A Randomized, Double-blind, Placebo-controlled Trial of DL-3-n-butylphthalide in Amyotrophic Lateral Sclerosis Patients

Background: To determine the ecacy and safety of DL-3-n-butylphthalide (NBP) for the treatment of amyotrophic lateral sclerosis (ALS). Methods: A randomized, double-blind, placebo-controlled trial was performed at 19 ALS clinical centers of the Chinese ALS Association. Patients with denite or probable ALS were randomly treated with NBP or placebo for 12 months. The Revised Amyotrophic Lateral Sclerosis Functional Rating Scale (ALSFRS-R) score was the primary endpoint and was evaluated every 3 months. Secondary endpoints included survival and tracheotomy incidence, total Medical Research Council (MRC) score, percentage of predicted forced vital capacity (FVC), and clinical global impression scale score assessed using the visual analog scale. Results: Between November 23, 2015 and November 22, 2017, 312 ALS patients were enrolled and randomly allocated to either the NBP group (156 patients) or placebo group (156 patients). Ninety-three patients in the NBP group and 92 patients in the placebo group were included in the primary end point analysis. There was no signicant difference in the ALSFRS-R score, total MRC score, or clinical global impression between the two groups after treatment. The NBP group exhibited a mild trend of less decrease in the percentage of predicted FVC between baseline and the 12-month visit than the placebo group (least-squares mean change from baseline ± standard error: -7.34±4.28, 95(cid:0)CI(-15.24,0.56), p=0.0335). Adverse events were reported in 56.5% of patients in the placebo group and 68.8% of patients in the NBP group (χ 2 =2.99, P=0.0838). No serious adverse event related to treatment occurred. Conclusion: we found no evidence that NBP improved the ALSFRS-R score in patients with ALS. The results suggest a mild trend in the percentage of predicted FVC decreased slowly in the NBP treatment group than in the placebo group.


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Results: Between November 23, 2015 and November 22, 2017, 312 ALS patients were enrolled and randomly allocated to either the NBP group (156 patients) or placebo group (156 patients). Ninety-three patients in the NBP group and 92 patients in the placebo group were included in the primary end point analysis. There was no signi cant difference in the ALSFRS-R score, total MRC score, or clinical global impression between the two groups after treatment. The NBP group exhibited a mild trend of less decrease in the percentage of predicted FVC between baseline and the 12-month visit than the placebo group (least-squares mean change from baseline ± standard error: -7.34±4.28, 95 CI(-15.24,0.56), p=0.0335). Adverse events were reported in 56.5% of patients in the placebo group and 68.8% of patients in the NBP group (χ 2 =2.99, P=0.0838). No serious adverse event related to treatment occurred.
Conclusion: we found no evidence that NBP improved the ALSFRS-R score in patients with ALS. The results suggest a mild trend in the percentage of predicted FVC decreased slowly in the NBP treatment group than in the placebo group. Background Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease in which motor neurons degenerate, resulting in progressive limb and bulbar paralysis and eventually leading to patients requiring mechanical ventilatory support and death. Riluzole and Edaravone have been approved by some countries as treatments to improve the survival of patients with ALS. However, their effects are mild, and a pressing need remains for more effective disease-modifying treatments.
DL-3-n-butylphthalide (NBP) is a synthesized drug that is extracted as a pure component from seeds of Apium graveolens Linn (Chinese Celery). Previous studies showed that NBP signi cantly reduces oxidative damage, improves mitochondrial function, reduces neuronal apoptosis, and inhibits in ammation (1). In SOD1-G93A transgenic mice, oral administration of 60 mg/kg/d NBP immediately after symptom onset signi cantly prolonged survival, decreased the progression rate of motor de cits, and suppressed body weight reduction. Furthermore, motor neurons were preserved in the anterior horns compared with mice given a vehicle control (2,3). Some evidence has indicated that NBP has protective effects in ALS mice; therefore, a placebo-controlled clinical trial was conducted to determine whether a similar effect could be observed in ALS patients.  (4). Included patients displayed onset of progressive weakness within 18 months prior to the study. The age at onset was between 20 and 75 years old. The forced vital capacity (FVC) was more than 70% of the predicted normal value. Patients had not taken Riluzole or Edaravone in the 3 months before recruitment. All patients were capable of understanding the information provided and giving full informed consent.
The exclusion criteria were as follows: electromyography showed motor nerve conduction block and abnormal sensory nerve conduction; computed tomography or magnetic resonance imaging revealed lesions that may explain the patient's clinical presentation; dementia or mental disorder; serious heart, liver, kidney, or other related disease; participation may endanger the patient's life; gastrointestinal disorders or gastrointestinal surgery that may affect gastrointestinal absorption; a history of allergies, especially medication allergies; patients requiring ventilator-assisted breathing or tracheotomy; patients who were breast feeding or pregnant; patients who had received or were currently receiving Riluzole treatment; presentation of bulbar or thoracic symptoms or signs at onset; or patients who had di culty taking medicine.

Sample size calculation
The sample size was calculated according to a previously published clinical trial of ALS patients (5). To provide 80% power to detect an adjusted mean difference between groups of 3 points on the ALSFRS-R score(30.2±8.9 vs 33.2±8.0) and considering a dropout rate of 25% at the last visit, with a 1:1 randomization ratio between NBP and placebo group, at a two-sided alpha level of 0.05, we calculated that a sample size of 312 patients would be required approximately (156 in the NBP group and 156 in the placebo group).

Treatment group
Patients eligible for admission were randomized, using SAS statistical analysis to generate A randomized arrangement of 312 subjects receiving treatment (study drug versus control drug) in A 1:1 ratio between group A and group B. That is, list the serial number, the corresponding treatment assignment, and follow this compilation blind.
Patients randomly assigned to groups A or B were treated with butylphthalide soft capsules (100mg/ tablet) or placebo (A placebo with physical characteristics such as appearance, size, color, dosage form, weight, taste and smell as similar as possible to the experimental drug, but without the active ingredients of the experimental drug).NBP group patients were given two soft capsules(200mg) three times a day for 12 months. Placebo group patients were given two placebo soft capsules three times a day for 12 months.

Evaluation of functional status
All patients were evaluated before treatment with NBP or placebo and then followed up for 12 months. A functional evaluation was conducted every 3 months after enrollment and included the ALSFRS-R, survival and tracheotomy incidence, total Medical Research Council (MRC) score, respiratory function, and clinical global impression (CGI) scale reported by the patient.
The ALSFRS-R score was evaluated according to a questionnaire. The maximum ALSFRS-R score was 48. The total MRC score was calculated as the sum of the muscle strength in 24 muscle movements, including neck exion, neck extension, bilateral shoulder abduction, elbow exion, elbow extension, wrist exion, wrist extension, thumb extension, hip exion, knee exion, knee extension, ankle dorsi exion, and ankle plantar exion. Each muscle movement was evaluated with the MRC scale (0-5); the total MRC score was 120. Respiration function was monitored with an FVC test and was expressed as a percentage of the expected value. The CGI scale was used to assess disease severity and was evaluated by patients using a visual analogue scale ranging from no symptoms to the most serious symptoms (0 to 7 points).

Outcomes
The primary endpoint was the ALSFRS-R score after 12 months of treatment. The secondary e cacy outcomes were survival, tracheotomy, total MRC score, FVC, and the CGI score reported by the patient.

Adverse events
The investigator observed patients for adverse events every 3 months after treatment and instructed patients to report any events at any time. After patients routinely or prematurely terminated the study, adverse events were monitored for 3 months.

Statistical analysis
The qualitative factors among the baseline data were analyzed using the χ2 test, and the quantitative data were analyzed using the two independent-samples t test. The outcome measures were the changes in the ALSFRS-R score, percentage of predicted FVC, total MRC score, and CGI score from baseline to 12 months. To estimate the treatment effects, comparisons between groups were performed using the Mann-Whitney U-test for two sets of independent samples, as the data did not follow a normal distribution. Statistical analyses were performed using SPSS 17.0 software (SPSS, Inc., Chicago, IL, USA).
All statistical tests were two-sided, and a value of P<0.05 was considered to indicate a statistically signi cant difference.  Table 1. Both groups had a similar sex ratio, age at rst visit, duration from disease onset, site of onset, bulbar involvement at rst visit, body mass index, ALSFRS-R score, and predicted FVC.

Primary outcome
Changes in the ALSFRS-R score between baseline and the last visit of both groups are listed in Table 2. There was no difference in the ALSFRS-R score between the two groups.

Secondary outcomes
Survival and tracheotomy: After 12 months of treatment, 13 of 93 patients in the NBP group had died and 2 had undergone tracheotomy; 6 of 92 patients in the placebo group had died and none had undergone tracheotomy (χ 2 =4. 24, P=0.0394).
After 12 months of treatment, the changes in endpoint parameters between baseline and the 12-month visit were determined, no difference was detected between the two group in total MRC score and GCI (see Table 2). There was a mild trend of less decrease in percentage of predicted FVC in the NBP group than in the placebo group although there was no signi cant statistical difference.

Adverse events
Adverse events were reported in 56.5% of patients in the placebo group and 68.8% of patients in the NBP group (χ 2 =2.99, P=0.0838). No severe adverse events related to the treatment were reported. Adverse events with an incidence rate greater than 1% included increases in creatinine kinase, alanine aminotransferase, and aspartate transaminase levels, gastrointestinal symptoms, lower limb edema, dizziness, respiratory symptoms, and rash (Table 3).

Discussion
In this randomized, placebo-controlled, double-blind trial of NBP therapy in ALS patients, no intergroup difference was observed in the change in ALSFRS-R score after 12 months of treatment. There was also no difference between the two groups for some secondary outcomes, including the total MRC score and CGI scale.
A comparison of the changes in the percentage of predicted FVC during 12 months of treatment showed that this parameter decreased more slowly in the NBP group than in the control group. This result suggested that NBP might have a potential effect on slowing the decrease of respiratory function. However, this result was inconsistent with the higher mortality and tracheotomy rate in the NBP group after 12 months of treatment. The potential mechanism responsible for slowing the decrease in FVC during the 12 months of treatment needs to be further explored. The results should be interpreted cautiously because more than half of the patients in the two groups could not complete the FVC test at the 12-month visit because of bulbar muscle weakness. Those patients might have also had more serious respiratory muscle weakness in addition to bulbar muscle weakness. Therefore, the % FVC might not be a good outcome parameter to evaluate respiratory muscle function in ALS patients. Although we only recruited patients with limb symptoms at onset, most patients developed bulbar muscle weakness during follow-up. Nasal sniff pressure has been suggested as a better assessment of respiratory function in ALS patients because it can be completed in patients with bulbar muscle weakness (6).
In ALS patients, earlier treatment results in a greater bene t. In this study, we recruited ALS patients with onset duration of less than 18 months. To avoid dropout of patients because of di culty in swallowing medicines and prevent the patients from aspiration, we only recruited patients with limb onset, and these patients exhibit longer survival. However, in this condition, it is di cult to determine the survival and tracheotomy incidence, which comprise the gold standard outcome for ALS patients. In this study, 88.6% of patients were surviving at the last visit. Although the mortality and tracheotomy rate was slightly higher in the NBP group, longer follow-up of survival and tracheotomy incidence should be performed to avoid sample bias.
In this clinical trial, we de ned the ALSFRS-R score as the primary endpoint, which has been adopted in other clinical trials (7)(8)(9)(10)(11)(12). However, the ALSFRS-R score might not be su ciently sensitive to re ect mild deterioration in ALS patients. In most studies, the ALSFRS-R score slowly decreased during follow-up among all patients, but for individual patients, 25% of 3,132 patients did not exhibit decline over 6 months. Over 12 months, 16% of 2,105 patients did not exhibit decline (13). A more e cient and accurate scale for evaluating the changes in ALS patients should be recommended for future clinical trials, especially for patients with slow progression. However, this study only compared the conditions after 12 months of treatment, which might affect the evaluation of the bene t of NBP in ALS patients, and a longer follow-up time might provide more information.
Because Riluzole and Edaravone are expensive and are not covered by the medical insurance in most provinces in China, many patients could not afford them. Furthermore, we had no information about whether NBP affect the pharmacokinetics and action of Riluzole or Edaravone, patients always doubt whether NBP decrease the effect of those medicine. So, no Patients received Riluzole or Edaravone treatment in this study.
Although NBP treatment of SOD1 mice showed bene ts including delayed disease onset and longer survival (2, 3), we found no de nite bene t of NBP treatment in ALS patients. This phenomenon was common in past clinical trials. The loss of motor neurons in ALS patients is insidious; almost one-third of motor neurons degenerate before weakness is presented. Therefore, initiation of treatment after several months of disease progression might be too late.

Conclusion
In summary, we found no effect of NBP treatment on ALS patients according to the primary and most secondary outcome parameters, except for a mild trend of less decrease in percentage of predicted FVC.

Consent for publication
All the authors have approved the manuscript.

Availability of data and materials
The datasets used and/or analysed in the current study are available from the corresponding authors on reasonable request except those with privacy or ethical restrictions.

Competing interests
The DL-NBP in ALS clinical trials were funded by CSPC NBP Pharmaceutical Co. Ltd.

Funding
This work was supported by CSPC NBP Pharmaceutical Co. Ltd.

Authors' Contributions
Liying Cui conceived and designed the experiments and reviewed and revised the manuscript; the clinical trial was performed at 19 ALS Clinical Centers; Jia He designed the protocol and input, checked, and analyzed the data; Mingsheng Liu wrote the manuscript.  Figure 1 Diagram of subject distribution