WO2022067405A1

WO2022067405A1 - Immunomodulatory, antioxidant, broad-spectrum antiviral food supplement based on natural products, and use thereof

Info

Publication number: WO2022067405A1
Application number: PCT/BR2020/050446
Authority: WO
Inventors: Marcelo Martins GRASTI; William Abrão SAAD
Original assignee: Grasti Marcelo Martins
Priority date: 2020-10-01
Filing date: 2020-10-30
Publication date: 2022-04-07
Also published as: BR102020020169A2

Abstract

The present invention describes an immunomodulatory, antioxidant, broad-spectrum antiviral food supplement comprising a combination of active ingredients of natural origin, namely quercetin, zinc sulphate, caffeine and Echinacea purpurea, as active ingredients, and a pharmaceutically acceptable excipient, namely soy lecithin, and the use thereof in systemic viral infections with immunological repercussions. As it is a supplement based on natural products, it is difficult for the product to be allergenic, and it can therefore be widely used, in addition to being cheap to produce and easy to transport, especially as there is no need for refrigeration. The present invention thus solves problems in the technical field relating to the availability of raw material, transport logistics and the provision of a supplement that assists in reducing the morbidity and mortality caused by idiosyncratic immunological reaction of carriers of infectious disease with immune system repercussions, in addition to providing a reduction in plasma viral concentrations.

Description

BROAD SPECTRUM IMMUNOMODULATING, ANTIOXIDANT AND ANTIVIRAL FOOD SUPPLEMENT BASED ON NATURAL PRODUCTS AND ITS USE

BRIEF DESCRIPTION

[001] This patent application deals with an "IMMUNOMODULATING, ANTIOXIDANT AND ANTIVIRAL FOOD SUPPLEMENT OF BROAD SPECTRUM BASED ON NATURAL PRODUCTS AND THEIR USE", which refers to a food supplement produced from natural products with recognized properties beneficial for health and of low cost, being liable to use on a large scale and hardly allergenic, as well as its use. In view of its immunomodulatory, antioxidant and antiviral activities, the proposed supplement aims to make it possible to reduce morbidity and mortality caused by an idiosyncratic immunological reaction in patients with an infectious disease with immunosystemic repercussions and to reduce plasma viral concentration titers.

APPLICATION FIELD

[002] The present invention belongs to the human need section, to the health field, more specifically, to the field of medical or pharmaceutical preparations, as it is a broad-spectrum immunomodulatory, antioxidant and antiviral food supplement based on natural products and their use to enable the reduction of morbidity and mortality caused by idiosyncratic immunological reaction, in systemic viral infections with immunological repercussion.

CONVICTION

[003] Each and every infectious disease is only successful when there is an imbalance between the resistance force of the organism or immunity and the force to cause disease, the pathogenicity of the etiological agent of the infection. Thus, a victorious viral agent is the result of a dysfunction between these antagonistic factors.

[004] The current pandemic scenario allows the use of low-cost tools that mitigate/alleviate, even if by in vitro evidence, the inflammatory process resulting from immunosystemic infections. In view of the lack of specific etiological treatment against the agents that cause exacerbated reactions, it remains to make the host as strong as possible.

[005] In order to understand the evolution of these diseases, it is necessary to understand that the biggest cause of the morbidity and mortality rate reached by viral agents or the direct cytotoxic action, is not due to the increase in plasma levels of viral products, capsids and other viral components alone, but rather from the over-stimulated and dysfunctional inflammatory chain, originating from intercellular and intracellular messengers of endocrine, paracrine and other unknown products.

[006] The pathophysiology of immunosystemic diseases of viral origin, in turn, is briefly described, using the example of the SARS-CoV-2 agent as a prototype, due to its current relevance and prevalence in our population. Viral agent -> exaggerated immune response -> release of cytokines in high titers (IL-6, etc) -> systemic inflammatory response syndrome -> cell death -> increased ferritin -> activation of the coagulation cascade -> microthrombosis -> formation of SHUNT in pulmonary microcirculation -> silent hypoxia -> ischemia and multiple organ dysfunction syndrome.

[007] Therefore, the reduction of the multisystem inflammatory state of the proposed supplement formulation can be achieved from five interconnected factors: i. Reduction in blood concentration of viral product (Ornar et al., 2020 - preprint: In-Silico Identification of Potent Inhibitors of COVID-19 Main Protease (Mpro) and Angiotensin Converting Enzyme 2 (ACE2) from Natural Products: Quercetin, Hispidulin, and Cirsimaritin Exhibited Better Potential Inhibition than Hydroxy-Chloroquine Against COVID-19 Main Protease Active Site and ACE2 Skalny et al., 2020: Zinc and respiratory tract infections: Perspectives for COVID-19 (Review); Kimet et al., 2020: Inhibition of influenza virus replication by plant-derived isoquercetin); ii. Blocking different inflammatory chains (Hulisz, 2004: Efficacy of zinc against common cold viruses: an overview; Zhou et al., 2020: Clinical course and risk factors for mortality of adult inpatients with COVID-19 in Wuhan, China: a retrospective cohort study); iii. Stimulation of the immune state; iv. Symptom support factors; v. Reestablishment of nutritional deficits (Himoto, 2018: Associations between Zinc Deficiency and Metabolic Abnormalities in Patients with Chronic Liver Disease; Braun et al., 2017: Prevalence of Zinc Deficiency in Cardiac Surgery Patients; Zhou et al., 2020: Clinical course and risk factors for mortality of adult inpatients with COVID-19 in Wuhan, China: a retrospective cohort study).

[008] In addition, it is worth noting that there are several points of intersection between the inflammatory process and hemostasis that configure an important worsening mechanism. [009] Relatively common and catastrophic results observed are: unwinding of thromboses, embolisms and various procoagulant processes; which are evidenced by the sudden increase in the dimer titers, in relation to the basal levels observed in the individual and, differently from what is observed in sepsis-induced coagulopathies.

[010] Added to this, the difficult monitoring in the reality of the Unified Health System (SUS), and other health systems around the world, corroborate for late hospital admissions. Important Brazilian medical institutions, however, advocate the use of telemonitoring to collect detailed clues of possible severe clinical alterations, often of sudden onset.

[011] In this context, as a possible integrative solution for the care and prevention of the current health crisis, references can be made to former allies: quercetin supplementation, which causes changes in steroid metabolism and heme synthesis (eg. . tetrahydrocortisone and androsterone), which are considered possible clues of concussions, caused by pathogens, in the functioning in the regulation of inflammation and hemostasis, considering that synthetic drugs of the same class, for example, dexamethasone, showed a large gain in the indices of mortality, in vivo, randomized and double-blind.

[012] In addition, quercetin metabolites are involved in inflammatory intracellular signaling and its use with other antioxidants can increase its activity, which is why it is employed as one of the actives in the present invention. [013] The results of a three-month study in 1083 patients by the University of Cambridge (Cialdella-Kam et al., 2013: Dose-response to 3 months of quercetin-containing supplements on metabolite and quercetin conjugate profile in adults ), indicated that the metabolites present in the systemic circulation after oral administration of quercetin can act as a potent antioxidant and anti-inflammatory, due to their bioavailability and the action of the -hydroxyl groups of their derivatives.

[014] Several authors have also suggested that another substance used as an active agent in the proposed supplement, zinc, affects a common step in cell replication cycles. Enzymatic studies using recombinant RNA-dependent RNA polymerases (RdRPs) (SARS-CoV nsp12) purified from Escherichia coll revealed that Zn ²⁺ directly inhibited SARS-CoV RdRp elongation and reduced template binding (Quiles et al., 2020). : Do nutrients and other bioactive molecules from foods have anything to say in the treatment against COVID-19?). It was described in vitro that the increase in the concentration of intracellular Zn ²⁺ is able to inhibit the replication of several viruses of RNA, including influenza virus, polio virus, and SARS-CoV virus (te Velthuis et al., 2010: Zn ²⁺ Inhibits Coronavirus and Arterivirus RNA Polymerase Activity In Vitro and Zinc Ionophores Block the Replication of These Viruses in Cell Culture).

[015] As if the potential of zinc in the present invention and its synergistic novelty were not enough, the component is used due to in vitro and in vivo evidence of facilitating action of independent composites, in a process called ionophoresis, which is capable of transporting specific ions , like zinc, across a lipid membrane in a cell. While zinc is critical to supporting the immune system response, it needs an ionophore in order to be able to support the immune system response in intracellular pathways.

[016] Zinc is required within virus-infected cells to inhibit viral RNA polymerase and prevent virus replication, as previously discussed. [017] Due to its ionic nature, zinc requires an ionophore to bind to it and transport it across the cell membrane more efficiently, as all cells tend to keep their differential potential (DDP) constant. Although pharmaceutical zinc ionophores such as hydroxychloroquine and chloroquine associated with ivermectin exist, natural ionophores such as quercetin represent an excellent zinc ionophore and do not have the same biosafety profile as these drugs (Qui et al., 2016: Prophylactic Efficacy). of Quercetin 3-/3-O-d-Glucoside against Ebola Virus Infection).

[018] It is public knowledge that quercetin, a potent antioxidant, demonstrates broad-spectrum antiviral activity, as well as the presence of zinc titers greater than normal supplementation, resulting in increased antiviral activity. [019] In addition, the decrease in the cytokine storm, caused by an exacerbated reaction of the affected patient's immune system, is achieved from these components that act synergistically in a process known as ionophoresis - one in which a substance, by having the chelating property of the electroactive species of zinc, it allows its entry more easily into the cell membrane, as the cell's DDP always tends to be preserved, therefore, the cell hardly admits the entry of charged species.

[020] The formulation of the proposed supplement is of natural origin, does not have an etiological treatment appeal, much less specific, since this would be very ambitious and imprudent, considering that there are no clinical tests that corroborate the certainty of such affirmation. Therefore, the claimed supplement formulation is intended to be used as a booster of the immune system, given that its components have action widely evidenced by several national and international literature, including clinical studies concluded; thus, the supplement of wide interest, for the uniqueness of the junction of these natural metabolites and for the synergism of its components.

[021] In view of the wide interest in the supplement, the present invention still has an important advantage in situations of scarcity or absence of inputs, since, as there are no synthetic substances in its composition, it does not depend on possibly scarce inputs in crises of any origin. Another important advantage is its low acquisition value and easy transport (no need for refrigeration); finally, there is still a marginal profit: substances of natural or semi-synthetic origin have less rejection by the body, which is known as the “Law of Similarity”. In this way, the proposed supplement would reach the general population, however difficult their access to it may be.

[022] To date, there is no knowledge of a low-cost solution of natural origin, capable of large-scale use and hardly allergenic, whose components have a safety profile well known in the literature, capable of helping against infection. by SARS-CoV-2, and its immuno-systemic consequences. [023] Nevertheless, the study conducted by FCMUSP, Department of Infectious Diseases, demonstrated inhibitory activity with titers from 0.12mg/dL, of 100% of the culture infected VERO E6 by the SARS-CoV-2 agent - plausible levels of the substances mentioned above, in view of their pharmacokinetic profile described in the international and national literature. Thus, it is observed that the technical contribution of the present invention is evident, as well as its originality and relevance for the population.

BACKGROUND OF THE INVENTION

[024] In the current state of the art, prior art is present that describes compositions based on zinc and quercetin for the production of antivirals, as well as compositions that comprise quercetin with anti-inflammatory and/or antioxidant action. However, none of the previous items recovered describes the formulation of a broad-spectrum immunomodulatory, antioxidant and antiviral food supplement based on natural products and its use with the ability to reduce morbidity and mortality caused by an idiosyncratic immunological reaction in systemic viral infections of immunological repercussion and that the synergistic action of the active components has enabled inhibitory activity, in vitro, for the SARS-CoV-2 agent. [025] Priority WO2018039755A1, entitled "Pharmaceutical formulation based on zinc and quercetin for the production of effective antiviral medication for dengue and zika", refers to compositions for the prevention or treatment of symptoms of infections caused by Flaviviruses, such as dengue . The compositions comprise a combination of a zinc compound, which may be chelated and/or non-chelated with the flavonoid quercetin or its analogues and derivatives, and other components may also enter the formulation according to the indication, age group, form of presentation, pathologies existing and weight. Antiviral activity is achieved through the synergistic effect of quercetin and zinc that act through different mechanisms of action, preventing the virus from creating resistance mechanisms.

[026] Priority US8420132B2, entitled "Synergistic anti-inflammatory and antioxidant dietary supplement compositions", describes dietary supplement compositions comprising enriched 3-O-acetyl-11-keto-[3-boswellic acid and enriched demethyl curcuminoids. The described composition exhibits anti-inflammatory, antiulcerogenic and antioxidant activities. Also disclosed are novel compositions that have a synergistic effect on the specific inhibition of COX-2 and 5-LOX activity and optionally contain glucosamine, resveratrol, garlic extract, chondroitin, methylsulfonmethane, bromelain, serrathiopeptidase, quercitin, gallic acid, caffeic acid, green tea extract, aspirin and ibuprofen.

[027] Priority US8440704B2, entitled "Quercetin-containing compositions", describes a composition containing quercetin, vitamin B3 and vitamin C as the only active ingredients, in which the ratio of quercetin, vitamin B3 and vitamin C is 1 : 0.02-1 : 0.2-2.5. The described composition can be administered both as a food supplement and as a pharmaceutical formulation to be used in the treatment of multiple sclerosis, thyroiditis, myositis, lupus, celiac disease, eczema, hives, psoriasis, prostatitis, inflammatory bowel disease, arthritis, leukemia, colon cancer, prostate cancer, kidney cancer, liver cancer, breast cancer, lung cancer, sexual dysfunction, chronic constipation, coronary heart disease, atherosclerosis, stroke or peripheral arterial disease.

[028] Priority US9446088B2, entitled "Preparation and use of a plant extract from Solanum glaucoohyllum with an enriched content of 1,25 dihydroxyvitamin D3 glycosides and quercetin glycosides", describes the preparation of an enriched and balanced plant extract of Solanum glaucophyllum with a enriched content of 1,25-dihydroxyvitamin D3, glycosides and flavonols, particularly quercetin, and its use for the prevention and treatment of diseases related to the reduction of bone mass, such as osteopenia or osteoporosis, for the prevention and treatment of tibial dyschondroplasia, preferably in poultry, for the treatment of milk fever, being, therefore, a dietary supplement for human and veterinary use.

[029] Although compositions that employ quercetin in association or not with zinc are present in the current state of the art, none of the foregoing describes the availability of a low-cost solution and associated natural components, capable of large-scale use and hardly allergenic, to be used against SARS-CoV-2 infection and its immuno-systemic consequences, thus solving problems present in the current state of the art in an innovative way.

OBJECTIVES OF THE INVENTION

[030] The present invention aims to make it possible, through the availability of a broad-spectrum immunomodulatory, antioxidant and antiviral food supplement, to reduce morbidity and mortality caused by an idiosyncratic immunological reaction in patients with infectious disease with immunosystemic repercussions and to reduce the titers of plasma viral concentrations.

OF THE INVENTION

[031] The present invention describes a broad-spectrum immunomodulatory, antioxidant and antiviral food supplement that comprises a combination of active agents of natural origin: quercitin, zinc sulfate, caffeine and Equinaceae purpureae, as active agents and a pharmaceutically acceptable excipient, the soy lecithin; and its use in systemic viral infections with immunological repercussions, such as H1 N1, Parvovirus, Coxsakievirus and Coronoviridae family infections.

ADVANTAGES OF THE INVENTION

[032] The present invention has the following main advantages: Availability of a broad spectrum immunomodulatory, antioxidant and antiviral food supplement based on components of natural origin and which, therefore, is hardly allergenic; Availability of a low-cost, broad-spectrum immunomodulatory, antioxidant and antiviral food supplement that can be used on a large scale; Availability of a broad-spectrum immunomodulatory, antioxidant and antiviral food supplement with inhibitory activity against SARS-CoV-2; Availability of a broad-spectrum immunomodulatory, antioxidant and antiviral food supplement with activity in infections caused by H1 N1, Parvovirus, Coxsakievirus and Coronoviridae family.

DESCRIPTION OF THE FIGURES

[033] The invention will be described in a preferred embodiment, so, for better understanding, references will be made to the related figures:

FIG. 1 : Positive control of a cell infected with SARS-CoV-2 virus at a concentration of 0.01 MOI. Cells in blue are intact, with cytoplasm and nucleus. Cells in green are infected in the cytoplasm with the SARS-CoV-2 virus. In red, computational analysis of cell integrity without the presence of the virus;

FIG. 2: Negative control from uninfected cell. Cells in blue are intact, with cytoplasm and nucleus. In red, computational analysis of cell integrity;

FIG. 3: Infected and treated cell (1.02 mg/mL supplement) containing: Cells + Supplement + Virus. In blue: cells treated with the supplement and infected with SARS-CoV-2 virus, concentration of 0.01 MOI. Blue cells: cytoplasm and nucleus integrity. In red: Computational analysis of cellular integrity, using the Operetta device. The result demonstrates the absence of the virus, which was inhibited by the supplement;

FIG. 4: Infected and treated cell (0.051 mg/mL supplement) containing: Cells + Supplement + Virus. In blue: cells treated with the supplement and infected with SARS-CoV-2 virus, concentration of 0.01 MOI. Blue cells: cytoplasm and nucleus integrity. In red: Computational analysis of cellular integrity, using the Operetta device. The result demonstrates the absence of the virus, which was inhibited by the supplement;

FIG. 5: Infected and treated cell (0.25 mg/mL supplement) containing: Cells + Supplement + Virus. In blue: cells treated with the supplement and infected with SARS-CoV-2 virus, concentration of 0.01 MOI. Blue cells: cytoplasm and nucleus integrity. In red: Computational analysis of cellular integrity, using the Operetta device. The result demonstrates the absence of the virus, which was inhibited by the supplement;

FIG. 6: Infected and treated cell (0.12 mg/mL supplement) containing: Cells + Supplement + Virus. In blue: cells treated with the supplement and infected with SARS-CoV-2 virus, concentration of 0.01 MOI. Cells in blue: cytoplasm and nucleus integrity. In red: Computational analysis of cellular integrity, using the Operetta device. The result demonstrates the absence of the virus, which was inhibited by the supplement; FIG. 7: Infected and treated cell (0.002 mg/mL supplement) containing: Cells + Supplement + Virus. In blue: cells treated with the supplement and infected with SARS-CoV-2 virus, concentration of 0.01 MOI. Cells in blue: cytoplasm and nucleus integrity. In red: Computational analysis of cellular integrity, using the Operetta device. The result demonstrates the absence of the virus, which was inhibited by the supplement;

FIG. 8: Positive control of a cell infected with SARS-CoV-2 virus, concentration of 0.02 MOI. Cells in blue are intact, with cytoplasm and nucleus. Cells in green are infected in the cytoplasm with the SARS-CoV-2 virus. In red, computational analysis of cell integrity without the presence of the virus;

FIG. 9: Infected and treated cell (0.075 mg/mL supplement) containing: Cells + Supplement + Virus. In blue: cells treated with the supplement and infected with SARS-CoV-2 virus, concentration of 0.02 MOI. Blue cells: cytoplasm and nucleus integrity. In red: Computational analysis of cellular integrity, using the Operetta device. The result demonstrates the absence of the virus, which was inhibited by the supplement;

FIG. 10: Graph demonstrating low toxicity of the supplement tested in VERO 6 cell culture;

FIG. 11 : Graph demonstrating cell survival in the presence of the supplement;

FIG. 12: Graphs comparing supplement activity and cell survival with chloroquine.

DETAILED DESCRIPTION OF THE INVENTION

[034] The broad spectrum immunomodulatory, antioxidant and antiviral food supplement based on natural products is characterized by comprising a combination of quercitin, zinc sulfate, caffeine and Equinaceae purpureae, as active agents and pharmaceutically acceptable excipient. As the proposed formulation consists of:

'Z Quercetin . 500mg; Zinc sulfate . 150mg; Caffeine. 50mg; Echinacea purpurae. 200mg; Soy lecithin (excipient). 300 mg.

[035] The use of the claimed supplement occurs in systemic viral infections of immunological repercussion, due to its inhibitory activity in infections by H1 N1, Parvovirus, Coxsakievirus and Coronoviridae family.

[036] The supplement is obtained at a temperature of 22°C, for that, the solid active principles, in powder, are weighed, in an analytical balance, and mixed in a plastic bag pharmaceutical, then, the powder mixture is packed in plates containing capsules, which are closed and filled. After filling, the average weight is made with a sample of 10 capsules, not exceeding 5% of difference between them, such analysis takes place in a period of 15 minutes.

[037] For in vitro testing to verify the efficiency of the supplement against the Sars-Cov-2 virus, after the isolation and titration of the SARS.CoV2/SP02.2020.HIAE.Br virus, the supplement efficiency tests were started in front of to the virus in VERO clone CCI-81 cells. After 24 hours, no cytotoxicity was observed in the dilutions of the supplement. The observation of the antiviral effect is carried out from 72 hours after the beginning of the experiment.

[038] The results obtained in the in vitro tests of the supplement against the SARS-CoV-2 virus, demonstrated the inhibition of the action of the SARS-CoV-2 virus, by the action of the supplement at concentrations from 1.02mg/ml_ to 0, 12mg/ml_ in VERO E6 cells, as seen in figures 1 to 7, where the viral concentration was 0.01 MOI. [039] Note the inhibition of the virus action at a concentration of 0.075mg/ml_, as seen in figures 8 and 9, where the viral concentration was 0.02 MOI. Absence of cytotoxicity was also observed from the 10 ² dilution of the supplement.

[040] The toxicity analysis of the supplement in VERO E6 cell culture showed that there was no toxicity to cells at the recommended concentrations, concentrations from 1.02mg/ml_ to 0.12mg/ml_ for viral concentration of 0.01 MOI and concentration of 0.075mg/ml_ for a viral concentration of 0.02 MOI, as seen in figures 10, 11 and 12.

[041] From the above, it is noted that the immunomodulatory, antioxidant and antiviral food supplement of broad spectrum based on natural products and its use to enable the reduction of morbidity and mortality caused by idiosyncratic immunological reaction, in systemic viral infections of immunological repercussion demonstrated in vitro activity against SARS-CoV-2, reducing its action on cell culture and innovatively solving problems present in the current state of the art.

Claims

1. BROAD SPECTRUM IMMUNOMODULATING, ANTIOXIDANT AND ANTIVIRAL FOOD SUPPLEMENT BASED ON NATURAL PRODUCTS characterized in that it comprises a combination of quercitin, zinc sulfate, caffeine and Equinaceae purpureae, as active agents and pharmaceutically acceptable excipient.

2. BROAD SPECTRUM IMMUNOMODULATING, ANTIOXIDANT AND ANTIVIRAL FOOD SUPPLEMENT BASED ON NATURAL PRODUCTS, according to claim 1, characterized in that its formulation is: v' Quercetin . 500mg; Zinc sulfate . 150mg; Caffeine. 50mg; Echinacea purpurae. 200mg; Soy lecithin (excipient). 300 mg.

3. USE OF FOOD SUPPLEMENT IMMUNOMODULATOR, ANTIOXIDANT AND ANTIVIRAL OF BROAD SPECTRUM BASED ON NATURAL PRODUCTS, as described in with any of claims 1 or 2, characterized in that it occurs in systemic viral infections of immunological repercussion.

4. USE OF FOOD SUPPLEMENT IMMUNOMODULATOR, ANTIOXIDANT AND ANTIVIRAL OF BROAD SPECTRUM BASED ON NATURAL PRODUCTS, according to claim 3, characterized by activity in infections by H1 N1, Parvovirus, Coxsakievirus and Coronoviridae family.

5. USE OF FOOD SUPPLEMENT IMMUNOMODULATOR, ANTIOXIDANT AND ANTIVIRAL OF BROAD SPECTRUM BASED ON NATURAL PRODUCTS, according to claim 3, characterized in that it is used in concentrations from 1.02mg/mL to 0.12mg/mL for viral concentration of 0 .01 MOI, and; concentration of 0.075mg/mL for viral concentration of 0.02 MOI, for inhibition of SARS-CoV-2 activity.