Effect of Ginkgolide in Ischemic Stroke patients with large Artery Atherosclerosis: Results from a randomized trial

Abstract Background Dual antiplatelet therapy is considered beneficial in acute ischemic stroke (AIS) patients with intracranial artery stenosis (ICAS), with more bleeding events. Ginkgolide is shown to reduce platelet activation after infarction, which might be of benefit in AIS. We aimed to explore the effect of Ginkgolide in AIS patients with ICAS. Methods This was a randomized, double‐blinded, placebo‐controlled trial conducted at 61 centers in China. Within 72 h after onset, consecutive patients diagnosed as AIS with ICAS were randomized to either Ginkgolide or placebo treatment. The primary outcome was the composite of mortality and recurrent stroke (ischemic or hemorrhagic) during first 4 weeks in an intention‐to‐treat analysis. Secondary functional outcome was assessed by modified Rankin Scale and improvement of stroke severity was assessed by National Institution of Health Stroke Scale at day 28. Safety outcome was measured by the rate of severe adverse event (SAE). Results There were 936 patients randomized to either Ginkgolide or placebo treatment. Their average age was 64.2 ± 10.4 years old and 36.0% of the patients were female. The composite index event occurred in six patients in placebo group, and none occurred in Ginkgolide group (risk ratio 1.01; 95% CI 1.00–1.02). There were more patients who achieved favorable outcome in Ginkgolide group, compared with that of the placebo group (OR 2.16, 95%CI 1.37–3.41). SAE occurred in five (1.1%) patients in the Ginkgolide group and three (0.6%) in the placebo group (OR0.60, 95CI% 0.14–2.53). Intracranial hemorrhage occurred in 1/473 (0.2%) in the placebo group. Conclusions Ginkgolide, working as PAF antagonist, may reduce recurrent stroke in AIS with ICAS patients within 72 hours after onset. It might be an optional treatment in moderate‐to‐severe AIS patients with ICAS. (http://www.chictr.org.cn Number as ChiCTR‐IPR‐17012310).


| INTRODUC TI ON
The aggressive medical management, including dual antiplatelets treatment, to prevent stroke in non-disabling stroke with intracranial stenosis (ICAS), has become the standard therapy since 2011. 1,2 The clopidogrel plus aspirin versus aspirin alone for reducing embolization in patients with acute symptomatic cerebral or carotid artery stenosis study (CLAIR) and Clopidogrel in High-Risk Patients with Acute Nondisabling Cerebrovascular Events (CHANCE) trial showed the benefit of dual antiplatelet therapy (DAPT) in reducing the rates of recurrence stroke and mortality within 28-90 days. 3,4 Additionally, CHANCE subgroup analysis showed the extra benefit of DAPT in patients with ICAS. 4,5 However, the use of dual antiplatelets treatment is limited due to its timing and its higher risk of bleeding. 6 The DAPT used could be one of the predictors to bleeding risk. 6 In platelet-oriented inhibition in new TIA and minor ischemic stroke (POINT) trial, the risk of hemorrhagic stroke or transformation increased if DAPT was used for 90 days. 7 Limited evidence showed that DAPT in non-minor stroke was not superior to monotherapy in ICAS patients during the acute phase. 8 Ginkgolide is a promising agent that enhances antiplatelet agent function via platelet aggregation factor (PAF) inhibition. [9][10][11] On the other hand, it might also have less bleeding risk. Therefore, we conducted this pilot study to compare the feasibility and efficacy of Ginkgolide in AIS patients with ICAS. The mechanism of Ginkgolide as an antagonist of PAF was also explored. Since common platelet aggregation is synergistically mediated by PAF, thromboxane A2 (TXA2), and adenosine-5'-diphosphate (ADP), all parameters were assessed in this study.

| Study Protocol and study design
The GISAA study protocol and data collection were approved by the ethics committee of Huashan Hospital (Ethical approval number: KY2016-199) and all of the study centers. 12 This study was conducted in accordance with the Declaration of Helsinki. The trial was a prospective, multicenter, randomized, open-label, active controlled, blinded endpoint trial (registered as http://www.chictr.org. cn; number as ChiCTR-IPR-17012310). All participants or their representatives provided written consent before randomization.

| Participates
From October 2016 to July 2018, patients with an acute moderateto-severe ischemic stroke were consecutively screened at 61 centers in China. Patients aged above 18 years, with National Institutes of Health Stroke Scale (NIHSS) scored >3 points at randomization, were included within 72 h of symptom onset. All patients had a baseline MRA or CTA scan to confirm their large vessel atherosclerotic changes. The ICAS in our study was defined as a stenosis or occlusion of one or more than one of these vessels: intracranial carotid artery, anterior cerebral artery, middle cerebral artery, and posterior cerebral artery or vertebrobasilar artery stenosis or occlusion on CT/MR angiogram.
Patients were excluded from the trial if they were candidates of thrombolysis and thrombectomy, or had a diagnosis of intracranial hemorrhage, acute coronary syndrome, rheumatic heart disease, or was assessed by National Institution of Health Stroke Scale at day 28. Safety outcome was measured by the rate of severe adverse event (SAE). score of more than 1 at randomization; or any contraindication to ginkgo, alcohol, and glycerin were excluded. 12

| Randomization and procedures
Immediately after signing the written informed consent, eligible patients were randomized at a ratio of 1:1 to receive either Ginkgolide or placebo. Randomized block was used to allocate patients, and the random allocation scheme of 1:1 for the treatment group and the control group was made using SAS 9.4 software. The generate random, encoding by using randomized method, block selection and the length of the random initial value parameters such as seeds of confidential data, are sealed together in blind bottom. Patients were also stratified by their baseline severity and risk factors. According to this random number, the drug is coded by the person who has nothing to do with the test. Each clinical research center was allocated according to the assigned drug number and selected according to the case order.
The trial included four visits: randomization (baseline), day 7 (±1 day), day 14 (±3 days), and day 28 (±3 day). All visits involved a face-to-face interview. Data were collected in a case report form.
The blood sample was drawn at baseline, day 7, and day 14 for the purpose of monitoring the dynamic changes in common plateletactivating pathways. Levels of PAF, TXA 2 , and ADP were analyzed by the core laboratory using ELISA Kit with standard protocol. Both the investigators and the patients were blinded to the platelet reactivity data until the end of the trial.

| Intervention
All patients who met the inclusion criteria were randomly assigned to receive aspirin 100 mg oral daily combined with intravenous ginkgolide 10 ml for 14 days or aspirin 100 mg oral daily combined with placebo after randomization immediately. Placebo was produced specifically as unidentical in appearance, and labeled with same drug label. Blinded details were demonstrated in a study protocol published previously. 12

| Outcome
Primary outcome was to measure the rate of stroke recurrence and mortality at day 28 after randomization. Recurrent stroke was defined as an index event (ischemic or hemorrhagic) after 24 h of randomization or NIHSS increasing >4 points. Ischemic stroke was F I G U R E 1 Trial profile. mRS, modified Rankin Scale; NIHSS, National Institution of Health Stroke Scale defined as an acute focal infarction of the brain or retina with clinical or imaging evidence of infarction lasting 24 h or more, or rapid worsening of an existing focal neurological deficits with NIHSS score increasing >4 points. Hemorrhagic stroke was defined as acute-onset brain parenchyma or subarachnoid hemorrhage with associated neurologic symptoms.
Secondary outcome included the following: (1) the improvement of clinical symptoms measured by NIHSS score at 14 and 28 days after randomization; and (2) the functional outcome assessed by mRS at 28 days after randomization.
The primary safety outcome included any severe adverse events (SAE), including major bleeding events, which were defined as that in the PLATO study: fatal or life-threatening hemorrhage, major hemorrhage, and other.

| Data availability
All individual data were de-identified from the GISAA trial. These data would be shared with qualified researchers upon request to the principal investigator (Dr. Qiang Dong).  Table 2). There was an association between the use of ginkgolide and favorable outcome (RR 1.492, 95% CI 1.013-2.198). Similar improvement trend was observed on neurological deficits measured by NIHSS ( Table 2). The distribution of mRS showed that the patients in the GISAA group were more likely to have a better functional outcome compared with those in the control group ( Figure 2).

The specified subgroups analysis of index events was conducted
and is demonstrated in Figure 3. There was no significant difference in specified subgroups.  The NIHSS improvement between the baseline NIHSS and the performance at 28 days and all levels of platelet aggregation pathway at 14 days were presented by 95% CI difference and analyzed by linear regression, presented with mean ± SD and coef.

F I G U R E 2 Function outcome measured by modified Rankin Scales of all patients
F I G U R E 3 Specified Subgroup analysis of the primary outcome. NIHSS, National Institution of Health Stroke Scale. Center-imaging assessment was performed among 299 in Ginkgolide group and 307 in placebo group who has DICOM information. According to NACET criteria, CTA or MRA imaging was classified into mild (0%-30%), moderate (30%-50%), and severe stenosis (>50%). The patients with 30%-50% stenosis had no events. Therefore, there is no RR for these specific patients concerning the level of PAF, TXA2, and ADP were tested for normality (Table S1). A total of 370, 367, and 352 patient samples in the ginkgolide group and 386, 380, and 366 patient samples in the placebo group were included in day 0, 7, and 14 analyses, respectively. Using linear regression model, we observed that there was a reduction in the PAF level in the ginkgolide group after adjustment of age, onset of time, sex, and the severity at baseline (p = 0.036, adjusted p = 0.035). There were no similar trends found in TXA2 and ADP pathway (Figure 4).

| DISCUSS ION
Our study showed that ginkgolide had a slight benefit in moderateto-severe AIS patients with ICAS. Furthermore, ginkgolide was also associated with functional outcome (mRS 0-2) improvement at day 28 follow-up compared with those in the placebo group. By assessing the various platelet-activating pathways, the effect of intravenous ginkgolide was associated with PAF inhibition in AIS patients.
Ginkgolide used in this study, playing a role as an inhabitation of PAF receptor, was induced by ischemic stroke. 10 The reduction in PAF and its pathway were reportedly helpful to reduce the volume of infarction in acute phase. [9][10][11] In our study, the dynamic changes in PAF of both groups were confirmed, which is consistent with the recurrent stroke risk. Ginkgolide helped slightly in decreasing accumulation of PAF after ischemic stroke, which might be one of the mechanisms.
AIS patients with ICAS had a higher risk of recurrent stroke in acute phase, 3,14 which was challenging 15 ]. Based on previous studies, ICAS is still one of the most challenging conditions in AIS patients. The standard treatment of ICAS, especially in acute phase, is debated. 16 Based on previous CHANCE trial, aspirin plus clopidogrel in mild stroke with ICAS is recommended. 5 However, this combination was not superior to aspirin monotherapy in patients with ICAS in the CHANCE substudy. 17 The POINT trial showed that the dual antiplatelet treatment might cause more bleeding events 7 ]. Moreover, only mild stroke patients were included in the above studies. In our study, we focused on those patients with moderate stroke, which might be more complicated.
On the one hand, other antithrombotic treatments were also explored. 16  with the CHANCE trial subgroup analysis. 5 The "ceiling effect" might be influenced on the effect of primary outcome, while there was no effect on the secondary functional outcome. Third, many other risk factors, as confounders, might be influenced on the recurrence in ICAS patients, such as the residual flow, leptomeningeal collateral flow, and hemispheric blood flow, which were supposed to be balanced at randomization; we failed to analyze these factors in our study. 25,26 Finally, the underlying mechanisms and their pathways of other components within ginkgolide need further studies on mechanism. Although our study showed a slight benefit in the composite index event prevention for the primary outcome, a larger scale with longer follow-up duration RCT trial is needed to confirm this finding.
In this trial, intravenous ginkgolide was safe and had a benefit in recurrent stroke prevention in AIS patients with ICAS. It might also be associated with functional improvement in a short-term follow-up, which could be considered as an optional choice in disabling stroke patients with ICAS at acute phase.

CO N FLI C T O F I NTE R E S T S
All authors report no disclosures.

E XCLUS I V E LI CE N S E
The corresponding author has the right to grant on behalf of all authors.

PATI E NT A N D PU B LI C I N VO LV E M E NT
Partly patient and public were involved in our study. We will write a plain language summary and design a leaflet for dissemination to their peers and distribute to patient groups after publishing.

DATA AVA I L A B I L I T Y S TAT E M E N T
The technical appendix, dataset, and statistical code are available from the corresponding author at dong_qiang@fudan.edu.cn.
The lead author affirms that this manuscript is an honest, ac-