WO2022173456A1 - Composition pharmaceutique de micronutriments et son utilisation pour inhiber simultanément de multiples mécanismes cellulaires d'infectivité provoqués par un coronavirus, ses variants et mutants - Google Patents

Composition pharmaceutique de micronutriments et son utilisation pour inhiber simultanément de multiples mécanismes cellulaires d'infectivité provoqués par un coronavirus, ses variants et mutants Download PDF

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WO2022173456A1
WO2022173456A1 PCT/US2021/024182 US2021024182W WO2022173456A1 WO 2022173456 A1 WO2022173456 A1 WO 2022173456A1 US 2021024182 W US2021024182 W US 2021024182W WO 2022173456 A1 WO2022173456 A1 WO 2022173456A1
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pharmaceutical
range
micronutrient composition
micronutrient
acid
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PCT/US2021/024182
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English (en)
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Matthias W. Rath
Aleksandra Niedzwiecki
Anna Goc
Vadimo IVANOV
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Rath Matthias W
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Priority to EP21801817.4A priority Critical patent/EP4064861A4/fr
Priority to DE212021000181.5U priority patent/DE212021000181U1/de
Publication of WO2022173456A1 publication Critical patent/WO2022173456A1/fr

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    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A23L33/105—Plant extracts, their artificial duplicates or their derivatives
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A23L33/15—Vitamins

Definitions

  • a pharmaceutical micronutrient composition and its use to simultaneously inhibit multiple cellular mechanisms of infectivity caused by coronavirus, its variants and mutants CROSS REFERENCE TO RELATED APPLICATION [001] The current application claims priority to pending US provisional application 63149633 filed on 15 th February 2021. The said US provisional application is hereby incorporated by reference in its entireties for all of its teachings. FIELD OF STUDY [002] This application discloses pharmaceutical micronutrient composition and shows that the said composition simultaneously inhibits multiple cellular mechanisms of infectivity caused by coronavirus and its variants and mitigates coronavirus infection in mammals.
  • coronavirus is a rapidly mutating virus and, within one year of the pandemic, several mutations of this virus have emerged in United Kingdom, South Africa, Brazil and other countries, with each of these mutations potentially giving rise to further coronavirus subtypes.
  • Clinical reports show that the British mutation of the coronavirus can infect patients who have received the vaccine developed against the original coronavirus SARS-CoV-2, thereby challenging any claim of a universal efficacy of the available vaccines against all coronavirus mutations.
  • coronaviruses including SARS-CoV-2
  • SARS-CoV-2 The cell entry mechanisms of coronaviruses, including SARS-CoV-2, have been extensively studied. To enter host cells, coronaviruses first bind to a cell surface receptor for viral attachment, subsequently enter cell endosomes, and eventually fuse viral and lysosomal membranes (Li et al., 2016). Coronavirus entry is mediated by a spike protein anchored on the surface of the virus.
  • the spike S1 protein on SARS-CoV-2 contains a receptor-binding domain (RBD) that specifically recognizes its cellular receptor, angiotensin-converting enzyme 2 (ACE2).
  • RBD receptor-binding domain
  • ACE2 angiotensin-converting enzyme 2
  • the receptor-binding domain on SARS-CoV-2 spike protein part S1 head binds to a target cell using the human ACE2 (hACE2) receptor on the cell surface and is proteolytically activated by human proteases.
  • hACE2 human ACE2
  • Coronavirus entry into host cells is an important determinant of viral infectivity and pathogenesis (Du et al, 2009, Du et al.2017).
  • the cellular receptor for the virus binding is angiotensin-converting enzyme 2 or ACE2, which is an integral membrane protein present on many cells throughout the human body, with strong expression in the heart, vascular system, gastrointestinal system and kidneys, as well as in type II alveolar cells in the lungs.
  • ACE2 angiotensin-converting enzyme 2
  • TMPRSS2 transmembrane protease
  • furin furin
  • cathepsins as well as RNA-dependent RNA polymerase (RdPp) catalyzing viral RNA multiplication.
  • COVID-19 infections have been associated with a high inflammatory response in the host, termed a “cytokine storm”, thrombosis and other patho-mechanisms that can trigger a fateful cascade of clinical events associated with advanced coronavirus infections.
  • cytokine storm a high inflammatory response in the host
  • thrombosis a high inflammatory response in the host
  • other patho-mechanisms that can trigger a fateful cascade of clinical events associated with advanced coronavirus infections.
  • the ability of such new approaches to ameliorate such infection-related complications should be an additional target.
  • preventive and therapeutic strategies for inhibiting the infective mechanisms of all coronaviruses – irrespective of mutation and/or subtype – thereby offering new avenues towards the global control of the pandemic.
  • the instant pharmaceutical micronutrient composition prevents, inhibits, treats and delays attachment, penetration, biosynthesis, maturation and release of a coronavirus SARS- Cov-2 virus in a mammal.
  • the phytochemicals in combination with other vitamins prevents various steps of infection in a mammal.
  • various combinations of individual micronutrients are called mixtures.
  • mixture D a pharmaceutical micronutrient composition is made up of resveratrol, cruciferous plant extract, curcumin, quercetin, naringenin, baicalein, theaflavin, vitamin C and N-actylcysteine.
  • a pharmaceutical micronutrient compound comprises an ascorbate in the range of 10 mg to 200,000 mg, N-acetylcysteine in the range of 2 mg to 30,000mg, theaflavins in the range 5 mg to 3,000 mg, resveratrol in the range of 10 mg to 5,000 mg, cruciferous plant extracts in the range of 5 mg to 5000 mg (or equivalent amount of its active compound, sulforaphane), curcumin in the range of 5 mg to 10,000mg, quercetin in the range of 5 mg to 2,000mg , naringenin in the range of 5 mg to 3,000 mg, and baicalein in the range of 5 mg to 3,000mg.
  • additional micronutrients are added to form a pharmaceutical micronutrient compound such as a phenolic acid, gallic acid, tannic acid, chlorogenic acid and rosmarinic acid; a flavonoid such as fisetin, morin, myricetin, kaempferol, rutin, luteolin, baicalin, scutellarin, naringenin, hesperidin, hesperetin, apigenin, genistein, phloroglucinol, schisandrin, urolithin A, punicalagin, brazilin, hispidulin, papaverine, silymarin, procyanidin B2, procyanidin B3, stilbenes and pterostilbene; an alkaloid such as palmatine, berberine, cannabidiol, castanospermine, usnic acid, malic acid, terpenes, D-limonene and carnosic acid.
  • a pharmaceutical micronutrient compound such as
  • 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 5 mg to 3,000 mg, resveratrol in the range of 10 mg to 5,000 mg, cruciferous plant extracts in the range of 5 mg to 5,000 mg (or equivalent amount of its active compound, sulforaphane), curcumin in the range of 5 mg to 10,000 mg, quercetin in the range of 5 mg to 2,000mg , naringenin in the range of 5 mg to 3,000 mg, and baicalein in the range of 5 mg to 3,000 mg.
  • 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 or another pharmaceutically acceptable form of ascorbate.
  • the pharmaceutical micronutrient composition further consists of at least one of the theaflavins in the range 5 mg to 3,000 mg, resveratrol in the range of 10 mg to 5,000 mg, cruciferous plant extracts in the range of 5 mg to 5,000mg, curcumin in the range of 5 mg to 10,000 mg, quercetin in the range of 5 mg to 2,000mg, and a combination thereof.
  • a pharmaceutically acceptable formulation for various forms of use, such as oral, injectable, absorbable, etc.
  • the pharmaceutical micronutrient composition is in the form of oral, non- invasive peroral, topical (for example, transdermal), enteral, transmucosal, targeted delivery, sustained-release delivery, delayed release, pulsed release and parenteral methods.
  • the viral infection and/or viral disease uses a cellular receptor for a viral entry on a surface of an epithelial cells, endothelial cells and/or other cell types.
  • the viral infection and/or viral disease is that which uses an angiotensin converting enzyme 2 (ACE2) receptor on the surface of an epithelial cell, endothelial cell and other cell types, for the viral entry, is treated, prevented and mitigated using pharmaceutical micronutrient composition.
  • the pharmaceutical micronutrient composition in one embodiment, is used to treat the human and other species with severe acute respiratory syndrome-related coronaviruses (SARS- CoV-1, SARS-CoV2 and their variants) that use 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 in one embodiment, is used to treat the human and other species with Middle East respiratory syndrome-related coronavirus (MERS- CoV), and its variants that use the angiotensin converting enzyme 2 (ACE2) receptors on the surface of epithelial cells, endothelial cells and other cell types, for viral entry.
  • MERS- CoV Middle East respiratory syndrome-related coronavirus
  • ACE2 angiotensin converting enzyme 2
  • the pharmaceutical micronutrient composition in one embodiment, is mixture D, which is used in humans to treat, prevent, inhibit and stop inflammation caused by severe acute respiratory syndrome-related coronaviruses (SARS-CoV-1, SARS-CoV-2 and their variants), and Middle East respiratory syndrome-related coronavirus (MERS-CoV) and its variants.
  • Figure 4A, 4B and 4C show viability of cells upon treatment with indicated polyphenols for 1h, 3h, and 48h.
  • Figures 5A and 5B show SARS-CoV-2 pseudo-virions binding to cells at different patterns of treatment.
  • Figures 6A and 6B show SARS-CoV-2 pseudo-virions’ entry to cells at different pattern of treatment.
  • Figures 7A, 7B, 7C, 7D, 7E, 7F, 7G, 7H, 7I, 7J, 7K show images of syncytia taken after treatment with indicated polyphenols.
  • Figure 8 shows quantification of syncytia after treatment with indicated polyphenols.
  • Figure 9 shows selection of the most effective formulation based on RBD to ACE2 binding inhibition of various micronutrient mixtures.
  • Figure 10 shows the test for safety for mixture D in human small alveolar epithelial cells.
  • Figure 11 shows inhibition of RBD binding and efficacy of the fMixture alone and its combination with Vitamin D.
  • Figure 12 shows inhibition of cellular internalization of the mutated forms of SARS- CoV-2: viral strains from the UK, Brazil, and South Africa.
  • Figure 13 shows inhibition of cellular entry of the mutated forms of SARS-CoV-2: viral strains from the UK, Brazil, and South Africa, upon application of different patterns of treatment.
  • Figure 14 shows inhibition of ACE2 expression under normal and pro-inflammatory conditions.
  • Figure 15 shows inhibition of viral RNA-dependent RNA polymerase (RdRp) activity by mixture D with and without vitamin D.
  • Figure 16 shows inhibition of furin activity by mixture D.
  • Figure 17 shows inhibition of cellular activity of native cathepsin L by mixture D applied individually and with vitamin D.
  • Figure 18 shows mixture D’s inhibitory effect on activity of recombinant cathepsin L and the effects of additional vitamin D.
  • Figure 19 shows anti-inflammatory effect: inhibition of IL6 secretion under normal and pro-inflammatory conditions by mixture D alone and combined with vitamin D.
  • the life cycle of the virus 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 through endocytosis or membrane fusion (penetration). Once viral contents are released inside the host cells, viral RNA enters the nucleus for replication. Viral messenger RNA (mRNA) is used to make viral proteins (biosynthesis). New viral particles are then made (maturation) and released. Coronaviruses consist of four structural proteins: spike (S), membrane (M), envelope (E) and nucleocapsid (N).
  • S spike
  • M membrane
  • E envelope
  • N nucleocapsid
  • Spike is composed of a transmembrane trimetric glycoprotein protruding from the viral surface, which determines the diversity of coronaviruses and host tropism.
  • the most effective approach to viral infectivity suppression is by identifying molecules that are able to safely regulate and/or inhibit the expression of infection-pathway-related proteins.
  • Figure 1A shows the cellular mechanism of viral entry and several entry points for the SARS-CoV-2 virus and others through ACE2 receptors, which, having entered, require furin and cathepsin L for replication, protein synthesis, maturation and release into the bloodstream.
  • Figure 1B shows the systemic effect of the release of interleukin 6 (IL-6) in response to inflammation caused by viral infection.
  • IL-6 may be a therapeutic target for inhibiting the cytokine storm and cytokine storm-associated organ damage. We would show that this is a good target to prevent organ damage.
  • the safest and most effective molecules able to exert such a regulatory role are natural compounds, namely micronutrients. These natural compounds are by their very nature able to affect simultaneously, multiple biochemical processes in cellular metabolism.
  • a “mammal” to be treated by the subject method may mean either a human or non- human animal, such as mice, primates and vertebrates.
  • the specific diseases that would be targets for a treatment using a pharmaceutical micronutrient composition are infections caused by SARS-CoV-2, SARS-CoV-2 variants (such as the UK, Nigeria, South Africa and Brazil variants, and 19 other 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 their subtypes, four main sub-groupings of coronaviruses, known as alpha, beta, gamma and delta.
  • 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 their subtypes, four main sub-groupings of coronaviruses, known as alpha, beta, gamma and delta.
  • Cell-Cell fusion assay was performed according to Ou et al. Briefly, A549 cells transduced with eGFP-luciferase-SARS-CoV-2 spike S1 lentivirus vector (GenScript, Piscataway, NJ) were detached with 1 mM EDTA, treated with indicated concentrations of selected polyphenols for 1h. at 37°C and overlaid on 80-95% confluent human A549 lung epithelial cells overexpressing hACE2. After 4h. incubation at 37oC, images of syncytia were captured with a Zeiss Axio Observer A1 fluorescence microscope (Carl Zeiss Meditec, Inc, Dublin, CA).
  • ACE-2 expression assay Human Small Airways Epithelial Cells (HSAEpC) were supplied by ATCC (American Type Culture Collection, Manassas, VA) and cultured in Small Airways Epithelial Cells culture medium (ATCC). HSAEpC cells were seeded in 96-well plates covered with collagen at 6 passage and grown to confluent layer. Cell culture medium was supplemented with indicated amounts of mixture D and 50 mcg/ml ascorbic acid in 100 mcl per well. After 72h. cells were supplemented with fresh medium and the same addition for another 72h.
  • ATCC American Type Culture Collection, Manassas, VA
  • ATCC Small Airways Epithelial Cells culture medium
  • Receptor binding and entry assays cell lines and pseudoviruses: Human alveolar epithelial cell line A549 was obtained from ATCC. Human alveolar epithelial cell line A549, stably overexpressing hACE2 receptor (hACE2/A549), was obtained from GenScript (Piscataway, NJ). Both cell lines were maintained in Dulbecco’s MEM containing 10% fetal bovine serum (FBS), 100 U/ml penicillin and 100 ⁇ g/ml streptomycin.
  • FBS fetal bovine serum
  • Pseudovirus particles with spike glycoprotein as the envelope protein with eGFP and luciferase (eGFP-luciferase- SARS-CoV-2 spike glycoprotein pseudotyped particles) and pseudotyped ⁇ G-luciferase (G* ⁇ G- luciferase) rVSV, were purchased from Kerafast (Boston, MA).
  • Bald pseudovirus particles with eGFP and luciferase (eGFP-luciferase-SARS-CoV-2 pseudo-typed 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 ammonia chloride were purchased from Sigma (St. Louis, MO). All other polyphenols and camostat mesylate were obtained from Cayman Chemical Company (Ann Arbor, MI). All antibodies were from Santa Cruz Biotechnology (Santa Cruz, CA).
  • TMPRSS2 recombinant protein was from Creative BioMart (Shirley, NY).
  • SARS-CoV-2 RBD binding to hACE2 Binding/neutralization reaction was performed using a SARS-CoV-2 surrogate virus neutralization test kit that can detect either antibody or inhibitors that block the interaction between the receptor binding domain (RBD) of the SARS- CoV-2 spike protein and the hACE2 cell surface receptor (GenScript, Piscataway, NJ).
  • RBD receptor binding domain
  • GenScript Piscataway, NJ
  • polyphenols at 100 ⁇ g/ml were incubated with either HRP-conjugated receptor-binding domain (RBD fragment) of SARS-CoV-2 spike S1 domain, or with hACE2 immobilized on 96-well plate for 30 min.
  • RBD binding This assay was performed using a GenScript SARS-CoV-2 surrogate virus neutralization test kit that can detect either antibody or inhibitors that block the interaction between the RBD of the viral spike protein with the ACE2 cell surface receptor. All test samples with indicated concentrations, and positive and negative controls (provided by the manufacturer) were diluted with the sample dilution buffer with a volume ratio of 1:9. In separate tubes, HRP- conjugated RBD was also diluted with the HRP dilution buffer with a volume ratio of 1:99. Binding/neutralization reaction was performed according to manufacturer’s protocol.
  • diluted positive and negative controls as well as the test samples with indicated concentrations were mixed with the diluted HRP-RBD solution with a volume ratio of 1:1 and incubated for 30 min. at 37oC.
  • 100 ⁇ L each of the positive control mixture, negative control mixture, and the test sample mixtures were added to the corresponding wells with immobilized ACE2 receptor and incubated for 15 min. at 37oC.
  • the plates were washed four times with 260 ⁇ l/well of the 1 x wash solution, and TMB solution was added to each well (100 ⁇ l/well). Plates were incubated in the dark at room temperature for up to 5 min.
  • binding of pseudo-typed virion mutants of SARS-CoV-2 to hACE2 receptor The experiment was conducted according to GenScript recommendations with small modifications. Briefly, eGFP-luciferase-SARS-CoV-2 spike protein encapsulated pseudo-virions were incubated at 37oC with 5 and 10 ⁇ g/ml of mixture D and simultaneously added to hACE2/A549 cells. Cells were incubated for an additional 1h. at 37oC.
  • Cathepsin L activity assay 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 with mixture D at 5 and 10 ⁇ g/ml concentrations for 24h. were washed with cold 1 x PBS, and lysed 100 ⁇ l with CL buffer for 8 min. After 3 minutes of centrifugion at 4°C, supernatants were collected and enzymatic reaction was set up by mixing 50 ⁇ l of treated sample, 50 ⁇ l of control sample, 50 ⁇ l of background control sample, 50 ⁇ l of positive and negative controls.
  • mixture D at 5.0 and 10 ⁇ g/ml concentrations was added to cathepsin L (0.2 mU/ ⁇ l) for 15 mins at 22oC, prior to fluorogenic substrate (Ac-FR-AFC) (10 ⁇ M) addition and incubation for 60 mins at RT.
  • Positive control contained only cathepsin L, and negative control containing cathepsin L and cathepsin L inhibitor E64d (25 ⁇ M).
  • Results were calculated with Microsoft Excel software and presented as a percentage of unsupplemented controls (an average of three repetitions +/- standard deviation).
  • In vitro RdRp activity was examined using a SARS-CoV-2 RNA Polymerase Assay Kit (ProFoldin, Hudson, MA) according to the manufacturer’s protocol.
  • IL-6 assay Human Small Airways Epithelial Cells (HSAEpC) were supplied by ATCC and cultured in Small Airways Epithelial Cells culture medium (ATCC). SAEC cells were seeded in six-well plates covered with collagen at 6 passage and grown to confluent layer.
  • Table 4 Binding ability of selected plant extracts and their major components, to RBD of SARS-CoV-2 and to ACE2 receptor.
  • the inhibitory effect of these most effective polyphenols, curcumin, theaflavin-3’3-digallate and brazilin, on RBD-hACE2 binding was dose dependent and ranged from 20% to 95% at the concentrations from 2.5-10 ⁇ g/ml, respectively.
  • test polyphenols showed different efficacy on cell transduction by the pseudo-virions.
  • curcumin showed significant inhibitory effect at lower concentrations compared with brazilin and theaflavin-3’3-digallate.
  • SARS-CoV-2 virions to curcumin for 1h. before and simultaneously with adding to hACE2/A549 cells resulted in inhibition of transduction starting from its 5.0 ⁇ g/ml concentration.
  • Higher (10 ⁇ g/ml) concentrations of brazilin and theaflavin-3,3’-digallate were required to achieve statistically significant inhibitory effects using the same patterns of exposure, All test polyphenols added 1h.
  • FIG. 7A, 7B, 7C, 7D, 7E, 7F, 7G, 7H, 7I, 7J, 7K and Figure 8 show the effect of test polyphenols on fusion to the human ACE2 receptor overexpressing A549 cells.
  • A549 cells expressing eGFP spike protein were pre-treated with indicated polyphenols at different concentrations for 1h. at 37oC and co-cultured for an additional 4h. at 37oC with A549 cells stably expressing human ACE2 receptor.
  • the scale bar indicates 250 ⁇ m.
  • Figure 9 shows Mixture D (resveratrol, cruciferous plant extract, curcumin, quercetin, naringenin, baicalin, theaflavin, vitamin C and N-acetylcysteine) gives the best inhibition of binding.
  • Figure 10 shows the safety of the mixture D on human alveolar cells. The pharmaceutical micronutrient composition mixture D was applied at 5 and 10 mcg/ml doses individually and in combinations with vitamin D and was safe to be used on human small alveolar epithelial cells.
  • Figure 11 shows inhibition of RBD binding of the mixture D alone and its combination with vitamin D.
  • Mixture D (10 mcg/ml) added simultaneously with mutated virions to cells overexpressing ACE2 was equally effective in inhibiting cellular entry of these mutated forms of SARS-CoV-2: by 48% for UK mutation, by 47% for Brazilian mutation, by 48% for South African mutation. These effects were concentration dependent. Exposure of viral particles to the mixture D for 1h, before combining them with cells also inhibited cellular entry of these viral mutants by up to 40%. These results not only show efficacy for inhibiting cellular entry by viral strains but also show that the direct exposure of viral particles to this pharmaceutical micronutrient compound helps to prevent the viral entry.
  • Figure 13 shows inhibition of cellular entry by mutated forms of SARS-CoV-2, viral strains from the UK, Brazil and South Africa, owing to the inhibitory effect of the mixture D when applied simultaneously with the virions and cells.
  • Figure 14 shows inhibition of ACE2 expression under normal and pro-inflammatory conditions. Exposure of human small alveolar epithelial cells to the mixture D for 6 days resulted in inhibition of ACE2 expression by 73% at 12 mcg/ml. This inhibitory effect of the mixture D on ACE2 expression persisted and was even enhanced under pro-inflammatory conditions (inhibition between 83-86%).
  • Figure 15 shows inhibition of viral RdRp activity and effects of vitamin D.
  • Figure 16 shows inhibition of furin activity in the cells, owing to mixture D activity.
  • Mixture D applied individually at 10 mcg/ml could decrease furin activity by 33%, and at 20 mcg/ml by 52%.
  • Figure 17 shows the test results of inhibition of cellular activity of cathepsin L by mixture D and the effects of vitamin D and Mixture D.
  • Mixture D applied to the cells individually and in combination with vitamin D shows 20% inhibition of cathepsin L activity.
  • Figure 18 shows anti-inflammatory effect: inhibition of IL-6 secretion under normal and pro-inflammatory conditions by the mixture D alone and combined with vitamin D.
  • Mixture D (10 mcg/ml) applied to small alveolar endothelial cells for 3 days decreased IL-6 secretion by 50%.
  • Exposure of HSAEpC to lipopolysaccharide (LPS, 5 mcg/ml) increased IL-6 secretion by 43%. Under this pro-inflammatory condition, the mixture D could inhibit IL-6 secretion by 55%.
  • Drug formulations suitable for these administration routes can be produced by adding one or more pharmacologically acceptable carrier to the agent and then treating the micronutrient composition through a routine process known to those skilled in the art.
  • the mode of administration includes, but is not limited to, non-invasive peroral, topical (for example, transdermal), enteral, transmucosal, targeted delivery, sustained-release delivery, delayed release, pulsed release and parenteral methods.
  • Peroral administration may be administered both in liquid and dry state.
  • pharmaceutical micronutrient composition would be more specifically mixture D.
  • Formulations suitable for oral administration may be in the form of capsules, cachets, pills, tablets, lozenges (using a flavored bases, usually sucrose and acacia or tragacanth), powders, granules, or as a solution or a 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 pastilles (using an inert base, such as gelatin and glycerin or sucrose and acacia), each containing a predetermined amount of a subject composition as an active ingredient.
  • Subject compositions may also be administered as a bolus, electuary or paste.
  • an oral solid drug product When an oral solid drug product is prepared, pharmaceutical micronutrient composition is mixed with an excipient (and, if necessary, one or more additives such as a binder, a disintegrant, a lubricant, a coloring agent, a sweetening agent, and a flavoring agent), and the resultant mixture is processed through a routine method, to thereby produce an oral solid drug product such as tablets, coated tablets, granules, powder or capsules.
  • Additives may be those generally employed in the art.
  • 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, carboxymethylcellulose, hydroxypropyl cellulose, hydroxypropyl starch, methyl cellulose, ethyl cellulose, shellac, calcium phosphate and polyvinyl pyrrolidone.
  • Disintegrants include dried starch, sodium arginate, powdered agar, sodium hydroxy carbonate, calcium carbonate, sodium lauryl sulfate, monoglyceryl stearate and lactose.
  • Lubricants include purified talc, stearic acid salts, borax and polyethylene glycol. Sweetening agents include sucrose, orange peel, citric acid and tartaric acid.
  • liquid drug product for oral administration When a liquid drug product for oral administration is prepared, pharmaceutical micronutrient composition is mixed with an additive such as a sweetening agent, a buffer, a stabilizer, or a flavoring agent, and the resultant mixture is processed through a routine method, to produce an orally administered liquid drug product such as an internal solution medicine, syrup or elixir.
  • a sweetening agent include vanillin
  • examples of the buffer include sodium citrate
  • stabilizer include tragacanth, acacia, and gelatin.
  • compositions containing pharmaceutical micronutrient composition for rectal or vaginal administration may be presented as a suppository, which may be prepared by mixing a subject composition with one or more suitable non-irritating carriers, comprising, for example, cocoa butter, polyethylene glycol, a suppository wax or a salicylate, which is solid at room temperature, but liquid at body temperature and, therefore, will melt in the appropriate body cavity and release the encapsulated compound(s) and composition(s).
  • suitable non-irritating carriers comprising, for example, cocoa butter, polyethylene glycol, a suppository wax or a salicylate, which is solid at room temperature, but liquid at body temperature and, therefore, will melt in the appropriate body cavity and release the encapsulated compound(s) and composition(s).
  • Formulations that are suitable for vaginal administration also include pessaries, tampons, creams, gels, pastes, foams or spray formulations containing such carriers as are known in the art to be appropriate.
  • a targeted-release portion for capsules containing pharmaceutical micronutrient composition can be added to the extended-release system by means of either applying an immediate-release layer on top of the extended release core; using coating or compression processes, or in a multiple-unit system such as a capsule containing extended- and immediate- release beads.
  • sustained release is art recognized.
  • a therapeutic composition that releases a substance over time may exhibit sustained-release characteristics, in contrast to a bolus type administration in which the entire amount of the substance is made biologically available at one time.
  • one or more of the pharmaceutically acceptable excipients may undergo gradual or delayed degradation (e.g., through hydrolysis), with concomitant release of any material incorporated therein, e.g., a therapeutic and/or biologically active salt and/or composition, for a sustained or extended period (as compared with the release from a bolus). This release may result in prolonged delivery of therapeutically effective amounts of any of the therapeutic agents disclosed herein.
  • sustained-release formulations in which the pharmaceutical micronutrient composition is released over a period of time in a controlled manner from a formulation.
  • sustained release formulations include liposomes, drug-loaded biodegradable microspheres and pharmaceutical micronutrient composition polymer conjugates.
  • Delayed-release dosage formulations are created by coating a solid dosage form with a film of a polymer, which is insoluble in the acid environment of the stomach, but soluble in the neutral environment of the small intestine.
  • the delayed-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 either a tablet or a capsule.
  • Preferred coating materials include bioerodible, gradually hydrolysable, gradually water-soluble, and/or enzymatically degradable polymers, and may be conventional “enteric” polymers.
  • Enteric polymers become soluble in the higher pH environment of the lower gastrointestinal tract, or slowly erode as the dosage form passes through the gastrointestinal tract, while enzymatically degradable polymers are degraded by bacterial enzymes present in the lower gastrointestinal tract, particularly in the colon.
  • a delayed-release tablet may be formulated by dispersing a drug within 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 ester, 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 for providing a delayed-release matrix.
  • Fatty compounds for use as a matrix material include, but are not limited to, waxes (e.g., carnauba wax) and glycerol tristearate.
  • a pulsed-release dosage is one that mimics a multiple dosing profile without repeated dosing, and typically allows at least a twofold reduction in dosing frequency as compared with the drug presented as a conventional dosage form (e.g., as a solution or prompt drug-releasing, conventional solid dosage form).
  • a pulsed-release profile is characterized by a time period of no release (lag time) or reduced release, followed by rapid drug release. These can be formulated for critically ill patients using the instant pharmaceutical micronutrient composition.
  • parenteral administration and “administered parenterally” 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, intra- arterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intra-articular, subcapsular, subarachnoid, intraspinal and intrastemal injection and infusion.
  • compositions disclosed herein suitable for parenteral administration, comprise one or more subject compositions in combination with one or more pharmaceutically acceptable sterile, isotonic, aqueous, or non-aqueous solutions, dispersions, suspensions or emulsions, or sterile powders, which may be reconstituted into sterile injectable solutions or dispersions just prior to use, and which may contain antioxidants, buffers, bacteriostats, solutes that render the formulation isotonic within the blood of the intended recipient, or suspending or thickening agents.
  • an injection product When an injection product is prepared, pharmaceutical micronutrient composition is mixed with an additive such as a pH regulator, a buffer, a stabilizer, an isotonicity agent or a local anesthetic, and the resultant mixture is processed through a routine method, to thereby produce an injection for subcutaneous injection, intramuscular injection, or intravenous injection.
  • an additive such as a pH regulator, a buffer, a stabilizer, an isotonicity agent or a local anesthetic
  • 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
  • examples of the isotonicity agent include sodium chloride and glucose.
  • Adjuvants are used to enhance the immune response. Various types of adjuvants are available. Haptens and Freund′s adjuvant may also be used to produce water-in-oil emulsions of immunogens.
  • pharmaceutically acceptable is art recognized. In certain embodiments, the term includes compositions, polymers and other materials and/or dosage forms that are within the scope of sound medical judgment, suitable for use in contact with the tissues of mammals, both human beings and animals, without excessive toxicity, irritation, allergic response or other problem or complication, commensurate with a reasonable benefit-risk ratio.
  • pharmaceutically acceptable carrier includes, for example, pharmaceutically acceptable materials, compositions or vehicles, such as a liquid or solid filler, diluent, solvent or encapsulating material involved in carrying or transporting any subject composition from one organ or portion of the body, to another organ or portion of the body.
  • a pharmaceutically acceptable carrier is non-pyrogenic.
  • materials that may serve as pharmaceutically acceptable carriers include: (1) sugars, such as lactose, glucose and sucrose; (2) starches, such as corn starch 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) pyr
  • the pharmaceutical micronutrient compositions described herein are formulated in a manner such that said compositions will be delivered to a mammal in a therapeutically effective amount, as part of a prophylactic, preventive or therapeutic treatment to overcome the infection caused by corona viruses (irrespective of the type).
  • the dosage of the pharmaceutical micronutrient compositions which may be referred to as therapeutic composition provided herein, may be determined by reference to the plasma concentrations of the therapeutic composition or other encapsulated materials. For example, the blood samples may be tested for their immune response to their corresponding viral load or lack thereof.
  • the therapeutic pharmaceutical micronutrient composition provided by this application may be administered to a subject in need of treatment by a variety of conventional routes of administration, including orally, topically, parenterally, e.g., intravenously, subcutaneously or intramedullary. Further, the therapeutic compositions may be administered intranasally, as a rectal suppository, or using a “flash” formulation, i.e., allowing the medication to dissolve in the mouth without the need to use water. Furthermore, the compositions may be administered to a subject in need of treatment by controlled-release dosage forms, site-specific drug delivery, transdermal drug delivery, patch-mediated drug delivery (active/passive), by stereotactic injection, or in nanoparticles.
  • an active ingredient can be present in the therapeutic compositions of the present invention for localized use via the cutis, intranasally, pharyngolaryngeally, bronchially, intravaginally, rectally or ocularly.
  • the active ingredients can be packaged in a pressurized aerosol container together with a gaseous or liquefied propellant, for example dichlorodifluoromethane, carbon dioxide, nitrogen, propane and the like, with the usual adjuvants such as cosolvents and wetting agents, as may be necessary or desirable.
  • the most common routes of administration also include the preferred transmucosal (nasal, buccal/sublingual, vaginal, ocular and rectal) and inhalation routes.
  • the subject pharmaceutical micronutrient composition of the present application may be lyophilized or subjected to another appropriate drying technique such as spray drying.
  • the subject compositions may be administered once, or may be divided into a number of smaller doses to be administered at varying intervals of time, depending in part on the release rate of the compositions and the desired dosage.
  • Formulations useful in the methods provided herein include those suitable for oral, nasal, topical (including buccal and sublingual), rectal, vaginal, aerosol and/or parenteral administration.
  • the formulations may conveniently be presented in unit dosage form and may be prepared by any methods well known in the art of pharmacy.
  • the amount of a subject pharmaceutical micronutrient composition that may be combined with a carrier material to produce a single dose may vary depending upon the subject being treated and the particular mode of administration.
  • the therapeutically acceptable amount described herein may be administered in inhalant or aerosol formulations.
  • the inhalant or aerosol formulations may comprise one or more agents, such as adjuvants, diagnostic agents, imaging agents, or therapeutic agents useful in inhalation therapy.
  • the final aerosol formulation may, for example, contain 0.005-90% w/w, for instance 0.005-50%, 0.005-5% w/w, or 0.01-1.0% w/w, of medicament relative to the total weight of the formulation.
  • aqueous and non-aqueous carriers examples 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.
  • polyols such as glycerol, propylene glycol, polyethylene glycol and the like
  • vegetable oils such as olive oil
  • injectable organic esters such as ethyl oleate.
  • Proper fluidity may be maintained, for example by the use of coating materials such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants.

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  • Life Sciences & Earth Sciences (AREA)
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  • Health & Medical Sciences (AREA)
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  • Chemical & Material Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Food Science & Technology (AREA)
  • Polymers & Plastics (AREA)
  • Botany (AREA)
  • Acyclic And Carbocyclic Compounds In Medicinal Compositions (AREA)

Abstract

La composition pharmaceutique de micronutriments comprenant le mélange D dans cette étude aide à atténuer, inhiber, prévenir et stopper des maladies provoquées par des infections virales. Le coronavirus associé au syndrome respiratoire du Moyen-Orient et le coronavirus associé au syndrome respiratoire aigu sévère, ainsi que leurs variants et mutants affectant les mammifères et provoquant une infection, sont traités avec succès à l'aide d'un mélange D. Le mélange D contient des micronutriments clés tels qu'un ascorbate, la N-acétylcystéine, des théaflavines, le resvératrol, des extraits de plantes crucifères, la curcumine, la quercétine, la naringénine et la baïcaline et une combinaison de ceux-ci. Des micronutriments supplémentaires ont été testés avec le mélange D et semblent avoir des effets bénéfiques.
PCT/US2021/024182 2021-02-15 2021-03-25 Composition pharmaceutique de micronutriments et son utilisation pour inhiber simultanément de multiples mécanismes cellulaires d'infectivité provoqués par un coronavirus, ses variants et mutants WO2022173456A1 (fr)

Priority Applications (2)

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EP21801817.4A EP4064861A4 (fr) 2021-02-15 2021-03-25 Composition pharmaceutique de micronutriments et son utilisation pour inhiber simultanément de multiples mécanismes cellulaires d'infectivité provoqués par un coronavirus, ses variants et mutants
DE212021000181.5U DE212021000181U1 (de) 2021-02-15 2021-03-25 Eine pharmazeutische Mikronährstoffzusammensetzung und ihre Verwendung zur gleichzeitigen Hemmung mehrerer zellulärer Mechanismen der Infektiosität, die durch Coronaviren, ihre Varianten und Mutanten verursacht werden

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US20070087063A1 (en) * 2001-02-02 2007-04-19 Metagenics, Inc. Medical composition for balancing bodily processes
US7238373B2 (en) * 2003-04-04 2007-07-03 Nutritox Llc Nutritional supplement
US20100150895A1 (en) * 2005-11-23 2010-06-17 Elizabeth Mazzio Nutraceutical agent for attenuating the Neurodegenerative process associated with Parkinson's disease
WO2011046423A1 (fr) * 2009-10-15 2011-04-21 Purecircle Sdn Bhd Rébaudioside d hautement pur, et applications correspondantes

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Publication number Priority date Publication date Assignee Title
US20070087063A1 (en) * 2001-02-02 2007-04-19 Metagenics, Inc. Medical composition for balancing bodily processes
US7238373B2 (en) * 2003-04-04 2007-07-03 Nutritox Llc Nutritional supplement
US20100150895A1 (en) * 2005-11-23 2010-06-17 Elizabeth Mazzio Nutraceutical agent for attenuating the Neurodegenerative process associated with Parkinson's disease
WO2011046423A1 (fr) * 2009-10-15 2011-04-21 Purecircle Sdn Bhd Rébaudioside d hautement pur, et applications correspondantes

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MAZZIO ELIZABETH A., CLOSE FRAN, SOLIMAN KARAM F.A.: "The Biochemical and Cellular Basis for Nutraceutical Strategies to Attenuate Neurodegeneration in Parkinson’s Disease", INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES, vol. 12, no. 1, 1 January 2011 (2011-01-01), pages 506 - 569, XP055963828, DOI: 10.3390/ijms12010506 *

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