KR20230128752A - COMPOSITION FOR ANTIVIRAL ACTIVITY COMPRISING Ipratropium - Google Patents

Abstract

The present invention relates to the use of ipratropium or a salt thereof for the prevention or treatment of COVID-19. According to the present invention, ipratropium inhibits SARS-CoV2 without cytotoxicity and inflammation caused by SARS-CoV2 infection. Relevant cytokines were significantly suppressed, and when treated in combination with remdesivir, its anti-inflammatory effect was shown to significantly increase, so it is used for antiviral purposes, in particular, for preventing or treating COVID-19 caused by SARS-CoV2 can be put to good use.

Description

Antiviral composition containing ipratropium {COMPOSITION FOR ANTIVIRAL ACTIVITY COMPRISING Ipratropium}

The present invention relates to the antiviral effect of ipratropium or a salt thereof, and to the use of ipratropium or a salt thereof for prevention or treatment of COVID-19.

Coronavirus is an enveloped virus that has single-stranded positive RNA as its genome and has been isolated from various animals, including humans, since it was first discovered in 1937. Coronaviruses are classified into four genera: alphaCoV, betaCoV, deltaCoV, and gammaCoV. AlphaCoV causing common respiratory infections are HCoV-229E, HCoV-NL63, and betaCoV are HCoV-OC43 and HCoV-HKU1, and upper respiratory tract infections such as colds belong to this category. It can also lead to infections and digestive problems. Of these, alpha-coronavirus and beta-coronavirus mainly infect mammals, while gamma-coronavirus and delta-coronavirus infect birds. Recently, there is a confirmed case of infection with Delta-coronavirus in pigs. SARS-CoV (B lineage) and MERS-CoV (C lineage), which belong to betaCoV, cause severe respiratory infections characterized by acute respiratory symptoms. Human coronaviruses that infect humans, reported to the Centers for Disease Control and Prevention (CDC), include HCoV-229E and HCoV-OC43, discovered in the 1960s, and Severe Acute Respiratory Syndrome (SARS-CoV), discovered in 2003. ) HCoVNL63 (2004) and HCoV-HKU1 (2005), which were discovered after the pandemic, and Middle East Respiratory Syndrome, which was first identified in Saudi Arabia ( MERS) has a coronavirus (MERS-CoV), and the genome sequence isolated from a patient with pneumonia in the early stage of COVID-19, which recently occurred in Wuhan and spread worldwide in December 2019, has nucleotide homology to bat SARS-likeCoVZXC21 89%, homology with SARS-CoV was analyzed to be 82%, and it was found to belong to the same betaCoV, and was named SARS-CoV2. As such, SARS-CoV2 has been reported as the 7th strain of human infecting coronavirus, different from the existing 6 strains of coronavirus.

Coronavirus disease-19 (COVID-19), a respiratory infection caused by the new type of SARS-CoV2, is caused by droplets from coughing or sneezing of an infected person penetrating the mucous membranes of the eyes, nose, or mouth. , it spreads through the process of touching the eyes, nose, or mouth after contacting contaminated objects with hands, etc., and the infectivity is also very high. When infected with SARS-CoV2, after an incubation period estimated to be about 2 to 14 days, fever, cough, shortness of breath, pneumonia, diarrhea, abdominal pain, and abdominal discomfort are the main symptoms, and symptoms vary from mild to severe. , symptoms such as sore throat, hemoptysis, and nausea sometimes appear, and cases of asymptomatic infection have also been reported. The entry of SARS-CoV2 into cells occurs when the spike (S) virus protein composed of 1273 amino acids, which is present in the outer envelope of the virus and protrudes outward like a 'corona', binds to the angiotensin converting enzyme 2 (ACE2) receptor It is done. ACE2 mediates cell entry of the three coronavirus strains, SARS-CoV, NL63 and SARSCoV-2, and in particular, SARS-CoV and SARS-CoV2 share 76% identity in amino acid sequences, indicating their binding to ACE2. Describe the trend of the virus. The first step in the viral entry process is the binding of the N-terminal portion of the viral protein unit S1 to the pocket of the ACE2 receptor. The second step, considered the most important for viral entry, is protein cleavage between the S1 and S2 units, known to be mediated by the receptor transmembrane protein serine 2 (TMPRSS2), a member of the Hepsin/TMPRSS subfamily (Polo V. et al. al., European Journal of Internal Medicine, 2020).

As described above, it has been confirmed that the novel coronavirus can spread to humans and cause major problems, and the development of novel coronavirus therapeutics is urgently needed. Type I interferon and ribavirin are non-specific antiviral agents that have shown efficacy in clinical practice, but they have been reported to have antiviral effects only in the early stages of infection (A. Zumla et.al, Nature Reviews Drug Discovery, 15:327). -347,2016), Remdesivir, a drug developed by Gilead Sciences in the US as an experimental treatment for Ebola, has been approved by the US Food and Drug Administration (FDA) for emergency use as a treatment for severely ill patients with COVID-19, but this is not a formal treatment For the treatment of severely ill patients, patients with low oxygen levels in the blood or those who need to be treated with oxygen therapy or artificial respirators are the targets. In addition, remdesivir has limitations as a treatment for COVID-19, such as that side effects have not yet been clinically verified and have the effect of reducing the patient's recovery period by 4 days, but there is no significant difference in patient mortality. Therefore, there is an urgent need to develop effective therapeutic agents for the treatment of COVID-19.

An object of the present invention is to provide an antiviral composition.

In addition, an object of the present invention is to provide a pharmaceutical composition for preventing or treating COVID-19.

In addition, an object of the present invention is to provide a food composition for preventing or improving COVID-19.

In order to solve the above problems, the present invention provides an antiviral composition comprising ipratropium or a salt thereof as an active ingredient.

In addition, the present invention provides a pharmaceutical composition for preventing or treating COVID-19 comprising ipratropium or a salt thereof as an active ingredient.

In addition, the present invention provides a food composition for preventing or improving COVID-19, comprising ipratropium or a salt thereof as an active ingredient.

According to the present invention, ipratropium inhibited SARS-CoV2 without cytotoxicity and significantly suppressed inflammation-related cytokines caused by SARS-CoV2 infection, and when treated in combination with remdesivir, its inflammation-related cytokine inhibitory effect Since it has been shown to significantly increase, it can be usefully used for antiviral purposes, especially for prevention or treatment of COVID-19 caused by SARS-CoV2.

1 is a schematic diagram of an experiment for confirming the SARS-CoV2 inhibitory ability (qRT-PCR analysis) and cytotoxicity (WST analysis) of ipratropium and/or remdesivir.
2 is a diagram confirming the cytotoxicity of ipratropium and/or remdesivir.
3 is a diagram confirming the SARS-CoV2 inhibitory effect by treatment with ipratropium alone or combined treatment with ipratropium and remdesivir.
Figure 4 is a diagram confirming the inflammation-related cytokine inhibitory effect by ipratropium alone treatment or combined treatment of ipratropium and remdesivir.
5 is a diagram illustrating the construction and confirmation of a cell line (A549-ACE2i) infected with SARS-CoV2 in an A549 cell line overexpressing ACE2 and TMPRSS2:
A549-ACE2: A549 cell line overexpressing human ACE2 and TMPRSS2 proteins;
A549-ACE2 (infection): A549 cell line overexpressing human ACE2 and TMPRSS2 proteins and infected with SARS-CoV2;
A: A549 cell line (A549-ACE2i) overexpressing ACE2 and TMPRSS2 infected with SARS-CoV2; and
B: Expression of ACE2 and TMPRSS2 in A549 cell line, ACE2 and TMPRSS2 overexpressing A549 cell line, and ACE2 and TMPRSS2 overexpressing A549 cell line infected with SARS-CoV2.
6 is a diagram confirming the effect of ipratropium alone or combined treatment of ipratropium and remdesivir on the inflammatory response caused by SARS-CoV2 infection.

Hereinafter, the present invention will be described in detail as an embodiment of the present invention with reference to the accompanying drawings. However, the following embodiments are presented as examples of the present invention, and if it is determined that detailed descriptions of well-known techniques or configurations may unnecessarily obscure the gist of the present invention, the detailed descriptions may be omitted. , the present invention is not limited thereby. Various modifications and applications of the present invention are possible within the scope of the claims described below and equivalents interpreted therefrom.

In addition, the terms used in this specification (terminology) are terms used to appropriately express preferred embodiments of the present invention, which may vary according to the intention of a user or operator or customs in the field to which the present invention belongs. Therefore, definitions of these terms will have to be made based on the content throughout this specification. Throughout the specification, when a certain component is said to "include", it means that it may further include other components without excluding other components unless otherwise stated.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice for testing the present invention, the preferred materials and methods are described herein. Also, the contents of all publications incorporated by reference herein are incorporated herein by reference.

In one aspect, the present invention relates to an antiviral composition comprising Ipratropium or a salt thereof as an active ingredient.

In one embodiment, ipratropium may be a compound represented by Formula 1 below:

In one embodiment, the salt of ipratropium may be ipratropium bromide represented by Formula 2 below:

The present invention includes not only ipratropium or its pharmaceutically acceptable salts, but also possible solvates, hydrates, racemates or stereoisomers that can be prepared therefrom.

As the salt of ipratropium of the present invention, an acid addition salt formed with a pharmaceutically acceptable free acid is useful. Acid addition salts are formed with inorganic acids such as hydrochloric acid, nitric acid, phosphoric acid, sulfuric acid, hydrobromic acid, hydroiodic acid, nitrous acid or phosphorous acid, and aliphatic mono- and dicarboxylates, phenyl-substituted alkanoates, hydroxy alkanoates and alkanes. Obtained from non-toxic organic acids such as dioates, aromatic acids, aliphatic and aromatic sulfonic acids. Such pharmaceutically non-toxic salts include sulfate, pyrosulfate, bisulphate, sulphite, bisulfite, nitrate, phosphate, monohydrogen phosphate, diiderogen phosphate, metaphosphate, pyrophosphate chloride, bromide, iodine. Id, fluoride, acetate, propionate, decanoate, caprylate, acrylate, formate, isobutyrate, caprate, heptanoate, propiolate, oxalate, malonate, succinate, suberate , sebacate, fumarate, maleate, butyne-1,4-dioate, hexane-1,6-dioate, benzoate, chlorobenzoate, methylbenzoate, dinitro benzoate, hydroxybenzoate, Toxybenzoates, phthalates, terephthalates, benzenesulfonates, toluenesulfonates, chlorobenzenesulfonates, xylenesulfonates, phenylacetates, phenylpropionates, phenylbutyrates, citrates, lactates, hydroxybutyrates, glycolates, Maleate, tartrate, methanesulfonate, propanesulfonate, naphthalene-1-sulfonate, naphthalene-2-sulfonate or mandelate.

The acid addition salt according to the present invention is prepared by a conventional method, for example, by dissolving ipratropium in an excess aqueous acid solution, and dissolving the salt in a water-miscible organic solvent such as methanol, ethanol, acetone or acetonitrile. It can be prepared by precipitating it. In addition, it may be prepared by evaporating the solvent or excess acid from the mixture and drying it, or by suction filtering the precipitated salt.

In addition, a pharmaceutically acceptable metal salt may be prepared using a base. An alkali metal or alkaline earth metal salt is obtained, for example, by dissolving the compound in an excess alkali metal hydroxide or alkaline earth metal hydroxide solution, filtering the undissolved compound salt, and evaporating and drying the filtrate. At this time, as a metal salt, it is pharmaceutically suitable to prepare a sodium, chlorine or calcium salt. In addition, the corresponding silver salt is obtained by reacting an alkali metal or alkaline earth metal salt with a suitable negative salt (eg, silver nitrate).

In one embodiment, the composition of the present invention may further include an antiviral agent, the antiviral agent is remdesivir, nirmatrelvir / ritonavir (nirmatrelvir / ritonavir), chloroquine (chloroquine), hydroxy hydroxychloroquine, interferon, niclosamide, favilavir, efavirenz, etravirine, nevirapine, doravirine , rilpivirine, delavirdine, zidovudine, didanosine, zalcitabine, stavudine, lamivudine, abacavir, te nofovir disoproxil fumarate, emtricitabine, tenofovir alafenamide fumarate, saquinavir, ritonavir, indinavir (indinavir), nelfinavir, amprenavir, lopinavir, atazanavir, fosamprenavir, tipranavir, darunavir ( darunavir), cobicistat, dolutegravir, raltegravir, enfuvirtide, maraviroc, elvitegravir, tenofovir alafenamide, tenofovir disoproxil, amantadine, rimantadine, oseltamivir, zanamivir, peramivir , baloxavir, laninamivir, aciclovir, valacyclovir, idoxuridine, vidarabine, penciclovir, famciclovir, trifluridine, cidofovir, foscarnet, clevudine, telbivudine, entecavir, adefovir , besifovir, ribavirin, boceprevir, dasabuvir, daclatasvir, asunaprevir, sofosbuvir, elbasvir, grazoprevir, glecaprevir, pibrentasvir, ombitasvir, paritaprevir and imiquimod ( imiquimod), and more preferably remdesivir.

In one embodiment, the virus may be a coronavirus, and may be one or more selected from the group consisting of alphacoronavirus, betacoronavirus, gammacoronavirus, and deltacoronavirus, and porcine epidemic diarrhea virus: PEDV), canine coronavirus (CCV), feline infectious peritonitis virus (FIPV), bovine coronavirus (BCV), avian infectious bronchitis virus (IBV), contagious Transmissible gastroenteritis coronavirus (TGEV), severe acute respiratory syndrome coronavirus (SARS-CoV), severe acute respiratory syndrome coronavirus (SARS-CoV2), Middle East respiratory Syndrome coronavirus (Middle East respiratory syndrome coronavirus: MERS-CoV), or may be a combination thereof, most preferably severe acute respiratory syndrome coronavirus (SARS-CoV2).

In one embodiment, the composition of the present invention is capable of reducing the copy number of a virus.

In one embodiment, the composition of the present invention can inhibit the inflammatory response caused by viral infection.

In one aspect, the present invention relates to a pharmaceutical composition for preventing or treating a viral infection comprising Ipratropium or a salt thereof as an active ingredient.

In one embodiment, the virus may be a coronavirus, and may be one or more selected from the group consisting of alphacoronavirus, betacoronavirus, gammacoronavirus, and deltacoronavirus, and porcine epidemic diarrhea virus: PEDV), canine coronavirus (CCV), feline infectious peritonitis virus (FIPV), bovine coronavirus (BCV), avian infectious bronchitis virus (IBV), contagious Transmissible gastroenteritis coronavirus (TGEV), severe acute respiratory syndrome coronavirus (SARS-CoV), severe acute respiratory syndrome coronavirus (SARS-CoV2), Middle East respiratory Middle East respiratory syndrome coronavirus (MERS-CoV), or a combination thereof.

In one aspect, the present invention relates to a pharmaceutical composition for preventing or treating coronavirus disease-19 (COVID-19) comprising ipratropium or a salt thereof as an active ingredient.

In one embodiment, ipratropium may be a compound represented by Formula 1 below:

[Formula 1]

.

In one embodiment, the salt of ipratropium may be ipratropium bromide represented by Formula 2 below:

[Formula 2]

.

In one embodiment, the composition of the present invention may include ipratropium bromide as an active ingredient.

In one embodiment, the composition of the present invention is capable of reducing the copy number of SARS-CoV2.

In one embodiment, the composition of the present invention can inhibit inflammation caused by SARS-CoV2 infection.

In one embodiment, the composition of the present invention may further include an antiviral agent, and the antiviral agent is remdesivir, nirmatrelvir/ritonavir, chloroquine, hydroxychloroquine, interferon, niclosamide, favira Vir, efavirenz, etravirine, nevirapine, doravirine, rilpivirine, delavirdine, zidovudine, didanosine, zalcitabine, stavudine, lamivudine, abacavir, tenofovir disoproxy fumarate, emtrici Tabine, tenofovir alafenamide fumarate, saquinavir, ritonavir, indinavir, nelfinavir, amprenavir, lovinavir, atazanavir, fosamprenavir, tipranavir, darunavir , cobicistat, dolutegravir, raltegravir, enfuvertide, maraviroc, elvitegravir, tenofovir alafenamide, tenofovir disoproxil, amantadine, rimantadine, oseltamivir, Zanamivir, peramivir, baloxavir, laninamivir, acyclovir, valacyclovir, idoxuridine, vidarabine, penciclovir, famciclovir, trifluridine, cidofovir, foscarnet , clevudine, telbivudine, entecavir, adefovir, besifovir, ribavirin, boceprevir, dasabuvir, daclatasvir, asunaprevir, sofosbuvir, elbasvir, grazoprevir, It may be any one or more selected from the group consisting of glecaprevir, pibrentasvir, ombitasvir, paritaprevir, and imiquimod, and most preferably remdesivir.

In the present invention, the term "prevention" refers to all activities that inhibit or delay the occurrence, spread, and recurrence of COVID-19 by administration of the pharmaceutical composition according to the present invention, and "treatment" refers to the use of the composition of the present invention It refers to all activities that improve or beneficially change the symptoms of COVID-19 by administration. Those of ordinary skill in the art to which the present invention pertains will be able to determine the degree of improvement, enhancement and treatment by knowing the exact criteria of the disease for which the composition of the present application is effective by referring to the data presented by the Korean Medical Association, etc. will be.

The term "therapeutically effective amount" used in combination with an active ingredient in the present invention means an amount effective for preventing or treating COVID-19, and the therapeutically effective amount of the composition of the present invention includes several factors, such as For example, it may vary depending on the administration method, the target site, the condition of the patient, and the like. Therefore, when used in the human body, the dosage should be determined in an appropriate amount considering both safety and efficiency. It is also possible to estimate the amount to be used in humans from the effective amount determined through animal experiments. These considerations in determining an effective amount can be found, for example, in Hardman and Limbird, eds., Goodman and Gilman's The Pharmacological Basis of Therapeutics, 10th ed. (2001), Pergamon Press; and E.W. Martin ed., Remington's Pharmaceutical Sciences, 18th ed. (1990), Mack Publishing Co.

The pharmaceutical composition of the present invention is administered in a pharmaceutically effective amount. As used herein, the term "pharmaceutically effective amount" means an amount that is sufficient to treat a disease with a reasonable benefit / risk ratio applicable to medical treatment and does not cause side effects, and the effective dose level is the patient's Health condition, severity of COVID-19, activity of drug, sensitivity to drug, method of administration, time of administration, route of administration and excretion rate, duration of treatment, factors including drugs used in combination or concurrently, and other medical fields well known It can be determined according to known factors. The composition of the present invention may be administered as an individual therapeutic agent or in combination with other therapeutic agents, may be administered sequentially or simultaneously with conventional therapeutic agents, and may be administered singly or in multiple doses. Considering all of the above factors, it is important to administer the amount that can obtain the maximum effect with the minimum amount without side effects, which can be easily determined by those skilled in the art.

The pharmaceutical composition of the present invention may include a carrier, diluent, excipient, or a combination of two or more commonly used in biological preparations. As used herein, the term "pharmaceutically acceptable" means exhibiting non-toxic properties to cells or humans exposed to the composition. The carrier is not particularly limited as long as it is suitable for in vivo delivery of the composition. For example, Merck Index, 13th ed., Merck & Co. Inc. , saline, sterile water, Ringer's solution, buffered saline, dextrose solution, maltodextrin solution, glycerol, ethanol, and one or more of these components may be mixed and used. Customary additives may be added. In addition, diluents, dispersants, surfactants, binders, and lubricants may be additionally added to formulate formulations for injection, such as aqueous solutions, suspensions, and emulsions, pills, capsules, granules, or tablets. Furthermore, it can be preferably formulated according to each disease or component by using an appropriate method in the art or by using a method disclosed in Remington's Pharmaceutical Science (Mack Publishing Company, Easton PA, 18th, 1990).

In one embodiment, the pharmaceutical composition may be one or more formulations selected from the group consisting of oral formulations, external preparations, suppositories, sterile injection solutions and sprays, and oral or injection formulations are more preferred.

As used herein, the term "administration" means providing a predetermined substance to an individual or patient by any suitable method, and parenteral administration (eg, intravenous, subcutaneous, intraperitoneal) according to the desired method Or it can be applied topically as an injection formulation) or administered orally, and the dosage varies depending on the patient's weight, age, sex, health condition, diet, administration time, administration method, excretion rate, and severity of the disease. Liquid formulations for oral administration of the composition of the present invention include suspensions, internal solutions, emulsions, syrups, etc., and various excipients such as wetting agents, sweeteners, aromatics, and preservatives in addition to water and liquid paraffin, which are commonly used simple diluents etc. may be included. Formulations for parenteral administration include sterilized aqueous solutions, non-aqueous solvents, suspensions, emulsions, freeze-dried formulations, suppositories, and the like. The pharmaceutical composition of the present invention may be administered by any device capable of transporting an active substance to a target cell. Preferred administration methods and formulations include intravenous injections, subcutaneous injections, intradermal injections, intramuscular injections, drip injections, and the like. Injections are formulated with aqueous solvents such as physiological saline and IV, non-aqueous solvents such as vegetable oil, higher fatty acid esters (e.g., ethyl oleate, etc.), alcohols (e.g., ethanol, benzyl alcohol, propylene glycol, glycerin, etc.). Stabilizers (e.g., ascorbic acid, sodium hydrogensulfite, sodium pyrosulfite, BHA, tocopherol, EDTA, etc.) to prevent deterioration, emulsifiers, buffers to control pH, A pharmaceutical carrier such as a preservative (eg, phenylmercuric nitrate, thimerosal, benzalkonium chloride, phenol, cresol, benzyl alcohol, etc.) may be included.

As used in the present invention, the term "individual" refers to monkeys, cows, horses, sheep, pigs, chickens, turkeys, quails, cats, dogs, mice, including humans who have or may develop COVID-19, All animals, including rats, rabbits or guinea pigs, can be effectively prevented or treated by administering the pharmaceutical composition of the present invention to a subject. The pharmaceutical composition of the present invention may be administered in parallel with existing therapeutic agents.

The pharmaceutical composition of the present invention may further include pharmaceutically acceptable additives, wherein the pharmaceutically acceptable additives include starch, gelatinized starch, microcrystalline cellulose, lactose, povidone, colloidal silicon dioxide, calcium hydrogen phosphate, Lactose, Mannitol, Taffy, Gum Arabic, Pregelatinized Starch, Corn Starch, Powdered Cellulose, Hydroxypropyl Cellulose, Opadry, Sodium Starch Glycolate, Carnauba Lead, Synthetic Aluminum Silicate, Stearic Acid, Magnesium Stearate, Aluminum Stearate, Stearic Acid Calcium, white sugar, dextrose, sorbitol, and talc may be used. The pharmaceutically acceptable additive according to the present invention is preferably included in an amount of 0.1 part by weight to 90 parts by weight based on the composition, but is not limited thereto.

In one aspect, the present invention relates to a food composition for preventing or improving COVID-19, comprising ipratropium or a pharmaceutically acceptable salt thereof as an active ingredient.

When the composition of the present invention is used as a food composition, the composition may be added as it is or used together with other foods or food ingredients, and may be appropriately used according to a conventional method. In addition to the active ingredient, the composition may include food additives acceptable in food science, and the mixing amount of the active ingredient may be appropriately determined depending on the purpose of use (prevention, health or therapeutic treatment).

The term "food supplement additive" used in the present invention refers to a component that can be added to food supplementally, and can be appropriately selected and used by those skilled in the art as being added to prepare a health functional food of each formulation. Examples of food additives include various nutrients, vitamins, minerals (electrolytes), flavors such as synthetic flavors and natural flavors, colorants and fillers, pectic acid and its salts, alginic acid and its salts, organic acids, protective colloidal thickeners , pH regulators, stabilizers, preservatives, glycerin, alcohol, carbonation agents used in carbonated beverages, etc. are included, but the types of food additives of the present invention are not limited by the above examples.

The food composition of the present invention may include health functional food. The term "health functional food" used in the present invention refers to food prepared and processed in the form of tablets, capsules, powders, granules, liquids and pills using raw materials or ingredients having useful functionalities for the human body. Here, 'functionality' means obtaining useful effects for health purposes, such as adjusting nutrients for the structure and function of the human body or physiological functions. The health functional food of the present invention can be manufactured by a method commonly used in the conventional technical field, and can be prepared by adding raw materials and components commonly added in the conventional technical field at the time of manufacture. In addition, the formulation of the health functional food may also be manufactured without limitation as long as the formulation is recognized as a health functional food. The composition for food of the present invention can be prepared in various types of formulations.

In addition, there is no limitation on the type of health food in which the composition of the present invention can be used. In addition, the composition of the present invention can be prepared by mixing suitable other auxiliary ingredients and known additives that may be contained in health functional foods according to the selection of those skilled in the art. Examples of foods that can be added include meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, chewing gum, dairy products including ice cream, various soups, beverages, tea, drinks, alcoholic beverages, and Vitamin complexes, etc.

The present invention will be described in more detail through the following examples. However, the following examples are only for specifying the content of the present invention, and the present invention is not limited thereto.

Example 1. Cytotoxicity

WST analysis was performed to confirm whether Ipratropium is toxic to cells. Specifically, vero E6 cells were inoculated with SARS-CoV2 and treated with ipratropium alone (0.001 or 0.01 μM), remdesivir alone (4 μM), and ipratropium and remdesivir at various concentrations, respectively. Vir combination treatment (remdesivir 4 μM + ipratropium 0.001 μM, remdesivir 4 μM + ipratropium 0.01 μM or remdesivir 4 μM + ipratropium 0.1 μM) was performed for 48 hours. Then, cell viability by each drug treatment was measured through WST analysis. As a result, it was confirmed that treatment with ipratropium and/or remdesivir did not affect cell viability (FIG. 2).

Example 2. SARS-CoV2 inhibition ability

qRT-PCR analysis was performed to confirm the ability of ipratropium to inhibit SARS-CoV2. Specifically, vero E6 cells were inoculated with SARS-CoV2 and treated with ipratropium alone (0.001 or 0.01 μM), remdesivir alone (4 μM), and ipratropium and remdesivir at various concentrations, respectively. Vir combination treatment (remdesivir 4 μM + ipratropium 0.001 μM, remdesivir 4 μM + ipratropium 0.01 μM or remdesivir 4 μM + ipratropium 0.1 μM) was performed for 48 hours. Then, RNA was extracted from the cells and the amount of E or RdRp gene was confirmed through qRT-PCR analysis to measure the genome copy number of SARS-CoV2. As a result, it was found that the copy number of SARS-CoV2 decreased in all of the ipratropium alone treatment group, the remdesivir alone treatment group, and the ipratropium and remdesivir combined treatment group (FIG. 3 ).

Example 3. Inhibitory effect of inflammatory response

In order to determine whether ipratropium and/or remdesivir can suppress the inflammatory response, microglia BV2 cells were treated with LPS to induce inflammation, and then ipratropium alone at various concentrations was used. treatment (0.01, 0.1 or 0.5 μM), remdesivir alone (2.5 μM), and ipratropium and remdesivir combined treatment (remdesivir 2.5 μM+ipratropium 0.01 μM, remdesivir 2.5 μM+if 0.1 μM of latropium or 2.5 μM of remdesivir + 0.5 μM of ipratropium) were treated, and changes in the expression of inflammation-related genes IL6, CCL2, CCL3, CXCL10, and GCSF were observed through qRT-PCR analysis. As a result, the expression of IL6, CCL2, CCL3, CXCL10, and GCSF increased by LPS was decreased in all of the ipratropium alone treatment group, remdesivir alone treatment group, and ipratropium and remdesivir combined treatment group. It was shown to do (Fig. 4).

Example 4. Inhibition of inflammatory response by SARS-CoV2 infection

4-1. Establishment of SARS-CoV2 infected cell lines

It has been reported that high expression of ACE2 and TMPRSS2, which bind to the spike protein on the surface of the coronavirus and allow the virus to attach to the cell membrane and enter the cell, induces more active infection of SARS-CoV2. It is one of the cell lines commonly used in research. In addition, since the SARS-CoV2 virus can only be used in BL3 facilities due to its infectious risk and transmissibility, an A549 cell line was engineered to overexpress human ACE2 and TMPRSS2 proteins (A549-ACE2), to enable use in BL2 laboratories. A cell line expressing SARS-CoV2 was established by infecting SARS-CoV2 lentivirus, a pseudotyped virus in which genes related to infection and transmission, which express the SARA-CoV2 spike protein, were deleted (A549-ACE2i). In order to confirm whether the A549-ACE2i cells were properly established, green fluorescence inserted into the virus was confirmed, and whether the virus was well inserted into the cell was confirmed under a fluorescence microscope. In addition, in order to confirm successful virus infection in A549-ACE2i cells, the expression of ACE2 and TMPRSS2 was confirmed by qRT-PCR analysis. As a result, it was confirmed that the expression of ACE2 and TMPRSS2 was increased in the A549-ACE2 cell line infected with SARS-CoV2 virus compared to A549 and A549-ACE2. By confirming that SARS-CoV2 virus infection was well induced in the ACE2-TMPRSS2 overexpressing A549 cell line (A549-ACE2) and that the expression of ACE2 and TMPRSS2 was increased, A549-ACE2i cells into which SARS-CoV2 virus was inserted were properly As a result, it was confirmed that the expression of ACE2 and TMPRSS2 was increased compared to A549 and A549-ACE2 in the A549-ACE2 cell line infected with SARS-CoV2 virus. By confirming that SARS-CoV2 virus infection was well induced in the ACE2-TMPRSS2 overexpressing A549 cell line (A549-ACE2) and that the expression of ACE2 and TMPRSS2 was increased, A549-ACE2i cells into which SARS-CoV2 virus was inserted were properly confirmed to have been established.

4-2. Inflammatory response to SARS-CoV2 infection

In order to determine the effect of ipratropium or the combined treatment of ipratropium and remdesivir on the inflammatory response caused by SARS-CoV2 infection, the SARS-CoV2 infected ACE2 and TMPRSS2 overexpressing A549 cell lines (A549-ACE2-TMPRSS2, A549-AACE2i) and monocyte THP1 cells were co-cultured, each treated with ipratropium alone (0.1uM), remdesivir After treatment alone (2.5 μM) and combined treatment with ipratropium and remdesivir, changes in the expression of inflammation-related genes IL6, IL8, and IL10 were confirmed by qRT-PCR analysis. As a result, it was found that the expression of IL6, IL8, and IL10, which increased due to co-culture, was reduced by ipratropium alone, remdesivir alone, and ipratropium and remdesivir combined treatment (FIG. 6). .

Claims (15)

An antiviral composition comprising Ipratropium or a salt thereof as an active ingredient. The antiviral composition according to claim 1, wherein ipratropium is represented by Formula 1 below:
[Formula 1]
. The antiviral composition according to claim 1, wherein the salt of ipratropium is ipratropium bromide represented by the following formula (2):
[Formula 2]
. The antiviral composition according to claim 1, further comprising an antiviral agent. The method of claim 4, wherein the antiviral agent is remdesivir, nirmatrelvir/ritonavir, chloroquine, hydroxychloroquine, interferon, niclosamide (niclosamide), favilavir, efavirenz, etravirine, nevirapine, doravirine, rilpivirine, delavirdine, zidovudine, didanosine, zalcitabine, stavudine, lamivudine, abacavir, tenofovir disoproxil fumarate, emtrici Emtricitabine, tenofovir alafenamide fumarate, saquinavir, ritonavir, indinavir, nelfinavir, amprenavir (amprenavir), lopinavir, atazanavir, fosamprenavir, tipranavir, darunavir, cobicistat, dolutegravir (dolutegravir), raltegravir, enfuvirtide, maraviroc, elvitegravir, tenofovir alafenamide, tenofovir disoproxil (tenofovir disoproxil), amantadine, rimantadine, oseltamivir, zanamivir, peramivir, baloxavir, laninamivir , aciclovir, valacyclovir, idoxuridine, vidarabine, penciclovir, famciclovir, trifluridine, cidofovir, foscarnet, clevudine, telbivudine, entecavir, adefovir, besifovir, ribavirin, bonded boceprevir, dasabuvir, daclatasvir, asunaprevir, sofosbuvir, elbasvir, grazoprevir At least one antiviral composition selected from the group consisting of glecaprevir, pibrentasvir, ombitasvir, paritaprevir, and imiquimod. . The antiviral composition according to claim 1, wherein the virus is a coronavirus. The antiviral composition according to claim 6, wherein the coronavirus is at least one selected from the group consisting of alphacoronavirus, betacoronavirus, gammacoronavirus, and deltacoronavirus. The method of claim 6, wherein the coronavirus is porcine epidemic diarrhea virus (PEDV), canine coronavirus (CCV), feline infectious peritonitis virus (FIPV), bovine coronavirus coronavirus: BCV), avian infectious bronchitis virus (IBV), transmissible gastroenteritis coronavirus (TGEV), severe acute respiratory syndrome coronavirus (SARS-CoV), severe acute An antiviral composition that is a severe acute respiratory syndrome coronavirus 2: SARS-CoV2, a Middle East respiratory syndrome coronavirus: MERS-CoV, or a combination thereof. A pharmaceutical composition for preventing or treating coronavirus disease-19 (COVID-19) comprising ipratropium or a salt thereof as an active ingredient. The pharmaceutical composition for preventing or treating COVID-19 according to claim 9, wherein ipratropium is represented by Formula 1 below:
[Formula 1]
. The pharmaceutical composition for preventing or treating COVID-19 according to claim 9, wherein the salt of ipratropium is ipratropium bromide represented by Formula 1 below:
[Formula 2]
. The pharmaceutical composition for preventing or treating COVID-19 according to claim 9, which suppresses inflammation caused by SARS-CoV2 infection. The pharmaceutical composition for preventing or treating COVID-19 according to claim 9, further comprising an antiviral agent. 14. The method of claim 13, wherein the antiviral agent is remdesivir, nirmatrelvir/ritonavir, chloroquine, hydroxychloroquine, interferon, niclosamide, faviravir, efavirenz, etravirin, nevirapine, dora fishin, rilpivirine, delavirdine, zidovudine, didanosine, zalcitabine, stavudine, lamivudine, abacavir, tenofovir disoproxi fumarate, emtricitabine, tenofovir alafenamide fumarate, saquinavir , ritonavir, indinavir, nelfinavir, amprenavir, lovinavir, atazanavir, fosamprenavir, tipranavir, darunavir, cobicistat, dolutegravir, raltegravir , enpubertide, maraviroc, elvitegravir, tenofovir alafenamide, tenofovir disoproxil, amantadine, rimantadine, oseltamivir, zanamivir, peramivir, baloxavir, raninami Vir, acyclovir, valacyclovir, idoxuridine, vidarabine, penciclovir, famciclovir, trifluridine, cidofovir, foscarnet, clevudine, telbivudine, entecavir, adefovir, bessie Povir, ribavirin, boceprevir, dasabuvir, daclatasvir, asunaprevir, sofosbuvir, elbasvir, grazoprevir, glecaprevir, pibrentasvir, ombitasvir, A pharmaceutical composition for preventing or treating COVID-19, which is any one or more selected from the group consisting of paritaprevir and imiquimod. A food composition for preventing or improving COVID-19, comprising ipratropium or a pharmaceutically acceptable salt thereof as an active ingredient.