2.1 Trial Design

This was a multicentre, randomised, placebo-controlled, double-blind, Phase III clinical trial of sovateltide in patients with acute cerebral ischaemic stroke receiving the best standard of care (SOC). The study was conducted in 18 centres across India. Patients were randomised in a 1:1 ratio to the sovateltide or the control group. The study drug manufactured by Pharmazz India Private Limited at Gufic Biosciences Limited was supplied to the investigators at the participating sites. The study drug (normal saline or sovateltide [0.3 µg/kg]) was administered intravenously in three doses at intervals of 3 ± 1 h on Days 1, 3, and 6 (total sovateltide dose of 0.9 µg/kg/day). The control group received saline plus SOC, while the sovateltide group received sovateltide treatment plus SOC post-randomisation. The study duration for an individual patient was 90 days, including five study visits. Visit 1 included screening, randomisation, baseline measurements, and treatment on Day 1, measurements on Day 2, and measurements and treatment on Days 3 and 6, after the first visit measurements were included in visit 2 on Day 12 ± 2, visit 3 on Day 30 ± 5, visit 4 on Day 60 ± 7 (by telephone) while visit 5 was at the end of study (Day 90 ± 7).

2.2 Regulatory Oversight

The study complied with the International Conference on Harmonisation guidelines for Good Clinical Practice (GCP), the Helsinki Declaration, and local regulatory requirements. The study protocol (PMZ-1620/CT-3.1/2019) dated April 29, 2019, was approved by the Drugs Controller General of India (DCGI) (DCGI CT NOC. No.: CT/ND/66/2019). This study was conducted in 18 hospitals across India. O the 18 sites, 12 had more than 300 beds with at least 40 being intensive care unit beds. All the centres had emergency medical facilities and uniform SOC to manage stroke patients. The first patient was randomised on November 13, 2019, and the last was randomised on February 6, 2022. The date of trial completion was May 4, 2022. The study protocol was reviewed by each institutional ethics committee and approved before initiating patient enrolment. The study protocol and statistical analysis plan are available (Supplementary Information). The statistical analysis plan (SAP) was faithfully followed and was written before having access to any results. The trial registration was done at the Clinical Trials Registry, India (CTRI/2019/09/021373) and the United States National Library of Medicine, ClinicalTrials.gov (NCT04047563). The ethics committee of each site was informed of any protocol amendment, deviation, subject exclusion or withdrawal, and serious adverse events (SAEs) (details of participating sites are in Supplementary Table 1).

2.3 Patient Population

Screening of patients was based on the following inclusion criteria: (1) first-time stroke in adult of either sex aged 18–78 years; (2) patient or legally authorised representative willing to give informed consent; 3) stroke is ischaemic in origin and radiologically confirmed by computed tomography (CT) scan or diagnostic magnetic resonance imaging (MRI); (4) no haemorrhage as proved by cerebral CT/MRI scan; (5) cerebral ischaemic stroke patients presenting up to 24 h after onset of symptoms with NIHSS score > 5 and mRS score of 3–4; and (6) patient is < 24 h from time of stroke onset when the first dose of investigational drug is administered. Exclusion criteria used for the eligibility were: (1) patients receiving endovascular therapy or is a candidate for any surgical intervention for the treatment of stroke; (2) patients classified as comatose; (3) evidence of intracranial haemorrhage; (4) known pregnancy; (5) confounding pre-existing neurological or psychiatric disease; and (6) evidence of any other major life-threatening or serious medical condition that would prevent completion or impair the assessment of outcome.

2.4 Consent

The patients included in this study were in a life-threatening condition. For those patients who were not fit to consent themselves at the time of initiation of treatment, informed consent was taken from their legally authorised representative (LAR). The investigator informed the patient or a LAR in writing and verbally of the details of the study relevant to deciding on participation. The informed consent form included all the requirements per the GCP recommendations and schedule Y. Informed consent forms in English and regional languages were approved by the respective Ethics Committee and DCGI. The consenting process was recorded through an audio-video recording, labelled, and stored at the study site in a secure place. Medical confidentiality and data protection were ensured per the regulatory requirements, and the investigator stored the signed informed consent forms.

2.5 Randomisation and Blinding

An Interactive Web Response System (IWRS) containing randomisation codes was used to randomise the eligible patients into treatment groups. The patient and all relevant personnel involved with the study's conduct and interpretation (including investigational site personnel, the investigator, and the sponsor or designee's staff) were blinded to the study drug's identity (sovateltide/normal saline) and the randomisation codes. Independent of the monitoring team, an unblinded pharmacist dispensed the drugs under confidentiality. The final randomisation list was kept confidential until study completion. Emergency unblinding through IWRS was available only to the investigator when medically needed. For those patients who were unblinded, the date, time, and reason for emergency unblinding were recorded in the patient's medical record. Any adverse event (AE) or SAE that required unblinding, the treatment was recorded and reported as specified in the protocol. Each patient was monitored closely throughout their hospitalisation and followed until discharge from randomisation. After meeting the eligibility criteria, patients were randomised 1:1 to the sovateltide or control groups, respectively.

2.6 Safety Evaluation

All patients in both groups received the SOC (airway maintenance, managing breathing, circulatory insufficiencies, thrombolytics [if indicated], antiplatelet drugs [if indicated], and fluid resuscitation) for cerebral stroke throughout the study, according to local institutional standard practice. After randomisation, sovateltide or normal saline was administered intravenously to cerebral ischaemic stroke patients. The treatment and dose selection were based on preclinical proof-of-concept studies conducted in our laboratory [24, 26, 27, 30, 33, 34]. In the present study, sovateltide (30 µg per vial dissolved in 5 mL of normal saline) was administered at a dose of 0.30 µg/kg bodyweight intravenously over one minute in the sovateltide group, while the control group received normal saline. The normal saline or sovateltide was administered in three doses at 3 ± 1 h intervals on Day 1, Day 3, and Day 6 (total sovateltide dose of 0.9 µg/kg/day).

The Data and Safety Monitoring Board (DSMB) was assembled before the study initiation and the members included a senior practicing physician with extensive experience in critical care medicine, a biostatistician, and a clinical neuropharmacologist. The DSMB reviewed the study data on safety and critical efficacy endpoints at pre-determined intervals.

Every patient who received treatment was included in the safety analysis. All patients were followed up for safety assessment from the randomisation until the end of the study on Day 90. The safety evaluation was carried out by the study investigator based on AEs, physical examination, vital signs (heart rate, systolic blood pressure [SBP], diastolic blood pressure [DBP], body temperature, and respiratory rate), ECG; and clinical laboratory parameters as per protocol. Various biochemical tests, serum chemistry tests, haematological variables, coagulation variables, urine output, and organ functions such as kidney and liver function tests were assessed. The AEs were coded by System Organ Class and Preferred Term using the Medical Dictionary for Regulatory Activities (MedDRA) version 21.0.

All AEs were listed and categorised by AEs with onset dates before dosing and treatment-emergent adverse events (TEAEs). The TEAEs were tabulated by indicating the number and percentage of patients and the number of events. The numbers of patients experiencing any adverse drug reactions were summarised for each treatment group. Adverse events were collected, evaluated, and tabulated in relation to the study drug, severity, seriousness, action taken, outcome, System Organ Class and Preferred Term for the treatment groups. Serious AEs were summarised according to System Organ Class and Preferred Term for the treatment groups. The AEs related to the severity, were presented as the number of occurrences and percent for the respective group. The SAEs related to the seriousness were presented as the percentage and number of occurrences for the respective group. In addition, the incidence of intracranial haemorrhage (ICH) was observed as an AE of special interest. Post-randomisation and throughout the study duration, repeat CT/MRI was done at the investigator's discretion if the patient deteriorated neurologically or ICH was suspected for any reason.

2.7 Efficacy Assessments

The primary objective was to determine the neurological outcome, as non-hierarchically assessed by (1) the number of patients with a mRS score 0–2, the number of patients with an NIHSS 0–5, and the number of patients with a BI 90–100 at 90 days; (2) the number of patients with NIHSS score < 6, mRS < 2, and BI > 40 change from baseline. For all these endpoints, changes were mean through 90 days. The study's secondary objectives included (1) change in quality-of-life as assessed by EuroQol-EQ-5D and Stroke-Specific Quality of Life (SSQOL); (2) incidence in the recurrence of ischaemic stroke; and (3) the proportion of patients with 90-day all-cause mortality. We also analysed our data with patients having baseline NIHSS scores of ≥ 8 (instead of ≥ 6) for the number of patients with an mRS of 0–2 at Day 90. Efficacy analysis was carried out in all intention-to-treat (ITT) patients. Any patient exposed to the drug but for any reason that led to the discontinuation of the study, was considered independent of the treatment effect. Multiple imputations were used to impute missing responses and the Multiple Imputation (MI) method, Multivariate Imputation by Chained Equations (MICE), was used to impute the missing assessments on the endpoints. Efficacy, safety, and ordinal shift analysis included all randomised patients.

2.8 Sample Size and Statistical Analysis

The statistical tests for efficacy endpoints were carried out as two-sided on a 5% significance level. The statistics for continuous and discrete variables with the number (n) of observations, mean and standard error of the mean (SEM) is described. For categorical data, the descriptive statistics are presented with the number of exposed patients and number (n) with the percentage of observations in the various endpoint categories, where the percentage was based on the exposed patients. An unpaired t-test was used to compare the discrete and continuous variables between the groups at baseline and follow-ups. A two-way analysis of variance (ANOVA) was used for computing treatment differences with adjustment to factors affecting continuous endpoints. Tukey's multiple comparison test was used to determine the differences in the mean. A p-value of less than 0.05 was considered to be significant. All available data were used in the analyses. The Chi-square test was used to evaluate the improvement in NIHSS score ≥ 6, BI score ≥ 40, and mRS score ≥ 2 between the treatment groups and to compare the percentage of patients with mRS scores of 0–2 between the treatment groups. The confidence interval (CI) of the odds ratio (OR) by the Baptista-Pike method [35] was used. The Log-Rank (Mantel-Cox) test for survival analysis was used. The analyses used all available data. Data that were unavailable were assessed as "missing." GraphPad Prism 9.0.2 (GraphPad, San Diego, CA) was used for the statistical analysis.

2.8.1 Determination of Sample Size

The mRS score is the most robust and prevalent measure of functional outcome used to determine treatment effects in stroke trials. In the Phase II study, we demonstrated that sovateltide treatment of cerebral ischaemic stroke patients improved functional outcomes (CTRI/2017/11/010654). We used these Phase II findings to determine the sample size for the present Phase III trial. The mRS data on Day 90 showed an OR of 5.250 (95% CI was 1.000 to 27.63 (p = 0.0519). These results were used to determine the sample size for this Phase III study. The study power was set to 80% (beta, 0.2), the enrolment ratio 1:1, and the significance level (alpha) used was 0.05. The power analysis indicated that a sample size of a minimum of 108 patients (54 in each study group) was sufficient to achieve a power of 80% with a significance level of 0.05 alpha. The sample size calculated from the Phase II study outcomes (i.e., mRS score) was 110 patients (i.e., 55 patients in each group), which was consistent with the previous Phase III clinical studies in patients of ischaemic stroke with similar endpoints: CTRI/2009/091/000251 (50 patients); CTRI/2011/07/001922 (100 patients) and CTRI/2015/02/005556 (75 patients).

We completed the enrolment of 110 patients in this Phase III study as per approved protocol No.: PMZ-1620/CT-3.1/2019, Version 1.0; however, COVID-19 affected clinical trial progress due to follow-up difficulties. We submitted the amended protocol to the Central Drugs Standard Control Organization (CDSCO) office and received approval to increase the sample size for this study. This amendment request was for an increase in the enrolment from 110 (as per previously approved protocol) to 158 patients (Protocol No.: PMZ-1620/CT-3.1/2019, Version 2.0). We increased the study power from 80% to 90%. The power of the study was at 90%, with a beta of 0.1 and an alpha of 0.05, and resulted in 132 patients. Each group required 66 patients to be enrolled. Considering the discontinuation rate of about 20%, 158 patients (79 in each study group) were enrolled in the study.