Trial design and patients
This randomized, double-blind, placebo-controlled study was conducted in Firoozgar hospital in Iran University of Medical Sciences. We recruited eligible patients from the outpatient clinic and participants were considered eligible if they were diagnosed with ALS according to the El-Escorial diagnostic criteria for ALS. Besides, eligible patients were aged 18 to 80 years, able to swallow tablets/capsules, and showed slow vital capacity (SVC) greater than or equal to 50% predicted for sex, age and height. We excluded participants if they had kidney or liver dysfunction, history of laryngeal dystonia and/or akathisia, history of moderate or severe brain injury or stroke, complicated diabetes mellitus, and history of cardiac arrhythmia.
Ethical Standards
All procedures contributing to this work comply with the ethical standards of the relevant national guidelines on human experimentation and with the Helsinki Declaration of 1975, as revised in 2008. The protocol of this study was reviewed and approved by the Institutional Review Board of the Iran University of Medical Sciences (IR.IUMS.REC.1399.904) and was registered in the Iranian Registry of Clinical Trials (IRCT20201020049089N1) on 15/12/2020.
Randomization
Participants were randomly assigned in a 1:1 ratio to groups of bumetanide or placebo by using a block randomization scheme. The sample size was calculated based on a previous study by Lemonnie et al reporting that bumetanide 2 mg significantly improves clinical global impressions (CGI) score in 38.5% of patients with ASD [17]. Accordingly, with the 95 % confidence level, power set at 80%, and a potential attrition rate of 10%, the calculated sample size was 18 for each group of trial. All participants and their caregivers, clinicians, researcher and data analysts were blinded to treatment assignments until final data analysis.
Treatment
This study consisted of two phases including treatment phase (4 months) and follow up phase (1 month). Choosing of bumetanide dose was based on our prior investigations, reporting the effectiveness of bumetanide 2 mg on the reduction of seizure frequency in patients with epilepsy [15]. Hence, bumetanide 2 mg tablet (Almus Pharmaceuticals, UK) was administrated daily in bumetanide group and patients in the placebo group revived daily placebo tablet which was identical in size, appearance and taste with bumetanide. Bumetanide side effects were monitored through monthly blood serum analysis during the study. All patients were visited weekly to monitor their physiological symptoms.
MUNIX procedure
MUNIX analysis was developed by Nandedkar for assessment of number and size of motor units in patients with motor neuron disease [19]. The primary endpoint measurement in this study was the MUNIX index. We performed the MUNIX in ALS patients before and after treatment in muscles from lower and upper extremities, including tibialis anterior (TA), abductor pollicis brevis (APB), abductor digiti minimi (ADM), and trapezius. Motor unit size index (MUSIX) as an indicator of the motor unit surface was also calculated by EDX machine. Participants were positioned appropriately and tests were performed by the same neurologist through a standard electromyography instrument.
ALSFRS-R
The secondary endpoint measurement was ALSFRS-R score which is a well-established and widely distributed score for assessment of physical function in the domains of gross and fine motor function, bulbar symptoms, and respiratory function in ALS patients [20, 21]. ALSFRS-R is based on 12 items, each of which is rated on a 0-4 point scale. The score of total functional disability thus ranges from 0 (maximum disability) to 48 (normal) points [22]. In this study, we used the ALSFRS-R instrument before and after treatment in both groups of trial.
Statistical Analysis
Data were analyzed using the GraphPad Prism 8.1.1 (GraphPad, San Diego, CA, USA) statistical software package. Continuous values were expressed as mean ± standard deviation (SD) and analyzed using the Student t-test. The Pearson chi-square test was used to examine the categorical data. In all analyses, the significance level was set at p < 0.05.