular Disease, Beijing, China 23 5 Department of Neurology and Institute of Neurology, First Affiliated Hospital, Fujian Medical 24 25 University, Fuzhou, China 6 26 Department of Neurology, Wuhan No.1 Hospital, Wuhan, China 27 7 Department of Neurology, Taizhou First People's Hospital, Huangyan Hospital of Wenzhou 28 Medical University, Taizhou, China 29 8 Department of Neurology, Tianjin Huanhu Hospital, Tianjin, China 30 9 Department of Neurology, North China University of Science and Technology Affiliated 31 Hospital, Tangshan, China 32 10 33 Department of Neurology, the First Hospital of Fangshan District, Beijing, China 11 34 Department of Neurology, Tangshan Gongren Hospital, Tangshan, China 35 12 Department of Neurology, the First Affiliated Hospital of Zhengzhou University, Zhengzhou, 36 China 37 13 Department of Neurology, the Second Hospital of Hebei Medical University, Shijiazhuang, 38 China 39 14 40 Department of Neurology, Northern Jiangsu People's Hospital, Clinical Medical School, 41 Yangzhou University, Yangzhou, China 42 15 Dell Medical School, University of Texas at Austin, USA 43 16 INI Stroke Network, OSF Healthcare System, University of Illinois College of Medicine, 44 Peoria, USA 45 46 Corresponding author: Yongjun Wang and Yilong Wang 47 48 E-mail address: [email protected] and [email protected] 49 Address: No.6 Tiantanxili, Dongcheng District, Beijing, China, 100050 50 Business telephone: 0086-010-67098350 51 Fax numbers: 0086-010-67013383 52 Tables: 3; Figures: 4; Word count: 4355 (not including title page, figure legends, references, 53 tables and figures) 54 55 56 57 58 59 60 https://mc.manuscriptcentral.com/bmj Page 3 of 36 BMJ
Wang Page 2 1 2 3 ABSTRACT 4 5 6 OBJECTIVE 7 8 To test the hypothesis that ticagrelor plus aspirin is safe and superior to clopidogrel plus aspirin 9 10 for reducing the 90-day high platelet reactivity and stroke recurrence in patients with minor 11 Confidential: For Review Only 12 stroke or transient ischemic attack (TIA), particularly in carriers of the CYP2C19 loss-of- 13 14 15 function allele and patients with large-artery atherosclerosis. 16 17 DESIGN 18 19 Randomized, open-label, controlled, blinded-endpoint trial. 20 21 22 SETTING 23 24 Prospective studies conducted at 26 centres in China. 25 26 PARTICIPANTS 27 28 29 A total of 675 acute minor stroke or TIA patients. 30 31 INTERVENTION 32 33 Ticagrelor (180 mg loading dose, 90 mg twice daily thereafter) or clopidogrel (300 mg loading 34 35 dose, 75 mg daily thereafter) on a background of aspirin (100 mg daily for the first 21 days) 36 37 38 within 24 h of symptom onset. 39 40 MAIN OUTCOME MEASURES 41 42 The primary outcome of the trial was the proportion of patients with high platelet reactivity at 90 43 44 45 days. High platelet reactivity was defined as a P2Y12 reaction unit of >208 measured using the 46 47 VerifyNow P2Y12 assay. The secondary outcomes included high platelet reactivity at 90±7 days 48 49 in patients carrying genetic variants that would affect clopidogrel metabolism and any stroke 50 51 52 (ischemic or haemorrhagic) recurrence at 90±7 days, 6 months, and 1 year. 53 54 RESULTS 55 56 57 58 59 60 https://mc.manuscriptcentral.com/bmj BMJ Page 4 of 36
Wang Page 3 1 2 3 At 90 days, high platelet reactivity occurred in 35 of 280 patients (12.5%) in the 4 5 6 ticagrelor/aspirin group and 86 of 290 patients (29.7%) in the clopidogrel/aspirin group (odds 7 8 ratio [OR], 0.34; 95% confidence interval [CI], 0.22-0.52; P<0.001), and 10.8% versus 35.4% 9 10 (OR, 0.22, 95% CI, 0.12-0.40; P<0.001) of patients carrying CYP2C19 loss-of-function alleles 11 Confidential: For Review Only 12 (P=0.04 for interaction). Stroke occurred in 21 of 336 patients (6.3%) in the ticagrelor/aspirin 13 14 15 group and 30 of 339 patients (8.8%) in the clopidogrel/aspirin group (hazard ratio, 0.70; 95% CI, 16 17 0.40-1.22; P=0.20). Patients with large-artery atherosclerosis in the ticagrelor/aspirin group had 18 19 lower stroke recurrence at 90 days (6.0%) than did those in the clopidogrel/aspirin group (13.1%, 20 21 22 hazard ratio, 0.45; 95% CI, 0.20-0.98; P=0.04). No difference was observed in the rates of major 23 24 or minor haemorrhagic events between the ticagrelor and clopidogrel groups (4.8% and 3.5%, 25 26 respectively; P=0.42). 27 28 29 CONCLUSION 30 31 Minor stroke or TIA patients who were treated with ticagrelor had a lower rate of high platelet 32 33 reactivity than did those who were treated with clopidogrel, particularly those who were carriers 34 35 of the CYP2C19 loss-of-function allele and lower recurrent stroke in patients with large-artery 36 37 38 atherosclerosis. 39 40 TRIAL REGISTRATION 41 42 Clinicaltrials.gov NCT02506140 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 https://mc.manuscriptcentral.com/bmj Page 5 of 36 BMJ
Wang Page 4 1 2 3 Introduction 4 5 6 Acute minor ischemic stroke and transient ischemic attack (TIA) patients are at high risk of 7 8 recurrent stroke and cardiovascular events.1 The Clopidogrel in High-risk patients with Acute 9 10 Non-disabling Cerebrovascular Events (CHANCE) trial indicated that combined clopidogrel and 11 Confidential: For Review Only 12 aspirin treatment is superior to aspirin alone in reducing the risk of stroke,2 but may increase the 13 14 15 risk of non-intracranial haemorrhage.1, 3 The CHANCE genetic sub-study demonstrated that 16 17 patients who were carriers of the cytochrome P450 (CYP) 2C19*2 and *3 loss-of-function 18 19 alleles benefitted more from using aspirin alone than from using dual antiplatelet therapy.4 20 21 22 Additionally, about 50% patients with acute ischemic stroke had a risk of intracranial large- 23 24 artery atherosclerosis (LAA) in Asia, and there was a higher rate of recurrent stroke in patients 25 26 with intracranial arterial stenosis with minor stroke or a high risk of TIA than in those without.5, 6 27 28 7 29 Ticagrelor is primarily metabolized via the CYP3A4 enzyme. A genetic sub-study of the 30 31 Platelet Inhibition and Patient Outcomes (PLATO) trial indicated that ticagrelor is more 32 33 efficacious for acute coronary syndromes than clopidogrel regardless of CYP2C19 genotype, but 34 35 was associated with an increased risk of haemorrhage in patients with a history of stroke.8 The 36 37 38 Acute Stroke or Transient Ischemic Attack Treated With Aspirin or Ticagrelor and Patient 39 40 Outcomes (SOCRATES) Trial revealed a trend towards better efficacy in reducing the risk of 41 42 vascular events in the ticagrelor treated group than in the aspirin group in an Asian 43 44 45 subpopulation. However, limited data are available on the safety and efficacy of ticagrelor for the 46 47 treatment of stroke as compared to those for clopidogrel on a background of aspirin in acute 48 49 stroke patients.4, 9, 10 50 51 52 53 54 55 56 57 58 59 60 https://mc.manuscriptcentral.com/bmj BMJ Page 6 of 36
Wang Page 5 1 2 3 High platelet reactivity is defined as resistance or non-responsiveness to antiplatelet agents.11, 4 5 12 6 High platelet reactivity is associated with poor cerebrovascular outcomes, and might be of 7 8 clinical value as a surrogate marker in the evaluation of the effects of antiplatelet agents.13-16 9 10 We conducted this Platelet Reactivity in Acute Stroke or Transient Ischemic Attack (PRINCE) 11 Confidential: For Review Only 12 trial to test the hypothesis that ticagrelor plus aspirin is safe and superior to clopidogrel plus 13 14 15 aspirin in reducing the 90 day high platelet reactivity and stroke recurrence in minor stroke or 16 17 TIA patients, particularly in carriers of the CYP2C19 loss-of-function allele and patients with 18 19 LAA.17 20 21 22 Methods 23 24 The PRINCE study protocol and data collection were approved by the ethics committee of 25 26 Beijing Tiantan Hospital (NO. KY2014-048-01) and all of the study centres. An interim analysis 27 28 17 29 was pre-planned in the published protocol. All participants or their representatives provided 30 31 written consent before being enrolled in the study. The design of the trial was published 32 33 previously.17 The trial was conducted in accordance with the Declaration of Helsinki. 34 35 Study design and participants 36 37 38 The trial was a prospective, multicentre, randomized, open-label, active-controlled, blinded- 39 40 endpoint trial. Patients were recruited at 26 study centres from August 2015 to March 2017. The 41 42 trial included six visits: randomization (baseline), 7+2 days, 21±2 days, 90±7 days, 6 months±14 43 44 45 days, and 1 year±14 days. Additional visits at 2 and 24 h after the first dose was administered 46 47 were optional. All visits involved face-to-face interviews, with the exception of the 6-month 48 49 follow-up, which was conducted by telephone, with data collected on electronic case report 50 51 52 forms. The general schedule of the trial and the collection times for blood and urine samples are 53 54 listed in the supplementary appendix (Blood and urine samples collecting schedule). 55 56 57 58 59 60 https://mc.manuscriptcentral.com/bmj Page 7 of 36 BMJ
Wang Page 6 1 2 3 The PRINCE trial enrolled patients aged 40-80 years who had experienced an acute minor 4 5 6 ischemic stroke (National Institutes of Health Stroke Scale score of ≤3 at the time of 7 8 randomization) or those with a moderate to high risk of TIA (ABCD2 stroke risk score of >4 at 9 10 the time of randomization or >50% stenosis of cervical or intracranial vessels that could account 11 Confidential: For Review Only 12 13 for the presentation. Meet any of the two criteria for patients with TIA could be enrolled) who 14 15 could be treated with the study drug within 24 h of symptom onset. 16 17 Patients were excluded from the trial if they were diagnosed with intracranial haemorrhage, 18 19 20 acute coronary syndrome, or other pathology that could account for the neurological symptoms; 21 22 had a modified Rankin scale score of >2 at randomization; or had a contraindication to ticagrelor, 23 24 clopidogrel, or aspirin. Details of the full inclusion and exclusion criteria are provided in the 25 26 supplementary appendix (Inclusion and exclusion criteria). 27 28 29 Randomization and masking 30 31 Immediately after signing the written informed consent form, eligible patients were randomized 32 33 in a 1:1 ratio to the ticagrelor and aspirin (intervention) or clopidogrel and aspirin (control) group 34 35 36 via a block randomization process by investigators at the clinical centres. The block 37 38 randomization sequence was provided by an independent statistician using computer-generated 39 40 random numbers with a block size of four. The block size was not listed in the Chinese version 41 42 43 of the protocol (which was provided to the investigators) in order to prevent the investigators 44 45 from speculating about the group assignment. 46 47 VerifyNow testing was conducted in each study centre by qualified personnel who were 48 49 blinded to the treatment allocation. Each reported composite clinical vascular event and safety 50 51 52 outcome was independently adjudicated by two members (Drs Kehui Dong and Jimei Li) of the 53 54 Clinical Event Adjudication Committee (CEC) who were blinded to the treatment group 55 56 57 58 59 60 https://mc.manuscriptcentral.com/bmj BMJ Page 8 of 36
Wang Page 7 1 2 3 assignments. All discrepancies were reviewed by all five members of the CEC and resolved by 4 5 6 consensus. 7 8 Procedures 9 10 The enrolled patients were randomly assigned to receive either ticagrelor (a loading dose of 180 11 Confidential: For Review Only 12 mg administered as two 90 mg tablets on day 1, followed by 90 mg twice daily until day 90) 13 14 15 combined with aspirin (a loading dose of 100-300 mg administered as 1-3 100 mg tablets on day 16 17 1, followed by 100 mg once daily until day 21) or clopidogrel (a loading dose of 300 mg 18 19 20 administered as four 75 mg tablets on day 1, followed by 75 mg once daily until day 90) 21 22 combined with aspirin (a loading dose of 100-300 mg administered as 1-3 100 mg tablets on day 23 24 1, followed by 100 mg once daily until day 21). Both the investigators and the patients were 25 26 27 aware of the study drug assignment, but were blinded to the platelet reactivity data until the end 28 29 of the trial. The study drugs were required to be administered within 1 h of randomization. The 30 31 loading dose and maintenance doses of ticagrelor were administered as in the SOCRATES and 32 33 PLATO studies.10, 18 34 35 36 Outcomes 37 38 The primary outcome of the PRINCE trial was the proportion of patients with high platelet 39 40 reactivity at 90 days. High platelet reactivity was defined as a P2Y12 reaction unit (PRU) 41 42 43 of >208 measured using the VerifyNow P2Y12 assay. The PRUs were measured in each study 44 45 centre by specially trained and qualified personnel according to a standardized procedure 46 47 manual. To make sure the validity and reproducibility of the assay, we held two separate training 48 49 50 course for all the testing personnel from each centre. Pre-specified secondary outcomes included 51 52 high platelet reactivity at 90±7 days in patients carrying genetic variants that would affect 53 54 clopidogrel metabolism; any stroke (ischemic or haemorrhagic); and composite clinical vascular 55 56 57 58 59 60 https://mc.manuscriptcentral.com/bmj Page 9 of 36 BMJ
Wang Page 8 1 2 3 events (ischemic/haemorrhagic stroke, TIA, myocardial infarction, or vascular death) at 90±7 4 5 6 days, 6 months, and 1 year. 7 8 The primary safety outcome was major bleeding, which was defined as that in the PLATO 9 10 study classification of haemorrhagic events; fatal/life-threatening bleed, major bleed, and other 11 Confidential: For Review Only 12 (see supplementary appendix PLATO Bleeding classification”). Secondary safety outcomes 13 “ 14 15 included the incidence of intracranial bleeding; dyspnoea events; and mortality at 90±7 days, 6 16 17 months, and 1 year. Complete information on pre-specified outcomes is available in the study 18 19 protocol.17 20 21 22 Genotyping 23 24 While several genetic variants affect clopidogrel metabolism, we designed the present study to 25 26 focus on the CYP2C19 (National Center for Biotechnology Information (NCBI) Genome build 27 28 29 37.1, NG_008384). The pre-specified analysis included the single-nucleotide polymorphisms 30 31 (SNPs) CYP2C19*2 (681G>A, rs4244285), CYP2C19*3 (636G>A, rs4986893), and 32 33 CYP2C19*17 (-806C>T, rs12248560), which were genotyped in all participants with adequate 34 35 36 blood samples. Most genotyping of the three SNPs was performed using the Sequenom 37 38 MassARRAY iPLEX platform (Sequenom, San Diego, CA). Sanger sequencing (ABI 3500 39 40 Genetic Analyzer [Applied Biosystems]) was used when the results were otherwise inconclusive. 41 42 We used star allele nomenclature to categorize patients by the CYP2C19 metabolizer status 43 44 19 45 based on *2, *3, and *17 genotypes. Patients with at least one gain-of-function allele (*17) 46 47 were classified as "gain-of-function allele carriers", and those with at least one loss-of-function 48 49 allele (*2 or *3) were classified as "loss-of-function allele carriers".20 Patients who carried at 50 51 52 least one *17 allele (*1/*17 or *17/*17) were classified as "ultra-metabolizers", those without 53 54 any *2, *3, or *17 allele (*1/*1) were classified as "extensive metabolizers", those with one *2 55 56 57 58 59 60 https://mc.manuscriptcentral.com/bmj BMJ Page 10 of 36
Wang Page 9 1 2 3 or *3 allele (*1/*2 or *1/*3) were classified as "intermediate metabolizers", and those with at 4 5 20 6 least two *2 or *3 alleles (*2/*2, *2/*3, or *3/*3) were classified as "poor metabolizers". 7 8 Patients with one *17 and a loss-of-function allele (*2/*17 or *3/*17) were classified as 9 10 "unknown metabolizers”, since the clinical effect of these alleles is uncertain.21, 22 11 Confidential: For Review Only 12 Based on our pre-planned aims, we compared the recurrence of stroke in patients whose stroke 13 14 15 subtype was intracranial large-artery atherosclerosis (LAA) with those whose stroke subtype was 16 17 non-LAA based on the SSS-TOAST stroke subtype. Non-LAA included cardioaortic embolism, 18 19 small-artery occlusion, other causes, and undetermined causes. 20 21 22 Statistical analysis 23 24 We calculated that a total of 952 patients (estimated 10% dropout rate) would be required to 25 26 achieve 90% power with a two-sided α=0.05 to detect a relative reduction of 24% in the rate of 27 28 29 the primary outcome in the ticagrelor/aspirin group compared to that in the clopidogrel/aspirin 30 31 group. The DSMB opted to terminate the study after the interim analysis based on 476 patients 32 33 (50% of the projected necessary sample size) who completed 90 days of follow-up based on 34 35 achieving a pre-specified threshold for efficacy (P<0.005). At the time of this decision, an 36 37 38 additional 199 patients had already been recruited and randomized into the trial, and these 39 40 patients were also followed-up to study completion. Therefore, a total of 675 patients were 41 42 included in the intention-to-treat analyses. 43 44 45 Proportions were presented for categorical variables and medians with interquartile ranges or 46 47 means ± standard deviation were presented for continuous variables. We compared the baseline 48 49 characteristics of patients between the two treatment groups using the non-parametric Kruskal- 50 51 2 52 Wallis test for nominal variables, and the χ test or Fisher’s exact test for dichotomous variables. 53 54 55 56 57 58 59 60 https://mc.manuscriptcentral.com/bmj Page 11 of 36 BMJ
Wang Page 10 1 2 3 The rates of high platelet reactivity at the 90-day follow-up (the primary outcome) were 4 5 6 compared between the two groups using genmod models adjusted by the baseline high platelet 7 8 reactivity status, reported as an risk ratio (RR) with 95% confidence intervals (CIs). To evaluate 9 10 the influence of missing data for the primary outcome, sensitivity analyses were performed 11 Confidential: For Review Only 12 assuming all the missing data were high platelet reactivity or not. 13 14 15 The differences in the rates of stroke, composite outcome, death, and bleeding events during 16 17 the 90-day follow-up period were assessed using Cox proportional-hazards regression and 18 19 reported as hazard ratios (HRs) with 95% CIs. The proportional hazard assumption for the Cox 20 21 22 models was examined by including a time-dependent covariate with interaction of treatment 23 24 group and a logarithmic function of survival time in the model. 25 26 Whether the treatment effect differed in certain genotype categories was assessed by testing 27 28 29 the treatment-by-genotype interaction effect in genmod models for the primary outcome and Cox 30 31 models for other outcomes, as described earlier. Whether the treatment effect in reducing stroke 32 33 recurrence differed between LAA and non-LAA patients was assessed by testing treatment-by- 34 35 stroke subtype interaction effect in a genmod model. 36 37 38 All statistical analyses were two-sided, and differences with a P-value of <0.05 were 39 40 considered to be statistically significant. Analyses were performed using SAS software, version 41 42 9.4 (SAS Institute, Cary, NC). This study is registered with ClinicalTrials.gov, number 43 44 45 NCT02506140. 46 47 Patient and public involvement 48 49 The design, outcome measurement, recruiting plans or implementation of the study were 50 51 52 independent of any patient. The gene and platelet reactivity testing results of every patient will 53 54 be delivered to the patient himself or his appointed relatives after the primary results of the 55 56 57 58 59 60 https://mc.manuscriptcentral.com/bmj BMJ Page 12 of 36
Wang Page 11 1 2 3 results published through email or telephone. The results of the research will be broadcasted to 4 5 6 all the participants and general public through internet news, popular science article, newspapers 7 8 and social media. 9 10 Results 11 Confidential: For Review Only 12 Between August 2015 and March 2017, a total of 5,644 stroke or TIA patients were screened at 13 14 15 26 hospitals, and 675 patients (mean age, 61.1±8.5 years) were enrolled before the DSMB opted 16 17 to terminate the trial after reviewing the results of the interim analysis. Thus, 336 patients were 18 19 randomly assigned to the ticagrelor/aspirin group and 339 patients were assigned to the 20 21 22 clopidogrel/aspirin group (table 1). The primary causes of patients who were excluded from the 23 24 trial was beyond 24-hour time window and refused consent (figure 1). The average age was 25 26 60.8±8.7 years, and 26.8% of the patients were women. At 90 days, 12 patients were lost to 27 28 29 follow-up owing to clinical events, and the VerifyNow P2Y12 assay data were not obtained from 30 31 105 patients(see supplementary table A for details of the baseline characteristics of patients with 32 33 and without platelet function data). 34 35 Among the 675 patients enrolled, 650 (mean age, 60.8±8.7 years) had complete genotype data 36 37 38 for all three SNPs, of whom 321 were randomly assigned to the ticagrelor/aspirin group and 329 39 40 were assigned to the clopidogrel/aspirin group (see supplementary table B, C and D for details of 41 42 the baseline characteristics of patients enrolled in the genetic sub-study). Of the 650 participants, 43 44 45 374 (57.5%) were classified as CYP2C19 loss-of-function carriers. 46 47 We obtained valid measurements in 627 (92.9%) and 570 (84.4%) patients for the VerifyNow 48 49 P2Y12 assay at the 7-day and 90-day follow-up periods, respectively. The PRUs before 50 51 52 receiving the study drugs were similar in the ticagrelor/aspirin group (256.4±61.3) and the 53 54 clopidogrel/aspirin group (246.9±53·7, P=0.13). The PRU in the ticagrelor/aspirin group 55 56 57 58 59 60 https://mc.manuscriptcentral.com/bmj Page 13 of 36 BMJ
Wang Page 12 1 2 3 (69.3±87.0) was significantly lower than that in the clopidogrel/aspirin group (173.5±67.6; 4 5 6 P<0.001) at the 90-day follow-up (figure 2A). The primary outcome (high platelet reactivity) 7 8 was observed in 35 of the 280 patients (12.5%) in the ticagrelor/aspirin group, and in 86 of the 9 10 290 patients (29.7%) in the clopidogrel/aspirin group at 90 days (RR, 0.40; 95% CI, 0.28-0.56; 11 Confidential: For Review Only 12 P<0.001) (figure 2B and table 2). Similar results were observed for primary outcome in 13 14 15 sensitivity analyses assuming all missing data of primary outcome were high platelet reactivity 16 17 or not (see supplementary table E). 18 19 Stroke, the main secondary outcome, occurred in 21 (6.3%) of the 336 patients in the 20 21 22 ticagrelor/aspirin group and 30 (8.8%) of the 339 patients in the clopidogrel/aspirin group (HR, 23 24 0.70; 95% CI, 0.40-1.22; P=0.20). Composite clinical vascular events occurred in 22 patients 25 26 (6.5%) in the ticagrelor/aspirin group and 32 patients (9.4%) in the clopidogrel/aspirin group 27 28 29 (HR, 0.68; 95% CI, 0.40-1.18; P=0.17) (table 2). All of the proportional hazard assumptions 30 31 were met (P=0.89 for stroke and P=0.85 for composite events). 32 33 Among the CYP2C19 loss-of-function carriers, high platelet reactivity at 90 days was 34 35 markedly less frequent in the ticagrelor/aspirin group than in the clopidogrel/aspirin group 36 37 38 (10.8% in ticagrelor/aspirin group versus 35.4% in the clopidogrel/aspirin group; RR, 0.31; 95% 39 40 CI, 0.18-0.49; P<0.001) (see supplementary table F). The event rates of stroke, composite events, 41 42 and haemorrhagic events varied by treatment assignment and phenotype (figure 3). Other 43 44 45 primary and secondary outcomes in patients with available genotype data are listed in the 46 47 supplementary table G. 48 49 Among the patients with LAA, stroke recurrence at 90 days was markedly lower in the 50 51 52 ticagrelor/aspirin group than in the clopidogrel/aspirin group (6.0% in ticagrelor/aspirin group 53 54 versus 13.1% in clopidogrel/aspirin group; HR, 0.45; 95% CI, 0.20-0.98; P=0.04) (table 3 and 55 56 57 58 59 60 https://mc.manuscriptcentral.com/bmj BMJ Page 14 of 36
Wang Page 13 1 2 3 figure 4). However, among the patients with non-LAA, the risk of stroke recurrence was similar 4 5 6 in the two groups (8.1% in ticagrelor/aspirin group versus 7.4% in the clopidogrel/aspirin group; 7 8 HR, 1.1; 95% CI, 0.46-2.63; P=0.84) (table 3 and figure 4). The proportional hazard assumption 9 10 was met (P=0.93). 11 Confidential: For Review Only 12 The primary safety outcome (PLATO major haemorrhagic event) occurred in five patients 13 14 15 (1.5%) in the ticagrelor/aspirin group and four patients (1.2%) in the clopidogrel/aspirin group 16 17 (HR, 1.27; 95% CI, 0.34-4.72) (table 2). Three patients (0.9%) in the ticagrelor/aspirin group and 18 19 two patients (0.6%) in the clopidogrel/aspirin group experienced intracranial haemorrhage. 20 21 22 However, the rate of any haemorrhagic events occurring was higher in the ticagrelor/aspirin 23 24 group (22.3%) than in the clopidogrel/aspirin group (14.2%; HR, 1.65; 95% CI, 1.15-2.37) (table 25 26 2). All of the proportional hazard assumptions were met (P=0.99 for major haemorrhagic event 27 28 29 and P=0.82 for any haemorrhagic events). 30 31 The rate of major bleeding did not significantly vary in the ticagrelor/aspirin and 32 33 clopidogrel/aspirin groups between the carriers (rate of 0.0% in the ticagrelor/aspirin group 34 35 versus 1.6% in the clopidogrel/aspirin group; HR, 0.40; 95% CI, 0.04-3.85; P=0.43) and non- 36 37 38 carriers (rate of 0.0% in the ticagrelor/aspirin group versus 0.7% in the clopidogrel/aspirin 39 40 group) (see supplementary table G). 41 42 Dyspnoea was more common in the ticagrelor/aspirin group (16.1%) than in the 43 44 45 clopidogrel/aspirin group (3.2%) (see supplementary table H). A total of 69 (20.5%) patients in 46 47 the ticagrelor/aspirin group and 47 (13.9%) patients in the clopidogrel/aspirin group stopped 48 49 receiving the study drug before 90 days (figure 1). Two of the most common reasons were 50 51 52 dyspnoea and epistaxis. The rate of permanent discontinuation caused by dyspnoea was 4.2% in 53 54 the ticagrelor/aspirin group and 0.0% in the clopidogrel/aspirin group, and the rate of permanent 55 56 57 58 59 60 https://mc.manuscriptcentral.com/bmj Page 15 of 36 BMJ
Wang Page 14 1 2 3 discontinuation caused by epistaxis was 1.8% in the ticagrelor/aspirin group and 0.0% in the 4 5 6 clopidogrel/aspirin group (see supplementary table I). Serious adverse events and adverse events 7 8 leading to permanent drug discontinuation within 90 days are listed in the supplementary table I. 9 10 Discussion 11 Confidential: For Review Only 12 This PRINCE trial indicated that the rate of high platelet reactivity at 90 days was significantly 13 14 15 reduced with ticagrelor plus aspirin treatment compared to that with clopidogrel plus aspirin 16 17 treatment in acute minor stroke patients and those at moderate to high risk of TIA, while it was 18 19 not powered to study clinical events. There were numerically fewer strokes and composite 20 21 22 outcomes at 90 days in patients treated with ticagrelor plus aspirin than in those treated with 23 24 clopidogrel plus aspirin. 25 26 There are limited data addressing the safety of dual anti-platelet therapy with ticagrelor and 27 28 29 aspirin in stroke patients. In this study, we found that the risk of major, minor, or intracranial 30 31 haemorrhage in patients who were treated within 24 h of the onset of minor stroke or TIA did not 32 33 increase with treatment with a combination of ticagrelor compared to the risk in those who were 34 35 treated with clopidogrel plus aspirin, but a higher rate of dyspnoea and minimal haemorrhagic 36 37 38 events was observed in the ticagrelor/aspirin group than in the clopidogrel/aspirin group. 39 40 Compared to that in the PLATO study, the major haemorrhage rate in our study was markedly 41 42 lower (ticagrelor/aspirin group, 11.6% in PLATO versus 1.5% in PRINCE; clopidogrel/aspirin 43 44 18 45 group, 11.2% in PLATO versus 1.2% in PRINCE). This could be partially due to the short- 46 47 term use of dual anti-platelet therapy in our study (277 days in PLATO versus 21 days in 48 49 PRINCE). Compared to that in the Asian subgroup in the SOCRATES trial, which used 50 51 52 monotherapy, our study found a slightly higher major haemorrhagic rate (0.6% in the ticagrelor 53 54 group in SOCRATES versus 1.5% in the ticagrelor/aspirin group in PRINCE).4 This may be 55 56 57 58 59 60 https://mc.manuscriptcentral.com/bmj BMJ Page 16 of 36
Wang Page 15 1 2 3 related to the combined use of two drugs in our study. Similar to that reported in the PLATO and 4 5 6 SOCRATES trials, dyspnoea occurred more commonly with ticagrelor than with clopidogrel 7 8 treatment.10, 18 9 10 The premature discontinuation of the study drug was more frequent in the ticagrelor/aspirin 11 Confidential: For Review Only 12 group than in the clopidogrel/aspirin group, although the rate of serious adverse events did not 13 14 15 differ between the two groups. One potential explanation is that our study was of an open-label 16 17 design, considering that ticagrelor had not yet been approved for treatment of acute stroke 18 19 patients at the time of the study. Once an adverse event occurred in a patient receiving ticagrelor, 20 21 22 the treating physician or patient had the option of temporarily or permanently discontinuing the 23 24 drug and may have taken this opportunity more frequently when using an unapproved drug. 25 26 Furthermore, early discontinuation and non-adherence likely contributed to the increase in 27 28 29 platelet reactivity observed at 90 days compared to that observed at 7 days in patients 30 31 randomized to the ticagrelor/aspirin treatment groups. 32 33 Although the clinical efficacy (recurrent stroke) was tested between the two groups, the 34 35 sample size is small and the trial was not powered to find an clinical effect. There were 36 37 38 numerically fewer recurrent stroke and composite events in patients treated with ticagrelor plus 39 40 aspirin than in those treated with clopidogrel plus aspirin, but the difference was not statistically 41 42 significant. Given the testing power of 90% and the significance level of 5% (two-sided), a total 43 44 45 of 4,690 patients would be required to detect the relative risk difference between the 46 47 ticagrelor/aspirin and clopidogrel/aspirin groups based on the event rates in our study. The 48 49 current study provided 25.5% power to detect a 30% relative risk difference with a two-sided test 50 51 52 at a 5% significance level. A previous study demonstrated that patients with LAA had a higher 53 54 risk of stroke recurrence even after using clopidogrel and aspirin.6 The stroke recurrent rate in 55 56 57 58 59 60 https://mc.manuscriptcentral.com/bmj Page 17 of 36 BMJ
Wang Page 16 1 2 3 LAA group was higher than that in the non-LAA group in our study. Minor stroke or TIA with 4 5 6 LAA patients might benefit more from ticagrelor plus aspirin than from clopidogrel and aspirin 7 8 therapy. Considering the large numbers required for the stroke sub typing analyses, these results 9 10 were exploratory and hypothesis generating and future studies were needed. Also ,we found that 11 Confidential: For Review Only 12 the stroke recurrent rate in patients in non-LAA were relatively high (7.7%). This might be 13 14 15 relevant with the overall recurrent stroke rate after minor stroke or TIA were relatively high 16 17 because the recurrent stroke included a new stroke and also rapid worsening(NIHSS ≥4) of an 18 19 existing focal neurological deficit in our study. 20 21 22 Understanding the relationship between CYP2C19 variants and the impact of clopidogrel on 23 24 platelet function is critically important, and prompted the genetic analysis in the present study. 25 26 Among patients randomized to clopidogrel, there was a dose-response relationship between the 27 28 29 number of CYP2C19 loss-of-function alleles and the proportion of high platelet reactivity, with 30 31 patients carrying more loss-of-function alleles having higher rates of platelet aggregation. The 32 33 relationship between CYP2C19 and the response to ticagrelor was less clear, and a larger relative 34 35 benefit of ticagrelor was observed in those carrying more loss-of-function alleles. While a similar 36 37 38 trend was observed in clinical outcomes among those randomized to clopidogrel, the numbers 39 40 were small, and the patients randomized to ticagrelor also had numerically higher rates of 41 42 clinical events among those carrying a greater number of loss-of-function alleles. Thus, the 43 44 45 relative clinical benefit of ticagrelor on platelet inhibition among those with a greater number of 46 47 CYP2C19 loss-of-function alleles was not confirmed in this trial. 48 49 This study had several limitations. First, the primary outcome was a surrogate endpoint, which 50 51 52 was not completely consistent with the clinical outcome of a 90 day stroke recurrence. Further 53 54 study is needed to evaluate the clinical efficacy as the primary endpoint. Second, approximate 55 56 57 58 59 60 https://mc.manuscriptcentral.com/bmj BMJ Page 18 of 36
Wang Page 17 1 2 3 15% patients were lost to follow-up for the primary outcome, and the surrogate endpoint (high 4 5 6 platelet reactivity) was susceptible to missing data, which could have introduced bias. However, 7 8 similar results were observed assuming all the missing data were high platelet reactivity or not. 9 10 Third, potential selection bias may exist as we enrolled patients from sites which were mostly 11 Confidential: For Review Only 12 urban hospitals and had more experts and medical resources. Fourth, the stroke aetiology and the 13 14 15 genetic difference of CYP2C19 gene of Chinese stroke patients are different from that in 16 17 European patients. The results of our study should be evaluated in different populations in the 18 19 future. Finally, the open-label design could have led to a placebo effect,23 which could have 20 21 22 caused potential bias in adverse events assessment, drug continuation, and even the physicians’ 23 24 or patients’ decisions. 25 26 Conclusion 27 28 29 In minor ischemic stroke or TIA patients, combined treatment with ticagrelor and aspirin, as 30 31 compared to clopidogrel and aspirin, significantly reduced the rate of high platelet reactivity, 32 33 particularly carriers of CYP2C19 loss-of-function alleles. With regard to the stroke recurrence 34 35 risk at 90 days, patients with LAA might benefit more from ticagrelor and aspirin than from 36 37 38 clopidogrel and aspirin. Larger clinical trials are required to evaluate the clinical efficacy of 39 40 ticagrelor with aspirin in this setting. 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 https://mc.manuscriptcentral.com/bmj Page 19 of 36 BMJ
Wang Page 18 1 2 3 We thank all patients and their families for providing blood, and the participating researchers at 4 5 6 all study centres. 7 8 Contributors: YW, WC, XM, and YL contributed to the study design, drafting, and revision of 9 10 the manuscript. YP and HL contributed to study design, implementation of the statistical 11 Confidential: For Review Only 12 analysis, and revision of manuscript. KD contributed to study design and the review of the 13 14 15 clinical events that occurred in all patients. YW, YL, XZ, LL, SJ, and DW were members of the 16 17 PRINCE Trial Steering Committee. JJ coordinated the central MRI analyses. JL coordinated the 18 19 central blood sample and genetic sequencing. GC, ZW, JW, DW, JL, YC, YX, GZ, and XL 20 21 22 contributed to the study design, revision of the manuscript, and approval of the report. 23 24 Funding: The study was supported by grants from National Key Technology Research and 25 26 Development Program of the Ministry of Science and Technology of The People’s Republic of 27 28 29 China (2013BAI09B14, 2015BAI12B04, and 2015BAI12B02), the Advanced Innovation Centre 30 31 for Human Brain Protection (117212), Beijing Municipal Science and Technology Commission 32 33 (D15110700200000/D151100002015001/D151100002015002/D151100002015003), Beijing 34 35 Municipal Commission of Health and Family Planning, and AstraZeneca provided the study 36 37 38 drugs and with no role in design and analysis of this trial. 39 40 Competing interests: Yilong Wang reports grants from the Beijing Municipal Science and 41 42 Technology Commission and the Beijing Municipal Commission of Health and Family Planning. 43 44 45 Yongjun Wang reports grants from the National Key Technology Research and Development 46 47 Program of the Ministry of Science and Technology of The People’s Republic of China. 48 49 Ethical approval: The trial was conducted in accordance with the guiding principles of the 50 51 52 Declaration of Helsinki and was approved by the local ethics committees. 53 54 55 56 57 58 59 60 https://mc.manuscriptcentral.com/bmj BMJ Page 20 of 36
Wang Page 19 1 2 3 Data sharing: The technical appendix, dataset and statistical code are available from the 4 5 6 corresponding author at [email protected]. 7 8 Transparency statement: The lead author (the manuscript’s guarantor) affirms that this 9 10 manuscript is an honest, accurate, and transparent account of the study being reported; that no 11 Confidential: For Review Only 12 important aspects of the study have been omitted; and that any discrepancies from the study as 13 14 15 planned (and, if relevant, registered) have been explained. 16 17 This is an Open Access article distributed in accordance with the terms of the Creative Commons 18 19 Attribution (CC BY 4.0) license, which permits others to distribute, remix, adapt and build upon 20 21 22 this work, for commercial use, provided the original work is properly cited. See: 23 24 http://creativecommons.org/licenses/by/4.0/. 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 https://mc.manuscriptcentral.com/bmj Page 21 of 36 BMJ
Wang Page 20 1 2 3 References 4 5 6 1 Amarenco P, Lavallee PC, Labreuche J, et al. One-year risk of stroke after transient 7 8 ischemic attack or minor stroke. N Engl J Med 2016; 374: 1533-42. doi: 9 10 10.1056/NEJMoa1412981 11 Confidential: For Review Only 12 2 Wang Y, Wang Y, Zhao X, et al. Clopidogrel with aspirin in acute minor stroke or 13 14 15 transient ischemic attack. N Engl J Med 2013; 369: 11-9. doi: 10.1056/NEJMoa1215340 16 17 3 Wang D, Gui L, Dong Y, et al. Dual antiplatelet therapy may increase the risk of non- 18 19 intracranial haemorrhage in patients with minor strokes: a subgroup analysis of the 20 21 22 CHANCE trial. Stroke Vasc Neurol 2016; 1: 29-36. doi: 10.1136/svn-2016-000008. 23 24 4 Wang Y, Minematsu K, Wong KS, et al. Ticagrelor in acute stroke or transient ischemic 25 26 attack in Asian patients: from the SOCRATES Trial (acute stroke or transient ischemic 27 28 29 attack treated with aspirin or ticagrelor and patient outcomes). Stroke 2017; 48: 167-73. 30 31 doi: 10.1161/STROKEAHA.116.014891 32 33 5 Wang Y, Zhao X, Liu L, et al. Prevalence and outcomes of symptomatic intracranial large 34 35 artery stenoses and occlusions in china: The Chinese intracranial atherosclerosis (CICAS) 36 37 38 study. Stroke 2014;45:663-9. doi: 10.1161/STROKEAHA.113.003508 39 40 6 Liu L, Wong KS, Leng X, et al. Dual antiplatelet therapy in stroke and ICAS: Subgroup 41 42 analysis of CHANCE. Neurology 2015; 85: 1154–62. doi: 43 44 45 10.1212/WNL.0000000000001972 46 47 7 Brilakis ES, Patel VG, Banerjee S. Medical management after coronary stent 48 49 implantation: a review. JAMA 2013; 310: 189-98. doi: 10.1001/jama.2013.7086 50 51 52 8 Wallentin L, James S, Storey RF, et al. Effect of cyp2c19 and abcb1 single nucleotide 53 54 polymorphisms on outcomes of treatment with ticagrelor versus clopidogrel for acute 55 56 57 58 59 60 https://mc.manuscriptcentral.com/bmj BMJ Page 22 of 36
Wang Page 21 1 2 3 coronary syndromes: a genetic substudy of the PLATO trial. Lancet 2010; 376: 1320-8. 4 5 6 doi: 10.1016/S0140-6736(10)61274-3 7 8 9 Amarenco P, Albers GW, Denison H, et al. Efficacy and safety of ticagrelor versus 9 10 aspirin in acute stroke or transient ischaemic attack of atherosclerotic origin: a subgroup 11 Confidential: For Review Only 12 analysis of SOCRATES, a randomised, double-blind, controlled trial. Lancet Neurol 13 14 15 2017; 16: 301-10. doi: 10.1016/s1474-4422(17)30038-8 16 17 10 Johnston SC, Amarenco P, Albers GW, et al. Ticagrelor versus aspirin in acute stroke or 18 19 transient ischemic attack. N Engl J Med 2016; 375: 35-43. doi: 10.1056/NEJMoa1603060 20 21 22 11 Gurbel PA, Bliden KP, Guyer K, et al. Platelet reactivity in patients and recurrent events 23 24 post-stenting: Results of the prepare post-stenting study. J Am Coll Cardiol 25 26 2005;46:1820-26. doi: 10.1016/j.jacc.2005.07.041 27 28 29 12 Cuisset T, Frere C, Quilici J, et al. High post-treatment platelet reactivity identified low- 30 31 responders to dual antiplatelet therapy at increased risk of recurrent cardiovascular events 32 33 after stenting for acute coronary syndrome. J Thromb Haemost 2006;4:542-9. doi: 34 35 10.1111/j.1538-7836.2005.01751.x. 36 37 38 13 Wenaweser P, Dorffler-Melly J, Imboden K, et al. Stent thrombosis is associated with an 39 40 impaired response to antiplatelet therapy. J Am Coll Cardiol 2005;45:1748-52. doi: 41 42 10.1016/j.jacc.2005.01.058 43 44 45 14 Breet NJ, van Werkum JW, Bouman HJ, et al. Comparison of platelet function tests in 46 47 predicting clinical outcome in patients undergoing coronary stent implantation. JAMA 48 49 2010;303:754-62. doi: 10.1001/jama.2010.181 50 51 52 53 54 55 56 57 58 59 60 https://mc.manuscriptcentral.com/bmj Page 23 of 36 BMJ
Wang Page 22 1 2 3 15 Zheng AS, Churilov L, Colley RE, et al. Association of aspirin resistance with increased 4 5 6 stroke severity and infarct size. JAMA Neurol 2013;70:208-13. doi: 7 8 10.1001/jamaneurol.2013.601 9 10 16 Fiolaki A, Katsanos AH, Kyritsis AP, et al. High on treatment platelet reactivity to aspirin 11 Confidential: For Review Only 12 and clopidogrel in ischemic stroke: A systematic review and meta-analysis. J Neurol Sci 13 14 15 2017;376:112-6. doi: 10.1016/j.jns.2017.03.010 16 17 17 Wang Y, Lin Y, Meng X, et al. Effect of ticagrelor with clopidogrel on high on-treatment 18 19 platelet reactivity in acute stroke or transient ischemic attack (PRINCE) trial: rationale 20 21 22 and design. Int J Stroke 2017; 12: 321-5. doi: 10.1177/1747493017694390 23 24 18 Wallentin L, Becker RC, Budaj A, et al. Ticagrelor versus clopidogrel in patients with 25 26 acute coronary syndromes. N Engl J Med 2009; 361: 1045-57. doi: 27 28 29 10.1056/NEJMoa0904327 30 31 19 Scott SA, Sangkuhl K, Stein CM, et al. Clinical pharmacogenetics implementation 32 33 consortium guidelines for CYP2C19 genotype and clopidogrel therapy: 2013 update. Clin 34 35 Pharmacol Ther 2013; 94: 317-23. doi: 10.1038/clpt.2013.105 36 37 38 20 Wang Y, Zhao X, Lin J, et al. Association between CYP2C19 loss-of-function allele 39 40 status and efficacy of clopidogrel for risk reduction among patients with minor stroke or 41 42 transient ischemic attack. JAMA 2016; 316: 70-8. doi: 10.1001/jama.2016.8662 43 44 45 21 Pare G, Mehta SR, Yusuf S, et al. Effects of CYP2C19 genotype on outcomes of 46 47 clopidogrel treatment. N Engl J Med 2010; 363: 1704-14. doi: 10.1056/NEJMoa1008410 48 49 22 Scott SA, Sangkuhl K, Gardner EE, et al. Clinical pharmacogenetics implementation 50 51 52 consortium guidelines for cytochrome P450-2C19 (CYP2C19) genotype and clopidogrel 53 54 therapy. Clin Pharmacol Ther 2011; 90: 328-32. doi: 10.1038/clpt.2011.132 55 56 57 58 59 60 https://mc.manuscriptcentral.com/bmj BMJ Page 24 of 36
Wang Page 23 1 2 3 23 Gupta A, Thompson D, Whitehouse A, et al. Adverse events associated with unblinded, 4 5 6 but not with blinded, statin therapy in the Anglo-Scandinavian cardiac outcomes trial— 7 8 lipid-lowering arm (ASCOT-LLA): a randomised double-blind placebo-controlled trial 9 10 and its non-randomised non-blind extension phase. Lancet 2017; 389: 2473-81. doi: 11 Confidential: For Review Only 12 10.1016/s0140-6736(17)31075-9 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 https://mc.manuscriptcentral.com/bmj Page 25 of 36 BMJ
Wang Page 24 1 2 3 Figure legends 4 5 6 Figure 1. Trial profile 7 8 9 10 11 Confidential: For Review Only 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 https://mc.manuscriptcentral.com/bmj BMJ Page 26 of 36
Wang Page 25 1 2 3 Figure 2. Platelet reactivity at different visits over 90 days 4 5 6 (a) P2Y12 reaction units; (b) proportion of patients with high platelet reactivity. 7 8 9 A total of 333, 306, and 280 patients in the ticagrelor/aspirin group and 336, 321, and 290 10 11 Confidential: For Review Only 12 patients in the clopidogrel/aspirin group were included in the 0 day, 7 days, and 90 days 13 14 analyses, respectively. High platelet reactivity was defined as a P2Y12 reaction unit (PRU) 15 16 of >208 as measured using the VerifyNow P2Y12 assay. 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 https://mc.manuscriptcentral.com/bmj Page 27 of 36 BMJ
Wang Page 26 1 2 3 Figure 3. Effect of ticagrelor/aspirin compared to that of clopidogrel/aspirin on the 90 day 4 5 6 high platelet reactivity and clinical outcome stratified by metabolizer status 7 8 9 Patients with two *2 or *3 alleles (i.e., *2/*2, *2/*3, or *3/*3) were classified as having a poor- 10 11 metabolizerConfidential: phenotype, those with one *2 Foror *3 allele Review (i.e., *1/*2 or *1/*3) Only were classified as 12 13 having an intermediate-metabolizer phenotype, those without a *2, *3, or *17 allele (i.e., *1/*1) 14 15 were classified as having an extensive-metabolizer phenotype, and those with a single *17 allele 16 17 18 (i.e., *1/*17) and *17 homozygotes were classified as having an ultra-metabolizer phenotype. 19 20 HOPR was defined as a P2Y12 reaction unit (PRU) of >208 measured using the VerifyNow 21 22 P2Y12 assay. A composite event was defined as a new clinical vascular event, including stroke, 23 24 25 transient ischemic attack, myocardial infarction, or death from cardiovascular causes. A total of 26 27 321 patients in the ticagrelor/aspirin group and 329 patients in the clopidogrel/aspirin group were 28 29 included in the genetic analysis. NA = not applicable, CI = confidence interval, OR = odds ratio, 30 31 32 HR = hazard ratio. 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 https://mc.manuscriptcentral.com/bmj BMJ Page 28 of 36
Wang Page 27 1 2 3 Figure 4. Stroke recurrence risk with ticagrelor/aspirin compared to that with 4 5 6 clopidogrel/aspirin at 90 days based on stroke aetiology 7 8 9 LAA = large-artery atherosclerosis, Non- LAA = non-large-artery atherosclerosis (including 10 11 cardioaorticConfidential: embolism, small-artery occlusion, For other Review causes, and undetermined Only causes). 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 https://mc.manuscriptcentral.com/bmj Page 29 of 36 BMJ
Wang Page 28 1 2 3 TABLES 4 5 6 Table 1. Baseline characteristics of the patients in the PRINCE trial 7 8 Characteristic Ticagrelor/Aspirin Clopidogrel/Aspirin 9 10 (n=336) (n=339) 11 Confidential: For Review Only 12 13 Age years (mean+/-SD) 61.1±8·5 60.5±9·0 14 15 Median 62.0 61.0 16 17 Interquartile range 55.0-67.0 54.0-67.0 18 19 20 Female sex n (%) 91 (27.1) 90 (26.5) 21 22 Systolic blood pressure (mmHg) 152.3±22.5 154.9±21.2 23 24 Median 150.0 154.0 25 26 Interquartile range 137.5-168.0 140.0-170.0 27 28 29 Diastolic blood pressure (mmHg) 87.7±13.0 89.4±12.8 30 31 Median 87.5 88.0 32 33 Interquartile range 80.0-96.0 80.0-97.0 34 35 2 36 Body mass index (kg/m ) * 25.0±3.8 25.0±3.8 37 38 Median 24.6 24.8 39 40 Interquartile range 22.6-27.0 22.7-27.3 41 42 43 Pulse rate (bpm) 75.1±10.1 76.3±11.5 44 45 Medical history n (%) 46 47 Hypertension 203 (60.4) 208 (61.4) 48 49 Dyslipidaemia 20 (6.0) 21 (6.2) 50 51 52 Diabetes mellitus 79 (23.5) 85 (25.1) 53 54 Ischemic stroke 59 (17.6) 62 (18.3) 55 56 57 58 59 60 https://mc.manuscriptcentral.com/bmj BMJ Page 30 of 36
Wang Page 29 1 2 3 TIA 8 (2.4) 10 (2.9) 4 5 6 Coronary artery disease 26 (7.7) 25 (7.4) 7 8 Known atrial fibrillation 0 (0.0) 4 (1.2) 9 10 Flutter valvular heart disease 1 (0.3) 0 (0.0) 11 Confidential: For Review Only 12 13 Pulmonary embolism 0 (0.0) 0 (0.0) 14 15 Smoking status n (%) 16 17 Non-smoker 150 (44.6) 155 (45.7) 18 19 Current smoker 160 (47.6) 159 (46.9) 20 21 22 Ex-smoker 26 (7.7) 25 (7.4) 23 24 Drug use before randomisation n (%) 25 26 Proton-pump inhibitor 2 (0.6) 3 (0.9) 27 28 29 Statin 36 (10.7) 30 (8.8) 30 31 Aspirin 77 (22.9) 69 (20.4) 32 33 Clopidogrel 5 (1.5) 10 (2.9) 34 35 36 Ticagrelor 0 (0.0) 0 (0.0) 37 38 Mean time to randomization after onset of symptoms h 14.0 (8.3-20.6) 13.8 (8.0-20.8) 39 40 (range) 41 42 Time to randomization after onset of symptoms n (%) 43 44 45 <12 h 139 (41.4) 144 (42.5) 46 47 ≥12 h 197 (58.6) 195 (57.5) 48 49 Qualifying event n (%) 50 51 52 Minor stroke 275 (81.8) 289 (85.3) 53 54 TIA † 61 (18.2) 50 (14.7) 55 56 57 58 59 60 https://mc.manuscriptcentral.com/bmj Page 31 of 36 BMJ
Wang Page 30 1 2 3 Baseline ABCD2 score among patients with TIA as the 4 5 6 qualifying event ‡ 7 8 Median 5.0 4.5 9 10 Interquartile range 4.0-5.0 4.0-5.0 11 Confidential: For Review Only 12 13 SSS-TOAST stroke subtype n (%)§ 14 15 Large-artery atherosclerosis 151 (54.9) 153 (52.9) 16 17 Cardioaortic embolism 8 (2.9) 5 (1.7) 18 19 Small-artery occlusion 104 (37.8) 109 (37.7) 20 21 22 Other causes 7 (2.5) 9 (3.1) 23 24 Undetermined causes 5 (1.8) 13 (4.5) 25 26 Unknown 2 (0.7) 7 (2.4) 27 28 29 Unclassified 3 (1.1) 6 (2.1) 30 31 * Body-mass index is the weight in kilograms divided by the square of the height in meters. 32 33 34 † TIA = transient ischemic attack. 35 36 2 37 ‡ ABCD stroke risk scores range from 0 to 7, with higher scores indicating higher risk; data 38 39 provided in the table are only for the group of 111 patients whose qualifying event was TIA for 40 41 inclusion in the trial. 42 43 44 § SSS-TOAST stroke subtype = Stop Stroke Study Trial of Org 10172 in Acute Stroke 45 46 Treatment stroke aetiology classification (supplementary appendix “SSS-TOAST Classification 47 48 49 criteria”); data provided in the table are only for the group of 564 patients whose qualifying 50 51 event was minor stroke for inclusion in the trial. 52 53 54 55 56 57 58 59 60 https://mc.manuscriptcentral.com/bmj BMJ Page 32 of 36
Wang Page 31 1 2 3 Table 2. Effect of ticagrelor/aspirin compared to that of clopidogrel/aspirin on the efficacy 4 5 6 and safety outcomes 7 8 Outcomes Ticagrelor/As Clopidogrel/A Hazard ratio/ P-value 9 10 pirin n/N. (%) spirin n/N. Risk ratio 11 Confidential: For Review Only 12 13 (%) (95% CI)* 14 15 Primary efficacy outcome † 16 17 Baseline 268/333 (80.5) 260/336 (77.4) 1.04 (0.96-1.13) 0.33 18 19 20 7+2 days 12/306 (3.9) 89/321 (27.7) 0.14 (0.07-0.23) <0.001 21 22 90±7 days 35/280 (12.5) 86/290 (29.7) 0.40 (0.28-0.56) <0.001 23 24 25 Secondary efficacy outcome 26 27 Stroke 21/336 (6.3) 30/339 (8.8) 0.70 (0.40-1.22) 0.20 28 29 Composite events ‡ 22/336 (6.5) 32/339 (9.4) 0.68 (0.40-1.18) 0.17 30 31 32 Ischemic stroke 18/336 (5.4) 28/339 (8.3) 0.64 (0.35-1.16) 0.14 33 34 Haemorrhagic stroke 3/336 (0.9) 2/339 (0.6) 1.52 (0.25-9.08) 0.65 35 36 37 Myocardial infarction 0/336 (0.0) 1/339 (0.3) - - 38 39 Death from cardiovascular causes 1/336 (0.3) 2/339 (0.6) 0.50 (0.05-5.55) 0.58 40 41 Death from any cause 3/336 (0.9) 2/339 (0.6) 1.50 (0.25-9.00) 0.65 42 43 44 Transient ischemic attack 1/336 (0.3) 2/339 (0.6) 0.50 (0.05-5.53) 0.57 45 46 Primary safety outcomes § 47 48 Major bleeding 5/336 (1.5) 4/339 (1.2) 0.72 49 1.27 (0.34-4.72) 50 51 Major, fatal/life-threatening 4/336 (1.2) 3/339 (0.9) 1.35 (0.30-6.03) 0.69 52 53 Fatal bleeding 1/336 (0.3) 1/339 (0.3) 1.01 (0.06-16.13) 1.00 54 55 56 57 58 59 60 https://mc.manuscriptcentral.com/bmj Page 33 of 36 BMJ
Wang Page 32 1 2 3 Intracranial haemorrhage 3/336 (0.9) 2/339 (0.6) 1.27 (0.34-4.72) 0.72 4 5 6 Major, other 1/336 (0.3) 1/339 (0.3) 1.01 (0.06-16.18) 0.99 7 8 Minor bleeding 11/336 (3.3) 8/339 (2.4) 1.40 (0.56-3.47) 0.47 9 10 11 Major or minorConfidential: bleeding 16/336 For (4.8) Review12/339 (3.5) Only1.36 (0.64-2.88) 0.42 12 13 Minimal bleeding 64/336 (19.0) 36/339 (10.6) 1.86 (1.24-2.80) 0.003 14 15 16 Any bleeding 75/336 (22.3) 48/339 (14.2) 1.65 (1.15-2.37) 0.007 17 18 Other safety outcomes 19 20 Respiratory, thoracic, and mediastinal 22/336 (6.5) 0/339 (0.0) - <0.0001 21 22 23 disorders 24 25 Dyspnoea 14/336 (4.2) 0/339 (0.0) - 0.0001 26 27 Epistaxis 6/336 (1.8) 0/339 (0.0) - 0.04 28 29 * CI = confidence interval. Risk ratio for the primary efficacy outcome and hazard ratio for 30 31 32 secondary efficacy outcome. 33 34 † Primary outcome indicates high platelet reactivity, which was defined as a P2Y12 reaction unit 35 36 of >208 as measured by the VerifyNow P2Y12 assay. 37 38 39 ‡ A composite event was defined as a new clinical vascular event, including stroke, transient 40 41 ischemic attack, myocardial infarction, or death from cardiovascular causes. 42 43 § Primary safety outcomes were defined according to the PLATO criteria (see supplementary 44 45 46 appendix “PLATO Bleeding classification”). All 675 patients were included in the analysis of 47 48 safety outcomes. 49 50 51 52 53 54 55 56 57 58 59 60 https://mc.manuscriptcentral.com/bmj BMJ Page 34 of 36
Wang Page 33 1 2 3 Table 3. Stroke recurrence at 90 days by stroke aetiology 4 5 6 Stroke aetiology* Ticagrelor/As Clopidogrel/As Hazard ratio P- P-value for 7 8 pirin n/N. (%) pirin n/N. (%) (95% CI)* value the 9 10 (336) (339) interaction 11 Confidential: For Review Only 12 13 Large-artery atherosclerosis 9/151 (6.0%) 20/153 (13.1%) 0.45 (0.20-0.98) 0.04 0.13 14 15 Non-large-artery atherosclerosis 10/124 (8.1%) 10/136 (7.4%) 1.10 (0.46-2.63) 0.84 16 17 18 * Stroke aetiology was classified by the SSS-TOAST stroke subtype. 19 20 SSS-TOAST = Stop Stroke Study Trial of Org 10172 in Acute Stroke Treatment stroke aetiology 21 22 classification (supplementary appendix “SSS-TOAST Classification criteria”). 23 24 25 Non-large-artery atherosclerosis included patients with cardioaortic embolism, small-artery 26 27 occlusion, other causes, and undetermined causes. 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 https://mc.manuscriptcentral.com/bmj Page 35 of 36 BMJ
1 2 3 4 5 6 7 8 9 10 11 Confidential: For Review Only 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 82x106mm (600 x 600 DPI) 48 49 50 51 52 53 54 55 56 57 58 59 60 https://mc.manuscriptcentral.com/bmj BMJ Page 36 of 36
1 2 3 4 5 6 7 8 9 10 11 Confidential: For Review Only 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 171x136mm (600 x 600 DPI) 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 https://mc.manuscriptcentral.com/bmj Page 37 of 36 BMJ
1 2 3 4 5 6 7 8 9 10 11 Confidential: For Review Only 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 82x63mm (600 x 600 DPI) 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 https://mc.manuscriptcentral.com/bmj