Trial Design and Participants
COVID-19 patients hospitalized in an ICU department of Shiraz University of Medical Sciences, Shiraz, Iran, were enrolled in a randomized, double-blind, placebo-controlled phase III clinical trial conducted during March 21- June 19,2021. The trial was registered on the Iranian registry of clinical trials (https://www.irct.ir) (Trial registration: IRCT20200509047373N2. Registered 13 April 2021).The trial site was at Ali Asghar hospital, as one of the ICU centers specialized for caring COVID-19 patients in Shiraz city, to enroll the eligible subjects. ICU-admitted patients were included in the trial with several eligibility criteria including having confirmed diagnosis of COVID-19, high pulmonary involvement in imaging, requirement to respiratory support but not mechanical ventilation (however, they may require mechanical ventilation during trial), aged 15 years or older, filled informed consent, and not participated in other clinical trials. Pregnant women and patients younger than 15 years were excluded from the study. COVID-19 definite diagnosis was made by the infectious diseases specialist according to the national COVID-19 committee’s guidelines for diagnosis and management of COVID-19 patients via SARS-CoV-2 reverse transcriptase polymerase chain reaction (RT-PCR) positive results or through characteristic radiographic presentations in chest computed tomography (CT) or radiographic imaging. Written informed consent was obtained from all patients or their legal representatives. The research was approved by the Ethics Committee of the Shiraz University of Medical Sciences (registration No. IR.SUMS.REC.1399.1367). Eligible patients were randomly assigned in a 1:1 ratio to either medication or placebo group.
No prior data was available on the efficacy of the antiviral preparation in the management of COVID-19, so we performed a pilot study with 30 participants (15 per group) with the same protocol on similar patients and recorded the death rate as the preliminary data on the clinical efficacy of the preparation. According to the results, three (20%) of the participants in the antiviral preparation group and seven (46.7%) in the placebo group had an event (death through day 14). By considering 90% power to detect a between-group difference and the alpha level of 5%, we estimated a population size of 120 patients (60 per group).
Randomization and Interventions
Randomization was conducted by the statistical analyst using the website Randomization.com (http://www.randomization.com) and the permuted block randomization method with a block size of 4. Patients were enrolled by a health worker rather than the research team and the statistical analyst performed the patient allocation. Allocation was masked to all individuals including patients, physicians and statistical analysts. Both groups received COVID-19 local standard treatments including remdesivir and glucocorticoids according to the guidelines. Those patients with the underlying conditions also received their medications according to their past prescriptions. Patients assigned to the medication group received the antiviral preparation. Each patient was administered 1 ml (containing 433 mg of active materials) antiviral preparation in drop form using dropper every 3 hours for up to two consecutive weeks. Moreover, patients who underwent mechanical ventilation received the medicine up to 14 days or till discharge. Subjects in the placebo group were given a matching placebo containing normal saline in the same packaging with similar appearance, volume, and dosing as the active compound to maintain blinding. Standard care was provided for all patients in both groups.
Antiviral preparation Composition and dosing
The novel antiviral preparation tested in this study has already been identified with antiviral properties in cell culture experiments (data not published), and comprised several active herbal ingredients. Plant materials included Zataria multiflora Boiss (5mg), Glycyrrhiza glabra (10mg), Cinnamomum Vermont (2mg), Allium sativuml (2mg), and Syzygium romaticum (1mg). Plant materials were purchased from a local botanical supplier in Iran, and were then approved by a plant classification specialist in the Department of Botany, Shiraz University of Medical Sciences, Shiraz, Iran. All plant materials were cleaned, dried, mechanically powdered, and extracted with 100% deionized water. The water was then evaporated using hot plate at 270°C to prepare the extract, followed by filteration. Plant material powders were mixed separately in 200 ml of distilled water. These suspensions were filtered by high-altitude centrifuge and then mixed using a magnetic stirrer. The final extract was placed in the refrigerator until it was ready for use. The extract was used in drops at a final concentration of 433±5 mg/ml to be used sublingually. The sterility testing was performed to check the bacterial contamination in each extract. For sterility testing, nutrient agar plates were used and 0.1 mL of each extract was poured on it and incubated at 37°C. The bacterial growth was observed after 24 hrs. Reverse phase chromatographic analyses were carried out under gradient conditions using C18 column (4.6 mm × 250 mm) packed with 5 μm diameter particles. The extract was dissolved in HPLC grade solvent. Then, the sample was sonicated using ultrasonicator for 10 min. The extract was filtered and injected to the HPLC column using a mobile phase comprising acetonitrile: 0.1% phosphoric acid micture in a 30:70 ratio (v/v). The preparation was standardized based on 5.82 mg glycyrrhizic acid, and 2.5 µg cinnamaldehyde per each gram of the final preparation. The above procedures were undertaken in the Shimi Teb Salamat Co., Shiraz, Iran.
Throughout the trial since days 0 to 14, patients were assessed for their clinical status. Any adverse effect or exacerbation in outcomes was recorded. Demographic characteristics, age, sex, BMI, and underlying diseases were collected at baseline. Several clinical and paraclinical parameters were evaluated in both groups on day 0, and for those survived in days 7 and 14. Vital signs including body temperature, blood pressure, respiratory rate, and blood oxygen saturation (SpO2), laboratory parameters such as hemoglobin (Hb) values, white blood cells (WBCs) count, and C-reactive protein (CRP) levels were measured. SARS-CoV RT-PCR was conducted at the first time point via standard mid-nasal sterile swab samples. Pulmonary involvement was investigated via chest CT-scans. Co-existing conditions were also compared between medication and placebo groups. No significant change was applied in the final protocol compared to the initial planned protocol.
The main primary outcome was the COVID-19-associated deaths, which was assessed through day 14 after enrollment. Other outcomes were clinical status, and changes in vital signs and laboratory parameters. Any possible adverse effect causing discontinuation of the study and impacting progression of the disease, as well as clinical or laboratory parameters were all recorded. No change was applied in the outcomes after trial was commenced.
The retrieved data was exported to SPSS software, version 18.0 (SPSS Statistics). To compare the qualitative and quantitative variables, Chi-square, independent samples t-test, paired samples t-test, and analysis of covariance (ANCOVA) were used and also, for non-normal variables the non-parametric Mann-Whitney U test and Wilcoxon signed-rank test were employed. Binary logistic regression was also used to estimate the odds ratio (OR) of death. Survival analysis was conducted using the Kaplan-Meier curve and Cox regression.