Pharmaceutical composition for preventing or treating eosinophilia or eosinophilic inflammatory diseases, comprising inotodiol or prodrug thereof

Abstract

The present invention relates to a pharmaceutical composition for preventing or treating eosinophilia or eosinophilic inflammatory diseases, the composition comprising inotodiol or a prodrug thereof. The composition comprising inotodiol or a prodrug thereof according to one aspect exhibits the effect of inducing apoptosis specifically in eosinophils, and is thus expected to be useful as an agent for preventing or treating eosinophilia or eosinophilic inflammatory diseases.

Description

Pharmaceutical composition for preventing or treating eosinophilia or eosinophilic inflammatory disease comprising inotodiol or a prodrug thereof

[0001] The present invention relates to a pharmaceutical composition for preventing or treating eosinophilia or eosinophilic inflammatory disease, comprising inotodiol or a prodrug thereof.

[0002] Eosinophilia or eosinophilic inflammatory disease, which is an eosinophil-mediated disease, is a group of diseases characterized by the presence of a large number of eosinophils within the lesion or by the assumption that eosinophils play an important pathological or physiological role in the development of the disease.

[0003] Eosinophilia or eosinophilic inflammatory disease can occur due to various causes, and the symptoms of the disease can vary depending on the site of inflammation. For example, eosinophilic pneumonia (EP), a representative type of eosinophilia, can be seen with numerous eosinophil infiltrations in the lung parenchyma, whereas allergic bronchopulmonary aspergillosis can occur confined to the airways (Literature [Hanyang Med Rev 2013;33:65-74]).

[0004] In addition, eosinophils are directly or indirectly related to the pathogenesis and physiology of common diseases such as asthma and rare diseases, and play an important role in the pathogenesis and physiology of intractable diseases accompanied by chronic severe inflammation. Specifically, eosinophils are known to play an important role in causing morbidity in allergic diseases such as chronic bronchial asthma and atopic dermatitis (literature [Adv. Immunol., 39, 177(1986), Immunol. Today, 13, 501(1992)]), and are known to be associated with diseases commonly referred to as eosinophilia, such as hypereosinophilic syndrome (HES), eosinophilic gastroenteritis, eosinophilic leukemia, eosinophilic sarcoma, and Kimura's disease (literature [Ann. Intern. Med. 97, 78(1982)]).

[0005] Effective control of excessive and activated eosinophils is crucial for the treatment of eosinophil-mediated diseases, as their byproducts can cause epithelial damage and tissue toxicity, such as bronchial stenosis, airway edema, and mucus plugging in asthmatic patients [Simon HU, Blaser K, 1995, Immun. Today 16, 53-55; Haslett C, 1997, British Med. Bull. 53, 669-683].

[0006] Glucocorticoids and theophylline are known as treatments that remove eosinophils, but they have the disadvantage of causing various steroid-related side effects, such as metabolic abnormalities and cataracts, when taken for a long period of time.

[0007] In addition, substances having inhibitory activity against cytokines or chemokines involved in the differentiation or proliferation of eosinophils have been used as eosinophil function inhibitors, but there is a problem in that they do not act on cytokine-independent eosinophils that have been activated and infiltrated into the site of inflammation.

[0008] Therefore, there is a need to develop safe and effective treatments for eosinophil-mediated diseases that specifically suppress eosinophils and induce apoptosis of activated eosinophils.

[0009] One aspect provides a pharmaceutical composition for preventing or treating eosinophilia or eosinophilic inflammatory disease, comprising inotodiol, a prodrug thereof or an acceptable salt thereof.

[0010] Another aspect is to provide a health functional food for preventing or improving eosinophilia or eosinophilic inflammatory disease, comprising inotodiol, a prodrug thereof or a pharmaceutically acceptable salt thereof as an active ingredient.

[0011] Another aspect provides an inhalation composition for the prevention or treatment of eosinophilia or eosinophilic inflammatory disease, comprising inotodiol, a prodrug thereof or a pharmaceutically acceptable salt thereof as an active ingredient.

[0012] Another aspect is to provide a pharmaceutical composition for preventing or improving eosinophilia or eosinophilic inflammatory disease, comprising inotodiol, a prodrug thereof, or a pharmaceutically acceptable salt thereof as an active ingredient.

[0013] Another aspect provides the use of inotodiol, a prodrug thereof, or a pharmaceutically acceptable salt thereof for the manufacture of a medicament for the prevention or treatment of eosinophilia or eosinophilic inflammatory diseases.

[0014] Another aspect provides a method of treating eosinophilia or an eosinophilic inflammatory disease comprising administering to a subject in need thereof inotodiol, a prodrug thereof, or a pharmaceutically acceptable salt thereof.

[0015] One aspect provides a pharmaceutical composition for preventing or treating eosinophilia or eosinophilic inflammatory disease, comprising inotodiol, a prodrug thereof or an acceptable salt thereof.

[0016] The term "Inotodiol" in this specification refers to a compound having the IUPAC name (3S,5R,10S,13R,14R,17R)-17-[(2S,3R)-3-hydroxy-6-methylhept-5-en-2-yl]-4,4,10,13,14-pentamethyl-2,3,5,6,7,11,12,15,16,17-decahydro-1H-cyclopenta[a]phenanthren-3-ol. ((3S,5R,10S,13R,14R,17R)-17-[(2S,3R)-3-hydroxy-6-methylhept-5-en-2-yl]-4,4,10,13,14 -pentamethyl-2,3,5,6,7,11,12,15,16,17-decahydro-1H-cyclopenta[a]phenanthren-3-ol).

[0017] The above inotodiol is a major physiologically active component of chaga mushroom (Inonotus obliquus), and can be chemically synthesized according to a conventional method, manufactured as a pharmaceutically acceptable salt, or separated and purified from a chaga mushroom extract.

[0018] The term "prodrug" as used herein may be biologically inactive in itself, but may exhibit effective pharmacological effects after undergoing chemical / biochemical structural modifications during its residence time in the body. In other words, it refers to a drug that, despite being useful, has unsuitable properties such as side effects, stability, solubility, absorbability, and duration of action, but has been chemically modified to enable clinical use.

[0019] The term "prodrug of inotodiol" as used herein means a compound which may be biologically inactive in itself, but undergoes chemical / biochemical structural transformation to inotodiol during its residence time in the body.

[0020] In one specific example, the prodrug of inotodiol may be an inotodiol ester derivative compound formed through an ester condensation reaction between a hydroxyl group of an inotodiol compound of the following chemical formula 1 and a carboxyl group of a fatty acid glucuronic acid, an alkyl succinic anhydride, or a phenolic acid:

[0021] [Chemical Formula 1]

[0022]

[0023] Specifically, the prodrug may be a compound represented by the following chemical formula 2:

[0024] [Chemical Formula 2]

[0025]

[0026] In the above formula, R1 and R2 are independently OH, or -OC(O)-R3, and R3 is a straight or branched alkyl, alkenyl or alkynyl chain having 1 to 30, 4 to 30, or 6 to 30 carbon atoms,

[0027] At least one of R1 and R2 is -OC(O)-R3.

[0028] In one specific example, R3 is CH3(CH2) a-, or CH3(CH2) b (CH=CH[CH2]) c (CH2) d - An unsubstituted straight alkyl or alkenyl chain having a, wherein a is an integer from 8 to 24, b is an integer from 1 to 5, b is an integer from 1 to 6, and d is an integer from 3 to 7.

[0029] In one specific example, an inotodiol derivative is a compound formed through an ester bond between inotodiol and a fatty acid, and not only has increased solubility in organic solvents and improved stability, but is also easily decomposed into inotodiol and fatty acid in the small intestine, so that it is easily absorbed in the small intestine. For example, the inotodiol derivative may be formed through an ester bond between inotodiol and succinic acid. Specifically, the inotodiol derivative can be synthesized by performing a condensation reaction between alkyl succinic anhydride and inotodiol in the presence of a p-toluenesulfonic acid catalyst, and alkyl succinic anhydride can be added in an excess amount compared to inotodiol to induce a succinic acid ester bond in all or at least one hydroxyl group of inotodiol. After completion of the reaction, p-toluenesulfonic acid is neutralized and removed with sodium bicarbonate. The inotodiol derivative compound synthesized by the above method has improved water solubility because both or at least one of R1 and R2 of the above chemical formula 2 forms a succinic acid ester bond, and the ester bond is easily broken down, so that it can be easily absorbed in the small intestine.

[0030] In addition, the inotodiol derivative according to another specific example not only increases the water solubility of inotodiol through the ester bond between inotodiol and fatty acid, but also decreases the hydrolysis in the small intestine and the absorption rate in the body, thereby increasing the probability that the inotodiol derivative can reach the large intestine, and when the inotodiol derivative is decomposed by various microorganisms in the large intestine, the inotodiol can directly exert its pharmacological action. For example, the inotodiol derivative may be formed through the ester bond between inotodiol and phenolic acid. Specifically, the inotodiol derivative is expected to exhibit various physiological activities of inotodiol and phenolic acid, respectively, by releasing inotodiol and phenolic acid through hydrolysis of the ester bond in the large intestine. Therefore, the prodrug of inotodiol according to one aspect can not only reduce the dosage of inotodiol, but also reduce side effects that may occur when ingested at a high concentration.

[0031] The above inotodiol derivatives can be synthesized through chemical or biological methods. Specifically, the inotodiol derivatives can be synthesized by an esterification reaction of inotodiol and a fatty acid. The fatty acid is C 10 Inland C 30 It can be an unsaturated or saturated fatty acid, C1 to C 10It may be selected from the group consisting of carboxylic acids or phenolic acids. The unsaturated fatty acids may be, for example, myristoleic acid, palmitoleic acid, sapienic acid, oleic acid, elaidic acid, vaccenic acid, linoleic acid, linoelaidic acid, arachidonic acid, eicosapentaenoic acid, erucic acid, and docosahexaenoic acid. The saturated fatty acids may be, for example, caprylic acid, capric acid, lauric acid, palmitic acid, stearic acid, arachidic acid, behenic acid, lignoceric acid, cerotic acid, and myrysric acid. The saturated carboxylic acids may be, for example, formic acid, acetic acid, propionic acid, butyric acid, succinic acid, and the like. The above phenolic acids may be, for example, p-coumaric acid, cinnamic acid, ferulic acid, 3,4-dihydroxy benzoic acid, p-hydroxy benzoic acid, vanillic acid, caffeic acid, syringic acid, sinapinic acids, etc.

[0032] In one specific example, the inotodiol derivative comprises inotodiol and an unsaturated fatty acid, a saturated fatty acid and a C1 to C 10It may be synthesized by mixing fatty acids selected from the group consisting of carboxylic acids in a molar ratio of 1:10 to 30. For example, it may be mixed in a molar ratio of 1:10 to 30, 1:10 to 25, 1:10 to 20, 1:10 to 15, 1:10 to 13, 5:10 to 30, or 5:10 to 15. At this time, if the mixing ratio of inotodiol and fatty acid is less than or exceeds the above range, there is a problem that the reaction does not occur sufficiently, the production efficiency of inotodiol derivatives decreases, or the reaction time increases. In addition, the esterification reaction may be carried out at 55 to 65°C for 48 to 96 hours. At this time, if the esterification reaction conditions are below or exceed the above range, there is a problem that the complete dissolution of the fatty acid, which is the reaction substrate, is difficult, so that the reaction hardly occurs or the enzyme activity is reduced, so that the esterification reaction efficiency is low, and the final product, inotodiol derivative, cannot be synthesized in high yield. After the esterification reaction is completed, the purity of the ester contained in the product can be further increased through various known distillation methods or purification methods.

[0033] In addition, the mixture of inotodiol and fatty acid can be synthesized by adding a biological enzyme, and the biological enzyme is an enzyme suitable for producing a highly efficient inotodiol derivative, for example, Candida Antarctica Lipase B (CalB). The Candida Antarctica Lipase B can be an immobilized enzyme rather than a general enzyme in order to increase the yield of the final product, the inotodiol derivative, and the immobilized enzyme can be a commercially available product or can be manufactured and used using a conventional method.

[0034] The above-mentioned antactic lipase B can synthesize an inotodiol derivative by adding 400 to 500 parts by weight based on 100 parts by weight of inotodiol. At this time, if the content of antactic lipase B is below the above range, there is a problem that the ester reaction does not occur sufficiently, so the efficiency of producing the ester is low, and if it exceeds the above range, there is a problem that the final product, an inotodiol derivative, cannot be synthesized in a high yield, so it is not economical.

[0035] In one specific example, the pharmaceutical composition may be a pharmaceutical composition further comprising a cyclodextrin (CD) derivative represented by the following chemical formula 3.

[0036] [Chemical Formula 3]

[0037]

[0038] In the above chemical formula 3, n is 4, 5 or 6, and R 11 , R 12 , R 13 , R 14 , R 15 , R 16 , R 17 , R 18 and R 19 is independently -OH, substituted or unsubstituted alkoxy having 1 to 4 carbons, or substituted or unsubstituted alkylthio having 1 to 4 carbons.

[0039] The term “cyclodextrin (CD)” used herein refers to a cyclic compound in which glucose is linked as a basic unit via an α-(1→4)-glycosidic bond. The cyclodextrin is a ring-shaped sugar particle with a cavity, and can be classified into alpha (α), beta (β), and gamma (γ) types depending on the structure. The internal pores of the cyclodextrin according to the structure are 5.3 Å for α-cyclodextrin, 6.5 Å for β-cyclodextrin, and 8.3 Å for γ-cyclodextrin. Since the outside of the cyclodextrin ring has a hydroxyl group, it is hydrophilic, whereas the inside is hydrophobic. Substances having a molecular structure suitable for the internal structure of the cyclodextrin can be incorporated into the hydrophobic internal cavity of the cyclodextrin to form an inclusion compound. In one specific example, the inotodiol may be enclosed in the internal cavity of the cyclodextrin.

[0040] In one specific example, the pharmaceutical composition may be one in which the inotodiol, a prodrug thereof, or a pharmaceutically acceptable salt thereof is encapsulated within a cyclodextrin derivative represented by the chemical formula 3.

[0041] The term “inclusion” as used herein refers to a stoichiometric molecular phenomenon in which a guest molecule is captured by interaction with the cavity of a cyclodextrin molecule.

[0042] In one specific example, the cyclodextrin derivative represented by the above chemical formula 3 may be γ-cyclodextrin represented by the following chemical formula 4.

[0043] [Chemical Formula 4]

[0044]

[0045] In one specific example, the pharmaceutical composition may be a mixture of inotodiol and a prodrug thereof or a pharmaceutically acceptable salt thereof; and a cyclodextrin derivative in a molar ratio of 1:1 to 10. Specifically, the molar ratio of inotodiol and a prodrug thereof or a pharmaceutically acceptable salt thereof; and a cyclodextrin derivative may be a mixture of 1:1 to 10, 1:1 to 9, 1:1 to 8, 1:1 to 7, 1:1 to 6, 1:1 to 5, 1:1 to 4, 1:1 to 3, or 1:1 to 2. For example, inotodiol and a cyclodextrin derivative may be mixed in a molar ratio of 1:3 to form an inclusion complex in which inotodiol is enclosed in a cavity of the cyclodextrin derivative.

[0046] As used herein, the term “pharmaceutically acceptable” may mean physiologically acceptable and, when administered to humans, does not typically cause allergic reactions such as gastrointestinal upset, dizziness, or similar reactions.

[0047] As used herein, the term “pharmaceutically acceptable salt” may refer to a salt according to one aspect of the present invention that is pharmaceutically acceptable and has the desired pharmacological activity of the parent compound. The salts of the parent compound may be synthesized from the parent compound containing a basic or acidic moiety by conventional chemical methods. Typically, such salts may be prepared by reacting the free acid form of these compounds with a stoichiometric amount of a suitable base, such as sodium, calcium, magnesium, or potassium, or by reacting the free base form of these compounds with a stoichiometric amount of a suitable acid. These reactions are typically carried out in water or in an organic solvent, or in a mixture of the two. Typically, when practical, non-aqueous media such as ether, ethyl acetate, ethanol, isopropanol, or acetonitrile may be used. The pharmaceutically acceptable salts include both acid or base addition salts and stereochemically isomeric forms thereof, and may be, for example, addition salts of organic or inorganic acids. The above salt includes any salt that maintains the activity of the parent compound in the subject of administration and does not cause undesirable effects, and is not particularly limited thereto.

[0048] These salts include inorganic and organic salts, for example, acetic acid, nitric acid, aspartic acid, sulfonic acid, sulfuric acid, maleic acid, glutamic acid, formic acid, succinic acid, phosphoric acid, phthalic acid, tannic acid, tartaric acid, hydrobromic acid, propionic acid, benzenesulfonic acid, benzoic acid, stearic acid, lactic acid, bicarboxylic acid, bisulfuric acid, bitartaric acid, oxalic acid, butyric acid, calcium idet, carbonic acid, chlorobenzoic acid, citric acid, idetic acid, toluenesulfonic acid, fumaric acid, gluceptic acid, esilinic acid, pamoic acid, gluconic acid, methylnitric acid, malonic acid, hydrochloric acid, hydroiodoic acid, hydroxynaphtholic acid, isethionic acid, lactobionic acid, mandelic acid, mucic acid, It can be naphthylic acid, muconic acid, p-nitromethanesulfonic acid, hexamic acid, pantothenic acid, monohydrogenphosphoric acid, dihydrogenphosphoric acid, salicylic acid, sulfamic acid, sulfanilinic acid, or methanesulfonic acid.

[0049] In addition, the salt forms include salts of alkali and alkaline earth metals, such as ammonium salts, lithium salts, sodium salts, potassium salts, magnesium salts, and calcium salts, salts with organic bases, such as benzathine, N-methyl-D-glucamine, and hydrabamine salts, and salts with amino acids, such as arginine and lysine. In addition, the salt forms can be converted into free forms by treatment with a suitable base or acid.

[0050] In one specific example, the inotodiol or a prodrug thereof may be a pharmaceutical composition that induces eosinophil apoptosis.

[0051] The term "eosinophil" as used herein refers to a type of white blood cell, a member of the immune system in mammals that plays a role in fighting multicellular parasites and certain infections. They are developed in the bone marrow through hematopoiesis and are released into the bloodstream, where they undergo terminal differentiation and cease to proliferate. Eosinophils are granulocytes, measuring 12-15 μm in diameter, with a bilobed nucleus and a cytoplasm filled with granules. The granules appear bright crimson when stained with the acid dye eosin due to the basic proteins present within them. The small granules in the cytoplasm contain various chemical mediators such as peroxidase, RNAse, DNAse, lipase, and plasminogen. These mediators are secreted through the degranulation process following eosinophil activation, leading to histological and functional changes that contribute to disease. Eosinophils are involved in inflammatory responses, and their numbers are known to increase in allergic inflammation, parasitic infections, and drug hypersensitivity reactions.

[0052] The term “apoptosis” as used herein refers to a form of programmed cell death that occurs in some eukaryotic organisms and unicellular microorganisms, and is morphologically characterized by cell shrinkage, cell membrane blebbing, nuclear fragmentation or karyorrhexis, chromatin condensation, and intranucleosomal cleavage of DNA. Unlike necrosis, which causes inflammatory changes due to the leakage of intracellular components during cell death, in the case of apoptosis, the entire process proceeds in a regulated state, and the cells are engulfed by macrophages after death, leaving no inflammation.

[0053] The term “eosinophilia” used in this specification refers to a disease caused by an increase in eosinophils.

[0054] In one specific example, the eosinophilia is eosinophilic cardiomyopathy, eosinophilic pneumonia (EP), idiopathic chronic eosinophilic pneumonia (ICEP), idiopathic acute eosinophilic pneumonia (IAEP), eosinophilic bronchitis, eosinophilic eosophagitis, eosinophilic gastroenteritis, eosinophilic gastritis, eosinophilic colitis, eosinophilic enteritis, ulcerative colitis, Crohn's disease (CD), eosinophilic fasciitis It may mean any one selected from the group consisting of, but is not limited to, eosinophilic fasciitis, idiopathic myositis, chronic eosinophilic leukemia (CEL), hypereosinophilic syndrome (HES), Idiopathic Hypereosinophilic Syndrome (IHES), Churg-Strauss syndrome, Loffler's Syndrome (Simple Pulmonary Eosinophilia), Paragonimiasis, Toxic-oil Syndrome, and Eosinophilia-myalgia Syndrome.

[0055] The term “inflammation” used herein refers to a response to protect the body against harmful factors, and may include a series of processes involving immune cells, blood vessels, and inflammatory mediators to suppress cell damage, remove damaged tissues and necrotic cells, and regenerate tissues.

[0056] The term "inflammatory disease" as used herein can be defined as a pathological symptom caused by an inflammatory response that is specific to a local or systemic biological defense response to external physical or chemical stimuli, infection by external infectious agents such as bacteria, fungi, viruses, and various allergens, or autoimmunity. This inflammatory response involves a series of complex physiological reactions such as activation of various inflammatory mediators and enzymes related to immune cells (e.g., iNOS, COX-2, etc.), secretion of inflammatory mediators (e.g., secretion of NO, TNF-α, IL-6, etc.), fluid infiltration, cell migration, tissue destruction, etc., and is externally manifested by symptoms such as erythema, pain, edema, fever, and decline or loss of specific bodily functions. Since the above inflammatory disease may be acute, chronic, ulcerative, allergic, or necrotic, as long as any disease is included in the above definition of inflammatory disease, it does not matter whether it is acute, chronic, ulcerative, allergic, or necrotic.

[0057] The term “eosinophilic inflammatory disease” used in this specification refers to an inflammatory disease caused by infiltration of eosinophils into tissues due to various causes.

[0058] In one specific example, the eosinophilic inflammatory disease may mean any one selected from the group consisting of allergic bronchopulmonary aspergillosis, eosinophilic drug allergy, eosinophilic asthma, and allergic rhinitis, but is not limited thereto.

[0059] In one specific example, the squamous cell inflammatory disease may be an inflammatory disease caused in the nasal cavity, paranasal sinuses, or bronchi. In particular, it may be a respiratory disease characterized by excessive mucus secretion in the respiratory tract. In this case, the squamous cell inflammatory disease characterized by excessive mucus secretion in the respiratory tract may include, but is not limited to, chronic rhinitis, chronic sinusitis, and chronic bronchitis.

[0060] As used herein, the term "pharmaceutical composition" may refer to a molecule or compound that, when administered to a subject, imparts several beneficial effects. Beneficial effects may include enabling diagnostic determination; improving a disease, symptom, disorder, or condition; reducing or preventing the onset of a disease, symptom, disorder, or condition; and generally combating a disease, symptom, disorder, or condition.

[0061] In addition to the active ingredient, the pharmaceutical composition may further include one or more auxiliary agents selected from the group consisting of pharmaceutically acceptable carriers, excipients, diluents, fillers, bulking agents, wetting agents, disintegrating agents, emulsifiers (surfactants), lubricants, sweeteners, flavoring agents, suspending agents, preservatives, etc.

[0062] The above-mentioned adjuvant can be appropriately adjusted according to the formulation to which the pharmaceutical composition is applied, and one or more adjuvants that can be commonly used in the pharmaceutical field can be selected and used. In one specific example, the pharmaceutically acceptable carrier is one that is commonly used in the formulation of drugs, and can be used by mixing one or more of saline solution, sterile water, Ringer's solution, buffered saline, dextrose solution, maltodextrin solution, glycerol, ethanol, liposome, and more than one component among these components, and other conventional additives such as antioxidants, buffers, and bacteriostatic agents can be added as needed. In addition, diluents, dispersants, surfactants, binders, and lubricants can be additionally added to formulate the composition into an injectable formulation such as an aqueous solution, suspension, or emulsion, pills, capsules, granules, or tablets, and a target organ-specific antibody or other ligand can be combined with the carrier to act specifically on the target organ. Furthermore, it can be preferably formulated according to each disease or ingredient using an appropriate method in the relevant technical field or a method disclosed in Remington's literature (Remington's Pharmaceutical Science (latest edition), Mack Publishing Company, Easton PA).

[0063] In one specific example, the pharmaceutical composition is administered orally or parenterally, and the parenteral administration may be any one selected from the group consisting of nasal inhalation, subcutaneous injection, intravenous injection, intramuscular injection, and intrathoracic injection. Specifically, the effective amount of the active ingredient or the pharmaceutical composition can be administered orally or parenterally during clinical administration and can be used in the form of a general pharmaceutical formulation. Parenteral administration may mean administration through a route other than oral administration, such as rectal, intravenous, peritoneal, intramuscular, intraarterial, transdermal, nasal, inhalation, ocular, or subcutaneous, and may be administered by local administration to a lesion site, etc. When administered orally, the pharmaceutical composition may be formulated in a form that coats the active ingredient to prevent the active ingredient from being decomposed in the stomach or protects it from being decomposed in the stomach. When the pharmaceutical composition of the present invention is used as a medicine, it may additionally contain one or more active ingredients having the same or similar function.

[0064] The pharmaceutical composition may be formulated in the form of a solution, suspension, syrup, or emulsion in an aqueous or oily medium, or in the form of a powder, granule, tablet, or capsule, and may additionally contain a dispersing agent or stabilizer for formulation. When formulating the pharmaceutical composition, it may be prepared using diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrants, and surfactants that are commonly used. Formulations for parenteral administration may include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, and suppositories. Non-aqueous solvents and suspensions may include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate. Suppository bases that can be used include Witepsol, Macrogol, Tween 61, cocoa butter, liurin butter, and glycerogelatin.

[0065] The above pharmaceutical composition may be used by mixing with various carriers acceptable as pharmaceuticals, such as saline solution or organic solvents, and carbohydrates such as glucose, sucrose or dextran, antioxidants such as ascorbic acid or glutathione, chelating agents, low-molecular-weight proteins or other stabilizers may be used as pharmaceuticals to increase stability or absorbability.

[0066] The above pharmaceutical composition may be administered in a pharmaceutically effective amount. There are no specific restrictions on the dosage, and it may vary depending on the absorption rate in the body, body weight, patient's age, sex, health condition, diet, administration time, administration method, excretion rate, and disease severity. The pharmaceutical composition of the present invention is manufactured considering the effective dosage range, and the unit dosage form prepared in this way may be administered multiple times at regular intervals using a specialized dosage method or according to the judgment of a specialist who monitors or observes the administration of the drug and the individual's needs, as needed.

[0067] The term "treatment" in this specification is used to mean alleviation or improvement of pathological symptoms, reduction of the extent of disease, delay or palliation of disease progression, improvement, alleviation or stabilization of disease state or symptoms, partial or complete recovery, prolongation of survival, and other beneficial treatment results.

[0068] The term "prevention" in this specification is used to mean all mechanisms and / or effects that act on a subject who does not have a specific disease to prevent the development of said specific disease, delay the onset of said disease, or reduce the frequency of said disease.

[0069]

[0070] Another aspect provides a health functional food for preventing or improving eosinophilia or eosinophilic inflammatory disease, comprising inotodiol, a prodrug thereof, or a pharmaceutically acceptable salt thereof as an active ingredient.

[0071] The term "health functional food" in this specification may refer to a food manufactured or processed using a specific ingredient as a raw material or by extracting, concentrating, refining, mixing, etc. a specific ingredient contained in a food raw material for the purpose of health supplementation, and may refer to a food designed and processed so that the above-mentioned ingredient can sufficiently exert a bioregulatory function on the body, such as biodefense, regulation of biological rhythm, and prevention and recovery of disease.

[0072] There are no specific restrictions on the types of the above foods. Examples of foods to which the above substances can be added include formulations selected from the group consisting of powders, granules, tablets, capsules, pills, gels, jellies, suspensions, emulsions, syrups, tea bags, infused teas, and health drinks, and can include all health foods in the conventional sense.

[0073] The above health functional food may include food additives that are food-related and acceptable, and may include an appropriate carrier commonly used in the manufacture of health functional foods.

[0074] In addition to the above, the composition of the present invention may include various nutrients, vitamins, electrolytes, flavoring agents, coloring agents, pectic acid and its salts, alginic acid and its salts, organic acids, protective colloid thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohol, carbonating agents used in carbonated beverages, etc.

[0075] The above "inotodiol", "inotodiol prodrug", "eosinophilia", "eosinophilic inflammatory disease", etc. are as described above. The matters mentioned in the pharmaceutical composition and health functional food of the present invention are equally applicable unless they are contradictory.

[0076]

[0077] Another aspect provides an inhalation composition for the prevention or treatment of eosinophilia or eosinophilic inflammatory disease, comprising inotodiol, a prodrug thereof or a pharmaceutically acceptable salt thereof as an active ingredient.

[0078] In the inhalant composition of the present invention, the active ingredient may be added directly to the inhalant or used in combination with other ingredients, and may be used appropriately according to conventional methods. The amount of active ingredient mixed may be appropriately determined depending on the intended use (preventive or therapeutic).

[0079] Inhalants for parenteral administration include aerosols, powders for inhalation, and liquids for inhalation. The liquids for inhalation may be in the form of being dissolved or suspended in water or another suitable medium at the time of use. These inhalants are manufactured according to known methods. For example, in the case of liquids for inhalation, preservatives (benzalkonium chloride, parabens, etc.), colorants, buffering agents (sodium phosphate, sodium acetate, etc.), isotonic agents (sodium chloride, concentrated glycerin, etc.), thickeners (carboxyvinyl polymers, etc.), absorption promoters, etc. are appropriately selected and prepared as needed. In the case of powders for inhalation, lubricants (stearic acid and its salts, etc.), binders (starch, dextrin, etc.), excipients (lactose, cellulose, etc.), colorants, preservatives (benzalkonium chloride, parabens, etc.), absorption promoters, etc. are appropriately selected and prepared as needed.

[0080] The above inhalation composition can be administered via an inhalation device, which is a device capable of delivering the composition to a subject, such as the subject's lung tissue, and includes, for example, an inhaler, a nebulizer, or a ventilator. When administering a liquid for inhalation, a nebulizer (atomizer, nebulizer) is typically used, and when administering a powder for inhalation, an inhalation injector for powder medication can typically be used, but is not limited thereto.

[0081]

[0082] Another aspect provides a pharmaceutical composition for preventing or improving eosinophilia or eosinophilic inflammatory disease, comprising inotodiol, a prodrug thereof, or a pharmaceutically acceptable salt thereof as an active ingredient.

[0083] The term "quasi-drug" in this specification refers to products that are used for the purpose of diagnosing, treating, improving, alleviating, managing or preventing diseases in humans or animals, and have a milder effect than pharmaceutical products. For example, according to the Pharmaceutical Affairs Act, quasi-drugs are products other than those used for pharmaceutical purposes, and include products used for treating or preventing diseases in humans or animals, products that have a mild effect on the human body or do not directly affect it.

[0084] When the composition of the present invention is included in a quasi-drug for the purpose of preventing or improving eosinophilic inflammatory disease, the composition may be included as is or used together with other quasi-drug ingredients, and may be used appropriately according to a conventional method. The amount of active ingredients mixed may be appropriately determined depending on the intended use. The quasi-drug of the present invention may contain various bases and additives necessary for formulation depending on its formulation, and the types and amounts of these ingredients may be easily selected by those skilled in the art. The quasi-drug composition of the present invention may be prepared in a formulation selected from the group consisting of, for example, disinfecting cleaners, detergents, kitchen cleaners, cleaning detergents, wet tissues, detergents, soaps, hand washes, humidifier fillers, masks, ointments, filter fillers, and portable products that directly or indirectly inhale air or oxygen to temporarily supply air or oxygen, but is not limited thereto.

[0085] The above “inotodiol”, “inotodiol prodrug”, “eosinophilia”, “eosinophilic inflammatory disease”, etc. are as described above.

[0086]

[0087] Another aspect provides a use of inotodiol, a prodrug thereof, or a pharmaceutically acceptable salt thereof for the prevention or treatment of eosinophilia or eosinophilic inflammatory diseases.

[0088] Another aspect provides the use of inotodiol, a prodrug thereof, or a pharmaceutically acceptable salt thereof for the manufacture of a medicament for the prevention or treatment of eosinophilia or eosinophilic inflammatory diseases.

[0089] Another aspect provides a method of treating eosinophilia or an eosinophilic inflammatory disease comprising administering to a subject in need thereof inotodiol, a prodrug thereof, or a pharmaceutically acceptable salt thereof.

[0090] The above “inotodiol”, “inotodiol prodrug”, “eosinophilia”, “eosinophilic inflammatory disease”, etc. are as described above.

[0091] A composition containing inotodiol or a prodrug thereof according to one aspect exhibits an effect of inducing apoptosis specifically in eosinophils, and is expected to be useful as a preventive or therapeutic agent for eosinophilia or eosinophilic inflammatory diseases.

[0092] Figure 1 is a photograph showing the results of H&E staining of EoL-1 cells treated with 5 μg / ml and 10 μg / ml of inotodiol (γ-CD + Inotodiol (3:1) inclusion complex) for 1 day, respectively, as a negative control group containing inotodiol solvent (DMSO 0.04%), a positive control group containing 1 μM dexamethasone, and inotodiol for 1 day.

[0093] Figure 2 shows photographs of fluorescent signals measured after fluorescent immunostaining of EoL-1 cells treated with 5 μg / ml and 10 μg / ml of inotodiol (γ-CD + Inotodiol (3:1) inclusion complex) for 1 day, as a negative control group containing inotodiol solvent (DMSO 0.04%), as a positive control group containing 1 μM dexamethasone, and as a negative control group containing 1 μM dexamethasone.

[0094] Figure 3 shows the expression levels of eosinophil apoptosis-related proteins:

[0095] Figure 3a shows the results of measuring the expression level of apoptosis-related proteins in EOL-1 cells treated with only 0.5 mM butyric acid for 2 days and in the case of treating 0.5 mM butyric acid for 1 day followed by 10 μg / ml inotodiol (γ-CD + Inotodiol (3:1) inclusion complex, solvent: DMSO) for 1 day, and Figure 3b is a graph showing the results of measuring the pixel density of the expression level of the apoptosis-related proteins of Figure 3a using the OptimEyes program.

[0096] Figure 4 is a schematic diagram showing the process of creating a mouse model of eosinophilic chronic rhinosinusitis (Eosinophilic CRS) induced by SEB (staphylococcal enterotoxins B).

[0097] Figure 5 is a graph showing the results of measuring the expression levels of IL-4, IL-5, and Mast cell tryptase in nasal lavage fluid of a mouse model of chronic rhinosinusitis (CRS) induced by SEB using ELISA.

[0098] Figure 6 is a graph showing the results of measuring the expression levels of Helper T cell-related cytokines IL-4, IL-5, IL-10, IL-13, IL-17A, and IFNγ in the paranasal mucosa of a mouse model of chronic rhinosinusitis (CRS) induced by SEB using q-PCR.

[0099] Figure 7 is a graph showing the results of measuring the expression levels of pro-inflammatory cytokines IL-6 and IL-1β in the paranasal mucosa of a mouse model of chronic rhinosinusitis (CRS) induced by SEB using q-PCR.

[0100] Figure 8 is a graph showing the results of measuring the expression levels of epithelial-derived cytokines IL-25 and IL-33 in the paranasal mucosa of a mouse model of chronic rhinosinusitis (CRS) induced by SEB using q-PCR.

[0101] Figure 9 is a graph showing the results of measuring the expression levels of the chemokines CCL1, CCL2, and CXCL2 in the paranasal mucosa of a mouse model of chronic rhinosinusitis (CRS) induced by SEB using q-PCR.

[0102] Figure 10 shows the expression results of cleaved caspase 3 and siglec F in the head tissue of a mouse model of eosinophilic chronic rhinosinusitis (Eosinophilic CRS) induced by SEB:

[0103] Figure 10a is a photograph showing the fluorescent signal expressed by cleaved caspase 3 and siglec F in a head tissue section of a mouse model of chronic eosinophilic rhinosinusitis (CRS) induced by SEB, and Figure 10b is a graph showing the ratio of cells co-expressing cleaved caspase 3 and siglec F (% of Cleaved casp3+ SiglecF+ / Siglec F+ cells) in a head tissue section of the mouse model.

[0104] Figure 11 shows the results of measuring the thickness of the respiratory epithelium in a paraffin block of head tissue from a mouse model of eosinophilic chronic rhinosinusitis (Eosinophilic CRS) using a slide scanner system:

[0105] Figure 11a is a photograph showing the results of H&E staining of mouse head tissue sections, Figure 11b is a graph quantifying the thickness of the respiratory epithelium, and Figure 11c is a graph showing the number of eosinophils per area (eosinophils / Fields) in three areas out of five tissue sections at 200x magnification.

[0106] Figure 12 shows goblet cells in a paraffin block of head tissue from a mouse model of eosinophilic chronic rhinosinusitis (Eosinophilic CRS):

[0107] Figure 12a is a photograph showing the result of PAS staining of a paraffin block of head tissue of a mouse model, and Figure 12b is a graph showing the number of goblet cells per area (goblet cells / field) quantified at 200x magnification of a slide stained with PAS of a paraffin block of head tissue of a mouse model.

[0108] Figure 13 is a graph showing the results of measuring cell viability after treating human eosinophilic leukemia cells (EoL-1), human mast cells (LUVA), mouse macrophage cells (Raw264.7), and human dermal fibroblast primary cells (HDF) with inotodiol:

[0109] Figure 13a is a graph measuring cell viability by treating human eosinophilic leukemia cells (EoL-1 cells) with inotodiol at different concentrations, Figure 13b is a graph measuring cell viability by treating human mast cells (LUVA) with inotodiol at different concentrations, Figure 13c is a graph measuring cell viability by treating mouse macrophage cells (Raw264.7) with inotodiol at different concentrations, and Figure 13d is a graph measuring cell viability by treating human dermal fibroblast primary cells (HDF cells) with inotodiol at different concentrations.

[0110] Hereinafter, the present invention will be described in more detail through examples. However, these examples are intended to exemplify the present invention, and the scope of the present invention is not limited by these examples.

[0111]

[0112] Example 1. EoL-1 cell line culture

[0113] EoL-1 cell line was cultured in RPMI-1640 supplemented with fetal bovine serum (FBS), 1% streptomycin, and penicillin. To differentiate EOL-1 cells into eosinophils, 0.5 mM butyric acid (BA) was treated for 1 day. Then, 5 μg / ml or 10 μg / ml inotodiol (γ-CD + Inotodiol (3:1) inclusion complex, solvent: DMSO) was treated for 1 day. The negative control group was treated with the same inotodiol solvent (DMSO 0.04%), and the positive control group was treated with 1 μM dexamethasone.

[0114] Example 2. Confirmation of the effect of inotodiol on inducing eosinophil apoptosis in cell lines.

[0115] 2.1. H&E staining

[0116] EoL-1 cells cultured in the same manner as in Example 1 were fixed using 4% paraformaldehyde, and the fixed cells were applied to slides and stained with H&E using hematoxylin and eosin.

[0117] The results of observing EoL-1 cells after H&E staining are shown in Figure 1.

[0118] Figure 1 shows the results of H&E staining of EoL-1 cells treated with 5 μg / ml and 10 μg / ml of inotodiol (γ-CD + Inotodiol (3:1) inclusion complex) for 1 day as a negative control group containing inotodiol solvent (DMSO 0.04%), a positive control group containing 1 μg / ml of dexamethasone, and the cell death event. Cell shrinkage, membrane blebbing, nuclear fragmentation, and cell death are indicated by red arrows.

[0119] As shown in Figure 1, compared to the negative control group containing only 0.04% DMSO, in the case of EoL-1 cells treated with 5 μg / ml and 10 μg / ml of inotodiol (γ-CD + Inotodiol (3:1) inclusion complex), more evidence of apoptosis, such as cell shrinkage, membrane blebbing, nuclear fragmentation, and cell death, was observed.

[0120] This means that inotodiol has the effect of inducing eosinophil apoptosis.

[0121] 2.2. Fluorescent Immunostaining

[0122] EoL-1 cells cultured in the same manner as in Example 1 were fixed using 4% paraformaldehyde, and the fixed cells were applied to slides. Fluorescent immunostaining was performed on the slides applied with the cells using a cleaved caspase 3 antibody conjugated to FITC (Fluorescein Isothiocyanate).

[0123] Specifically, the slides on which cells were fixed were treated with FITC-conjugated cleaved caspase 3 antibody (Alexa Fluor® 488 Conjugate) and incubated overnight at 4°C. The experiment was performed according to the method provided by the manufacturer, and the fluorescence signal was observed using an upright fluorescence microscope (Leica, DM2500).

[0124] The results of observing fluorescence signals in EoL-1 cells are shown in Figure 2.

[0125] Figure 2 shows photographs of fluorescent signals measured after fluorescent immunostaining of EoL-1 cells treated with 5 μg / ml and 10 μg / ml of inotodiol (γ-CD + Inotodiol (3:1) inclusion complex) for 1 day, as a negative control group containing inotodiol solvent (DMSO 0.04%), as a positive control group containing 1 μM dexamethasone, and as a negative control group containing 1 μM dexamethasone.

[0126] As confirmed in Fig. 2, cleaved caspase 3, an enzyme activated during the apoptosis process, was confirmed to be more activated in EoL-1 cells treated with 5 μg / ml and 10 μg / ml of inotodiol (γ-CD + Inotodiol (3:1) inclusion complex), respectively, compared to the negative control group containing only 0.04% DMSO.

[0127] This means that inotodiol has the effect of inducing eosinophil apoptosis, and that inotodiol is effective in preventing and treating eosinophilia.

[0128]

[0129] 2.3 Eosinophil apoptosis-related protein antibody array

[0130] An antibody array of apoptosis-related proteins was performed on EoL-1 cells cultured in the same manner as in Example 1.

[0131] Specifically, the Proteome Profiler Human Apoptosis Array Kit (R&D #ARY009) was used, and the proteins of EOL-1 cells were reacted with a membrane bound to apoptosis-related gene-specific capture antibodies at 4℃ overnight. After washing with a washing solution (washing buffer), biotinylated detection antibodies were reacted at room temperature for 1 hour. After washing with a washing solution, the membrane was reacted with a chemiluminescent detection reagent, and then exposed to an X-ray film for 5 minutes. The pixel density was analyzed using the OptimEyes program, and the results are shown in Fig. 3.

[0132] Figure 3 shows the expression level of eosinophil apoptosis-related proteins: Figure 3a shows the results of measuring the expression level of apoptosis-related proteins in EOL-1 cells treated with only 0.5 mM butyric acid for 2 days and in the case of treating 0.5 mM butyric acid for 1 day followed by 10 μg / ml inotodiol (γ-CD + Inotodiol (3:1) inclusion complex, solvent: DMSO) for 1 day, and Figure 3b is a graph showing the results of measuring the pixel density of the expression level of the apoptosis-related proteins of Figure 3a using the OptimEyes program.

[0133] As shown in Figure 3, it was confirmed that the expression level of proteins related to eosinophil apoptosis increased more when inotodiol and butyric acid were administered together than when butyric acid (BA) was administered alone.

[0134] This means that inotodiol has the effect of inducing eosinophil apoptosis, and that inotodiol is effective in preventing and treating eosinophilia.

[0135]

[0136] Example 3. Creation of a mouse model of eosinophilic chronic rhinosinusitis (CRS) induced by SEB.

[0137] A mouse model of eosinophilic chronic rhinosinusitis (CRS) was created using staphylococcal enterotoxin B (SEB), a toxic substance that can cause inflammatory diseases. The process of creating a mouse model of eosinophilic CRS induced by SEB is shown in Figure 4.

[0138] Figure 4 is a schematic diagram showing the process of creating a mouse model of eosinophilic chronic rhinosinusitis (Eosinophilic CRS) induced by SEB.

[0139] Specifically, Balb / c 4-week-old mice were purchased (Orient Bio TM) and a mixture of 25 mg of ovalbumin (OVA) and 2 mg of aluminum hydroxide (Alu) dissolved in phosphate-buffered saline (PBS) was injected intraperitoneally into the animals on the day of the experiment and on day 5. From day 21 of the experiment, 3% OVA was dissolved in phosphate-buffered saline (PBS) and administered intranasally for 5 days. From day 28 of the experiment, 3% ovalbumin solution was instilled intranasally (IN) three times a week for 4 weeks, and SEB was instilled intranasally three times a week from week 8 to week 12 of the experiment. One hour after SEB instillation, inotodiol (γ-CD + Inotodiol (3:1) inclusion complex, 20 mg / kg) and dexamethasone (Dexmethasone 10 mg / kg) were injected intraperitoneally (IP) once a week. The negative control group was administered (IP) PBS (vehicle).

[0140] Mice with eosinophilic chronic rhinosinusitis (Eosinophilic CRS) induced by SEB were divided into “CON”, “NP”, “NP+Ino”, and “NP+Dex” groups for the experiment.

[0141] "CON" refers to the group that received PBS instead of intraperitoneal injection or nasal instillation of ovalbumin solution, intraperitoneal instillation of SEB, or intraperitoneal injection of therapeutic drugs (inotodiol and dexamethasone) throughout the entire process of creating the ECRS mouse model; "NP" refers to the group that received intraperitoneal injection of a solvent (Tween 80% + NaCl 0.9%, 20 ul) as a therapeutic drug in the ECRS mouse model; "NP+Ino" refers to the group that received intraperitoneal injection of inotodiol (inotodiol 20 mg / kg); and "NP+Dex" refers to the group that received intraperitoneal injection of dexamethasone (dexmethasone 10 mg / kg).

[0142]

[0143] Example 4. Confirmation of the effect of inotodiol on reducing inflammation-related gene expression in mice.

[0144] 4.1 Confirmation of the effect of inotodiol on reducing the expression of type 2 cytokines and mast cell tryptase in nasal irrigation fluid.

[0145] Nasal lavage fluid was collected from a mouse model prepared in the same manner as in Example 3.

[0146] Specifically, the mouse model was anesthetized, and tracheotomy was performed in the anesthetized mouse model. A 24-gauge catheter was inserted from the trachea to the posterior nares of the mice that underwent tracheotomy. 400 μl of PBS was injected through the catheter, collected from the nostrils, and the collected solution was centrifuged. The supernatant was used to measure the expression levels of IL-4, IL-5, and mast cell tryptase using an enzyme-linked immunosorbent assay (ELISA).

[0147] The results are shown in Figure 5.

[0148] Figure 5 is a graph showing the results of measuring the expression levels of IL-4, IL-5, and Mast cell tryptase in nasal lavage fluid of a mouse model of chronic rhinosinusitis (CRS) induced by SEB using ELISA.

[0149] As shown in Fig. 5, when nasal inflammation was induced in mice with SEB (NP), the expression levels of type 2 cytokines IL-4, IL-5, and Mast cell tryptase (MCT) increased compared to the control (CON) that did not induce nasal inflammation. In contrast, when inotodiol (γ-CD + Inotodiol (3:1) inclusion complex, 20 mg / kg) was administered after nasal inflammation was induced (NP), the expression levels of IL-4, IL-5, and Mast cell tryptase (MCT) were significantly reduced to levels similar to those of the control (CON).

[0150] This means that when inotodiol was administered after inducing inflammation, the expression levels of inflammation-related factors IL-4, IL-5, and Mast cell tryptase (MCT) were reduced, indicating that inotodiol is effective in preventing or treating eosinophilic inflammatory diseases.

[0151]

[0152] 4.2 Confirmation of the effect of inotodiol on reducing mRNA expression of cytokines related to Helper T cells (Th) in the mouse paranasal sinus mucosa.

[0153] mRNA was extracted from the paranasal sinus mucosa of a mouse model prepared in the same manner as Example 3 using TRIzol, and cDNA was synthesized. The expression levels of Th (Helper T cell)-related cytokines IL-4, IL-5, IL-10, IL-13, IL-17A, and IFNγ were measured using q-PCR.

[0154] The results are shown in Figure 6.

[0155] Figure 6 is a graph showing the results of measuring the expression levels of Helper T cell-related cytokines IL-4, IL-5, IL-10, IL-13, IL-17A, and IFNγ in the paranasal mucosa of a mouse model of chronic rhinosinusitis (CRS) induced by SEB using q-PCR.

[0156] As shown in Fig. 6, when nasal inflammation was induced in mice with SEB (Staphylococcal Enterotoxin B) (NP), the expression levels of Th-related cytokines IL-4, IL-5, IL-10, IL-13, IL-17A, and IFNγ increased compared to the control (CON) that did not induce nasal inflammation. In contrast, when inotodiol (γ-CD + Inotodiol (3:1) inclusion complex, 20 mg / kg) was administered after nasal inflammation was induced (NP), the expression levels of Th-related cytokines IL-4, IL-5, IL-10, IL-13, IL-17A, and IFNγ were significantly reduced.

[0157] This means that when inotodiol was administered after inducing inflammation, the expression levels of inflammation-related factors IL-4, IL-5, IL-10, IL-13, IL-17A, and IFNγ were reduced, indicating that inotodiol is effective in preventing or treating eosinophilic inflammatory diseases.

[0158]

[0159] 4.3. Confirmation of the effect of inotodiol on reducing the mRNA expression of inflammatory cytokines in the mouse paranasal sinus mucosa.

[0160] mRNA was extracted from the paranasal sinus mucosa of a mouse model prepared in the same manner as Example 3 using TRIzol, and cDNA was synthesized. Then, the expression levels of pro-inflammatory cytokines IL-6 and IL-1β were measured using q-PCR.

[0161] The results are shown in Figure 7.

[0162] Figure 7 is a graph showing the results of measuring the expression levels of pro-inflammatory cytokines IL-6 and IL-1β in the paranasal mucosa of a mouse model of chronic rhinosinusitis (CRS) induced by SEB using ELISA.

[0163] As shown in Fig. 7, when nasal inflammation was induced in mice with SEB (NP), the expression levels of pro-inflammatory cytokines IL-6 and IL-1β increased compared to the control (CON) that did not induce nasal inflammation. On the other hand, when nasal inflammation was induced in mice with SEB and inotodiol (γ-CD + Inotodiol (3:1) inclusion complex, 20 mg / kg) was administered, the expression levels of pro-inflammatory cytokines IL-6 and IL-1β were significantly reduced to a level similar to that of the control (CON).

[0164] This means that when inotodiol was administered after inflammation was induced, the expression levels of inflammatory cytokines IL-6 and IL-1β were reduced, indicating that inotodiol is effective in preventing or treating eosinophilic inflammatory diseases.

[0165]

[0166] 4.4. Confirmation of the effect of inotodiol on reducing the mRNA expression of epithelium-derived associated cytokines in the mouse paranasal sinus mucosa.

[0167] mRNA was extracted from the paranasal sinus mucosa of a mouse model prepared in the same manner as Example 3 using TRIzol, and cDNA was synthesized. Then, the expression levels of epithelial-derived cytokines IL-25 and IL-33 were measured using q-PCR.

[0168] The results are shown in Figure 8.

[0169] Figure 8 is a graph showing the results of measuring the expression levels of epithelial-derived cytokines IL-25 and IL-33 in the paranasal mucosa of a mouse model of chronic rhinosinusitis (CRS) induced by SEB using ELISA.

[0170] As shown in Fig. 8, when nasal inflammation was induced in mice with SEB (NP), the expression levels of epithelial cytokines IL-25 and IL-33 increased compared to the control (CON) that did not induce nasal inflammation. On the other hand, when inotodiol (γ-CD + Inotodiol (3:1) inclusion complex, 20 mg / kg) was administered after nasal inflammation was induced in mice with SEB, the expression levels of epithelial cytokines IL-25 and IL-33 were significantly reduced to a level similar to that of the control (CON).

[0171] This means that when inotodiol was administered after inducing inflammation, the expression levels of IL-25 and IL-33, which are epithelial-derived cytokines that promote inflammation, were reduced, indicating that inotodiol is effective in preventing or treating eosinophilic inflammatory diseases.

[0172]

[0173] 4.5. Confirmation of the effect of inotodiol on reducing chemokine mRNA expression in the mouse paranasal sinus mucosa.

[0174] mRNA was extracted from the paranasal sinus mucosa of a mouse model prepared in the same manner as Example 3 using TRIzol, and cDNA was synthesized. Then, the expression levels of the chemokines CCL1, CCL2, and CXCL2 were measured.

[0175] The results are shown in Figure 9.

[0176] Figure 9 is a graph showing the results of measuring the expression levels of chemokines CCL1, CCL2, and CXCL2 in the paranasal mucosa of a mouse model of chronic rhinosinusitis (CRS) induced by SEB using ELISA.

[0177] As shown in Fig. 9, when nasal inflammation was induced in mice with SEB (NP), the expression levels of chemokines CCL1, CCL2, and CXCL2 increased compared to the control (CON). On the other hand, when inotodiol (γ-CD + Inotodiol (3:1) inclusion complex, 20 mg / kg) was administered after nasal inflammation was induced in mice with SEB, the expression levels of chemokines CCL1, CCL2, and CXCL2 were significantly reduced to a level similar to the control (CON).

[0178] This means that when inotodiol was administered after inducing inflammation, the expression levels of CCL1, CCL2, and CXCL2, which are chemokines that induce and maintain inflammation, were reduced, indicating that inotodiol is effective in preventing or treating eosinophilic inflammatory diseases.

[0179]

[0180] Example 5. Confirmation of the effect of inotodiol on inducing eosinophil apoptosis in the mouse paranasal sinus mucosa.

[0181] The head of the mouse model, prepared in the same manner as in Example 3, was prepared as a paraffin block. Specifically, the head of the mouse model was fixed and decalcified, and the mouse head was embedded in paraffin to prepare a paraffin block, and 4 μm-thick sections were prepared for staining. The paraffin sections were deparaffinized and dehydrated using a sequential ethanol series. The slides were boiled in 10 mM citrate buffer (pH 6.0) for 4 minutes to expose the antigen, and endogenous peroxidase activity was blocked by reacting in 3% H2O2 for 10 minutes and then washing with PBS.

[0182] Immunofluorescence staining was performed using cleaved caspase 3, an enzyme activated during the apoptosis process, and Siglec F, a protein specifically expressed on the surface of eosinophils. For cleaved caspase 3 antibody, cell signaling #9669 (Alexa Fluor® 488 Conjugate) was purchased and used, and for Siglec F antibody, Thermo fisher scientific, #14-1702-82 was purchased and used. After reacting the antibodies with deparaffinized slides overnight, they were incubated with Goat anti-Rat IgG (H+L) Cross-Adsorbed Secondary Antibody (Alexa Fluor TM 594 A-11007) for 2 hours. Counterstaining was performed using 300 nM DAPI (4,6-diamidino-2-phenylindole, Invitrogen) for nuclear staining.

[0183] The fluorescence signals of cleaved caspase 3 and Siglec F and the percentage of cells co-expressing caspase 3 and Siglec F (% of Cleaved casp3+ SiglecF+ / Siglec F+ cells) in two areas of the two mouse head tissue sections at 200x magnification are shown in Figure 10.

[0184] Figure 10 shows whether Cleaved caspase 3 and Siglec F are expressed in the paranasal mucosa of a mouse model of chronic eosinophilic rhinosinusitis (CRS) induced by SEB: Figure 10a is a photograph showing the fluorescent signal caused by the expression of Cleaved caspase 3 and Siglec F in the paranasal mucosa of a mouse model of chronic eosinophilic rhinosinusitis (CRS) induced by SEB, and Figure 10b is a graph showing the ratio of cells co-expressing Cleaved caspase 3 and Siglec F (% of Cleaved casp3+ SiglecF+ / Siglec F+ cells) in a head tissue section of the mouse model.

[0185] As shown in Fig. 10a, in the case of the control (CON) that did not induce nasal inflammation, the fluorescence signal of Siglec F, which is specifically expressed on the cell surface of eosinophils, was barely visible, whereas in the case of inducing nasal inflammation in the mouse (NP), the fluorescence signal of Siglec F was confirmed to be more visible than in the control (CON).

[0186] Meanwhile, as shown in Fig. 10b, when inotodiol (20 mg / kg) was administered after inducing inflammation in the mouse nasal cavity, it was confirmed that the ratio of cells co-expressing caspase 3 and Siglec F (% of Cleaved casp3+ SiglecF+ / Siglec F+ cells) was high.

[0187] This means that when inotodiol is administered after inflammation is induced, the expression rate of cleaved caspase 3, an enzyme activated in the process of apoptosis, is high, which means that inotodiol has the effect of inducing apoptosis of eosinophils and is effective in preventing and treating eosinophilia. It also means that inotodiol can be usefully used in the prevention or treatment of eosinophilic inflammatory diseases.

[0188]

[0189] Example 6. Confirmation of the effect of inotodiol on improving SEB-induced pathology in a mouse model of eosinophilic chronic rhinosinusitis (CRS).

[0190] 6.1. Alleviating effect of increased thickness of respiratory epithelium

[0191] After deparaffinization of the paraffin blocks prepared by the method of Example 5, H&E staining was performed. The thickness of the respiratory epithelium in four areas out of five tissue sections was measured at high magnification (400x) using a slide scanner system.

[0192] The results are shown in Figure 11.

[0193] Figure 11 shows the results of measuring the thickness of the respiratory epithelium in paraffin blocks of head tissue of a mouse model of eosinophilic chronic rhinosinusitis (Eosinophilic CRS) using a slide scanner system: Figure 11a is a photograph showing the results of H&E staining of mouse head tissue sections, Figure 11b is a graph quantifying the thickness of the respiratory epithelium, and Figure 11c is a graph showing the number of eosinophils per area (eosinophils / Fields) in three areas out of five tissue sections at 200x magnification.

[0194] As shown in Figure 11, when nasal inflammation was induced in mice with SEB (NP), it was confirmed that the respiratory epithelial thickness and the number of eosinophils per area increased rapidly compared to the control (CON).

[0195] Meanwhile, after inducing inflammation in the nasal cavity of mice with SEB, when inotodiol (20 mg / kg) was administered, the respiratory epithelial thickness was improved to a level similar to that of the control (CON), and the number of eosinophils per area was also confirmed to be reduced.

[0196] This suggests that when inotodiol is administered after inflammation is induced, the inflammatory response-induced proliferation of respiratory epithelial cells and the resulting increase in epithelial tissue thickness are alleviated. Therefore, inotodiol is effective in the prevention or treatment of eosinophilic inflammatory diseases.

[0197]

[0198] 6.2. Measurement of the number of goblet cells

[0199] Paraffin blocks prepared using the method of Example 5 were deparaffinized and then PAS stained. Specifically, goblet cells were stained using Periodic Acid Schiff (PAS) Stain Kit (Mucin Stain) #ab150680. After 10 minutes of reaction with PAS reagent, the sections were washed and then reacted with Schiff's reagent for 30 minutes. After that, hematoxylin staining for counterstaining was performed, and then 30 seconds of reaction with Bluing reagent was performed to convert soluble red to insoluble blue. Dehydration was then performed.

[0200] Among the photographs showing the above PAS staining results, the number of goblet cells, which increase in number due to inflammation in the nasal cavity, paranasal sinuses, and bronchi, was counted at 200x magnification.

[0201] The results are shown in Figure 12.

[0202] Figure 12 shows goblet cells in a paraffin block of head tissue from a mouse model of eosinophilic chronic rhinosinusitis (Eosinophilic CRS): Figure 12a is a photograph showing the results of PAS staining of a paraffin block of head tissue from a mouse model, and Figure 12b is a graph showing the number of goblet cells per area of ​​a slide stained with PAS of a paraffin block of head tissue from a mouse model at 200x magnification.

[0203] As shown in Fig. 12a, when nasal inflammation was induced in mice with SEB (NP), the number of goblet cells was confirmed to increase rapidly compared to the control (CON). On the other hand, when inotodiol (γ-CD + Inotodiol (3:1) inclusion complex, 20 mg / kg) was administered after nasal inflammation was induced in mice with SEB, the number of goblet cells in the mouse nasal cavity was confirmed to decrease.

[0204] Goblet cells, responsible for mucus secretion in respiratory epithelial tissue, are characterized by increased numbers and activity when inflammatory responses persist or are chronic. A decrease in the number of goblet cells in the mouse nasal cavity indicates an alleviation of the inflammatory response.

[0205] Therefore, when inotodiol is administered after inflammation is induced, it can be confirmed that inotodiol is effective in preventing or treating eosinophilic inflammatory diseases through the phenomenon of a decrease in the number of goblet cells.

[0206]

[0207] Example 7. Measurement of cell viability of various cells upon treatment with inotodiol.

[0208] To determine whether inotodiol is specific for eosinophilic disease, human eosinophilic leukemia cells (EoL-1), human mast cells (LUVA), mouse macrophage cells (Raw264.7), and human dermal fibroblast primary cells (HDF) were treated with inotodiol, and cell viability was measured. The results are shown in Fig. 13.

[0209] 7.1 Cell viability of human eosinophilic leukemia cells (EoL-1)

[0210] Human eosinophilic leukemia cells (EoL-1 cells) were seeded in 96-well plates at 0.01 × 106 cells / ml and cultured overnight. EoL-1 cells (200 uL) were treated with 2 uL of 50 mM butyric acid (BA) stock (in RPMI1640) (final concentration 500 uM), and CO2 incubation was performed for 24 hr. Inotodiol (γ-CD + Inotodiol(3:1) inclusion complex, in DMSO) 1 mM stock was treated, and the remaining concentrations were serially diluted (final concentration 2.5, 5, and 10 uM), and CO2 incubation was performed for 24 hr. The results measured at 450 nm using a plate reader using the Quantimax cell viability assay kit (20 uL / well, 1 hr) are shown in Figure 13a.

[0211] Figure 13a is a graph measuring cell viability by treating human eosinophilic leukemia cells (EoL-1 cells) with inotodiol at various concentrations.

[0212] As shown in Fig. 13a, when inotodiol was treated on EoL-1 cells, it was confirmed that cell viability decreased.

[0213] This means that inotodiol has the effect of inducing eosinophil death, and that inotodiol is effective in preventing and treating eosinophilia.

[0214] 7.2 Cell viability of human mast cells (LUVA)

[0215] Human mast cells (LUVA) were seeded at 0.1X106 cells / ml in a 96-well plate and cultured overnight.

[0216] LUVA cells (200 uL) were treated with 2 uL of a 10 mM stock of inotodiol (γ-CD + Inotodiol(3:1) inclusion complex, in DMSO) (final concentration 100 uM), and the remaining concentrations were treated by serial dilution (50, 25, 12.5, 6.25 uM) and CO2 incubation was performed for 24 h. 10 uL of MTT solution (5 mg / mL) was added and incubated for 2 h, after which the solution was aspirated, 100 uL of DMSO was added, and the spectrum was measured at 450 nm using a plate reader. The results are shown in Fig. 13b.

[0217] Figure 13b is a graph measuring cell viability by treating human mast cells (LUVA) with inotodiol at various concentrations.

[0218] As shown in Fig. 13b, when inotodiol was treated on human mast cells (LUVA), it was confirmed that the cytotoxicity of inotodiol could appear from 50 to 100 uM. Compared to the results on human eosinophilic leukemia cells (EoL-1 cells), this indicates that cell viability decreased at a concentration more than 20 times higher than the concentration (2.5 uM) at which inotodiol exhibited cytotoxicity in human eosinophilic leukemia cells, indicating that inotodiol acts specifically on eosinophils.

[0219]

[0220] 7.3 Cell viability of mouse macrophage cells (Raw264.7)

[0221] Mouse macrophage cells (Raw264.7) were seeded in a 96-well plate at 0.1X106 cells / ml and cultured overnight.

[0222] Raw264.7 cells (200 μL) were treated with 2 μL of a 10 mM stock of inotodiol (γ-CD + Inotodiol(3:1) inclusion complex, in DMSO) (final concentration 100 μM). The remaining concentrations were serially diluted (50, 25, 12.5, 6.25 μM) and CO2 incubated for 24 hours. 10 μL of MTT solution (5 mg / mL) was added and incubated for 2 hours. The solution was aspirated, and 100 μL of DMSO was added, followed by measurement at 450 nm using a plate reader. The results are shown in Fig. 13c.

[0223] Figure 13c is a graph measuring cell viability by treating mouse macrophage cells (Raw264.7) with inotodiol at various concentrations.

[0224] As shown in Fig. 13c, when inotodiol was treated to mouse macrophage cells (Raw264), it was confirmed that treatment with 50 uM inotodiol had no effect on the cell viability of macrophages. In addition, when treatment with 100 uM inotodiol, the cell viability decreased to 80%. However, since cytotoxicity is usually judged to be absent when the cell viability is 80% or higher, it was confirmed that inotodiol had no cytotoxicity to macrophages. When compared with the results of human eosinophilic leukemia cells (EoL-1 cells), no cytotoxicity was observed even at a high concentration that was more than 20 times the concentration (2.5 uM) at which inotodiol exhibited cytotoxicity in human eosinophilic leukemia cells, meaning that inotodiol acts specifically on eosinophils.

[0225]

[0226] 7.4 Cell viability of human dermal fibroblast primary cells (HDF)

[0227] Human dermal fibroblast primary cells (HDF cells) were seeded at 0.05X106 cells / ml in a 96-well plate and cultured overnight.

[0228] 1 uL of a 2 mM stock of inotodiol (γ-CD + Inotodiol(3:1) inclusion complex, in DMSO) was treated (final concentration 20 uM) to Raw264.7 cells (100 uL), and the remaining concentrations were serially diluted (10, 5, 2.5, 1.25, 0.6 uM) and CO2 incubated for 24 hours. 10 uL of MTT solution (5 mg / mL) was added, and after 2 hours of incubation, the solution was aspirated, and 100 uL of DMSO was added, and the spectrum was measured at 450 nm using a plate reader. The results are shown in Fig. 13d.

[0229] Figure 13d is a graph measuring cell viability by treating human dermal fibroblast primary cells (HDF cells) with inotodiol at various concentrations.

[0230] As shown in Fig. 13d, when human dermal fibroblast primary cells (HDF) were treated with inotodiol, it was confirmed that there was no cytotoxicity even when 20 uM of inotodiol was treated.

[0231] This means that inotodiol does not exhibit cytotoxicity even at a high concentration (20 uM), which is more than eight times the concentration (2.5 uM) at which it exhibited cytotoxicity in human eosinophilic leukemia cells, compared to the results in human eosinophilic leukemia cells (EoL-1 cells), indicating that inotodiol acts specifically on eosinophils.

Claims

1. A pharmaceutical composition for preventing or treating eosinophilia or eosinophilic inflammatory disease, comprising inotodiol, a prodrug thereof or a pharmaceutically acceptable salt thereof.

2. In claim 1, the prodrug is a pharmaceutical composition wherein the prodrug is an inotodiol ester derivative compound formed through an ester condensation reaction between a hydroxyl group of an inotodiol compound of the following chemical formula 1 and a carboxyl group of a fatty acid, glucuronic acid alkyl anhydride succinic acid, or phenolic acid: [Chemical Formula 1] .

3. In claim 2, the pharmaceutical composition wherein the inotodiol ester derivative compound is represented by the following chemical formula 2: [Chemical formula 2] In the above formula, R1 and R2 are independently OH, or -OC(O)-R3, and R3 is a straight or branched alkyl, alkenyl or alkynyl chain having 1 to 30 carbon atoms, At least one of R1 and R2 is -OC(O)-R3.

4. In claim 3, R3 is CH3(CH2) a -, or CH3(CH2) b (CH=CH(CH2)) c (CH2) d A pharmaceutical composition having an unsubstituted straight alkyl or alkenyl chain, wherein a is an integer from 8 to 24, b is an integer from 1 to 5, b is an integer from 1 to 6, and d is an integer from 3 to 7.

5. In claim 1, the pharmaceutical composition further comprises a cyclodextrin derivative represented by the following chemical formula 3: [Chemical Formula 3] In the above chemical formula 3, n is 4, 5 or 6, and R 11 , R 12 , R 13 , R 14 , R 15 , R 16 , R 17 , R 18 and R 19 is independently -OH, a substituted or unsubstituted alkoxy having C1 to C4, or a substituted or unsubstituted alkylthio having C1 to C4.

6. A pharmaceutical composition according to claim 5, wherein the inotodiol, a prodrug thereof, or a pharmaceutically acceptable salt thereof is enclosed within a cyclodextrin derivative represented by the chemical formula 3.

7. A pharmaceutical composition according to claim 5, wherein the cyclodextrin derivative represented by the chemical formula 3 is γ-cyclodextrin of the following chemical formula 4: [Chemical Formula 4] .

8. A pharmaceutical composition according to claim 1, wherein the inotodiol or a prodrug thereof induces eosinophil apoptosis.

9. In claim 1, the eosinophilia is eosinophilic cardiomyopathy, eosinophilic pneumonia (EP), idiopathic chronic eosinophilic pneumonia (ICEP), idiopathic acute eosinophilic pneumonia (IAEP), eosinophilic bronchitis, eosinophilic eosophagitis, eosinophilic gastroenteritis, eosinophilic gastritis, eosinophilic colitis, eosinophilic enteritis, eosinophilic fasciitis, idiopathic myositis, chronic eosinophilic A pharmaceutical composition, wherein the pharmaceutical composition is any one selected from the group consisting of chronic eosinophilic leukemia (CEL), hypereosinophilic syndrome (HES), idiopathic hypereosinophilic syndrome (IHES), Churg-Strauss syndrome, Loffler's Syndrome, Simple Pulmonary Eosinophilia, Paragonimiasis, Toxic-oil Syndrome, and Eosinophilia-myalgia Syndrome.

10. A pharmaceutical composition according to claim 1, wherein the eosinophilic inflammatory disease is an inflammatory disease caused in the nasal cavity, paranasal sinuses, or bronchial tubes.

11. A pharmaceutical composition according to claim 10, wherein the eosinophilic inflammatory disease is any one selected from the group consisting of allergic bronchopulmonary aspergillosis, eosinophilic drug allergy, and eosinophilic asthma.

12. A pharmaceutical composition according to claim 1, wherein the pharmaceutical composition is administered orally or parenterally, and the parenteral administration is any one selected from the group consisting of nasal inhalation, subcutaneous injection, intravenous injection, intramuscular injection, and intrathoracic injection.

13. A health functional food containing inotodiol, a prodrug thereof, or a pharmaceutically acceptable salt thereof as an active ingredient for preventing or improving eosinophilia or eosinophilic inflammatory disease.

14. Use of inotodiol, a prodrug thereof, or a pharmaceutically acceptable salt thereof for the manufacture of a medicament for the prevention or treatment of eosinophilia or eosinophilic inflammatory disease.

15. A method for treating eosinophilia or an eosinophilic inflammatory disease, comprising administering to a subject in need thereof inotodiol, a prodrug thereof, or a pharmaceutically acceptable salt thereof.

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