Plant formulation ATRICOV 452 in improving the level of COVID-19 specific inflammatory markers in patients.

Importance: Due to the huge demand for healthcare facilities, there is a need for safe therapeutic intervention which can reduce the need for extensive healthcare support. Objective: In phase 1, to determine the safety of plant formulation in healthy volunteers and in phase 2, to validate its potential to improve the level of COVID-19 specific inflammatory markers. Design: Setting, and Participants: The phase 1 single fasting dose study was conducted on 24 healthy adult human volunteers to evaluate the safety and tolerability of plant formulation. The usage of plant formulation in humans was found safe and encouraged conducting Phase 2 clinical trial to evaluate the efficacy in 100 COVID-19 patients along with the standard of care. Interventions: Phase 1: plant formulation capsule of 500 mg single dose; Phase 2: plant formulation 1 gm thrice a day for 14 days.
Results: During the Phase 1 trial no adverse event was observed and all organ systems were normal in function. During the Phase 2 trial, 100 patients underwent randomization, 50 were assigned to receive plant formulation, and 50 to receive placebo. Three patients in the placebo and two patients in the plant formulation group had dropped out of the study. Hence, the primary analysis population included 95 patients (48 allocated to plant formulation and 47 to placebo). The COVID-19-specific inflammatory markers improved faster and became normal in the plant formulation treatment group. No one needed hospital care or oxygen. Conclusions: and Relevance: The investigational product, plant formulation (ATRICOV 452) has been found to be safe in phases 1 and 2. Further, in the phase 2 trial, ATRICOV 452 was effective at the dose of 1 mg, three times a day dose frequency to improve the level of COVID-19 specific inflammatory markers. Trial registration: Phase 1: CTRI Registration number: CTRI 2020/09/027660 Phase 2: CTRI Registration number: CTRI/2021/01/030795.

Introduction

During this growing COVID-19 pandemic situation, as of September 30th, 2020, the World Health Organization (WHO) has reported that 33,502,430 and 1,004,421 death cases have been confirmed worldwide, and it has spread to 235 countries and the demand for supportive care facilities is also increasing [1]. Currently, there is no effective cure for SARS-CoV-2 infection and the most common treatment for patients with COVID-19 is supportive care [2]. Meanwhile, the existing supportive care facility for COVID-19 treatment is insufficient to anticipate the growing demand [3]. Hence, efforts are being made globally towards identifying the need for supportive care on the basis of severity, spectrum, and impact of the disease and extending the facility to the needy.

Multiple repurposed anti-viral drugs, including Remdesivir and Lopinavir plus Ritonavir, have been used as adjunct drugs in COVID-19 treatment and their intravenous administration requires the clinician's assistance [4,5,6]. As SARS-CoV-2 is highly contagious, that can be transmitted from person to person in a hospital with high traffic. Hence, reducing hospital visits and hospitalisation by using oral anti-SARS-CoV-2 drugs is also an option to reduce traffic in hospitals [7]). In that regard, effective strategies for prophylaxis and holistic management are of paramount importance in curtailing the progress of the disease and reducing the burden on hospitals.

Contemporary reports on clinical experiences/research have also suggested that inflammatory responses are also playing a critical role in the COVID-19 progression [8,9]. Inflammatory responses induced by rapid SARS-CoV-2 replication and cellular destruction can recruit macrophages and monocytes to induce the release of cytokines and chemokines [10]. These cytokines and chemokines then attract immune cells and activate immune responses, leading to cytokine storms and aggravations [11,12]. The clinical observations have reported some inflammatory markers, which certainly help in tracing and measuring the disease severity and fatality [13]. Inflammatory markers such as procalcitonin (PCT), serum ferritin, erythrocyte sedimentation rate (ESR), C-reactive protein (CRP) and interleukin-6 (IL-6) have been reported to be significantly associated with the high risks of the development of severe COVID-19 [14].

The clinical trials of curcumin and ashwagandha extract against SARS-CoV-2 have suggested their beneficial use in boosting immunity against SARS-CoV-2 and demonstrated the potential act of phytochemicals in COVID-19 management. The phytochemicals in Garlic extract, Green tea extract, Sulforaphane and Ginseng applied in the clinical trial against virus-causing respiratory diseases also indicate a possibility for their application in managing SARS-CoV-2 [15].

The anti-SARS-CoV-2 activity of Zanthoxylum piperitum (E1), Withania somnifera (E2), Calophyllum inophyllum (E3), Andrographis paniculata (E4), and Centella asiatica (E5) ethanol extracts was evaluated in vitro and the E4, E5 and E2 were found promising [16]. Hence, the plant formulation was prepared by mixing the E4, E5 and E2 extracts in equal proportion and labelled as ATRICOV 452. The plant formulation was evaluated for acute and sub-chronic toxicity and found safe at 2000 mg/kg body weight (Latha et al., 2021). Motivated by these preclinical studies, in this study, phase 1 and phase 2 clinical trials were conducted to evaluate the safety and potential of ATRICOV 452 to improve the level of COVID-19 specific inflammatory markers. The ATRICOV 452 was orally administered to 24 healthy volunteers and 100 COVID-19 patients in phase 1 and phase 2 respectively.

Materials and methods

Phase 1 clinical trial

In spite of known safety, the CTRI approved Phase-1 study entitled “A Prospective, Interventional, Open-label, Phase 1, single-centre, single fasting dose study to evaluate the safety and tolerability of ATRICOV 452 capsule of 500 mg in healthy adult human subjects” was conducted in the CDSO-DCGI approved CRO ICBio Clinical Research Pvt. Ltd., Bangalore, located at Bangalore. The study was interventional, single fasting dose, single-arm with the objective of assigning the safety of plant formulation (ATRICOV 452) in 24 healthy volunteers. The study protocol was approved by an in-house ethical committee constituted by CRO and conducted from Sep 17, 2020 to Sep. 24, 2020.

Phase 2 clinical trial

The single-blind, placebo-controlled, randomized clinical trial was designed to evaluate the potential of plant formulation (ATRICOV 452) in improving the level of COVID-19 specific inflammatory markers in adult COVID-19 patients under the standard of care also. The study was conducted in compliance with the Declaration of Helsinki,13 the Good Clinical Practice ICH guidelines, and local regulatory requirements.

Study design

This trial was conducted for 14 Days in two different trial sites namely, Vedant Hospital, Thane and Life point Multispeciality Hospital, Pune. The IEC approval for the study was obtained from Vedant Hospital Institutional Ethics Committee (VHIEC) and Lifepoint Research Ethics committee on January 13, 2021, and March 13, 2021. The enrolment process was started from March 19, 2021, to June 16, 2021, without regard to sex, race, ethnicity, or religion. Patients were randomized based on a stratified randomization method where the randomization was based on mild or moderate symptoms and the presence of co-morbidity like diabetes & hypertension over both active and placebo arms. Diagnosis of SARS-CoV-2 infection was done using RT-PCR and COVID-19 (Blood test: Total leucocyte count, NLR, CRP, Ferritin, D-Dimer, LDH, IL-6, PCT and ESR, Chest X-Ray and CT-Scan) specific investigations were carried out to assess the severity of the disease. Medical history, clinical examination, and vital signs were recorded carefully. The patients were classified into mild, moderate and severe according to the following criteria: Patients with uncomplicated upper respiratory tract infection possessing SpO2 ≥94% in room air, RR ≤ 24/m and no evidence of hypoxemia or breathlessness were considered as mild. The patients with pneumonia with no signs of severe disease, SpO2 within the range of 94%–90% in room air RR in the range of 24–30/m were considered as moderate and the patients with severe pneumonia with SpO2 < 90% in room air, RR > 30/m were considered severe and not recruited for the study. The subjects (100 patients) recruited for the study were grouped into Group A and B using a randomization table. Group A was given standard of care along with a placebo and Group B was given standard of care along with plant formulation (ATRICOV 452). Bitterness in the mouth, abdominal pain and nausea were identified as expected adverse drug reactions associated with plant formulation (ATRICOV 452). Patients with co-morbidity were treated with concomitant treatment. Periodic investigations were made as listed in the schedule of events (Table 1). The patients were discharged as per the protocol and monitored for signs and symptoms over the telephone.

Table 1

Schedule of events detailing the investigations performed during different treatment visits.

	Screening	Treatment visits								FVa	UVa
Visit	1	2	3	4	5	6	7	8	9	10
Screening Day	0	1	4	5	6	7	8	11	15	23 ± 2
Obtaining Informed Consent	X
Demography	X
Medical/Surgical History	X
Chest X-ray/CT scan	X								X
Pregnancy test (if adult female)	X
Covid-19 specific Lab investigation	X					X		X	X
Covid-19 related laboratory test (IgG)	X
Routine Lab investigation	X								X
Vital Signs (BP, Pulse rate, Spo2/RR/Temp)	X	X	X	X	X	X	X	X	X	X	X
Physical Examination	X	X	X	X	X	X	X	X	X	X	X
Covid-19 related common sign and symptoms	X	X	X	X	X	X	X	X	X	X	X
Investigator assessments	X	X	X	X	X	X	X	X	X	X	X
Inclusion/Exclusion Criteria	X
IP Dispensing		X	X	X	X	X	X	X
Issue Dairy card		X	X	X	X	X	X	X
Dosing		X	X	X	X	X	X	X	X
Checking for concomitant medications (if any)		X	X	X	X	X	X	X	X	X	X
Compliance check		X	X	X	X	X	X	X	X
AE/SAE assessment monitoring		X	X	X	X	X	X	X	X	X	X

^ Follow Up visit, Unscheduled Visit, * Chest X-Ray/CT if suggested by the Physician # RT PCR positive patients selected for study.

Participants

The study included adults with SARS-CoV-2 infection confirmed by RT-PCR. The patients were excluded if the physician finds vulnerability based on their history, nutritional status, physical appearance, or any other reason and did not comply with any of the inclusion and exclusion criteria (Fig. 1).

Fig. 1

Flowchart illustrating the trial protocol, inclusion and exclusion criteria.

Randomization and stratification

Patients were randomized 1:1 to ATRICOV 452 or identical placebo capsules. Randomization schedules were generated that were stratified by signs and symptoms (mild and moderate) and co-morbidity (present and absent). Treatments were randomly allocated using a randomization table. All outcome assessors, investigators, and research staff who were involved in the trial were blinded to participant treatment assignment. The trial was carried out as per the flowchart (Fig. 1).

Intervention

Group A was given standard of care along with a placebo containing inert starch (Identical looking 500 mg) 2 capsules thrice a day for 14 days. Group B was given standard of care along with plant formulation (ATRICOV 452) (500 mg) 2 capsules thrice a day for 14 days. The standard of care for mild patients was Tab-Favipiravir 1800 mg 1-0-1 on Day I and 800 mg 1-0-1 for 6 days (total 7 days). The standard of care for moderate patients was Oseltamavir 75 mg Cap 1-0-1 for 5 days. This dose range was determined based on the preclinical and phase 1 findings.

Outcome measures

In the phase 1 trial, the parameters like SGOT, ALPI, vital and hematopoietic parameters were measured to ensure drug tolerance and safety. Pathologically, the level of inflammatory markers such as CRP, leucocyte count, NLR, D-dimer, PCT, LDH, and IL-6 is expected to be increased in COVID-19 patients with severe symptoms than in patients with mild and moderate symptoms [4,17,18,19,20,21]. Hence in the phase 2 trial, the levels of these inflammatory markers were measured as the indicators of recovery due to ATRICOV 452 treatment.

Results and discussion

Clinical trial phase 1

The close observation was performed for safety parameters on days 5, 6 and 7 after administration of ATRICOV 452 through IP mode. As per the clinical assessment, there were no allergic reactions, neurological and musculoskeletal abnormalities observed during the study period. Subjects have not experienced any gastrointestinal disorders during the study period.

The results of the T-Test revealed that there was no statistically significant difference between means (p = 0.3403 and p = 0.0103) for SGOT and ALPI. The results of the Wilcoxon signed-rank-test revealed that there was no statistically significant difference between Means (p = 0.2373 and p = 0.3594) for SGPT and Bilirubin.

The Means of Vital Parameters (Pulse Rate, Blood Pressure, and Temperature) for different time points were not normally distributed and did not follow the assumptions for repeated measures One-way ANOVA, non-parametric mixed-methods ANOVA was used to compare the means overall different time point.

The results of all vital parameters by using non-parametric oneway repeated measures ANOVA shows that all parameters were not statistically significant main effect of means at different time points for pulse rate (p = Page of 0.994), Temperature (p = 0.9952), Systolic Blood Pressure (p = 0.9948) and Diastolic Blood Pressure (p = 0.9943).

Since the hematopoietic parameters of haemoglobin, WBC, neutrophils, and platelet count were normally distributed, the T-Test was used to compare the means for Pre-Dose and Post-Dose. Whereas the rest of all other parameters i.e., RBC, Lymphocytes, Eosinophil, Monocytes, PCV were normally distributed and, the Wilcoxon signed-rank-test was used to compare the means for Pre-Dose and Post-Dose.

The results of using the T-Test revealed that there was no statistically significant difference between Means for Neutrophils (p = 0.4924), for WBC (p = 0.0543), for Platelet Count (p = 0.3584). Whereas the results of using the T-Test revealed that there was a statistically significant difference between Means for Hemoglobin (p = 0.0034). The results of using the Wilcoxon signed-rank-test revealed that there was no statistically significant difference between Means for Lymphocytes (p = 0.9197), eosinophils (p = 0.2852), for Monocytes (p = 0.1514). Whereas the results of using the Wilcoxon signed-rank- test revealed that there was a statistically significant difference between means for RBC (p = 0.0008) and for PCV (p = 0.0005).

There was no adverse event observed, all organ system was normal in function so no need for any additional intensive investigation. IP Tolerances were considered in the study for the tolerability of investigational products. IP tolerance evaluation was performed on days 5, 6 and after 7 days of IP administration, After evaluation on days 5, 6 and after 7 days IP administration there was no side effect on any organ system.

Clinical trial phase 2

Patients

Patients were randomized, 50 were assigned to receive ATRICOV 452 and 50 to receive a placebo. Three patients in the placebo and two patients in ATRICOV 452 group had dropped out of the study due to unknown reasons. Hence, the primary analysis population included 95 patients (48 allocated to ATRICOV 452 and 47 to placebo).

Patients in both groups were balanced in demographic and disease characteristics at baseline. The mean age of patients in the primary analysis population was 39 years (interquartile range [IQR], 22–59), 46 (52%) were women, and 44(48%) were men, they did not have any known comorbidities at baseline. The most common were, fever (94 patients, 98.94%) and cough (95 patients, 100%), followed by respiratory rate (baseline mean 13.16) and SPO2 (baseline mean 93.244).

Primary outcome

The time to resolution of symptoms in patients assigned to ATRICOV 452 vs placebo was not significantly different (median, 6 days vs 7 days). In the ATRICOV 452 and placebo groups, symptoms resolved in 100% of patients by day 15. The type of placebo that patients received did not affect the results.

Swab test results for all (100) subjects were found positive at the screening visit (visit 1) and after EOT (visit 9) test results were performed for 95, and all 95 subjects were negative.

Subject-wise data was observed from visit 1 to EOT and mean values of all COVID-19-specific inflammatory markers (NLR, CRP, Ferritin, D-dimer, IL-6, and PCT) levels were significantly improved compared to the placebo group.

A retrospective study found several differences in leucocyte count between severe and non-severe COVID-19 patients [18]. In that reported study, both groups experienced an increase in leucocytes with the severe group having a significantly greater increase (5.6 vs 4.9 × 109/L; P < 0.001). Neutrophils were predominantly driving this increase as the severe set (4.3 vs 3.2 × 109/L; P < 0.001). Interestingly, the levels of lymphocytes, monocytes, basophils and eosinophils were less, resulting in a greater neutrophil-to-lymphocyte ratio (NLR; 5.5 vs 3.2; P < 0.001). NLR is an infamous biomarker, high in widespread inflammatory conditions and can be used to reflect disease severity. However, a larger study is needed to clarify NLR's effectiveness as a biomarker. Another conducted in China concludes similar findings of high NC and low LC count in severely affected patients, suggesting NLR could be a potential biomarker for early detection of severe COVID-19 [6].

In the current study, the mean percentage improvement in NLR levels on visits 6, 8, and 9 with reference to visit 1 were −28%, −24%, and −19% respectively in ATRICOV 452 treated group. Whereas in the placebo group the mean percentage improvement in NLR levels on visits 6, 8, and 9 with reference to visit 1 were 7%, 9%, and −5% respectively. This has illustrated the level of NLR was improved from a visit to visit while it was worsened in the placebo-treated group.

Pathologically, computed tomography scans (CT-Scans) can identify lung lesions relating to COVID-19. Nevertheless, a study conducted in China revealed that the CT scores could not differentiate mild cases from severe. However, compared to erythrocyte sedimentation rate (ESR), CRP levels were significantly greater during early periods of severe cases and proved to be a more sensitive biomarker in reflecting disease development [19]. The mean percentage improvement in CRP levels on visits 6, 8, and 9 with reference to visit 1 were −76%, −75%, and −55% respectively in ATRICOV 452 treated group. Whereas in the placebo-treated group the mean percentage improvement in CRP levels on visits 6, 8, and 9 with reference to visit 1 were −94%, −66%, and −36% respectively. This has illustrated the proportionate improvement in CRP levels from a visit to visit in both ATRICOV 452 and placebo-treated groups. However, the mean CRP level during visit 1 was less in ATRICOV 452 treated group, it has further improved and the CRP level was normal during visit 8. The mean percentage improvement in ferritin levels on visits 6, 8, and 9 with reference to visit 1 were −2%, −9%, and 0.5% respectively in ATRICOV 452 treated group. Whereas in the placebo group the mean percentage improvement in CRP levels on visits 6, 8, and 9 with reference to visit 1 were −198%, −175%, and −17% respectively. This has illustrated the proportionate improvement in ferritin levels from a visit to visit in both ATRICOV 452 and placebo-treated groups.

A retrospective cohort study composed of 191 patients found that D-dimer levels >1.0 μg/mL(p = 0.0033) were associated with increased mortality among COVID-19 patients. Furthermore, they found that levels of 2.0 μg/mL or more on admission were the optimum cut-off to predict in-hospital mortality for COVID-19 [21]. Studies have reported that nearly 90% of inpatients with pneumonia had increased coagulation activity marked by rising D-dimer levels [22]. The mean percentage improvement in D-Dimer levels on visits 6, 8, and 9 with reference to visit 1 were −22%, −11%, and 2% respectively in ATRICOV 452 treated group. Whereas in the placebo-treated group the mean percentage improvement in D-Dimer levels on visits 6, 8, and 9 with reference to visit 1 were −6%, −1%, and 4% respectively. This has illustrated the proportionate improvement in ferritin levels from a visit to visit in both ATRICOV 452 and placebo-treated groups.

Studies have revealed that levels of IL-6, the most common type of cytokine released by activated macrophages, rise sharply in severe manifestations of COVID-19 and a proportionate rise of IL-6 is correlated with disease severity [4,23]. In the ATRICOV 452 treated group, the mean percentage improvement in IL-6 levels on visits 6, 8, and 9 with reference to visit 1 were 27%, 30%, and 34% respectively. Whereas in the placebo-treated group the mean percentage improvement in IL-6 levels on visits 6, 8, and 9 with reference to visit 1 were 21%, 36%, and 40% respectively. This has illustrated the proportionate improvement in ferritin levels from a visit to visit in both ATRICOV 452 and placebo-treated groups.

A retrospective cohort study that included 140 COVID-19 patients found significantly increased IL-6, CRP, and PCT levels in the severe group than in the moderate group [17]. The mean percentage improvement in PCT levels on visits 6, 8, and 9 with reference to visit 1 were −34%, −24%, and 9% respectively in ATRICOV 452 treated group. Whereas in the placebo-treated group the mean percentage improvement in PCT levels on visits 6, 8, and 9 with reference to visit 1 were −27%, −40%, and 0.2% respectively. This has illustrated the proportionate improvement in ferritin levels from a visit to visit in both ATRICOV 452 and placebo-treated groups.

The mean scores of systemic safety Biochemical test (RBS, HbAlc) from visit 1 to visit 9 were normal and also at EOT.

During the treatment period, no patients were in the requirement for supplemental oxygen, non-invasive ventilation, mechanical ventilation and Intensive Care Unit (ICU).

Secondary outcomes

There was no requirement for escalation of care in both the treatment groups. The proportions of patients who developed a fever during the study period were not significantly different between the 2 treatment groups.

Considering the exploratory nature and analysis of study outcomes, the safety and potential of ATRICOV 452 in improving the level of COVID-19 specific inflammatory markers in adult COVID-19 patients were assessed. No serious adverse events occurred during the study and ATRICOV 452 was found well tolerated. Although this is an exploratory phase 2 study, the study has demonstrated the safety and efficiency of ATRICOV 452 (along with the standard of care) in improving the level of COVID-19-specific inflammatory markers.

Conclusion

The investigational product, ATRICOV 452 capsule of 500 mg, has been found to be safe and well-tolerated for single-dose study, without any safety concerns. The CTRI registered Phase 2 trial performed with two arms (Placebo & ATRICOV 452) has significantly reduced the level of COVID-19 specific inflammatory markers and no adverse events were reported. This proved the safety and efficacy of plant formulation ATRICOV 452 along with the standard of care in improving the level of COVID-19 specific inflammatory markers and consequently in reducing COVID-19-associated morbidity.

Key points

Question: Can an active intervention with plant formulation in COVID patients is safe and improve the level of COVID-19 specific inflammatory markers?

Findings: Current study has demonstrated the utility of plant formulation (ATRICOV 452) in COVID-19 patients. The clinical trial registry of India (CTRI) registered Phase 1 trial has shown the safe usage of plant formulation in healthy human volunteers. Further, a single-blind, randomized, placebo-controlled, exploratory phase 2 clinical trial that included 100 patients, has shown that the plant formulation (ATRICOV 452) can improve the level of COVID-19 specific inflammatory markers compared with the placebo group. No adverse drug reactions or events are recorded.

Meaning: Results and outcomes demonstrate the safe usage of plant formulation (ATRICOV 452) in humans and it can improve the level of COVID-19 specific inflammatory markers in COVID-19 patients.

Declaration of interests

The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Latha Damle is the founder of Atrimed Biotech LLP and holds equity in Atrimed Pharmaceuticals. Hrishikesh Damle hold equity shares in Atrimed Pharmaceuticals.

Grant information: The study was sponsored by Atrimed Pharmaceuticals Pvt. Ltd and no external funding was received for the study.