Anti-allergy composition comprising nitrofurazone or nitrofurantoin

Abstract

The present invention relates to a composition for improving, preventing, or treating allergic diseases, wherein nitrofurazone or nitrofurantoin inhibits degranulation in mast cells, inhibits the expression of the inflammatory cytokine TNF-α, and reduces Syk phosphorylation, thereby effectively blocking the progression of allergic diseases induced by antigens, and can be usefully utilized as a fundamental therapeutic composition.

Description

Technology Field

[0001] The present invention relates to a composition for the prevention, treatment, or improvement of allergic diseases, comprising nitrofurazone or nitrofurantoin as an active ingredient. Background Technology

[0002] Allergy is an overreaction of the immune system caused by foreign antigens (allergens) in a harmless environment. Symptoms such as itching, fever, and pain, which are the main symptoms of asthma, atopic dermatitis, and allergic rhinitis, manifest through allergic reactions, and these symptoms are caused by the excessive activation of mast cells.

[0003] Mast cells play a crucial role in the initiation of allergic reactions, and mast cell activation is closely associated with specific immunoglobulin E (IgE) antibodies, which act as typical activators. IgE binds to Fcε receptor I (FcεRI) expressed on the surface of mast cells. When FcεRI cross-links with an antigen on mast cells, it triggers an intracellular signaling cascade, activating downstream mediators such as Lyn, Syk, protein kinase C (PKC), and mitogen-activated protein kinase (MAPK), and intracellular Ca 2+It increases the concentration of calcium ions. Furthermore, the aggregation of IgE-FcεRI induces the exocytosis of preformed granules, including histamine, chemokines, cytokines, and serine proteases, and stimulates the novel synthesis of leukotrienes and prostaglandins. These inflammatory mediators cause various pathophysiological symptoms in allergic reactions, including contraction of airway smooth muscle, mucus secretion, and the recruitment of other inflammatory cells. Therefore, the regulation of inflammatory mediator production is important for the control of allergic reactions.

[0004] One of the key characteristics of mast cells is the secretion of histamine and various other inflammatory substances that trigger allergies. Histamine is involved in allergic and inflammatory responses and plays a crucial role in mediating interactions between different cells. Amid various reports regarding these allergy-related mechanisms, much research is being conducted to develop more effective anti-allergic substances. Prior art literature

[0005] Patent Document 1. Korean Published Patent Application No. 10-2022-0091528 The problem to be solved

[0006] The present invention has been devised in consideration of the above-mentioned problems, and the objective of the present invention is to provide a pharmaceutical composition for the improvement, prevention, or treatment of allergic diseases.

[0007] Another objective of the present invention is to provide a food composition for the improvement or prevention of allergic diseases. means of solving the problem

[0008] To achieve the above objective, the present invention provides a pharmaceutical composition for the prevention or treatment of allergic diseases comprising, as an active ingredient, a compound represented by the following chemical formula 1 or chemical formula 2 or a pharmaceutically acceptable salt thereof.

[0009] [Chemical Formula 1]

[0010]

[0011] [Chemical Formula 2]

[0012]

[0013] The above allergic disease may be one or more selected from the group consisting of asthma, hypersensitivity, allergic rhinitis, allergic conjunctivitis, allergic dermatitis, contact dermatitis, urticaria, atopy, insect allergy, food allergy, and drug allergy.

[0014] To achieve the above other objectives, the present invention provides a food composition for improving or preventing allergic diseases comprising, as an active ingredient, a compound represented by the following chemical formula 1 or chemical formula 2 or a food-grade acceptable salt thereof.

[0015] [Chemical Formula 1]

[0016]

[0017] [Chemical Formula 2]

[0018]

[0019] To achieve the other objectives mentioned above, the present invention provides a method for inhibiting the degranulation of mammalian-derived mast cells using a compound represented by the following chemical formula 1 or chemical formula 2.

[0020] [Chemical Formula 1]

[0021]

[0022] [Chemical Formula 2]

[0023] Effects of the invention

[0024] The present invention provides a pharmaceutical composition or food composition for the improvement, prevention, or treatment of allergic diseases.

[0025] Nitrofurazone represented by Chemical Formula 1 or nitrofurantoin represented by Chemical Formula 2, discovered in the present invention, inhibits degranulation in mast cells, inhibits the expression of the inflammatory cytokine TNF-α, and reduces Syk phosphorylation, thereby effectively blocking the progression of allergic diseases induced by antigens, and can be usefully utilized as a fundamental therapeutic composition.

[0026] In addition, it was confirmed that nitrofurazone and nitrofurantoin discovered in this invention effectively suppress allergic reactions in vivo. Brief explanation of the drawing

[0027] Figure 1 is a graph showing the results of evaluating the efficacy of nitrofurazone in inhibiting mast cell degranulation at different concentrations (0, 1, 5, 10, 20 μM). Figure 2 is a graph showing the results of evaluating the efficacy of nitrofurantoin in inhibiting mast cell degranulation at different concentrations (0, 1, 5, 10, 20 μM). Figure 3 is a graph showing the results of evaluating the efficacy of nifuroxazide in inhibiting mast cell degranulation at different concentrations (0, 1, 5, 10, 20 μM). Figure 4 confirms the effect on local skin allergic reactions and is an image confirming the inhibitory effect on dye exudation from blood vessels into tissues caused by allergic reactions in experimental animals administered with a control group, antigen, nitrofurazone, nitrofurantoin, nifuroxazide, and cetirizine. Figure 5 is a graph showing the change in dye concentration due to allergic reactions in the ears of experimental animals administered with a control group, antigen, nitrofurazone, nitrofurantoin, nifuroxazide, and cetirizine. Figure 6 shows the RT-PCR results analyzing TNF-α expression in mast cells (BMMC) treated with nitrofurazone. Figure 7 shows the RT-PCR results analyzing TNF-α expression in mast cells (BMMC) treated with nitrofurantoin. Figure 8 is a Western blot result analyzing the phosphorylation of Syk in mast cells (BMMC) treated with nitrofurazone. Figure 9 is the result of a Western blot analysis of the phosphorylation of Syk in mast cells (BMMC) following nitrofurantoin treatment. FIG. 10 is a diagram showing the mechanism by which a compound of Formula 1 (nitrofurazone) according to the present invention inhibits the intracellular FcεRI signaling pathway. Specific details for implementing the invention

[0028] Below, various aspects and embodiments of the present invention will be examined in more detail.

[0029] The objects, other objects, features, and advantages of the present invention will be easily understood through the following preferred embodiments associated with the accompanying drawings. However, the present invention is not limited to the embodiments described herein and may be embodied in other forms. Rather, the embodiments introduced herein are provided to ensure that the disclosed content is thorough and complete and that the spirit of the invention is sufficiently conveyed to a person skilled in the art.

[0030] In this specification, terms such as “comprising” or “having” are intended to specify the existence of the features, numbers, steps, actions, components, parts, or combinations thereof described in the specification, and should not be understood as precluding the existence or addition of one or more other features, numbers, steps, actions, components, parts, or combinations thereof.

[0031] In this specification, where a range is described for a variable, it will be understood that the variable includes all values ​​within the described range, including the described endpoints of the range. For example, the range “5 to 10” will be understood to include not only the values ​​5, 6, 7, 8, 9, and 10, but also any sub-ranges such as 6 to 10, 7 to 10, 6 to 9, 7 to 9, etc., and any values ​​between integers valid for the category of the described range, such as 5.5, 6.5, 7.5, 5.5 to 8.5, and 6.5 to 9. Also, for example, the range “10% to 30%” will be understood to include all integers including values ​​such as 10%, 11%, 12%, 13%, etc. and up to 30%, as well as any sub-range such as 10% to 15%, 12% to 18%, 20% to 30%, etc., and any value between valid integers within the stated range category such as 10.5%, 15.5%, 25.5%, etc.

[0032] The present invention will be described in detail below.

[0033] One aspect of the present invention provides a pharmaceutical composition for the prevention or treatment of allergic diseases, comprising a compound represented by the following Formula 1 or Formula 2 or a pharmaceutically acceptable salt thereof as an active ingredient, having anti-allergic activity.

[0034] [Chemical Formula 1]

[0035]

[0036] [Chemical Formula 2]

[0037]

[0038] Even if the backbone is the same nitrofuran derivative, nifuroxazide represented by the following chemical formula 3 exhibits an allergy-inhibiting effect only in cells and does not show any effect in actual animal models. Therefore, the active ingredient of the present invention may be nitrofurazone or nitrofurantoin represented by the following chemical formula 1 or 2, and most preferably may be nitrofurazone represented by chemical formula 1.

[0039] [Chemical Formula 3]

[0040]

[0041] Chemical formula 1 above represents nitrofurazone, which has conventionally been used to treat infections caused by some bacteria. Chemical formula 2 above represents nitrofurantoin, which has conventionally been used to treat simple urinary tract infections such as Escherichia coli, enterococci, Staphylococcus aureus, Klebsiella, Enterobacter, and acute cystitis. Therefore, there was no known information regarding the effects of nitrofurazone or nitrofurantoin on allergic diseases.

[0042] In the present invention, "pharmaceuticalally acceptable salt" refers to a salt that is generally safe, non-toxic, and not unsuitable in biological or other aspects, and is therefore usable in the preparation of a pharmaceutical composition. Specifically, it refers to an inorganic salt, an organic salt, or an addition salt of a metal salt of a compound. The inorganic salt may be a hydrochloride, a bromate, a phosphate, a sulfate, or a disulfate. The organic salt may be a formate, an acetate, a propionate, a lactate, an oxalate, a tartrate, a malate, a maleate, a citrate, a fumarate, a besylate, a camsylate, an edicyl, a trichloroacetic acid, a trifluoroacetic acid, a benzoate, a gluconate, a methanesulfonate, a glycolate, a succinate, a 4-toluenesulfonate, a galacturonate, an emvonate, a glutamate, a methanesulfonic acid, an ethanesulfonic acid, a benzenesulfonic acid, a p-toluenesulfonic acid, or an aspartate. The metal salt may be a calcium salt, sodium salt, magnesium salt, strontium salt, or potassium salt.

[0043] In the present invention, "allergic disease" refers to a disease caused by allergy, and specifically includes diseases caused by an immune hypersensitivity reaction resulting from exposure to various allergens that induce an immune hypersensitivity reaction. The disease caused by the allergic disease may be at least one selected from the group consisting of asthma, hypersensitivity, allergic rhinitis, allergic conjunctivitis, allergic dermatitis, contact dermatitis, urticaria, atopy, insect allergy, food allergy, and drug allergy. In addition to these, various allergic diseases exist, and the presented diseases are exemplary and are not limited thereto.

[0044] In this invention, "asthma" is an inflammatory airway obstruction disease that causes difficulty breathing. Asthma patients complain of symptoms such as wheezing, shortness of breath, and coughing due to airway obstruction, and these symptoms tend to worsen at night than during the day. The causes of asthma involve a complex interplay of environmental and genetic factors. Mast cells and eosinophils are primarily involved in the airway obstruction caused by asthma, and the typical symptoms of asthma resulting from their inflammatory response in the airways manifest as airway obstruction.

[0045] In the present invention, "atopic dermatitis" is a skin eczema disease accompanied by severe itching and recurring chronically, and is used interchangeably with terms such as "atopy" and "atopic disease." It is known that the onset of atopic dermatitis is caused by a complex interplay of various factors, including genetic predisposition, environmental factors, immunological abnormalities in the patient, and abnormalities in the skin's protective barrier. The most significant characteristic of atopic dermatitis is severe itching and a highly sensitive reaction to external stimuli. The itching usually worsens in the evening, and it is characterized by scratching the skin, which leads to eczematous changes; furthermore, as the eczema worsens, a vicious cycle is repeated in which the itching becomes even more severe.

[0046] In the present invention, "treatment" refers to any act of improving or beneficially altering the symptoms of an allergic disease by administering the composition of the present invention.

[0047] In the present invention, "prevention" refers to any act in which the possibility of developing an allergic disease is suppressed or delayed by the administration of the composition of the present invention.

[0048] The above composition can prevent, treat, or improve allergic diseases by inhibiting the signal transduction of the initial signal that activates mast cells. Specifically, the above composition can prevent, treat, or improve allergic diseases by inhibiting the signal transduction and / or phosphorylation of Syk.

[0049] The above composition can inhibit the expression of any one selected from the group consisting of TNF-α, IL-2, IL-3, and IL-4, and preferably inhibits the expression of TNF-α. This can prevent, treat, or improve allergic diseases.

[0050] The above composition may prevent, treat, or improve allergic diseases through an inhibitory action on mast cell degranulation, and specifically, may inhibit the degranulation of mast cells. That is, it may prevent or treat allergic diseases through a mechanism that inhibits the secretion / expression level of beta-hexosaminidase or inhibits the secretion / expression level of histamine cytokines.

[0051] In the present invention, "containing as an active ingredient" means containing a sufficient amount to achieve the efficacy or activity of a compound represented by Formula 1 or Formula 2 of the present invention.

[0052] The above composition may be a pharmaceutical composition. The above pharmaceutical composition may include a pharmaceutically acceptable carrier. For oral administration, at least one selected from the group consisting of binders, lubricants, disintegrants, excipients, solubilizers, dispersants, stabilizers, suspending agents, colorants, and flavorings may be used as the pharmaceutically acceptable carrier, but is not limited thereto. For injectables, a buffer, preservative, analgesic, solubilizer, isotonic, or stabilizer may be mixed and used, and for topical administration, a base, excipient, lubricant, or preservative may be mixed and used.

[0053] The formulation of the pharmaceutical composition according to the present invention can be prepared in various ways by mixing it with a pharmaceutically acceptable carrier as described above. For example, when administered orally, it can be prepared in the form of a tablet, troche, capsule, elixir, suspension, syrup, wafer, etc., and in the case of an injectable, it can be prepared in the form of a unit dosing ampoule or a multi-dose formulation, and can also be formulated as a solution, suspension, tablet, capsule, sustained-release formulation, etc.

[0054] Meanwhile, examples of carriers, excipients, or 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.

[0055] The 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, local, sublingual, or rectal. Oral or parenteral administration is preferred. The administration may be systemic or local.

[0056] The above "parenteral" includes subcutaneous, intradermal, intravenous, intramuscular, intra-articular, intrasynovial, intrasternal, intradural, intralesional, and intracranial injection or infusion techniques. The pharmaceutical composition according to the present invention may also be administered in the form of a suppository for rectal administration. The above administration may be a single dose of 0.1 mg to 1,000 mg of a compound represented by Formula 1 or Formula 2, for example, 0.1 mg to 500 mg, 0.1 mg to 100 mg, 0.1 mg to 50 mg, 0.1 mg to 25 mg, 1 mg to 1,000 mg, 1 mg to 500 mg, 1 mg to 100 mg, 1 mg to 50 mg, 1 mg to 25 mg, 5 mg to 1,000 mg, 5 mg to 500 mg, 5 mg to 100 mg, 5 mg to 50 mg, 5 mg to 25 mg, 10 mg to 1,000 mg, 10 mg to 500 mg, 10 mg to 100 mg, 20 mg to 100 mg, or 50 mg to 100 mg.

[0057] The dosage of the above composition may be, for example, about 0.001 mg / kg to about 1000 mg / kg based on an adult, for example, about 0.01 mg / kg to about 100 mg / kg, or for example, about 0.1 mg / kg to about 100 mg / kg. The frequency of administration may be once a day, multiple times a day, 2 to 3 times a week, 1 to 4 times a month, or 1 to 12 times a year.

[0058] Another aspect of the present invention relates to a food composition for improving or preventing allergic diseases comprising, as an active ingredient, a compound represented by the following chemical formula 1 or chemical formula 2 or a food-grade acceptable salt thereof.

[0059] [Chemical Formula 1]

[0060]

[0061] [Chemical Formula 2]

[0062]

[0063] The specific details of the compound represented by Chemical Formula 1 or Chemical Formula 2 are as described above, so they are omitted to avoid excessive duplication.

[0064] In the present invention, "food-acceptable salt" refers to a salt that is generally safe, non-toxic, and not unsuitable in biological or other aspects, and is therefore usable in the preparation of food compositions. Specifically, it refers to an inorganic salt, an organic salt, or an addition salt of a metal salt of a compound. The inorganic salt may be a hydrochloride, a bromate, a phosphate, a sulfate, or a disulfate. The organic salt may be a formate, an acetate, a propionate, a lactate, an oxalate, a tartrate, a malate, a maleate, a citrate, a fumarate, a besylate, a camsylate, an edicyl, a trichloroacetic acid, a trifluoroacetic acid, a benzoate, a gluconate, a methanesulfonate, a glycolate, a succinate, a 4-toluenesulfonate, a galacturonate, an emvonate, a glutamate, a methanesulfonic acid, an ethanesulfonic acid, a benzenesulfonic acid, a p-toluenesulfonic acid, or an aspartate. The metal salt may be a calcium salt, sodium salt, magnesium salt, strontium salt, or potassium salt.

[0065] In the 'food composition' of the present invention, there are no special restrictions on the types of food. Examples of foods to which the compound represented by Chemical Formula 1 or Chemical Formula 2 may be added include drinks, meat, sausage, bread, biscuits, rice cakes, chocolate, candies, snacks, confectionery, pizza, ramen, other noodles, chewing gum, dairy products including ice cream, various soups, beverages, alcoholic beverages, vitamin complexes, dairy products and dairy processed products, and include all health functional foods or health supplements in the conventional sense.

[0066] In the food composition of the present invention, the compound represented by Formula 1 or Formula 2 may be added directly to food or used together with other food or food ingredients, and may be appropriately used according to conventional methods. The amount of compound represented by Formula 1 or Formula 2 added to the food composition of the present invention may be appropriately determined according to its purpose of use (for prevention or improvement). It is preferable, but not limited to, to include the compound represented by Formula 1 or Formula 2 in an amount of 0.1 to 50 weight% with respect to the total weight of the food composition of the present invention. However, in the case of long-term consumption for the purpose of health and hygiene or health control, the above amount may be less than the above range, and since there are no issues regarding safety, the active ingredient may be used in an amount greater than the above range.

[0067] When manufacturing a beverage, there are no special restrictions on other ingredients other than containing a compound represented by Chemical Formula 1 or Chemical Formula 2, and various flavoring agents or natural carbohydrates, etc., may be included as additional ingredients, as in conventional beverages. The natural carbohydrates include monosaccharides, e.g., glucose, fructose, etc.; disaccharides, e.g., maltose, sucrose, etc.; polysaccharides, e.g., dextrin, cyclodextrin, etc., and conventional sugars, and sugar alcohols such as xylitol, sorbitol, erythritol, etc. As for the flavoring agents, natural flavoring agents (taumatin, stevia extract (e.g., rebaudioside A, glycyrrhizin, etc.)) and synthetic flavoring agents (saccharin, aspartame, etc.) may be advantageously used. The proportion of the natural carbohydrates is about 1 to 20 weight%, preferably about 5 to 12 weight%, based on the total weight of the food composition of the present invention.

[0068] In addition to the above, 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 and thickening agents (cheese, chocolate, etc.), 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. Furthermore, the food composition of the present invention may contain fruit pulp for the production of natural fruit juices, fruit juice beverages, and vegetable beverages. These ingredients may be used independently or in combination. Although the proportion of these additives is not particularly important, it is generally selected in the range of 0.1 to about 20 weight percent with respect to the total weight of the food composition of the present invention.

[0069] In another aspect, the present invention relates to a method for inhibiting the degranulation of mammalian-derived mast cells using a compound represented by the following chemical formula 1 or chemical formula 2.

[0070] [Chemical Formula 1]

[0071]

[0072] [Chemical Formula 2]

[0073]

[0074] In the present invention, mammals may be humans or mammals other than humans, and preferably may include mammals including dogs, cats, rats, livestock, etc., birds, reptiles, farmed fish, etc. without limitation, which may exclude humans. In addition, the method for inhibiting the degranulation of mammalian-derived mast cells in the present invention may be performed "in vitro."

[0075] The present invention is to be explained in more detail below through examples, etc.; however, the scope and content of the present invention shall not be interpreted as being narrowed or limited by the examples, etc. below. Furthermore, based on the disclosure of the present invention including the examples below, it is evident that a person skilled in the art can easily practice the present invention even without specific experimental results presented, and it is natural that such variations and modifications fall within the scope of the appended claims.

[0076] <Experimental Materials>

[0077] Nitrofurazone, nitrofurantoin, and nifuroxazide were purchased from MedChemExpress (Monmouth Junction, NJ, USA) and can be represented by the following chemical formulas 1 to 3.

[0078] [Chemical Formula 1] : nitrofurazone

[0079]

[0080] [Chemical Formula 2] : nitrofurantoin

[0081]

[0082] [Chemical Formula 3] : Nifuroxazide

[0083]

[0084] Monoclonal dinitrophenol (DNP)-specific IgE, human serum albumin (DNP-HSA), Evans blue, toluidine blue O, and cetirizine were purchased from Sigma-Aldrich (St. Louis, MO, USA). The antibody against the phosphorylated form of Syk (Tyr352; dilution fold, 1:1000) and beta-actin were purchased from Cell Signaling Technology, Inc. (Danvers, MA, USA). The antibody against Syk (dilution fold, 1:200) was obtained from Santa Cruz Biotechnology (Dallas, Texas, USA).

[0085] <Statistical Analysis>

[0086] Experimental results were obtained by repeating the experiment three times and expressed as the mean and standard error (mean ± SEM). For animal experiments, a minimum of 5 animals were used per experimental group. For in vitro experiments, each experiment was performed three times.

[0087] Statistical analysis was performed using one-way ANOVA and independent sample Student's t-test. All statistical calculations (*p<0.05 and *p<0.01) were performed using GraphPad Prism 7 software (GraphPad Software Inc., San Diego, CA). IC50 values ​​were calculated using the AAT bioquest IC50 calculator (https: / / www.aatbio.com / tools / ic50-calculator).

[0088] <Experimental Example 1> Inhibitory effect of antigen stimulation on degranulation in mast cells

[0089] Culture of BMMC (bone marrow-derived mast cells)

[0090] Bone marrow cells were isolated from the femurs and tibias of 5-week-old mice and cultured in RPMI (Roswell Park Memorial Institute) 1640 culture medium containing 4 mM L-glutamine, 100 Units / ml penicillin, 100 μg / ml streptomycin, 1 mM sodium pyruvate, 0.1 mM non-essential amino acids, 25 mM HEPES, 50 mM 2-mercaptoethanol, 10% FBS, and 10 ng / ml interleukin (IL)-3. After 4–5 weeks, FcεRⅠ and c-Kit expression were used as maturation markers for mast cells to assess the purity of the BMMCs. Cells with a purity of 95% or higher (>95%) were used for subsequent experiments.

[0091] Observation of allergic reactions by treating BMMC cells with antigens of varying concentrations

[0092] Nitrofurazone, nitrofurantoin, and nifuroxazide were prepared as candidate substances, and each of them was dissolved in Tyrode buffer to prepare solutions of various concentrations (0, 1, 5, 10, 20 μM).

[0093] BMMCs(3.0×10 5Cells (cells / well) were seeded into a 96-well plate and cultured in RPMI (Roswell Park Memorial Institute) 1640 for 24 hours. The culture medium was removed, and cells were sensitized by treating with medium containing 100 ng / ml DNP-specific IgE for 4 hours. Next, the solution was removed from the cells, and after treatment with Tyrode buffer (pH 7.4, 135 mM NaCl, 5 mM KCl, 20 mM HEPES, 5.6 mM glucose, 1.8 mM CaCl2, 1 mM MgCl2, and 0.5% BSA), solutions of various concentrations of nitrofurazone, nitrofurantoin, or nifuroxazide were added for 30 minutes, and an immunoreaction was induced for 15 minutes by adding 50 ng / ml of DNP-HSA (Dinitrophenol-Human Serum Albumin). Then, 30 μL of the supernatant was taken and transferred to a 96-well plate, 30 μL of p-nitrophenyl N-acetyl-beta-D-glucosamine (p-NAG) was added, and the mixture was incubated at 37 °C for 1 hour. Then, 250 μL of stop buffer (0.1 M carbonate buffer) was added to terminate the reaction, and absorbance was measured at 405 nm. The degree of degranulation of mast cells was expressed as the percentage (%) of released β-hexosaminidase out of total β-hexosaminidase.

[0094] Figure 1 is a graph showing the results of the evaluation of the efficacy of nitrofurazone in inhibiting mast cell degranulation at different concentrations (0, 1, 5, 10, 20 μM), Figure 2 is a graph showing the results of the evaluation of the efficacy of nitrofurantoin in inhibiting mast cell degranulation at different concentrations (0, 1, 5, 10, 20 μM), and Figure 3 is a graph showing the results of the evaluation of the efficacy of nifuroxazide in inhibiting mast cell degranulation at different concentrations (0, 1, 5, 10, 20 μM).

[0095] Looking at Figures 1 to 3, it was confirmed that the candidate substances nitrofurazone, nitrofurantoin, and nifuroxazide all exhibited inhibitory effects on degranulation. In particular, it was confirmed that nitrofurazone had a significant effect at 5 μM or higher, while nitrofurantoin and nifuroxazide had a significant effect at 1 μM or higher.

[0096] Based on the above results, the nitrofuran derivative of the present invention inhibited degranulation caused by antigen stimulation in BMMC in a concentration-dependent manner, and it was confirmed that it has a novel efficacy as an effective mast cell-dependent allergy treatment.

[0097] <Experimental Example 2> Confirmation of effect on local skin allergic reactions

[0098] Animal experiments were approved by Konkuk University's Institutional Animal Care and Use Committee (IACUC) and were conducted in accordance with institutional guidelines.

[0099] The skin allergy effects of nitrofurazone, nitrofurantoin, and nifuroxazide were tested using passive cutaneous anaphylaxis (PCA), which is used when developing drugs with anti-allergic effects. Cetirizine was prepared as a positive control. It is a second-generation antihistamine used to treat allergic rhinitis, dermatitis, and urticaria.

[0100] Specifically, mice (Balb / c, 5 weeks old, male) were purchased from Orient Bio, and after an acclimatization period of one week, 50 ng of DNP-specific IgE was injected intradermally into 10 μl of PBS. After 12 hours, a nitrofuran derivative (100 mg / kg) or cetirizine (100 mg / kg) dissolved in 5% Arabic gum (Sigma-Aldrich, St. Louis, MO, USA) was administered orally. After one hour, 100 μg of DNP-HSA was mixed with 250 μl of Evans blue at a concentration of 5 mg / ml and administered intravenously. The antigen administered was physiological saline (100 mg / kg) instead of the nitrofuran derivative or cetirizine, and the control group consisted of normal animals that were not treated with anything.

[0101] After 1 hour, the experimental animals were euthanized and ear tissue was collected. To extract the dye from the collected ear tissue, 1 ml of formamide was treated at 63°C for 24 hours. The absorbance of the dye was measured at 620 nm.

[0102] Figure 4 is an image confirming the effect on local skin allergic reactions, showing changes in dye exuded from blood vessels into tissues due to allergic reactions in experimental animals administered with a control group, antigen, nitrofurazone, nitrofurantoin, nifuroxazide, and cetirizine, and Figure 5 is a graph confirming changes in dye concentration due to allergic reactions in the ears of experimental animals administered with a control group, antigen, nitrofurazone, nitrofurantoin, nifuroxazide, and cetirizine.

[0103] As shown in Figures 4 and 5, no dye was detected in the control group of experimental animals, but 12-15 μg / ml of dye was detected in the antigen group of experimental animals. In other words, it was confirmed that allergic skin inflammatory disease can be easily induced in experimental animals through the PCA experiment.

[0104] Meanwhile, it was found that the amount of Evans blue in experimental animals orally administered nitrofurazone was significantly suppressed to a level similar to that of the control group. In experimental animals orally administered nitrofurantoin, it was confirmed that the amount of Evans blue was significantly suppressed to a level similar to that of the commercially available antihistamine cetirizine, although not to the same extent as nitrofurazone.

[0105] On the other hand, no significant decrease in the amount of Evans blue was observed in experimental animals orally administered nifuroxazide.

[0106] Although it was confirmed that the three types of nitrofuran derivatives in the present invention have excellent degranulation effects in cell experiments on mast cells, actual in vivo experiments revealed that nifuroxazide had no inhibitory effect on passive skin allergies, and nitrofurantoin showed only an effect similar to that of conventional antihistamines. However, while nitrofurazone showed a weaker degranulation effect than other nitrofuran derivatives in cell experiments, it was confirmed that the degree of passive skin allergic reaction was significantly inhibited in in vivo experiments.

[0107] <Experimental Example 3> Analysis of Anti-Allergy Effect via RT-PCR

[0108] Culture of BMMC (bone marrow-derived mast cells)

[0109] Bone marrow cells were isolated from the femurs and tibias of 5-week-old mice and cultured in RPMI (Roswell Park Memorial Institute) 1640 culture medium containing 4 mM L-glutamine, 100 Units / ml penicillin, 100 μg / ml streptomycin, 1 mM sodium pyruvate, 0.1 mM non-essential amino acids, 25 mM HEPES, 50 mM 2-mercaptoethanol, 10% FBS, and 10 ng / ml interleukin (IL)-3. After 4–5 weeks, FcεRⅠ and c-Kit expression were used as maturation markers for mast cells to assess the purity of the BMMCs. Cells with a purity of 95% or higher (>95%) were used for subsequent experiments.

[0110] Analysis of TNF-α secretion inhibitory ability in BMMC cells

[0111] BMMC(8.0×10 5Cells (1 / well) were seeded into a 96-well plate, 20 ng / ml DNP-specific IgE was added, and the cells were incubated overnight. 0 or 10 μM nitrofurazone and nitrofurantoin, respectively, were added and incubated for 30 minutes, after which 25 ng / ml DNP-HSA was added and incubated for 15 minutes. Total RNA was extracted from each cell using the Easy-spin™ Total RNA Extraction Kit (iNtRON Biotechnology, Inc., Gyeonggi-do, Korea). Total RNA was reverse transcribed for cDNA synthesis using AccuPower® RT PreMix & Master Mix (Bioneer Co., Daejeon, Korea) according to the manufacturer's protocol. PCR was performed under the following conditions. The cycle was set to 94 ℃ for 30 seconds, 58 ℃ for 20 seconds, and 72 ℃ for 30 seconds, and this was repeated a total of 32 times. The primer sequences used for PCR analysis are as follows.

[0112] Sequence No. 1: mouse TNF-α forward

[0113] 5'-CACCACGCTCTTCTGTCTACTGAAC-3'

[0114] Sequence No. 2: mouse TNF-α reverse

[0115] 5'-CCGGACTCCGTGATGTCTAAGTACT-3'

[0116] Sequence No. 3 : rat GAPDH, forward

[0117] 5'- TCCCTCAAGATTGTCAGCAA-3'

[0118] Sequence No. 4: rat GAPDH, reverse

[0119] 5'- AGATCCACAACGCATACATT-3' for

[0120] Figure 6 is the RT-PCR result of analyzing TNF-α expression in mast cells (BMMC) treated with nitrofurazone, and Figure 7 is the RT-PCR result of analyzing TNF-α expression in mast cells (BMMC) treated with nitrofurantoin.

[0121] As shown in Figures 6 and 7, TNF-α expression was not detected in mast cells that were not treated, while TNF-α expression was increased in mast cells treated with the antigen (DNP-HSA). At this time, it was observed that TNF-α expression decreased to a normal level when nitrofurazone and nitrofurantoin were treated.

[0122] In addition to degranulation, one of the reactions occurring in mast cells during IgE-mediated allergic reactions is the expression of inflammatory cytokines. Cytokines produced by mast cells in response to antigen stimulation include TNF-α (tumor necrosis facto) and IL-4. Among these, TNF-α is a very important cytokine in allergic inflammatory reactions, which induces a late-phase allergic reaction by causing leukocytes such as neutrophils and eosinophils to enter the inflammatory site.

[0123] Since nitrofurazone and nitrofurantoin inhibited the expression of TNF-α in mast cells in response to antigen stimulation, it can be seen that nitrofurazone and nitrofurantoin have the effect of suppressing delayed allergic symptoms by inhibiting the expression of inflammatory cytokines in response to antigen stimulation.

[0124] <Experimental Example 4> Analysis of Anti-allergic Effect via Western Blot

[0125] Culture of BMMC (bone marrow-derived mast cells)

[0126] Bone marrow cells were isolated from the femurs and tibias of 5-week-old mice and cultured in RPMI (Roswell Park Memorial Institute) 1640 culture medium containing 4 mM L-glutamine, 100 Units / ml penicillin, 100 μg / ml streptomycin, 1 mM sodium pyruvate, 0.1 mM non-essential amino acids, 25 mM HEPES, 50 mM 2-mercaptoethanol, 10% FBS, and 10 ng / ml interleukin (IL)-3. After 4–5 weeks, FcεRⅠ and c-Kit expression were used as maturation markers for mast cells to assess the purity of the BMMCs. Cells with a purity of 95% or higher (>95%) were used for subsequent experiments.

[0127] Analysis of Syk's phosphorylation inhibitory ability via Western blot

[0128] BMMC(2.0×10 6Cells (in a well) were seeded into a 96-well plate. 100 ng / ml DNP-specific IgE was added and the cells were stimulated for 4 hours. Afterward, the cells were washed with clean culture medium, and 0 or 10 μM of nitrofurazone and nitrofurantoin, respectively, were added and incubated for 30 minutes. Next, the cells were stimulated with 50 ng / ml DNP-HSA antigen for 15 minutes, and the reaction was terminated using ice. The cells were lysed using RIPA buffer (Thermo Fisher Scientific, Waltham, MA, USA) containing 1 mM PMSF (phenylmethylsulfonyl fluoride) and a protease inhibitor cocktail (Sigma Aldrich, St. Louis, MO, USA). Cell lysates were centrifuged at 13,000 rpm for 10 minutes, the supernatant was mixed with 4×Nu-PAGE™ sample buffer (Thermo Fisher Scientific, Waltham, MA, USA), and denatured by heating at 96 °C for 5 minutes. Proteins were separated by SDS (sodium dodecyl sulfate-polyacrylamide) electrophoresis and transferred to a PVDF (polyvinylidene fluoride) membrane using transfer cassettes (Bio-Rad, Hercules, CA, USA). They were incubated with a specific primary antibody in TBS-T buffer containing 5% BSA or skim milk (Tris-buffered saline containing 0.1% Tween 20). Next, the samples were cultured with a secondary antibody labeled with horseradish peroxidase, and protein bands were detected using Luminata Crescendo Western HRP substrate (EMD Millipore Corp., Billerica, MA, USA).

[0129] Figure 8 is the result of a Western blot analysis of the phosphorylation of Syk and Syk in mast cells (BMMC) treated with nitrofurazone, and Figure 9 is the result of a Western blot analysis of the phosphorylation of Syk and Syk in mast cells (BMMC) treated with nitrofurantoin.

[0130] Inflammatory mediators activate MMPs to destroy the tissue extracellular matrix. In some cells, this inhibits the cross-linking of cell adhesion molecules and promotes the influx of inflammatory leukocytes and lymphocytes. Various cellular signals regulate this process, and the FcεRI signaling pathway plays a crucial role. Signals that induce immune activation are regulated by recognizing the outer membrane binding site of FcεRI and stimulating Lyn and Syk signals (Fig. 10). Specifically, when mast cells are activated by an antigen, Syk binds to the ITAM of the γ-subunit of FcεRI, becoming phosphorylated and activated. The activated Syk signal causes mast cells to degranulate and secrete cytokines, thereby inducing an allergic response.

[0131] As a result of examining the intracellular protein-level FcεRI signaling pathway using nitrofurazone and nitrofurantoin, it was confirmed that they significantly reduced Syk phosphorylation.

[0132] In other words, nitrofurazone and nitrofurantoin appear to inhibit Syk phosphorylation, thereby reducing PI3K levels, and also reduce phosphorylation of the MAPK signaling pathway.

[0133] Therefore, it was confirmed that treatment with nitrofurazone and nitrofurantoin alleviates inflammatory stimulation of IgE-mediated mast cells (BMMCs), reduces TNF-α expression, and regulates FcεRI signaling by controlling cytokine secretion through the inhibition of Syk phosphorylation. Through these mechanisms, it can be seen that nitrofurazone and nitrofurantoin can be utilized as therapeutic agents to suppress allergic reactions.

Claims

Claim 1 A pharmaceutical composition for the prevention or treatment of allergic diseases comprising, as an active ingredient, a compound represented by the following Chemical Formula 1 or Chemical Formula 2 or a pharmaceutically acceptable salt thereof. [Chemical Formula 1] [Chemical Formula 2] Claim 2 A pharmaceutical composition for the prevention or treatment of allergic diseases according to claim 1, characterized in that the allergic disease is one or more selected from the group consisting of asthma, hypersensitivity, allergic rhinitis, allergic conjunctivitis, allergic dermatitis, contact dermatitis, urticaria, atopy, insect allergy, food allergy, and drug allergy. Claim 3 A food composition for the improvement or prevention of allergic diseases comprising, as an active ingredient, a compound represented by the following Chemical Formula 1 or Chemical Formula 2 or a food-grade acceptable salt thereof. [Chemical Formula 1] [Chemical Formula 2] Claim 4 Using a compound represented by the following Chemical Formula 1 or Chemical Formula 2 Mammals other than humans Method for inhibiting degranulation of derived mast cells.[Chemical Formula 1] [Chemical Formula 2]

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