Efficacy of aspirin and clopidogrel combined with Butylphthalide on frequent transient ischemic attack of middle-aged and elderly patients with ABCD2

Background: Transient ischemic attack (TIA) is the most important independent risk factor for cerebral infarction.The incidence of secondary cerebral infarction in the early stages of TIA,especially within 7 days after onset.Butylphthalide (NBP) is the only clinically approved emerging anti-ischemic drug in China,with clinical significance close to artemisinin and bicyclol. Methods: The trial was a randomised,double-blind,controlled trial of frenquent TIA in patients aged 40 years or older from mar 1,2019 to Dec 31,2019 in Heze municipal hospital.We randomly assigned 238 patients within 24 hours after onset of TIA to both groups (loading dose of 300 mg of clopidogrel on day 1,followed by 75 mg of clopidogrel per day for 90 days,plus loading dose of 300 mg of aspirin on day 1,followed by 100 mg of aspirin per day for 90 days).In addition,the test group (NBP soft capsule 200mg three times a day for 90 days).And all patients also were divided into the low-risk layer (ABCD 2 <4) and the medium-high-risk layer(ABCD 2 ≥4) by the ABCD 2 scale.The primary outcome was stroke occurred(ischemia or bleeding),TIA recurrence,Acute coronary syndrome (ACS) or death on days 7 and 90.Differences in outcomes between groups were assessed by using the Cox proportional hazards model. Results: Aspirin and clopidogrel combined with NBP was significantly better in reducing the incidence of stroke,TIA,ACS or death on the 7th and 90th days of frequent TIA than the control group (P <0.05).But in the low-risk group,the incidences of the two groups of stroke,TIA,ACS or death were lower on days 7 and 90,with no significant difference (P> 0.05).The incidence of stroke,TIA,ACS or death in the test group was significantly lower than the control group on days 7 and 90 (P <0.05). Conclusion: In this prespecified exploratory analysis,aspirin and clopidogrel combined with NBP was superior to which aspirin

Transient ischemic attack (TIA) is the most important independent risk factor for Cerebral Infarction (CI).The incidence of secondary CI is very high in the early stages of TIA,especially within 7 days after onset,reaching 8%-10.5% [1] .According to reports,the risk of stroke or Acute Coronary Syndrome (ACS) in the 3 months before the onset of TIA is 12-20% [2,3] ,and the frequency TIA [4] are more likely to develop a complete stroke.Other studies have shown that TIA is not only a precursor to ischemic stroke,but also an important risk factor for Alzheimer's disease (AD) and vascular demenTIA (VD) [5] ,and its severity and prognosis cannot be ignored. Therefore,early detection, intervention and effective control of TIA are decisive in preventing and avoiding secondary cerebral infarction,AD and VD.Although inhibitors of platelet aggregation are formally recognized as effective drugs for TIA [6] ,but the etiology of TIA is complex,and the specific pathogenesis is not completely clear.It's hard control the progress of various types of TIA timely and effectively just by one category drugs.Therefore,the exploration of more effective drugs is still the focus of current research.
In the beginning,3-N-butyphthalide is a kind of biological agent originally extracted from seed of NBP,as an emerging anti-ischemic drug,inhibits platelet aggregation,reduces thrombosis,improves microcirculation,reduces cerebral infarction volume,resists oxidative stress,protects mitochondrial function,reduces neuronal apoptosis,and reduces inflammation,response and mediate autophagy of neurons,promote neurogenesis,etc in stroke animal models.It exerts multi-targeting effects on a variety of pathophysiological mechanisms and shows obvious neuroprotective effects [7][8][9][10][11][12][13][14][15][16][17][18][19][20] .It has been found that its beneficial effect is far beyond the therapeutic scope of stroke [21,22] after further research and application of its application.Especially in the field of neurodegenerative diseases,people have found that NBP and its derivatives are used in AD,VD,Parkinson's disease (PD) and Amyotrophic Lateral Sclerosis (ALS) all have unique effects and efficacy [7,[23][24][25] .
In addition,relevant clinical studies have shown that short-term NBP has a good clinical effect on decreasing the incidence of cerebral infarction of the patients with TIA [12] .Here,we used the ABCD 2 scale [26] and performed a study to evaluate the efficacy and safety of the short-term Aspirin and clopidogrel combined with NBP in the treatment of frequent TIA of middle-aged and elderly patients.Thus,appropriate intervention measures should be selected for the prevention and treatment for patients with frequent TIA in different risk groups,so as to reduce the occurrence of stroke.

Patients And Methods 2.1 Study Design And Patients
This trial was a randomised,double-blind,controlled trial of frequent TIA of middle-aged and elderly patients from mar 1,2019 to Dec 31,2019 in Heze municipal hospital.The informed consent in written form was formally obtained from all participants,and oue study was conducted in accordance with the Declaration of Helsinki.If any patients have passed away,consent is required from the patient's next of kin.
Eligible patients with frequent TIA [4] of middle-aged and elderly patients.Exclusion criteria:1.Head CT or MRI plain scan found intracranial hemorrhage,aneurysm or other neurological symptoms which cannot accept the treatment;2.Thrombolysis,anticoagulation therapy,carotid or cerebral or coronary artery stent implantation or reconstruction in Plan;3.Being allergic to NBP,clopidogrel and aspirin;4.A history of ventricular aneurysm,atrial fibrillation or suspected cardiovascular embolism causing TIA;5.Take in NSAIDS for more than 7 days recently;6.A history of gastrointestinal bleeding within 6 months;7.A history of a major surgery within 30 days;8.Coagulation dysfunction;9.Previous history of non-traumatic intracranial hemorrhage;10.Severe liver and kidney dysfunction;11.Pregnancy or lactation;12.Incomprehension or incooperation with the study or treatment or followup.Additionally,standard treatments such as hypoglycemic and antihypertensive therapy may be prescribed to all patients at the discretion of the investigators and clinicians.

Randomization And Treatments
Patients enrolled were randomly assigned to 1 of the 2 treatment groups with the use of a doubleblind,double-dummy design.The site investigator called into an automated system that randomly assigned a number corresponding to a medication kit stored at the research site,and the medication in the kit was administered to the patient.NBP soft capsule and the matching placebo were purchased from Shijiazhuang Pharmaceutical Group Co.Ltd,which had no other role in the study.We randomly assigned 238 patients within 24 hours after onset of TIA to test group (loading dose of 300 mg of clopidogrel on day 1,followed by 75 mg of clopidogrel per day for 90 days,plus loading dose of 300 mg of aspirin on day 1,followed by 100 mg of aspirin per day for 90 days;NBP soft capsule 200 mg three times a day for 90 days) or control group (loading dose of 300 mg of clopidogrel on day 1,followed by 75 mg of clopidogrel per day for 90 days,plus loading dose of 300 mg of aspirin on day 1,followed by 100 mg of aspirin per day for 90 days;NBP soft capsule placebo 200 mg three times a day for 90 days).After day 90,treatment was at the choice of the clinician and the patient.
Additionally,standard treatments such as hypoglycemic and antihypertensive therapy may be prescribed to all patients at the discretion of the investigators and clinicians.Previous studies [27] indicated that 90% of strokes occurred in the layer with ABCD 2 ≥ 4,so all the patients were divided into the low-risk layer (85,39.7%) and the medium-high-risk layer (129,60.3%) by the ABCD 2 scale.
All patients ought to return to hospital or keep in contact with the researchers by telephone on days 7 and 90 for follow-up,and the occurrence of possible outcome events,changes of test drugs and possible adverse events should be recorded.

Outcome Criteria
Patients were evaluated clinically or followed up on days 7 and 90.The endpoint of the trial was a compound event of Stroke,TIA,ACS or death (death from cerebrovascular disease) on days 7 and 90,which conforms to a standard definition [28] ,or the occurrence of serious or uncoordinated nonserious adverse events [29] .

Statistical Methods
All data were analyzed by using SPSS 22.0 (SPSS Inc,Chicago,IL) software and tabulated using Microsoft office software (word,excel).All data were expressed as the mean standard deviation (SD).Differences in baseline factors (age,sex,concomitant medications,risk factors) among the groups in the different layers were compared using the T-test or chi-square test.COX regression analysis on the influence factors for resolving time of the two groups in all patients and the different layers.The treatment effects were compared with analysis of variance and 95% confidence intervals (CIs).Statistical analysis of safety data was done with Pearson chi-square test.P < 0.05 was regarded significant difference.

Conclusion 3.1 patient characteristics
The baseline characteristics of the patients in this study are shown in Table 1.In all 228 patients with frequent TIA,14 patients who quit in process,and the remaining 214 patients completed the study smoothly.There was 90 (42.1%) patients were female and there were no significant difference in age,sex,risk factors or concomitant medication.The greatest risk factors for these two groups were smoking (test group:54.1%;control group:49.5%),hypertension (Test group:48.6%;Control group:51.5%),and hypercholesterolemia (Test group:55.0%;Control group:48.5%).There was no significant difference in these risk features.Similarly,there was also no significant difference in these risk features in the low-risk layer and the medium-high-risk layer by the ABCD 2 scale.There was only one death in each of the two groups,and COX regression analysis the resolving time of the two groups was also no significant difference (P > 0.05).

3.2.1
Follow-up results of all patients (ABCD 2 = 0-7) on the 7th and 90th day after treatment As shown in Table 2,on the 7th day of treatment,there were 2 cases (1.8%) of stroke,TIA,ACS or death in the test group and 12 cases (11.7%) in the control group.On the 90th day of treatment,there were 8 cases (7.2%) of stroke,TIA,ACS or death in the test group and 20 cases (19.4%) in the control group.The incidence of stroke,TIA,ACS or death in the test group was significantly lower than that in the control group on the 7th and 90th day of treatment,all showed obvious difference (P < 0.05).

3.2.2
Follow-up results of Low-risk group (ABCD 2 < 4) on the 7th and 90th day after treatment As shown in Table 2,there were 45 cases in the test group and 40 cases in the control group.On the 7th day of treatment,1 (2.2%) case of total cases of stroke,TIA,ACS or death occurred in the test group;and 3 (7.5%)cases in the control group.Treatment follow-up on the 90th day showed that 2 (4.4%) cases of stroke,TIA,ACS or death occurred in the test group and 5 (12.5%) cases in the control group.The incidence of stroke,TIA,ACS or death were lower in the two groups on the 7th and 90th days of treatment. showed no significant difference in efficacy (P > 0.05).

3.2.3
Follow-up results of the medium and high-risk group (ABCD 2 ≥ 4) on the 7th and 90th day after treatment As shown in Table 2,there were 66 cases in the test group and 63 cases in the control group.On the 7th day of treatment,2 (3.0%) cases of stroke,TIA,ACS or death occurred in the test group;and 9 (14.3%) cases in the control group;follow-up after 90 days of treatment,it was found that 6 (9.1%) cases of stroke,TIA,ACS or death occurred in the test group and 15 (23.8%) cases in the control group.No matter on the 7th or 90th day of treatment,the incidence of stroke,TIA,ACS or death in the test group was significantly lower than that in the control group,and the efficacy was significantly different (P < 0.05).

Safety Results
As shown in Table 2,1(0.9%) case with subcutaneous bleeding,4 (3.6%) cases with nasal and gum bleeding in the test group;and 3 (2.9%) cases with subcutaneous bleeding,2 (1.9%) cases with nasal and gum bleeding in the control group.However,there was no significant difference in the incidence of bleeding between the two groups.No primary intracranial hemorrhage or gastrointestinal hemorrhage was observed in either groups.

Discussion
The purpose of this study was to evaluate the efficacy and safety of the 90-day NBP in the treatment of patients with frequent TIA.First of all,we discuss the specific performance of two groups under the low-risk and medium-high-risk layers with ABCD 2 scale. Table 2 showed that on the 7th and 90th day of treatment,no matter which plan was used,the disease progression can be well controlled in the low-risk group.It might be that the low-risk group had a lower risk of stroke,TIA,ACS or death,which was easier to control. Table 2 showed that on the 7th and 90th day of treatment,the incidence of stroke,TIA,ACS or death in the test group was significantly lower than that in the control group,indicating that the efficacy of Aspirin and clopidogrel combined with NBP in the treatment of frequent TIA in the medium-high-risk group was more accurate than that in the double antibody group In this study,all patients with frequent TIA at 90 days' NBP treatment were more effective than the 90 days' dual antibody plan.This might be due to the proportion of patients (129,60.3%) with ABCD 2 ≥ 4 in the study population was large,thus the overall distribution,and the results showed the larger proportion of the medium-high-risk group of the results.
Previous studies [16,55] have found that the changes of ALT and AST induced by NBP were not significantly different from those in the control group,thus monitoring on serum Alanine transaminase (ALT) and Aspartate transaminase (AST) levels wasn't included in this study. Table 2 in this study showed that the incidence of adverse events was similar and the difference was not statistically significant.This finding suggested that NBP was safe for patients with frequent TIA.

Conclusion
An understanding of frequent TIA of middle-aged and elderly patients whose mechanisms and causes is important to deliver safe and efficacious treatments for early stroke,TIA recurrence,ACS or death prevention.In this prespecified exploratory analysis,our results indicated that the long-term and continuous aspirin and clopidogrel combined with NBP was superior to which aspirin combined with clopidogrel at preventing frequent TIA of middle-aged and elderly patients,which is safe surely.Especially for the medium-high-risk layer with ABCD 2 .Our study is a newly finished,and requires further inspection,verification and improvement,so there is need to carefully interpret the results.Although the distribution of baseline factors that can affect functional outcomes in this study are similar,there is no significant difference.And due to the relatively small sample size,so multicenter and large-sample trials are urgently needed to further confirm that long-term NBP treatment of frequent TIA of middle-aged and elderly patients is effective and reliable without significant side effect.We all look forward to NBP,which is from China and is used like artemisinin Drugs that serve the world can have broader prospects.

Ethics approval and consent to participate
The Institutional Review Board of Heze municipal hospital reviewed and approved the purpose and design of our work.Informed consent in written or oral form was formally obtained from all participants,and our was conducted in accordance with the Declaration of Helsinki.If any patients have passed away,consent was required from the patient's next of kin.

Availability of data and materials
All data generated or analysed during this study are included in this published article [and its supplementary information files].

Competing interests
The authors declare that they have no competing interests

Authors' contributions
QW and HL carried out the experimental work and the data collection and interpretation.YW participated in the design and coordination of experimental work, and acquisition of data.HZ and YG participated in the study design,data collection,analysis of data and preparation of the manuscript.YC carried out the study design,the analysis and interpretation of data and drafted the manuscript.All authors read and approved the final manuscript.    Figure 1 Profile of the clinicle trial