WO2023059099A1 - Pharmaceutical composition for preventing, improving, alleviating, or treating pulmonary fibrosis, comprising ezetimibe as active ingredient - Google Patents

Pharmaceutical composition for preventing, improving, alleviating, or treating pulmonary fibrosis, comprising ezetimibe as active ingredient Download PDF

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WO2023059099A1
WO2023059099A1 PCT/KR2022/015046 KR2022015046W WO2023059099A1 WO 2023059099 A1 WO2023059099 A1 WO 2023059099A1 KR 2022015046 W KR2022015046 W KR 2022015046W WO 2023059099 A1 WO2023059099 A1 WO 2023059099A1
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pulmonary fibrosis
ezetimibe
bleomycin
preventing
alleviating
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PCT/KR2022/015046
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French (fr)
Korean (ko)
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김송이
이찬호
배수한
김영삼
이진구
한지수
임범진
곽세현
신주혜
이유설
박정수
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연세대학교 산학협력단
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/397—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having four-membered rings, e.g. azetidine
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P11/00—Drugs for disorders of the respiratory system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
    • A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
    • A23V2200/00—Function of food ingredients
    • A23V2200/30—Foods, ingredients or supplements having a functional effect on health
    • A23V2200/314—Foods, ingredients or supplements having a functional effect on health having an effect on lung or respiratory system
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
    • A23V2250/00—Food ingredients
    • A23V2250/30—Other Organic compounds

Definitions

  • composition for preventing, improving, alleviating or treating pulmonary fibrosis comprising ezetimibe or a salt thereof as an active ingredient, and uses thereof.
  • Pulmonary fibrosis is a disease in which the lungs do not function normally because lung tissue is damaged and scarred to become thick and hard. As pulmonary fibrosis worsens, breathing becomes progressively shorter. In many cases, the exact cause of pulmonary fibrosis cannot be identified. This fibrosis is called idiopathic pulmonary fibrosis (IPF).
  • IPF idiopathic pulmonary fibrosis
  • Pulmonary fibrosis includes idiopathic pulmonary fibrosis; pulmonary fibrosis due to autoimmune diseases such as rheumatoid arthritis, viral infections, diseases such as gastroesophageal reflux disease (GERD); familial pulmonary fibrosis (familial PF); There are various types of pulmonary fibrosis caused by harmful substances such as asbestos, silica, dust, radiation treatment, and smoking. Treatment strategies for pulmonary fibrosis are diverse and individual depending on the condition and symptoms of the patient, but a cure that can cure pulmonary fibrosis has not yet been found.
  • Ezetimibe is a lipid-lowering agent that inhibits cholesterol absorption and is used as a treatment for hyperlipidemia and hypercholesterolemia.
  • Easytrol ® as a single ezetimibe product
  • Vitorin ® ezetimibe + simvastatin
  • Rosuzet ® ezetimibe + rosuvastatin
  • Atojet as combination products of ezetimibe and other hyperlipidemia drugs.
  • ® (ezetimibe + atorvastatin) (Patent Document 1).
  • Patent Document 1 US 2014-0287042 A1
  • compositions for preventing, improving, alleviating or treating pulmonary fibrosis comprising ezetimibe or a pharmaceutically acceptable salt thereof as an active ingredient.
  • a health functional food composition for preventing, improving, or alleviating pulmonary fibrosis comprising ezetimibe or a food chemically acceptable salt thereof as an active ingredient.
  • a method for preventing, ameliorating, alleviating or treating pulmonary fibrosis comprising administering an effective amount of ezetimibe or a pharmaceutically acceptable salt thereof to a subject in need thereof.
  • ezetimibe or a pharmaceutically acceptable salt thereof for use in the manufacture of a medicament for preventing, ameliorating, alleviating or treating pulmonary fibrosis.
  • ezetimibe or a pharmaceutically acceptable salt thereof for preventing, improving, alleviating or treating pulmonary fibrosis.
  • One aspect provides a pharmaceutical composition for preventing, improving, alleviating or treating pulmonary fibrosis, comprising Ezetimibe or a pharmaceutically acceptable salt thereof as an active ingredient.
  • the “Ezetimibe” has a structural formula represented by Formula 1 below:
  • the ezetimibe was approved by the US Food and Drug Administration (FDA) in 2002, and its substance patent expired in 2016.
  • Ezetimibe can reduce pulmonary fibrosis-related factors induced by TGF- ⁇ , specifically TGF- ⁇ 1, in lung fibroblasts.
  • Ezetimibe can inhibit the differentiation of lung fibroblasts into myofibroblasts or reduce collagen expression.
  • the ezetimibe can inhibit the differentiation of lung fibroblasts into myofibroblasts or reduce the expression of collagen protein and/or mRNA in lung fibroblasts.
  • the collagen may be COL1A1 (collagen, type I, alpha 1).
  • the ezetimibe may be in any pharmaceutically acceptable form.
  • pharmaceutically acceptable means an amount sufficient to exhibit a therapeutic effect and not causing side effects, such as the type of disease, the sensitivity of the patient to the drug, the route of administration, the method of administration, the number of administrations, the duration of treatment, etc. It can be readily determined by a person skilled in the art according to factors well known in the medical arts.
  • the ezetimibe may be in the form of a pharmaceutically acceptable salt thereof.
  • These salts include acid addition salts used in the pharmaceutical field, for example salts derived from inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, sulfamic acid, phosphoric acid or nitric acid; and acetic acid, propionic acid, succinic acid, glycolic acid, stearic acid, citric acid, maleic acid, malonic acid, methanesulfonic acid, tartaric acid, malic acid, phenylacetic acid, glutamic acid, benzoic acid, salicylic acid, 2-acetoxybenzoic acid, fumaric acid, toluenesulfonic acid, oxalic acid, salts derived from organic acids such as trifluoroacetic acid or glucuronic acid.
  • inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, sulfamic acid, phosphoric acid or n
  • the salt may be a base addition salt such as ammonium, dimethylamine, monomethylamine, monoethylamine, or diethylamine.
  • the salt includes a common metal salt form, for example, a salt derived from a metal such as lithium, sodium, potassium, magnesium, or calcium.
  • the acid addition salt, base addition salt or metal salt may be prepared according to a conventional method.
  • Pharmaceutically acceptable salts and general methodologies for their preparation are well known in the art. See, for example, P. Stahl, et al. Handbook of Pharmaceutical Salts: Properties, Selection and Use, 2nd Revised Edition (Wiley-VCH, 2011)]; [S.M. Berge, et al., "Pharmaceutical Salts," Journal of Pharmaceutical Sciences, Vol. 66, no. 1, January 1977].
  • Ezetimibe is included as an active ingredient in the composition.
  • the term “included as an active ingredient” means that ezetimibe is added to the extent that the above-mentioned effects can be exhibited, and various ingredients are added as subcomponents for drug delivery and stabilization, etc. to be formulated in various forms. It means to include being.
  • the ezetimibe may be included in the composition in a pharmaceutically effective amount.
  • an effective amount refers to that amount or dose of ezetimibe that, when administered to a patient in single or multiple doses, provides a desired effect in a patient under diagnosis or treatment.
  • the effective amount can be readily determined by the attending diagnostician as a person skilled in the art by using known techniques or by observing results obtained under similar circumstances.
  • the mammalian species When determining an effective amount for a patient, the mammalian species; his size, age and general health; the specific disease or disorder involved; degree or severity of involvement of the disease or disorder; individual patient response; the specific compound being administered; administration mode; bioavailability characteristics of the administered agent; the selected dosing regimen; use of concomitant medication; A number of factors are taken into account by the attending physician diagnostician, including but not limited to, and other relevant circumstances.
  • the ezetimibe daily dose is 0.001 to 20 mg, 0.001 to 15 mg, 0.001 to 10 mg, 0.01 to 20 mg, 0.01 to 15 mg, 0.01 to 10 mg, 0.1 to 20 mg, 0.1 to 20 mg It may be included in a dose of 15 mg, or 0.1 to 10 mg, but is not limited thereto.
  • the dose of ezetimibe can be appropriately adjusted according to the individual.
  • pulmonary fibrosis refers to a phenomenon in which a large amount of extracellular matrix including collagen is accumulated in the interstitium of the lung and all diseases in which such a phenomenon appears.
  • the cause of the pulmonary fibrosis is not limited.
  • the pulmonary fibrosis may include pulmonary fibrosis caused by idiopathic interstitial pneumonia of unknown cause (eg, idiopathic pulmonary fibrosis, pulmonary fibrosis caused by idiopathic nonspecific interstitial pneumonia, etc.); pulmonary fibrosis caused by autoimmune diseases or connective tissue diseases such as rheumatoid arthritis, lupus, systemic sclerosis, myositis, Sjogren's syndrome; Pulmonary fibrosis due to diseases such as infectious diseases (eg, coronavirus, pneumocystis pneumonia, etc.), gastroesophageal reflux disease (GERD); familial pulmonary fibrosis (familial PF); pulmonary fibrosis caused by harmful substances such as asbestos, silica, beryllium, dust, and smoking; pulmonary fibrosis due to radiation therapy or radiation exposure; Pulmonary fibrosis caused by drugs such as anticancer drugs (eg Bleomycin), antibiotics, anticancer
  • the pulmonary fibrosis may be idiopathic pulmonary fibrosis.
  • the pulmonary fibrosis may be pulmonary fibrosis caused by administration of bleomycin or sequelae after COVID-19 infection.
  • bleomycin is an anticancer drug used for squamous cell carcinoma of the head and neck, lung, skin, etc., and has been reported to exhibit side effects such as pulmonary fibrosis.
  • the pulmonary fibrosis may include pulmonary fibrosis caused by the use of other drugs such as bleomycin.
  • coronavirus disease is an infectious disease caused by a coronavirus.
  • the coronavirus infection may be COVID-19, but is not limited thereto.
  • coronavirus infection-19 (COVID-19) is an infectious disease caused by severe acute respiratory syndrome coronavirus 2: SARS-CoV-2. Therefore, the pulmonary fibrosis may be pulmonary fibrosis caused by COVID-19.
  • the pulmonary fibrosis may be idiopathic pulmonary fibrosis.
  • the pulmonary fibrosis may be pulmonary fibrosis caused by administration of bleomycin or sequelae after COVID-19 infection.
  • bleomycin is an anticancer drug used for squamous cell carcinoma of the head and neck, lung, skin, etc., and has been reported to exhibit side effects such as pulmonary fibrosis.
  • the pulmonary fibrosis may include pulmonary fibrosis caused by the use of other drugs such as bleomycin.
  • prevention refers to any action that inhibits or delays the onset of pulmonary fibrosis by administration of the composition.
  • improvement refers to any process in which symptoms of pulmonary fibrosis are improved by administration of the composition.
  • Alleviation means that the symptoms of pulmonary fibrosis are alleviated, the progression of pulmonary fibrosis is delayed, or the cause(s) of pulmonary fibrosis itself is alleviated or eliminated by administration of the composition. Alleviation within this specification may also include mitigating the progression of pulmonary fibrosis.
  • treatment refers to any action that improves or benefits the symptoms of pulmonary fibrosis by administration of the composition.
  • the pharmaceutical composition may further include a pharmaceutically acceptable carrier.
  • Pharmaceutically acceptable carriers may include binders, lubricants, disintegrants, excipients, solubilizers, dispersants, stabilizers, suspending agents, pigments and flavors for oral administration, and buffers, preservatives, and painless agents for injections.
  • a topical agent, a solubilizing agent, an isotonic agent, and a stabilizer may be mixed and used, and in the case of topical administration, a base, an excipient, a lubricant, and a preservative may be used.
  • Formulations of the pharmaceutical composition may be variously prepared by mixing with a pharmaceutically acceptable carrier as described above.
  • a pharmaceutically acceptable carrier for example, for oral administration, it can be prepared in the form of tablets, troches, capsules, elixirs, suspensions, syrups, wafers, etc., and in the case of injections, it can be prepared in the form of unit dose ampoules or multiple doses.
  • it can be formulated into solutions, suspensions, tablets, pills, capsules, and sustained-release preparations.
  • examples of carriers, excipients and diluents suitable for formulation include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl Cellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate or mineral oil may be used.
  • fillers, anti-coagulants, lubricants, wetting agents, flavoring agents, emulsifiers and preservatives may be further included.
  • the pharmaceutical composition may further include one or more other agents for treating pulmonary fibrosis.
  • the other agent may be a therapeutic agent for pulmonary fibrosis, but is not limited thereto.
  • the therapeutic agent for pulmonary fibrosis known substances may be used.
  • the therapeutic agent for pulmonary fibrosis may be pirfenidone, nintedanib, and the like.
  • Another aspect provides a health functional food composition for preventing, improving, or alleviating pulmonary fibrosis, comprising Ezetimibe or a food chemically acceptable salt thereof as an active ingredient.
  • the health functional food composition may be formulated into a conventional health functional food formulation known in the art.
  • the health functional food composition may use ezetimibe alone or together with other foods or food ingredients, and may be appropriately used according to conventional methods.
  • the mixing amount of the active ingredient may be appropriately determined depending on the purpose of use (prevention, health or therapeutic treatment).
  • beverage compositions may contain various flavoring agents or natural carbohydrates as additional components, like conventional beverages.
  • the natural carbohydrates include monosaccharides such as glucose and fructose; disaccharides such as maltose and sucrose; and polysaccharides such as dextrins and cyclodextrins; It is a sugar alcohol, such as xylitol, sorbitol, and erythritol.
  • sweetener natural sweeteners such as thaumatin and stevia extract, or synthetic sweeteners such as saccharin and aspartame may be used.
  • the food composition is also used in nutrients, vitamins, electrolytes, flavors, colorants, pectic acid and its salts, alginic acid and its salts, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohols, carbonated beverages carbonation agent, or a combination thereof.
  • the food composition may also contain natural fruit juice, fruit juice beverages, fruit flesh for preparing vegetable beverages, or a combination thereof.
  • Another aspect provides a method for preventing, ameliorating, alleviating or treating pulmonary fibrosis, comprising administering an effective amount of ezetimibe or a pharmaceutically acceptable salt thereof to a subject in need thereof.
  • the term "individual” means a subject in need of treatment for a disease, and more specifically, mammals such as humans or non-human primates, mice, rats, dogs, cats, horses, and cattle.
  • the "subject in need thereof” refers to an individual in need of prevention, improvement or treatment of pulmonary fibrosis.
  • administration means the introduction of a substance into a patient by any suitable method.
  • the route of administration may be any general route capable of reaching a target in vivo in a patient.
  • the administration may be, for example, intraperitoneal administration, intravenous administration, intramuscular administration, subcutaneous administration, intradermal administration, oral administration, topical administration, intranasal administration, or intrarectal administration, but is not limited thereto.
  • the administration may be oral administration.
  • the dosage may be prescribed in various ways depending on factors such as formulation method, administration method, patient's age, weight, sex, medical condition, food, administration time, administration route, excretion rate and reaction sensitivity, and those skilled in the art can determine these factors Dosage can be appropriately adjusted in consideration of these factors.
  • the dosage ranges from 0.001 mg to 1,000 mg, 0.001 mg to 500 mg, 0.001 mg to 100 mg, 0.001 mg to 50 mg, 0.001 mg to 20 mg, 0.001 mg to 10 mg, ezetimibe per day per subject. It may be 0.1 mg to 100 mg, 0.1 mg to 50 mg, 0.1 mg to 20 mg, or 0.1 mg to 10 mg, but is not limited thereto.
  • the number of administrations can be once a day or two or more times within the range of clinically acceptable side effects, and administration can be performed at one or two or more sites, daily or at intervals of 2 to 5 days.
  • the number of administration days may be administered from 1 day to 30 days per treatment. If necessary, the same treatment can be repeated after a titration period.
  • the same dosage per kg as for humans is used, or the above dosage is converted by the volume ratio (eg, average value) of the organ (heart, etc.) between the target animal and the human.
  • a single dose can be administered.
  • an effective amount of ezetimibe or a pharmaceutically acceptable salt thereof may be administered simultaneously, separately, or sequentially with an effective amount of one or more other active ingredients.
  • the one or more other active ingredients may be one or more other agents for treating pulmonary fibrosis, but are not limited thereto.
  • Another aspect provides the use of ezetimibe or a pharmaceutically acceptable salt thereof for use in the manufacture of a medicament for preventing, ameliorating, alleviating or treating pulmonary fibrosis.
  • Another aspect provides the use of ezetimibe or a pharmaceutically acceptable salt thereof for preventing, ameliorating, alleviating or treating pulmonary fibrosis.
  • Redundant content is omitted in consideration of the complexity of the present specification, and terms not defined otherwise in the present specification have meanings commonly used in the technical field to which the present invention belongs.
  • composition according to one aspect may have an effect of preventing, improving, alleviating or treating pulmonary fibrosis by including ezetimibe or a pharmaceutically acceptable salt thereof. Therefore, ezetimibe can be used as a pharmaceutical composition for preventing, improving, alleviating or treating pulmonary fibrosis.
  • 1 is a result of treating mouse lung fibroblasts with ezetimibe at the indicated concentration for 24 hours in a cell culture medium of 15% FBS, and performing an MTT assay.
  • Figure 2 shows the results of MTT assay after treatment with ezetimibe at the indicated concentration for 24 hours in mouse lung fibroblasts in FBS 0.1% cell culture medium conditions.
  • Figure 3 shows the results of treating human lung fibroblasts with ezetimibe at the indicated concentrations for 24 hours in a cell culture medium of 15% FBS, and performing the MTT assay.
  • Figure 4 is a result of comparing the TGF ⁇ 1 untreated state and the TGF ⁇ 1 treated state, and showing the Col1A1 protein expression level according to the treatment concentration of ezetimibe in the TGF ⁇ 1 treated state at the same time.
  • FIG. 5 is a result showing the mRNA expression level of Col1A1 and Acta2 for each treatment concentration of ezetimibe.
  • FIG. 6 is a result showing the level of Col1A1 protein expression according to ezetimibe treatment time when the concentration of ezetimibe was fixed at 20 ⁇ M.
  • FIG. 7 is a result showing the mRNA expression levels of Col1A1 and Acta2 according to ezetimibe treatment time when the concentration of ezetimibe was fixed at 20 ⁇ M.
  • FIG. 10 is a result showing the level of Col1A1 protein expression according to ezetimibe treatment time when the concentration of ezetimibe was fixed at 20 ⁇ M.
  • FIG. 11 shows the mRNA expression levels of Col1A1 and Acta2 according to ezetimibe treatment time when the concentration of ezetimibe was fixed at 20 ⁇ M.
  • FIG. 12 is a schematic view showing the administration method of each group in an experiment for confirming the pulmonary fibrosis alleviating effect of ezetimibe in a bleomycin-induced mouse pulmonary fibrosis model.
  • 13 is a graph showing the change in survival rate of mice according to the number of days after administration of bleomycin.
  • 15 is a diagram confirming the results of comparing COL1A1 and fibronectin, which are fibrosis markers of each group, using Western blot and quantifying the result of Western blot through Image J.
  • 16 is a diagram showing the results of confirming the expression of fibrosis markers Col1a1, Col1a2, Acta2, Col3a1 and Eda-fn mRNA through RT-qPCR.
  • 18 is a diagram showing the results of quantification of collagen-stained areas in slides of lung pathological tissues for each mouse using the Aperio Imagescope program.
  • mice In the case of mouse lung tissue, adult mice between 7 and 10 weeks of age were euthanized, and both lungs were aseptically collected.
  • lung tissue of about 2 cm 3 obtained in a general surgical procedure was aseptically removed and used only for patients who agreed to the lung tissue collection study that passed the Institutional Review Board (IRB).
  • IRS Institutional Review Board
  • the culture medium was replaced, and 2 weeks after separation, subculture was performed, and the cell culture medium was changed to MEM with 15% FBS and 1X P/S (penicillin/streptomycin) added.
  • the tissue was removed using a 100 ⁇ m cell filter, and only the isolated lung fibroblasts were cultured. Thereafter, the cells were subcultured at intervals of about 5 days to increase the purity of the cells and increase the number of cells. All experiments were performed while changing to normoxic conditions in the third passage.
  • TGF ⁇ 1 a solution containing 0.1% (w/v) BSA (Bovine Serum Albumin) in a 20 mM citrate solution (pH 3.0) containing 100 mM NaCl was used as a vehicle, and ezetimibe was DMSO ( Dimethyl Sulfoxide) was used as a vehicle. All drugs and their solvents were applied to the cells at 1:1000 (v/v).
  • BSA Bovine Serum Albumin
  • DMSO Dimethyl Sulfoxide
  • Bleomycin is an anticancer drug used for squamous cell carcinoma of the head and neck, lung, skin, etc., and has been reported to exhibit side effects such as pulmonary fibrosis. Based on these characteristics, bleomycin is the most representative drug used in animal models of pulmonary fibrosis. Therefore, pulmonary fibrosis was induced using bleomycin. Experiments were conducted in the following order using C57BL/6J mice between 7 and 10 weeks of age.
  • mice were given a 15-minute fasting period, and anesthesia was performed through respiratory anesthesia.
  • control group control, normal saline
  • 50 ul of aseptically treated physiological saline was aspirated through the pharynx to the mouse, and for the bleomycin-administered group and bleomycin and ezetimibe-administered group, 50 ul of bleomycin 2U/kg was orally administered to the mouse. was aspirated
  • the group administered with bleomycin and ezetimibe received 2 mg/kg of ezetimibe diluted in 0.5% sodium carboxymethyl cellulose prepared in distilled water, and bleomycin and ezetimibe.
  • the group administered with Nintedanib received 40 mg/kg of Nintedanib diluted in 0.5% sodium carboxymethyl cellulose, and the control and bleomycin groups received oral administration of 0.5% sodium carboxymethyl cellulose five times a week, daily.
  • the primary antibody was reacted at 4°C for 18 to 24 hours, washed with TTBS (Tween-Tris buffered saline), and the secondary antibody was reacted at room temperature for more than 1 hour.
  • TTBS Tetween-Tris buffered saline
  • Trizol 1000 ul of Trizol and 200 ul of chloroform were added to the tissues or cells, mixed well, and centrifuged at 13,000 rpm for 15 minutes using a centrifuge at 4 °C.
  • RNA in the solution was quantified using a trace spectrophotometer.
  • cDNA complementary DNA
  • This experiment is an experiment to confirm cytotoxicity by reflecting the number of viable cells by measuring ATP released while lysing cells.
  • the concentration of ezetimibe that causes toxicity to the cells was confirmed using the MTT assay.
  • experiments were conducted in FBS 0.1% culture medium and FBS 15% culture medium, respectively.
  • 1 is a result of treating mouse lung fibroblasts with ezetimibe at the indicated concentration for 24 hours in a cell culture medium of 15% FBS, and performing an MTT assay.
  • Figure 2 shows the results of MTT assay after treatment with ezetimibe at the indicated concentration for 24 hours in mouse lung fibroblasts in FBS 0.1% cell culture medium condition.
  • Figure 3 shows the results of treating human lung fibroblasts with ezetimibe at the indicated concentrations for 24 hours in a cell culture medium of 15% FBS, and performing the MTT assay.
  • ezetimibe did not cause cytotoxicity in human and mouse lung fibroblasts at a concentration of less than 20 ⁇ M. Based on this, in subsequent experiments, 20 ⁇ M of ezetimibe was used at the maximum concentration.
  • Collagen 1a1 (Col1A1), one of the final products of fibrosis produced by treating fibroblasts with TGF ⁇ 1 to differentiate into myofibroblasts, was identified as a major detection target.
  • Figure 4 is a result of comparing the TGF ⁇ 1 untreated state and the TGF ⁇ 1 treated state, and showing the Col1A1 protein expression level according to the treatment concentration of ezetimibe in the TGF ⁇ 1 treated state at the same time.
  • FIG. 5 shows the mRNA expression levels of Col1A1 and Acta2 for each treatment concentration of ezetimibe under the same experimental conditions as in FIG. 4 .
  • FIG. 6 is a result showing the level of Col1A1 protein expression according to ezetimibe treatment time when the concentration of ezetimibe was fixed at 20 ⁇ M.
  • FIG. 7 is a result showing the mRNA expression levels of Col1A1 and Acta2 according to the treatment time of ezetimibe when the concentration of ezetimibe was fixed at 20 ⁇ M under the same experimental conditions as in FIG. 6 .
  • Collagen 1a1 (Col1A1), one of the final products of fibrosis produced by treating fibroblasts with TGF ⁇ 1 to differentiate into myofibroblasts, was identified as a major detection target.
  • FIG. 9 shows the mRNA expression levels of Col1A1 and Acta2 for each treatment concentration of ezetimibe under the same experimental conditions as in FIG. 8 .
  • FIG. 10 is a result showing the level of Col1A1 protein expression according to ezetimibe treatment time when the concentration of ezetimibe was fixed at 20 ⁇ M.
  • FIG. 11 is a result showing the mRNA expression levels of Col1A1 and Acta2 according to ezetimibe treatment time when the concentration of ezetimibe was fixed at 20 ⁇ M under the same experimental conditions as in FIG. 10.
  • the TGF ⁇ 1-treated group also increased COL1A1 protein and mRNA expression by TGF ⁇ stimulation, but such an increase in COL1A1 protein and mRNA expression was effectively expressed by ezetimibe treatment It was confirmed that this decreased.
  • ezetimibe has a therapeutic effect on pulmonary fibrosis in human and mouse pulmonary fibrosis cell models by TGF ⁇ 1 stimulation of pulmonary fibroblasts.
  • mice In order to confirm the effect of ezetimibe on alleviating pulmonary fibrosis in a bleomycin-induced mouse pulmonary fibrosis model, the survival rate of mice was confirmed.
  • FIG. 12 is a schematic view showing the administration method of each group in an experiment for confirming the pulmonary fibrosis alleviating effect of ezetimibe in a bleomycin-induced mouse pulmonary fibrosis model.
  • 13 is a graph showing the change in survival rate of mice according to the number of days after administration of bleomycin.
  • mice administered with ezetimibe exhibited an excellent survival effect compared to mice administered with bleomycin, which was found to be similar to that of nintedanib, an existing idiopathic pulmonary fibrosis drug.
  • 15 is a diagram confirming the results of comparing COL1A1 and fibronectin, which are fibrosis markers of each group, using Western blot and quantifying the result of Western blot through Image J.
  • 16 is a diagram showing the results of confirming the expression of fibrosis markers Col1a1, Col1a2, Acta2, Col3a1 and Eda-fn mRNA through RT-qPCR.
  • 18 is a diagram showing the results of quantification of collagen-stained areas in slides of lung pathological tissues for each mouse using the Aperio Imagescope program.
  • ezetimibe showed efficacy in inhibiting fibrosis in mice administered with bleomycin, especially when compared to mice using standard dose of nintedanib, a drug already marketed and used internationally for patients with idiopathic pulmonary fibrosis. However, it was confirmed that similar or superior effects appeared. Accordingly, it was confirmed that ezetimibe can alleviate and treat pulmonary fibrosis.

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Abstract

Provided are: a pharmaceutical composition for preventing, improving, alleviating, or treating pulmonary fibrosis, the composition comprising Ezetimibe or a salt thereof as an active ingredient; a health functional food composition; a method for preventing, improving, alleviating, or treating pulmonary fibrosis using same; and a use thereof. The composition according to one aspect of the present invention contains Ezetimibe and may thus have the effect of preventing, improving, alleviating, or treating pulmonary fibrosis.

Description

에제티미브를 유효성분으로 포함하는 폐섬유증의 예방, 개선, 완화 또는 치료용 약학적 조성물Pharmaceutical composition for preventing, improving, alleviating or treating pulmonary fibrosis containing ezetimibe as an active ingredient
에제티미브 또는 이의 염을 유효성분으로 포함하는 폐섬유증의 예방, 개선, 완화 또는 치료용 조성물 및 이의 용도에 관한 것이다.It relates to a composition for preventing, improving, alleviating or treating pulmonary fibrosis comprising ezetimibe or a salt thereof as an active ingredient, and uses thereof.
폐섬유증(pulmonary fibrosis)은 폐 조직이 손상되고 상처를 입어 두껍고 딱딱하게 변성되어 폐가 정상적으로 작동하지 않는 질환이다. 폐섬유증이 악화됨에 따라 점차 호흡이 짧아진다. 폐섬유증을 유발하는 원인을 정확히 찾아 낼 수 없는 경우가 많은데, 이러한 섬유증을 특발성 폐섬유증(idiopathic pulmonary fibrosis, IPF)이라고 한다. 폐섬유증에는 특발성 폐섬유증; 류마티스성 관절염 같은 자가 면역 질환, 바이러스 감염, 위식도 역류성 질환(gastroesophageal reflux disease, GERD) 등의 질병으로 인한 폐섬유증; 가족성 폐섬유증(familial PF); 석면, 실리카, 먼지, 방사선 치료, 흡연과 같은 유해물질에 의한 폐섬유증 등 다양한 종류가 있다. 폐섬유증의 치료 전략은 환자의 상태, 증상에 따라 다양하고 개별적이나, 아직까지 폐섬유증을 완치할 수 있는 치료제는 찾지 못하고 있다.Pulmonary fibrosis is a disease in which the lungs do not function normally because lung tissue is damaged and scarred to become thick and hard. As pulmonary fibrosis worsens, breathing becomes progressively shorter. In many cases, the exact cause of pulmonary fibrosis cannot be identified. This fibrosis is called idiopathic pulmonary fibrosis (IPF). Pulmonary fibrosis includes idiopathic pulmonary fibrosis; pulmonary fibrosis due to autoimmune diseases such as rheumatoid arthritis, viral infections, diseases such as gastroesophageal reflux disease (GERD); familial pulmonary fibrosis (familial PF); There are various types of pulmonary fibrosis caused by harmful substances such as asbestos, silica, dust, radiation treatment, and smoking. Treatment strategies for pulmonary fibrosis are diverse and individual depending on the condition and symptoms of the patient, but a cure that can cure pulmonary fibrosis has not yet been found.
에제티미브(Ezetimibe)는 콜레스테롤의 흡수를 억제하는 지질저하제이며, 고지혈증, 고콜레스테롤혈증의 치료제로 사용되고 있다. 에제티미브 단일제 제품으로는 이지트롤®이 있고, 에제티미브와 다른 고지혈증 치료제의 복합제 제품으로는 바이토린®(에제티미브+심바스타틴), 로수젯®(에제티미브+로수바스타틴), 아토젯®(에제티미브+아토르바스타틴)이 있다(특허문헌 1).Ezetimibe is a lipid-lowering agent that inhibits cholesterol absorption and is used as a treatment for hyperlipidemia and hypercholesterolemia. There is Easytrol ® as a single ezetimibe product, and Vitorin ® (ezetimibe + simvastatin), Rosuzet ® (ezetimibe + rosuvastatin), and Atojet as combination products of ezetimibe and other hyperlipidemia drugs. ® (ezetimibe + atorvastatin) (Patent Document 1).
그러나, 에제티미브의 폐섬유증 치료 용도 등 다른 의약 용도에 대한 연구는 부족한 실정이다.However, studies on other medicinal uses of ezetimibe, such as use in the treatment of pulmonary fibrosis, are lacking.
[선행기술문헌][Prior art literature]
[특허문헌][Patent Literature]
(특허문헌 1) US 2014-0287042 A1(Patent Document 1) US 2014-0287042 A1
에제티미브 또는 이의 약학적으로 허용가능한 염을 유효성분으로 포함하는 폐섬유증의 예방, 개선, 완화 또는 치료용 약학적 조성물을 제공한다.Provided is a pharmaceutical composition for preventing, improving, alleviating or treating pulmonary fibrosis comprising ezetimibe or a pharmaceutically acceptable salt thereof as an active ingredient.
에제티미브 또는 이의 식품학적으로 허용가능한 염을 유효성분으로 포함하는 폐섬유증의 예방, 개선 또는 완화용 건강기능식품 조성물을 제공한다.Provided is a health functional food composition for preventing, improving, or alleviating pulmonary fibrosis, comprising ezetimibe or a food chemically acceptable salt thereof as an active ingredient.
유효량의 에제티미브 또는 이의 약학적으로 허용가능한 염을 이를 필요로 하는 개체에게 투여하는 단계를 포함하는 폐섬유증의 예방, 개선, 완화 또는 치료 방법을 제공한다.Provided is a method for preventing, ameliorating, alleviating or treating pulmonary fibrosis, comprising administering an effective amount of ezetimibe or a pharmaceutically acceptable salt thereof to a subject in need thereof.
폐섬유증의 예방, 개선, 완화 또는 치료용 약제의 제조에 사용하기 위한 에제티미브 또는 이의 약학적으로 허용가능한 염의 용도를 제공한다.Provided is the use of ezetimibe or a pharmaceutically acceptable salt thereof for use in the manufacture of a medicament for preventing, ameliorating, alleviating or treating pulmonary fibrosis.
폐섬유증의 예방, 개선, 완화 또는 치료를 위한 에제티미브 또는 이의 약학적으로 허용가능한 염의 용도를 제공한다.Provided is the use of ezetimibe or a pharmaceutically acceptable salt thereof for preventing, improving, alleviating or treating pulmonary fibrosis.
일 양상은 에제티미브(Ezetimibe) 또는 이의 약학적으로 허용가능한 염을 유효성분으로 포함하는 폐섬유증의 예방, 개선, 완화 또는 치료용 약학적 조성물을 제공한다.One aspect provides a pharmaceutical composition for preventing, improving, alleviating or treating pulmonary fibrosis, comprising Ezetimibe or a pharmaceutically acceptable salt thereof as an active ingredient.
상기 “에제티미브(Ezetimibe)”는 하기 화학식 1로 표시되는 구조식을 갖는다:The “Ezetimibe” has a structural formula represented by Formula 1 below:
Figure PCTKR2022015046-appb-img-000001
(화학식 1)
Figure PCTKR2022015046-appb-img-000001
(Formula 1)
상기 에제티미브는 2002년 미국식품의약국(FDA)의 승인을 받았으며, 이의 물질특허는 2016년 만료되었다.The ezetimibe was approved by the US Food and Drug Administration (FDA) in 2002, and its substance patent expired in 2016.
상기 에제티미브는 폐 섬유모세포에서 TGF-β, 구체적으로 TGF-β1에 의해 유도된 폐섬유증 관련 인자를 감소시킬 수 있다.Ezetimibe can reduce pulmonary fibrosis-related factors induced by TGF-β, specifically TGF-β1, in lung fibroblasts.
상기 에제티미브는 폐 섬유모세포에서 근섬유모세포로의 분화를 억제하거나 콜라겐 발현을 감소시킬 수 있다. 구체적으로, 상기 에제티미브는 폐 섬유모세포에서 근섬유모세포로의 분화를 억제하거나 폐 섬유모세포에서 콜라겐 단백질 및/또는 mRNA의 발현을 감소시킬 수 있다. 상기 콜라겐은 COL1A1 (collagen, type I, alpha 1)일 수 있다. Ezetimibe can inhibit the differentiation of lung fibroblasts into myofibroblasts or reduce collagen expression. Specifically, the ezetimibe can inhibit the differentiation of lung fibroblasts into myofibroblasts or reduce the expression of collagen protein and/or mRNA in lung fibroblasts. The collagen may be COL1A1 (collagen, type I, alpha 1).
상기 에제티미브는 약학적으로 허용가능한 임의의 형태일 수 있다.The ezetimibe may be in any pharmaceutically acceptable form.
상기 "약학적으로 허용가능한"이란 치료 효과를 나타낼 수 있을 정도의 충분한 양과 부작용을 일으키지 않는 것을 의미하며, 질환의 종류, 환자의 약물에 대한 민감도, 투여 경로, 투여 방법, 투여 횟수, 치료 기간 등 의학 분야에 잘 알려진 요소에 따라 당업자에 의해 용이하게 결정될 수 있다.The term "pharmaceutically acceptable" means an amount sufficient to exhibit a therapeutic effect and not causing side effects, such as the type of disease, the sensitivity of the patient to the drug, the route of administration, the method of administration, the number of administrations, the duration of treatment, etc. It can be readily determined by a person skilled in the art according to factors well known in the medical arts.
상기 에제티미브는 이의 약학적으로 허용가능한 염의 형태일 수 있다. 상기 염은 약학 분야에서 사용되는 산 부가염, 예를 들면 염산, 브롬산, 황산, 술팜산, 인산 또는 질산과 같은 무기산으로부터 유도된 염; 및 아세트산, 프로피온산, 숙신산, 글리콜산, 스테아르산, 시트르산, 말레산, 말론산, 메탄술폰산, 타르타르산, 말산, 페닐아세트산, 글루탐산, 벤조산, 살리실산, 2-아세톡시벤조산, 푸마르산, 톨루엔술폰산, 옥살산, 트리플루오로아세트산 또는 글루쿠론산과 같은 유기산으로부터 유도된 염을 포함한다. 또한, 상기 염은, 암모늄, 디메틸아민, 모노메틸아민, 모노에틸아민, 디에틸아민과 같은 염기 부가 염일 수 있다. 또한, 상기 염은 통상의 금속 염 형태, 예를 들면 리튬, 소듐, 칼륨, 마그네슘, 또는 칼슘과 같은 금속으로부터 유도된 염을 포함한다. 상기 산 부가염, 염기 부가염 또는 금속염은 통상의 방법에 따라 제조될 수 있다. 약학적으로 허용가능한 염 및 이를 제조하는 일반 방법론은 관련 기술 분야에 널리 공지되어 있다. 예를 들어, 문헌 [P. Stahl, et al. Handbook of Pharmaceutical Salts: Properties, Selection and Use, 2nd Revised Edition (Wiley-VCH, 2011)]; [S.M. Berge, et al., "Pharmaceutical Salts," Journal of Pharmaceutical Sciences, Vol. 66, No. 1, January 1977]을 참조할 수 있다.The ezetimibe may be in the form of a pharmaceutically acceptable salt thereof. These salts include acid addition salts used in the pharmaceutical field, for example salts derived from inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, sulfamic acid, phosphoric acid or nitric acid; and acetic acid, propionic acid, succinic acid, glycolic acid, stearic acid, citric acid, maleic acid, malonic acid, methanesulfonic acid, tartaric acid, malic acid, phenylacetic acid, glutamic acid, benzoic acid, salicylic acid, 2-acetoxybenzoic acid, fumaric acid, toluenesulfonic acid, oxalic acid, salts derived from organic acids such as trifluoroacetic acid or glucuronic acid. In addition, the salt may be a base addition salt such as ammonium, dimethylamine, monomethylamine, monoethylamine, or diethylamine. In addition, the salt includes a common metal salt form, for example, a salt derived from a metal such as lithium, sodium, potassium, magnesium, or calcium. The acid addition salt, base addition salt or metal salt may be prepared according to a conventional method. Pharmaceutically acceptable salts and general methodologies for their preparation are well known in the art. See, for example, P. Stahl, et al. Handbook of Pharmaceutical Salts: Properties, Selection and Use, 2nd Revised Edition (Wiley-VCH, 2011)]; [S.M. Berge, et al., "Pharmaceutical Salts," Journal of Pharmaceutical Sciences, Vol. 66, no. 1, January 1977].
상기 조성물에 상기 에제티미브는 유효성분으로 포함된다. 용어 “유효성분으로 포함”은 상기에서 언급한 효과를 나타낼 수 있을 정도로 에제티미브가 첨가되는 것을 의미하고, 약물전달 및 안정화 등을 위하여 다양한 성분을 부성분으로 첨가하여 다양한 형태로 제형화(formulation)되는 것을 포함하는 의미이다.Ezetimibe is included as an active ingredient in the composition. The term “included as an active ingredient” means that ezetimibe is added to the extent that the above-mentioned effects can be exhibited, and various ingredients are added as subcomponents for drug delivery and stabilization, etc. to be formulated in various forms. It means to include being.
따라서, 상기 에제티미브는 약학적 유효량으로 상기 조성물에 포함될 수 있다. Thus, the ezetimibe may be included in the composition in a pharmaceutically effective amount.
용어 "유효량" 또는 "약학적 유효량"은 환자에게 단일 또는 다회 용량으로 투여되었을 때, 진단 또는 치료 하에 환자에서 원하는 효과를 제공하는, 상기 에제티미브의 양 또는 용량을 지칭한다. 유효량은 공지 기술을 사용함으로써 또는 유사 환경 하에서 수득한 결과를 관찰함으로써 관련 기술분야의 통상의 기술자로서 주치의 진단의에 의해 쉽게 결정될 수 있다. 환자에 대한 유효량을 결정할 때, 포유동물종; 그의 크기, 연령 및 일반적인 건강 상태; 연루된 구체적인 질환 또는 장애; 질환 또는 장애의 연루 정도 또는 중증도; 개별 환자의 반응; 투여되는 특정 화합물; 투여 모드; 투여되는 제제의 생체이용성 특징; 선택된 투약 요법; 동시 약물처치 사용; 및 다른 관련된 환경을 포함하나, 이에 제한되지 않는 다수의 인자가 주치의 진단의에 의해 고려된다.The term "effective amount" or "pharmaceutically effective amount" refers to that amount or dose of ezetimibe that, when administered to a patient in single or multiple doses, provides a desired effect in a patient under diagnosis or treatment. The effective amount can be readily determined by the attending diagnostician as a person skilled in the art by using known techniques or by observing results obtained under similar circumstances. When determining an effective amount for a patient, the mammalian species; his size, age and general health; the specific disease or disorder involved; degree or severity of involvement of the disease or disorder; individual patient response; the specific compound being administered; administration mode; bioavailability characteristics of the administered agent; the selected dosing regimen; use of concomitant medication; A number of factors are taken into account by the attending physician diagnostician, including but not limited to, and other relevant circumstances.
일 구체예에서, 상기 에제티미브는 일일 투여량 0.001 내지 20 mg, 0.001 내지 15 mg, 0.001 내지 10 mg, 0.01 내지 20 mg, 0.01 내지 15 mg, 0.01 내지 10 mg, 0.1 내지 20 mg, 0.1 내지 15 mg, 또는 0.1 내지 10 mg의 용량으로 포함될 수 있으나, 이에 제한되지 않는다. 상기 에제티미브의 용량은 개체에 따라 적절히 조절할 수 있다.In one embodiment, the ezetimibe daily dose is 0.001 to 20 mg, 0.001 to 15 mg, 0.001 to 10 mg, 0.01 to 20 mg, 0.01 to 15 mg, 0.01 to 10 mg, 0.1 to 20 mg, 0.1 to 20 mg It may be included in a dose of 15 mg, or 0.1 to 10 mg, but is not limited thereto. The dose of ezetimibe can be appropriately adjusted according to the individual.
상기 “폐섬유증(pulmonary fibrosis, PF)”은 폐의 간질(Interstitium)에 에 콜라겐을 포함한 세포외 기질(Extracellular matrix)가 다량 축적되는 현상 및 이러한 현상이 나타나는 모든 질환을 총칭한다. 상기 폐섬유증은 그 원인에 제한이 없다. 따라서, 상기 폐섬유증은 원인을 정확히 알 수 없는 특발성 간질성 폐렴(Idiopathic interstitial pneumonia)에 의한 폐섬유증(예: 특발성 폐섬유증, 특발성 비특이 간질성 폐렴에 의한 폐섬유증 등); 류마티스성 관절염, 루푸스, 전신경화증, 근염, 쇼그렌 증후군과 같은 결체조직질환 또는 자가면역질환에 의한 폐섬유증; 감염 질환(예: 코로나바이러스감염증, 폐포자충 폐렴 등), 위식도 역류성 질환(Gastroesophageal reflux disease, GERD) 등의 질병으로 인한 폐섬유증; 가족성 폐섬유증(familial PF); 석면, 실리카, 베릴륨, 먼지, 흡연과 같은 유해물질에 의한 폐섬유증; 방사선 치료나 방사선 노출로 인한 폐섬유증; 항암제(예: Bleomycin), 항생제, 항부정맥제 등 약물에 의한 폐섬유증; 급성 호흡부전 증후군(Acute respiratory distress syndrome, ARDS)의 후유증으로 인한 폐섬유증 등을 모두 포함할 수 있다.The "pulmonary fibrosis (PF)" refers to a phenomenon in which a large amount of extracellular matrix including collagen is accumulated in the interstitium of the lung and all diseases in which such a phenomenon appears. The cause of the pulmonary fibrosis is not limited. Accordingly, the pulmonary fibrosis may include pulmonary fibrosis caused by idiopathic interstitial pneumonia of unknown cause (eg, idiopathic pulmonary fibrosis, pulmonary fibrosis caused by idiopathic nonspecific interstitial pneumonia, etc.); pulmonary fibrosis caused by autoimmune diseases or connective tissue diseases such as rheumatoid arthritis, lupus, systemic sclerosis, myositis, Sjogren's syndrome; Pulmonary fibrosis due to diseases such as infectious diseases (eg, coronavirus, pneumocystis pneumonia, etc.), gastroesophageal reflux disease (GERD); familial pulmonary fibrosis (familial PF); pulmonary fibrosis caused by harmful substances such as asbestos, silica, beryllium, dust, and smoking; pulmonary fibrosis due to radiation therapy or radiation exposure; Pulmonary fibrosis caused by drugs such as anticancer drugs (eg Bleomycin), antibiotics, antiarrhythmics; Pulmonary fibrosis due to sequelae of acute respiratory distress syndrome (ARDS) may all be included.
일 구체예에서, 상기 폐섬유증은 특발성 폐섬유증일 수 있다.In one embodiment, the pulmonary fibrosis may be idiopathic pulmonary fibrosis.
일 구체예에서, 상기 폐섬유증은 폐섬유증은 블레오마이신(Bleomycin)의 투여로부터 발생하는 것 또는 COVID-19 감염 후 후유증으로 인한 폐섬유증일 수 있다. 예를 들어, 블레오마이신은 두경부, 폐, 피부 등의 편평상피암 등에 사용하는 항암제인데, 폐섬유증 등의 부작용을 나타내는 것으로 보고되어 있다. 따라서, 상기 폐섬유증은 블레오마이신과 같은 다른 약물의 사용에 의해 발생한 폐섬유증을 포함할 수 있다.In one embodiment, the pulmonary fibrosis may be pulmonary fibrosis caused by administration of bleomycin or sequelae after COVID-19 infection. For example, bleomycin is an anticancer drug used for squamous cell carcinoma of the head and neck, lung, skin, etc., and has been reported to exhibit side effects such as pulmonary fibrosis. Thus, the pulmonary fibrosis may include pulmonary fibrosis caused by the use of other drugs such as bleomycin.
상기 "코로나바이러스감염증(coronavirus disease)"은 코로나바이러스에 의해 감염되는 감염성 질환이다. 상기 코로나바이러스감염증은 코로나바이러스감염증-19일 수 있으나, 이에 제한되지 않는다. 상기 "코로나바이러스감염증-19(COVID-19)"는 중증 급성 호흡기 증후군 코로나바이러스 2(severe acute respiratory syndrome coronavirus 2: SARS-CoV-2)에 의해 감염되는 감염성 질환이다. 따라서, 상기 폐섬유증은 COVID-19에 의한 폐섬유증일 수 있다.The "coronavirus disease" is an infectious disease caused by a coronavirus. The coronavirus infection may be COVID-19, but is not limited thereto. The "coronavirus infection-19 (COVID-19)" is an infectious disease caused by severe acute respiratory syndrome coronavirus 2: SARS-CoV-2. Therefore, the pulmonary fibrosis may be pulmonary fibrosis caused by COVID-19.
일 구체예에서, 상기 폐섬유증은 특발성 폐섬유증일 수 있다.In one embodiment, the pulmonary fibrosis may be idiopathic pulmonary fibrosis.
일 구체예에서, 상기 폐섬유증은 폐섬유증은 블레오마이신(Bleomycin)의 투여로부터 발생하는 것 또는 COVID-19 감염 후 후유증으로 인한 폐섬유증일 수 있다. 예를 들어, 블레오마이신은 두경부, 폐, 피부 등의 편평상피암 등에 사용하는 항암제인데, 폐섬유증 등의 부작용을 나타내는 것으로 보고되어 있다. 따라서, 상기 폐섬유증은 블레오마이신과 같은 다른 약물의 사용에 의해 발생한 폐섬유증을 포함할 수 있다.In one embodiment, the pulmonary fibrosis may be pulmonary fibrosis caused by administration of bleomycin or sequelae after COVID-19 infection. For example, bleomycin is an anticancer drug used for squamous cell carcinoma of the head and neck, lung, skin, etc., and has been reported to exhibit side effects such as pulmonary fibrosis. Thus, the pulmonary fibrosis may include pulmonary fibrosis caused by the use of other drugs such as bleomycin.
용어 "예방"은 상기 조성물의 투여로 폐섬유증의 발병을 억제 또는 지연시키는 모든 행위를 의미한다. The term "prevention" refers to any action that inhibits or delays the onset of pulmonary fibrosis by administration of the composition.
용어 "개선"은 상기 조성물의 투여로 폐섬유증의 증세가 개선되는 모든 과정을 의미한다.The term "improvement" refers to any process in which symptoms of pulmonary fibrosis are improved by administration of the composition.
용어 "완화"는 상기 조성물의 투여로 폐섬유증의 증상이 완화되거나, 폐섬유증의 진행이 지연되거나, 또는 폐섬유증 자체의 원인(들)의 완화 또는 소멸을 포함하는 것을 의미한다. 또한 본 명세서 내에서 완화는 폐섬유증의 진행을 완화하는 것을 포함할 수 있다. The term "alleviation" means that the symptoms of pulmonary fibrosis are alleviated, the progression of pulmonary fibrosis is delayed, or the cause(s) of pulmonary fibrosis itself is alleviated or eliminated by administration of the composition. Alleviation within this specification may also include mitigating the progression of pulmonary fibrosis.
용어 "치료"는 상기 조성물의 투여로 폐섬유증의 증세가 호전되거나 이롭게 되는 모든 행위를 의미한다.The term "treatment" refers to any action that improves or benefits the symptoms of pulmonary fibrosis by administration of the composition.
상기 약학적 조성물은 약학적으로 허용가능한 담체를 더 포함할 수 있다. 약학적으로 허용가능한 담체는 경구 투여 시에는 결합제, 활택제, 붕해제, 부형제, 가용화제, 분산제, 안정화제, 현탁화제, 색소 및 향료 등을 사용할 수 있으며, 주사제의 경우에는 완충제, 보존제, 무통화제, 가용화제, 등장화제 및 안정화제 등을 혼합하여 사용할 수 있으며, 국소 투여용의 경우에는 기제, 부형제, 윤활제 및 보존제 등을 사용할 수 있다.The pharmaceutical composition may further include a pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers may include binders, lubricants, disintegrants, excipients, solubilizers, dispersants, stabilizers, suspending agents, pigments and flavors for oral administration, and buffers, preservatives, and painless agents for injections. A topical agent, a solubilizing agent, an isotonic agent, and a stabilizer may be mixed and used, and in the case of topical administration, a base, an excipient, a lubricant, and a preservative may be used.
상기 약학적 조성물의 제형은 상술한 바와 같은 약학적으로 허용가능한 담체와 혼합하여 다양하게 제조될 수 있다. 예를 들어, 경구 투여 시에는 정제, 트로키, 캡슐, 엘릭서, 서스펜션, 시럽 및 웨이퍼 등의 형태로 제조할 수 있으며, 주사제의 경우에는 단위 투약 앰플 또는 다수회 투약 형태로 제조할 수 있다. 그 외에도 용액, 현탁액, 정제, 환약, 캡슐 및 서방형 제제 등으로 제형화할 수 있다.Formulations of the pharmaceutical composition may be variously prepared by mixing with a pharmaceutically acceptable carrier as described above. For example, for oral administration, it can be prepared in the form of tablets, troches, capsules, elixirs, suspensions, syrups, wafers, etc., and in the case of injections, it can be prepared in the form of unit dose ampoules or multiple doses. In addition, it can be formulated into solutions, suspensions, tablets, pills, capsules, and sustained-release preparations.
한편, 제제화에 적합한 담체, 부형제 및 희석제의 예로는 락토즈, 덱스트로즈, 수크로즈, 솔비톨, 만니톨, 자일리톨, 에리스리톨, 말티톨, 전분, 아카시아, 알지네이트, 젤라틴, 칼슘 포스페이트, 칼슘 실리케이트, 셀룰로즈, 메틸 셀룰로즈, 미정질 셀룰로즈, 폴리비닐피롤리돈, 물, 메틸히드록시벤조에이트, 프로필히드록시벤조에이트, 탈크, 마그네슘 스테아레이트 또는 광물유 등이 사용될 수 있다. 또한, 충진제, 항응집제, 윤활제, 습윤제, 향료, 유화제 및 방부제 등을 추가로 포함할 수 있다.On the other hand, examples of carriers, excipients and diluents suitable for formulation include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl Cellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate or mineral oil may be used. In addition, fillers, anti-coagulants, lubricants, wetting agents, flavoring agents, emulsifiers and preservatives may be further included.
상기 약학적 조성물은 폐섬유증을 치료하기 위한 하나 이상의 다른 제제를 더 포함할 수 있다. 상기 다른 제제는 폐섬유증 치료제일 수 있으나, 이에 제한되지 않는다. 상기 폐섬유증 치료제는 공지의 물질을 사용할 수 있다. 예를 들어, 상기 폐섬유증 치료제는 피르페니돈(pirfenidone), 닌테다닙 (nintedanib) 등일 수 있다.The pharmaceutical composition may further include one or more other agents for treating pulmonary fibrosis. The other agent may be a therapeutic agent for pulmonary fibrosis, but is not limited thereto. As the therapeutic agent for pulmonary fibrosis, known substances may be used. For example, the therapeutic agent for pulmonary fibrosis may be pirfenidone, nintedanib, and the like.
다른 양상은 에제티미브(Ezetimibe) 또는 이의 식품학적으로 허용가능한 염을 유효성분으로 포함하는 폐섬유증의 예방, 개선 또는 완화용 건강기능식품 조성물을 제공한다.Another aspect provides a health functional food composition for preventing, improving, or alleviating pulmonary fibrosis, comprising Ezetimibe or a food chemically acceptable salt thereof as an active ingredient.
상기 건강기능식품 조성물은 당해 기술분야에 공지되어 있는 통상적인 건강기능식품의 제형으로 제제화될 수 있다.The health functional food composition may be formulated into a conventional health functional food formulation known in the art.
상기 건강기능식품 조성물은 에제티미브 단독을 사용하거나, 또는 다른 식품 또는 식품 성분과 함께 사용될 수 있고, 통상적인 방법에 따라 적절하게 사용될 수 있다. 유효성분의 혼합 양은 사용 목적(예방, 건강 또는 치료적 처치)에 따라 적합하게 결정될 수 있다. 상기 건강기능식품의 종류에는 특별한 제한은 없다. 건강기능식품의 종류 중 음료 조성물은 통상의 음료와 같이 여러 가지 향미제 또는 천연 탄수화물 등을 추가 성분으로서 함유할 수 있다. 상기 천연 탄수화물은 포도당, 과당과 같은 모노사카라이드; 말토스, 슈크로스와 같은 디사카라이드; 및 덱스트린, 사이클로덱스트린과 같은 폴리사카라이드; 자일리톨, 소르비톨, 에리트리톨 등의 당알콜이다. 감미제로서는 타우마틴, 스테비아 추출물과 같은 천연 감미제나, 사카린, 아스파르탐과 같은 합성 감미제 등을 사용할 수 있다. 상기 식품 조성물은 또한 영양제, 비타민, 전해질, 풍미제, 착색제, 펙트산 및 그의 염, 알긴산 및 그의 염, 유기산, 보호성 콜로이드 증점제, pH 조절제, 안정화제, 방부제, 글리세린, 알콜, 탄산 음료에 사용되는 탄산화제, 또는 그 조합을 함유할 수 있다. 상기 식품 조성물은 또한, 천연 과일쥬스, 과일쥬스 음료, 야채 음료의 제조를 위한 과육, 또는 그 조합을 함유할 수 있다.The health functional food composition may use ezetimibe alone or together with other foods or food ingredients, and may be appropriately used according to conventional methods. The mixing amount of the active ingredient may be appropriately determined depending on the purpose of use (prevention, health or therapeutic treatment). There is no particular limitation on the type of health functional food. Among the types of health functional foods, beverage compositions may contain various flavoring agents or natural carbohydrates as additional components, like conventional beverages. The natural carbohydrates include monosaccharides such as glucose and fructose; disaccharides such as maltose and sucrose; and polysaccharides such as dextrins and cyclodextrins; It is a sugar alcohol, such as xylitol, sorbitol, and erythritol. As the sweetener, natural sweeteners such as thaumatin and stevia extract, or synthetic sweeteners such as saccharin and aspartame may be used. The food composition is also used in nutrients, vitamins, electrolytes, flavors, colorants, pectic acid and its salts, alginic acid and its salts, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohols, carbonated beverages carbonation agent, or a combination thereof. The food composition may also contain natural fruit juice, fruit juice beverages, fruit flesh for preparing vegetable beverages, or a combination thereof.
또 다른 양상은 유효량의 에제티미브(Ezetimibe) 또는 이의 약학적으로 허용가능한 염을 이를 필요로 하는 개체에게 투여하는 단계를 포함하는 폐섬유증의 예방, 개선, 완화 또는 치료 방법을 제공한다.Another aspect provides a method for preventing, ameliorating, alleviating or treating pulmonary fibrosis, comprising administering an effective amount of ezetimibe or a pharmaceutically acceptable salt thereof to a subject in need thereof.
용어 "개체"란 질환의 치료를 필요로 하는 대상을 의미하고, 보다 구체적으로 인간 또는 비-인간인 영장류, 마우스, 래트, 개, 고양이, 말 및 소 등의 포유류를 의미한다. 상기 "이를 필요로 하는 개체"란 폐섬유증의 예방, 개선 또는 치료를 필요로 하는 개체를 의미한다.The term "individual" means a subject in need of treatment for a disease, and more specifically, mammals such as humans or non-human primates, mice, rats, dogs, cats, horses, and cattle. The "subject in need thereof" refers to an individual in need of prevention, improvement or treatment of pulmonary fibrosis.
용어 "투여"는 어떠한 적절한 방법으로 환자에게 소정의 물질을 도입하는 것을 의미한다. 투여 경로는 환자의 생체 내 표적에 도달할 수 있는 어떠한 일반적인 경로일 수 있다. 상기 투여는, 예를 들어, 복강 내 투여, 정맥 내 투여, 근육 내 투여, 피하 투여, 피내 투여, 경구 투여, 국소 투여, 비내 투여, 직장 내 투여일 수 있으나, 이에 제한되지 않는다. 일 구체예에서, 상기 투여는 경구 투여일 수 있다.The term "administration" means the introduction of a substance into a patient by any suitable method. The route of administration may be any general route capable of reaching a target in vivo in a patient. The administration may be, for example, intraperitoneal administration, intravenous administration, intramuscular administration, subcutaneous administration, intradermal administration, oral administration, topical administration, intranasal administration, or intrarectal administration, but is not limited thereto. In one embodiment, the administration may be oral administration.
투여량은 제제화 방법, 투여 방식, 환자의 연령, 체중, 성별, 병적 상태, 음식, 투여 시간, 투여 경로, 배설 속도 및 반응 감응성과 같은 요인들에 의해 다양하게 처방될 수 있고, 당업자라면 이러한 요인들을 고려하여 투여량을 적절히 조절할 수 있다. 예를 들어, 상기 투여량은 개체당 일당 에제티미브 0.001 mg 내지 1,000 mg, 0.001 mg 내지 500 mg, 0.001 mg 내지 100 mg, 0.001 mg 내지 50 mg, 0.001 mg 내지 20 mg, 0.001 mg 내지 10 mg, 0.1 mg 내지 100 mg, 0.1 mg 내지 50 mg, 0.1 mg 내지 20 mg, 0.1 mg 내지 10 mg일 수 있으나, 이에 제한되지 않는다. 투여 횟수는 1일 1회 또는 임상적으로 용인가능한 부작용의 범위 내에서 2회 이상이 가능하고, 투여 부위에 대해서도 1개소 또는 2개소 이상에 투여할 수 있으며, 매일 또는 2 내지 5일 간격으로 총 투여 일수는 한번 치료 시 1일에서 30일까지 투여될 수 있다. 필요한 경우, 적정 시기 이후에 동일한 치료를 반복할 수 있다. 인간 이외의 동물에 대해서도, kg당 인간과 동일한 투여량으로 하거나, 또는 예를 들면 목적의 동물과 인간과의 기관(심장 등)의 용적비(예를 들면, 평균값) 등으로 상기의 투여량을 환산한 양을 투여할 수 있다.The dosage may be prescribed in various ways depending on factors such as formulation method, administration method, patient's age, weight, sex, medical condition, food, administration time, administration route, excretion rate and reaction sensitivity, and those skilled in the art can determine these factors Dosage can be appropriately adjusted in consideration of these factors. For example, the dosage ranges from 0.001 mg to 1,000 mg, 0.001 mg to 500 mg, 0.001 mg to 100 mg, 0.001 mg to 50 mg, 0.001 mg to 20 mg, 0.001 mg to 10 mg, ezetimibe per day per subject. It may be 0.1 mg to 100 mg, 0.1 mg to 50 mg, 0.1 mg to 20 mg, or 0.1 mg to 10 mg, but is not limited thereto. The number of administrations can be once a day or two or more times within the range of clinically acceptable side effects, and administration can be performed at one or two or more sites, daily or at intervals of 2 to 5 days. The number of administration days may be administered from 1 day to 30 days per treatment. If necessary, the same treatment can be repeated after a titration period. For non-human animals, the same dosage per kg as for humans is used, or the above dosage is converted by the volume ratio (eg, average value) of the organ (heart, etc.) between the target animal and the human. A single dose can be administered.
상기 방법에서, 유효량의 에제티미브 또는 이의 약학적으로 허용가능한 염은 유효량의 하나 이상의 다른 활성 성분과 동시에, 개별적으로, 또는 순차적으로 투여할 수 있다. 상기 하나 이상의 다른 활성 성분은 폐섬유증을 치료하기 위한 하나 이상의 다른 제제일 수 있으나, 이에 제한되지 않는다.In the method, an effective amount of ezetimibe or a pharmaceutically acceptable salt thereof may be administered simultaneously, separately, or sequentially with an effective amount of one or more other active ingredients. The one or more other active ingredients may be one or more other agents for treating pulmonary fibrosis, but are not limited thereto.
또 다른 양상은 폐섬유증의 예방, 개선, 완화 또는 치료용 약제의 제조에 사용하기 위한 에제티미브 또는 이의 약학적으로 허용가능한 염의 용도를 제공한다.Another aspect provides the use of ezetimibe or a pharmaceutically acceptable salt thereof for use in the manufacture of a medicament for preventing, ameliorating, alleviating or treating pulmonary fibrosis.
또 다른 양상은 폐섬유증의 예방, 개선, 완화 또는 치료를 위한 에제티미브 또는 이의 약학적으로 허용가능한 염의 용도를 제공한다.Another aspect provides the use of ezetimibe or a pharmaceutically acceptable salt thereof for preventing, ameliorating, alleviating or treating pulmonary fibrosis.
중복되는 내용은 본 명세서의 복잡성을 고려하여 생락하며, 본 명세서에서 달리 정의되지 않은 용어들은 본 발명이 속하는 기술분야에서 통상적으로 사용되는 의미를 갖는 것이다.Redundant content is omitted in consideration of the complexity of the present specification, and terms not defined otherwise in the present specification have meanings commonly used in the technical field to which the present invention belongs.
일 양상에 따른 조성물은 에제티미브 또는 이의 약학적으로 허용가능한 염을 포함함으로써 폐섬유증의 예방, 개선, 완화 또는 치료 효과를 가질 수 있다. 따라서, 에제티미브를 폐섬유증의 예방, 개선, 완화 또는 치료를 위한 약학적 조성물 등으로 이용할 수 있다. The composition according to one aspect may have an effect of preventing, improving, alleviating or treating pulmonary fibrosis by including ezetimibe or a pharmaceutically acceptable salt thereof. Therefore, ezetimibe can be used as a pharmaceutical composition for preventing, improving, alleviating or treating pulmonary fibrosis.
도 1은 FBS 15% 세포 배양액 조건에서 마우스 폐 섬유모세포에 에제티미브를 24시간 동안 표시된 농도 대로 처리하고, MTT assay를 진행한 결과이다.1 is a result of treating mouse lung fibroblasts with ezetimibe at the indicated concentration for 24 hours in a cell culture medium of 15% FBS, and performing an MTT assay.
도 2는 FBS 0.1% 세포 배양액 조건에서 마우스 폐 섬유모세포에 에제티미브를 24시간 동안 표시된 농도 대로 처리하고, MTT assay를 진행한 결과이다.Figure 2 shows the results of MTT assay after treatment with ezetimibe at the indicated concentration for 24 hours in mouse lung fibroblasts in FBS 0.1% cell culture medium conditions.
도 3은 FBS 15% 세포 배양액 조건에서 사람 폐 섬유모세포에 에제티미브를 24시간 동안 표시된 농도 대로 처리하고, MTT assay를 진행한 결과이다.Figure 3 shows the results of treating human lung fibroblasts with ezetimibe at the indicated concentrations for 24 hours in a cell culture medium of 15% FBS, and performing the MTT assay.
도 4는 TGFβ1 미처리 상태와 TGFβ1 처리 상태를 비교하고, 동시에 TGFβ1 처리 상태에서 에제티미브의 처리 농도별 Col1A1 단백질 발현 정도를 나타낸 결과이다. Figure 4 is a result of comparing the TGFβ1 untreated state and the TGFβ1 treated state, and showing the Col1A1 protein expression level according to the treatment concentration of ezetimibe in the TGFβ1 treated state at the same time.
도 5는 상기 에제티미브의 처리 농도별 Col1A1과 Acta2의 mRNA 발현 정도를 나타낸 결과이다.5 is a result showing the mRNA expression level of Col1A1 and Acta2 for each treatment concentration of ezetimibe.
도 6은 에제티미브의 농도를 20 μM으로 고정하였을 때, 에제티미브 처리 시간에 따른 Col1A1 단백질 발현 정도를 나타낸 결과이다.6 is a result showing the level of Col1A1 protein expression according to ezetimibe treatment time when the concentration of ezetimibe was fixed at 20 μM.
도 7은 상기 에제티미브의 농도를 20 μM으로 고정하였을 때, 에제티미브 처리 시간에 따른 Col1A1과 Acta2의 mRNA 발현 정도를 나타낸 결과이다.7 is a result showing the mRNA expression levels of Col1A1 and Acta2 according to ezetimibe treatment time when the concentration of ezetimibe was fixed at 20 μM.
도 8은 TGFβ1 미처리 상태와 TGFβ1 처리 상태를 비교하고, 동시에 TGFβ1 처리 상태에서 에제티미브의 농도별 COL1A1 단백질 발현 정도를 나타낸 결과이다. 8 is a result of comparing the TGFβ1 untreated state and the TGFβ1 treated state, and showing the COL1A1 protein expression level according to the concentration of ezetimibe in the TGFβ1 treated state at the same time.
도 9는 에제티미브의 처리 농도별 Col1A1과 Acta2의 mRNA 발현 정도를 나타낸 결과이다.9 is a result showing the mRNA expression level of Col1A1 and Acta2 for each treatment concentration of ezetimibe.
도 10은 에제티미브의 농도를 20 μM으로 고정하였을 때, 에제티미브 처리 시간에 따른 Col1A1 단백질 발현 정도를 나타낸 결과이다.10 is a result showing the level of Col1A1 protein expression according to ezetimibe treatment time when the concentration of ezetimibe was fixed at 20 μM.
도 11은 에제티미브의 농도를 20 μM으로 고정하였을 때, 에제티미브 처리 시간에 따른 Col1A1과 Acta2의 mRNA 발현 정도를 나타낸 결과이다.11 shows the mRNA expression levels of Col1A1 and Acta2 according to ezetimibe treatment time when the concentration of ezetimibe was fixed at 20 μM.
도 12는 블레오마이신 유발 마우스 폐섬유증 모델에서 에제티미브의 폐섬유증 완화 효과를 확인하기 위한 실험에 있어서 각 군의 투여 방법을 간단한 모식도로 확인한 도이다.FIG. 12 is a schematic view showing the administration method of each group in an experiment for confirming the pulmonary fibrosis alleviating effect of ezetimibe in a bleomycin-induced mouse pulmonary fibrosis model.
도 13은 블레오마이신 투여 후 일수(Days)에 따른 마우스의 생존율 변화를 나타낸 그래프이다.13 is a graph showing the change in survival rate of mice according to the number of days after administration of bleomycin.
도 14은 블레오마이신 미투여군(Control, n=14), 블레오마이신 투여군(Bleomycin, n=13), 블레오마이신 + 에제티미브 투여군(Bleo+Ezetimibe, n=17) 및 블레오마이신 + 닌테다닙 투여군(Bleo+Nintedanib, n=7)에서 콜라겐 양을 측정한 결과이다. 도 14의 그래프 상의 각각의 점은 각각 한 마리의 마우스의 실험 결과를 표시하며, 평균 ± 표준오차로 표기하였고, one-way ANOVA로 분석하였다.14 is a bleomycin non-administration group (Control, n = 14), a bleomycin-administered group (Bleomycin, n = 13), a bleomycin + ezetimibe-administered group (Bleo + Ezetimibe, n = 17), and a bleomycin + nintedanib-administered group ( This is the result of measuring the amount of collagen in Bleo+Nintedanib, n=7). Each point on the graph of FIG. 14 represents the experimental results of one mouse, expressed as mean ± standard error, and analyzed by one-way ANOVA.
도 15는 웨스턴 블랏을 이용하여 각 군의 섬유화 마커인 COL1A1과 피브로넥틴(Fibronectin)을 비교한 결과 및 Image J를 통해 웨스턴 블랏의 결과를 정량한 결과를 확인한 도이다.15 is a diagram confirming the results of comparing COL1A1 and fibronectin, which are fibrosis markers of each group, using Western blot and quantifying the result of Western blot through Image J.
도 16은 RT-qPCR을 통해 섬유화 마커인 Col1a1, Col1a2, Acta2, Col3a1 및 Eda-fn mRNA의 발현을 확인한 결과를 나타낸 도이다. 16 is a diagram showing the results of confirming the expression of fibrosis markers Col1a1, Col1a2, Acta2, Col3a1 and Eda-fn mRNA through RT-qPCR.
도 17은 Masson-trichrome stain으로 섬유화 정도를 비교한 결과를 나타낸 것이다.17 shows the results of comparing the degree of fibrosis with Masson-trichrome stain.
도 18은 마우스별 폐 병리조직의 슬라이드에서 콜라겐이 염색된 부위를 Aperio Imagescope 프로그램을 이용하여 정량한 결과를 나타낸 도이다.18 is a diagram showing the results of quantification of collagen-stained areas in slides of lung pathological tissues for each mouse using the Aperio Imagescope program.
도 19는 블레오마이신 투여 후 일수(Days)에 따른 마우스 체중 변화를 나타낸 그래프이다. 평균 ± 표준오차로 표기하였고, Two-way ANOVA (측정 날짜와 투약군)로 분석하였다. 각 군당 대조군으로 n=23, 블레오마이신 투여군(Bleomycin)으로 n=18 / 블레오마이신 + 에제티미브 투여군(Bleo+Eze)으로 n=21 마리가 분석에 활용되었다.19 is a graph showing the change in body weight of mice according to the number of days after administration of bleomycin. It was expressed as mean ± standard error and analyzed by two-way ANOVA (measurement date and administration group). For each group, n = 23 as a control group, n = 18 as a bleomycin group (Bleomycin) / n = 21 animals as a bleomycin + ezetimibe group (Bleo + Eze) were used for analysis.
이하 본 발명을 실시예를 통하여 보다 상세하게 설명한다. 그러나, 이들 실시예는 본 발명을 예시적으로 설명하기 위한 것으로 본 발명의 범위가 이들 실시예에 한정되는 것은 아니다.Hereinafter, the present invention will be described in more detail through examples. However, these examples are intended to illustrate the present invention by way of example, and the scope of the present invention is not limited to these examples.
재료 및 방법Materials and Methods
1. 인간 및 마우스 폐 섬유모세포의 분리 및 배양1. Isolation and culture of human and mouse lung fibroblasts
본 실험에는 타 논문(Seluanov et al., “Establishing Primary Adult Fibroblast Cultures From Rodents”. J Vis Exp. 2010 Oct 5;(44):2033. doi: 10.3791/2033)을 참고하고, 부분적으로 수정하여 적용하였다. 모든 약품 및 비품은 상기 논문과 동일하게 사용하였다. In this experiment, another paper (Seluanov et al., “Establishing Primary Adult Fibroblast Cultures From Rodents”. J Vis Exp. 2010 Oct 5;(44):2033. doi: 10.3791/2033) was referred to and partially modified and applied. did All chemicals and supplies were used in the same way as in the above paper.
마우스 폐 조직의 경우, 7~10주령 사이의 성체 마우스를 안락사하여 무균적으로 양쪽 폐를 채취하였다. In the case of mouse lung tissue, adult mice between 7 and 10 weeks of age were euthanized, and both lungs were aseptically collected.
인간 폐 조직의 경우, IRB(Institutional Review Board)를 통과한 폐 조직 채취 연구에 동의한 환자에 한하여, 일반적인 수술 과정에서 얻어지는 폐 조직 약 2 cm3 크기를 무균적으로 떼어와서 사용하였다.In the case of human lung tissue, lung tissue of about 2 cm 3 obtained in a general surgical procedure was aseptically removed and used only for patients who agreed to the lung tissue collection study that passed the Institutional Review Board (IRB).
21번 blade를 이용하여 폐 조직을 1 mm3 미만의 크기로 잘게 자른 뒤, DMEM/F12 배지에 Liberase™ TM Research Grade를 0.14 Wunsch units/mL로 녹여서, 37℃에서 천천히 교반기로 저어주며 40분간 인큐베이션을 진행하였다. 이후 혈청이 포함된 세포 배양액으로 조직을 씻어주고, 100 mm 세포 배양 디쉬 5개에 나누어서 배양하였다. O2 3%의 저산소 조건에서 배양을 진행하였다.After cutting the lung tissue into small pieces of less than 1 mm 3 using a No. 21 blade, dissolve Liberase™ TM Research Grade in DMEM/F12 medium at 0.14 Wunsch units/mL, and incubate for 40 minutes at 37℃ while slowly stirring with a stirrer. proceeded. Thereafter, the tissue was washed with a serum-containing cell culture medium, divided into five 100 mm cell culture dishes, and cultured. The culture was performed under hypoxic conditions of 3% O 2 .
분리 1주 뒤 배양액을 새로 갈아주며, 분리 2주 뒤 계대배양을 시행하고, FBS 15% 및 1X P/S (penicillin/streptomycin)를 추가한 MEM 세포배양액으로 변경하였다. 이때 100 μm 세포 필터를 이용해서 조직을 제거하여, 분리된 폐 섬유모세포만을 배양하였다. 이후 약 5일 간격으로 계대배양하여 세포의 순도를 높이고 세포 수를 늘렸다. 3번째 계대(passage)에서 정상 산소 조건으로 변경하면서 모든 실험을 시행하였다.After 1 week of separation, the culture medium was replaced, and 2 weeks after separation, subculture was performed, and the cell culture medium was changed to MEM with 15% FBS and 1X P/S (penicillin/streptomycin) added. At this time, the tissue was removed using a 100 μm cell filter, and only the isolated lung fibroblasts were cultured. Thereafter, the cells were subcultured at intervals of about 5 days to increase the purity of the cells and increase the number of cells. All experiments were performed while changing to normoxic conditions in the third passage.
2. 세포 실험 조건2. Cell Experiment Conditions
모든 실험은 계대(passage) 3의 세포를 사용하였다. TGFβ1 처리 전에 세포 배양액을 FBS의 농도가 0.1%로 감소된 MEM 세포 배양액으로 변경하여 1시간 30분간 처리하였다. 이후에 비히클(vehicle) 또는 TGFβ1 2 ng/ml과 에제티미브를 24시간 동안 처리하였다. TGFβ1는 100 mM NaCl이 포함된 20 mM 시트레이트(citrate) 용액(pH 3.0)에 0.1%(w/v) BSA(Bovine Serum Albumin)이 포함된 용액을 비히클로 사용하였고, 에제티미브는 DMSO(Dimethyl Sulfoxide)를 비히클로 사용하였다. 모든 약물 및 그 용매는 세포에 1:1000 (v/v)으로 처리되었다.Cells at passage 3 were used in all experiments. Prior to TGFβ1 treatment, the cell culture medium was changed to MEM cell culture medium in which the concentration of FBS was reduced to 0.1%, and treated for 1 hour and 30 minutes. Thereafter, vehicle or TGFβ1 2 ng/ml and ezetimibe were treated for 24 hours. For TGFβ1, a solution containing 0.1% (w/v) BSA (Bovine Serum Albumin) in a 20 mM citrate solution (pH 3.0) containing 100 mM NaCl was used as a vehicle, and ezetimibe was DMSO ( Dimethyl Sulfoxide) was used as a vehicle. All drugs and their solvents were applied to the cells at 1:1000 (v/v).
3. 블레오마이신(Bleomycin)을 이용한 마우스 폐 섬유증 유발 및 약물 투여3. Induction of pulmonary fibrosis in mice using bleomycin and drug administration
블레오마이신은 두경부, 폐, 피부 등의 편평상피암 등에 사용하는 항암제로, 폐섬유증 등의 부작용을 나타내는 것으로 보고되어 있다. 이러한 특성을 바탕으로 블레오마이신은 폐 섬유증의 동물모델에 사용하는 가장 대표적인 약물이다. 따라서, 블레오마이신을 사용하여 폐섬유증을 유발하였다. 7~10주령 사이의 C57BL/6J 마우스를 이용하여 아래의 순서로 실험을 진행하였다.Bleomycin is an anticancer drug used for squamous cell carcinoma of the head and neck, lung, skin, etc., and has been reported to exhibit side effects such as pulmonary fibrosis. Based on these characteristics, bleomycin is the most representative drug used in animal models of pulmonary fibrosis. Therefore, pulmonary fibrosis was induced using bleomycin. Experiments were conducted in the following order using C57BL/6J mice between 7 and 10 weeks of age.
1) 인두 흡인 과정이 원활할 수 있도록 마우스에 15분의 공복시간 주고, 호흡마취를 통해 마취를 진행하였다.1) To facilitate the pharyngeal aspiration process, the mice were given a 15-minute fasting period, and anesthesia was performed through respiratory anesthesia.
2) 마우스는 바닥과 수직이 되게 세워주고, 코와 혀를 잡아주어 기도를 통해 호흡할 수 있게 위치하였다.2) The mouse was placed perpendicular to the floor and positioned so that it could breathe through the airway by holding the nose and tongue.
3) 대조군(control, normal saline)은 무균적으로 처리된 생리식염수 50 ul를 마우스에 인두 흡인 시켜주었고, 블레오마이신 투여군과 블레오마이신 및 에제티미브 투여군은 블레오마이신 2U/kg 50 ul를 마우스에 인두 흡인 시켜주었다.3) For the control group (control, normal saline), 50 ul of aseptically treated physiological saline was aspirated through the pharynx to the mouse, and for the bleomycin-administered group and bleomycin and ezetimibe-administered group, 50 ul of bleomycin 2U/kg was orally administered to the mouse. was aspirated
4) 주입 직후 마우스 무게를 측정하고, 일주일에 3번 마우스 희생 전까지 무게를 측정하며 관찰하였다.4) The weight of the mouse was measured immediately after injection, and the weight was measured and observed three times a week until the mouse was sacrificed.
5) 블레오마이신 주입 7일 후부터, 블레오마이신과 에제티미브를 투여한 군은 증류수로 제작한 0.5% 소듐 카르복시메틸 셀룰로오스(Sodium Carboxymethyl Cellulose)에 희석한 에제티미브 2 mg/kg를, 블레오마이신과 닌테다닙(Nintedanib)을 투여한 군은 0.5% 소듐 카르복시메틸 셀룰로오스에 희석한 닌테다닙 40mg/kg를, 대조군과 블레오마이신 군은 0.5% 소듐 카르복시메틸 셀룰로오스를 일주일에 5번, 매일 경구 투여하였다.5) From 7 days after bleomycin injection, the group administered with bleomycin and ezetimibe received 2 mg/kg of ezetimibe diluted in 0.5% sodium carboxymethyl cellulose prepared in distilled water, and bleomycin and ezetimibe. The group administered with Nintedanib received 40 mg/kg of Nintedanib diluted in 0.5% sodium carboxymethyl cellulose, and the control and bleomycin groups received oral administration of 0.5% sodium carboxymethyl cellulose five times a week, daily.
6) 블레오마이신 주입 3주 뒤 안락사하여 폐 검체를 획득하였다.6) Euthanasia was performed 3 weeks after bleomycin injection, and lung samples were obtained.
7) 실험 과정에서 사망한 마우스로부터는 검체를 획득하지 않았고, 생존 분석을 제외한 모든 분석에서 제외하였다. 7) Specimens were not obtained from mice that died during the experiment, and were excluded from all analyzes except survival analysis.
4. 단백질 정량 및 웨스턴 블랏4. Protein quantification and western blot
1) 세포에 류펩틴(Leupeptin), 아프로티닌(Aprotinin) 및 PMSF(phenylmethylsulfonyl fluoride)가 함유된 용해 혼합액을 넣은 뒤, 4℃에서 5분 동안 유지시켰다.1) A lysis mixture containing Leupeptin, Aprotinin, and PMSF (phenylmethylsulfonyl fluoride) was added to the cells, and maintained at 4°C for 5 minutes.
2) 스크래퍼로 세포를 모아준 뒤, 강한 진동으로 세포가 더욱 잘 용해될 수 있도록 해주고, 4℃에서 10분 이상 유지시켰다.2) After collecting the cells with a scraper, strong vibration was used to better dissolve the cells, and the cells were maintained at 4°C for more than 10 minutes.
3) 4℃에서 13500 rpm의 속도로 15분 동안 원심분리하여, 상층액만 획득하였다.3) Centrifuged at 4° C. at 13500 rpm for 15 minutes to obtain only the supernatant.
4) 브래드포드 시약 500 ul에 상기 획득한 용해물 1 ul을 넣고, 잘 섞어주어 200 ul씩 96웰 플레이트에 분주하였다.4) 1 ul of the above-obtained lysate was added to 500 ul of Bradford reagent, mixed well, and dispensed in 200 ul each into a 96-well plate.
5) VERSAmax™ microplate reader 기기를 이용하여 595 nm로 농도를 측정하였다.5) The concentration was measured at 595 nm using a VERSAmax™ microplate reader.
6) 각 샘플은 5X SDS 시약과 함께 1 mg/ml의 농도로 일정하게 맞추어주고, 100℃에서 10분 동안 가열하였다.6) Each sample was uniformly adjusted to a concentration of 1 mg/ml with 5X SDS reagent and heated at 100°C for 10 minutes.
7) 확인하고자 하는 단백질의 크기에 따라 각기 다른 폴리아크릴아마이드 농도를 가진 젤을 이용하여 전기영동 상에서 분리 시켜주며, 약 80V에서 30분 전개 후, 120V에서 1시간 전개하였다.7) Depending on the size of the protein to be identified, gels with different polyacrylamide concentrations were used to separate them on electrophoresis, and development was performed at about 80V for 30 minutes and then at 120V for 1 hour.
8) 분리가 끝난 젤을 나일론 막에 옮겨 75V에서 2시간 전사하였다.8) The separated gel was transferred to a nylon membrane and transferred at 75V for 2 hours.
9) 5% 탈지우유 혹은 소혈청알부민 용액으로 15분 이상 블로킹하여 불특정 항원항체 반응을 막아주었다.9) Unspecific antigen-antibody reaction was prevented by blocking with 5% skim milk or bovine serum albumin solution for more than 15 minutes.
10) 1차 항체는 4℃에서 18~24시간 반응 시키고, TTBS (Tween-Tris buffered saline)로 세척 후, 2차 항체를 상온에서 1시간 이상 반응시켰다.10) The primary antibody was reacted at 4℃ for 18 to 24 hours, washed with TTBS (Tween-Tris buffered saline), and the secondary antibody was reacted at room temperature for more than 1 hour.
11) TTBS로 세척 후, 화학발광기질이 포함된 용액을 막 위에 고르게 분주한 뒤, 노출 시간에 따라 X-ray 필름 또는 ImageQuant800 기기로 그 발광양을 측정하였다.11) After washing with TTBS, a solution containing a chemiluminescent substrate was evenly dispensed on the membrane, and the amount of emission was measured with an X-ray film or ImageQuant800 device according to the exposure time.
12) Image J 프로그램을 이용하여 정량적으로 비교하였다.12) Quantitative comparison was made using the Image J program.
5. qPCR 5.qPCR
1) 조직 혹은 세포에 트라이졸 1000 ul과 클로로포름 200 ul를 넣고 잘 섞어준 뒤, 4℃의 원심분리기를 이용하여 13,000 rpm 속도로 15분 동안 원심분리하였다.One) 1000 ul of Trizol and 200 ul of chloroform were added to the tissues or cells, mixed well, and centrifuged at 13,000 rpm for 15 minutes using a centrifuge at 4 °C.
2) 분리된 상층액 200 ul과 동량의 아이소프로필 알코올 200 ul을 넣은 뒤, 4℃의 원심분리기를 이용하여 13,000 rpm 속도로 15분 동안 원심분리하였다.2) 200 ul of the separated supernatant and 200 ul of isopropyl alcohol were added thereto, followed by centrifugation at 13,000 rpm for 15 minutes using a centrifuge at 4°C.
3) 상층액은 제거하고, 75% 에탄올 1 ml을 넣은 뒤, 4℃의 원심분리기를 이용하여 13,000 rpm 속도로 15분 동안 원심분리시켜 침전물을 세척하였다.3) The supernatant was removed, 1 ml of 75% ethanol was added, and the precipitate was washed by centrifugation at 13,000 rpm for 15 minutes using a centrifuge at 4°C.
4) 모든 상층액을 깨끗하게 제거한 뒤, 적당량의 0.1% DEPC (Diethylpyrocarbonate)가 처리된 증류수로 침전물을 녹이고, 미량 분광광도계를 이용하여 용액 내의 RNA를 정량하였다.4) After removing all the supernatant, the precipitate was dissolved in distilled water treated with an appropriate amount of 0.1% DEPC (Diethylpyrocarbonate), and RNA in the solution was quantified using a trace spectrophotometer.
5) 정량한 농도를 기반으로 cDNA (complementary DNA)를 합성하였다.5) Based on the quantified concentration, cDNA (complementary DNA) was synthesized.
6) SYBR GREEN의 형광 표식을 사용하며, QuantStudio3 혹은 StepOnePlus Real time PCR (55℃:2분 - 95℃:10분 - 95℃:15초 - 60℃:1분)을 이용하여, 증폭량에 따라 내부제어유전자인 18S 리보솜 RNA의 Ct값으로 보정하여 상대값으로 결과를 도출하였다. 6) Using the fluorescent label of SYBR GREEN, using QuantStudio3 or StepOnePlus Real time PCR (55℃: 2 minutes - 95℃: 10 minutes - 95℃: 15 seconds - 60℃: 1 minute), internal The result was derived as a relative value by correcting with the Ct value of 18S ribosomal RNA, which is a control gene.
6. MTT assay 6. MTT assay
본 실험은 세포를 용해시키며 나온 ATP를 측정하여 생존 가능한 세포 수를 반영하여 세포 독성을 확인하는 실험이다.This experiment is an experiment to confirm cytotoxicity by reflecting the number of viable cells by measuring ATP released while lysing cells.
1) 96웰 플레이트에 세포 1.25×104 개를 분주 후 하루 배양하였다.1) 1.25 × 10 4 cells were seeded in a 96-well plate and cultured for one day.
2) 기존 배양액을 제거하고, 실험용 배양액에 에제티미브를 농도에 따라 처리하여 24시간 배양을 진행하였다.2) The existing culture medium was removed, and ezetimibe was treated according to the concentration in the experimental culture medium, followed by 24-hour culture.
3) 상온에서 30분 유지시켜 온도 평형을 맞춰주었다.3) It was maintained at room temperature for 30 minutes to balance the temperature.
4) CellTiter-Glo 기질 시약 25 ul을 주입 후, Centro XS3 LB 960 High Sensitivity Microplate Luminometer를 이용하여 발광 세포 생존력 분석을 진행하였다. 4) After injecting 25 ul of CellTiter-Glo substrate reagent, luminescent cell viability was analyzed using a Centro XS 3 LB 960 High Sensitivity Microplate Luminometer.
7. Sircol 콜라겐 분석(Collagen assay)7. Sircol collagen assay
Sircol™ Soluble Collagen Assay (Biocolor Ltd., S1000)를 이용하였으며, 제조사의 매뉴얼에 따라 아래 과정대로 실험을 진행하였다.Sircol ™ Soluble Collagen Assay (Biocolor Ltd., S1000) was used, and the experiment was conducted according to the manufacturer's manual according to the following procedure.
1) 0.5 M 아세트산으로 희석한 0.1 mg/ml 펩신을 1 ml 분주하고 물리적으로 분쇄하여 마우스의 우측 폐 상엽을 용해시켰다.1) 1 ml of 0.1 mg/ml pepsin diluted with 0.5 M acetic acid was dispensed and physically ground to dissolve the right upper lobe of the mouse's lung.
2) 4℃의 원심분리기를 이용하여 13500 rpm 속도로 15분 동안 분리하여, 용해된 상층액 200 ul을 획득하였다.2) Separation at 13500 rpm for 15 minutes using a centrifuge at 4° C. to obtain 200 ul of the dissolved supernatant.
3) 용해된 조직 샘플을 1/10로 희석시켜 100 ul을 만들고, Sircol 염색시약 1 ml 넣어주었다.3) The dissolved tissue sample was diluted 1/10 to make 100 ul, and 1 ml of Sircol staining reagent was added.
4) 실온에서 30분 동안 쉐이커에 두어 잘 섞이도록 하였다.4) Place in a shaker at room temperature for 30 minutes to mix well.
5) 12,000 rpm의 속도로 10분간 원심분리한 뒤 상층액은 버리고, 침전물을 획득하였다.5) After centrifugation at 12,000 rpm for 10 minutes, the supernatant was discarded and the precipitate was obtained.
6) 차가운 산-염 세척 시약 750 ul을 조심스럽게 첨가하여, 침전물과 튜브 표면의 결합하지 않은 염색약을 세척하였다.6) 750 ul of cold acid-salt washing reagent was carefully added to wash the precipitate and unbound dye on the tube surface.
7) 12,000 rpm의 속도로 10분간 다시 한번 원심분리 하여 상층액은 제거하고, 최대한 용액이 남아있지 않게 하였다.7) The supernatant was removed by centrifugation again at 12,000 rpm for 10 minutes, and no solution remained as much as possible.
8) 1 ml의 알칼리 시약을 넣고, 10초 이상 강한 진동으로 혼합액을 섞어주었다.8) 1 ml of alkaline reagent was added, and the mixed solution was mixed with strong vibration for more than 10 seconds.
9) 96웰 플레이트에 200 ul의 샘플을 넣고 3~4번 반복으로 분주하여, VERSAmax microplate reader 기기를 이용하여 555 nm로 측정하였다.9) 200 ul of sample was put in a 96-well plate and dispensed repeatedly 3 to 4 times, and measured at 555 nm using a VERSAmax microplate reader.
8. 조직학적 분석8. Histological analysis
1) 마우스 해부 과정에서 좌측 폐엽을 기관지와 함께 10% Formalin으로 부풀린 상태로 적출하고, 10% Formalin 용액에서 24시간 동안 고정하였다.1) During mouse dissection, the left lobe of the lung was excised along with the bronchi in a state inflated with 10% Formalin, and fixed in 10% Formalin solution for 24 hours.
2) 고정된 조직을 파라핀 절편으로 제작하여 슬라이드글라스에 붙인 뒤, Masson's trichrome 염색으로 염색하였다.2) The fixed tissues were made into paraffin sections, attached to slide glass, and then stained with Masson's trichrome staining.
3) 제작된 염색 슬라이드를 Aperio AT2 기기로 400배 고해상도 디지털 이미지로 스캔하였다. 3) The fabricated stained slide was scanned as a 400-fold high-resolution digital image with an Aperio AT2 machine.
4) 스캔한 이미지를 Aperio ImageScope 프로그램을 활용하여 정량 및 정성적으로 분석하였다.4) The scanned images were quantitatively and qualitatively analyzed using the Aperio ImageScope program.
9. 통계처리9. Statistical processing
R version 4.0.2 및 Prism 9를 이용하였고, 3개 이상의 그룹이 포함된 모든 실험에서 one-way ANOVA 및 다중 비교에 대해 Tukey 검증을 이용하여 통계적으로 분석하였다. 마우스 체중 변화의 분석에는 two-way ANOVA를 사용하였다. 통계적 유의성은 * p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001 순으로 표시하였으며, 일부 data에서는 p-value를 직접 기재하였다.R version 4.0.2 and Prism 9 were used, and statistical analysis was performed using one-way ANOVA and Tukey's test for multiple comparisons in all experiments involving three or more groups. Two-way ANOVA was used for analysis of mouse body weight change. Statistical significance was indicated in the order of * p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001, and in some data, the p-value was directly described.
결과result
실험예 1: MTT assay를 이용한 에제티미브의 세포 투여 농도 결정Experimental Example 1: Determination of cell administration concentration of ezetimibe using MTT assay
MTT assay를 이용하여 세포에 독성이 발생하는 에제티미브의 농도를 확인하였다. 마우스 폐 섬유모세포에서는 FBS 0.1% 배양액과 FBS 15% 배양액에서 각각 실험을 진행하였다.The concentration of ezetimibe that causes toxicity to the cells was confirmed using the MTT assay. In mouse lung fibroblasts, experiments were conducted in FBS 0.1% culture medium and FBS 15% culture medium, respectively.
도 1은 FBS 15% 세포 배양액 조건에서 마우스 폐 섬유모세포에 에제티미브를 24시간 동안 표시된 농도 대로 처리하고, MTT assay를 진행한 결과이다.1 is a result of treating mouse lung fibroblasts with ezetimibe at the indicated concentration for 24 hours in a cell culture medium of 15% FBS, and performing an MTT assay.
도 2는 FBS 0.1% 세포 배양액 조건에서 마우스 폐 섬유모세포에 에제티미브를 24시간 동안 표시된 농도 대로 처리하고, MTT assay를 진행한 결과이다.Figure 2 shows the results of MTT assay after treatment with ezetimibe at the indicated concentration for 24 hours in mouse lung fibroblasts in FBS 0.1% cell culture medium condition.
도 3은 FBS 15% 세포 배양액 조건에서 사람 폐 섬유모세포에 에제티미브를 24시간 동안 표시된 농도 대로 처리하고, MTT assay를 진행한 결과이다.Figure 3 shows the results of treating human lung fibroblasts with ezetimibe at the indicated concentrations for 24 hours in a cell culture medium of 15% FBS, and performing the MTT assay.
도 1 내지 3에 나타낸 바와 같이, 사람 및 마우스 폐 섬유모세포에서 에제티미브는 20 μM 농도 미만에서는 세포 독성이 발생하지 않는 것으로 확인되었다. 이를 바탕으로, 이후 실험은 에제티미브 20 μM를 최대 농도로 사용하였다.As shown in FIGS. 1 to 3 , it was confirmed that ezetimibe did not cause cytotoxicity in human and mouse lung fibroblasts at a concentration of less than 20 μM. Based on this, in subsequent experiments, 20 μM of ezetimibe was used at the maximum concentration.
실험예 2: 마우스 폐 섬유모세포에서의 에제티미브의 폐섬유증 치료 효능 확인Experimental Example 2: Confirmation of pulmonary fibrosis treatment efficacy of ezetimibe in mouse lung fibroblasts
마우스 폐 섬유모세포에서의 에제티미브의 폐섬유증 치료 효능을 확인하기 위한 실험을 하였다. 섬유모세포에 TGFβ1를 처리하여 근섬유모세포로 분화시키며 생성되는 섬유화의 최종 산물 중 하나인 Collagen 1a1 (Col1A1)을 주요 검출 대상으로 확인하였다.An experiment was conducted to confirm the therapeutic effect of ezetimibe on pulmonary fibrosis in mouse lung fibroblasts. Collagen 1a1 (Col1A1), one of the final products of fibrosis produced by treating fibroblasts with TGFβ1 to differentiate into myofibroblasts, was identified as a major detection target.
도 4는 TGFβ1 미처리 상태와 TGFβ1 처리 상태를 비교하고, 동시에 TGFβ1 처리 상태에서 에제티미브의 처리 농도별 Col1A1 단백질 발현 정도를 나타낸 결과이다. Figure 4 is a result of comparing the TGFβ1 untreated state and the TGFβ1 treated state, and showing the Col1A1 protein expression level according to the treatment concentration of ezetimibe in the TGFβ1 treated state at the same time.
도 5는 상기 도 4와 같은 실험 조건에서 에제티미브의 처리 농도별 Col1A1과 Acta2의 mRNA 발현 정도를 나타낸 결과이다.FIG. 5 shows the mRNA expression levels of Col1A1 and Acta2 for each treatment concentration of ezetimibe under the same experimental conditions as in FIG. 4 .
도 6은 에제티미브의 농도를 20 μM으로 고정하였을 때, 에제티미브 처리 시간에 따른 Col1A1 단백질 발현 정도를 나타낸 결과이다.6 is a result showing the level of Col1A1 protein expression according to ezetimibe treatment time when the concentration of ezetimibe was fixed at 20 μM.
도 7은 상기 도 6과 같은 실험 조건에서 에제티미브의 농도를 20 μM으로 고정하였을 때, 에제티미브 처리 시간에 따른 Col1A1과 Acta2의 mRNA 발현 정도를 나타낸 결과이다.7 is a result showing the mRNA expression levels of Col1A1 and Acta2 according to the treatment time of ezetimibe when the concentration of ezetimibe was fixed at 20 μM under the same experimental conditions as in FIG. 6 .
도 4 및 5에 나타낸 바와 같이, 에제티미브는 농도가 증가함에 따라 Col1A1의 단백질 및 mRNA 발현을 감소시키는 것으로 확인되었고, 보조적으로 함께 측정한 Acta2 mRNA도 함께 감소시키는 것을 확인하였다.As shown in FIGS. 4 and 5, it was confirmed that ezetimibe decreased the protein and mRNA expression of Col1A1 as the concentration increased, and also decreased Acta2 mRNA measured together.
도 6 및 7에 나타낸 바와 같이, 에제티미브의 농도를 20 μM로 고정하였을 때, 처리 시간이 24시간으로 충분할 경우 Col1A1과 Acta2를 감소시키는 것을 확인하였다.As shown in FIGS. 6 and 7 , when the concentration of ezetimibe was fixed at 20 μM, it was confirmed that Col1A1 and Acta2 were reduced when the treatment time was sufficient for 24 hours.
따라서, 마우스 폐 섬유모세포를 이용한 폐섬유증 세포 모델에서 에제티미브의 폐섬유증 치료 효과를 확인하였다.Therefore, the therapeutic effect of ezetimibe on pulmonary fibrosis was confirmed in a pulmonary fibrosis cell model using mouse lung fibroblasts.
실험예 3: 인간 폐 섬유모세포에서의 에제티미브의 폐섬유증 치료 효능 확인Experimental Example 3: Confirmation of pulmonary fibrosis treatment efficacy of ezetimibe in human pulmonary fibroblasts
인간 폐 섬유모세포에서의 에제티미브의 폐섬유증 치료 효능을 확인하기 위한 실험을 하였다. 섬유모세포에 TGFβ1를 처리하여 근섬유모세포로 분화시키며 생성되는 섬유화의 최종 산물 중 하나인 Collagen 1a1 (Col1A1)을 주요 검출 대상으로 확인하였다.Experiments were conducted to confirm the efficacy of ezetimibe in treating pulmonary fibrosis in human lung fibroblasts. Collagen 1a1 (Col1A1), one of the final products of fibrosis produced by treating fibroblasts with TGFβ1 to differentiate into myofibroblasts, was identified as a major detection target.
도 8은 TGFβ1 미처리 상태와 TGFβ1 처리 상태를 비교하고, 동시에 TGFβ1 처리 상태에서 에제티미브의 농도별 COL1A1 단백질 발현 정도를 나타낸 결과이다. 8 is a result of comparing the TGFβ1 untreated state and the TGFβ1 treated state, and showing the COL1A1 protein expression level according to the concentration of ezetimibe in the TGFβ1 treated state at the same time.
도 9는 상기 도 8와 같은 실험 조건에서 에제티미브의 처리 농도별 Col1A1과 Acta2의 mRNA 발현 정도를 나타낸 결과이다.FIG. 9 shows the mRNA expression levels of Col1A1 and Acta2 for each treatment concentration of ezetimibe under the same experimental conditions as in FIG. 8 .
도 10은 에제티미브의 농도를 20 μM으로 고정하였을 때, 에제티미브 처리 시간에 따른 Col1A1 단백질 발현 정도를 나타낸 결과이다.10 is a result showing the level of Col1A1 protein expression according to ezetimibe treatment time when the concentration of ezetimibe was fixed at 20 μM.
도 11은 상기 도 10과 같은 실험 조건에서 에제티미브의 농도를 20 μM으로 고정하였을 때, 에제티미브 처리 시간에 따른 Col1A1과 Acta2의 mRNA 발현 정도를 나타낸 결과이다.11 is a result showing the mRNA expression levels of Col1A1 and Acta2 according to ezetimibe treatment time when the concentration of ezetimibe was fixed at 20 μM under the same experimental conditions as in FIG. 10.
도 8 내지 11에 나타낸 바와 같이, 인간 폐 섬유모세포에 있어서도 TGFβ1 처리군은 TGFβ 자극에 의하여 COL1A1 단백질과 mRNA 발현이 증가하나, 이와 같은 COL1A1 단백질과 mRNA 발현증가는 에제티미브 처리에 의해 효과적으로 그 발현이 감소되는 것을 확인하였다.As shown in FIGS. 8 to 11, in human lung fibroblasts, the TGFβ1-treated group also increased COL1A1 protein and mRNA expression by TGFβ stimulation, but such an increase in COL1A1 protein and mRNA expression was effectively expressed by ezetimibe treatment It was confirmed that this decreased.
따라서, 상기 실험예 2 및 3의 결과를 종합해보면, 폐 섬유모세포의 TGFβ1 자극에 의한 인간과 마우스의 폐섬유증 세포 모델에서, 에제티미브는 폐섬유증 치료 효과를 가짐을 확인하였다.Therefore, taking the results of Experimental Examples 2 and 3 together, it was confirmed that ezetimibe has a therapeutic effect on pulmonary fibrosis in human and mouse pulmonary fibrosis cell models by TGFβ1 stimulation of pulmonary fibroblasts.
실험예 4: 블레오마이신 유발 마우스 폐섬유증 모델에서 에제티미브의 폐섬유증 완화 효과 확인 - 마우스 생존율의 비교를 통하여Experimental Example 4: Confirmation of Ezetimibe's Pulmonary Fibrosis Alleviation Effect in Bleomycin-Induced Mouse Pulmonary Fibrosis Model - Through Comparison of Mouse Survival Rate
블레오마이신 유발 마우스 폐섬유증 모델에서 에제티미브의 폐섬유증 완화 효과를 확인하기 위하여, 마우스의 생존율을 확인하였다.In order to confirm the effect of ezetimibe on alleviating pulmonary fibrosis in a bleomycin-induced mouse pulmonary fibrosis model, the survival rate of mice was confirmed.
도 12는 블레오마이신 유발 마우스 폐섬유증 모델에서 에제티미브의 폐섬유증 완화 효과를 확인하기 위한 실험에 있어서 각 군의 투여 방법을 간단한 모식도로 확인한 도이다.FIG. 12 is a schematic view showing the administration method of each group in an experiment for confirming the pulmonary fibrosis alleviating effect of ezetimibe in a bleomycin-induced mouse pulmonary fibrosis model.
도 13은 블레오마이신 투여 후 일수(Days)에 따른 마우스의 생존율 변화를 나타낸 그래프이다.13 is a graph showing the change in survival rate of mice according to the number of days after administration of bleomycin.
도 12에 나타난 바와 같이, 모든 약물은 블레오마이신 주입 후 7일차부터 투여되어 염증에 대한 효과를 배제하고 항섬유화 효과를 확인하였다. 도 13에서 확인한 바와 같이, 에제티미브를 투여한 마우스에서 블레오마이신을 투여한 마우스 대비 뛰어난 생존 효과가 나타났으며, 이는 기존 특발폐섬유증의 약제인 닌테다닙과 유사한 수준으로 나타나는 것을 확인할 수 있었다.As shown in Figure 12, all drugs were administered from the 7th day after bleomycin injection to exclude the effect on inflammation and confirm the anti-fibrotic effect. As confirmed in FIG. 13, mice administered with ezetimibe exhibited an excellent survival effect compared to mice administered with bleomycin, which was found to be similar to that of nintedanib, an existing idiopathic pulmonary fibrosis drug.
따라서, 마우스 생존율의 확인을 통한 블레오마이신 유발 마우스 폐섬유증 모델에서 에제티미브의 폐섬유증 치료 또는 완화 효과를 확인하였다.Therefore, the treatment or alleviation effect of ezetimibe on pulmonary fibrosis was confirmed in a bleomycin-induced mouse pulmonary fibrosis model through confirmation of mouse survival rate.
실험예 5: 블레오마이신 유발 마우스 폐섬유증 모델에서 에제티미브의 폐섬유증 치료 효능 확인- 콜라겐 발현양 비교를 통하여Experimental Example 5: Confirmation of Ezetimibe's Treatment of Pulmonary Fibrosis in a Bleomycin-Induced Mouse Pulmonary Fibrosis Model - Through Comparison of Collagen Expression
블레오마이신 유발 마우스 폐섬유증 모델에서 에제티미브의 폐섬유증 치료 효과를 확인하기 위하여, 콜라겐 양을 측정하였다.In order to confirm the therapeutic effect of ezetimibe on pulmonary fibrosis in a bleomycin-induced mouse pulmonary fibrosis model, the amount of collagen was measured.
도 14는 블레오마이신 미투여군(Control, n=14), 블레오마이신 투여군(Bleomycin, n=13), 블레오마이신 + 에제티미브 투여군(Bleo+Ezetimibe, n=17) 및 블레오마이신 + 닌테다닙 투여군(Bleo+Nintedanib, n=7)에서 콜라겐 양을 측정한 결과이다. 도 14의 그래프 상의 각각의 점은 각각 한 마리의 마우스의 실험 결과를 표시하며, 평균 ± 표준오차로 표기하였고, one-way ANOVA로 분석하였다.14 is a bleomycin non-administration group (Control, n = 14), a bleomycin-administered group (Bleomycin, n = 13), a bleomycin + ezetimibe-administered group (Bleo + Ezetimibe, n = 17), and a bleomycin + nintedanib-administered group ( This is the result of measuring the amount of collagen in Bleo+Nintedanib, n=7). Each point on the graph of FIG. 14 represents the experimental results of one mouse, expressed as mean ± standard error, and analyzed by one-way ANOVA.
도 14에서 확인한 바와 같이, 에제티미브를 투여한 군에서 블레오마이신 투여 군 대비 폐 내의 콜라겐 양이 더 적은 것으로 확인되어, 폐 섬유화를 억제하여 치료하는 효과가 있는 것을 확인하였다. 또한, 그 효과가 기존 특발폐섬유증 약제인 닌테다닙과 유사한 수준임을 확인하였다. 따라서, 콜라겐 발현 양 억제를 확인을 통한 블레오마이신 유발 마우스 폐섬유증 모델에서 에제티미브의 폐섬유증 치료 또는 완화 효과를 확인하였다.As confirmed in FIG. 14 , it was confirmed that the amount of collagen in the lungs was lower in the group administered with ezetimibe than in the group administered with bleomycin, and thus, it was confirmed that there was an effect of inhibiting and treating lung fibrosis. In addition, it was confirmed that the effect was similar to that of nintedanib, an existing idiopathic pulmonary fibrosis drug. Therefore, the treatment or alleviation effect of ezetimibe on pulmonary fibrosis was confirmed in a bleomycin-induced mouse pulmonary fibrosis model by confirming the suppression of the amount of collagen expression.
실험예 6: 블레오마이신 유발 마우스 폐섬유증 모델에서 에제티미브의 폐섬유증 치료 효능 확인- 섬유화 마커 단백질 및 mRNA 변화를 통하여Experimental Example 6 Confirmation of Ezetimibe's Pulmonary Fibrosis Treatment Efficacy in Bleomycin-Induced Mouse Pulmonary Fibrosis Model - Through Changes in Fibrosis Marker Protein and mRNA
블레오마이신 유발 마우스 폐섬유증 모델에서 에제티미브의 폐섬유증 치료 효과를 확인하기 위하여, 섬유화 마커 단백질 및 mRNA 변화를 측정하였다.In order to confirm the therapeutic effect of ezetimibe on pulmonary fibrosis in a bleomycin-induced mouse pulmonary fibrosis model, changes in fibrosis marker protein and mRNA were measured.
도 15는 웨스턴 블랏을 이용하여 각 군의 섬유화 마커인 COL1A1과 피브로넥틴(Fibronectin)을 비교한 결과 및 Image J를 통해 웨스턴 블랏의 결과를 정량한 결과를 확인한 도이다.15 is a diagram confirming the results of comparing COL1A1 and fibronectin, which are fibrosis markers of each group, using Western blot and quantifying the result of Western blot through Image J.
도 16은 RT-qPCR을 통해 섬유화 마커인 Col1a1, Col1a2, Acta2, Col3a1 및 Eda-fn mRNA의 발현을 확인한 결과를 나타낸 도이다. 16 is a diagram showing the results of confirming the expression of fibrosis markers Col1a1, Col1a2, Acta2, Col3a1 and Eda-fn mRNA through RT-qPCR.
도 15 및 도 16의 그래프 상의 각각의 점은 각각 한 마리의 마우스의 실험 결과를 표시하며, 평균 ± 표준오차로 표기하였고, one-way ANOVA로 분석하였다. 각 군 당 n=4~5 마리가 분석에 사용되었다.Each point on the graphs of FIGS. 15 and 16 represents the experimental results of one mouse, respectively, expressed as mean ± standard error, and analyzed by one-way ANOVA. n=4-5 animals in each group were used for analysis.
도 15에 확인한 바와 같이, 에제티미브를 투여한 군에서 블레오마이신을 투여한 군 대비 섬유화 마커의 감소를 확인하였다.As confirmed in FIG. 15, it was confirmed that the fibrosis markers were decreased in the group administered with ezetimibe compared to the group administered with bleomycin.
도 16에 나타난 바와 같이, 에제티미브를 투여한 군에서 블레오마이신을 투여한 군 대비 모든 폐 섬유화 마커인 Col1a1, Col1a2, Acta2, Col3a1 및 Eda-fn 의 감소가 현저하게 나타나는 것을 확인하였다.As shown in FIG. 16, it was confirmed that all pulmonary fibrosis markers, Col1a1, Col1a2, Acta2, Col3a1, and Eda-fn, were significantly reduced in the ezetimibe-administered group compared to the bleomycin-administered group.
실험예 7: 블레오마이신 유발 마우스 폐섬유증 모델에서 에제티미브의 폐섬유증 치료 효능 확인- 폐 병리조직 비교를 통하여Experimental Example 7: Confirmation of pulmonary fibrosis treatment efficacy of ezetimibe in bleomycin-induced mouse pulmonary fibrosis model - through comparison of lung pathological tissue
블레오마이신 유발 마우스 폐섬유증 모델에서 에제티미브의 폐섬유증 치료 효과를 확인하기 위하여, 폐 병리조직을 통한 치료 효과를 확인하였다.In order to confirm the therapeutic effect of ezetimibe on pulmonary fibrosis in a bleomycin-induced mouse pulmonary fibrosis model, the therapeutic effect was confirmed through lung pathology.
도 17은 Masson-trichrome stain으로 섬유화 정도를 비교한 결과를 나타낸 것이다.17 shows the results of comparing the degree of fibrosis with Masson-trichrome stain.
도 18은 마우스별 폐 병리조직의 슬라이드에서 콜라겐이 염색된 부위를 Aperio Imagescope 프로그램을 이용하여 정량한 결과를 나타낸 도이다.18 is a diagram showing the results of quantification of collagen-stained areas in slides of lung pathological tissues for each mouse using the Aperio Imagescope program.
도 17에서 확인한 바와 같이, 블레오마이신 투여 군 대비 에제티미브 투여 군에서 섬유화가 호전된 것을 확인할 수 있었다.As confirmed in FIG. 17, it was confirmed that fibrosis was improved in the ezetimibe-administered group compared to the bleomycin-administered group.
도 18에 나타난 바와 같이, 블레오마이신 투여 군 대비 에제티미브 투여 군에서 콜라겐 염색부위의 면적이 감소한 것을 확인하여, 에제티미브 투여를 통한 폐 섬유증 치료 효과를 확인하였다. As shown in FIG. 18, it was confirmed that the area of the collagen-stained area was reduced in the ezetimibe-administered group compared to the bleomycin-administered group, confirming the treatment effect of ezetimibe for pulmonary fibrosis treatment.
실험예 8: 블레오마이신 유발 마우스 폐섬유증 모델에서 에제티미브의 폐섬유증 치료 효능 확인- 마우스 체중 회복 비교를 통하여Experimental Example 8: Confirmation of pulmonary fibrosis treatment efficacy of ezetimibe in bleomycin-induced mouse pulmonary fibrosis model - through comparison of mouse weight recovery
블레오마이신 유발 마우스 폐섬유증 모델에서 에제티미브의 폐섬유증 치료 효과를 확인하기 위하여, 마우스 체중 회복을 통한 치료 효과를 확인하였다.In order to confirm the therapeutic effect of ezetimibe on pulmonary fibrosis in a bleomycin-induced mouse pulmonary fibrosis model, the therapeutic effect was confirmed through weight recovery of the mouse.
도 19는 블레오마이신 투여 후 일수(Days)에 따른 마우스 체중 변화를 나타낸 그래프이다. 평균 ± 표준오차로 표기하였고, Two-way ANOVA (측정 날짜와 투약군)로 분석하였다. 각 군당 대조군으로 n=23, 블레오마이신 투여군(Bleomycin)으로 n=18 / 블레오마이신 + 에제티미브 투여군(Bleo+Eze)으로 n=21 마리가 분석에 활용되었다.19 is a graph showing the change in body weight of mice according to the number of days after administration of bleomycin. It was expressed as mean ± standard error and analyzed by two-way ANOVA (measurement date and administration group). For each group, n = 23 as a control group, n = 18 as a bleomycin group (Bleomycin) / n = 21 animals as a bleomycin + ezetimibe group (Bleo + Eze) were used for analysis.
도 19에서 확인한 바와 같이, 블레오마이신에 의해 7일차까지 동일한 수준의 염증이 유도되었으나, 에제티미브를 투여한 마우스에서는 블레오마이신 투여 후 16일째부터 유의하게 체중 회복 효과가 더 크게 나타나는 것을 확인하였다.As confirmed in FIG. 19 , although the same level of inflammation was induced by bleomycin until the 7th day, it was confirmed that in the mice administered with ezetimibe, the weight recovery effect was significantly greater from the 16th day after bleomycin administration.
종합하면, 에제티미브는 블레오마이신 투여한 마우스에서 섬유화를 억제하는 효능을 보였으며, 특히 이미 기존 시판되어 특발폐섬유증 환자에게 국제적으로 사용되고 있는 약물인 닌테다닙을 표준 용량으로 사용한 마우스와 비교한 경우에도, 이와 유사하거나 더 우월한 효과가 나타나는 것을 확인하였다. 이에 에제티미브는 폐 섬유화를 완화 및 치료할 수 있음을 확인하였다.In summary, ezetimibe showed efficacy in inhibiting fibrosis in mice administered with bleomycin, especially when compared to mice using standard dose of nintedanib, a drug already marketed and used internationally for patients with idiopathic pulmonary fibrosis. However, it was confirmed that similar or superior effects appeared. Accordingly, it was confirmed that ezetimibe can alleviate and treat pulmonary fibrosis.
이상의 설명은 본 발명의 기술 사상을 예시적으로 설명한 것에 불과한 것으로서, 본 발명이 속하는 기술 분야에서 통상의 지식을 가진 자라면 본 발명의 본질적인 특성에서 벗어나지 않는 범위에서 다양한 수정 및 변형이 가능할 것이다.The above description is merely an example of the technical idea of the present invention, and various modifications and variations can be made to those skilled in the art without departing from the essential characteristics of the present invention.

Claims (9)

  1. 에제티미브(Ezetimibe) 또는 이의 약학적으로 허용가능한 염을 유효성분으로 포함하는 폐섬유증의 예방, 개선, 완화 또는 치료용 약학적 조성물.A pharmaceutical composition for preventing, improving, alleviating or treating pulmonary fibrosis, comprising Ezetimibe or a pharmaceutically acceptable salt thereof as an active ingredient.
  2. 청구항 1에 있어서, 상기 에제티미브는 폐 섬유모세포에서 근섬유모세포로의 분화를 억제하거나 콜라겐 발현을 감소시키는 것인 약학적 조성물. The pharmaceutical composition according to claim 1, wherein the ezetimibe inhibits differentiation from lung fibroblasts to myofibroblasts or reduces collagen expression.
  3. 청구항 1에 있어서, 상기 에제티미브의 일일 투여량은 0.001 내지 20 mg의 용량으로 포함되는 것인 약학적 조성물.The pharmaceutical composition according to claim 1, wherein the daily dose of ezetimibe is contained in a dose of 0.001 to 20 mg.
  4. 청구항 1에 있어서, 상기 폐섬유증은 특발성 폐섬유증 및 특발성 비특이 간질성 폐렴에 의한 폐섬유증을 포함하는 특발성 간질성 폐렴에 의한 폐섬유증; 류마티스성 관절염, 루푸스, 전신경화증, 근염 및 쇼그렌 증후군을 포함하는 결체조직질환 또는 자가면역질환에 의한 폐섬유증; 감염 질환 및 위식도 역류성 질환을 포함하는 질병으로 인한 폐섬유증; 가족성 폐섬유증; 석면, 실리카, 베릴륨, 먼지 및 흡연을 포함하는 유해물질에 의한 폐섬유증; 방사선 치료 또는 방사선 노출로 인한 폐섬유증; 항암제, 항생제 및 항부정맥제를 포함하는 약물에 의한 폐섬유증; 급성 호흡부전 증후군의 후유증으로 인한 폐섬유증으로 이루어진 군에서 선택된 것인 약학적 조성물.The method according to claim 1, wherein the pulmonary fibrosis is pulmonary fibrosis caused by idiopathic interstitial pneumonia including pulmonary fibrosis caused by idiopathic pulmonary fibrosis and idiopathic nonspecific interstitial pneumonia; pulmonary fibrosis caused by connective tissue diseases or autoimmune diseases including rheumatoid arthritis, lupus, systemic sclerosis, myositis and Sjogren's syndrome; pulmonary fibrosis due to diseases including infectious diseases and gastroesophageal reflux disease; familial pulmonary fibrosis; pulmonary fibrosis caused by harmful substances including asbestos, silica, beryllium, dust and smoking; pulmonary fibrosis due to radiation therapy or exposure to radiation; pulmonary fibrosis caused by drugs including anticancer drugs, antibiotics and antiarrhythmics; A pharmaceutical composition selected from the group consisting of pulmonary fibrosis due to sequelae of acute respiratory failure syndrome.
  5. 청구항 1에 있어서, 상기 폐섬유증은 블레오마이신(Bleomycin)의 투여로부터 발생하는 것 또는 COVID-19 감염 후 후유증으로 인한 폐섬유증인 것인 약학적 조성물.The pharmaceutical composition according to claim 1, wherein the pulmonary fibrosis is pulmonary fibrosis caused by administration of bleomycin or sequelae after COVID-19 infection.
  6. 에제티미브(Ezetimibe) 또는 이의 식품학적으로 허용가능한 염을 유효성분으로 포함하는 폐섬유증의 예방 또는 개선용 건강기능식품 조성물.A health functional food composition for preventing or improving pulmonary fibrosis, comprising Ezetimibe or a food chemically acceptable salt thereof as an active ingredient.
  7. 유효량의 에제티미브 또는 이의 약학적으로 허용가능한 염을 이를 필요로 하는 개체에게 투여하는 단계를 포함하는 폐섬유증의 예방, 개선, 완화 또는 치료 방법.A method for preventing, improving, alleviating or treating pulmonary fibrosis comprising administering an effective amount of ezetimibe or a pharmaceutically acceptable salt thereof to a subject in need thereof.
  8. 폐섬유증의 예방, 개선, 완화 또는 치료용 약제의 제조에 사용하기 위한 에제티미브 또는 이의 약학적으로 허용가능한 염의 용도.Use of ezetimibe or a pharmaceutically acceptable salt thereof for use in the manufacture of a medicament for preventing, ameliorating, alleviating or treating pulmonary fibrosis.
  9. 폐섬유증의 예방, 개선, 완화 또는 치료를 위한 에제티미브 또는 이의 약학적으로 허용가능한 염의 용도.Use of ezetimibe or a pharmaceutically acceptable salt thereof for the prevention, improvement, alleviation or treatment of pulmonary fibrosis.
PCT/KR2022/015046 2021-10-08 2022-10-06 Pharmaceutical composition for preventing, improving, alleviating, or treating pulmonary fibrosis, comprising ezetimibe as active ingredient WO2023059099A1 (en)

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KR101845862B1 (en) * 2015-11-11 2018-04-06 (주)나디안바이오 Pharmaceutical compositions for treatment or prevention of idiopathic pulmonary fibrosis
JP2017203013A (en) * 2016-05-13 2017-11-16 エルメッド エーザイ株式会社 Ezetimibe-containing pharmaceutical composition and production method thereof, hardness decreasing inhibitor of ezetimibe-containing pharmaceutical composition and method of inhibiting decrease of hardness thereof, as well as moisture-absorption inhibitor of ezetimibe-containing pharmaceutical composition and moisture-absorption inhibiting method thereof
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