Antiviral compositions and methods of use thereof

Abstract

Provided are antiviral compositions containing a cationic antiviral agent (cationic agent) and a copper salt for preventing, controlling, or treating viral infections in the nasopharynx and throat area of ​​mammals, particularly humans and animals, and methods for preventing, controlling, or treating viral infections in mammals.

Description

[Technical Field]

[0001] REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Patent Application No. 63 / 103,881, filed August 31, 2020.

[0002] The present invention relates to antiviral compositions containing a cationic antiviral agent (cationic drug) and a copper salt for controlling viral infections in the nasopharyngeal and throat region of humans and animals. [Background technology]

[0003] The Severe Acute Respiratory Syndrome (SARS) virus and the Covid (COVID) virus were first discovered in China and have spread rapidly throughout Asia, Europe, North America, etc. It is generally believed that the viruses are transmitted through breathing in particles of saliva or phlegm from patients with these diseases.

[0004] The Covid virus infection pandemic has demonstrated the complexity of effective treatment.Recently, the Covid and SAR infection pandemics have led to an urgent search for optimal tools to limit the spread of the disease.

[0005] The coronavirus family includes alpha coronavirus 229E, NL63, beta coronavirus 43, HKU1 and human coronavirus MER 6-COV C, Middle East respiratory syndrome. SAR-COV beta coronavirus, which causes the respiratory syndrome SARS, and SAR-COV-2, a novel coronavirus, which causes coronavirus 2019, Covid-19.

[0006] Populations worldwide are frequently infected with human coronaviruses 229E, NL63, OC43, and HKU1. Covid-19 is believed in the art to be an excellent experimental model for determining the biological activity of synergistic test combinations against the Covid virus.

[0007] The antibacterial function of cationic surfactants is well known in the art for various applications as antibacterial (or anti-germ) agents, such as in water / oil emulsions in nanoparticles as disclosed in U.S. Pat. No. 8,877,208.

[0008] Copper salts are used to fight infections (Gadi, Borkov Current Chemical Biology 2012, 6; Borkov, G et al 2007, Antimicrobial Agents Chemotherapy Vol 51 p 2605).

[0009] Cationic surfactants derived from lauric acid and arginine, particularly the ester of lauramide of arginine monohydrate, hereinafter designated LAE, can be used to protect against viruses. LAE and its derivatives are described in WO 2008 / 014824, the entirety of which is incorporated herein by reference.

[0010] The most common cationic antibacterial or antiviral agents are the quaternary ammonium compounds disclosed in U.S. Pat. Nos. 2,984,639, 3,325,402, 3,431,208 and British Patent No. 1,319,396, each of which is incorporated herein by reference in its entirety.

[0011] Safe and effective antiviral products for the treatment of viral infections are urgently needed, especially in light of the current global Covid-19 outbreak. There is an urgent need in the art for safe and effective new treatments for viral infections. DISCLOSURE OF THE INVENTION [Problem to be solved by the invention]

[0012] The present inventors have discovered that compositions containing a cationic antiviral agent, a copper salt, and water exhibit surprising and remarkably strong synergistic antiviral activity. In particular, compositions containing the cationic surfactant LAE or benzalkonium chloride (BAC) in combination with a copper salt exhibit synergistically improved antiviral activity, which was unexpected from each component when used alone. Thus, the present invention relates to the direct administration of a formulation of a cationic surfactant together with a copper salt to animals or humans for the prophylactic or therapeutic treatment of viral diseases.

[0013] One aspect of the present invention relates to an antiviral composition comprising a cationic antiviral agent, a copper salt, and water.

[0014] The cationic agent in the composition may be present in an amount of 2 ppm to 20,000 ppm, and the copper salt may be present in an amount of 1 ppm to 10,000 ppm. The solvent in the composition may be selected from one or more of water, alcohol, propylene glycol, ethyl acetate, methyl isobutyl ketone, acetone, tetrahydrofuran, isopropyl ether, and combinations thereof. The cationic agent may be selected from the group consisting of ethyl auroyl arginate (LAE), quaternary ammonium compounds, benzalkonium chloride, benzethonium chloride, methylbenzethonium chloride, cetalkonium chloride, cetylpyridinium chloride, cetrimonium, guanidine, and combinations thereof. The copper salt may include gluconate, citrate, acetate, amino acid, or peptide.

[0015] The antiviral composition may further comprise 0.01% to 20% of one or more plasticizers selected from glycol, glycerin, xylitol, ethanol, and combinations thereof.

[0016] In the antiviral composition, a cationic agent and copper can be used simultaneously, each satisfying Equation 1, where FICI stands for Fractional Inhibitory Concentration (FIC) index, FICA stands for the FIC of agent A, and FICB stands for the FIC of agent B. Here, agent A is a cationic agent, and agent B is a copper agent.

[0017] [Formula 1]: FICI = FICA + FICB

[0018] The formula is FICA = [CA]sy / [CA]al and FCB = [CS]sy / [CS]al, where [CA]al is the minimum inhibitory concentration (MIC) of the cationic agent, [CS]al is the minimum inhibitory concentration (MIC) of the copper salt, [CA]sy is the minimum inhibitory concentration (MIC) of the cationic agent when both the cationic agent and the copper agent are used together, and [CS]sy is the minimum inhibitory concentration (MIC) of the copper salt when both the cationic agent and the copper salt are used together.

[0019] In the antiviral compositions according to the invention, the composition has a fractional inhibition index (FICI) of less than 0.5. An FICI < 0.5 indicates synergy, an FICI > 1 indicates additivity, and an FICI > 2 indicates indifference (Hollander et al: Antimicrobial agents Chemotherapy 1998, Vol. 42, pages 744-748).

[0020] The antiviral composition has a pH between pH 4 and pH 8.

[0021] The antiviral composition may be formulated or may be in the form of, for example, but not limited to, a nasal spray, nasal gel, aerosol, throat lozenge, gargle, rectal suppository, buccal strip, transdermal formulation, topical formulation, or external formulation.

[0022] Non-limiting examples of formulations of antiviral compositions for use in application include capsules, microcapsules, tablets, enteric coatings, granules, powders, pills, ointments, suppositories, suspensions, syrups, emulsions, liquids, spray inhalants, oral strips, eye drops, or nasal drops. For surface applications, the dosage is 0.01 to 100 mg / dm 2 , preferably 0.5 to 50 mg / dm 2 , more preferably 1 to 19 mg / dm 2 It may also be used in

[0023] Another aspect of the present invention relates to a method of preventing, suppressing, controlling or treating bacterial or viral infections in a subject in need thereof, comprising administering or applying a composition containing a cationic antiviral agent, a copper salt, as described above, in the nasopharynx or throat, particularly in humans and animals.

[0024] The foregoing summary of the invention and the following brief description of the drawings and detailed description of the invention are all exemplary and explanatory and are intended to provide further details of the invention as claimed. Other objects, advantages and novel features will become readily apparent to those skilled in the art from the following detailed description of the invention. The invention is described in detail below. [Brief explanation of the drawings]

[0025] Figure 1 shows the antiviral effect of Solution 1 against rVSG-dG 2019-CoV-2-18AA S in Vero cells on day 1.

[0026] Figure 2 shows the antiviral effect of solution 2 against rVSG-dG 2019-CoV-2-18AA S in Vero cells on day 1. DETAILED DESCRIPTION OF THE INVENTION

[0027] The antiviral compositions provided herein include a cationic agent having antiviral activity, a copper salt, and water. The cationic agent may be any one or more selected from ethyl lauroyl arginate (LAE), a quaternary ammonium compound, and a guanidine compound.

[0028] The cationic antiviral agent may be a cationic surfactant derived from lauric acid and arginine, and in particular, the ester of lauramide with arginine monohydrate (hereinafter referred to as LAE) may be used to protect against viruses. Details of LAE and its derivatives are described in WO2008 / 014824, the contents of which are incorporated herein in their entirety.

[0029] Cationic antiviral agents may be quaternary ammonium compounds as disclosed in U.S. Patents 2,984,639, 3,325,402, 3,431,208, and British Patent 1,319,396, each of which is incorporated herein by reference. Cationic antiviral quaternary ammonium compounds may include those in which one or two substituents on the quaternary nitrogen have alkyl groups, typically with a carbon chain length of 8 to 20, typically 10 to 18, while the remaining substituents have lower carbon atoms, typically alkyl or benzyl groups, e.g., 1 to 7 atoms, typically methyl or ethyl groups. These include benzalkonium chloride and cetylpyridinium chloride.

[0030] The cationic antiviral agent may be a guanidine compound as disclosed in German Patent Application No. 2,233,383, incorporated herein.

[0031] The copper salts used are copper salts that release copper ions into the water and include gluconate, citrate, acetate, amino acids, peptides and copper / polymer complexes.

[0032] Non-limiting examples of copper(II) salts include copper(II) sulfate, copper(II) chloride, copper(II) hydroxide, copper(II) perchlorate, copper(II) selenate, copper(II) sulfide, copper(II) thiocyanate, copper(II) triflate, copper(II) tetrafluoroborate, copper(II) triarsenite acetate (Paris Green (or Paris Green)), copper(II) benzoate, C (Scheele Green), copper(II) chromite, copper(II) gluconate, copper(II) peroxide, and copper(II) usnate.

[0033] Copper salts of amino acids and peptides are disclosed by PA Kober and K. Surguira (J. Bio. Chem., vol. X111 no. 1 pages 1-11), which may include salts of glycine, alanine, aminobutyric acid, valine, leucine, isoleucine, and amino acid dipeptides and polypeptides.

[0034] Copper polymer complexes such as acrylic acid, polymers, oligomers, copolymers of maleic acid and / or anhydride and copolymers of olefins having one or more carbon atoms per molecule (polymers, oligomers, copolymers of olefins having one or more carbon atoms per molecule and maleic acid and / or anhydride, acrylic acid) can be used. Preferred are the polymeric polymaleates, polymethyl methacrylate, vinyl methyl ether copolymers and other carboxylic acid polymers disclosed in U.S. Patent No. 4,217,343, the contents of which are incorporated herein by reference.

[0035] The most preferred combination of ingredients is LAE or benzalkonium chloride in combination with a copper salt to achieve a synergistic antiviral effect.

[0036] The composition further comprises 0.01% to 20% of a plasticizer, wherein the plasticizer is one or more selected from glycol, glycerin, ethanol and / or glycol.

[0037] It is preferable to directly dissolve the compounds in one of the following solvents before use: food-grade water, ethanol, glycerin, propylene glycol, and a mixture of glycol and water. For treatments at specific pH values ​​(pH 4-8), the use of a corresponding buffer solution is recommended. Alternatively, the synergistic combination can be easily used as a solid. Surfaces such as masks, solid furniture, and protective clothing can be protected.

[0038] The present invention relates to the use of a combination of the cationic surfactants LAE or BAC with a copper salt to achieve a synergistic antiviral effect against viral infections.

[0039] The present invention further relates to the direct administration of the cationic surfactant in a formulation with a copper salt to a subject, particularly an animal or human, in need thereof for the prophylactic or therapeutic treatment of viral diseases. "Subject in need" means a human or animal at risk of or suffering from a viral infection.

[0040] The cationic surfactant plus copper salt formulation disclosed in WO 2008 / 0014824 can be applied to surfaces as a solution. This is an easy and suitable method for treating surfaces of ground, vehicles, animals, and people. For other applications, the cationic surfactant plus copper salt applied as a solid is more suitable and may be equally effective.

[0041] Treatment of products to prevent any type of viral infection can involve the presence of a formulation of cationic surfactant LAE or BAC with a certain concentration of copper salt. More specifically, according to embodiments, the formulation contains approximately 2 to 20,000 ppm of LAE or BAC and 1 to 10,000 ppm, preferably 100 to 10,000 ppm, and more preferably 200 to 2000 ppm of the protected copper salt. This is the same concentration as described for achieving a bactericidal effect. Products treated with the cationic surfactant and copper salt in the above concentration ranges can be, for example, food or cosmetics.

[0042] Treatment of virus-infected surfaces, such as surfaces of food preparations, cosmetic surfaces, ground surfaces, surfaces of any type of vehicle, and surfaces of any equipment used in handling virus-infected animals, requires the presence of a cationic surfactant LAE or BAC plus a copper salt. More specifically, according to preferred embodiments of the LAE or BAC plus copper salt, a sufficient level of copper salt is required to achieve the desired antiviral activity on such surfaces. Such concentration levels are considered to be 2 to 20,000 ppm, more preferably 100 to 10,000 ppm, even more preferably 100 to 10,000 ppm, and even more preferably 200 to 2,000 ppm, including the surfactant plus copper salt according to the preferred claimed formulation containing LAE, BAC, and a copper salt. These concentrations are given in terms of the concentration of the cationic surfactant plus copper salt applied to the surface to be treated. When treating a surface with a solid preparation of the cationic surfactant of this formulation, the amount applied is 0.01 to 100 mg / dm3 of the amount of cationic surfactant LAE or BAC plus copper salt. 2 range, preferably 0.5 to 50 mg / dm 2 More preferably, the amount is 1 to 19 mg / dm 2 The amount must be

[0043] Treatment of liquid preparations, such as drinking fluids or natural water sources like lakes or ponds, more specifically, according to preferred embodiments of LAE or BAC plus copper salt, requires the presence of a cationic surfactant at a concentration level sufficient to achieve the desired antiviral effect in the drinking fluid or water. According to preferred embodiments containing LAE or BAC plus copper salt, such concentration levels are believed to be in the range of 0.2 to 20,000 ppm, more preferably 2 to 15,000 ppm, even more preferably 100 to 10,000 ppm, and most preferably 200 to 2000 ppm, containing the cationic surfactant LAE or BAC containing the copper salt. These concentrations are provided in terms of the concentration of the cationic surfactant in the liquid or water being treated.

[0044] Treatment of animals or humans refers to administering the cationic surfactant in a manner suitable for absorption of the compounds used in accordance with one aspect of the present invention. The compounds can be administered orally, parenterally (including intraperitoneally, subcutaneously, and intramuscularly), or externally (topically, e.g., rectally, transdermally, intravenously, or intranasally). The administered preparations may have the form of conventional pharmaceutical preparations, such as capsules, microcapsules, tablets, enteric-coated formulations, granules, powders, pills, ointments, suppositories, oral strips, suspensions, syrups, emulsions, liquids, spray inhalants, eye drops, and nasal drops.

[0045] The above pharmaceutical preparations may contain excipients (sucrose, starch, mannitol, sorbitol, lactose, glucose, cellulose, talc, calcium phosphate, calcium carbonate, etc.), binders (cellulose, methylcellulose, hydroxymethylcellulose, polyvinylpyrrolidone, PVP, gelatin, gum arabic, polyethylene glycol, sucrose, starch, starch, etc.), disintegrants (starch, carboxymethylcellulose, hydroxypropyl starch, sodium bicarbonate, calcium phosphate, calcium citrate, etc.), lubricants (magnesium stearate, aerosol, talc, lauric acid, etc.), and / or granules (sodium stearate, lauric acid ... These preparations can be manufactured in accordance with conventional methods using various organic or inorganic carriers conventionally used in pharmaceutical preparations, such as ethanol, glycerin, sodium methylsulfate, caustics (citric acid, menthol, glycine, orange powder, etc.), preservatives (sodium benzoate, sodium bisulfite, methylparaben, propylparaben), stabilizers (citric acid, sodium citrate, acetic acid, etc.), suspending agents (methylcellulose, polyvinylpyrrolidone, aluminum stearate, etc.), dispersing agents (hydroxypropylmethylcellulose, etc.), diluents (water, etc.), base waxes (cocoa butter, white petrolatum, polyethylene glycol, etc.), and other suitable agents.

[0046] The dosage of the cationic surfactant and copper salt according to one embodiment of the present invention must be determined based on the dosage required to achieve the desired preventive or therapeutic effect. Typically, a single dose of 0.1 mg / kg to 10 mg / kg is administered orally or parenterally. The usual dosage for humans is a unit dose of 0.1 to 1000 mg per individual, more preferably 0.5 to 500 mg per individual. It can be administered 1 to 4 times a day depending on the severity of symptoms. The usual dosage for animals is 0.1 to 100 mg per dose, preferably 0.5 to 50 mg per dose.

[0047] Example The following examples are provided to illustrate the present invention. However, it should be understood that the present invention is not limited to the specific conditions or details set forth in these examples. Throughout the specification, any citation to a publicly available document, including a U.S. patent, is specifically incorporated by reference. Example 1

[0048] VSV pseudotype neutralization assay for SARS-CoV2

[0049] Integrated Biologic Testing (IBT) conducted the study using an established VSV neutralization assay similar to the system IBT and others have previously reported for filoviruses and SARS-CoV2. Briefly, G-deleted VSV was pseudotyped (or pseudotyped) with the SARS-CoV2 spike protein and produced in HEK293T cells. The system includes a luciferase reporter gene that is used to read out the assay.

[0050] Specifically, four dilutions of the test mixture of LAE or BAC and copper gluconate (200, 100, 50, and 25 μg / ml) and a control were prepared and mixed with VSV virus at a 1:1 ratio at room temperature for 1 hour, followed by incubation on Vero cells at 37°C. The following day, cells were lysed, and luciferase activity was measured to evaluate the antiviral effect of the test compound on blocking virus entry in Vero cells. All samples were run in triplicate. Data analysis was performed using XLFit and Graphedpad Prism.

[0051] Estimating synergistic effects between cationic agents (e.g., LAE or BAC) and copper using the fractional inhibition index (FICI)

[0052] The inventors have found that the antiviral (Covid 19) effect of the combined use of a copper salt and a cationic antibacterial agent is greater than the sum of the copper salt and the cationic antibacterial agent when used separately. The antiviral composition of the present invention can simultaneously use a cationic agent and a copper salt that satisfy the following formula 1:

[0053] [Formula 1] FICI = FICA + FICB Here, FICA = [CA]sy / [CA]al, FiCB = [CS]sy / [CS]al, [CA]al is the minimum inhibitory concentration (MIC) of the cationic agent (e.g., LAE or BAC) alone, [CS]al is the minimum inhibitory concentration (MIC) of the copper salt alone, [CA]sy is the minimum inhibitory concentration (MIC) of the cationic agent (e.g., LAE or BAC) when used simultaneously with the copper agent, and [CS]sy is the minimum inhibitory concentration (MIC) of the copper agent when used simultaneously with the cationic agent.

[0054] The partial inhibitory index (FICI) of the composition is less than 0.5, demonstrating that the composition has a synergistic antiviral effect. According to established principles of synergy between two active agents, if the partial inhibitory concentrations of the two agents, when added, are less than 0.5, synergy is indicated. That is, an FICI < 0.5 is synergistic, an FICI > 1 is additive, and an FICI > 2 is indifferent (Hollander et al: Antimicrobial agents Chemotherapy 1998, Vol. 42 p. 744-748).

[0055] Example 2 Spray solution used to evaluate antiviral efficacy To evaluate the antiviral effect of the composition of the present invention, Solution 1 was prepared according to a conventional method for preparing a spray solution, containing the active LAC and other ingredients as shown in the table below.

[0056] solution 1 TIFF2023540466000002.tif115149

[0057] solution 2 TIFF2023540466000003.tif131169

[0058] Results Antiviral test

[0059] The results of viral inhibition of SV Covid-19 by the LAE / copper gluconate combination (Solution 1) at concentrations of 25, 50, 100, and 200 μg / ml are shown in Figure 1. The reported effect of LAE alone on viral inhibition is 300 μg / ml (WO 2008 / 014824) and 300 μg / ml for copper alone (Sagripanti, JC et al. Applied environ. microbiol: 1993: vol 59: 4374-4376). Therefore, the FICI for the LAE / copper combination is LAE 30 / Cu 30 / 300 = 0.10 + 0.10 = 0.20 (inhibitory concentration = 30 ppm), which is less than 0.5, thus demonstrating an unexpected synergistic effect between LAE and copper against SAR-Covid.

[0060] The results of viral inhibition of SV Covid-19 by BAC plus copper salt (Solution 2) are shown in Figure 2. The figure shows that BAC plus copper gluconate had 100% inhibition of SV Covid-2 at 20 μg / ml. However, BAC alone has been reported to exhibit antiviral activity at 100 μg / ml (Eric G Romanoswki et al. J. Occult. Pharmacol Therapy. 2019: vol 35: pp 311-314). Therefore, the combination: BAC 20 / 100 + copper 20 / 300 = 0.2 + 0.07 = 0.27, which is lower than 0.5, demonstrating an unexpected synergistic effect between BAC and copper salt against SAR-CoVid2.

[0061] Example 3 This example provides formulations prepared using compositions according to the present invention. Using the ingredients listed in the table below, various formulations were prepared using conventional methods for administering compositions according to the present invention.

[0062] nasal spray TIFF2023540466000004.tif66169

[0063] Nasal gel (or gel) TIFF2023540466000005.tif58169

[0064] Lozenges TIFF2023540466000006.tif50169

[0065] Mouse Gargle TIFF2023540466000007.tif73169

[0066] Surface Treatment TIFF2023540466000008.tif73169

[0067] It will be apparent to those skilled in the art that various modifications and variations can be made in the methods and compositions of the present invention without departing from the spirit or scope of the invention. Thus, it is intended that the present invention cover the modifications and variations of this invention provided they come within the scope of the appended claims and their equivalents.

Claims

1. 200 ppm to 20,000 ppm of ethyl lauroyl arginate (LAE), 1 ppm to 10,000 ppm of a copper salt, and A solvent that has improved antiviral activity against the COVID virus, the composition has a pH range of 4 to 8; The antiviral composition further comprises 0.01% to 20% of a plasticizer selected from the group consisting of glycol, glycerin, xylitol, ethanol, and combinations thereof.

2. 2. The antiviral composition of claim 1, wherein the solvent is selected from the group consisting of water, alcohol, propylene glycol, ethyl acetate, methyl isobutyl ketone, acetone, tetrahydrofuran, isopropyl ether, and combinations thereof.

3. 2. The antiviral composition of claim 1, wherein the copper salt comprises a gluconate, a citrate, an acetate, an amino acid salt, or a peptide salt.

4. FICA=[CA]sy / [CA]al and FCB=[CS]sy / [CS]al, [CA]al is the minimum inhibitory concentration (MIC) of the cationic agent and [CS]al is the minimum inhibitory concentration (MIC) of the copper salt; [CA]sy is the minimum inhibitory concentration (MIC) of the cationic agent when the cationic agent and the copper salt are used together, [CS]sy is the minimum inhibitory concentration (MIC) of the copper salt when the cationic agent and the copper salt are used together, The composition has a fractional inhibition index (FICI) of less than 0.

5.

2. The antiviral composition of claim 1, wherein the cationic agent and the copper salt satisfy Formula 1. [Formula 1] FICI=FICA+FICB

5. 2. The antiviral composition of claim 1, wherein the composition is a pharmaceutical formulation including a nasal spray, a nasal gel, an aerosol, a throat lozenge, a gargle, a rectal suppository, an oral strip, a transdermal formulation, a topical formulation, or an external formulation for surface treatment.

6. 2. The antiviral composition of claim 1, wherein the composition is a pharmaceutical formulation selected from the group consisting of capsules, microcapsules, tablets, enteric coatings, granules, powders, pills, ointments, suppositories, oral strips, suspensions, syrups, emulsions, liquids, spray inhalants, eye drops or nasal drops.

7. The composition is 0.01 to 100 mg / dm ２ 13. The antiviral composition of claim 1, wherein the antiviral composition is applied to a surface in an amount of

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