Composition for treating rhinovirus infectious diseases, comprising GPR40 agonist as active ingredient

Abstract

The present invention relates to a composition for treating rhinovirus infectious diseases, comprising a GPR40 agonist as an active ingredient. Viruses causing cold symptoms include coronaviruses, picomaviruses, rhinoviruses, coxsackieviruses, adenoviruses, parainfluenza viruses, respiratory syncytial viruses, enteroviruses and the like, but since there is no universal antiviral agent capable of treating all types of viruses, application of the most appropriate therapeutic agent should vary depending on the virus causing the cold symptoms. The GPR40 agonist provided in the present invention exhibits a remarkable therapeutic effect on cold diseases caused by rhinovirus infection, and thus is expected to be actively used in the medical and healthcare fields.

Description

A composition for treating rhinovirus infection diseases comprising a GPR40 agonist as an active ingredient

[0001] The present invention relates to a composition for the prevention or treatment of rhinovirus infection, comprising a GPR40 agonist as an active ingredient.

[0002] The term "cold" is a well-known term used by both medical professionals and the general public to collectively refer to diseases caused by viral infections that may involve the sinuses, nose, ears, and bronchi. Symptoms of a cold range from common ones such as sneezing, runny nose, nasal congestion or stuffiness, pain or itching, coughing, and hoarseness, to symptoms requiring treatment, such as headache, fever, chills, and asthma exacerbation. Generally, the common cold is known to be a group of diseases caused by members of various viral families rather than a single viral entity; it is estimated that over 200 viruses cause cold symptoms, including coronavirus, picomavirus, rhinovirus, coxsackievirus, adenovirus, parainfluenza virus, respiratory syncytial virus, and enterovirus. However, since there is no universal antiviral drug capable of treating all types of viruses, the most appropriate treatment must be applied depending on the specific virus causing the cold symptoms.

[0003] Meanwhile, GPR40 is known as a G protein-coupled receptor of pancreatic beta cells, and because it has the effect of enhancing glucose-dependent insulin secretion, there have been attempts to develop GPR40 agonists as treatments for diabetes.

[0004] However, the inventors of the present invention completed the invention by discovering that the GPR40 agonist has a significant therapeutic effect on cold diseases caused by rhinovirus infection. Since the composition containing the GPR40 agonist of the present invention as an active ingredient has a significant therapeutic effect on rhinovirus infection diseases, which account for the majority of common colds, it is expected to be actively utilized in the medical and healthcare fields.

[0005] One objective of the present invention is to provide a composition capable of preventing, improving, or treating rhinovirus infection diseases by including a GPR40 agonist as an active ingredient.

[0006] Another objective of the present invention is to provide a method for preventing, improving, or treating rhinovirus infection using the above-described composition.

[0007] In a preferred embodiment of the present invention, the rhinovirus infection disease may be a cold disease caused by rhinovirus infection, but is not limited thereto.

[0008] However, the technical problems that the present invention aims to solve are not limited to those mentioned above, and other unmentioned problems will be clearly understood by those skilled in the art from the description below.

[0009] Various embodiments described herein are described with reference to the drawings. In the following description, for a complete understanding of the invention, various specific details, such as specific forms, compositions, and processes, are described. However, specific embodiments may be practiced without one or more of these specific details, or in combination with other known methods and forms. In other examples, known processes and manufacturing techniques are not described as specific details so as not to unnecessarily obscure the invention. Reference throughout this specification to "one embodiment" or "an embodiment" means that the particular features, forms, compositions, or characteristics described in association with the embodiment are included in one or more embodiments of the invention. Accordingly, the context of "in one embodiment" or "an embodiment" expressed at various places throughout this specification does not necessarily represent the same embodiment of the invention. Additionally, particular features, forms, compositions, or characteristics may be combined in any suitable way in one or more embodiments.

[0010] Unless otherwise specifically defined in the specification, all scientific and technical terms used herein have the same meaning as commonly understood by those skilled in the art to which the present invention pertains.

[0011] In the present invention, “GPR40 agonist” means a substance that enhances the synthesis or activity of GPR40 (G-protein coupled receptor 40) protein, or enhances the expression of the GPR40 gene itself. Although not limited thereto, the agonist may specifically be a substance represented by the following chemical formula 1.

[0012] In the present invention, as a result of screening various candidate substances, a compound of the following chemical formula 1 was derived as a candidate substance for treating rhinovirus infection disease.

[0013] [Chemical Formula 1]

[0014]

[0015] Hereinafter, the compound of the present invention, or the selected compound of the present invention, refers to the compound of Chemical Formula 1.

[0016] In one aspect of the present invention, the present invention provides a compound selected from the compound represented by Formula 1, its pharmaceutically acceptable salts, crystalline forms, co-crystals, optical isomers, derivatives, hydrates, and solvates.

[0017] The above-mentioned pharmaceutically acceptable salt is a salt generally considered by those skilled in the art to be suitable for medical application (e.g., because such salt is not harmful to the subject to be treated with said salt), or a salt that causes acceptable side effects within each treatment. Generally, the above-mentioned pharmaceutically acceptable salt is a salt considered acceptable by regulatory authorities such as the U.S. Food and Drug Administration (FDA), the European Medicines Agency (EMA), or the Pharmaceuticals and Medical Devices Agency (PMDA) of the Japanese Ministry of Health, Labour and Welfare. However, in principle, the present invention also includes, for example, an intermediate in the preparation of a compound according to the present invention or a physiologically active derivative thereof, or a salt of a compound according to the present invention that is not pharmaceutically acceptable in itself as a pharmaceutically acceptable salt of the compound according to the present invention or as an intermediate in the preparation of a physiologically active derivative thereof. The above-mentioned salt includes water-insoluble salts, and in particular, includes water-soluble salts.

[0018] In each case, a person skilled in the art can easily determine whether a specific compound according to the present invention or its physiologically functional derivative can form a salt, that is, whether the compound according to the present invention or its physiologically functional derivative has a charged group such as, for example, an amino group, a carboxylic acid group, etc.

[0019] Exemplary salts of the compounds of the present invention are acid addition salts or salts with bases, in particular pharmaceutically acceptable inorganic and organic acid addition salts and salts with bases commonly used in pharmaceuticals, which are water-insoluble or particularly water-soluble acid addition salts. Depending on the substituents of the compounds of the present invention, salts with bases may also be suitable. Acid addition salts may be formed, for example, by mixing a solution of the compounds of the present invention with a solution of a pharmaceutically acceptable acid such as hydrochloric acid, sulfuric acid, fumaric acid, maleic acid, succinic acid, acetic acid, benzoic acid, citric acid, tartaric acid, carbonic acid, or phosphoric acid. Likewise, pharmaceutically acceptable base addition salts are alkali metal salts (e.g., sodium or potassium salts); alkaline earth metal salts (e.g., calcium or magnesium salts); It may include salts formed with suitable organic ligands (e.g., ammonium, quaternary ammonium, and amine cations formed using counteranions such as halides, hydroxides, carboxylates, sulfates, phosphates, nitrates, alkyl sulfons, and aryl sulfons).Exemplary examples of pharmaceutically acceptable salts include acetate, adipate, alginate, arginate, ascorbate, aspartate, benzenesulfonate, benzoate, bicarbonate, bisulfate, bitartrate, borate, bromide, butyrate, calcium edetate, camphorate, camphosulfonate, camsylate, carbonate, chloride, citrate, digluconate, dihydrochloride, dodecyl sulfate, edetate, edicylate, ethanesulfonate, formate, fumarate, galactate, galacturonate, gluconate, glutamate, glycerophosphate, hemisulfate, heptanoate, hexanoate, hexylresorcinate, hydrobromide, hydrochloride, hydroiodide, 2-hydroxy-ethanesulfonate. Hydroxynaphthoate, iodide, isobutyrate, isothionate, lactate, laurate, lauryl sulfate, maleate, maleate, malonate, mandelate, methanesulfonate (mesylate), methyl sulfate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate, pantothenate, pectinate, persulfate, 3-phenylpropionate, phosphate / diphosphate, phthalate, picrate, pivalate, polygalacturonate, propionate, salicylate, stearate, sulfate, subverate, succinate, tannate, tartrate, tosylate, undecanoate, valerate, etc. are included but not limited thereto.

[0020] A salt that is not pharmaceutically acceptable and, for example, may be obtained as a process product during the manufacture of a compound according to the present invention on an industrial scale is also included in the present invention, and if desired, can be converted into a pharmaceutically acceptable salt by a method known to those skilled in the art.

[0021] Meanwhile, since the compounds according to the present invention may have asymmetric carbon centers, they may exist as R or S isomers or racemic compounds, and all of these optical isomers and mixtures may be included within the scope of the present invention.

[0022] In addition, the compounds of the present invention as well as their salts may contain varying amounts of solvent, for example, when separated into crystalline forms. Accordingly, solvates of the compounds of the present invention, particularly hydrates, as well as solvates of the salts of the compounds of the present invention, particularly hydrates, may be included within the scope of the present invention. More particularly, the present invention may include hydrates of compounds, salts, and / or physiologically functional derivatives according to the present invention, comprising one, two, or half molecules of water with respect to stoichiometry.

[0023] Hereinafter, the compound of the present invention refers to the compound described above.

[0024] In another aspect of the present invention, the present invention provides a pharmaceutical composition for the prevention or treatment of rhinovirus infection disease comprising, as an active ingredient, an agonist of the GPR40 (G-protein coupled receptor 40) protein or the gene encoding it. In this case, the agonist of the GPR40 protein or the gene encoding it may specifically be a compound selected from the compound represented by Formula 1, the pharmaceutically acceptable salt, crystalline form, co-crystal, optical isomer, derivative, hydrate, and solvate thereof, and may act as a co-agonist of the GPR120 (G-protein coupled receptor 120) protein or the gene encoding it.

[0025] The above rhinovirus infection disease may be a respiratory disease and may be selected from the group consisting of the common cold, asthma, chronic obstructive pulmonary disease (COPD), pneumonia, pulmonary tuberculosis, allergic rhinitis, anxiety, depression, episodic cluster headache, migraine, diabetes, chronic kidney disease / diabetic nephropathy, diabetic retinopathy, diabetic neuropathy, cachexia, anorexia nervosa, inflammation, inflammatory bowel disease, ulcerative colitis, Crohn's disease, irritable bowel syndrome, dyspepsia, and vomiting, but is not limited thereto.

[0026] The above pharmaceutical composition is intended for the purpose of preventing or treating rhinovirus infection. The prevention or treatment described herein comprehensively includes activities that suppress the occurrence or exacerbation of rhinovirus infection or improve the symptoms of rhinovirus infection.

[0027] In the present invention, the pharmaceutical composition may be characterized in that it is in the form of a capsule, tablet, granule, injection, ointment, powder, or beverage, and the pharmaceutical composition may be characterized in that it is intended for humans.

[0028] The pharmaceutical composition of the present invention is not limited to these, but may be formulated and used in the form of oral formulations such as powders, granules, capsules, tablets, and aqueous suspensions, as well as topical preparations, suppositories, and sterile injectable solutions, according to conventional methods. The pharmaceutical composition of the present invention may include a pharmaceutically acceptable carrier. For oral administration, the pharmaceutically acceptable carrier may include binders, lubricants, disintegrants, excipients, solubilizers, dispersants, stabilizers, suspending agents, colorants, flavorings, etc. For injectable preparations, it may include buffers, preservatives, analgesics, solubilizers, isotonic agents, stabilizers, etc., in combination; and for topical administration, a base, excipients, lubricants, preservatives, etc. may be used. The formulations of the pharmaceutical composition of the present invention may be prepared in various ways by mixing with the pharmaceutically acceptable carriers described above. For example, for oral administration, it can be manufactured in the form of tablets, troches, capsules, elixirs, suspensions, syrups, wafers, etc., and for injectables, it can be manufactured in the form of unit dosing ampoules or multiple dosing ampoules. In addition, it can be formulated as a solution, suspension, tablet, capsule, sustained-release formulation, etc.

[0029] Meanwhile, examples of carriers, excipients, and diluents suitable for formulation include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, or mineral oil. Additionally, fillers, anticoagulants, lubricants, wetting agents, fragrances, emulsifiers, preservatives, etc. may be additionally included.

[0030] Routes of administration of the pharmaceutical composition according to the present invention include, but are not limited to, oral, intravenous, intramuscular, intra-arterial, intramedullary, intradural, intracardiac, transdermal, subcutaneous, intraperitoneal, intranasal, intestinal, topical, sublingual, or rectal. Oral or parenteral administration is preferred. Parenteral administration includes subcutaneous, intradermal, intravenous, intramuscular, intra-articular, intrasynovial, intrasternal, intradural, intralesional, and intracranial injection or infusion techniques. The pharmaceutical composition of the present invention may also be administered in the form of a suppository for rectal administration.

[0031] The pharmaceutical composition of the present invention may vary depending on several factors including the activity of the specific compound used, age, body weight, general health, gender, diet, time of administration, route of administration, release rate, drug combination, and the severity of the specific disease to be prevented or treated, and the dosage of the pharmaceutical composition may be appropriately selected by a person skilled in the art, depending on the patient's condition, body weight, degree of disease, drug form, route of administration, and duration, and may be administered at a dose of 0.0001 to 50 mg / kg or 0.001 to 50 mg / kg per day. Administration may be administered once a day or divided into several doses. The dosage does not limit the scope of the present invention in any way. The pharmaceutical composition according to the present invention may be formulated as a pill, coated tablet, capsule, liquid, gel, syrup, slurry, or suspension.

[0032] The pharmaceutical composition of the present invention may be used alone or in combination with methods using surgery, hormone therapy, chemotherapy, and biological response modifiers.

[0033] The present invention also provides a method for preventing, improving, or treating a rhinovirus infection, comprising the step of administering to a subject requiring administration (e.g., a human) a pharmaceutically effective amount of a pharmaceutical composition comprising, as an active ingredient, a compound selected from the compound of the present invention, its pharmaceutically acceptable salts, crystalline forms, co-crystals, optical isomers, derivatives, hydrates, and solvates. In this case, "administration" means providing a predetermined compound of the present invention to the subject by any appropriate method.

[0034] Specific details regarding the rhinovirus infection, the pharmaceutical composition, and the route of administration in the above-mentioned method for the prevention, improvement, or treatment of the rhinovirus infection are omitted to avoid excessive complexity of the specification, as they overlap with those described in the above-mentioned “a pharmaceutical composition comprising as an active ingredient a compound selected from the compounds of the present invention, pharmaceutically acceptable salts, crystalline forms, co-crystals, optical isomers, derivatives, hydrates, and solvates thereof.”

[0035] In the present invention, the "subject" requiring administration may include both mammals and non-mammalians. Here, examples of mammals may include, but are not limited to, humans, non-human primates such as chimpanzees, and other ape and monkey species; domesticated animals such as cattle, horses, sheep, goats, and pigs; domesticated animals such as rabbits, dogs, and cats; and laboratory animals such as rodents such as rats, mice, and guinea pigs. Additionally, examples of non-mammalians in the present invention may include, but are not limited to, birds and fish.

[0036] In the present invention, the “pharmaceuticalally effective amount” refers to a sufficient amount of agent to provide a desirable biological result. Such result may be a reduction and / or alleviation of the signs, symptoms, or causes of a disease, or any other desirable change in the biological system. For example, the “effective amount” for therapeutic use is the amount of the compound disclosed in the present invention required to provide a clinically significant reduction in the disease. In any individual case, an appropriate “effective” amount may be determined by a person skilled in the art using routine experiments. Accordingly, the expression “effective amount” generally refers to an amount of the active substance that has a therapeutic effect.

[0037] In another aspect of the present invention, the present invention provides a food composition for the prevention or improvement of rhinovirus infection disease comprising, as an active ingredient, an agent of the GPR40 (G-protein coupled receptor 40) protein or the gene encoding it. In this case, the agent of the GPR40 protein or the gene encoding it may specifically be a compound selected from the compound represented by Formula 1, the pharmaceutically acceptable salt, crystalline form, co-crystal, optical isomer, derivative, hydrate, and solvate thereof, and may act as a co-agent of the GPR120 (G-protein coupled receptor 120) protein or the gene encoding it.

[0038] The above rhinovirus infection disease may be a respiratory disease and may be selected from the group consisting of the common cold, asthma, chronic obstructive pulmonary disease (COPD), pneumonia, pulmonary tuberculosis, allergic rhinitis, anxiety, depression, episodic cluster headache, migraine, diabetes, chronic kidney disease / diabetic nephropathy, diabetic retinopathy, diabetic neuropathy, cachexia, anorexia nervosa, inflammation, inflammatory bowel disease, ulcerative colitis, Crohn's disease, irritable bowel syndrome, dyspepsia, and vomiting, but is not limited thereto.

[0039] The above food composition is intended for the purpose of preventing or improving rhinovirus infection. The prevention or improvement described herein comprehensively includes activities that suppress the occurrence or exacerbation of rhinovirus infection or improve the symptoms of rhinovirus infection.

[0040] In the present invention, the food composition is used in various ways for the indications intended in the present invention, namely, the prevention or improvement of rhinovirus infection diseases. The food composition containing the compound of the present invention may be manufactured in the form of various food products, such as beverages, chewing gum, tea, vitamin complexes, powders, granules, tablets, capsules, jellies, confectionery, rice cakes, bread, etc. When the composition of the present invention is included in the food composition, the amount may be added in a ratio of 0.1 to 100% of the total weight. Here, when the food composition is manufactured in the form of a beverage, there are no special limitations other than containing the food composition in the indicated ratio, and it may contain various flavoring agents or natural carbohydrates as additional ingredients, as in ordinary beverages. That is, as natural carbohydrates, it may include monosaccharides such as glucose, disaccharides such as fructose, polysaccharides such as sucrose, conventional sugars such as dextrin and cyclodextrin, and sugar alcohols such as xylitol, sorbitol, and erythritol. Examples of the above-mentioned flavoring agents include natural flavoring agents (taumatin, stevia extract (e.g., rebaudioside A, glycyrrhizin, etc.)) and synthetic flavoring agents (saccharin, aspartame, etc.). In addition, the food composition of the present invention may contain various nutritional agents, vitamins, minerals (electrolytes), flavoring agents such as synthetic and natural flavoring agents, coloring agents, pectic acid and its salts, alginic acid and its salts, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohol, carbonating agents used in carbonated beverages, etc. These ingredients may be used independently or in combination. The proportion of such additives is generally selected in the range of 0.1 to 100 parts by weight per 100 parts by weight of the composition of the present invention, but is not limited thereto.

[0041] In another aspect of the present invention, the present invention provides a cosmetic composition for the prevention or improvement of rhinovirus infection disease comprising, as an active ingredient, an agent of the GPR40 (G-protein coupled receptor 40) protein or the gene encoding it. In this case, the agent of the GPR40 protein or the gene encoding it may specifically be a compound selected from the compound represented by Formula 1, the pharmaceutically acceptable salt, crystalline form, co-crystal, optical isomer, derivative, hydrate, and solvate thereof, and may act as a co-agent of the GPR120 (G-protein coupled receptor 120) protein or the gene encoding it.

[0042] The above rhinovirus infection disease may be a respiratory disease and may be selected from the group consisting of the common cold, asthma, chronic obstructive pulmonary disease (COPD), pneumonia, pulmonary tuberculosis, allergic rhinitis, anxiety, depression, episodic cluster headache, migraine, diabetes, chronic kidney disease / diabetic nephropathy, diabetic retinopathy, diabetic neuropathy, cachexia, anorexia nervosa, inflammation, inflammatory bowel disease, ulcerative colitis, Crohn's disease, irritable bowel syndrome, dyspepsia, and vomiting, but is not limited thereto.

[0043] The above cosmetic composition is intended for the purpose of preventing or improving rhinovirus infection. The prevention or improvement described herein comprehensively includes activities of suppressing the occurrence or exacerbation of rhinovirus infection or improving the symptoms of rhinovirus infection.

[0044] In the present invention, the cosmetic composition is used in various ways for the indications intended in the present invention, namely, the prevention or improvement of rhinovirus infection diseases, and the cosmetic composition containing the compound of the present invention may be manufactured in the form of a lotion, nourishing lotion, nourishing essence, massage cream, beauty bath additive, body lotion, body milk, bath oil, baby oil, baby powder, shower gel, shower cream, sunscreen lotion, sunscreen cream, tanning cream, skin lotion, skin cream, UV protection cosmetic, concealer, cleansing milk, hair loss treatment cosmetic, face and body lotion, face and body cream, skin whitening cream, hand lotion, hair lotion, cosmetic cream, jasmine oil, bath soap, liquid soap, beauty soap, shampoo, hand sanitizer (hand cleaner), medicinal soap (non-medical), cream soap, facial wash, body cleanser, scalp cleanser, hair rinse, cosmetic soap, teeth whitening gel, toothpaste, etc. It can be manufactured in the form of a cream, preferably. To this end, the composition of the present invention may further include a solvent or a suitable carrier, excipient, or diluent that is commonly used in the manufacture of cosmetic compositions.

[0045] In the present invention, the type of solvent that may be further added to the cosmetic composition is not particularly limited, but, for example, water, saline solution, DMSO, or a combination thereof may be used. In addition, carriers, excipients, or diluents include, but are not limited to, purified water, oil, wax, fatty acid, fatty acid alcohol, fatty acid ester, surfactant, humectant, thickener, antioxidant, viscosity stabilizer, chelating agent, buffer, lower alcohol, etc. Additionally, whitening agents, moisturizers, vitamins, sunscreens, perfumes, dyes, antibiotics, antibacterial agents, and antifungal agents may be included as needed.

[0046] In the cosmetic composition of the present invention, hydrocarbon oil, silicone oil, hydrogenated vegetable oil, castor oil, cottonseed oil, olive oil, palm oil, jojoba oil, and avocado oil may be used as the oil, and beeswax, spermaceti, carnauba, candelilla, montan, ceresin, liquid paraffin, and lanolin may be additionally used as the wax.

[0047] In the cosmetic composition of the present invention, stearic acid, linoleic acid, and oleic acid may be used as the fatty acid, cetyl alcohol, octyl dodecanol, oleyl alcohol, panthenol, lanolin alcohol, stearyl alcohol, and hexadecanol may be used as the fatty acid alcohol, and isopropyl myristate, isopropyl palmitate, and butyl stearate may be used as the fatty acid ester. Cationic surfactants, anionic surfactants, and nonionic surfactants known in the art may be used as surfactants, and surfactants derived from natural products are preferred as much as possible.

[0048] In addition to the above, the cosmetic composition of the present invention may include hygroscopic agents, thickeners, antioxidants, etc., which are widely known in the field of cosmetics, and the types and amounts thereof are as known in the art.

[0049] In addition, the cosmetic composition of the present invention may include a color tone.

[0050] In addition, in the present invention, the cosmetic composition may be included in the powder in the form of an emulsion, and the powder may be a mixture of one or more selected from the group consisting of talc, kaolin, mica, sericite, silica, silica dimethyl silylate, nylon-12, methyl methacrylate crosspolymer, polymethyl methacrylate, boron nitride, alumina, titanium dioxide, zinc oxide, iron oxide, silica, pearl, organic dye, lake, and organic pigment, and is not limited thereto as long as it corresponds to powders commonly used in the art.

[0051] In another aspect of the present invention, the present invention provides a method for preventing or treating rhinovirus infection by administering a pharmaceutically effective amount of GPR40 (G-protein coupled receptor 40) protein or an agonist of the gene encoding it to an individual requiring treatment. In this case, the GPR40 protein or the agonist of the gene encoding it may specifically be a compound selected from the compound represented by Formula 1, its pharmaceutically acceptable salt, crystalline form, co-crystal, optical isomer, derivative, hydrate, and solvate, and may act as a co-agonist of GPR120 (G-protein coupled receptor 120) protein or the gene encoding it.

[0052] The above rhinovirus infection disease may be a respiratory disease and may be selected from the group consisting of the common cold, asthma, chronic obstructive pulmonary disease (COPD), pneumonia, pulmonary tuberculosis, allergic rhinitis, anxiety, depression, episodic cluster headache, migraine, diabetes, chronic kidney disease / diabetic nephropathy, diabetic retinopathy, diabetic neuropathy, cachexia, anorexia nervosa, inflammation, inflammatory bowel disease, ulcerative colitis, Crohn's disease, irritable bowel syndrome, dyspepsia, and vomiting, but is not limited thereto.

[0053] The above prevention or treatment comprehensively includes activities to suppress the occurrence or exacerbation of rhinovirus infection or to improve the symptoms of rhinovirus infection.

[0054] In another aspect of the present invention, the present invention provides a use for the prevention or treatment of rhinovirus infection disease of an agent of the GPR40 (G-protein coupled receptor 40) protein or the gene encoding it. In this case, the agent of the GPR40 protein or the gene encoding it may specifically be a compound selected from the compound represented by Formula 1, the pharmaceutically acceptable salt, crystalline form, co-crystal, optical isomer, derivative, hydrate, and solvate thereof, and may act as a co-agent of the GPR120 (G-protein coupled receptor 120) protein or the gene encoding it.

[0055] The above rhinovirus infection disease may be a respiratory disease and may be selected from the group consisting of the common cold, asthma, chronic obstructive pulmonary disease (COPD), pneumonia, pulmonary tuberculosis, allergic rhinitis, anxiety, depression, episodic cluster headache, migraine, diabetes, chronic kidney disease / diabetic nephropathy, diabetic retinopathy, diabetic neuropathy, cachexia, anorexia nervosa, inflammation, inflammatory bowel disease, ulcerative colitis, Crohn's disease, irritable bowel syndrome, dyspepsia, and vomiting, but is not limited thereto.

[0056] The above prevention or treatment comprehensively includes activities to suppress the occurrence or exacerbation of rhinovirus infection or to improve the symptoms of rhinovirus infection.

[0057] The GPR40 agonist provided in this invention exhibits a significant therapeutic effect on cold diseases caused by rhinovirus infection, and is therefore expected to be actively utilized in the medical and healthcare fields.

[0058] Figure 1 shows the results confirming that the metabolite profile of human nasal lavage fluid is correlated with the presence of rhinovirus, according to one embodiment of the present invention. (Figure 1A) A schematic diagram of detecting metabolites using human nasal lavage fluid (NLF). Human NLF was collected from HRV-positive (+) or HRV-negative (-) patients (n = 9 for HRV-, n = 6 for HRV-), and each human NLF was analyzed via LC-MS / MS-based targeted metabolite analysis. (Figure 1B) Quantification results of metabolite concentrations in human NLF. Results are expressed as mean ± SEM. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001 (Student's test used).

[0059] Figure 2 shows the results confirming that HRV16 infection in BEAS-2B cells is reduced by linoleic acid treatment according to one embodiment of the present invention. BEAS-2B cells were infected with HRV16 with or without metabolites. (Figure 2A) Schematic diagram of an in vitro experiment using BEAS-2B cells. (Figure 2B) Time-dependent HRV16 mRNA expression levels in BEAS-2B cells were measured by quantitative PCR (qPCR) (n=4). (Figure 2C) Relative HRV16 mRNA expression levels in BEAS-2B cells treated with each metabolite were measured by qPCR at 24 hpi (n=4). Concentrations of LA (1, 10, 25, 50 μM), uridine (1, 10, 25, 50 μM), and nicotinamide (1, 10, 25, 50 μM). (Fig. 2D) Cytotoxicity was measured by the LDH assay at 24 hpi (n=3). (Fig. 2E) Representative immunoblot analysis results of HRV16 capsid proteins (VP0, VP2) in BEAS-2B cell lysates treated with Mock, HRV16, and LA (50 μM) + HRV16 at 24 hpi. The relative intensity of viral protein levels was quantified using image J, and the intensity of VP0 and VP2 proteins was normalized using GAPDH (n=4). (Fig. 2F) Viral load of HRV16-infected BEAS-2B cells was measured by TCID50 of the culture supernatant (n=3). (Fig. 2G) Schematic diagram of the analysis of LA treatment timing using BEAS-2B cells. (H) Relative HRV16 mRNA expression levels in the LA (50 μM) treated group (n=4). Results are expressed as mean ± SEM. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001 (using one-way ANOVA with Tukey's multiple comparisons test or Student's st-test).

[0060] Figure 3 shows the results confirming that the GPR40 / GPR120 dual agonist GW9508, according to one embodiment of the present invention, exhibits an antiviral effect in BEAS-2B cells infected with HRV16. (Figure 3A) A schematic diagram of the action model between GPR40 / GPR120, GW9508, and LA in airway epithelial cells. (Figure 3B) Relative HRV16 mRNA expression levels in BEAS-2B cells treated with LA (50 μM) or GW9508 (50 μM) were measured by qPCR at 24 hpi (n=3). (Fig. 3C) Representative immunoblot analysis results of HRV16 capsid proteins (VP0, VP2) in BEAS-2B cell lysates treated with Mock, HRV16, LA (50 μM) + HRV16, and GW9508 (50 μM) + HRV16 at 24 hpi. The relative intensity of viral protein levels was quantified using Image J, and the intensity of VP0 and VP2 proteins was normalized to GAPDH (n=4). (Fig. 3D) Viral load of HRV16-infected BEAS-2B cells was measured by TCID50 of the culture supernatant at 24 hpi (n=5). Results are expressed as mean ± SEM. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001 (one-way ANOVA with Tukey's multiple comparisons test).

[0061] Figure 4 shows the results confirming that LA treatment induces transcriptional changes in HRV-infected BEAS-2B cells according to one embodiment of the present invention. (Figure 4A) Principal Coordinate Analysis (PCA) plots of the Mock, HRV16, LA, or LA+HRV16 groups (n=4). (Figure 4B) The heatmap displays the normalized expression levels of genes within the Defense Response to Virus (GO: 0051607) gene set. The top 30 genes with the highest Signal-to-Ratio (SNR) values ​​are listed in decreasing order. V-virus infection, LV-LA-infected virus. (Figure 4C) The relative mRNA expression levels of each group were measured by qPCR at 4 hpi (n = 4). Results are expressed as mean ± SEM. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001(using Student'sst-test).

[0062] Figure 5 shows the results confirming that the upregulation of ISG20 by LA according to one embodiment of the present invention depends on the NF-κB pathway in BEAS-2B cells. (Figure 5A) Representative immunoblot analysis results for the time-dependent phosphorylation of p65 (p-p65) protein by LA treatment (50 μM) in BEAS-2B cells (n=3). (Figure 5B) Representative immunoblot analysis results for p-p65 protein at the NF-κB inhibitor CAPE (20 μM) 30 minutes prior to LA (50 μM) treatment in BEAS-2B cells (n=3). (Figure 5C) Representative immunoblot analysis results for ISG20 protein at 24 hpi in BEAS-2B cells (n=3). The relative intensity of each protein level was quantified using Image J, and the protein intensity was normalized to GAPDH. Results are expressed as mean ± SEM. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001 (using one-way ANOVA with Tukey's multiple comparisons test).

[0063] Figure 6 shows the results confirming that the antiviral effect of LA in BEAS-2B cells depends on Isg20, according to one embodiment of the present invention. (Figure 6A) Relative expression levels of ISG20 mRNA between groups treated with scrambled shRNA (sc-shRNA) or ISG20-specific shRNA (sh-ISG20) (n=3). (Figure 6B) Representative immunoblot analysis results of ISG20 protein in scrambled BEAS-2B cells and shISG20-treated BEAS-2B cells (n=3). Relative intensity of protein levels was quantified using image J. (Figure 6C) Viral load of HRV16-infected BEAS-2B cells was measured by TCID50 of the culture supernatant (n=8). Results are expressed as mean ± SEM. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001(using Student'sst-test).

[0064] Figure 7 shows the results confirming that nasal administration of Na-LA according to one embodiment of the present invention increases Isg20 in vivo and induces antiviral activity. (Figure 7A) Schematic diagram of an in vivo HRV1B infection experiment. (Figure 7B) The viral load of HRV1B in mouse BAL fluid was measured by TCID50 in a time-dependent manner. (Figure 7C) Schematic diagram of an in vivo GPR40 / 120 antagonist treatment experiment. (Figure 7D) The viral load of HRV1B in BAL fluid was measured by TCID50 at 8 hpi (n=5). (Figure 7E) The viral load of HRV1B in mouse lungs was measured by TCID50 at 8 hpi (n=5). (Figure 7F) Representative hematoxylin and eosin (H&E) staining results of lung sections (40X) at 16 hpi (n=3). (Fig. 7G) Histological scores are displayed as a bar graph (n=3). (Fig. 7H) Representative immunoblot analysis results for Isg20 protein in the Mock, UV-HRV1B, HRV1B, Na-LA + HRV1B, and GW1100 + AH7614 + Na-LA + HRV1B groups at 8 hpi are shown (n=4). The relative intensity of protein levels was quantified using Image J, and the intensity of ISG20 protein was normalized to GAPDH. Results are expressed as mean ± SEM. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001 (one-way ANOVA with Tukey's multiple comparisons test or Student's test).

[0065] The inventors of the present invention screened various candidate substances to discover a therapeutic substance effective for rhinovirus infection diseases, specifically cold diseases caused by rhinovirus infection, and as a result, derived a compound of the following chemical formula 1 as a useful candidate substance.

[0066] [Chemical Formula 1]

[0067]

[0068] GPR40 (FFAR1) and GPR120 (FFAR4) are known as long-chain fatty acid receptors. Based on this, the researchers investigated whether GPR40 / 120 is involved in the antiviral effects of LA in airway epithelial cells. The compound of Formula 1 of the present invention (GW9508) was used as a dual agonist for GPR40 / GPR120 to activate the receptors. Treatment with GW9508 reduced HRV16 mRNA expression levels at 24 hpi, an effect similar to that observed with LA. Additionally, treatment with GW9508 reduced HRV16 capsid protein in cell lysates and viral load in the medium, with both decreasing to levels similar to those observed in the LA-treated group. These results suggest that the antiviral effects of LA may be mediated through GPR40 / GPR120.

[0069] The present invention will be described in more detail below through examples. These examples are intended solely to explain the present invention more specifically, and it will be obvious to those skilled in the art that the scope of the present invention is not limited by these examples according to the gist of the invention.

[0070] [Experimental Method]

[0071] 1. Screening of compounds for the treatment of rhinovirus infections

[0072] The inventors of the present invention screened various candidate substances to discover a therapeutic substance effective for rhinovirus infection diseases, specifically cold diseases caused by rhinovirus infection, and as a result, derived a compound of the following chemical formula 1 as a useful candidate substance.

[0073] [Chemical Formula 1]

[0074]

[0075] The compound represented by the above chemical formula 1 was identified as an agonist of GPR40 (G protein-coupled receptors FFA1), and the compound name was identified as 4-(3-phenoxybenzylamino)phenylpropionic acid.

[0076] 2. Collection of human nasal lavage fluid samples

[0077] Human nasal lavage fluids (NLF) from patients exhibiting cold symptoms were collected and diluted with saline to obtain a fixed total volume of 5 ml. All samples were immediately centrifuged at 5,000 xg for 35 minutes at 4°C to remove cells and impurities, and the supernatant was carefully collected and filtered using a 0.2 μm syringe filter (SARTORIUS, S6534). Viral RNA was extracted from each sample using the QIAamp Viral RNA Kit (QIAGEN, 52906), and to confirm the presence of human rhinovirus in each sample, a one-step RT-qPCR was performed using the Luna® Universal Probe One-Step RT-qPCR Kit (NEW ENGLAND Biolabs, E3006L) with human rhinovirus 16 (HRV16) primers according to the manufacturer's instructions. Subsequently, RNA levels were quantified using HRV16 specific sequences (HRV16 forward, 5′- GTG AAG AGC CSC RTG TGC T -3′; HRV16 reverse, 5′- GCT SCA GG TTA AGG TTA CC -3′; and HRV16 probe, 5′-(6-FAM TM )-TGA GTC CTC CGG CCC CTG AAT G-(TAMRA TM)-3′). Afterwards, each sample was classified based on the presence or absence of rhinovirus infection and stored at -80℃ until further analysis. All studies in this invention were approved by the Institutional Review Board (IRB) of Yonsei University College of Medicine (4-2021-0573), and samples were used from participants who agreed to provide information.

[0078] 3. Target Liquid Chromatography-Mass Spectrometry (LC-MS) Method

[0079] For the target LC-MS, reduced L-glutathione (Sigma, G6013), N-acetylneuraminic acid (Sigma, A2388), nicotinamide (Sigma, N0636), uric acid (Sigma, U0881), uridine (Sigma, U3003), linoleic acid (Sigma, L1012), citric acid (Sigma, C2404), L-(+)-lactic acid (Sigma, L6402), and (+) sodium-L-ascorbic acid (Sigma, A4034) were used as standards, and all standards were diluted in distilled water (DW) or ethanol (EtOH) from 1 ppm to 1 ppb.

[0080] Mouse bronchoalveolar lavage (BAL) liquid samples were diluted 20 to 200 times in methanol (MetOH) and analyzed in a Thermo Vanquish using a Unison C18 column (3.0 x 150 mm). Each sample was analyzed twice in total: once in cation mode and once in anion mode. For LC, mobile phase A was set to 100% water containing 0.1% formic acid, and mobile phase B was set to 0.1% formic acid contained in MetOH:acetonitrile (1:1). The slopes were set to 10% B from 0 to 0.1 min, 1 to 90% B from 0.1 to 2.5 min, 90% B from 2.5 to 3 min, 90 to 5% B from 3 to 3.1 min, and maintained at 5% B until 5 min. The flow rate was 0.4 ml / min, the column temperature was 45°C, and the positive mode injection volume was 1 µL; mass spectrometry was performed using a Thermo TSQ Altis. Source parameters are shown in Table 1.

[0081] DescriptionParametersScan type (m / z)5-2,000Sheath gas flow rate (Arb)50Aux gas flow rate (Arb)10Heater temp (℃)350Capillary temp (℃)325Spray Voltage (+) (V)3,500Spray Voltage (-) (V)-2,500RF Lens (V)49, 52, 57, 70Normalized collision energy (V)7-15Resolution (Full MS)30,000Resolution (MS 2 )600

[0082] 4. Cell Culture

[0083] H1HeLa cells were purchased from the American Type Culture Collection (ATCC) (CRL-1958) and cultured in MEM (Gibco, 11095-080) containing 10% (v / v) heat-inactivated fetal bovine serum (FBS) (Gibco, 16000-044) and 1% (v / v) penicillin / streptomycin (Gibco, 15140-122). Human lung epithelial cell line BEAS-2B cells were purchased from the ATCC (CRL-9609) and cultured in RPMI1640 (Gibco, 22400-089) containing 10% (v / v) heat-inactivated fetal bovine serum and 1% (v / v) penicillin / streptomycin. Cells were cultured at 175 cm² in a 37°C, 5% CO2 incubator. 2 They were cultured in flasks (Corning, 431080), and the number of passages for all cells used in this study was set to less than 30.

[0084] 5. Viral Infection and Metabolite Treatment

[0085] BEAS-2B cells were placed in a 12-well plate (Corning, 3513) at a rate of 1.5 x 10⁶ BEAS-2B cells 24 hours prior to infection. 5Cells were inoculated into wells, and the next day, the medium was replaced with RPMI1640 containing 2% FBS for viral infection at a multiplicity of infection (MOI) of 5. Cells were cultured in a wet incubator at 33°C and 5% CO2 for 1 hour. After inoculation, cells were washed with PBS, and the medium was replaced with fresh RPMI1640 containing 2% FBS for 24 hours. For metabolite treatment experiments, cells were treated with each concentration of linoleic acid (Sigma, L1012), uridine (Sigma, U3003), or nicotinamide (Sigma, N0636) from 24 hours before inoculation to 24 hours after inoculation. For agent experiments, the compound of Formula 1 (GW9508) was treated with the virus at a concentration of 50 μM for 1 hour. For the linoleic acid (LA) treatment timing assay, cells were treated with 50 μM LA in RPMI 1640 containing 2% FBS 24 hours before HRV16 inoculation, simultaneously with HRV16 inoculation for 1 hour, or up to 24 hours after HRV16 inoculation. To inhibit nuclear factor kappa B (NF-κB), cells were pretreated with 20 μM caffeic acid phenethyl ester (Sigma, C8221) for 30 minutes, washed, and then treated with LA and virus for 1 hour.

[0086] 6. Virus replication

[0087] Human rhinovirus 16 (HRV16; VR-283) and human rhinovirus 1B (HRV1B; VR-1645) were purchased from ATCC, and 175 cm³ of H1HeLa cells were used for viral replication. 2 6x10 in flask (Corning, 431080) 6Cells were inoculated with [the appropriate amount], washed with PBS the next day, and inoculated with 5 ml of virus stock diluted in serum-free 1x MEM (Gibco, 11095-080) for 90 minutes in a high-humidity incubator at 33°C and 5% CO2. The flask was gently shaken every 15 minutes to evenly distribute the virus inoculum across the entire monolayer, and after inoculation, the cells were washed with PBS and the medium was replaced with 1x MEM containing 5% FBS. Once appropriate cytotoxicity was detected, the flask was immediately frozen at -80°C. After freezing and thawing twice, the supernatant was collected and centrifuged at 3,000 xg for 30 minutes; the supernatant was filtered through a 0.2 μm syringe filter (Sartorius, S6534) and titrated by plaque analysis. The supernatant derived from HV1B proliferation was concentrated and centrifuged at 2,000 rpm for 2 hours using a 100 kDa cutoff filter (Millipore, UFC9100).

[0088] 7. Tissue culture infection dose 50% (TCID50)

[0089] Viruses in cell medium, mouse lung lysate, or mouse BAL body fluid were titrated using the TCID50 method. Briefly, 8 x 10⁶ H1HeLa cells were placed in a 96-well plate (Corning, 3596). 3 The next day, the virus was serially diluted in 1x MEM containing 1% (v / v) penicillin / streptomycin and inoculated into the cells, which were then cultured in a high-humidity incubator at 33°C and 5% CO2 for 7 days. If cytotoxic effects were observed in the wells, the TCID50 was calculated using the Spearman-Karber formula.

[0090] 8. Plaque test

[0091] For plaque assay, 5 x 10⁶ H1Hela cells were placed in a 12-well plate (Corning, 3513). 5Cells were inoculated into wells, and the following day, the cells were washed with PBS and treated with serially diluted viruses. Specifically, the viruses were inoculated into the cells in a wet incubator at 33°C and 5% CO2 for 90 minutes. During inoculation, the plate was gently shaken every 15 minutes to ensure even distribution of the virus inoculum across the entire monolayer. After inoculation, the cells were washed with PBS and replaced with plaque assay medium (1x MEM containing 0.5% low-melting point agarose (Promega, V2111) and 2% FBS), and the plates were cultured in a wet incubator at 33°C and 5% CO2 for 5 days. Subsequently, the cells were fixed with 4% paraformaldehyde for 1 hour and stained with 0.5% crystal violet (Sigma, C0775). Viral titers were calculated by counting the plaques and multiplying by the dilution factor (Plaque-Forming units / ml).

[0092] 9. Western Blotting

[0093] Proteins were extracted using Radioimmunoprecipitation Assay (RIPA) buffer (Thermo Scientific, R0278) and Halt™ Protease & Phosphatase Inhibitor Single-Use Cocktail, EDTA-free (100x) (Thermo Scientific, 78443). An equal amount of protein (15 μg) was separated onto a 10% SDS-PAGE gel and transferred to a polyvinylidene difluoride (PVDF) membrane (Merck Millipore, IPVH00010). The membrane was then blocked for 1 hour at room temperature in Tris-buffered saline (50 mM Tris-Cl, pH 7.5, 150 mM NaCl) containing 0.5% Tween 20 (TTBS) and 5% skim milk. Subsequently, the membrane was incubated overnight at 4°C with the primary antibody in 5% skim milk containing TTBS. The following day, the membrane was washed three times with TTBS and incubated with the secondary antibody in 5% skim milk containing TTBS at room temperature for 1 hour. The blots were visualized using Pierce ECL Western Blotting Substrate (Thermo Scientific, 32106) and exposed to X-ray film. The list of antibodies used for immunoblotting is as follows: for the primary antibody, mouse anti-human rhinovirus antibody (QED Bioscience, 18758), rabbit anti-phosphorylated NF-κB p65 antibody (Cell Signaling Technology, 3033S), rabbit anti-NF-κB p65 antibody (Cell Signaling Technology, 4764S), mouse anti-GAPDH antibody (Santa Cruz, sc-32233), and rabbit anti-ISG20 antibody (Invitrogen, PA5-30073). For the secondary antibodies, goat anti-mouse IgG (H+L)-HRP (GenDEPOT, SA001) and goat anti-rabbit IgG (H+L)-HRP (Jackson ImmunoResearch Laboratory, AB-2337913).

[0094] 10. Real-time Quantitative Polymerase Chain Reaction (RT-qPCR)

[0095] Total RNA was isolated from BEAS-2B cells using Hybrid-R™ (GeneAll Biotechnology, 305-101), and complementary DNA (cDNA) was synthesized from 500 ng of RNA using random hexamer primers (Invitrogen, N8080127), RNase inhibitors (Applied Biosystems, N8080119), dNTPs (Applied Biosystems, N8080260), and M-MLV reverse transcriptase (Invitrogen, 28025013). For quantitative PCR (qPCR), KAPA SYBR FAST qPCR Master Mix (2X) (Roche, KK4605) was used according to the manufacturer's instructions, and qPCR was performed using the QuantStudio 3 Real-Time PCR System (Thermo Scientific). Gene expression levels were evaluated using the comparative Ct method (2-ΔΔCt method). The primer sequences used for real-time qPCR in this study are shown in Table 2 below.

[0096] Sequence number definition sequence (5' - 3') 1Human 18s rRNA(F)GCT TAA TTT GAC TCA ACA CGG GA2Human 18s rRNA(R)AGC TAT CAA TCT GCT AAT CCT GTC3HRV16(F)TCT CTA CAG GGC CCT TAC TCG4HRV16(R)CCA CTC TTC TCT CGG GAA CTT5HRV1B(F)CCA TCG CTC ACT ATT CAG CAC6HRV1B(R)TCT ATC CCG AAC ACA CTG TCC7Human ISG20(F)AGC GGC TAC ACA ATC TAC GA8Human ISG20(R)AGG CTG TTC TGG ATG CTC TT9Human DMBT1(F)ACT ACG ACA GAT TGG TGG CA10Human DMBT1(R)GTT GGG GTA GTA TGC AGG GT11Human TRIM35(F)GCT TCG CGA GTT CTT GAG AG12Human TRIM35(R)GCT GCT TCA TCT TCT CGT CG13Human TRIM27(F)TGT TTG GGA GTT TGA GCA GC14Human TRIM27(R)AAG AGA ACT GGG TGA TGG CA15Human IRF1(F)AGG GGA AAA GGA GCC AGA TC16Human IRF1(R)CCT TGT TCC TGC TCT GGT CT

[0097] 11. Lactate Dehydrogenase (LDH) Analysis

[0098] Cytotoxicity was measured using the CytoTox 96 non-radiotoxicity assay kit (Promega, G1780) according to the manufacturer's instructions. Briefly, the culture supernatant of BEAS-2B cells was collected and incubated with the reagent in a 96-well plate (SPL, 31096) for 30 minutes, and results were obtained at 490 nm using a spectrophotometer after incubation. The positive control was provided with the kit, and the culture medium was used as the negative control.

[0099] 12. Bulk mRNA Sequencing and Data Analysis

[0100] Total RNA was isolated from BEAS-2B cells using Hybrid-R™ (GeneAll Biotechnology, 305-101), and total RNA concentration was calculated using Quant-IT RiboGreen (Invitrogen, R11490). To evaluate the integrity of total RNA, samples were run on TapeStation RNA screen tape (Agilent, 5067-5576), and only high-quality RNA preparations with a RIN of 7.0 or higher were used for RNA library construction. Libraries were prepared independently with 1 µg of total RNA per sample using the Illumina TruSeq Stranded mRNA Sample Prep Kit (Illumina Inc., RS-122-2101). As the first step of the workflow, mRNA molecules containing poly-A were purified using poly-T-attached magnetic beads. After purification, the mRNA was fragmented into small pieces using divalent cations at high temperature, and the cleaved RNA fragments were copied into first-strand cDNA using SuperScript II reverse transcriptase (Invitrogen, 18064014) and random primers. Subsequently, second-strand cDNA was synthesized using DNA Polymerase I, RNase H, and dUTP. The cDNA fragments then underwent end repair, single 'A' base addition, and adapter ligation. The product was then purified and concentrated by PCR to prepare the final cDNA library. The library was quantified using the KAPA Library Quantification Kit for the Illumina Sequencing Platform according to the qPCR Quantification Protocol Guide (KAPA BIOSYSTEMS, KK4854) and qualified using TapeStation D1000 ScreenTape (Agilent Technologies, 5067-5582). Then, the indexed library is Illumina NovaSeqX(Illumina, Inc.It was submitted to San Diego, CA, USA, and bidirectional (2×100 bp) sequencing was performed by Macrogen Incorporated.

[0101] RNA sequencing reads were aligned to the reference genome GRCh38 using the STAR aligner (version 2.5.4b). For batch correction, the ComBat-seq function of the Bioconductor package sva (version 3.54.0) was used. Differential gene expression analysis was performed using the Bioconductor DEseq2 package (version 1.44.0) (Love et al., 2014), and gene count data were imported and normalized using the DESeq2 default normalization method. Gene Set Enrichment Analysis (GSEA) was performed using the GSEA module of the GenePattern public server (https: / cloud.genepattern.org). The C5 gene set, containing the gene ontology (GO) gene set from the MSigDB collection (version 2024.1), was used with the default settings of the GSEA module.

[0102] 13. Enzyme-linked immunosorbent assay (ELISA)

[0103] The supernatant of BEAS-2B cells was collected at 24 hpi and stored at -80°C. For mouse bronchoalveolar lavage (BAL), samples were collected by washing the lungs with 1 ml of ice-cold PBS at 8 hpi, centrifuged at 1700×g for 5 minutes, and the supernatant was collected and stored at -80°C for future use. Human IFN-β (R&D Systems, DY814-05) levels in cell medium, and mouse IFN-β (R&D Systems, DY8234-05) and mouse IL-28A / B (IFN-lambda 2 / 3, R&D Systems, DY1789B-05) levels in mouse BAL fluid were quantified using the corresponding DuoSet® ELISA kit according to the manufacturer's instructions. Absorbance was measured at 450 nm using a VersaMax™ microplate reader (Molecular Devices), and the data were analyzed using SoftMax® Pro Software v5.2 (Molecular Devices).

[0104] 14. Short hairpin RNA (shRNA) transfection using lentivirus

[0105] For the knockdown of human ISG20, a lentivirus starter kit was purchased from Lugen Sci (Seoul, Korea) and performed according to the manufacturer's instructions. Briefly, 1 x 10⁶ BEAS-2B cells were placed in a 12-well plate prior to lentivirus infection. 5BEAS-2B cells were inoculated into wells, and the following day, the cells were washed with PBS transfected with lentiviral shRNA (MOI 20) using 1 X Lenti-TD-MAX (Lugen Sci, LS-001). After 24 hours of inoculation, the cells were washed with PBS and replaced with fresh culture medium containing 1 μM puromycin (Invivogen, ant-pr-1) for positive selection. ISG20 expression was verified in all knockdown cells used in this study via RT-qPCR and Western blot. The sequence of the ISG20-specific shRNA was 5'-GAC ATG AGC GGC TAC ACA ATC-3', and for the scrambled shRNA, the sequence 5'-GCA CTA CCA GAG CTA ACT CAG ATA GTA CT-3' was used.

[0106] 15. Preparation of an in vivo mouse model

[0107] Six-week-old female Balb / c mice were purchased from Orient Bio Inc. (Seongnam, South Korea) and housed in an Animal Biosafety Level 2 (ABSL-2) facility. All animal experiments were approved by the Institutional Animal Care and Use Committee (IACUC) of Yonsei University College of Medicine in accordance with the guidelines described by the Association for the Evaluation and Accreditation of Laboratory Animal Care (AAALAC) International (Facility No. 001071) (Protocol No. 2022-0282). 5 x 10 to Balb / c mice 6 Inoculate TCID50 (50 µl) of HRV1B intranasally, and administer 1,200 mJ / cm² for 30 minutes to inactivate HRV1B. 2The mice were exposed to UV light. For local administration of LA, 2.5 mg / kg of sodium linoleate (Sigma Aldrich, L8134) was inoculated simultaneously with HRV1B. To inactivate GPR120 and GPR40, GPR120 antagonists (AH7614, 2.5 mg / kg) and GPR40 antagonists (GW1100, 2.5 mg / kg) were injected intraperitoneally into Balb / c mice 30 minutes prior to HRV1B infection. BAL fluid was collected by washing with ice-cold PBS through a catheter (BD, 382423), and for histological analysis, mouse lung tissue was collected at 16 hpi after systemic perfusion, fixed in 4% PFA for 24 hours, and then embedded in paraffin.

[0108] 16. Histological analysis

[0109] Paraffin-embedded tissue sections were stained with hematoxylin and eosin (H&E) to evaluate inflammatory cell infiltration. To assess lung damage, H&E-stained lung sections were scored by a blind reviewer for the presence and severity of inflammation. Scores were assigned based on the degree of perivascular and peribronchial infiltration, edema, alveolar septal infiltration, hemorrhage, and epithelial damage as follows: 0: None; 1: Mild; 2: Moderate; 3: Severe.

[0110] [Experimental Results]

[0111] 1. The metabolite profile of human nasal lavage fluid is correlated with the presence of rhinovirus.

[0112] According to prior art, various metabolites are associated with antiviral effects against viral infections. In the present invention, researchers sought to verify the effects of nine potential metabolites capable of inhibiting HRV infection, including linoleic acid (LA), uridine, nicotinamide, uric acid, ascorbic acid, lactic acid, citric acid, N-acetylneuraminic acid, and glutathione. To this end, a one-step qPCR was first performed to confirm the presence of the human rhinovirus 16 (HRV16) gene in human nasal lavage fluid (NLF) (Fig. 1A). Through the one-step qPCR, the HRV16 gene was detected in the HRV16 positive group but not in the HRV16 negative group. Next, a targeted LC-MS / MS-based metabolite analysis was performed on the NLF. Interestingly, LC-MS / MS analysis showed that the amounts of metabolites such as LA, uridine, and nicotinamide were increased in HRV16-positive human NLF compared to HRV16-negative human NLF (Fig. 1B). However, no differences were observed in other metabolites of human NLF (Fig. 1B). These results suggest that the concentrations of specific metabolites in NLF are associated with the presence of HRV.

[0113] 2. Reduction of HRV16 infection in BEAS-2B cells by LA treatment

[0114] To characterize the metabolites of human NLF against HRV infection, HRV16 was induced in human lung epithelial cell line BEAS-2B cells at an infection multiplicity (MOI) of 5 (Fig. 2A). Compared to control medium, HRV16 mRNA expression levels significantly increased at 16 hours post-infection (hpi), peaking at 24 hpi (Fig. 2B). Subsequently, HRV16 mRNA expression levels gradually decreased and were undetectable at 48 hpi (Fig. 2B). To confirm the antiviral effects of LA, uridine, and nicotinamide, which were significantly increased in HRV-positive human NLF, these metabolites were treated with HRV16 in BEAS-2B cells (Fig. 2A). Interestingly, LA significantly reduced HRV16 mRNA expression levels in a dose-dependent manner at 24 hpi (Fig. 2C). However, uridine and nicotinamide showed no effect at 24 hpi (Fig. 2C). There were no cytotoxic effects upon treatment with each metabolite (Fig. 2D). Since LA was the only drug to exhibit an antiviral effect against HRV16 infection, it was selected as a potential target for future investigation. Consistent with the effect of LA on HRV16 mRNA expression levels, treatment with LA reduced the levels of the HRV16 capsid proteins VP0 and VP2 in cell lysates (Fig. 2E) and viral titers in cell medium at 24 hpi (Fig. 2F). Next, experiments were conducted to determine whether the timing of LA treatment affected the antiviral effect (Fig. 2G). Interestingly, the simultaneous treatment group showed the greatest antiviral effect, but the pre-treatment and post-treatment groups also reduced RNA levels (Fig. 2H). Therefore, the antiviral effect of LA was confirmed in additional experiments using the simultaneous treatment group.

[0115] 3 GPR40 / GPR120 dual agonist GW9508 shows antiviral effects in BEAS-2B cells infected with HRV16.

[0116] GPR40 (FFAR1) and GPR120 (FFAR4) are known as long-chain fatty acid receptors. Based on this, the researchers investigated whether GPR40 / 120 is involved in the antiviral effect of LA in airway epithelial cells. The compound of Formula 1 of the present invention (GW9508) was used as a dual agonist for GPR40 / GPR120 and was used for receptor activation (Fig. 3A). Treatment with GW9508 reduced HRV16 mRNA expression levels at 24 hpi, and this effect was similar to that observed with LA (Fig. 3B). In addition, treatment with GW9508 reduced HRV16 capsid protein in cell lysates and viral load in the medium, with both decreasing to levels similar to those observed in the LA-treated group (Figs. 3C, D). These results suggest that the antiviral effect of LA may be mediated through GPR40 / GPR120.

[0117] 4. LA treatment induces transcriptional changes in BEAS-2B cells infected with HRV.

[0118] To confirm transcriptional changes in BEAS-2B cells in response to LA treatment, high-throughput mRNA sequencing at 4 hpi was performed on Mock, HRV16-infected (V), LA-treated (L), and LA-treated and HRV16-infected (LV) samples. Interestingly, there was little difference between Mock and V, indicating that viral infection did not induce changes in gene expression in BEAS-2B cells (Fig. 4A). In contrast, LA treatment induced significant changes in gene expression compared to Mock or HRV16 infection (Fig. 4A). Therefore, the altered genes were compared by comparing the V group and the LV group. The gene set GOBP: Defense Response to Virus (GO: 0051607), consisting of 312 genes related to the antiviral response protecting cells, was investigated (Fig. 4B). The top five genes identified in the gene set, including Deleted in Malignant Brain Tumors 1 (DMBT1), Interferon-stimulated Gene 20 kDa (ISG20), Tripartite Motif Containing 35 (TRIM35), Interferon Regulatory Factor 1 (IRF1), and Tripartite Motif Containing 27 (TRIM27), were validated via RT-qPCR. Among these, DMBT1, ISG20, and TRIM27 were upregulated in the LV group compared to the V group, whereas TRIM35 and IRF1 showed no significant difference (Fig. 4C). DMBT1 is known to influence viral entry by directly binding to envelope proteins. However, since HRV is an envelope-less virus, it is unlikely to exhibit antiviral activity against HRV. Consequently, DMBT1 was excluded from further experiments. TRIM27 was significantly upregulated in the LV group, but the fold change was not large (less than 1.5-fold, Fig. 4C).On the other hand, ISG20 showed significant upregulation in the LV group, exhibited the second-highest signal-to-noise ratio (Fig. 4B), and the fold change exceeded 3.0 (Fig. 4C). These results suggest that ISG20 can be used as a target gene mediating the antiviral effect of LA in BEAS-2B cells.

[0119] 5. Upregulation of ISG20 by LA depends on the NF-κB pathway in BEAS-2B cells.

[0120] Several previous studies have revealed that HRV infection does not increase the production of type I interferon (IFN-I) in airway epithelial cells. Similar to previous studies, this study also found that HRV16 infection did not increase IFN-I secretion in BEAS-2B cells. Furthermore, ISG20 mRNA levels did not increase in the HRV16-infected group at 4 hpi. This may be due to the upregulation of ISG20 induced by LA treatment rather than HRV infection. Therefore, based on studies regarding the regulatory mechanisms underlying ISG20 upregulation after LA treatment, the contribution of IFN-I to the BEAS-2B cell system is likely minimal.

[0121] Previous studies have shown that NF-κB activation directly induces ISG20 expression in endothelial cells. In this study as well, higher NF-κB p65 phosphorylation was observed in LA-treated cells compared to HRV16-infected cells at 30 minutes post-infection (Fig. 5A). To determine whether NF-κB inhibition affects ISG20 expression, caffeic acid phenethyl ester (CAPE) was used as an NF-κB inhibitor; as a result, p65 phosphorylation decreased with CAPE treatment (Fig. 5B). Inhibition of NF-κB using CAPE reduced ISG20 expression at 24 hpi (Fig. 5C). These results indicate that the upregulation of ISG20 expression in response to LA treatment depends on NF-κB activation in BEAS-2B cells.

[0122] 6. ISG20 is responsible for the antiviral effect of LA in BEAS-2B cells.

[0123] Next, the researchers investigated whether the antiviral effect of LA treatment is directly mediated by ISG20. Using a lentivirus-based knockdown system in BEAS-2B cells, it was confirmed that the transcriptional level of ISG20 was reduced in the ISG20-specific short hairpin RNA (sh-ISG20) lentivirus treatment group compared to the scrambled short hairpin RNA (sc-shRNA) lentivirus treatment group (Fig. 6A). Additionally, protein expression was reduced in the sh-ISG20 lentivirus treatment group compared to the sc-shRNA group (Fig. 6B). In cell culture medium, HRV16 titers were significantly higher in the sh-ISG20 group compared to the sc-shRNA group, both in the absence and presence of LA (Fig. 6C). Collectively, these results suggest that the antiviral effect of LA against HRV16 is dependent on ISG20.

[0124] 7. Respiratory administration of Na-LA enhances antiviral effects in HRV-infected mouse models through a mechanism dependent on GPR40 / 120.

[0125] To investigate the antiviral effects of LA in an in vivo HRV infection model, HRV1B was inoculated intranasally (in) into Balb / c mice regardless of treatment with sodium linoleate (Na-LA) (Fig. 7A). HRV1B titers peaked in bronchoalveolar lavage fluid (BAL) at 8 hpi, whereas viral titers were undetectable for UV-inactivated HRV1B (Fig. 7B). Notably, the viral load in BAL fluid was significantly reduced at 8 hpi in the HRV1B-infected group treated with Na-LA compared to the HRV1B-infected group not treated with Na-LA (Fig. 7B). Additionally, Na-LA treatment improved lung histopathology at 16 hpi (Figs. 7F, G).

[0126] Previous studies conducted on BEAS-2B cells demonstrated that the antiviral effect of LA is mediated through GPR40 / GPR120 signaling (Fig. 3). To evaluate whether this mechanism is relevant in an HRV-infected mouse model, HRV1B titers and lung histopathology were assessed in mice pretreated with GPR40 and GPR120 antagonists (intraperitoneally injected AH7614 inhibits GPR120 and GW1100 inhibits GPR40) (Fig. 7C). Interestingly, treatment with these antagonists reversed the decrease in HRV1B titers observed with Na-LA treatment, restoring viral loads in BAL fluid (Fig. 7D), lung lysates (Fig. 7E), and lung histopathology (Fig. 7F, G) to levels similar to those of the untreated HRV1B-infected group. Furthermore, the increase in ISG20 expression induced by Na-LA was attenuated by a GPR40 / 120 antagonist (Fig. 7H).

[0127] To determine whether the upregulation of ISG20 by Na-LA treatment is interferon-dependent, IFN-β and IFN-λ2 / 3 levels were measured in BAL fluid. Na-LA treatment did not increase the expression of IFN-β or IFN-λ2 / 3. Taken together, these results demonstrate that inhaled administration of LA enhances the antiviral effect against HRV through a GPR40 / 120-dependent mechanism. Furthermore, the results of this study show that the antiviral activity of LA is mediated through the GPR40 / 120-ISG20 signaling axis rather than the interferon signaling pathway. These findings suggest that the antiviral effect of LA may be mediated via GPR40 / GPR120, and that GW9508 could be utilized as a therapeutic agent for rhinovirus infections as a GPR40 / GPR120 agonist.

[0128] Foregoing, specific parts of the present invention have been described in detail. It is evident to those skilled in the art that such specific descriptions are merely preferred embodiments and do not limit the scope of the invention. Accordingly, the actual scope of the invention is defined by the appended claims and their equivalents.

[0129] The inventors of the present invention completed the invention by discovering that GPR40 agonists have a significant therapeutic effect on cold diseases caused by rhinovirus infection. Since the composition containing the GPR40 agonist of the present invention as an active ingredient has a significant therapeutic effect on rhinovirus infections, which account for the majority of common colds, it is expected to be actively utilized in the medical and healthcare fields.

Claims

1. A pharmaceutical composition for the prevention or treatment of rhinovirus infection, comprising as an active ingredient a GPR40 (G-protein coupled receptor 40) protein or an agonist of the gene encoding it.

2. In Paragraph 1, A pharmaceutical composition wherein the above agent is a compound selected from a compound represented by the following chemical formula 1, its pharmaceutically acceptable salts, crystalline forms, co-crystals, optical isomers, derivatives, hydrates, and solvates: [Chemical Formula 1] .

3. In Paragraph 1, A pharmaceutical composition in which the agonist of the above GPR40 (G-protein coupled receptor 40) protein or the gene encoding it acts as a co-agonist of the GPR120 (G-protein coupled receptor 120) protein or the gene encoding it.

4. In Paragraph 1, A pharmaceutical composition in which the above-mentioned rhinovirus infection is a respiratory disease.

5. In Paragraph 1, A pharmaceutical composition wherein the above-mentioned rhinovirus infection disease is selected from the group consisting of the common cold, asthma, chronic obstructive pulmonary disease (COPD), pneumonia, pulmonary tuberculosis, allergic rhinitis, anxiety, depression, episodic cluster headache, migraine, diabetes, chronic kidney disease / diabetic nephropathy, diabetic retinopathy, diabetic neuropathy, cachexia, anorexia nervosa, inflammation, inflammatory bowel disease, ulcerative colitis, Crohn's disease, irritable bowel syndrome, dyspepsia, and vomiting.

6. In Paragraph 1, The above pharmaceutical composition is prepared in the form of an oral formulation of a powder, granule, capsule, tablet, powder, or aqueous suspension; an external preparation; a suppository; or a sterile injectable solution.

7. A food composition for the prevention or improvement of rhinovirus infection, comprising a GPR40 (G-protein coupled receptor 40) protein or an agonist of the gene encoding it.

8. In Paragraph 7, A food composition wherein the above agent is a compound selected from a compound represented by the following chemical formula 1, its pharmaceutically acceptable salts, crystalline forms, co-crystals, optical isomers, derivatives, hydrates, and solvates: [Chemical Formula 1] .

9. In Paragraph 7, A food composition in which the agonist of the above GPR40 (G-protein coupled receptor 40) protein or the gene encoding it acts as a co-agonist of the GPR120 (G-protein coupled receptor 120) protein or the gene encoding it.

10. In Paragraph 7, A food composition in which the above-mentioned rhinovirus infection is a respiratory disease.

11. In Paragraph 7, A food composition in which the above-mentioned rhinovirus infection disease is selected from the group consisting of the common cold, asthma, chronic obstructive pulmonary disease (COPD), pneumonia, pulmonary tuberculosis, allergic rhinitis, anxiety, depression, incidental cluster headache, migraine, diabetes, chronic kidney disease / diabetic nephropathy, diabetic retinopathy, diabetic neuropathy, cachexia, anorexia nervosa, inflammation, inflammatory bowel disease, ulcerative colitis, Crohn's disease, irritable bowel syndrome, dyspepsia, and vomiting.

12. In Paragraph 7, The above food composition is a food composition manufactured in the form of a beverage, gum, tea, vitamin complex, powder, granule, tablet, capsule, jelly, confectionery, rice cake, or bread.

13. A cosmetic composition for the prevention or improvement of rhinovirus infection, comprising a GPR40 (G-protein coupled receptor 40) protein or an agonist of a gene encoding the same.

14. In Paragraph 13, A cosmetic composition wherein the above agent is a compound selected from a compound represented by the following chemical formula 1, pharmaceutically acceptable salts, crystalline forms, co-crystals, optical isomers, derivatives, hydrates, and solvates thereof: [Chemical Formula 1] .

15. In Paragraph 13, A cosmetic composition in which the agonist of the above GPR40 (G-protein coupled receptor 40) protein or the gene encoding it acts as a co-agonist of the GPR120 (G-protein coupled receptor 120) protein or the gene encoding it.

16. In Paragraph 13, A cosmetic composition in which the above-mentioned rhinovirus infection is a respiratory disease.

17. In Paragraph 13, A cosmetic composition wherein the above-mentioned rhinovirus infection disease is selected from the group consisting of the common cold, asthma, chronic obstructive pulmonary disease (COPD), pneumonia, pulmonary tuberculosis, allergic rhinitis, anxiety, depression, episodic cluster headache, migraine, diabetes, chronic kidney disease / diabetic nephropathy, diabetic retinopathy, diabetic neuropathy, cachexia, anorexia nervosa, inflammation, inflammatory bowel disease, ulcerative colitis, Crohn's disease, irritable bowel syndrome, dyspepsia, and vomiting.

18. In Paragraph 13, The above cosmetic composition is manufactured in the form of a toner, nourishing lotion, nourishing essence, massage cream, beauty bath additive, body lotion, body milk, bath oil, baby oil, baby powder, shower gel, shower cream, sunscreen lotion, sunscreen cream, tanning cream, skin lotion, skin cream, UV protection cosmetic, concealer, cleansing milk, hair loss treatment cosmetic, face and body lotion, face and body cream, skin whitening cream, hand lotion, hair lotion, cosmetic cream, jasmine oil, bath soap, liquid soap, beauty soap, shampoo, hand sanitizer (hand cleaner), medicinal soap (non-medical), cream soap, facial wash, body cleanser, scalp cleanser, hair rinse, cosmetic soap, teeth whitening gel, or toothpaste.

19. A method of preventing or treating rhinovirus infection by administering a pharmaceutically effective amount of GPR40 (G-protein coupled receptor 40) protein or an agonist of the gene encoding it to an individual requiring treatment.

20. In Paragraph 19, A method for prevention or treatment in which the above agent is a compound selected from compounds represented by the following chemical formula 1, pharmaceutically acceptable salts, crystalline forms, co-crystals, optical isomers, derivatives, hydrates, and solvates thereof: [Chemical Formula 1] .

21. Use of GPR40 (G-protein coupled receptor 40) protein, or an agonist of the gene encoding it, for the prevention or treatment of rhinovirus infection.

22. In Paragraph 21, For preventive or therapeutic use, the above agent is a compound selected from compounds represented by the following chemical formula 1, pharmaceutically acceptable salts, crystalline forms, co-crystals, optical isomers, derivatives, hydrates, and solvates thereof: [Chemical Formula 1] .

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