KR20220000371A - Composition for preventing or treating fibrosis - Google Patents

Abstract

When using a composition of the present invention, various fibrosis-related diseases, including lung fibrosis and renal fibrosis, can be more effectively treated or prevented by inhibiting the activity of histone acetyltransgerase (HAT).

Description

Composition for preventing or treating fibrosis

The present invention relates to a composition that can effectively prevent or treat fibrosis-related diseases.

Fibrosis or fibrosis (Fibrosis) is an abnormal accumulation of collagen matrix according to injury or inflammation that changes the structure and function of various tissues. In the case of fibrosis, regardless of the location of its occurrence, the most etiological factor is the excessive accumulation of fibrous connective tissue such as collagen matrix replacing normal tissue. Progressive fibrosis in the kidneys, liver, fat, lungs, heart, bone or bone marrow, and skin is a major cause of death or suffering. Among fibrosis, pulmonary fibrosis, in particular, is a disease that causes serious structural changes in the lung tissue by inducing tissue fibrosis as chronic inflammatory cells infiltrate the alveolar wall within the lung tissue. one of the diseases. Among them, idiopathic pulmonary fibrosis is a type of interstitial pneumonia in which fibrosis of the lung parenchyma is progressively progressing and is known to have the worst prognosis. Since the cause of idiopathic pulmonary fibrosis is currently unknown, smoking, viral infection, environmental toxic substances, or a family history are only presumed to be triggers of pulmonary fibrosis (Am J Manag Care. 2017 Jul; 23 (11 Suppl)). :S176-S182.). Clinical symptoms include a gradual increase in dyspnea during exercise, usually cough without sputum. Human lungs have a lot of extra lung capacity so that they can live without much inconvenience even if one side is removed, so it is difficult for pulmonary fibrosis patients to recognize the symptoms, so it is often diagnosed after the symptoms worsen. Because there are few methods to repair lung tissue that has undergone fibrosis due to lung fibrosis, the prognosis of pulmonary fibrosis varies considerably depending on the patient, but in general, the prognosis is so poor that the average survival period after diagnosis is within 3 to 5 years. Not good.

Treatment methods for pulmonary fibrosis found in the early stages of progression include steroids, azathioprine, and cyclophosphamide, such as steroids, and antioxidants, such as acetylcysteine. There is a treatment method using a treatment method and a treatment method through the administration of growth factors such as cytokine IFN-γ. Treatment methods using steroid drugs and antioxidants have been continuously studied and reported since 2000, but none have been proven as a clear drug efficacy. It has been reported that When pulmonary fibrosis progresses to complete fibrosis, it is difficult for any drug to be effective. Accordingly, recent studies on methods to treat pulmonary fibrosis by regulating transcription factors that play a direct role in transcription have been actively conducted to more effectively treat pulmonary fibrosis, but currently there are few drugs approved as therapeutic agents for lung fibrosis. . As the best method is to prevent pulmonary fibrosis from progressing to lung fibrosis by diagnosing it at an early stage when fibrosis is not severely advanced, it is urgently necessary to develop a therapeutic agent that can prevent pulmonary fibrosis in advance as well as development of a therapeutic agent to treat it after the onset of pulmonary fibrosis. .

The present inventors have completed the present invention by confirming that it is possible to prevent or improve various fibrosis-related diseases by excavating specific compounds obtained from natural products.

One object of the present invention is to provide a pharmaceutical composition that can effectively prevent or treat fibrosis-related diseases.

Another object of the present invention is to provide a food composition that can effectively prevent or improve fibrosis-related diseases.

Another object of the present invention is to provide a cosmetic composition that can effectively prevent or improve fibrosis-related diseases.

However, the technical problem to be achieved by the present invention is not limited to the above-mentioned problems, and other problems not mentioned will be clearly understood by those of ordinary skill in the art from the following description.

Hereinafter, various embodiments described herein are described with reference to the drawings. In the following description, various specific details are set forth, such as specific forms, compositions and processes, and the like, for a thorough understanding of the present invention. However, certain embodiments may be practiced without one or more of these specific details, or in conjunction with other known methods and forms. In other instances, well-known processes and manufacturing techniques have not been described in specific detail in order not to unnecessarily obscure the present invention. Reference throughout this specification to “one embodiment” or “an embodiment” means that a particular feature, form, composition, or characteristic described in connection with the embodiment is included in one or more embodiments of the invention. Thus, references to "in one embodiment" or "an embodiment" in various places throughout this specification do not necessarily refer to the same embodiment of the invention. Additionally, the particular features, forms, compositions, or properties may be combined in any suitable way in one or more embodiments.

Unless specifically defined in the present invention, all scientific and technical terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

According to an embodiment of the present invention, 2,6-dimethoxy-4-phenylphenol represented by Formula 1 below or a pharmaceutically acceptable salt thereof is included as an active ingredient. It relates to a composition for preventing, ameliorating or treating fibrosis, wherein:

[Formula 1]

In the present invention, 2,6-dimethoxy-4-phenylphenol (2,6-dimethoxy-4-phenylphenol) is a kind of phenyl-based compound and is also called acuparin. It is an active ingredient contained in plants of the Rosaceous subtribe Pyrinae/Maloideae, and is mainly known to have antimicrobial and antioxidative effects.

In the present invention, the pharmaceutically acceptable salt may include an addition salt of an acid or a base. For example, the compound may be in the form of an addition salt of an organic or inorganic acid. Salts have a desirable effect in a patient when administered to a patient, and include, but may not be particularly limited to, any salts that retain the activity of their parent compound. These salts include inorganic and organic salts such as acetic acid, nitric acid, aspartic acid, sulfonic acid, sulfuric acid, maleic acid, glutamic acid, formic acid, succinic acid, phosphoric acid, phthalic acid, tannic acid, tartaric acid, hydrobromic acid, propionic acid, benzenesulfonic acid, Benzoic acid, stearic acid, lactic acid, bicarboxylic acid, bisulfuric acid, bitartaric acid, oxalic acid, butyric acid, calcium idet, carbonic acid, chlorobenzoic acid, citric acid, idetic acid, toluenesulfonic acid, fumaric acid, gluceptic acid, ecylline Acid, pamoic acid, gluconic acid, methyl nitric acid, malonic acid, hydrochloric acid, hydroiodic acid, hydroxynaphtolic acid, isethionic acid, lactobionic acid, mandelic acid, mucoic acid, nafsilic acid, muconic acid, p -nitromethanesulfonic acid, hexamic acid, pantothenic acid, monohydrogenphosphoric acid, dihydrogenphosphoric acid, salicylic acid, sulfamic acid, sulfanilic acid, salts of methanesulfonic acid, and the like. Addition salts of bases include salts of alkali metals or alkaline earth metals, such as salts of ammonium, lithium, sodium, potassium, magnesium, calcium and the like; salts with organic bases, such as salts of benzathine, N-methyl-D-glucamine, hydrabamine and the like; and salts with amino acids, such as arginine, lysine, and the like. In addition, these salts can be converted to their free form by treatment with an appropriate base or acid.

The composition of the present invention is characterized in that it inhibits the activity of histone acetyltransferase (HAT), and thus has a remarkably excellent inhibitory effect on fibrosis-related diseases that have occurred or are likely to develop in an individual.

In the present invention, the "histone acetylation enzyme" is an enzyme that acetylates a histone substrate, resulting in an important regulatory effect on chromatin structure and assembly, and gene transcription. Modification of these proteins by HAT plays an important role in the regulation of gene expression, and dysregulation thereof is known to cause cancer, neurodegeneration and several other diseases. Examples of diseases associated with deregulation of histone deacetylase activity include tumor diseases, Huntington's disease caused by triplet amplification, degenerative diseases, ischemia (ischemia), oxidative stress, nervous system inflammatory response of epilepsy, diseases caused by protein aggregates, HIV infection, malaria, leishmaniosis, protozoa, fungi, phytotoxic agent , infections caused by viruses and parasites, autoimmune diseases, chronic host-directed immune reactions, hypertrophy and heart disease, skin fibrotic diseases, spinal and medulla oblongata ( spinal and bulbar muscular atrophy, bipolar disorder, psychiatric disorder, X-fragile syndrome, arthritis, kidney disease, psoriasis, intestinal-colitis disease, beta thalassemia (beta thalassemia), respiratory disease, and Rubinstein-Taybi syndrome.

In the present invention, the "individual" refers to mammals including humans, for example, humans, rats, mice, guinea pigs, hamsters, rabbits, monkeys, dogs, cats, cattle, horses, pigs, sheep and goats. It may be selected from, and preferably may be a human, but is not limited thereto.

Diseases to be prevented or treated in the pharmaceutical composition of the present invention, "fibrotic diseases" that have occurred or are likely to develop in an individual are abnormal production, accumulation and deposition of extracellular matrix by fibroblasts. As a disease that occurs, fibrosis of any organ may be included as long as it is an organ in which collagen matrix due to damage or inflammation that can change the structure and function of various tissues can be abnormally accumulated, preferably kidney, liver, lung, heart , bone, bone marrow, and may be fibrosis occurring in at least one organ selected from the group consisting of skin, but is not limited thereto.

For the purpose of the present invention, the fibrosis is a phenomenon in which the expression of genes related to fibrosis, for example, genes such as collagen, is promoted by TGF-β (Transforming growth factor-β), whose expression level is increased through p300, an acetylation enzyme. It may be induced by fibrosis or may be induced by the absence of an enzyme capable of recovering cells from damage that may be induced by fibrosis, but is not limited thereto.

The fibrosis of the present invention is lung fibrosis (Pulmonary fibrosis), uterine fibroids, myelofibrosis (myelofibrosis), liver fibrosis (liver fibrosis), heart fibrosis (Heart fibrosis), multiple sclerosis, kidney fibrosis (kidney fibrosis), cystic fibrosis ( cystic fibrosis), neutropenia, skeletal muscle fibrosis, skin sclerosis, dermatomyositis, mediastinal fibrosis and splenic fibrosis caused by sickle cell anemia may be at least one selected from the group consisting of, preferably pulmonary fibrosis or kidney It may be fibrosis, but is not limited thereto.

In one embodiment of the present invention, the pulmonary fibrosis is a type of respiratory disease that causes severe respiratory problems due to hardening of the lung tissue, and is a chronic disease that causes swelling and scarring of the alveoli and interstitial tissues of the lungs. Development of scarred (fibrous) tissue due to the formation or development of excess fibrous connective tissue in the lungs (fibrosis), which scar tissue replaces healthy tissue and causes inflammation, chronic inflammation is a sign of fibrosis can be perceived as a precursor. It is a disease characterized by diffuse fibrous proliferation in the alveolar wall and the main symptoms are coughing or shortness of breath. As fibrosis progresses and the lung wall thickens, the amount of oxygen supplied to the blood decreases, causing the patient to constantly feel short of breath.

In the present invention, pulmonary fibrosis refers to the terminal form of interstitial pneumonia in a narrow sense, but refers to the coexistence of pulmonary fibrosis and interstitial pneumonia in a narrow sense. Any interstitial pneumonia can cause pulmonary fibrosis. Interstitial pneumonia is a generic term for diseases that cause inflammation in the interstitium of the lungs, and includes those caused by specific causes such as infection, collagen disease, radiation, drugs, and dust, and idiopathic interstitial pneumonia of unknown cause. Idiopathic interstitial pneumonia is classified as idiopathic pulmonary fibrosis (IPF) or nonspecific interstitial pneumonia (NSIP) based on findings from thoracoscopic lung biopsy (VATS) or high-resolution computed tomography (HRCT). , acute interstitial pneumonia (AIP), cryptogenic organizing pneumonia (COP), respiratory bronchiolitis-associated interstitial lung disease (RB-ILD), dissociative interstitial pneumonia It is classified into (desquamative interstitial pneumonia: DIP) and lymphoid interstitial pneumonia (LIP).

Pulmonary fibrosis in the present invention is idiopathic pulmonary fibrosis (Idiopathic Pulmonary Fibrosis), nonspecific interstitial pneumonia (Nonspecific Interstitial Pneumonia), acute interstitial pneumonia (Acute Interstitial Pneumonia), idiopathic organizing pneumonia (Cryptogenic Organizing Pneumonia), respiratory bronchitis-associated epilepsy It may be Respiratory Bronchiolitisassociated Interstitial Lung, Desquamative Interstitial Pneumonia, Lymphoid Interstitial Pneumonia, interstitial pulmonary fibrosis and diffuse pulmonary fibrosis, preferably idiopathic pulmonary fibrosis. may be, but is not limited thereto.

The lung fibrosis of the present invention is caused by various causes, for example, microscopic damage to the lungs induced by inhalation of fine particles (asbestos, stone powder, metal dust, particles present in cigarette smoke, silica dust, etc.) can do. Pulmonary fibrosis can also occur as a secondary effect of other diseases (such as autoimmune diseases, viral or bacterial infections) and include cytotoxic agents (such as bleomycin, busulfan and methotrexate); antibiotics (such as nitrofurantoin and sulfasalazine); antiarrhythmic drugs (such as amiodarone and tocainide); anti-inflammatory drugs (such as gold and penicylamine); It may be caused by certain drugs such as illegal drugs (drugs, cocaine, heroin, etc.), and in the case of idiopathic pulmonary fibrosis, it may be caused by other unknown causes.

In another embodiment of the present invention, the renal fibrosis is a type of disease caused by the accumulation of extracellular matrix in the kidney, and is a chronic kidney disease that reduces renal function or causes loss of renal function (renal failure) in severe cases. It results from the formation or development of excessive fibrous connective tissue in the kidneys (fibrosis). In the present invention, renal fibrosis is accompanied by an inflammatory response caused by endothelial cell disorders, and fibrosis may occur in the excessively produced extracellular matrix (ECM).

In the present invention, renal fibrosis may correspond to glomerular sclerosis, diabetic renal fibrosis, tubular interstitial fibrosis, hypertensive renal fibrosis, and the like, but is not limited thereto if it corresponds to a disease causing fibrosis in kidney tissue.

In the present invention, the factor involved in the fibrosis is selected from the group consisting of ACTA2 (actin alpha 2; alpha smooth muscle actin; α-SMA), COL1A1 (alpha-1 type I collagen), FN1 (fibronectin 1), and the like. It may be any one or more genes or proteins encoded by them, but is not limited thereto.

In the present invention, the ACTA2 is a protein also called alpha-smooth muscle actin (α-SMA), and corresponds to a multifunctional protein family that forms microfilaments. ACTA2 is one of six actin isoforms and is involved in smooth muscle contraction and is found in almost all mammals. Mutations in the ACTA2 gene cause various vascular diseases such as thoracic aortic disease, coronary artery disease, stroke, and multiorgan smooth muscle dysfunction syndrome. In addition, the ACTA2 is commonly called α-SMA, which is a marker of myofibroblast formation and is mainly used in the study of anti-fibrotic effects and fibrosis mechanisms. In particular, the neosynthesis of α-SMA changes lung fibroblasts into myofibroblast phenotype, and is up-regulated at the site where lung fibrosis progresses, and plays a major role in the development and progression of lung fibrosis. is known

In the present invention, COL1A1 encodes a pro-alpha 1 chain of type I collagen including two alpha 1 chains and one alpha 2 chain in a triple helix structure. Type I is fibrinogenic collagen found in most connective tissues and is known to be abundant in bones, corneas, dermis and tendons. When the gene is mutated, osteogenic imperfecta types I-IV, Ehlers-Danlos syndrome type VIIA, and Ehlers-Danlos syndrome classic type (Ehlers) -Danlos syndrome Classical type), Caffey Disease, and idiopathic osteoporosis.

In the present invention, the FN1 encodes fibronectin, a glycoprotein that exists as a soluble dimer in plasma and in a dimer or multimeric form on the cell surface and extracellular matrix. The fibronectin is well known to be involved in cell adhesion and migration processes, including embryogenesis, wound healing, blood clotting, host defense and metastasis.

In one embodiment of the present invention beta-transforming growth factor (Transforming growth factor β; TGF-β) epithelial cells-mesenchymal cell transition to the main TGF-β subtype produced in bronchial epithelial cells and fibroblasts of normal lung tissue ( It is a major factor inducing epithelial-mesenchymal transition (EMT), which is known to induce neosynthesis of α-SMA and change the phenotype of epithelial cells to myofibroblasts. Since fibronectin secretion and α-SMA are upregulated by TGF-β, it is mainly used to study the mechanism of tissue fibrosis.

The composition of the present invention may be used as a pharmaceutical composition, a food composition or a cosmetic composition, but is not limited thereto.

In the present invention, "prevention" may include, without limitation, any act of blocking symptoms caused by fibrosis or suppressing or delaying symptoms of respiratory disease using the composition of the present invention.

In the present invention, "improvement" may include, without limitation, any action that improves or beneficially changes symptoms that have worsened, such as alleviation or reduction of symptoms caused by fibrosis by ingesting or irradiating the composition of the present invention.

In the present invention, "treatment" may include, without limitation, any action in which symptoms due to fibrosis are improved or beneficial using the composition of the present invention.

In the present invention, the pharmaceutical composition may be characterized in the form of capsules, tablets, granules, injections, ointments, powders or beverages, and the pharmaceutical composition may be characterized in that it is targeted to humans.

The pharmaceutical composition of the present invention is not limited thereto, but may be formulated in the form of oral dosage forms such as powders, granules, capsules, tablets, aqueous suspensions, external preparations, suppositories, and sterile injection solutions according to conventional methods, respectively. have. The pharmaceutical composition of the present invention may include a pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers may include binders, lubricants, disintegrants, excipients, solubilizers, dispersants, stabilizers, suspending agents, dyes, fragrances, etc. for oral administration, and in the case of injections, buffers, preservatives, pain-freezing agents A topical agent, solubilizer, isotonic agent, stabilizer, etc. can be mixed and used, and in the case of topical administration, a base, excipient, lubricant, preservative, etc. can be used. The dosage form of the pharmaceutical composition of the present invention can be prepared in various ways by mixing with a pharmaceutically acceptable carrier as described above. For example, in the case of 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. have. In addition, it can be formulated as a solution, suspension, tablet, capsule, sustained release formulation, and the like.

Meanwhile, examples of suitable carriers, excipients and diluents for formulation include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, malditol, starch, acacia gum, 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, it may further include a filler, an anti-agglomeration agent, a lubricant, a wetting agent, a flavoring agent, an emulsifier, a preservative, and the like.

The route of administration of the pharmaceutical composition according to the present invention is, but not limited to, oral, intravenous, intramuscular, intraarterial, intramedullary, intrathecal, intracardiac, transdermal, subcutaneous, intraperitoneal, intranasal, intestinal, topical, sublingual or rectal. Oral or parenteral administration is preferred.

In the present invention, "parenteral" includes subcutaneous, intradermal, intravenous, intramuscular, intraarticular, intrasynovial, intrasternal, intrathecal, intralesional and intracranial injection or infusion techniques. The pharmaceutical composition of the present invention may also be administered in the form of a suppository for rectal administration.

The pharmaceutical composition of the present invention varies depending on several factors including the activity of the specific compound used, age, weight, general health, sex, formula, administration time, administration route, excretion rate, drug formulation, and the severity of the specific disease to be prevented or treated The dosage of the pharmaceutical composition may vary depending on the patient's condition, weight, disease severity, drug form, administration route and period, but may be appropriately selected by those skilled in the art, and may be 0.0001 to 50 mg/kg per day or It may be administered at 0.001 to 50 mg/kg. Administration may be administered once a day, or may be administered in several divided doses. The above dosage does not limit the scope of the present invention in any way. The pharmaceutical composition according to the present invention may be formulated as pills, dragees, capsules, solutions, gels, syrups, slurries, and suspensions.

In the present invention, the food composition is variously used for the prevention or improvement of indications for the purpose of the present invention, and the food composition comprising the composition of the present invention as an active ingredient is a variety of foods, for example, beverages, It can be prepared in the form of gum, tea, vitamin complex, powder, granule, tablet, capsule, confectionery, rice cake, bread, and the like. Since the food composition of the present invention is improved from the existing food intake with little toxicity and side effects, it can be safely used even when taken for a long period of time for the purpose of prevention. When the composition of the present invention is included in the food composition, the amount may be added in a proportion of 0.1 to 100% of the total weight. Here, when the food composition is prepared in the form of a beverage, there is no particular limitation other than containing the food composition in the indicated ratio, and it may contain various flavoring agents or natural carbohydrates as additional ingredients like a conventional beverage. That is, as natural carbohydrates, monosaccharides such as glucose, disaccharides such as fructose, polysaccharides such as sucrose, and common sugars such as dextrin and cyclodextrin, and sugar alcohols such as xylitol, sorbitol and erythritol are included. can do. Examples of the flavoring agent include natural flavoring agents (taumartin, stevia extract (eg, rebaudioside A, glycyrrhizin, etc.) and synthetic flavoring agents (saccharin, aspartame, etc.). Others of the present invention of various nutrients, vitamins, minerals (electrolytes), flavoring agents such as synthetic and natural flavoring agents, coloring agents, pectic acid and salts thereof, alginic acid and salts thereof, organic acids, protective colloidal thickeners, pH adjusting agents, It may contain stabilizer, preservative, glycerin, alcohol, carbonation agent used in carbonated beverage, etc. These components can be used independently or in combination.The proportion of these additives is usually per 100 parts by weight of the composition of the present invention It is generally selected in the range of 0.1 to 100 parts by weight, but is not limited thereto.

In the present invention, the cosmetic composition is a lotion, nutritional lotion, nutritional essence, massage cream, cosmetic bath water additive, body lotion, body milk, bath oil, baby oil, baby powder, shower gel, shower cream, sunscreen lotion, sun Screen cream, suntan cream, skin lotion, skin cream, sunscreen cosmetics, cleansing milk, depilatory makeup, face and body lotion, face and body cream, skin whitening cream, hand lotion, hair lotion, cosmetic cream, jasmine oil , bath soap, water soap, beauty soap, shampoo, hand sanitizer (hand cleaner), medicated soap, medical, cream soap, facial wash, whole body cleaner, scalp cleaner, hair conditioner, makeup soap, tooth whitening gel, toothpaste, etc. can be manufactured with To this end, the composition of the present invention may further include a solvent or an appropriate carrier, excipient or diluent commonly used in the preparation of cosmetic compositions.

In the present invention, the type of solvent that can be further added to the cosmetic composition is not particularly limited, but, for example, water, saline, DMSO, or a combination thereof may be used. In addition, as the carrier, excipient or diluent, purified water, oil, wax, fatty acid, fatty alcohol, fatty acid ester, surfactant, humectant, thickener, antioxidant, viscosity stabilizer, chelating agent, buffer, lower alcohol, etc. included, but not limited to. In addition, if necessary, it may include a whitening agent, a moisturizer, a vitamin, a sunscreen, a perfume, a dye, an antibiotic, an antibacterial agent, an antifungal agent.

In the present invention, hydrogenated vegetable oil, castor oil, cottonseed oil, olive oil, palm seed oil, jojoba oil, and avocado oil may be used as the oil in the present invention, and as the wax, beeswax, spermaceti, carnauba, candelilla, montan, ceresin, Liquid paraffin, lanolin can be used.

In the present invention, as the fatty acid, stearic acid, linoleic acid, linolenic acid, and oleic acid may be used, and as the fatty acid alcohol, cetyl alcohol, octyl dodecanol, oleyl alcohol, panthenol, lanolin alcohol, stearyl alcohol, and hexadecane may be used. All may be used, and as the fatty acid ester, isopropyl myristate, isopropyl palmitate, and butyl stearate may be used. As the surfactant, cationic surfactants, anionic surfactants and nonionic surfactants known in the art can be used, and surfactants derived from natural products are preferred as far as possible. In addition, it may include a desiccant, a thickener, an antioxidant, etc. widely known in the cosmetic field, and the types and amounts thereof are as known in the art.

When the composition of the present invention is used, by inhibiting the activity of histone acetyltransgerase (HAT), various fibrosis-related diseases, including lung fibrosis and renal fibrosis, can be more effectively treated or improved, or the onset thereof can be prevented.

1 is a diagram showing the results of confirming the histone acetylation inhibitory efficacy according to the concentration of acufarin using the HAT activity assay kit, according to an embodiment of the present invention.
2 is a diagram showing the results of confirming cytotoxicity through MTT analysis for each concentration of acufarin in a lung cell line (MRC5 cell) according to an embodiment of the present invention.
3 is a diagram showing the results of comparing and confirming the expression levels of α-SMA, COL1A1, and fibronectin, which are cell fibrosis target markers, using qPCR, according to an embodiment of the present invention.
4 is a view showing the results confirmed by Western blot to measure the protein level of α-SMA of acuparin, according to an embodiment of the present invention.
5 is a comparison of the gene expression levels of COL1A1 and COL3A1, which are cell fibrosis target markers, when acuparin is treated in a kidney-derived cell line (Human proximal tubule cell line; HK2 cell line) according to an embodiment of the present invention using qPCR. A diagram showing the results.
6 is a diagram showing the results of confirming cytotoxicity through MTT analysis for each concentration of acufarin in a kidney-derived cell line (HK2 cell) according to an embodiment of the present invention.
7 is a diagram confirming the antifibrotic effect of acuparin using a bleomycin-induced lung fibrosis mouse model according to an embodiment of the present invention.
8A and 8B are diagrams showing the results of a collagen assay according to the presence or absence of acufarin treatment and the treatment concentration in the control group and the lung fibrosis mouse model according to an embodiment of the present invention.
9 is a diagram showing the results of comparing and confirming the percentage BAL fluid cell differential coefficient of bronchoalveolar lavage (BAL fluid) analysis cells after acuparin treatment in a lung fibrosis mouse model according to an embodiment of the present invention.

Hereinafter, the present invention will be described in more detail through examples. These examples are only for illustrating the present invention in more detail, and it will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples according to the gist of the present invention. .

The program used for statistical analysis of all data of the present invention was GraphPad Prism 6.0 version of GraphPad Software (La Jolla, CA, USA), and the experimental data in the following Examples were analyzed by analysis of variance (ANOVA) Following the analysis, Bonferroni post-hoc test was performed. Data values were expressed including ± error range in the mean, and p value < 0.05 when significant correlation was observed.

Example 1: Confirmation of the inhibitory efficacy of histone acetylation of acuparin (Aucuparin)

1.1 Cell culture

The inventors of the present invention performed all experiments with the approval of the Yonsei University School of Medicine Evaluation Committee (Institutional Review Board, IRB). For the experiment, a lung fibroblast cell line (MRC-5), which has already been confirmed and used in the experiment, was obtained from the American Type Culture Collection (ATCC), and 5% fetal bovine serum according to the guide of the American cell line bank. streptomycin (Gibco) 37 ℃, 5% CO 2 incubator (HERAcell 150i, Thermo Scientific, Waltham, MA, USA) by adding - the contained 5.6mM glucose-containing Dulbecco'smodified Eagle's medium (DMEM) 1 % penicillin in the culture medium It was cultured in and used for the experiment.

1.2 Confirmation of inhibitory efficacy

In order to confirm the efficacy of the acuparin active substance of the present invention, an experiment according to the acuparin concentration was performed using a HAT activity assay kit (Biovision Inc. USA).

The HAT activity assay kit uses acetyl CoA and H3 histone peptides as substrates as a fluorescence assay to analyze a sample for histone acetyltransferase (HAT) activity or screen for HAT inhibitors. In this assay, HAT enzyme is an acetyl group -Catalyze the transfer of -CoA to histone peptide to produce two substances, an acetylated peptide and CoA-SH HAT activity can be detected. Nuclear protein was extracted from the cell line cultured in Example 1.1, and after further treatment with acufarin in the nuclear extract (50ug) of each group, HAT activity was measured according to the manufacturer's guide, and absorbance was measured at 450 nm. The results were converted into percentages and are shown in FIG. 1 .

As shown in FIG. 1 , in the case of the acuparin-treated group, the HAT activity was significantly reduced in a concentration-dependent manner compared to the negative control, and it was found that the inhibitory efficacy of the HAT activity was excellent.

1.3 Identification of cytotoxicity

Next, a cytotoxicity test was performed to confirm the effect of acufarin on the cells of MRC5 cells, which are lung cells. The experiment was conducted by administering the same concentration of acuparin as in the HAT activity inhibition confirmation experiment, and MTT analysis was performed accordingly. The MTT assay corresponds to one of the methods for measuring mitochondrial activity, and this assay is specifically, the cells cultured in Example 1.1 were isolated and cultured in RPMI-1640 medium with 10% fetal bovine serum. (Cells were obtained repeatedly in a short cycle, and confirmed through karyotype analysis and isoenzyme analysis). MTT assay was used to determine the extent of cell proliferation. Cells were seeded in a 96-well plate at 6 x 10 ³ cells per well and incubated overnight to achieve 80% cell confluency. After incubating the cells, check the cell viability using the MTT reagent ((3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium) according to the manufacturer's protocol (Roche, Basel, Switzerland; 114650007001). At this time, absorbance was measured at 550 nm, and viable cells were counted through the trypan blue dye removal method.The data are expressed as a percentage of the signal observed in the control group, which is untreated cells (vehicle-treated cells), shown in FIG. and was shown as the mean ± error range (SEM) of three experiments, as shown in Fig. 2, it was confirmed that there was no cytotoxicity even when acuparin was treated at a concentration of 50 uM.

Example 2: Effect of acufarin on various cell fibrosis (1) - lung cells

2.1 Confirmation of expression level of target markers through qPCR

In order to confirm the effect of acuparin on cell fibrosis, the lung fibroblast line (MRC-5) was dispensed in a 6-well plate and then treated with acuparin by concentration. After treatment, stimulation was performed with TGF-β1 (1uM) for up to 96 hours, and if necessary, an antioxidant N-acetylcysteine (NAC) or an NADPH oxidase inhibitor diphenyleniodonium (DPI) was added to TGF It was pre-treated 1 hour before -β1 administration. After 24 hours, RNA was extracted and cDNA was synthesized to qPCR the expression levels of target markers, alpha-smooth muscle actin (α-SMA), alpha-1 type I collagen (COL1A1) and fibronectin. (quantitative real time polymerase chain reaction, qPCR) was confirmed. The results for the three target factors are shown in FIG. 3 .

As a result of comparing FC (fold change) obtained by dividing the value of the sample treatment group by the value of the negative control group, which is the reference condition value in FIG. 3 , when acufarin was treated, alpha smooth muscle actin (α-SMA) increased by TGF-β1, It was confirmed that the expression levels of both alpha-1 type I collagen (COL1A1) and fibronectin were significantly reduced in a concentration-dependent manner.

2.2 Confirmation of expression level of α-SMA by Western blotting

In order to confirm whether the effect shown in Example 2.1 is the same at the actual protein level, the α-SMA protein level according to the acufarin concentration among the three target factors was confirmed through western blotting, and the results are shown in FIG. 4 . . As a specific experimental method, cells were washed twice with cold PBS and lysed on ice with protein extraction buffer (Pro-Prep, iNtRON Biotechnology) according to the manufacturer's protocol. Protein concentration was determined by BCA assay (Pierce Biotechnology). The same amount of protein (20ug) was separated on an 8-10% sodium dodecyl sulfate-polyacrylamide gel. The digested protein was subjected to electricity on a polyvinylidene fluoride membrane (Millipore). The membrane was then blocked with 5% nonfat milk in TBS-T for 1 h at room temperature, with primary antibodies appropriately diluted to optimal concentrations against α-SMA and β-actin (Santa Cruz Biotechnology). Incubated overnight at 4°C. Then, the membrane was washed 3 to 5 times with TBST, and a secondary antibody corresponding to horse radish peroxidase (Santa Cruz) was conjugated for 1 hour at room temperature. After washing, the blot was developed with ECL reagent (Pierce) and exposed for 3-5 minutes using Kodak X-OMAT AR Film (Eastman Kodak).

As shown in FIG. 4 , it was confirmed that the expression level of alpha smooth muscle actin (α-SMA) increased by TGF-β1 was significantly decreased in a concentration-dependent manner when acuparin was treated. As such, the reduced α-SMA protein level suggests that acufarin can inhibit TGF-β-induced fibrosis, and thus can be used as a therapeutic agent for lung fibrosis.

Example 3: Effect of acuparin on various cellular fibrosis (2) - kidney cells

In order to confirm the effect of acuparin on various cell fibrosis, an experiment using a kidney-derived cell line was additionally performed in vitro. Serum starvation was performed on the kidney-derived (human kidney 2; HK2) cells 2 hours earlier, and about 70% of the plate was treated with 20ng/ml of TGF-β, specifically, acuparin during serum starvation. (1 or 5 uM), treated with TGF-β after serum starvation for 2 hours, and again after 24 hours to obtain cells and qRT-PCR analysis results are shown in FIG. 5 . As a result of comparing and confirming the gene expression levels of COL1A1 and COL3A1, which are cell fibrosis target markers after treatment with acuparin in a kidney-derived cell line, it was confirmed that the collagen gene expression increased by TGF-b was inhibited by acuparin. Additionally, HK2 cell line was treated with acuparin for each concentration, and cytotoxicity was confirmed from cell viability by MTT assay 24 hours later, and the results are shown in FIG. 6 . These results suggest that acuparin can be used as a formulation capable of obtaining a therapeutic effect for renal fibrosis.

Example 4: Effect of acuparin on fibrosis in vivo (3) - In vivo lung fibrosis mouse model experiment

To verify the effect on fibrosis in vivo, a bleomycin-induced lung fibrosis mouse model was first prepared. To induce lung fibrosis, bleomycin (2mg/kg) was intratracheally inserted into C57BL/6 mice to cause lung fibrosis, and then acuparin (8 or 12 mg/kg) was administered every other day for 2 weeks from the next day. It was injected subcutaneously. Two weeks later, the mice were sacrificed, and samples of lung and bronchoalveolar lavage (BAL Fluid) were obtained and analyzed.

As a result, as shown in FIG. 7 , pulmonary fibrosis was induced in mice by treatment with bleomycin (BLM). In addition, referring to FIGS. 8A and 8B , as a result of confirming through a collagen assay according to the presence or absence and treatment concentration of acuparin treatment in the control group and the lung fibrosis-induced mouse model, when acuparin was treated, it was concentration-dependently It was confirmed that the amount of collagen production decreased. Furthermore, as a result of comparatively confirming the differential coefficient of BAL fluid cells through cellular analysis of bronchoalveolar lavage (BAL fluid) (see FIG. 9 ), the percentage of bronchoalveolar lavage cells that are high in pulmonary fibrosis after subcutaneous administration of acufarin It was confirmed that this concentration-dependently decreased significantly.

Taken together, the above results indicate that fibrosis caused by TGF-β signals can be inhibited in various organs when acuparin, which has an HAT inhibitory effect, is treated, and from this, acuparin can be used in various organs including lungs and kidneys. It can be expected that there is a therapeutic effect on fibrotic diseases in which fibrosis occurs in organs.

As described above in detail a specific part of the present invention, for those of ordinary skill in the art, this specific description is only a preferred embodiment, and it is clear that the scope of the present invention is not limited thereto. Accordingly, the substantial scope of the present invention will be defined by the appended claims and their equivalents.

Claims (10)

2,6-dimethoxy-4-phenylphenol (2,6-dimethoxy-4-phenylphenol) represented by the following formula (1) or a pharmaceutically acceptable salt thereof as an active ingredient, a pharmaceutical for the prevention or treatment of fibrosis Composition:
[Formula 1]

The method of claim 1,
The composition is a histone acetylase (histone acetlytransgerase; HAT) that inhibits the activity, a pharmaceutical composition for the prevention or treatment of fibrosis. The method of claim 1,
The fibrosis is lung fibrosis (Pulmonary fibrosis), uterine fibroids, myelofibrosis (myelofibrosis), liver fibrosis (liver fibrosis), heart fibrosis (Heart fibrosis), multiple sclerosis (kidney fibrosis), cystic fibrosis (cystic fibrosis) , neutropenia, skeletal muscle fibrosis, skin sclerosis, dermatomyositis, mediastinal fibrosis (mediastinal fibrosis) and at least one selected from the group consisting of splenic fibrosis caused by sickle cell anemia, a pharmaceutical composition for the prevention or treatment of fibrosis. The method of claim 1,
The pharmaceutical composition is for oral, intravenous, intramuscular, intraarterial, intramedullary, intrathecal, intracardiac, transdermal, subcutaneous, intraperitoneal, intranasal, intestinal, topical, sublingual or rectal administration, for the prevention or treatment of fibrosis pharmaceutical composition. Food for preventing or improving fibrosis, comprising 2,6-dimethoxy-4-phenylphenol represented by the following formula (1) or a pharmaceutically acceptable salt thereof as an active ingredient composition.
[Formula 1]

6. The method of claim 5,
The composition inhibits histone acetylase activity, the food composition for the prevention or improvement of fibrosis. 6. The method of claim 5,
The fibrosis is lung fibrosis (Pulmonary fibrosis), uterine fibroids, myelofibrosis (myelofibrosis), liver fibrosis (liver fibrosis), heart fibrosis (Heart fibrosis), multiple sclerosis (kidney fibrosis), cystic fibrosis (cystic fibrosis) , Neutropenia, skeletal muscle fibrosis, skin sclerosis, dermatomyositis, mediastinal fibrosis (mediastinal fibrosis) and at least one selected from the group consisting of splenic fibrosis caused by sickle cell anemia, food composition for preventing or improving fibrosis. Cosmetics for preventing or improving fibrosis, comprising 2,6-dimethoxy-4-phenylphenol represented by the following formula (1) or a pharmaceutically acceptable salt thereof as an active ingredient Composition:
[Formula 1]

9. The method of claim 8,
The composition inhibits histone acetylase activity, the cosmetic composition for the prevention or improvement of fibrosis. 9. The method of claim 8,
The fibrosis is lung fibrosis (Pulmonary fibrosis), uterine fibroids, myelofibrosis (myelofibrosis), liver fibrosis (liver fibrosis), heart fibrosis (Heart fibrosis), multiple sclerosis (kidney fibrosis), cystic fibrosis (cystic fibrosis) , neutropenia, skeletal muscle fibrosis, skin sclerosis, dermatomyositis, mediastinal fibrosis (mediastinal fibrosis) and at least one selected from the group consisting of splenic fibrosis caused by sickle cell anemia, cosmetic composition for preventing or improving fibrosis.

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