Randomized double-blind clinical study in patients with COVID-19 to evaluate the safety and efficacy of a phytomedicine (P2Et)

Abstract

Background: It has been proposed that polyphenols can be used in the development of new therapies against COVID-19, given their ability to interfere with the adsorption and entrance processes of the virus, thus disrupting viral replication. Seeds from Caesalpinia spinosa, have been traditionally used for the treatment of inflammatory pathologies and respiratory diseases. Our team has obtained an extract called P2Et, rich in polyphenols derived from gallic acid with significant antioxidant activity, and the ability to induce complete autophagy in tumor cells and reduce the systemic inflammatory response in animal models.

Methods: In this work, a phase II multicenter randomized double-blind clinical trial on COVID-19 patients was designed to evaluate the impact of the P2Et treatment on the clinical outcome and the immunological parameters related to the evolution of the disease. The Trial was registered with the number No. NCT04410510*. A complementary study in an animal model of lung fibrosis was carried out to evaluate in situ lung changes after P2Et in vivo administration. The ability of P2Et to inhibit the viral load of murine and human coronaviruses in cellular models was also evaluated.

Results: Patients treated with P2Et were discharged on average after 7.4 days of admission vs. 9.6 days in the placebo group. Although a decrease in proinflammatory cytokines such as G-CSF, IL-15, IL-12, IL-6, IP10, MCP-1, MCP-2 and IL-18 was observed in both groups, P2Et decreased to a greater extent G-CSF, IL-6 and IL-18 among others, which are related to lower recovery of patients in the long term. The frequency of T lymphocytes (LT) CD3+, LT double negative (CD3+CD4-CD8-), NK cells increased in the P2Et group where the population of eosinophils was also significantly reduced. In the murine bleomycin model, P2Et also reduced lung inflammation and fibrosis. P2Et was able to reduce the viral replication of murine and human coronaviruses in vitro, showing its dual antiviral and anti-inflammatory role, key in disease control.

Conclusions: Taken together these results suggest that P2Et could be consider as a good co-adjuvant in the treatment of COVID-19.

Clinical trail registration: https://ichgcp.net/clinical-trials-registry/NCT04410510, identifier: NCT04410510.

Keywords: COVID-19; complementary and alternative medicine; immunomodulation; phytomedicine; polymolecular drugs; polyphenols.

Conflict of interest statement

Authors SF and CU are inventors of a granted patent related to P2Et. Authors SF, CU, and RB-R are partners of the DreemBio company who was a licensee of related patents. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2022 Urueña, Ballesteros-Ramírez, Gomez-Cadena, Barreto, Prieto, Quijano, Aschner, Martínez, Zapata-Cardona, El-Ahanidi, Jandus, Florez-Alvarez, Rugeles, Zapata-Builes, Garcia and Fiorentino.

Figures

Figure 1

Screening, Randomization, and Outcomes. COVID-19 patients were included in the study. 1,042 patients were screened and 91 patients underwent randomization. 47 patients received placebo and 44 patients received P2Et extract. A blood sample was taken from each patient before and after treatment to assess safety parameters, clinical parameters, immune innate cell populations by flow cytometry and cytokines for a MAGPIX® System. This figure was created using BioRender (https://Biorender.com/).

Figure 2

Absolute counts of Innate lymphocytes cells in the peripheral blood of COVID-19 patients. (A) Absolute counts of total Innate lymphocytes cells (ILCs) in COVID-19 patients (moderate and severe) treated with placebo or P2Et. Healthy donors (HD) were included. (B) Absolute counts of Innate type 2 lymphocytes cells (ILC2s) in COVID-19 patients (moderate and severe) treated with placebo or P2Et. HD were included. (C) Absolute counts of Natural killer Bright cells (NKBr) in COVID-19 patients (moderate and severe) treated with placebo or P2Et. HD were included. (D) Absolute counts of Natural killer Dim cells (NKDim) in COVID-19 patients (moderate and severe) treated with placebo or P2Et. HD were included. Data are represented as the mean ± SEM. The p values between Placebo and P2Et group or within groups were calculated using a Mann-Whitney test. *p < 0.05, **p < 0.01. The p values between COVID-19 patients and HD were calculated using a Mann-Whitney test. ♦p < 0.05, ♦♦p < 0.01.

Figure 3

P2Et treatment modulate of inflammatory response in COVID-19 patients. Samples from placebo, P2Et and Healthy donors (HD) groups were analyzed by Milliplex to identify the quantity of cytokines present in the serum. (A) IL-5, (B) IL-6, (C) G.CSF, (D) IL-10, (E). IL-18, (F) IFN-γ, (G) MCP-2, and (H) MCP-1 cytokines. Each dot represent one human sample. The p values between placebo and P2Et group or within groups were calculated using a Wilconxon test. *p < 0.05, **p < 0.01, ***p < 0.001.

Figure 4

P2Et exhibit antiviral activity against SARS-CoV-2 with a concentration-depending effect. (A) Vero E6 viability after 48 h of P2Et treatment was evaluated by MTT assay (6.25-−200 μg/mL). Data were presented as Mean ± SEM. The viability percentages of the treated cell were calculated based on untreated control. Two independent experiments with three replicates each were performed (n = 6). (B) The SARS-CoV-2 titer (PFU/mL) was reduced in Vero E6 supernatants after pre-post treatment with P2Et (n = 2). CQ (100 μM) was used as a positive control of viral inhibition. Data are represented as the mean ± SEM. The p values were calculated using a Mann-Whitney test, **p < 0.01, ***p < 0.001. Representative plaques of each treatment condition are shown.

Figure 5

P2Et treatment reduces ILC2 the pro-fibrotic factors in the lung of bleomycine challenged mice. (A) Lungs from naïve animals were used as negative controls compared and to bleomycine challenged animals treated either with PBS or P2Et. Absolut counts from lung ex vivo samples are shown. (B) Frequency of PD1 and CD69 positive cells among the ILC2 subset is presented. (C) Part of the lung cell suspension was used for an overnight re-stimulation with IL-33 and IL-25 in vitro. Intracellular cytokine staining was performed, and the frequencies of IL-5 and IL-13 positive ILC2 are shown. (D) From the ex-vivo lung cell suspension eosinophils and neutrophils were identified, and absolute numbers are represented. (E) Samples from all groups were analyzed by LegendPlex to identify the quantity of cytokines present in the serum of each animal. (F) Lung tissue was analyzed by qPCR to check the mRNA expression of pro-fibrotic factors. All graphs are presented as box and whiskers graphs showing the min and max. The middle line is plotted at the median. Each dot represent one mouse. The p values were calculated using a Kruskall-Wallis test, *p < 0.05, **p ≤ 0.01, ***p < 0.001, ****p < 0.001.

Figure 6

Proposed model for the P2Et effect in COVID-19. COVID-19 patients (moderate or severe) were treated with P2Et or Placebo. P2Et treatment (upper left) decreases ex vivo viral replication as well as proinflammatory cytokines such as IL-5, IL-6, IL-10, IL-18, G-CSF, and increases IFN-γ in peripheral blood. A decrease in eosinophils and an increase in other cell populations such as LT CD3+CD4-CD8-, NKBr, NKDim, and ILC2s are observed. The upper right image shows the effect of P2Et treatment in a mouse model of bleomycin-induced pulmonary fibrosis. In the lung, it decreases CD45+ cells, total ILC, ILC-2, eosinophils, and neutrophils. In peripheral blood it increases the production of IL-22. In summary, P2Et treatment regulates the inflammation induced by SARS-CoV-2 infection, modulating factors related to long-term clinical manifestations. The lower left image shows the effect of Placebo treatment in patients with COVID-19. A slight decrease in IL-6 and IL-10 is observed in moderate patients but an increase in IL-10 in severe patients. For the other cytokines (IL-5, IL-18, G-CSF, and IFN-γ) no significant changes are observed. On the contrary, in this group of patients, an increase in eosinophils and a slight increase in NKDim cells, as well as a decrease in LT CD3+CD4−CD8−, NKBr and ILC2s, are observed. In the PBS mouse model (negative control) there is evidence of an increase in CD45+ cells, total ILCs, ILC-2s, eosinophils, and neutrophils as well as a slight increase in markers related to pulmonary fibrosis in the lung that show the exaggerated inflammation. Image created using BioRender (https://Biorender.com/).