Acute stroke is the second leading cause of death in industrialized countries and is one of the major causes of death in China.1 Thrombolysis using alteplase can limit neurological damage and is approved to be the most important therapy in rapid intervention after the onset of stroke.2 However, only a few patients with ischemic stroke can benefit from this treatment because of the very brief time window and the necessity for the training of doctors in the use of alteplase.3,4 Chinese herbal medicine has been used for many centuries for the treatment of acute stroke patients in Asia. However, two major issues limit its application. Firstly, Chinese herbal medicine is commonly administered as a mixed compound; the difficulty in the determination and purification of active ingredients restrains pharmacodynamic and pharmacokinetic studies. Secondly, randomized clinical trials for the evaluation of Chinese herbal medicine are very few, resulting in poor evidences on their efficacy and safety profile.5–7
Dl-3-n-butylphthalide (NBP), first isolated from the seeds of celery, is now synthesized and found to exert protective effects against ischemic brain in animal models of acute ischemic stroke. Several mechanisms may relate to the therapeutic effects. Firstly, NBP can promote the NO production of endothelial cells and result in vasodilative effects. Secondly, NBP may have an aspirin like effect on the PGI2/TXA2 pathway, which may result in anti-thrombotic activities. Thirdly, NBP can increase the number of cerebral microvessels via up-regulating expressions of vascular endothelial growth factor (VEGF) and hypoxia induced factor-1α (HIF-1α).8–10 The NBP capsule has shown efficacy in ischemic stroke patients in a randomized double-blind placebo-controlled trial,11 which indicated that a 20-day treatment using NBP capsule significantly improved the functionality outcome measured by modified Rankin scale at day 21 compared with placebo. Chinese Guidelines for the Management of Stroke 2010, therefore recommended NBP capsules for the treatment of acute ischemic stroke.12 However, further studies are needed to confirm its efficacy since the previous study had a small sample size. In addition, although effective, the bioavailability being as low as 15% (data not published) limits the application of NBP capsule in acute ischemic stroke patients. The recommended eight capsules per day are difficult to swallow for many patients, especially at the onset of stroke.
Hereby, we report the results of a clinical trial to assess the efficacy and safety of NBP injection compared with sodium ozagrel, a thromboxane A-2 synthase inhibitor, in patients with acute ischemic stroke. In addition, we assessed NBP injection followed by small-dose NBP capsules as a continuous dose regimen for 90-day after stroke.
We conducted a randomized, double-blind, double-dummy trial in which patients were enrolled from March 2007 to December 2009 at 38 hospitals in China. The trial was approved by National Institutional Review Boards, ethics approval was obtained at each center and written informed consent was obtained from the patients or from legally acceptable surrogates (Clinical Trial Registration-URL: http://www.chictr.org, Unique identifier: ChiCTRTRC-09000483).
Patients were eligible for inclusion if they were 35–75 years of age, had a clinical diagnosis of acute ischemic stroke that had commenced within 48 hours before entry into the study, and had a score between 6 and 25 on the National Institutes of Health Stroke Scale (NIHSS). A cerebral computed tomographic (CT) scan was required before randomization to exclude patients who had intracranial hemorrhage. Patients were excluded if they had cardiogenic embolism, a stroke history with a score ≥2 on the modified Rankin score (mRS), severe liver or renal dysfunction, major disorders associated with an increased risk of bleeding, contraindications to antiplatelet treatments or the use of other antiplatelet drugs during the trial period.
Randomization and treatment schedule
Treatments included 2 stages: (1) initial treatment for 14 days with intravenous infusion of either NBP or sodium ozagrel, and (2) oral treatment with either NBP capsule or aspirin for a further 76 days. Drugs were packaged into 90- day treatment kits. Patients were randomly assigned into three groups (Figure 1).
Group A1, NBP injection 25 mg b.i.d. + sodium ozagrel placebo b.i.d. (Stage 1), NBP capsule 200 mg b.i.d. + aspirin placebo q.d. (Stage 2); group A2, NBP injection 25 mg b.i.d. + sodium ozagrel placebo b.i.d. (Stage 1), NBP capsule placebo b.i.d + aspirin 100 mg q.d. (Stage 2); group B, NBP injection placebo b.i.d. + sodium ozagrel 80 mg b.i.d. (Stage 1), NBP capsule placebo b.i.d + aspirin 100 mg q.d. (Stage 2).
For both NBP injection (or placebo) and sodium ozagrel (or placebo), the infusion lasted between 50 and 70 minutes and the two infusions had an interval of 6–8 hours.
Randomization was performed by telephone using a central interactive voice-response system that was accessed by the investigators. Patients undergoing randomization were stratified according to site and NIHSS score at baseline. A dynamic algorithm was used to maintain the balance of NIHSS scores among the study groups.
Patients were evaluated at enrollment and on days 8, 15, 30, and 90 after administration of the drug. Initial assessments included a physical examination (e.g., blood pressure, heart rate and electrocardiogram (EKG)), brain CT or MRI, the quantification of neurological deficit using NIHSS (scores range from 0 to 42, with higher scores indicating increasing severity), and the functional measures using mRS (with a range from 0, indicating no residual symptoms, to 5, indicating bedbound, requiring constant care). During follow-up, patients were accessed with the NIHSS on days 8, 15 and 30. The mRS and the Barthel Index (with a range from 0, indicating complete dependence on help with activities of daily living, to 100, indicating independence) were applied to measure disability on days 8, 15, 30, and 90. The flexible period of follow-up was 1 day for visit on day 8 and 3 days for visit on days 15, 30, and 90. Investigators were trained for the appropriate use of the NIHSS, mRS, and Barthel Index in the investigators meeting.
Vital signs and adverse events were regularly recorded at each visit. Routine laboratory tests and EKG were preformed on days 1 and 15 (the first day of drug administration and the first day after infusions).
The statistical analyses of efficacy followed the intention-to-treat principle, which included all randomly assigned patients, whether or not they were treated. For those who had missing data in outcome assessment and were known to be alive, the data of last observation were carried forward. Analyses were also processed in per protocol population, including those who completed the study without any major protocol violations. The primary end points were the disability at day 90 assessed by the mRS and the Barthel Index. Fisher's exact test was used to assess the group differences of qualitative variables at baseline, while one-way analysis of variance (ANOVA) was used for quantitative variables.
Patients were dichotomized as favorable outcome (mRS 0–1) or unfavorable outcome (mRS 2–6). Differences among the three groups on the proportion of patients with favorable outcome were compared by using χ2 test of proportions (with two-sided α=0.05). Using group B as reference, a Logistic regression analysis was further performed and baseline NIHSS score was included in the model to examine the efficacy for 90-day treatment of NBP. For the safety analysis, differences among groups were compared using Fisher's exact test. The adverse events and laboratory values collected in Stage 1 (days 1–14) or Stage 2 (days 15–90) were evaluated separately as the medication differed in these stages.
Of the 573 patients who underwent randomization, 535 patients who received study infusion and had at least one outcome data available were included in the efficacy analysis. Of the 535 patients, 179 were assigned to receive NBP injection and NBP capsule (group A1), 177 were assigned to receive NBP injection and aspirin (group A2), and 179 were assigned to receive sodium ozagrel and aspirin (group B). Baseline demographic and clinical characteristic of the three groups are listed in Table 1. All three groups were well-balanced in terms of baseline characteristics.
Functional outcome was measured on the mRS at day 90 and dichotomized as a favorable outcome (a score of 0 or 1) or an unfavorable outcome (a score of 2 to 6). The χ2 test of proportions showed that 63.80% patients in group A1 had a favorable outcome at 90 days, significantly higher than group A2 (49.10%) or group B (45.51%, P=0.002 in the intention-to-treat population, P=0.004 in per-protocol population, Table 2). Logistic regression showed that patients with 90-day treatment with NBP (group A1) were significantly more likely to have a favorable outcome than patients with treatment of sodium ozagrel (group B) (P <0.001, OR=2.40, 95% CI 1.49–3.86), adjusted for baseline NIHSS score. However, the difference between those who received NBP injection and aspirin (group A2) and those who received sodium ozagrel and aspirin (group B) was not significant (P=0.377, OR=1.29, 95% CI 0.81–2.06, Table 3). No significant difference was found on the increase of Barthel index score at day 90 among the three groups (P=0.179, Table 2).
Of the 573 patients who underwent randomization, 565 patients who received study infusion and had at least one safety data available were included in the safety analysis. Of the total of four patients three died in group A (0.79%) and one in group B (0.53%). Of these four patients, two died between days 1 and 14, and both were in NBP injection group. Of the two patients who died between days 15 and 90, one was in group A1 (NBP injection + NBP capsule), and the other was in group B (ozagrel + aspirin). No significant difference in mortality was found among treatment groups (P=1.000).
During 90 days of treatment, 163 patients had adverse events (AE) and the AE rates were similar among the three groups (group A1=28.19%, group A2=33.16%, group B=25.13%, P=0.229). Of the 109 patients who had adverse events between days 1 and 14, 74 (19.58%) were in NBP injection group and 35 (18.72%) were in sodium ozagrel group (P=0.910). Between days 15 and 90, adverse events occurred in 65 patients with 23 (12.23%) in group A1, 28 (14.74%) in group A2 and 14 (7.49%) in group B, respectively (P=0.077). There was no significant difference among the three groups in any specific adverse event or in any routine laboratory testing values (Table 4).
Among the 62 patients who discontinued the treatment, 42 (11.5%) received NBP and 20 (11.1%) received ozagrel (P=0.882).
In this randomized, double-blind, double-dummy trial, the 90-day treatment of NBP was found to be superior to the treatment of sodium ozagrel and aspirin for acute ischemic stroke patients. A 14-day regimen of NBP injection followed by 76 days of NBP capsule appeared to be more effective than that of a 14-day NBP injection followed by aspirin. This benefit was mainly seen at the disability end point as measured by mRS, but not for NIHSS and Barthel index. Both oral and intravenous treatment of NBP for as long as 3 months was found to be safe.
This study was designed to compare two intravenous administration drugs to treat patients with acute ischemic stroke. Ozagrel, a selective inhibitor of thromboxane A2 synthase and an inhibitor of platelet aggregation, was approved in 1993 to be effective and safe for noncardioembolic patients by the Japanese Ministry of Health and Welfare, and is currently widely used in China as a therapeutic agent to treat acute ischemic stroke patients.13 We therefore chose ozagrel as the comparison drug. In addition, the molecular mechanisms of NBP were studied in multiple in-vivo or in-vitro models of stroke, revealing therapeutic effects via anti-thrombotic and anti-inflammatory activities,10 protecting endothelial cells against oxidative/nitrosative stress and mitochondrial damage,14 and enhancing angiogenesis associated with up regulation of vascular endothelial growth factor and HIF-1 alpha expressions.8 According to these mechanisms, patients were expected to have further benefits from the continuous administration of NBP. The purpose of this trial was also to test the safety and efficacy of a 90-day regimen of NBP.
In the present study, a persistent regimen of NBP, as long as 90 days, was found to be more effective as compared with ozagrel and aspirin or with 14-day NBP injection and aspirin. Although the sample size was relatively small, significant differences were found in mRS, the widely accepted functional outcome measure.15 As the stroke evolution or recurrence rate was found to be similar between groups, the benefits in functional consequence seemed not to relate to the prevention of recurrence of stroke. In the previous randomized double-blind placebo-controlled trial of NBP capsule, acute ischemic stroke patients were found to benefit from 20-day of NBP capsule treatment and the dose was 400 mg per day, twice as much as that of the present study. Further studies with larger sample are needed to assess the differences between various doses in long-term therapy.
Because only patients with moderate severity of stroke were enrolled, only 4 deaths happened in the present study. This moderate severity patients approach was decided because the study had relatively small sample size, and complications associated with severe stroke patients, which are strong predictors of functional outcome, may be difficult to balance in a small sample study. Although previous in-vivo study found anti-thrombotic effect of NBP, the rate of intracranial hemorrhage and other bleeding was similar among the groups, suggesting that bleeding complications of NBP were not different from classic anti-thrombotic treatments. Overall, the rate of serious as well as nonserious adverse events was similar among the study groups, and both NBP injection and capsule appears to be relatively safe. In phase III trial of NBP capsule, recoverable mild to moderate elevated alanine aminotransferase (ALT) was found in 17.5% of the patents who took 800 mg NBP capsules per day and in 5.9% patients who took placebos. ALT and glutamic-oxalacetic transaminease (AST) were therefore monitored in the present study. At the dose of 50 mg/d NBP injection and 400 mg/d NBP capsule, no significant difference was found between the NBP and control group. This result was consistent with phase II trial of NBP injection, in which elevation of ALT was found in 2% patients who used 50 mg of NBP injection per day. These results suggested that the elevation of ALT or AST was more likely to be dose related, and the present study did not find significant difference of ALT or AST elevation at the dose of 50 mg/d NBP injection and 400 mg/d NBP capsule intake as compared with the placebo.
There are several strengths to our study. The randomized, double-blind, double-dummy design of this study attested the strict unawareness of the treatment assignments among patients and investigators. Among study groups, the baseline factors that could influence the functional outcome were similarly distributed. The main limitation of the present study is the relatively small sample size, so the efficacy results of this study need to be interpreted with caution.
In summary, the results of this study suggest that a 90- day treatment with NBP might be both effective and safe for patients with acute noncardioembolic ischemic stroke, especially for those with moderate severity. Further trials with larger sample size are needed to confirm the efficacy of long-term NBP therapy in stroke.
Appendix: Participating sites: KE Kai-fu, Affiliated Hospital of Nantong University; WANG Bao-jun, Baotou Central Hospital; FENG Lian-yuan, Bethune International Peace Hospital of PLA; LI Ji-mei, Beijing Friendship Hospital of Capital Medical University; YANG Qi-ming, Chenzhou No.1 People's Hospital; NIU Jun-ying, Chinese PLA Second Artillery General Hospital; ZHOU Hua-dong, Daping Hospital of third military medical university; ZENG Jinsheng, First Affiliated Hospital of Sun Yat-sen University; QIN Xin-yue, First Affiliated Hospital of Chongqing Medical University; DONG Wan-li, First Affiliated Hospital of Soochow University; ZHENG Rong-yuan, First Affiliated Hospital of Wenzhou Medical College; WU Jiang, First Hospital of JiLin University; ZHANG Chao-dong, First Hospital of China Medical University; PENG Kairun, General Hospital of Guangzhou Military Command of PLA; PAN Xiao-ping, Guangzhou First Municipal People's Hospital; LI Ling, Hebei Provincial People's Hospital; DONG Qiang, Huashan Hospital Affiliated to Fudan University; DING Xin-sheng, Jiangsu Province Hospital; CAO Bing-zhen, Jinan Military General Hospital; XU Yun, Nanjing Drum Tower Hospital; CHEN Ying-zhu, Northern Jiangsu People's Hospital; FAN Dong-sheng, Peking University Third Hospital; GAO Shan, Peking Union Medical College Hospital; CHI Zhao-fu, Qilu Hospital of Shandong University; ZHANG Zhao-hui, LU Zu-neng, Renmin Hospital of Wuhan University; BI Jian-zhong, Second Hospital of Shandong University; HE Jun-ying, Second Hospital of Hebei Medical University; LIU Jun-yan, Third Hospital of Hebei Medical University; SUN Hong-bin, Sichuan Provincial People's Hospital; HU Xin-yue, Sir Run Run Shaw Hospital; WANG Wei-zhen, Shanghai First People's Hospital; QU Fang, Shenyang Military General Hospital; CHEN Kang-ning, Southwest Hospital; WANG Wei, Wuhan Tongji Hospital; HE Li, West China Hospital, Sichuan University; ZHAN Qing, Shanghai Tongji Hospital; JIA Jian-ping, Xuanwu Hospital of Capital Medical University; ZHANG Jun-jian, LIU Yu-min, Zhongnan Hospital of Wuhan University.
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Keywords:© 2013 Chinese Medical Association
ischemic stroke; medical treatment; dl-3-n-butylphthalide