The Efficacy and Safety of Imusil® Tablets in the Treatment of Adult Patients With Mild COVID-19: A Prospective, Randomized, Multicenter, Open-Label Study

Introduction Coronavirus disease 2019 (COVID-19) is a serious concern of the new era. Along with antiviral synthetic medications, there is a need to discover efficacious herbal antiviral medicines with minimum side effects in patients against COVID-19. This study aimed to assess the efficacy and safety of Imusil® among patients with mild COVID-19. Methods A prospective, randomized, multicenter, open-label, interventional study was conducted in patients with mild COVID-19 infection. Patients received either Imusil one tablet four times a day (seven days) along with the standard of care (SoC) or only SoC. The study endpoints were reverse transcription-polymerase chain reaction (RT-PCR) negativity, changes in cycle threshold (CT), clinical improvement, change in blood inflammatory indexes, and safety assessment. Results A total of 100 patients were enrolled, and 98 received at least one dose of treatment. The median age of patients was 36.0 years, and 58 were males. By day 4, 85.4% of patients in the Imusil+SoC group tested negative for RT-PCR compared to 64% of patients exhibiting the same outcome in the SoC group (P=0.0156). After eight days, clinical improvement was observed in all patients from the Imusil+SoC group, while in the SoC group, clinical improvement was observed in 94.0% of patients (P=0.4947). During follow-up visits, the average C-reactive protein (CRP) levels decreased from baseline in both treatment groups. The decrease in the levels of CRP (-7.3 mg/dL versus -5.5 mg/dL), D-dimer (-231.0 ng/mL versus -151.6 ng/mL), and interleukin 6 (IL-6) (-2.3 pg/mL versus -2.0 pg/mL) at eight days was comparatively higher in the Imusil+SoC group versus the SoC group. There were no serious treatment-emergent adverse events in the drug arm. Conclusion Imusil provides effective antiviral activity and safety in mild COVID-19 patients. Imusil ensures faster RT-PCR negativity and clinical improvement and ensures effective reduction of inflammatory markers such as CRP, D-dimer and interleukin 6.


Introduction
The global pandemic coronavirus disease 2019 (COVID-19) is a serious concern of the new era that comprises a broad spectrum of severity, ranging from asymptomatic to severe. Severe cases of COVID-19 can be complicated by acute respiratory distress syndrome and lung failure leading to death [1]. Cytokine storm syndrome leading to hyper-inflammation is associated with poor prognosis in COVID- 19. Evidence suggests that elevated levels of pro-inflammatory cytokine interleukin 6 (IL-6) were associated with the disease severity of COVID-19 [2,3]. The infection induces pathological responses, including increased inflammation, oxidative stress, and apoptosis [4].
Many interventions and therapeutics have been proposed to combat the viral infection-induced inflammation and oxidative stress contributing to the etiology and pathogenesis of COVID-19. However, 1 2 3 4 5 6 6 these methods have not significantly improved treatment outcomes [4]. Antiviral synthetic medications such as favipiravir and remdesivir are initially effective for the treatment of COVID-19 infection [5,6]. Nevertheless, they are linked with several adverse effects that are harmful to patients with heart disease, diabetes, and kidney disease [7][8][9]. Therefore, the need for inherently safe biologically active therapeutics of plant origin is required for reducing and managing the risk of viral infections including COVID- 19. Traditional medicines and their combination had shown to result in the recovery of patients with COVID-19 [10]. The Ministry of Ayurveda, Yoga and Naturopathy, Unani, Siddha and Homeopathy (AYUSH), India, has identified neem, kutki (Picrorhiza kurroa), guduchi (Tinospora cordifolia), amla (Emblica officinalis), tulsi (Ocimum sanctum), ashwagandha (Withania somnifera), ginger (Zingiber officinale), turmeric (Curcuma longa), licorice (Glycyrrhiza glabra), aloe, shatavari (Asparagus racemosus), almond, cinnamon, black pepper, drumstick, and broccoli as important first-level ayurvedic herbs, which play a crucial role in the defense against this novel viral infection [11].
Imusil® (kutki extract 200 mg+guduchi extract 60 mg+amla extract 60 mg), a novel polyherbal formulation, is being explored in a recently published article for its anti-inflammatory and anti-COVID-19 effect in alleviating the inflammatory responses and thereby improving survival outcomes [12]. Kutki, guduchi, and amla possess immunomodulatory and antioxidant activities. Kutki suppresses the production of reactive oxygen species by inhibiting Nicotinamide adenine dinucleotide phosphate oxidase and pro-inflammatory cytokines. A molecular docking study of guduchi revealed a good activity against the main protease of the virus. Currently, amla is essentially utilized to boost immunity against COVID-19 infection [11].
The previous article elucidated Imusil as a potent anti-inflammatory and antioxidant agent [12]; however, the efficacy and safety of such a combinatorial drug in treating patients with COVID-19 are yet to be explained. Therefore, the present study aimed to evaluate the efficacy and safety of Imusil in mild COVID-19 patients.

Inclusion criteria
Adult patients (≥18 years old) of either sex, with confirmed mild COVID-19 infection (World Health Organization {WHO} ordinal score: 2-3), and patients with one or more of the symptoms of fever of ≥100.4°F, cough, sore throat, headache, nasal congestion, malaise, diarrhea, loss of smell, or loss of taste were included in the present study.

Exclusion criteria
Patients with a history of severe infections, including pneumonia and septicemia, or patients who failed to control systemic fungal, bacterial, or viral infection were excluded from the study. Patients with cardiac or pulmonary disease, renal and hepatic impairment (creatine of ≥2 mg/dL, liver enzymes and bilirubin 2.5 times the upper limits of normal (ULN), and alkaline phosphatase 1.5 times ULN), diabetes, obesity, metabolic syndrome, human immunodeficiency virus (HIV) or hepatitis B or C, or neurological or psychiatric disorders were excluded from the study. Patients who received herbal immunosuppressive therapy or other investigational drugs within the previous 30 days of screening or who were hypersensitive to any herbal medication containing kutki, guduchi, or amla extracts were also excluded from the study.

Randomization
The eligible patients were randomized (1:1) in two groups. Group

Treatment regimen
Imusil was administered at a dose of one tablet four times daily for seven days. The study involved a screening period (day 1) and a treatment period, which included seven days of dosing. Patients were monitored from the start of treatment. The procedures and assessments were performed during the followup period (day 4) and at the end of the study (day 8). Recording of vital signs including saturation of peripheral oxygen (SpO 2 ), physical examination, real-time reverse transcription-polymerase chain reaction (RT-PCR) report, detection of inflammatory markers (C-reactive protein {CRP}, D-dimer, and IL-6), clinical assessment based on a WHO ordinal scale (Table 1), and recording of concomitant medications and adverse events (AEs)/serious AEs (SAEs), if any, were done during the study period. Patients were followed for eight days after randomization.

Endpoints
The primary endpoints were evaluated on day 4 and day 8 and involved efficacy outcomes such as the rate of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RT-PCR negativity and changes in cycle threshold (CT) value in nasopharyngeal and/or oropharyngeal swab, the proportion of patients with a twopoint decrease in a WHO ordinal scale, and changes in blood inflammatory indexes for C-reactive protein, IL-6, and D-dimer. The clinical improvement was assessed using a WHO ordinal scale. The secondary endpoints, such as AEs and SAEs, compared to placebo as an add-on to the SoC, were also evaluated during the study.

Sample size
This study planned to enroll 100 patients with mild COVID-19. Each treatment group was intended to have 50 randomized patients in order to achieve at least 90 evaluable patients (excluding 10% dropout), with about 85% power at the 5% one-sided level of significance for the efficacy endpoint comparison (the proportion of patients getting RT-PCR negative by the end of treatment test plus SoC over SoC).

Definition
The WHO ordinal scale is the WHO ordinal severity scale that has been used to predict mortality and guide trials in COVID-19, which includes the following nine points: 0, no clinical or virological evidence of infection; 1, ambulatory and no activity limitation; 2, ambulatory and activity limitation; 3, hospitalized and no oxygen therapy; 4, hospitalized and oxygen mask or nasal prongs; 5, hospitalized and non-invasive mechanical ventilation (NIMV) or high-flow nasal cannula (HFNC); 6, hospitalized, intubation, and invasive mechanical ventilation (IMV); 7, hospitalized and IMV+additional support such as pressors or extracardiac membranous oxygenation (ECMO); and 8, death [13].

Statistical analysis
Data were analyzed using Statistical Analysis System (SAS) version 9.4 (SAS Institute Inc., Cary, NC). Descriptive statistics were used to describe categorical variables (frequency and percentages) and continuous variables (mean and standard deviation {SD} or median and range {depending on the normality of data}). A comparison of quality between the groups was made using one-way analysis of variance (ANOVA) and Kruskal-Wallis test for parametric and non-parametric variables, respectively. A comparison of quantity between the groups was made using the chi-square test. A paired non-parametric Wilcoxon signedrank test was used to compare variables pre-and post-treatment. A P<0.05 was considered statistically significant.

Patient disposition
A total of 100 patients were enrolled, of which two withdrew consent. A total of 98 patients received at least one dose of the investigational product and were included in the safety population ( Figure 1).

FIGURE 1: Patient disposition
*Two patients who withdrew consent were randomized but did not receive treatment drug SoC, standard of care; WHO, World Health Organization

Baseline characteristics
The median age was 36.0 years, and 58 (59.2%) patients were males. The average body mass index (BMI) of the patient was 23.8 kg/m 2 . The mean height and weight of the patients were 161.5 cm and 61.8 kg, respectively ( Table 2).

Change in inflammatory markers from baseline to day 8
Over the subsequent follow-up visits, the C-reactive protein (CRP) levels, level of D-dimer, and IL-6 levels decreased from baseline in both treatment groups, with no statistical difference between the groups at four days and at eight days (Figures 2-4). The decrease in the levels of CRP at eight days was comparatively higher in the Imusil+SoC group versus the SoC group (-7.3 mg/dL versus -5.5 mg/dL; P=0.2457). The decrease in the levels of D-dimer at eight days was also comparatively higher in the Imusil+SoC group versus the SoC group (-231.0 ng/mL versus -151.6 ng/mL; P=0.2689). The IL-6 levels had comparatively higher reduction with the Imusil+SoC group over the SoC group (-2.3 pg/mL versus -2.0 pg/mL; P=0.7685) at eight days.

Treatment-emergent AEs
There were no serious treatment-emergent AEs in the drug arm. Two patients from the placebo (SoC) arm reported vomiting (n=1) and pruritus (n=1).

Vital signs, physical examination, and laboratory evaluations
Patients from both groups reported normal pulse rate, respiratory rate, SpO 2 , temperature, and blood pressure during vital examinations. There was no statistical difference in the change in laboratory parameters between the Imusil+SoC and SoC groups ( Table 6).

Discussion
Viral load in the respiratory tract and the duration of viral shedding are important determinants of COVID-19 transmission [14]. Viral load in the upper respiratory tract usually appears to peak around the time of symptom onset, and viral shedding begins approximately 2-3 days prior to the onset of symptoms [15]. Asymptomatic and pre-symptomatic COVID-19 patients can transmit COVID-19 infection [16][17][18]. Evidence indicates that viral load leading to systemic inflammation is one of the important predictors of morbidity and mortality in patients with COVID-19 [19]. Furthermore, viral load determines the duration of infectiousness; therefore, the early identification of infection and the timely use of treatment could benefit patients in achieving effective control [14].
Remdesivir, lopinavir/ritonavir, lopinavir/ritonavir with interferon beta-1α, and chloroquine or hydroxychloroquine are some of the chosen study drugs for the treatment of COVID-19 [20]. For patients with comorbidities, it is inevitable to take daily medication along with COVID-19 medications. Therefore, it is required to have safe pharmacotherapy for COVID-19 treatment along with antihypertensive, antiasthmatic, and antidiabetic drugs. However, the abovementioned antiviral agents seem to have limited beneficial evidence in patients with comorbidities [7][8][9]. Thus, there is indeed a great rush to find the ayurvedic formulation against COVID-19.
Imusil is a proprietary ayurvedic medicine where the individual herbal ingredients have a variety of medicinal properties, including anti-inflammatory, antiviral, and immunomodulatory properties. The present study evaluated the antiviral efficacy and safety of a novel formulation Imusil, which is composed of kutki, guduchi, and amla.
The principal observation of the study is that RT-PCR negativity was faster and higher in the patients receiving Imusil. Patients in the Imusil+SoC group reported an improvement and recovery by 8+2 days of enrollment. However, comparatively lesser improvement was noted in the placebo arm.
It is interesting to note that a study by Patankar  Schulte et al. revealed that tumor necrosis factor-alpha (TNF-α) and IL-6 play a major role in the amplification of inflammatory response in COVID-19 infection [23]. Over the subsequent follow-up visits, the average CRP, D-dimer, and IL-6 levels decreased effectively in the Imusil+SoC group as compared to the SoC group. The previous study has shown a significant reduction in TNF-α and IL-6 levels post Imusil treatment [12]. In the previous study, an Imusil-treated cell showed significant reduction in TNF-α and IL-6 levels. Therefore, the results mentioned above, along with the present study, support the potent antiinflammatory activity of Imusil.
In this study, no serious treatment-emergent AEs were reported in the drug arm. In a systematic review and meta-analysis of 32 randomized controlled studies (n=3177), patients treated with herbal medicines demonstrated better clinical improvement with no adverse effects during the treatment period. None of the patients had SAEs requiring prolonged hospitalization or death (n=665; hazard ratio: 0.93; 95% CI: 0.76-1.14) [26]. Similarly, no drug-related AEs or SAEs were observed in patients utilizing the polyherbal AYUSH 64 formulation. None of the participants required invasive or non-invasive oxygen ventilation or developed complications such as acute respiratory distress syndrome, sepsis, or arrhythmia during the study period [27]. Thus, the results mentioned above, along with the present study, suggest that herbal interventions were relatively safe for the management of COVID-19.

Limitations
The pre-and post-analysis of clinical signs and symptoms within the group was not performed, which could have been valuable in understanding the impact on symptoms. Clinical trials with larger sample sizes and more extended follow-up periods can be conducted in the future. Lastly, another study on severely affected COVID-19 patients would be worthwhile to explore the efficacy and safety of Imusil among severe COVID-19 patients.

Conclusions
Imusil, a polyherbal combination drug, provides effective antiviral activity in mild to moderate COVID-19 patients. Imusil ensures faster RT-PCR negativity, and it aids in the comparable reduction of markers such as CRP, D-dimer, and IL-6. It can be safely given along with SoC to enhance its therapeutic activity in patients with mild to moderate COVID-19 disease.

Additional Information
Disclosures