DE212021000181U1 - A pharmaceutical micronutrient composition and its use for the simultaneous inhibition of multiple cellular mechanisms of infectivity caused by coronaviruses, their variants and mutants - Google Patents

Abstract

Pharmaceutical micronutrient composition comprising:
an ascorbate in the range of 10 mg to 200,000 mg, N-acetylcysteine in the range of 2 mg to 30,000 mg, theaflavin in the range of 5 mg to 3,000 mg, resveratrol in the range of 10 mg to 5,000 mg, extracts of cruciferous vegetables in the range of 5 mg to 5,000 mg, curcumin in the range of 5 mg to 10,000 mg, quercetin in the range of 5 mg to 2,000 mg, naringenin in the range of 5 mg to 3,000 mg, green tea polyphenol extract in the range of 1 mg to 10,000 mg, brasilin in the range of 1 mg to 5,000 mg and baicalin in the range of 5 mg to 3,000 mg.

Description

CROSS REFERENCE TO A RELATED APPLICATION

The present application claims priority from the pending US provisional application 63149633 , filed on February 15, 2021, and the utility model application 17212727 , filed March 25, 2021. The US provisional application and US utility model application are hereby incorporated by reference in their entirety for all of their teachings.

FIELD OF STUDY

This application discloses a pharmaceutical micronutrient composition and shows that the composition simultaneously inhibits multiple cellular mechanisms of infectivity caused by the coronavirus and its variants and attenuates coronavirus infection in mammals.

BACKGROUND

The emergence and rapid spread of the coronavirus pandemic has caused millions of deaths and is threatening human health and the economy on a global scale. Whole genome sequencing of the virus from patient samples from Wuhan, China (Zhu et al., 2020) identified a novel coronavirus classified by the Coronavirus Study Group (CSG) of the International Committee on Taxonomy of Viruses as severe acute Respiratory syndrome coronavirus-2 (SARS-CoV-2) (Gorbalenya et al., 2020). The disease caused by the virus has been named coronavirus disease 2019 (COVID-19) by the World Health Organization (WHO).

The coronavirus is a rapidly mutating virus and within a year of the outbreak of the pandemic, multiple mutations of this virus have emerged in the UK, South Africa, Brazil and other countries, each of which mutations can potentially lead to the emergence of additional coronavirus subtypes. Clinical reports show that the UK mutation of coronavirus can infect patients who have received the vaccine developed against the original coronavirus SARS-CoV-2, calling into question the claim of universal efficacy of available vaccines against all mutations of coronavirus .

It is therefore foreseeable that the ultimate control of the ongoing pandemic caused by the rapidly mutating coronavirus will be hampered by the need to develop a new vaccine for each new mutation of the coronavirus and the associated scientific, economic and social implications of such a strategy will.

A promising scientific route to this goal is to focus on the cell-surface "docking structure" of the coronavirus, the angiotensin-converting enzyme-2 (ACE-2) receptor. receptor). Significantly, all known corona viruses, including SARS CoV-2 and its mutations, use exactly this receptor as a docking structure and portal of entry for infections. This fact makes a detailed understanding of the regulation of the production/expression of this receptor on the surface of human cells - as well as the related cellular mechanisms - a priority goal for the development of global public health strategies to control the pandemic caused by a variety of current and future viral mutations is marked.

The mechanisms of cell entry of coronaviruses, including SARS-CoV-2, have been extensively studied. To enter host cells, coronaviruses first bind to a receptor on the cell surface for viral attachment, then enter cell dosomes and finally fuse viral and lysosomal membranes (Li et al., 2016). The entry of the coronavirus is mediated by a spike protein anchored to the surface of the virus. In mature viruses, the spike protein is trimeric, with three receptor-binding S1 heads sitting on a trimeric membrane fusion S2 stalk.

The SARS-CoV-2 spike S1 protein contains a receptor-binding domain (RBD) that specifically binds its cellular receptor, angiotensin-converting enzyme 2 (ACE2). The receptor-binding domain on the S1 head of the SARS-CoV-2 spike protein binds to a target cell via the human ACE2 receptor (hACE2) on the cell surface and is proteolytically activated by human proteases. The entry of coronaviruses into host cells is an important factor in viral infectivity and pathogenesis (Du et al, 2009, Du et al. 2017).

The cellular receptor for binding the virus is angiotensin-converting enzyme 2 or ACE2, which is an integral membrane protein found on many cells throughout the human body, with strong expression in the heart, vasculature, gastrointestinal system, and in the kidneys and in type II alveolar cells in the lungs. (Zhu et al., 2019, Li et al., 2003, Hoffman et al., 2005). Cellular coronavirus infections and intracellular viral replication are promoted by several host enzymatic proteins, including transmembrane serine protease 2 (TMPRSS2), furin, cathepsins, and RNA-dependent RNA polymerase (RdPp), which catalyzes viral RNA replication.

COVID-19 infections are associated with an intense host inflammatory response, a so-called "cytokine storm", thrombosis and other pathomechanisms that can trigger a fatal cascade of clinical events associated with advanced coronavirus infections. When evaluating new approaches to inhibiting coronavirus infectivity, the ability of these new approaches to improve such infection-related complications should be an additional goal. There is therefore an urgent need for preventive and therapeutic strategies to inhibit the infection mechanisms of all coronaviruses - regardless of mutation and/or subtype - and thus for new ways to control the pandemic globally.

SUMMARY

The present micronutrient pharmaceutical composition prevents, inhibits, treats and retards attachment, penetration, biosynthesis, maturation and release of a coronavirus SARS-Cov-2 in a mammal. In one embodiment, the phytochemicals, in combination with other vitamins, prevent various steps of infection in a mammal. In one embodiment, different combinations of individual micronutrients are referred to as mixtures. In one embodiment, Blend D, is a pharmaceutical micronutrient composition composed of resveratrol, extracts of cruciferous vegetables, curcumin, quercetin, naringenin, baicalein, theaflavin, vitamin C and N-actylcysteine.

In another embodiment, a pharmaceutical micronutrient composition comprises an ascorbate in the range of 10 mg to 200,000 mg, N-acetylcysteine in the range of 2 mg to 30,000 mg, theaflavins in the range of 5 mg to 3000 mg, resveratrol in the range of 10 mg to 5000 mg , extracts of cruciferous vegetables in the range of 5 mg to 5,000 mg (or an equivalent amount of their active ingredient sulforaphane), curcumin in the range of 5 mg to 10,000 mg, quercetin in the range of 5 mg to 2,000 mg, naringenin in the range of 5 mg to 3,000 mg and baicalein ranging from 5 mg to 3,000 mg.

In another embodiment, additional micronutrients are added to form a pharmaceutical micronutrient composition, such as a phenolic acid, gallic acid, tannic acid, chlorogenic acid, and rosmarinic acid; a flavonoid such as fisetin, morin, myrizetin, kaempferol, rutin, luteolin, baicalin, scutellarin, naringenin, hesperidin, hesperetin, apigenin, genistein, phloroglucinol, schisandrin, urolithin A, punicalagin, brasilin, hispidulin, papaverine, silymarin, procyanidin B2, procyanidin B3, stilbene and pterostilbene; an alkaloid such as palmatin, berberine, cannabidiol, castanospermine, usnic acid, malic acid, terpenes, d-limonene and carnosic acid.

In another embodiment, a pharmaceutical micronutrient mixture consists of an ascorbate in the range of 10 mg to 200,000 mg, N-acetylcysteine in the range of 2 mg to 30,000 mg, theaflavins in the range of 5 mg to 3,000 mg, resveratrol in the range of 10 mg to 5,000 mg, extracts of cruciferous vegetables ranging from 5 mg to 5,000 mg (or an equivalent amount of their active ingredient sulforaphane), curcumin ranging from 5 mg to 10,000 mg, quercetin ranging from 5 mg to 2,000 mg, naringenin ranging from 5 mg to 3,000 mg and baicalein ranging from 5 mg to 3,000 mg. In another embodiment, the ascorbates are at least one of or a combination of L-ascorbic acid, magnesium ascorbate, calcium ascorbate, ascorbyl palmitate, ascorbyl phosphate, sodium ascorbyl phosphate, and/or another pharmaceutically acceptable form of ascorbate.

In another embodiment, the pharmaceutical micronutrient composition further consists of at least one of theaflavins in the range of 5 mg to 3,000 mg, resveratrol in the range of 10 mg to 5,000 mg, extracts of cruciferous vegetables in the range of 5 mg to 5,000 mg, curcumin in the range of 5 mg to 10,000 mg, quercetin in the range of 5 mg to 2,000 mg, and a combination thereof.

In another embodiment, several additional ingredients are added to form a pharmaceutically acceptable formulation for various application forms, such as oral, injectable, absorbable, etc.. The pharmaceutical micronutrient composition is in the form of oral, non-invasive peroral, topical (e.g. transdermal ), enteral, transmucosal, targeted, sustained-release, delayed, pulsed, and parenteral delivery methods.

In one embodiment, the viral infection and/or viral disease uses a cellular receptor for virus entry on a surface of epithelial cells, endothelial cells, and/or other cell types.

In another embodiment, the viral infection and/or viral disease that uses an angiotensin converting enzyme 2 (ACE2) receptor on the surface of an epithelial cell, an endothelial cell and other cell types for viral entry is treated with a pharmaceutical micronutrient composition, prevented and weakened.

The pharmaceutical micronutrient composition is used in one embodiment for treating humans and other species with severe acute respiratory syndrome-associated coronaviruses (SARS-CoV-1, SARS-CoV2 and their variants) that express angiotensin-converting enzyme 2 (ACE2) receptors on the surface of epithelial cells, endothelial cells and other cell types for viral entry.

The pharmaceutical micronutrient composition is used in one embodiment for treating humans and other species with the Middle East Respiratory Syndrome-associated coronavirus (MERS-CoV) and its variants, the angiotensin-converting enzyme 2 (ACE2) receptors on the surface of Utilize epithelial cells, endothelial cells and other cell types for viral entry. In one embodiment, the pharmaceutical micronutrient composition is Blend D, which is used in humans to treat, prevent, inhibit, and disrupt inflammation caused by severe acute respiratory syndrome-associated coronaviruses (SARS-CoV-1, SARS -CoV-2 and its variants) and by the Middle East Respiratory Syndrome-associated coronavirus (MERS-CoV), and its variants.

Other features of the relevant embodiments will be apparent from the accompanying drawings and from the detailed description below.

character list

• 1A und 1B verschiedene zelluläre und systemische Mechanismen der Coronavirusinfektion zeigen.
• 2 die Ergebnisse der Bindung der rezeptorbindende Domäne (RBD) von SARS-CoV-2 an den humanen ACE2-Rezeptor zeigt.
• 3 eine dosisabhängige Bindung von SARS-CoV-2-Pseudovirionen an immobilisierte Epithelzellen, die hACE2 überexprimieren, zeigt.
• 4A, 4B und 4C die Viabilität der Zellen nach Behandlung mit angegebenen Polyphenolen für 1 h, 3 h und 48 h zeigen.
• 5A und 5B die Bindung von SARS-CoV-2-Pseudovirionen an Zellen bei unterschiedlichen Behandlungsmustern zeigen.
• 6A und 6B das Eindringen der SARS-CoV-2-Pseudovirionen in die Zellen bei unterschiedlichen Behandlungsmustern zeigen.
• 7A, 7B, 7C, 7D, 7E, 7F, 7G, 7H, 71, 7J, 7K Bilder von Synzytien, die nach Behandlung mit angegebenen Polyphenolen aufgenommen wurden, zeigen.
• 8 die Quantifizierung der Synzytien nach Behandlung mit angegebenen Polyphenolen zeigt.
• 9 die Auswahl der wirksamsten Formulierung auf der Grundlage der Hemmung der Bindung von RBD an ACE2 bei verschiedenen Mikronährstoffmischungen zeigt.
• 10 den Test zur Sicherheit für Mischung D in humanen kleinen Alveolarepithelzellen zeigt.
• 11 die Hemmung der RBD-Bindung und die Wirksamkeit der Mischung allein und ihrer Kombination mit Vitamin D zeigt.
• 12 die Hemmung der zellulären Internalisierung der mutierten Formen von SARS-CoV-2: Virusstämme aus dem Vereinigten Königreich, Brasilien und Südafrika zeigt.
• 13 die Hemmung des Zelleintritts der mutierten Formen von SARS-CoV-2: Virusstämme aus dem Vereinigten Königreich, Brasilien und Südafrika nach Anwendung verschiedener Behandlungsmuster zeigt.
• 14 die Hemmung der ACE2-Expression unter normalen und pro-inflammatorischen Bedingungen zeigt.
• 15 die Hemmung der Aktivität der viralen RNA-abhängigen RNA-Polymerase (RdRp) durch Mischung D mit und ohne Vitamin D zeigt.
• 16 die Hemmung der Furin-Aktivität durch Mischung D zeigt.
• 17 die Hemmung der zellulären Aktivität von nativem Cathepsin L durch Mischung D, die einzeln und mit Vitamin D verabreicht wird zeigt.
• 18 die hemmende Wirkung von Mischung D auf die Aktivität von rekombinantem Cathepsin L und die Auswirkungen von zusätzlichem Vitamin D zeigt.
• 19 die entzündungshemmende Wirkung: Hemmung der IL6-Sekretion unter normalen und pro-inflammatorischen Bedingungen durch die Mischung D allein und in Kombination mit Vitamin D zeigt.

Exemplary embodiments are illustrated by way of non-limiting example in the figures of the accompanying drawings, in which like references indicate similar elements and in which:

• 1A and 1B demonstrate different cellular and systemic mechanisms of coronavirus infection.
• 2 shows the results of binding of the receptor binding domain (RBD) of SARS-CoV-2 to the human ACE2 receptor.
• 3 shows dose-dependent binding of SARS-CoV-2 pseudovirions to immobilized epithelial cells overexpressing hACE2.
• 4A , 4B and 4C show the viability of the cells after treatment with indicated polyphenols for 1 h, 3 h and 48 h.
• 5A and 5B show the binding of SARS-CoV-2 pseudovirions to cells under different treatment regimens.
• 6A and 6B show the penetration of the SARS-CoV-2 pseudovirions into the cells with different treatment patterns.
• 7A , 7B , 7C , 7D , 7E , 7F , 7G , 7H , 71 , 7y , 7K Show images of syncytia taken after treatment with indicated polyphenols.
• 8th shows the quantification of syncytia after treatment with indicated polyphenols.
• 9 shows the selection of the most effective formulation based on inhibition of binding of RBD to ACE2 in different micronutrient mixtures.
• 10 shows the safety test for mixture D in human small alveolar epithelial cells.
• 11 shows the inhibition of RBD binding and the effectiveness of the mixture alone and its combination with vitamin D.
• 12 showing the inhibition of cellular internalization of the mutant forms of SARS-CoV-2: virus strains from the United Kingdom, Brazil and South Africa.
• 13 showing inhibition of cell entry of the mutant forms of SARS-CoV-2: virus strains from the UK, Brazil and South Africa after application of different treatment regimens.
• 14 shows inhibition of ACE2 expression under normal and pro-inflammatory conditions.
• 15 shows the inhibition of viral RNA-dependent RNA polymerase (RdRp) activity by Mixture D with and without vitamin D.
• 16 shows inhibition of furin activity by mixture D.
• 17 shows the inhibition of native cathepsin L cellular activity by mixture D administered alone and with vitamin D.
• 18 Figure 12 shows the inhibitory effect of Mixture D on recombinant cathepsin L activity and the effects of supplemental vitamin D.
• 19 shows the anti-inflammatory effect: inhibition of IL6 secretion under normal and pro-inflammatory conditions by mixture D alone and in combination with vitamin D.

Other features of the subject embodiments will become apparent from the detailed description that follows.

DETAILED DESCRIPTION

The virus life cycle with the host consists of the following five steps: attachment, penetration, biosynthesis, maturation and release. Once viruses bind to host receptors (attachment), they enter host cells by endocytosis or membrane fusion (penetration). Once the viral contents are released into the host cells, viral RNA travels to the cell nucleus for replication. The viral messenger RNA (mRNA) is used to produce viral proteins (biosynthesis). Subsequently, new viral particles are formed (maturation) and released. Corona viruses consist of four structural proteins: spike (S), membrane (M), envelope (E) and nucleocapsid (N). The spike consists of a transmembrane trimetric glycoprotein that protrudes from the virus surface and determines coronavirus diversity and host tropism.

Since multiple mechanisms are involved in the pathogenicity of SARS-CoV-2, all of which are ultimately regulated at the level of cellular metabolism, the most effective approach to suppressing viral infectivity is to identify molecules capable of inhibiting the expression of safely regulate and/or inhibit proteins associated with the infection pathway.

1A shows the cellular mechanism of viral entry and multiple entry sites for SARS-CoV-2 virus and others via ACE2 receptors, which require furin and cathepsin L for replication, protein synthesis, maturation, and release into the bloodstream after entry. 1B Figure 12 shows the systemic effect of interleukin 6 (IL-6) release in response to inflammation caused by viral infection. IL-6 could be a therapeutic target for inhibition of cytokine storm and organ damage associated with cytokine storm. We would show that this is a good target to prevent organ damage.

The safest and most effective molecules that can perform such a regulatory role are natural compounds, namely micronutrients. These natural compounds are naturally able to influence several biochemical processes in cell metabolism at the same time.

A "mammal" to be treated by the subject method can be either human or non-human animals such as mice, primates and vertebrates. The specific diseases eligible for treatment with a pharmaceutical micronutrient composition are infections caused by SARS-CoV-2, SARS-CoV-2 variants (such as the British, Nigerian, South African and Brazilian variants and 19 others mutations), MERS-CoV (the beta coronavirus that causes Middle East Respiratory Syndrome or MERS), SARS-CoV (the beta coronavirus that causes Severe Acute Respiratory Syndrome or SARS), SARS-CoV-2 and all of theirs Subtypes, four main subgroups of coronaviruses known as alpha, beta, gamma and delta.

Our previous study demonstrated that a natural micronutrient composition containing vitamin C, certain minerals, amino acids and plant extracts was effective in significantly decreasing cellular ACE2 expression in human lung alveolar epithelial and vascular endothelial cells. A combination of phytobiological compounds also demonstrated efficacy in inhibiting viral binding to cellular ACE2 receptors and affecting other mechanisms associated with viral infectivity.

Here we claim the effectiveness of certain combinations of micronutrients in significantly inhibiting the infectivity of the coronavirus, including binding of the virus to the ACE2 receptor, entry of the virus into the cell, intracellular virus replication and other mechanisms. In this study, we tested the efficacy of a specific nutrient composition containing vitamin C, N-acetylcysteine, resveratrol, theaflavins, curcumin, quercetin, naringenin, baicalin and extracts from cruciferous vegetables (broccoli, cabbage, cauliflower) on key aspects of CoV infectivity : Inhibition of viral RBD binding to the ACE2 receptors, cellular expression of the ACE2 receptors, inhibition of key enzymes involved in coronavirus activity, and anti-inflammatory and anticoagulant effects of this formulation.

The results show that this micronutrient composition was effective in inhibiting RBD binding of the SARS-CoV-2 spike protein to the ACE2 receptor (approximately 75% at 5 mcg/mL and 85% inhibition at 10 mcg/mL). At these concentrations, this micronutrient composition should be considered a safe and affordable approach to managing the current COVID-19 pandemic.

MATERIAL AND METHODS

Cell cultures: Human small airways epithelial cells (HSAEpC, purchased from ATCC) were cultured in airway epithelial cell growth medium (ATCC) in plastic flasks at 37°C and 5% CO ₂ . For the experiment, passage 5-7 HSAEpC were plated in collagen-coated 96-well plastic plates (Corning) in 100 µl of growth medium and grown to a confluent layer for 4-7 days. The human lung epithelial cell line A549 (from ATCC) was cultured in DMEM supplemented with 10% fecal bovine serum.

Micronutrient composition: The micronutrient combination used in our experiments was developed at Dr. Rath Research Institute (San Jose, Ca). The composition of all five tested mixtures is shown in Table 1. Table 1: All micronutrients were used in different combinations as mixtures: Micronutrient Blend D vitamin C N-acetylcysteine Theaflavin-3,3'-digallate resveratrol Cruciferous vegetable extracts curcumin quercetin naringenin baicalin

Cell-cell fusion assay: The cell-cell fusion assay was carried out according to Ou et al. accomplished. Briefly, A549 cells transduced with eGFP-luciferase-SARS-CoV-2 Spike S1 lentivirus vector (GenScript, Piscataway, NJ) were detached with 1 mM EDTA, with the indicated concentrations of selected polyphenols for 1 hour at 37 °C treated and layered onto 80-95% confluent A549 human lung epithelial cells overexpressing hACE2. After 4 hours of incubation at 37°C, images of syncytia were taken with a Zeiss Axio Observer A1 fluorescence microscope (Carl Zeiss Meditec, Inc, Dublin, CA). The positive control was 20 µg/ml anti-ACE2 antibodies. Results are expressed as a percentage of the polyphenol-free control (mean +/- SD, n=3).

Supplementation of the cells: The micronutrient mixture was dissolved in DMSO as either a 1 mg/ml or 10 mg/ml stock solution. For ACE2 expression experiments, HSAEpC cells were supplemented with the indicated doses of the formulation in 100 μl/well cell growth medium for 3-7 days. The nutrient concentrations used were given in micrograms per ml (µg/ml).

ACE-2 Expression Assay (ELISA): Human small airway epithelial cells (HSAEpC) were provided by ATCC (American Type Culture Collection, Manassas, VA) and cultured in small airway epithelial cell (ATCC) culture medium. HSAEpC cells were seeded into 96-well plates which were coated with collagen at passage 6 and grown to a confluent layer. The cell culture medium was supplemented with the indicated amounts of Mixture D and 50 mcg/ml ascorbic acid at 100 mcl per well. After 72 hours, the cells were supplemented with fresh medium and the same addition continued for another 72 hours. After 6 days of incubation, the cell layers were washed twice with phosphate-buffered saline (PBS) and fixed with 3% formaldehyde in PBS containing 0.5% Triton X100 for 1 hour at 4°C. The fixed cells were washed four times with PBS and incubated with 1% bovine serum albumin (BSA) in PBS overnight at 4°C. ACE2 expression was measured by an ELISA assay using primary anti-ACE2 polyclonal antibodies (SIGMA) and goat anti-mouse IgG secondary antibodies conjugated to horseradish peroxidase (HRP, Rockland). , measured. The amounts of retained HRP were determined by HRP substrate stained reaction as optical density at 450 nm with a microplate reader. The results were analyzed using the software Micro soft Excel and expressed as a percentage of unsupplemented controls (mean of three replicates +/- standard deviation).

Receptor Binding and Entry Experiments: Cell Lines and Pseudoviruses: The A549 human alveolar epithelial cell line was obtained from ATCC. The A549 human alveolar epithelial cell line, which stably overexpresses the hACE2 receptor (hACE2/A549), was obtained from GenScript (Piscataway, NJ). Both cell lines were maintained in Dulbecco's MEM with 10% fetal bovine serum (FBS), 100 U/ml penicillin and 100 μg/ml streptomycin. Pseudovirus particles with spiked glycoprotein as the coat protein, with eGFP and luciferase (eGFP-luciferase-SARS-CoV-2 spike glycoprotein pseudotyped particles) and pseudotyped ΔG-luciferase (G*ΔG-luciferase) rVSV were obtained from Kerafast (Boston, MA) acquired. Bald pseudovirus particles with eGFP and luciferase (eGFP-luciferase-SARS-CoV-2 pseudotyped particles) were purchased from BPS Bioscience (San Diego, CA). Lentiviral particles carrying human TMPRSS2 were from Addgene (Watertown, MA).

Test Compounds, Antibodies, Recombinant Proteins and Inhibitors: Curcumin, Tea Extract Standardized to 85% Theaflavins, Theaflavin-3,3'-Digallate, Gallic Acid, Tannic Acid, Andrographis Paniculata Extract, Andrographolide, Licorice Extract, Glycyrrhizic Acid, Broccoli Extract, L-Sulforaphane , usnic acid, malic acid, D-limonene and ammonium chloride were purchased from Sigma (St. Louis, MO). All other polyphenols and camostat mesylate were purchased from Cayman Chemical Company (Ann Arbor, MI). All antibodies were from Santa Cruz Biotechnology (Santa Cruz, CA). The recombinant TMPRSS2 protein was from Creative BioMart (Shirley, NY).

SARS-CoV-2 RBD binding to hACE2: The binding/neutralization reaction was performed using a SARS-CoV-2 surrogate virus neutralization assay kit containing either antibodies or inhibitors that target the interaction between the receptor-binding domain (RBD) of the SARS-CoV -2 spike protein and the hACE2 cell surface receptor (GenScript, Piscataway, NJ). For the screening assay, the tested polyphenols were mixed at a concentration of 100 µg/mL with either the HRP-conjugated receptor-binding domain (RBD fragment) of the SARS-CoV-2 spike S1 domain or with hACE2, which was coated on a plate 96-well was immobilized for 30 minutes at 37°C. Subsequently, the samples incubated with the RBD fragment were transferred to a 96-well plate with immobilized hACE2 receptor and incubated for another 15 minutes at 37 °C, while the hACE2-immobilized plates already incubated with different polyphenols were washed four times with washing buffer and washed with HRP -conjugated RBD fragments and then incubated at 37°C for 15 minutes. All plates were then washed four times with wash buffer and developed with tetramethylbenzidine (TMB) substrate solution for up to 5 minutes, followed by the addition of stop buffer. Optical density was immediately measured at 450 nm with a plate reader (Molecular Devices, San Jose, CA). Positive and negative controls were provided by the manufacturer. Results are expressed as a percentage of the polyphenol-free control (mean +/- SD, n=5).

RBD Binding: This assay was performed using a GenScript SARS-CoV-2 Surrogate Virus Neutralization Test Kit capable of detecting either antibodies or inhibitors that block the interaction between the viral spike protein RBD and the ACE2 cell surface receptor . All test samples at the indicated concentrations, as well as the positive and negative controls (supplied by the manufacturer) were diluted 1:9 by volume with the sample dilution buffer. In separate tubes, HRP-conjugated RBD was also diluted 1:99 by volume with the HRP dilution buffer. The binding/neutralization reaction was performed according to the manufacturer's protocol. Briefly, diluted positive and negative controls and test samples at the indicated concentrations were mixed with the diluted HRP-RBD solution 1:1 by volume and incubated at 37°C for 30 minutes. Thereafter, 100 μl each of the positive control mixture, the negative control mixture and the test sample mixtures were added to the corresponding wells with immobilized ACE2 receptor and incubated at 37° C. for 15 minutes. The plates were then washed four times with 260 µl/well of 1x wash solution and TMB solution added to each well (100 µl/well). Plates were incubated in the dark at room temperature for up to 5 minutes. Then 50 µl/well of stop solution was added to quench the reaction and the absorbance was immediately read in the plate reader at 450 nm. The experiment was performed three times in duplicate. Data are given as % of control.

Binding of pseudotyped mutant virions of SARS-CoV-2 to the hACE2 receptor: The experiment was performed according to GenScript recommendations with minor modifications. Briefly, eGFP-luciferase-SARS-CoV-2 spike protein encapsulated pseudo-virions were cultured at 37 °C with 5 and 10 µg/ml of Mixture D and simultaneously added to hACE2/A549 cells. Subsequently, the cells were washed three times with washing buffer and the primary antibody against the SARS-CoV-2 spike protein at a dilution of 1:1000, followed by an HRP-conjugated secondary antibody at a dilution of 1:2500 in the ELISA assay deployed. Transduction efficiency was quantified by monitoring luciferase activity with a luciferase assay system (Promega, Madison, WI) and a spectrofluorometer (Tecan Group Ltd., Switzerland). Positive and negative controls were provided by the manufacturer. The data are given in % of the control with no mixture addition (mean +/- SD, n=6).

Cathepsin L Activity Assay: The experiment was performed in cell lysates using a Cathepsin L Activity Assay Kit (Abcam, Cambridge, MA) according to the manufacturer's protocol. Briefly, 5 x 10 ⁶ A549 cells treated for 24 hours with Mixture D at concentrations of 5 and 10 µg/ml were washed with cold 1x PBS and 100 µl lysed with CL buffer for 8 minutes. After centrifugation at 4°C for 3 min, the supernatant was collected and the enzymatic reaction initiated by mixing 50 µl of treated sample, 50 µl of control sample, 50 µl of background control sample, and 50 µl of positive and negative controls. Then 50 µl of CL buffer and 1 µl of 1 mM DTT were added, followed by the addition of 2 µl of 10 mM CL substrate Ac-FR-AFC, except for the background control. The samples were incubated for 1 hour at 37°C and the fluorescence was recorded at extension/emission = 400/505 nm with a fluorescence spectrometer (Tecan Group Ltd., Switzerland). Data are presented as % of control with no PB addition (mean +/- SD, n=6).

The effect of Mixture D on isolated Cathepsin L activity was tested using a Cathepsin L Activity Screening Assay Kit (BPS Bioscience, San Diego, CA) according to the manufacturer's protocol. Briefly, Mixture D at concentrations of 5.0 and 10 µg/mL was added to cathepsin L (0.2 mU/µL) at 22°C for 15 minutes before fluorogenic substrate (Ac-FR-AFC) (10th µM) was added and incubated for 60 minutes at RT. The positive control contained only cathepsin L, the negative control contained cathepsin L and the cathepsin L inhibitor E64d (25 µM). The fluorescence was recorded with a fluorescence spectrometer (Tecan Group Ltd., Switzerland) at extension/emission = 360/480 nm. Data are presented as percentage of control with no PB addition (mean +/- SD, n=6).

Furin Activity Assay: The effects of Mixture D on furin enzymatic activity were evaluated using a SensoLyte Rh110 Furin Activity Assay Kit (AnaSpec, Fremont, CA) according to the manufacturer's protocol. Briefly, Mixture D at concentrations of 10 and 20 ng/ml was mixed with recombinant furin protein for 15 minutes, followed by the addition of the fluorogenic furin substrate Rh110. The samples were incubated for 1 h at 22°C and the fluorescence was recorded with a fluorescence spectrometer (Perceptive Biosystems Cytofluor 4000) at extension/emission = 490/520 nm. Results were calculated using Microsoft Excel software and expressed as a percentage of unsupplemented controls (mean of three replicates +/- standard deviation).

In vitro RdRp activity: In vitro RdRp activity was assayed using a SARS-CoV-2 RNA polymerase assay kit (ProFoldin, Hudson, MA) following the manufacturer's protocol. Briefly, 0.5 µl of 50x recombinant RdRp was incubated with 2.5 µl of 50x buffer and 21 µl of Mixture D at concentrations of 5 and 10 µg/ml for 15 minutes at RT, followed by the addition of the master mix containing 0.5 µl Contains 50x NTPs and 0.5 µl 50x template (as single-stranded polyribonucleotide). The reaction (25 µl) was incubated for 2 hours at 34 °C and then stopped by adding 65 µl of 10x fluorescent dye and the fluorescence signal was measured within 10 minutes at extension/emission=488/535 nm with a fluorescence spectrometer (Tecan, Group Ltd., Switzerland). Results are expressed as percentage of control with no PB addition (mean +/- SD, n=6).

Interleukin 6 (IL-6) Assay: Human small airway epithelial cells (HSAEpC) were supplied by ATCC and cultured in small airway epithelial cell (ATCC) culture medium. The SAEC cells were seeded in six-well plates coated with collagen at passage 6 and grown to a confluent layer. The cell culture medium was supplemented with the indicated amounts of Mixture D, 50 mcg/ml ascorbic acid and vitamin D3 in 3 ml per well. After 72 hours of incubation, the conditioned medium was removed and the IL-6 content measured using the R&D Systems Human IL6 ELISA assay according to the manufacturer's protocol. Results were calculated using Microsoft Excel software and expressed as a percentage of unsupplemented controls (mean of three replicates +/- standard deviation).

RESULTS

Our study helps unravel previously unknown but important antiviral mechanisms of natural products and advances our understanding of the biology of SARS-CoV-2. A clinical evaluation of their effectiveness in the pathophysiology of SARS-CoV-2 would be particularly interesting at later stages of the infection process. This should also include their impact on host response after SARS-CoV-2 infection, as well as whether their antiviral potential could support or complement current pharmacological treatments.

Efficacy of polyphenols and plant extracts in preventing the binding of the RBD sequence of SARS-CoV-2 and the hACE2 receptor. We investigated the ability of different classes of polyphenols to inhibit the binding of the SARS-CoV-2 spike protein RBD sequence to the hACE2 receptor using a two-step approach. In the first step, we analyzed 51 different polyphenols and plant extracts for their ability to mediate the binding of an HRP-conjugated RBD fragment of the SARS-CoV-2 spike protein to the immobilized hACE2 receptor and its direct binding to the hACE2 receptor itself inhibit.

As shown in Table 3 and Table 4, three polyphenols, brazilin, theaflavin-3,3'-di-gallate and curcumin, showed the highest potency (100%) in inhibiting RBD binding to hACE2 when administered at concentrations of 100 µg/ml were used. At the same time, these and other tested polyphenols did not bind significantly to the ACE2 receptor itself.

Here we provide in vitro experimental evidence that of the 51 polyphenols selected in this study, brasilin, theaflavin-3,3'-digallate and curcumin have the highest affinity for binding to the RBD spike protein of SARS-CoV-2. While curcumin showed moderate binding to the hACE2 receptor at very low concentrations, neither brasilin nor theaflavin-3,3'-digallate showed any binding affinity to this receptor.

We further examined this effect with hA549 cells expressing spike protein. By using spike protein coated pseudo-virions and a different exposure pattern to polyphenols, we could observe that all three polyphenolic compounds inhibit viral attachment to the cell surface ACE2 receptors after both short-term (1 h and 3 h) and long-term (48 h) exposure or incubation patterns. When the SARS-CoV-2 virions were preincubated for 1 hour with these compounds, added simultaneously, or added 1 hour after infection, the ability of the virions to bind to the cell surface ACE2 receptors and transduce cells was enhanced by all Test compounds decreased dose-dependently. Interestingly, the same inhibitory effect of the polyphenols, albeit at higher but still non-toxic concentrations, was observed when the SARS-CoV-2 pseudo-virions were forcibly bound to the cells by spin-infection. In addition, we found that brazilin, theaflavin-3,3'digallate, and curcumin can reduce cell-cell fusion between spike-expressing cells and hACE2-overexpressing cellular monolayers. Taken together, these results suggest that all three compounds have inhibitory properties specifically targeting RBD-SARS-CoV-2. Table 3. Effects of different classes of polyphenols on preventing binding of RBD of SARS-CoV-2 and binding of ACE2 receptor. Tested polyphenols and alkaloids (0.1 mg/ml) Binding with RBD (% of control ± SD) Binding with ACE2 (% of control ± SD) phenolic acids gallic acid 18.3±4.5 6.5±1.3 tannic acid 79.4±2.3 7.2±2.3 curcumin 100±0.2 4.6±2.4 chlorogenic acid 25.5±2.5 4.7±1.6 rosmarinic acid 22.5±3.8 7.9±1.8 flavonoids fisetin 22.4±1.9 6.0±2.4 quercetin 22.4±6.5 7.8±3.3 morin 30.5±5.8 5.6±3.1 myricetin 45.5±5.4 5.6±2.1 kaempfer oil 15.6±2.9 6.2±2.5 routine 20.6±6.3 4.8±2.0 luteolin 10.4±4.7 4.8±1.6 Baicalein 22.5±5.1 7.4±1.4 baicalin 10.3±2.9 4.9±1.9 scutellarine 8.1±3.7 7.5±1.7 Naringin 23.6±6.4 3.7±1.1 naringenin 20±5.1 8.3±1.6 hesperidin 90.3±3.8 8.3±2.3 hesperetin 42.5±4.6 4.9±2.7 apigenin 17.1±4.1 8.3±1.9 genistein 22.1±2.8 9.4±2.7 phloroglucinol 69.5±3.6 5.9±3.4 schisandrine 22.4±.3.3 5.1±2.7 Urolithin A 31.1±4.6 8.8±1.6 punicalagin 32.3±5.9 5.4±2.3 Brazilian 100±0.1 4.6±2.2 hispidulin 20.1±6.0 7.4±2.1 papaverine 1.6±0.2 6.5±3.7 silymarin 30.0±2.6 8.8±3.8 Procyanidin B2 31.1±3.6 5.8±2.7 Procyanidin B3 32.3±3.7 7.8±2.7 stilbenes trans resveratrol 22.3±2.9 5.5±2.4 pterostilbene 23.1±2.8 9.4±2.5 alkaloids palmatin 40.4±6.1 8.5±2.7 berberine 17.3±2.7 9.4±2.4 cannabidiol 1.4±0.3 5.8±2.0 castanospermine 8.2±2.3 5.5±3.1 usnic acid 22.0±3.4 5.7±1.7 malic acid 1.2±3.7 5.8±1.4 terpenes D-limonene 27.2±6.4 6.4±1.5 carnosic acid 27.1±5.1 6.9±4.1 Table 4. Binding ability of selected plant extracts and their main components to RBD of SARS-CoV-2 and to the ACE2 receptor. Tested plant extracts (0.1 mg/ml) Binding to RBD (% of Control±DS) Binding to ACE2 (% of control±DS) Tea extract (85% standardized catechin) 88.3±3.7 5.4±1.2 (+)-gallocatechin 69.5±2.8 5.7±1.6 (-)-Catechin Gallate 37.4±4.7 8.6±1.5 (-)-gallocatechin gallate 75.4±5.6 7.5±1.7 (-)-gallocatechin 73.5±6.7 3.9±2.3 (+)-Epigallocatechin gallate 87.5±6.8 5.9±2.0 Tea Extract (85% Theaflavins standardized) 100±0.3 5.6±2.1 Theafalvine 27.3±1.4 7.9±1.9 Theaflavin-3-'3-digallate 100±0.1 5.6±2.3 Broccoli Extract L-Sulforaphane 28.6±2.6 9.7±1.8 30.2±3.6 6.7±1.5 Andrographis paniculata extract andrographolide 18.4±1.8 5.8±3.6 22.1±2.5 5.6±2.4 Liquorice Extract Glycyrrhizic Acid 18.3±3.6 5.7±1.4 22.2±2.3 10.1±2.8

As in 2 , the inhibitory effect of these most potent polyphenols, curcumin, theaflavin-3'3-digallate and brazilin, on RBD-hACE2 binding was dose-dependent and ranged from 20% to 95% at concentrations of 2.5-10 µg/ml, respectively .

In a second step, we incubated A549 cells expressing the SARS-CoV-2 spike protein with these three test polyphenols for 1 hour and then exposed them to the soluble hACE2 receptor. In this experiment, we also observed a dose-dependent impairment of spike protein-hACE2 binding ranging from 15% to 95% at 2.5-10 µg/mL, consistent with the results previously obtained ( 3 ).

Cell viability tests revealed that short-term incubation (i.e. 1 h and 3 h) with these polyphenols at concentrations up to 25 µg/mL showed no cytotoxicity, as described in 4A , 4B and 4C shown. As in 5A shown, brasilin, theaflavin-3,3'-digallate, and curcumin similarly inhibited the binding of pseudotyped SARS-CoV-2 spike protein virions to hACE2/A549 in a dose-dependent manner, independent of exposure time and application pattern . Statistically significant inhibition of pseudovirion binding by all test polyphenols was observed as early as 5.0 µg/mL and 10 µg/mL when administered 1 h before ( 5A ) and at the same time ( 5B ) have been tested.

Another set of experiments also showed that brazilin, theaflavin-3,3'-digallate, and curcumin at non-toxic concentrations (i.e., 5.0-25 µg/mL) had a similar dose-dependent inhibitory effect on SARS-CoV-2 spike binding - Protein pseudotyped virions A549 attached to hACE2/A549. Inhibition of virion transduction ranged from 20% to 80% without spininfection and from 20% to 40% when spininfection was performed ( 6A ). Without spin-infection, statistically significant inhibition by test polyphenols was observed from a concentration of 5.0 µg/ml, both when SARS-CoV-2 spike pseudo-virions 1 hour before exposure of hACE2/A549 cells with selected polyphenols were incubated, as well as when added concomitantly with test polyphenols ( 3A ). When the test polyphenols were added 1 hour after exposure of SARS-CoV-2 spike pseudovirions to hACE2/A549 cells, a significant inhibitory effect of the polyphenols was observed from a concentration of 10 µg/ml.

The tested polyphenols showed different effectiveness on the cell transduction by the pseudovirions. When viral transduction of hACE2/A549 cells was forced through the use of spin-infection, curcumin showed significant inhibitory activity at lower concentrations compared to brazilin and theaflavin-3'3-digallate. Thus, the exposure of SARS-CoV-2 virions to curcumin for 1 h before and simultaneously with the addition to hACE2/A549 cells led to an inhibition of transduction from a concentration of 5.0 µg/ml. Higher (10 µg/mL) concentrations of brasililine and theaflavin-3,3'-digallate were required to achieve statistically significant inhibitory effects at the same exposure patterns. All test polyphenols, administered 1 h after the SARS-CoV-2 virions were applied to the cells, resulted in significant inhibition of transduction at a concentration of 10 µg/mL of each compound ( 6B ).

The effect of the test polyphenols on the fusion of A549 cells expressing pseudotyped SARS-CoV-2 spike protein virions with lung epithelial cells expressing hACE2 is in 4 shown. A549 pseudovirion-expressing cells preincubated with test polyphenols and then layered on hCE2/A549 cells for 4 hours showed markedly reduced attachment. Preincubation with brasilin at 25 µg/mL reduced cell attachment by 40%, with theaflavin-3'3-digallate by 40% to 70% at 10-25 µg/mL, and with curcumin by 70% to 95% at the same concentrations ( 10-25 µg/ml). These results were consistent with previously obtained datasets.

7A , 7B , 7C , 7D , 7E , 7F , 7G , 7H , 71 , 7y , 7K and 8th show the effect of test polyphenols on fusion with A549 cells overexpressing the human ACE2 receptor. A. Cell-cell fusion of A549 cells expressing eGFP spike protein with A549 cells stably expressing the human ACE2 receptor. A549 cells expressing eGFP spike protein were pretreated with the indicated polyphenols at different concentrations for 1 hour at 37 °C and for an additional 4 hours at 37 °C with A549 cells stably expressing the human ACE2 receptor , co-cultivated. The scale bar indicates 250 µm. B. Quantitative analysis of the syncytia formed. The experiments were performed in triplicate and repeated three times. Data are presented as percentage of control ± SD; Δp ≤ 0.01, * p ≤ 0.001. Control - 0.025% DMSO, positive control - 20 µg/ml anti-ACE2 antibody.

9 shows that Mixture D (resveratrol, cruciferous vegetable extract, curcumin, quercetin, naringenin, baicalin, theaflavin, vitamin C and N-acetylcysteine) has the best binding inhibition.

10 shows the safety of Mixture D on human alveolar cells. The pharmaceutical micronutrient composition Blend D was used at doses of 5 and 10 mcg/ml alone and in combination with vitamin D and was safe for use on human small alveolar epithelial cells.

11 Figure 1 shows the inhibition of RBD binding by Mixture D alone and its combination with vitamin D. Mixture D is effective in inhibiting the binding of RBD to ACE2 receptors by 75% at 5 mcg/ml and by 85% at 10 mcg/ml compared to the control. Mixture D in combination with vitamin D did not further enhance this inhibitory effect. We can safely say that Mixture D alone has high potency and inhibits RBD binding.

12 shows the results of the inhibition of cellular internalization of the mutant forms of SARS-CoV-2: virus strains from the United Kingdom, Brazil and South Africa. Mixture D (10 mcg/mL) co-administered with mutant virions to cells overexpressing ACE2 was equally effective in inhibiting cellular entry of these mutant forms of SARS-CoV-2: by 48% for British mutation, um 47% for the Brazilian mutation and 48% for the South African mutation. These effects were concentration dependent. Exposure of viral particles to Mixture D for one hour prior to combining with cells also inhibited cellular entry of these viral mutants by up to 40%. These results not only demonstrate the effectiveness of inhibiting cell entry of viral strains, but also demonstrate that direct exposure of viral particles to this pharmaceutical micronutrient compound helps prevent viral entry.

13 Figure 1 shows inhibition of cell entry by mutant forms of SARS-CoV-2, virus strains from UK, Brazil and South Africa, due to the inhibitory effect of Mixture D when co-administered with the virions and cells.

14 shows the inhibition of ACE2 expression under normal and pro-inflammatory conditions. Exposure of human small alveolar epithelial cells to Mixture D for 6 days resulted in a 73% inhibition of ACE2 expression at 12 mcg/ml. This inhibitory effect of Mixture D on ACE2 expression persisted and was even enhanced under pro-inflammatory conditions (inhibition between 83-86%).

15 shows the inhibition of RdRp viral activity and the effects of vitamin D. It shows that Mixture D alone can inhibit RdRp activity by 53% when used at 10 mcg/ml and by 30% at 5 mcg /ml compared to the control. Combinations of Mixture D with vitamin D did not further enhance RdRp inhibition.

16 shows the inhibition of furin activity in the cells due to the effect of mixture D. Mixture D administered individually at a concentration of 10 mcg/ml increased the furin activity by 33% and at a concentration of 20 mcg/ml by 52 % reduce. 17 Figure 1 shows the test results of inhibition of cellular cathepsin L activity by Mixture D and the effects of vitamin D and Mixture D. Mixture D applied to cells alone and in combination with vitamin D inhibits cathepsin L activity by 20% . Mixture D in combination with vitamin D does not further increase this inhibitory effect.

18 Figure 1 shows the anti-inflammatory effect: reduced inhibition of IL-6 secretion under normal and pro-inflammatory conditions by Mixture D alone and in combination with vitamin D. Mixture D (10 mcg/ml) applied to small alveolar endothelial cells for three days IL-6 secretion by 50%. Exposure of HSAEpC to lipopolysaccharide (LPS, 5 mcg/ml) increased IL-6 secretion by 43%. Under these pro-inflammatory conditions, Mixture D was able to inhibit IL-6 secretion by 55%. This inhibitory effect was increased to 83% by combining Mixture D (10 mcg/ml) with 10 mcg/ml vitamin D.

Pharmaceutical formulations suitable for these routes of administration can be prepared by adding one or more pharmacologically acceptable carriers to the active ingredient and then treating the micronutrient composition by routine methods known to those skilled in the art. Modes of administration include, but are not limited to, non-invasive peroral, topical (e.g., transdermal), enteral, transmucosal, targeted, sustained-release, delayed, pulsed, and parenteral methods. The peroral administration can take place both in the liquid and in the dry state. In one embodiment, the pharmaceutical micronutrient composition is in particular the mixture D.

Formulations suitable for oral administration may be in the form of capsules, cachets, pills, tablets, lozenges (using a flavored base, usually sucrose and acacia or tragacanth), powders, granules, or as a solution or suspension in an aqueous or non- aqueous liquid, or as an oil-in-water or water-in-oil liquid emulsion, or as an elixir or syrup, or as lozenges (using an inert base such as gelatin and glycerin or sucrose and acacia), each one previously specified amount of a composition of interest as an active ingredient. The preparations can also be administered as a bolus, electuary (lick) or paste.

If an oral solid drug is prepared, the pharmaceutical micronutrient composition is mixed with an excipient (and optionally one or more additives such as a binder, a disintegrant, a lubricant, a color, a sweetener and a flavor) and the resulting mixture is in a routine procedure processed to thereby produce an oral solid drug product such as tablets, dragees, granules, powder or capsules. As the additives, those generally used in the art can be used. Examples of excipients include lactate, sucrose, sodium chloride, glucose, starch, calcium carbonate, kaolin, microcrystalline cellulose and silicic acid. Binders include water, ethanol, propanol, simple syrup, glucose solution, starch solution, liquefied gelatin, carboxymethyl cellulose, hydroxypropyl cellulose, hydroxypropyl starch, methyl cellulose, ethyl cellulose, shellac, calcium phosphate, and polyvinylpyrrolidone. Disintegrants include dried starch, sodium arginate, agar powder, sodium hydroxycarbonate, calcium carbonate, sodium lauryl sulfate, monoglyceryl stearate, and lactose. Lubricants include purified talc, stearic acid salts, borax, and polyethylene glycol. Sweeteners include sucrose, orange peel, citric acid and tartaric acid.

When preparing a liquid drug for oral administration, the micronutrient pharmaceutical composition is mixed with an additive such as a sweetener, a buffer, a stabilizer or a flavoring agent and the resulting mixture is processed in a routine procedure to prepare an orally administered liquid drug product such as an internal drug Solution to make a syrup or elixir. Examples of the sweetener include vanillin; Examples of the buffer include sodium citrate; and examples of the stabilizer include tragacanth, acacia and gelatin.

For transdermal (e.g., topical) administration, dilute, sterile, aqueous or partially aqueous solutions (usually at a concentration of about 0.1% to 5%), otherwise similar to the parenteral solutions mentioned above, can be prepared with a micronutrient pharmaceutical composition .

Formulations containing a pharmaceutical micronutrient composition for rectal or vaginal administration may be presented as a suppository which may be prepared by mixing a composition of interest with one or more suitable non-irritating excipients, e.g. cocoa butter, polyethylene glycol, a suppository wax or a salicylate which is solid at room temperature but liquid at body temperature and will therefore melt in the appropriate body cavity and release the encapsulated compound(s) and composition(s). Formulations suitable for vaginal administration also include pessaries, tampons, creams, gels, pastes, foams or spray formulations containing such excipients as are known in the art to be appropriate.

A controlled release portion for capsules containing a pharmaceutical micronutrient composition can be added to the extended release system either by applying an immediate release layer to the extended release core; by coating or compression processes or in a multi-unit system such as e.g. B. a capsule with extended and immediate release beads.

In the context of a pharmaceutical micronutrient composition, the term “sustained release” is used in technical jargon. For example, a therapeutic composition that releases a substance over time may have sustained-release properties, as opposed to a bolus-type administration where the entire amount of the substance is made bioavailable at once. In certain embodiments, upon contact with body fluids, including blood, spinal fluid, mucous secretions, lymph or the like, one or more of the pharmaceutically acceptable excipients may undergo gradual or delayed degradation (e.g., by hydrolysis) with concomitant release of any material contained therein , e.g. a therapeutic and/or biologically active salt and/or a therapeutic and/or biologically active composition, for a sustained or prolonged period of time (compared to release from a bolus). This release can result in prolonged delivery of therapeutically effective amounts of any of the therapeutic agents disclosed herein.

Current efforts in the field of drug delivery include the development of targeted delivery, in which the drug is only effective in the target area of the body (e.g. mucous membranes such as in the nasal cavity), and sustained-release formulations, in which the pharmaceutical micronutrient composition is delivered over a specific Period of time is released from a formulation in a controlled manner. Types of sustained release formulations include liposomes, drug-loaded biodegradable microspheres, and pharmaceutical micronutrient composition-polymer conjugates.

Sustained release dosage formulations are prepared by coating a solid dosage form with a film of a polymer that is insoluble in the acidic environment of the stomach but soluble in the neutral environment of the small intestine. The sustained release dosage units can be prepared, for example, by coating a pharmaceutical micronutrient composition with a selected coating material. The pharmaceutical micronutrient composition may be a tablet for incorporation into a capsule, a tablet for use as an inner core in a "coated core" dosage form, or a plurality of drug-containing beads, particles or granules for incorporation into a tablet or capsule. Preferred coating materials include bioerodible, gradually hydrolyzable, gradually water soluble and/or enzymatically degradable polymers, which are conventional "enteric" polymers can delve Enteric polymers, as those skilled in the art know, become soluble or slowly erode in the higher pH environment of the lower gastrointestinal tract as the dosage form passes through the gastrointestinal tract, while enzymatically degradable polymers are degraded by bacterial enzymes in the lower gastrointestinal tract, particularly in the colon. Alternatively, a sustained release tablet can also be formulated by dispersing a drug in a matrix of a suitable material such as a hydrophilic polymer or a fatty compound. Suitable hydrophilic polymers include, but are not limited to, polymers or copolymers of cellulose, cellulose esters, acrylic acid, methacrylic acid, methyl acrylate, ethyl acrylate, and vinyl, or enzymatically degradable polymers or copolymers as described above. These hydrophilic polymers are particularly useful to form a sustained release matrix. Fatty compounds that can be used as matrix material include, but are not limited to, waxes (e.g., carnauba wax) and glycerol tristearate. Once the active ingredient is mixed with the matrix material, the mixture can be compressed into tablets.

Pulsed-release dosing mimics a multiple-dose profile without repetitive dosing and typically allows for at least a two-fold reduction in dosing frequency compared to a traditional dosage form of the drug (e.g., solution or fast-release drug, traditional solid dosage form). A pulsed release profile is characterized by a period of no release (lag time) or reduced release followed by rapid drug release. These can be formulated for critically ill patients using the subject pharmaceutical micronutrient composition.

The terms "parenteral administration" and "parenterally administered" as used herein refer to modes of administration other than enteral and topical, such as injections, and include, without limitation, intravenous, intramuscular, intrapleural, intravascular, intrapericardial, intraarterial, intrathecal, intracapsular , intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, and intrastemal injection and infusion.

Certain pharmaceutical compositions disclosed herein which are suitable for parenteral administration comprise one or more of the subject compositions in combination with one or more pharmaceutically acceptable sterile, isotonic, aqueous or non-aqueous solutions, dispersions, suspensions or emulsions, or sterile powders, immediately prior to may be reconstituted into sterile injectable solutions or dispersions before use and which may comprise antioxidants, buffers, bacteriostats, solutes to render the formulation isotonic in the blood of the intended recipient, or suspending or thickening agents.

When preparing an injection product, the micronutrient pharmaceutical composition is mixed with an additive such as a pH regulator, a buffer, a stabilizer, an isotonizing agent or a local anesthetic, and the resulting mixture is processed in a routine procedure so as to prepare an injection for subcutaneous, intramuscular or to make an intravenous injection. examples of the pH regulator or buffer include sodium citrate, sodium acetate and sodium phosphate; examples of the stabilizer include sodium pyrosulfite, EDTA, thioglycolic acid and thiolactic acid; examples of the local anesthetic include procaine hydrochloride and lidocaine hydrochloride; and examples of the isotonicity agent include sodium chloride and glucose.

Adjuvants are used to boost the immune response. There are different types of adjuvants. Haptens and Freund's adjuvants can also be used to prepare water-in-oil emulsions of immunogens.

The phrase "pharmaceutically acceptable" is recognized in the art. In certain embodiments, the term includes compositions, polymers and other materials and/or dosage forms suitable, within reasonable medical judgment, for use in contact with the tissues of mammals, both human and animal, without undue toxicity, irritation, allergic reaction or other problems or complications that correspond to a reasonable risk-benefit ratio.

The term "pharmaceutically acceptable carrier" is recognized in the art and includes, for example, pharmaceutically acceptable materials, compositions or agents such as liquid or solid fillers, diluents, solvents or encapsulating materials which may be used during carriage or transporting a drug from one organ or part of the body to another organ or part of the body. Any carrier must be "acceptable" in the sense that it is compatible with the other ingredients of a drug and not harmful to the patient. In certain embodiments, a pharmaceutically acceptable carrier is nonpyrogenic. Some examples of materials that can serve as pharmaceutically acceptable carriers are: (1) sugars such as lactose, glucose, and sucrose; (2) starches such as corn and potato starch; (3) cellulose and its derivatives such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) talc; (8) cocoa butter and suppository waxes; (9) oils such as peanut oil, cottonseed oil, sunflower oil, sesame oil, olive oil, corn oil, and soybean oil; (10) glycols such as propylene glycol; (11) polyols such as glycerin, sorbitol, mannitol and polyethylene glycol; (12) esters such as ethyl oleate and ethyl laurate; (13) agar; (14) buffering agents such as magnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16) pyrogen-free water; (17) isotonic saline; (18) Ringer's solution; (19) ethyl alcohol; (20) phosphate buffer solutions; and (21) other non-toxic, compatible substances used in pharmaceutical formulations.

In certain embodiments, the pharmaceutical micronutrient compositions described herein are formulated to be delivered to a mammal in a therapeutically effective amount as part of a prophylactic, preventive, or therapeutic regimen for overcoming infection caused by coronaviruses (regardless of type).

In certain embodiments, the dosage of the micronutrient pharmaceutical compositions, which may be referred to herein as a therapeutic composition, can be determined by reference to the plasma concentrations of the therapeutic composition or other encapsulated materials. For example, the blood samples can be tested for their immune response to the appropriate viral load or lack thereof.

The therapeutic micronutrient pharmaceutical composition provided by this application can be administered to a subject in need of treatment via a variety of conventional routes of administration, including oral, topical, parenteral, e.g. B. intravenously, subcutaneously or intramedullary. In addition, the therapeutic compositions can be administered intranasally, as a rectal suppository, or using a "flash" formulation, i. That is, the drug can dissolve in the mouth without the need to use water. In addition, the compositions may be administered to a subject in need of treatment in controlled release, site-directed drug delivery, transdermal drug delivery, patch-mediated drug delivery (active/passive), by stereotactic injection, or in nanoparticles.

In terms of concentrations, an active ingredient may be present in the therapeutic compositions of the subject invention for topical application via the cutis, intranasal, pharyngolaryngeal, bronchial, intravaginal, rectal or ocular.

For use as an aerosol, the active ingredients can be used together with a gaseous or liquefied propellant, e.g. As dichlorodifluoromethane, carbon dioxide, nitrogen, propane and the like, as needed or desired with the usual auxiliaries such as co-solvents and wetting agents, in an aerosol container under pressure. The most common routes of administration also include the preferred transmucosal (nasal, buccal/sublingual, vaginal, ocular, and rectal) and inhaled routes of administration.

Furthermore, in certain embodiments, the subject pharmaceutical micronutrient composition of the subject application may be freeze dried or subjected to another suitable drying technique such as spray drying. The subject compositions may be administered once or may be divided into a series of smaller doses administered at varying intervals, depending in part on the release rate of the compositions and the dosage desired.

Formulations useful in the methods presented herein include those suitable for oral, nasal, topical (including buccal and sublingual), rectal, vaginal, aerosol, and/or parenteral administration. The formulations can conveniently be presented in the form of unit doses and can be prepared by any of the methods known in pharmacy. The amount of a pharmaceutical micronutrient composition that may be combined with a carrier material to produce a single dose may vary depending on the subject being treated and the particular mode of administration.

The therapeutically acceptable amount described herein can be administered in inhalation or aerosol formulations. The inhalation or aerosol formulations may comprise one or more agents useful in inhalation therapy, such as adjuvants, diagnostic agents, imaging agents, or therapeutic agents. The final aerosol formulation may, for example, contain 0.005-90% w/w, e.g. 0.005-50%, 0.005-5% w/w or 0.01-1.0% w/w of the drug based on the total weight of the formulation.

Examples of suitable aqueous and non-aqueous carriers that can be used in the pharmaceutical micronutrient composition include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol and the like) and suitable mixtures thereof, vegetable oils such as olive oil and injectable organic esters such as ethyl oleate. Adequate flowability can be ensured, for example, by using coating materials such as lecithin, by maintaining the required particle size in dispersions and by using surfactants.

QUOTES INCLUDED IN DESCRIPTION

This list of documents cited by the applicant was generated automatically and is included solely for the better information of the reader. The list is not part of the German patent or utility model application. The DPMA assumes no liability for any errors or omissions.

Patent Literature Cited

• US63149633 [0001]
• US17212727 [0001]

Claims (23)

Pharmaceutical micronutrient composition comprising: an ascorbate in the range of 10 mg to 200,000 mg, N-acetylcysteine in the range of 2 mg to 30,000 mg, theaflavin in the range of 5 mg to 3,000 mg, resveratrol in the range of 10 mg to 5,000 mg, extracts of cruciferous vegetables in the range of 5 mg to 5,000 mg, curcumin in the range of 5 mg to 10,000 mg, quercetin in the range of 5 mg to 2,000 mg, naringenin in the range of 5 mg to 3,000 mg, green tea polyphenol extract in the range of 1 mg to 10,000 mg, brasilin in the range of 1 mg to 5,000 mg and baicalin in the range of 5 mg to 3,000 mg. Pharmaceutical micronutrient composition claim 1 wherein the ascorbate is at least one of or a combination of L-ascorbic acid, magnesium ascorbate, calcium ascorbate, ascorbyl palmitate, ascorbyl phosphate, sodium ascorbyl phosphate, and/or another pharmaceutically acceptable form of ascorbate. Pharmaceutical micronutrient composition claim 1 wherein the pharmaceutical micronutrient composition comprises theaflavin in the range of 5 mg to 3,000 mg, resveratrol in the range of 10 mg to 5,000 mg, extracts of cruciferous vegetables in the range of 5 mg to 5,000 mg, curcumin in the range of 5 mg to 10,000 mg, quercetin im range from 5 mg to 2,000 mg and a combination thereof. Pharmaceutical micronutrient composition claim 1 wherein the micronutrient pharmaceutical composition comprises green tea polyphenol extract in the range of 5 mg to 3,000 mg, resveratrol in the range of 10 mg to 5,000 mg, extracts of cruciferous vegetables in the range of 5 mg to 5,000 mg, curcumin in the range of 5 mg to 10,000 mg, quercetin in the range of 5 mg to 2,000 mg and a combination thereof, for the prevention and treatment of coronavirus infections and other viral infections. Pharmaceutical micronutrient composition claim 1 wherein the micronutrient pharmaceutical composition consists of brasilin in the range of 1 mg to 5,000 mg, theaflavin in the range of 5 mg to 3,000 mg, and curcumin in the range of 5 mg to 10,000 mg. Pharmaceutical micronutrient composition claim 1 wherein the baicalein is from a natural and synthetic source, theaflavin is from a plant source and/or chemical derivative, curcumin is from a plant source and/or chemical derivative, resveratrol is from a plant source and/or chemical derivative , quercetin derived from a plant source and/or chemical derivative, cruciferous vegetable extract derived from a plant source and/or chemical derivative, naringenin derived from a plant source and/or chemical derivative, and N-acetylcysteine from a plant source and /or a chemical derivative. Pharmaceutical micronutrient composition claim 1 , further comprising: A phenolic acid such as: gallic acid, tannic acid, chlorogenic acid and rosmarinic acid, a flavonoid such as fisetin, morin, myrizetin, kaempferol, rutin, luteolin, baicalin, scutellarin, naringin, hesperidin, hesperetin, apigenin, genistein, phloroglucinol, Schisandrine, Urolithin A, Punicalagin, Brasiline, Hispidulin, Papaverine, Silymarin, Procyanidin B2, Procyanidin B3, Stilbene and Pterostilbene, an alkaloid such as Palmatine, Berberine, Cannabidiol, Castanospermine, Usnic Acid, Malic Acid, Terpenes, D-Limonene and Carnosic Acid. Pharmaceutical micronutrient composition claim 1 wherein the pharmaceutical micronutrient composition is used to treat an infectious disease. Pharmaceutical micronutrient composition claim 1 wherein the pharmaceutical micronutrient composition is used in the treatment of a viral infection and/or viral disease in humans and other species. Pharmaceutical micronutrient composition claim 9 wherein the viral infection and/or viral disease is one that utilizes a cellular receptor for viral entry on a surface of epithelial cells, endothelial cells, and/or other cell types. Pharmaceutical micronutrient composition claim 9 wherein the viral infection and/or viral disease is one that affects an angiotensin converting enzyme 2 (ACE2) receptor on the surface of an epithelial cell, an endothelial cell, and other cell types used for viral entry. Pharmaceutical micronutrient composition claim 11 , wherein the pharmaceutical micronutrient composition is used to treat humans and other species with severe acute respiratory syndrome-related coronaviruses (SARS-CoV-1), SARS-CoV2 and their variants or to treat mutants that utilize angiotensin converting enzyme 2 (ACE2) receptors on the surface of epithelial cells, endothelial cells and other cell types for viral entry. Pharmaceutical micronutrient composition claim 11 wherein the pharmaceutical micronutrient composition is used to treat humans and other species infected with Middle East Respiratory Syndrome-associated coronavirus (MERS-CoV), and its variants or mutants, that express the angiotensin-converting enzyme 2 (ACE2) receptor on the surface of epithelial cells, endothelial cells and other cell types for viral entry. A pharmaceutical micronutrient composition consisting of an ascorbate, N-acetylcysteine, theaflavin, resveratrol, extracts of cruciferous vegetables, curcumin, quercetin, naringenin and baicalin and a combination thereof. Pharmaceutical micronutrient composition Claim 14 where the concentration of each micronutrient is an ascorbate ranging from 10 mg to 200,000 mg, N-acetylcysteine ranging from 2 mg to 30,000 mg, theaflavin ranging from 5 mg to 3,000 mg, resveratrol ranging from 10 mg to 5,000 mg, extracts of cruciferous vegetables ranging from 5 mg to 5,000 mg, curcumin ranging from 5 mg to 10,000 mg, quercetin ranging from 5 mg to 2,000 mg, naringenin ranging from 5 mg to 3,000 mg, and baicalin ranging from 5 mg to 3,000 mg is. Pharmaceutical micronutrient composition Claim 14 , wherein the pharmaceutical micronutrient composition is used to treat humans and other species with Middle East Respiratory Syndrome related coronavirus (MERS-CoV), SARS-CoV, SARS-CoV2 and their variants or mutants, which the receptor of the angiotensin -converting enzyme 2 (ACE2) on the surface of epithelial cells, endothelial cells and other cell types for viral entry. Pharmaceutical micronutrient composition comprising: an ascorbate in the range of 10 mg to 200,000 mg, N-acetylcysteine in the range of 2 mg to 30,000 mg, theaflavin in the range of 5 mg to 3,000 mg, resveratrol in the range of 10 mg to 5,000 mg, extracts of cruciferous vegetables in the range of 5 mg to 5,000 mg, curcumin in the range of 5 mg to 10,000 mg, quercetin in the range of 5 mg to 2,000 mg, naringenin in the range of 5 mg to 3,000 mg and baicalin in the range of 5 mg to 3,000 mg, the ascorbate being at least one of or a combination of L-ascorbic acid, magnesium ascorbate, calcium ascorbate, ascorbyl palmitate, ascorbyl phosphate, sodium ascorbyl phosphate and/or another pharmaceutically acceptable form of ascorbate and a combination thereof. Pharmaceutical micronutrient composition Claim 17 , further comprising: a phenolic acid such as: gallic acid, tannic acid, chlorogenic acid and rosmarinic acid, a flavonoid such as fisetin, morin, myrizetin, kaempferol, rutin, luteolin, baicalin, scutellarin, naringin, hesperidin, hesperetin, apigenin, genistein, phloroglucinol, Schisandrine, urolithin A, punicalagin, brasilin, hispidulin, papaverine, silymarin, procyanidin B2, procyanidin B3, stilbene and pterostilbene, an alkaloid such as palmatin, berberine, cannabidiol, castanospermine, usnic acid, malic acid, terpenes, D-limonene and carnosic acid. Pharmaceutical micronutrient composition Claim 17 wherein the pharmaceutical micronutrient composition is in the form of oral, non-invasive peroral, topical (e.g., transdermal), enteral, transmucosal, targeted, sustained-release, delayed, pulsed, and parenteral delivery methods. Pharmaceutical micronutrient composition Claim 17 , wherein the pharmaceutical micronutrient composition is used to treat humans and other species infected with Middle East Respiratory Syndrome-associated coronavirus (MERS-CoV), SARS-CoV, SARS-CoV2 and their variants and mutants that express the angiotensin receptor -converting enzyme 2 (ACE2) on the surface of epithelial cells, endothelial cells and other cell types for viral entry. Pharmaceutical micronutrient composition Claim 18 , wherein the pharmaceutical micronutrient composition is used to treat humans and other species with Middle East Respiratory Syndrome related coronavirus (MERS-CoV), SARS-CoV, SARS-CoV2 and their variants or mutants, which the receptor of the angiotensin -converting enzyme 2 (ACE2) on the surface of epithelial cells, endothelial cells and other cell types for viral entry. Pharmaceutical micronutrient composition Claim 18 wherein the pharmaceutical micronutrient composition is used for treating a viral infection and/or viral disease in a human and other species. Pharmaceutical micronutrient composition Claim 17 wherein the pharmaceutical micronutrient composition is used for treatment by being introduced with food, drinking water, tube feeding and as an adjunct to another medical treatment.

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