Effects of precision antiplatelet therapy based on CYP2C19/11-dhTxB2 variability in prognosis of ischemic stroke/TIA patients: study protocol for a multicenter randomized controlled trial CURRENT STATUS: UNDER REVIEW

Clopidogrel and aspirin are routine drugs for the treatment of ischemic stroke (IS) and transient ischemic attack (TIA). However, with the increase of clinical application, a large number of patients have displayed clopidogrel resistance and/or aspirin resistance. At present, the effect of genetically tailored medication has not been prospective evaluated in a large sample. This study is an investigator-initiated, multicentre, large sample, prospectively, randomized controlled study evaluating the effects of precision antiplatelet medication based on the cytochrome P450 2C19 (CYP2C19) genetic test and the 11-dehydroxetane B2 (11-dhTxB2) test on IS/TIA patients over a duration of 12 months. Outcomes of interest including stroke recurrence, neurologic disabilities defined by the Modified Rankin Scale (mRS), bleeding events, other adverse events, and all-cause mortality will be assessed at the 1st, 3rd, 6th and 12th month post discharge. Demographics, risk factors, laboratory investigations, medications, physiological tests, and brain imaging will be assessed as well.


Introduction
The study is being conducted at 14 comprehensive first-class hospitals in Jiangxi Province.
Participation by eligible patients is voluntary and assignment into control and intervention groups is random. Patients in the control group are treated routinely according to the current clinical guidelines. Patients in the intervention group are tested for CYP2C19 polymorphisms and urine . Finally, personalized medication is developed for intervention group patients according to the test results.

Participants and recruitment
Inpatients are screen by a researcher in the neurology department of the participating hospital.
Eligible patients are then approached in the ward by a member of the research staff, who thoroughly explains the study and consents patients who choose to participate. Patients who voluntarily participate must sign the informed consent document and are asked to leave one to two phone numbers for a one-year follow-up. Researchers then include the participant in the study cohort and fill in the visit information.

Eligibility criteria
Patient eligibility criteria include: (1) age 18 to 85 years, (2) diagnosis of ischemic stroke (atherosclerotic cerebral infarction) or transient ischemic attack (diagnosis meets the criteria established by the Cerebrovascular Diseases Group of the Chinese Medical Association Neurology Society in 2014) [25] , (3) acute period of onset (2 weeks post event), (4) NIHSS score ≤ 15 points, (5) voluntarily participation and informed consent.
Termination of research standards are (1) misdiagnosed or misrepresented cases that do not meet the inclusion criteria and meet the exclusion criteria, (2) patients who voluntarily withdraw from the study, (3) the investigator determines that an effective clinical observer cannot be continued for medical or other reasons, (4) patients whose test results are double resistance (clopidogrel resistance and aspirin resistance), (5) those who switched to other antiplatelet drugs and stopped taking drugs in the middle of the study, or switched to anticoagulants in the middle.

Randomization
Once eligible patients sign informed consent form voluntarily researchers then register each patient in the queue and perform simple randomization. The proportion of control group to intervention group is 1:1.

Participant timeline
During the study period, patients who meet the criteria are required to voluntarily sign the informed consent document in order to participate. Participants are randomly assigned into the control group or the intervention group by the researchers. Baseline data such as BMI, NIHSS, mRS, imaging examination, and laboratory test results is collected during hospitalization. When discharged the NIHSS and mRS scores for each participant is assessed and recorded in addition to individualized medications and medical expenses. All patients are asked to attend follow-up visits at the first, third, sixth, and twelfth month post discharge and to report medication adherence and prognosis (including stroke recurrence, adverse events, neurological impairment, etc.). A participant timeline of the study is detailed in Table 1.
In addition, aspirin is widely used in secondary prevention of stroke because it inhibits cyclooxygenase (cox) and thus reduces thromboxane A (TXA2) production. Urine 11-dhTxB2 is a stable metabolite of TXA2, which reflects the activation state of platelets in the body and the function of aspirin. In this study, aspirin resistance is tested for using 11-dhTxB2 concentration in urine with 11-dhTxB2/urinary creatinine≥1500 pg/mg used to characterize the presence of aspirin resistance [34] .
Once available, results from the genetic and urine testing are used to develop individualized medication options.

Treatments
In 1-8th day, patients in the experimental group are taken 3-5 ml whole blood samples and 3-10 ml urine samples for CYP2C19 genotype and 11-dhTxB2 test, the results are usually obtained within 7-9th day. After testing, doctors adjust the anti-platelet medication according to the results. If the patient is only resistant aspirin, then only clopidogrel is prescribed; if the patient is only resistant to clopidogrel, then only aspirin is prescribed. If the patient displays no resistance to aspirin and clopidogrel, we will not change the patient's treatment plan. If the patient has dual resistance to clopidogrel and aspirin, we do not make medication interventions due to the risk of bleeding, and only recommend that the doctor increase the dose or use substitutive drugs. Patients in the control group do not receive anti-platelet testing. There are no blinding in this study. The harm in this study is to extract a small amount of blood samples from patients.If adverse events occur during follow-up, researchers will record, report, and dispose them.

Endpoints
The primary outcome of interest is stroke recurrence. Secondary outcomes include disabilities, bleeding events, other adverse events, and all-cause mortality.

Sample size
In Wang et al.'s study population 58.8% of participants were carriers of CYP2C19 loss-of-function alleles. Stroke recurrence in Non-carriers only : 6.7% in treatment (clopidogrel plus aspirin) vs. 12.4% control (aspirin only). Based on above results, 417 cases are needed for each group, for a total of 834 cases. We assume a 15% loss to follow-up rate and a design effect of 1.25 [34] , therefore our final calculated sample size is 1199, approximately 1200. Our goal is to recruit 1,200 to 6,000 patients.

Data collection and management
We have designed the data collection table on the Electronic Data Capture (EDC) platform according to the project requirements, and clarified the research process, the data table name, the collected data items, and formed the corresponding data collection guide. Finally, eCRF is constructed based on the research of medical records. Once all participants complete the trial, all medical records will be entered into the system. After the primary investigator, sponsor, statistical analyst, and data stewards confirm that the data in the database are correct, the data stewards will check the data integrity. Finally, all the data will be locked, the data administrator will import it into the specified database and send it to the statisticians for statistical analysis. The locked data can no longer be edited, and the problems found after the data is locked can be corrected in the statistical analysis program after confirmation. After the trial is over, the data administrator will file an EDC closure request and cancel all account access rights after obtaining the permission of the sponsor. Finally, when the data is completely backed up, the EDC will be turned off.

Statistical analysis
We will describe general characteristics of the study population using the mean, median, standard deviation (SD), range and interquartile range (IQR) for continuous and ordinal data, and counts or percentages for categorical data. The normality of variable distribution will be assessed prior to data presentation. For tests of independence the Pearson's χ 2 test will be used for categorical variables, and for the analysis of variance and Mann-Whitney U test will be used for numeric variables, as appropriate. Multiple imputation will be used to impute missing values, under a missing at random assumption, so as to reduce bias and avoid excluding participants from the analysis. Comparison of endpoint incidence between intervention and control groups will be assessed by risk rate (RR).
Multivariate Cox regression analysis will be used for the time-to-event data with adjustment of known confounders. In this study, the estimates of missing values for the primary variables are carriedforward to the absence of test data using the results of the closest observation. All statistical tests will be two-sided and P <0.05 is considered statistically significant. All of the above statistical analysis will use Statistical Analysis System (SAS Institute, Inc., Cary, NC) V.9.3.

Quality control measures
This study consists of sponsor commissions and contract research organization to complete the audit work, to ensure that the rights and interests of the subjects in the clinical trials are guaranteed, the recorded data are accurate and complete, and to ensure the test follows the follows the approved program, Quality Management Standards for Drug Clinical Trials [35] and related regulations.
If the program has problems during the actual implementation of the clinical trial, the program needs to be revised and will be presented to the sponsor. After consultation and discussion by the multicenter coordination committee, the clinical research unit will revise the plan, then submit to the sponsor and the participating units for signature approval. Finally, it is submitted to the ethics committee for approval and implementation. If important new information related to the test drug is found, the informed consent form must be modified in writing and sent to the ethics committee for approval.

Objectives
In order to verify the effect of genetically tailored medication based on CYP2C19 genotyping and 11-dhTxB2 testing, our study is establishing a prospective cohort study of 1200-6000 patients to collecting datas and exploring potential risk factors of neurological impairment. Finally, we will establish individualized treatment standards for stroke/TIA patients, and build an intelligent decisionmaking platform to provide a reference for clinical treatment.

Ethics and informed consent
Medical Research Ethics Committee in the Second Affiliated Hospital of Nanchang University approved the final agreement. The study is conducted in strict accordance with the relevant regulations and the requirements of the committee. All patients are voluntarily sign the informed consent form approved by the committee, indicating that they agree to participate. Consent includes the nature, objectives, and potential benefits, risks, and consequences of the study. In addition, the consent details the research background, the required follow-up time, privacy issues, and the patient's right to withdraw from the study at any time during the treatment period. All collected data will remain private and will not reveal any information about the patient.

Dissemination
The study findings will be disseminated in national and international conferences, and peer-reviewed publications.

Discussion
Stroke is the second most common cause of death, and the leading cause of long-term disability worldwide [36] . Clopidogrel and aspirin are routine drugs for the treatment of ischemic stroke and TIA patients [37] however a large number of patients have a genetic predisposition to clopidogrel resistance and/or aspirin resistance [38,39] . The effects of individualized medication based on CYP2C19 genetic test and 11-dhTxB2 test is still unknown. Currently, there is a lack of large-scale study for the evaluation of the clinically accurate dual antiplatelet therapy standards based on genetic and drug metabolite tests.
The CYP2C19 genetic test is expensive and not widely available in Chinese hospitals. Our study collects economic information such as family annual income, hospitalization expenses, and drug costs for the enrolled patients. Using this information, we will conduct economic analysis to evaluate the long-term benefits of CYP2C19 genetic/11-dhTxB2 testing for IS/TIA patients, to provide evidence for general genetic/11-dhTxB2 test.
Our proposed study has several strengths. First, this is the first study to assess the effect of individualized medication based on the CYP2C19 genetic test and 11-dhTxB2 test for IS/TIA patients.
Additionally, this is a multicentre prospective cohort study with multilateral evaluations and analyses of gene polymorphism, and prognosis of IS/TIA. There are several potential limitations. First, no prescribing guidelines standards for patients resistant to both clopidogrel and aspirin currently exist. Since forcibly increasing antiplatelet dosage may lead to a higher risk of bleeding this study did not adjust the medication for this group of patients, but only provided a reference for clinicians. Second, there is still the possibility of unrecognised confounders despite the multicentre design. Inconsistent levels of investigators and patient medication compliance in different hospitals may affect the effectiveness of personalized medicine.
Developing personalized antiplatelet medications and reducing stroke recurrence rates are pressing issues for Chinese society. We hope that our study will contribute to a better understanding of the association between personalized antiplatelet medications and stroke prognosis, and develop a standardized stratified treatment plan for IS/TIA patients. Not commissioned; externally peer reviewed. After the publication of research results such as articles and patents, research data other than patient privacy can be disseminated through inational and international conferences, and peer-reviewed publications. Table   Table 1. Schedule of assessments for the strategy to obtain CYP2C19/11-dhTxB2 testing's benefit by evaluating registry study