Use for preventing, improving or treating fibrotic CHP

Abstract

The present invention relates to the use of CHP for preventing, improving or treating fibrosis, and more specifically, to a pharmaceutical composition for preventing or treating fibrosis comprising CHP, a health functional food composition for preventing or improving fibrosis, an anti-fibrosis composition, a method for preventing, improving or treating fibrosis using CHP, and / or the use of CHP in preparing a pharmaceutical composition for preventing or treating fibrosis.

Description

Technical Field

[0001] The present invention relates to the use of CHP for preventing, improving or treating fibrosis, and more specifically, to a pharmaceutical composition for preventing or treating fibrosis, an anti-fibrotic composition, a method for preventing or treating fibrosis using CHP, and / or the use of CHP in preparing a pharmaceutical composition for preventing or treating fibrosis. Background Art

[0002] Fibrosis is a disease in which abnormal production, accumulation and deposition of extracellular matrix occur by fibroblasts, and the disease is caused by fibrosis of organs or tissues. Fibrosis is a very fatal disease that induces organ damage. For example, idiopathic pulmonary fibrosis (IPF) is the result of recurrent alveolar epithelial cell damage associated with fibroblast accumulation and myofibroblast differentiation. It is a chronic, progressive and fatal disease that causes excessive accumulation of extracellular matrix (ECM) while causing irreversible damage to lung parenchyma tissue.

[0003] In existing treatment studies, corticosteroids and immunosuppressive drugs are used, usually targeting the inflammatory process of fibrosis. However, such agents have shown little effect in clinical trials, so new drugs are needed to treat fibrosis.

[0004] In addition, Korean Patent Publication No. 10-2013-0006170 discloses a composition for regulating blood sugar containing soybean hydrolyzate containing CHP (CYCLO (His-Pro)) at a high concentration, but the anti-fibrotic effect of CHP is not yet clear. Summary of the invention

[0005] Technical issues

[0006] An object of the present invention is to provide a pharmaceutical composition comprising CHP for preventing or treating fibrosis.

[0007] Another object of the present invention is to provide an anti-fibrosis composition comprising CHP.

[0008] Another object of the present invention is to provide a method for preventing or treating fibrosis using CHP.

[0009] Another object of the present invention is to provide the use of CHP in preparing a pharmaceutical composition for preventing or treating fibrosis.

[0010] Technical Solution

[0011] In order to solve the above-mentioned problems, the present invention provides a pharmaceutical composition for preventing or treating fibrosis, comprising CHP or a pharmaceutically acceptable salt thereof.

[0012] Furthermore, the present invention provides a method for preventing or treating fibrosis, comprising the step of administering an effective amount of CHP to an individual in need thereof.

[0013] Furthermore, the present invention provides the use of CHP in the preparation of a pharmaceutical composition for preventing or treating fibrosis.

[0014] According to a preferred embodiment of the present invention, the above-mentioned fibrosis may occur in one or more of the group consisting of kidney, liver, lung, skin, heart, pancreas, urinary system, reproductive system, sweat glands, nerves, brain, bone marrow, muscles and joints.

[0015] According to another preferred embodiment of the present invention, the above-mentioned fibrosis is one or more selected from the group consisting of pulmonary fibrosis, idiopathic pulmonary fibrosis, radiation-induced lung injury or pulmonary fibrosis, pulmonary edema, cystic fibrosis, liver fibrosis, endomyocardial fibrosis, myocardial infarction, atrial fibrosis, glial scar, renal fibrosis, myelofibrosis, arthrofibrosis, adipose fibrosis, skin fibrosis, neural fibrosis and muscle fibrosis.

[0016] Furthermore, the present invention provides an anti-fibrotic composition comprising CHP.

[0017] Effects of the Invention

[0018] The composition comprising CHP of the present invention inhibits fibrosis occurring in various tissues or organs such as kidney, liver, lung, skin, heart, pancreas, urinary system, reproductive system, sweat glands, nerves, brain, bone marrow, muscles and joints, thereby effectively preventing, improving or treating fibrosis. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 The diagram shows the isolation and primary culture process of cells from human glomeruli and tubules (left: glomeruli, right: tubules).

[0020] Figure 2The graph shows changes in cell morphology according to CHP treatment concentrations (4 μg / ml, 20 μg / ml, and 100 μg / ml, respectively) in a human-derived proximal tubular epithelial cell fibrosis model.

[0021] Figure 3 Western blotting was used to confirm the changes in the expression levels of E-cadherin, fibronectin, and pSTAT3 proteins according to the CHP treatment concentrations (4 μg / ml, 20 μg / ml, and 100 μg / ml, respectively) in the human proximal tubular epithelial cell fibrosis model.

[0022] Figure 4 The graph shows changes in cell morphology according to CHP treatment concentrations (40 μg / ml and 100 μg / ml, respectively) in a human-derived glomerular endothelial cell fibrosis model.

[0023] Figure 5a Western blotting was used to confirm the changes in the expression level of fibronectin according to the CHP treatment concentrations (62.5 ng / ml, 125 ng / ml, and 250 ng / ml, respectively) in the human hepatocyte fibrosis model. Figure 5b The band sizes were quantified and the results were graphed.

[0024] Figure 6a Western blotting was used to confirm the changes in the expression level of fibronectin according to the CHP administration concentration (5 mg / kg and 35 mg / kg, respectively) in the animal model of liver fibrosis. Figure 6b The band sizes were quantified and the results were graphed.

[0025] Figure 7a Western blotting was used to confirm the changes in the expression level of fibronectin according to the CHP treatment concentration (62.5 ng / ml and 125 ng / ml, respectively) in the human lung cell fibrosis model. Figure 7b The band sizes were quantified and graphed.

[0026] Figure 8 The results are graphed showing changes in transforming growth factor β (TGFβ) and collagen 3 gene expression levels according to CHP administration concentrations (5 mg / kg and 35 mg / kg, respectively) in an animal model of pulmonary fibrosis.

[0027] Figure 9aWestern blotting was used to confirm the changes in the expression level of fibronectin according to the CHP treatment concentrations (62.5 ng / ml, 125 ng / ml, 250 ng / ml, and 500 ng / ml, respectively) in the human skin cell fibrosis model. Figure 9b The band sizes were quantified and graphed.

[0028] Fig.10 The results are graphically plotted showing changes in gene expression levels of fibrosis markers transforming growth factor-β, fibronectin, collagen 1, collagen 2, collagen 3, and collagen 4 according to CHP administration concentrations (5 mg / kg and 35 mg / kg, respectively) in an animal model of cardiac fibrosis.

[0029] Fig.11 The results are graphed showing changes in gene expression levels of fibrosis markers transforming growth factor-β, fibronectin, collagen 3, and connective tissue growth factor (CTGF) according to CHP administration concentrations (5 mg / kg and 35 mg / kg, respectively) in an animal model of adipose fibrosis. DETAILED DESCRIPTION

[0030] Hereinafter, the present invention will be described in more detail.

[0031] As mentioned above, in existing fibrosis-related treatment studies, corticosteroids and immunosuppressive drugs are used, usually targeting the inflammatory process of fibrosis, but such preparations have shown little effect in clinical trials, so new drugs for the treatment of fibrosis are needed.

[0032] Therefore, the present inventors confirmed that CHP inhibits fibrosis occurring in various tissues or organs and is effective in preventing, improving or treating fibrosis, thereby completing the present invention.

[0033] Therefore, the present invention provides a pharmaceutical composition for preventing or treating fibrosis, comprising CHP or a pharmaceutically acceptable salt thereof.

[0034] Furthermore, the present invention provides an anti-fibrotic composition comprising CHP.

[0035] In the present invention, "CHP (Cyclo-HisPro)" refers to a naturally occurring cyclic dipeptide composed of histidine-proline which is a metabolite of thyrotropin-releasing hormone (TRH) or a physiologically active dipeptide synthesized in vivo through the metabolic process of thyrotropin-releasing hormone and de novo, and refers to a substance widely distributed throughout the brain, spinal cord, gastrointestinal tract, etc.

[0036] In the composition of the present invention, the CHP may be synthesized or commercially available, and may be purified from substances containing CHP, such as prostate extracts and soybean hydrolyzates, for use.

[0037] The term "purified" is used to mean that CHP is in a concentrated form compared to the form that can be obtained from natural sources such as prostate extract. Purified components can be concentrated from their natural sources or obtained by chemical synthesis methods.

[0038] In the present invention, the term "fibrosis" is used interchangeably with "fibrotic disorder", "fibroproliferative disorder", "fibrotic disease", "fibroproliferative disease", "fibrotic disorder" and "fibroproliferative disorder" to refer to a disorder, disease or disorder characterized by dysregulated proliferation or activity of fibroblasts, abnormal accumulation of fibronectin and / or pathological or excessive accumulation of collagen tissue. Generally, such a disorder, disease or disorder can be treated by administering a compound having anti-fibrotic activity.

[0039] For example, the fibrosis may occur in one or more organs selected from the group consisting of kidney, liver, lung, skin, heart, pancreas, urinary system, reproductive system, sweat glands, nerves, brain, bone marrow, muscles, and joints.

[0040] For example, the fibrosis of the present invention can be one or more selected from the group consisting of pulmonary fibrosis, idiopathic pulmonary fibrosis, radiation lung injury or pulmonary fibrosis, pulmonary edema, cystic fibrosis, liver fibrosis, endomyocardial fibrosis, myocardial infarction, atrial fibrosis, glial scar, renal fibrosis, myelofibrosis, arthrofibrosis, adipose fibrosis, skin fibrosis, neural fibrosis and muscle fibrosis, but is not limited thereto.

[0041] Specifically, the CHP of the present invention has therapeutic effects on various fibrosis described in this application.

[0042] Among the uses of the composition of the present invention for preventing, improving, or treating fibrosis, the first embodiment relates to renal fibrosis, which is fibrosis occurring in the kidney.

[0043] Kidney diseases are classified into acute renal failure and chronic renal failure according to the state of progression, or into glomerulonephritis caused by the deposition of vascular complexes, diabetic nephropathy accompanied by diabetes or hypertensive nephropathy accompanied by hypertension, toxic nephropathy caused by the administration of drugs such as antibiotics or anticancer agents, bacterial infection, etc. Regardless of the cause of kidney disease, if the glomerular filtration rate decreases to less than 50% due to chronic renal insufficiency, in most cases, the glomerular filtration rate continues to decrease, eventually leading to late-stage renal failure, and inducing complications such as blood abnormalities, nervous system complications, gastrointestinal complications, immune complications, infections or bone dystrophy, and in severe cases, death.

[0044] Chronic renal failure refers to a state in which renal function gradually declines in one direction (irreversibility) and cannot maintain the homeostasis of the organism. All kidney diseases are accompanied by renal fibrosis, which eventually leads to late-stage renal failure. In particular, chronic renal function decline is closely related to the progression of renal fibrosis. Therefore, when the progression of fibrosis is inhibited, the progression of chronic renal failure can be inhibited.

[0045] The term "renal fibrosis" in the present invention includes diseases in which fibrosis occurs in the kidney due to various reasons. Fibrosis may include one or more selected from the group consisting of catheter installation, glomerular sclerosis, glomerulonephritis, nephritis, acute renal failure, chronic renal failure, end-stage renal disease and metabolic diseases, but is not limited to this.

[0046] The type of nephritis mentioned above may be any interstitial nephritis, for example, streptococcal nephritis, staphylococcal nephritis, pneumococcal nephritis, viral nephritis associated with chickenpox, hepatitis B, hepatitis C, HIV, etc., nephritis caused by parasitic infections such as malaria, infectious interstitial nephritis associated with fungal nephritis, mycoplasma nephritis, etc., interstitial nephritis associated with systemic lupus erythematosus (lupus nephritis), systemic scleroderma (collagenous kidney), Sjögren's syndrome and other collagen diseases, nephritis associated with vascular immune diseases such as purpura nephritis, polyarteritis, rapidly progressive glomerulonephritis, interstitial nephritis associated with radiation exposure, nephritis caused by gold preparations, NSAIDs, penicillamine (penicillin) and other drugs. The invention also includes drug-induced interstitial nephritis caused by anticancer drugs such as lamine and bleomycin, antibiotics, paraquat, etc., allergic nephritis caused by insect stings, pollen, Anacardiaceae plants, etc., nephritis accompanied by amyloidosis, diabetic nephropathy, chronic glomerulonephritis, malignant nephrosclerosis, polycystic kidney disease, etc., tubulointerstitial nephritis, nephritis accompanied by pregnancy poisoning or cancer, membranous proliferative glomerulonephritis, IgA nephropathy, mixed cryoglobulinemia nephritis, Goodpasture's syndrome nephritis, Wegener's granulomatosis nephritis, idiopathic interstitial nephritis such as acute interstitial nephritis, but is not limited to these.

[0047] As in Figure 2 As demonstrated in Figure 4, CHP restored the expression of E-cadherin, a junction marker, and reduced the expression of fibronectin and pSTAT3 proteins in fibrosis-induced proximal tubular epithelial cells, thereby showing a fundamental anti-fibrotic effect and can be used to treat various renal fibrosis regardless of the cause of fibrosis induction.

[0048] In the use of the composition of the present invention for preventing, improving or treating fibrosis, the second embodiment relates to liver fibrosis, which is fibrosis occurring in the liver.

[0049] The term "liver fibrosis" in the present invention refers to the symptoms of fibrous tissue proliferation caused by chronic liver damage, which may include one or more selected from the group consisting of chronic liver disease, hepatitis B virus infection, hepatitis C virus infection, hepatitis D virus infection, schistosomiasis, alcoholic liver disease or non-alcoholic fatty liver disease, metabolic diseases, protein deficiency, coronary artery disease, autoimmune hepatitis, cystic fibrosis, α-1 antitrypsin deficiency, primary biliary cirrhosis, drug reaction and toxins, but is not limited thereto.

[0050] Liver fibrosis is a precursor of cirrhosis and is the result of severe liver damage that leads to chronic liver disease. It is caused by the action of various cytokines and growth factors. Generally, liver fibrosis is reversible and consists of thin fibers. When nodules are formed and the cause of liver damage is temporary, the increased extracellular matrix is ​​decomposed by apoptosis and matrix metalloproteinases (MMP), thereby returning to normal. However, if the liver fibrosis process continues repeatedly, thick fibers are formed and develop into nodular cirrhosis. In addition, due to various inflammatory inducing factors, liver cells are damaged and cirrhosis is induced by the process of liver fibrosis in which abnormal extracellular matrix proteins including collagen accumulate. In order to regulate the expression of cirrhosis, it is important to regulate the accumulation of extracellular matrix. The inflammatory response in the case of liver cell damage activates hepatic stellate cells in the resting stage to secrete extracellular matrix and various cytokines and chemokines, among which transforming growth factor-β1 plays a role as a potent growth inhibitor. Transforming growth factor-β1 is a 25 kD substance that is secreted in an inactive latent form by binding to latent TGF-β1 binding protein. It exists in a state of binding to extracellular matrices such as type 1 collagen, type 4 collagen, laminin, and decorin, and is activated by various stimuli. Transforming growth factor-β1 regulates collagen expression by reducing the production of collagenase or increasing the production of collagenase inhibitors, and increases the production of tumor necrosis factor-α (TNF-α), interleukin-1 (IL-1), and platelet-derived growth factor (PDGF) in macrophages, playing an important role in the fibrosis process. Currently, transforming growth factor-β1 is only expressed in the location where fibrosis progresses, and is not expressed in normal liver tissue or inactive areas. It is well known that transforming growth factor-β1 plays an important role in liver fibrosis.

[0051] In addition, non-alcoholic fatty liver disease can be induced by obesity, diabetes, hyperlipidemia, drugs and the like regardless of alcohol consumption, and refers to a wide range of diseases including simple fatty liver (steatosis) not accompanied by inflammatory response, non-alcoholic steatohepatitis (NASH) accompanied by hepatocellular inflammation, advanced fibrosis and liver cirrhosis, depending on the progression.

[0052] Nonalcoholic fatty liver disease (NAFLD) is an increasing adult disease caused by a high-fat and high-calorie diet in modern society. According to reports, based on developed countries, 20% to 30% of the adult population exhibits nonalcoholic fatty liver disease, of which 2% to 3% will develop nonalcoholic steatohepatitis. In particular, histologically, patients with steatohepatitis that exhibits fibrosis and inflammation have a very high risk of developing cirrhosis, liver failure, and liver cancer.

[0053] Thus, in one embodiment of the present invention, an animal model of liver fibrosis induced by a high-fat diet was established, and it was confirmed that CHP showed an effect of reducing zonulin expression in the corresponding model.

[0054] Fatty liver itself is not a pathological condition, but a reversible symptom that will naturally recover after the pathogen is removed. However, if excessive fat accumulation in liver tissue continues, fatty hepatitis will occur, resulting in repeated liver cell necrosis and regeneration. During this process, the fibrous extracellular matrix increases and develops into liver fibrosis. When liver damage reaches a certain stage, regardless of the type of pathogen, the accumulation of extracellular matrix increases, and regenerative nodules are formed due to the continuous destruction and regeneration of liver cells, which deteriorates into irreversible liver cirrhosis.

[0055] Therefore, in the present invention, liver fibrosis is a disease that is clearly different from fatty liver, which is a reversible symptom, and refers to all diseases in which liver tissue is transformed into fibrotic tissue such as regenerative nodules, thereby reducing liver function.

[0056] As confirmed in FIG. 5 , CHP reduced the expression of fibronectin, a fibrogenic protein, in fibrosis-induced hepatocytes, and as confirmed in FIG. 6 , CHP reduced the expression of fibronectin in an animal model of induced liver fibrosis, showing a fundamental anti-fibrotic effect. Regardless of the cause of induced fibrosis, the present invention can also be applied to liver failure and liver cancer developed from cirrhosis, including various liver fibrosis.

[0057] Among the uses of the composition of the present invention for preventing, improving or treating fibrosis, the third embodiment relates to pulmonary fibrosis, which is fibrosis occurring in the lungs.

[0058] Among fibrosis, pulmonary fibrosis refers to a disease in which chronic inflammatory cells infiltrate the alveolar walls of lung tissue to induce tissue fibrosis and cause serious structural changes in lung tissue. Once fibrosis is caused by any reason, the lung tissue becomes hard and the alveolar walls become thicker, thereby reducing the amount of oxygen supplied by the blood and causing difficulty breathing. Currently, there is no treatment that can completely restore fibrotic lung tissue. Therefore, except for early detection of fibrosis or lung transplantation, patients usually die 3 to 5 years after the onset of symptoms.

[0059] Specifically, in the present invention, the term "pulmonary fibrosis" refers to the development of scar (fibrotic) tissue due to excessive formation of fibrous connective tissue or development of fibrous connective tissue (fibrosis) in the lungs. Specifically, pulmonary fibrosis refers to a chronic disease that induces swelling and scarring of alveoli and interstitial tissue of the lungs. Scar tissue as described above replaces healthy tissue to induce inflammation, and chronic inflammation can be determined as a precursor to fibrosis. Due to damage to the lung tissue as described above, the lungs may become stiff, and the individual may have difficulty breathing on their own.

[0060] In the present invention, pulmonary fibrosis may include one or more selected from the group consisting of idiopathic pulmonary fibrosis, radiation lung injury, nonspecific interstitial pneumonia, acute interstitial pneumonia, cryptogenic organizing pneumonia, respiratory bronchiolitis associated interstitial lung disease, desquamative interstitial pneumonia, lymphoid interstitial pneumonia, interstitial pulmonary fibrosis and diffuse pulmonary fibrosis, pulmonary edema, cystic fibrosis and pulmonary fibrosis caused by metabolic diseases, but is not limited thereto.

[0061] The above-mentioned pulmonary fibrosis can be caused by various reasons, for example, inhalation of fine particles (asbestos, stone dust, metal dust, particles in cigarette smoke, silica dust, etc.) causes microscopic damage to the lungs. In addition, pulmonary fibrosis also occurs as a secondary effect of other diseases (autoimmune diseases, viral or bacterial infections, etc.), and can also be induced by specific drugs such as cytotoxic agents (bleomycin, busulfan and methotrexate, etc.), antibiotics (nitrofurantoin and sulfasalazine, etc.), arrhythmia treatment agents (amiodarone and tocainide, etc.), anti-inflammatory drugs (gold and penicillamine, etc.), and illegal drugs (drugs, cocaine and heroin, etc.). The above-mentioned idiopathic pulmonary fibrosis can be caused by other unknown causes in addition to the above-mentioned reasons. In addition, long-term intake of a high-fat diet can induce pulmonary fibrosis, which is caused by the increase in inflammation levels after high-fat intake.

[0062] As confirmed in FIG. 7 , CHP reduced the expression of fibronectin, a fibrogenic protein, in fibrosis-induced lung cells. Figure 8 It was confirmed in the literature that CHP reduced the expression of transforming growth factor-β and collagen 3 genes, which are major fibrosis markers, in an animal model of induced pulmonary fibrosis, showing a fundamental anti-fibrotic effect. Therefore, it can be used to treat various pulmonary fibrosis regardless of the cause of fibrosis induction.

[0063] Among the uses of the composition of the present invention for preventing, improving or treating fibrosis, the fourth embodiment relates to skin fibrosis.

[0064] In the present invention, the term "skin fibrosis" is excessive scarring of the skin and is the result of a pathological wound healing response. The range of fibrotic skin diseases is very wide: scleroderma, renal fibrosing skin disease, mixed connective tissue disease, sclerosing myxedema, sclerosing edema and eosinophilic fasciitis. Exposure to chemical substances or physical factors (mechanical trauma, burns) is also a potential cause of fibrotic skin diseases. Skin fibrosis can be dominated by immune, autoimmune and inflammatory mechanisms. The balance of collagen production and decomposition of fibroblasts plays an important role in the pathophysiological process of skin fibrosis. Specific cytokines such as transforming growth factor-β (TGB-β) and interleukin-4 (IL-4) promote wound healing and fibrosis. Fibroblasts in normal skin are quiescent. They control the amount of connective tissue proteins and have low proliferation activity. After the skin is damaged, these cells are activated, that is, they express α-smooth muscle actin (α-SMA) and synthesize a large amount of connective tissue proteins. Activated cells are usually called muscle fibroblasts.

[0065] In the present invention, the term "skin fibrosis" is also intended to include "scleroderma".

[0066] Scleroderma is a chronic autoimmune connective tissue disease of unknown cause, characterized by excessive accumulation of collagen in the dermis, causing sclerotic changes and abnormalities in the vascular system that harden and thicken a part of the skin or the entire skin. Collagen constitutes connective tissue and plays a role in supporting and connecting body tissues. Scleroderma has several forms, some showing symptoms only in specific parts of the body, and some showing symptoms throughout the body including internal organs. That is, it is divided into localized scleroderma, which hardens only a part of the skin, and systemic scleroderma, which shows fibrosis symptoms in internal organs such as the lungs, digestive system, kidneys and heart including the skin due to increased collagen. Systemic scleroderma is divided into localized and extensive types according to the degree of invasion of the skin and internal organs, prognosis, and immunological test results.

[0067] In the prevention, improvement or treatment of skin fibrosis of the present invention, skin fibrosis may include one or more selected from the group consisting of scars, hypertrophic scars, keloids, localized scleroderma and systemic scleroderma, but is not limited thereto.

[0068] The cause of scleroderma is still unknown, but fibrosis of the tissue is thought to play an important role.

[0069] The early symptoms of scleroderma are very diverse, and in particular, skin symptoms become apparent in the later stages. Common symptoms of scleroderma include joint pain, morning stiffness, fatigue, and weight loss. Also, when exposed to cold, the blood supply to the fingers, toes, nose, and ears is temporarily restricted. This symptom is an early symptom of scleroderma patients and is one of the common symptoms. The skin of patients with scleroderma becomes hardened. This phenomenon of hardening of the skin usually occurs on both sides of the body while spreading widely. Eventually, tissues are damaged and skin pigmentation occurs.

[0070] The incidence of "localized scleroderma" in women is 2.6 times that in men. 75% of patients with localized scleroderma are between 20 and 50 years old, and linear scleroderma tends to develop at an earlier age. Localized scleroderma is divided into localized scleroderma, systemic localized scleroderma, linear localized scleroderma, or subcutaneous localized scleroderma.

[0071] Localized scleroderma usually starts with a red or purple-red spot with a clear boundary from the surrounding normal skin. The skin in a certain area loses elasticity and becomes hard. The area is brown, and in most cases, it fades and turns white. Most of the time, it occurs in one place, but there are cases where multiple spots occur. The sizes vary from coin size to adult palm size. Although it causes cosmetic problems, it does not develop into systemic sclerosis. In some patients, the hard skin can soften and recover on its own even without treatment.

[0072] Systemic localized scleroderma is a severe form of localized scleroderma, a condition in which a large area of ​​the skin hardens and hyperpigmentation is observed. The skin of the trunk, buttocks, and legs hardens extensively. However, it is distinguished from systemic sclerosis because there is no Raynaud's phenomenon or symptoms of invasion of internal organs.

[0073] Linear localized scleroderma is a condition where the skin hardens in a straight line, often in the order of legs, arms, forehead, and chest. In particular, the condition where the forehead is sunken in a vertical line is called en coup desabre, which causes cosmetic problems. Unlike localized scleroderma, linear scleroderma not only invades the skin, but also invades the muscles and periosteum tissues under the skin to fix it to the underlying tissues. It is more common in children and can sometimes cause severe dysplasia in the limbs and face.

[0074] Subcutaneous localized scleroderma shows hardening of the fat layer, fascia, muscle, and bone, and joint movement may be limited. Because the lesions occur deep, the characteristic skin pigment changes of scleroderma are not observed.

[0075] The incidence of systemic scleroderma in women is four times that in men. It can occur at any age, but is most common between the ages of 30 and 50. Compared with the localized type, the age of onset of the generalized type is younger. Raynaud's phenomenon is the first symptom of systemic scleroderma, and then sclerosis symptoms begin to occur in the skin and internal organs. Systemic scleroderma is divided into skin symptoms and internal organ symptoms.

[0076] In systemic sclerosis, the first skin symptoms begin in the fingers and hands. At first, only Raynaud's phenomenon is manifested, and then the fingers and hands become swollen, stiff, and red. The skin gradually hardens and spreads to the arms and face, and in the case of the generalized type, the skin continues to harden all over the body including the trunk. When hardening occurs on the face, wrinkles disappear overall and it becomes difficult to make expressions. The nose looks pointed, it is difficult to open the mouth, the lips become thin, and radial wrinkles appear around the mouth, making it look like an old man's mouth. Finger and toe sclerosis manifests as limited joint movement, inability to hold the hand all the time, and fingers bending and becoming pointed. Painful and difficult-to-heal ulcers appear on the fingertips and joints.

[0077] Generalized hyperpigmentation and focal hypopigmentation may be present, with hair loss and decreased sweating in affected areas. Telangiectasias may appear as circular macules on the face and upper trunk or may be observed around the nail creases. If the capillaries in the nail creases of patients with Raynaud's disease are abnormal, systemic sclerosis may later develop, and changes in the capillaries are important in determining prognosis. Skin calcifications may be observed and are relatively common around the finger joints.

[0078] Visceral organ symptoms occur in the gastrointestinal tract, lungs, heart, or kidneys. In the gastrointestinal tract, the esophagus is the most commonly affected site, with more than 90% of cases being affected. Reduced motility of the esophagus, dysphagia, and reflux esophagitis often occur. Due to reduced intestinal motility, symptoms such as constipation, diarrhea, and malabsorption may occur. The most important cause of death in patients with systemic sclerosis is lung symptoms, which occur in about 70% of patients. Due to pulmonary fibrosis, when exercising, patients complain of dyspnea and cough. Alveolitis often occurs, causing a continuous decline in lung function. In the heart, symptoms of cardiac conduction disorders, heart failure, and pericarditis may occur, and myocardial sclerosis may occur in 50% to 70% of patients. Kidney symptoms occur in about 45% of systemic sclerosis patients. It manifests as sudden acute renal failure, hypertension, etc., and slowly progressive uremia may also occur.

[0079] Therefore, in the use of the present invention for preventing, improving or treating skin fibrosis, skin fibrosis includes without limitation the lumen of organs or glands such as blood vessels and veins, ducts of submandibular gland, gall bladder, thyroid follicles, sweat gland ducts, ovaries, kidneys; epithelial cells of gums, tongue, palate, nose, throat, esophagus, stomach, small intestine, rectum, anus and vagina; fibrosis of any skin tissue and epithelial cells in dermis, scars, skin and scalp.

[0080] As confirmed in FIG. 9 , CHP exhibits a fundamental anti-fibrotic effect by reducing the expression of fibronectin, a fibrotic protein, in fibrosis-inducing skin cells, and therefore can be applied to the treatment of various skin fibrosis (scleroderma) regardless of the cause of fibrosis induction.

[0081] Among the uses of the composition of the present invention for preventing, improving or treating fibrosis, the fifth embodiment relates to cardiac fibrosis, which is fibrosis occurring in the heart.

[0082] In the present invention, the term "cardiac fibrosis" refers to the phenomenon that the heart becomes hard due to excessive deposition of matrix proteins between cardiac cells, which mainly occurs in the hearts of patients with myocardial infarction and refers to the main cause of reduced cardiac function. It may include one or more selected from the group consisting of endomyocardial fibrosis, atrial fibrosis, heart failure, myocardial infarction and cardiac fibrosis caused by metabolic diseases, but is not limited to this.

[0083] Fibrosis is characterized by the uneven accumulation of fibrotic collagen that occurs following myocardial cell death, inflammation, increased workload, hypertrophy, and stimulation by multiple hormones, cytokines, and growth factors.

[0084] Cardiac fibrosis can also be abnormal thickening of the heart valves caused by the unbalanced proliferation of cardiac fibroblasts, and further refers to the proliferation of fibroblasts in the heart muscle. Fibrocytes (Fibrocyte cells) usually refer to collagen and play a role in providing structural support for the heart. If overactivated, this process becomes the cause of valve thickening and fibrosis.

[0085] Cardiac fibrosis is the main cause of heart failure and myocardial infarction, and thus the term "cardiac fibrosis" may be interpreted as including heart failure and / or myocardial infarction induced by cardiac fibrosis.

[0086] like Fig.10 It was confirmed that CHP reduced the expression of fibronectin, collagen 1, collagen 2, collagen 3 and collagen 4 genes, which are major fibrosis markers, in an animal model of cardiac fibrosis induced by CHP, showing a fundamental anti-fibrotic effect. Therefore, it can be used to treat various cardiac fibrosis regardless of the cause.

[0087] Among the uses of the composition of the present invention for preventing, improving or treating fibrosis, the sixth embodiment relates to adipose fibrosis.

[0088] In the present invention, the term "adipose fibrosis" refers to all diseases involving fibrosis in adipose tissue.

[0089] It has been reported that when normal mice are fed a high-fat diet containing 60% fat for 16 to 24 weeks, fibrosis of adipose tissue occurs (Hu, M et al., Evidence-Based Complementary and Alternative Medicine, 1-12, 2018; Kwon, EY, & Choi, MS, Nutrients, 10(10), 1415, 2018; Nakazeki, F et al., Scientific Reports, 8(1), 2018; Muniappan, L et al..Scientific Reports, 7(1), 2017; Lancha, A et al., PLoS ONE, 9(5), e98398, 2014; Velázquez, KT et al., Physiological Reports, 5(18), e13412, 2017; Wang, L., Ye, X., Hua, Y., & Song, Y., Biomedicine & Pharmacotherapy, 105, 121-129, 2018).

[0090] Therefore, the present inventors established an animal model of adipose fibrosis induced by a high-fat diet and confirmed the changes in the expression of transforming growth factor-β, fibronectin, collagen 3, and connective tissue growth factor genes, which are fibrosis markers, according to the administration of CHP. Fig.11 It was confirmed in the literature that CHP exhibits anti-fibrotic effects by significantly reducing the expression of the above-mentioned fibrosis markers and can therefore be used to treat adipose fibrosis.

[0091] As described above, in the present invention, it is confirmed that in various tissue cells and animal models that induce fibrosis, CHP exhibits anti-fibrotic activity by reducing the expression of various fibrosis marker genes or proteins, and it can be predicted that it can be effectively applied to the prevention, improvement or treatment of fibrosis occurring in various tissues and / or organs in the body including the kidney, liver, lung, skin, heart, pancreas, urinary system, reproductive system, sweat glands, nerves, brain, bone marrow, muscles and joints.

[0092] In the composition for preventing, improving or treating fibrosis of the present invention, the terms "prevention", "improvement" and / or "treatment" refer to all actions that inhibit or delay a disease or condition, all actions that improve or beneficially change the state of a disease or condition, and all actions that delay, stop or reverse the progression of a disease or condition.

[0093] In the present application, the term "pharmaceutically acceptable" means that it is physiologically acceptable and generally does not cause allergic reactions or similar reactions when administered to the human body. Preferably, the above salt is an acid addition salt formed by a pharmaceutically acceptable free acid.

[0094] The above-mentioned pharmaceutically acceptable salt can be an acid addition salt formed by an organic acid or an inorganic acid, and the above-mentioned organic acid includes formic acid, acetic acid, propionic acid, lactic acid, butyric acid, isobutyric acid, trifluoroacetic acid, malic acid, maleic acid, malonic acid, fumaric acid, succinic acid, succinic acid monoamide, glutamic acid, tartaric acid, oxalic acid, citric acid, glycolic acid, glucuronic acid, ascorbic acid, benzoic acid, phthalic acid, salicylic acid, anthranilic acid, dichloroacetic acid, aminooxyacetic acid, benzenesulfonic acid, p-toluenesulfonic acid or methylsulfonic acid. Inorganic acid includes for example hydrochloric acid, bromic acid, sulfuric acid, phosphoric acid, nitric acid, carbonic acid or boric acid. The acid addition salt is preferably a chlorate or acetate form, more preferably a chlorate form.

[0095] Besides these, other examples of acceptable salts include γ-aminobutyric acid (GABA) salts, gabapentin salts, pregabalin salts, nicotinate salts, adipate salts, hemi-malonate salts, cysteine ​​salts, acetylcysteine ​​salts, methionine salts, arginine salts, lysine salts, ornithine salts or aspartate salts and the like.

[0096] In addition, the pharmaceutical composition of the present invention may also include a pharmaceutically acceptable carrier. A pharmaceutically acceptable carrier may include, for example, a carrier for oral administration or a carrier for parenteral administration. Carriers for oral administration may include lactose, starch, cellulose derivatives, magnesium stearate, stearic acid, etc. Carriers for parenteral administration include water, appropriate oils, saline solutions, aqueous glucose solutions, ethylene glycol, etc. In addition, the above-mentioned carrier may also include a stabilizer and a preservative. Suitable stabilizers include antioxidants such as sodium bisulfite, sodium sulfite or ascorbic acid. Suitable preservatives include benzalkonium chloride, methyl parahydroxybenzoate or propyl parahydroxybenzoate and chlorobutanol. Other pharmaceutically acceptable carriers may refer to the following literature (Remington's Pharmaceutical Sciences, 19th ed., Mack Publishing Company, Easton, PA, 1995).

[0097] The pharmaceutical composition of the present invention can be administered to mammals including humans by any method. For example, it can be administered orally or parenterally. The method of parenteral administration can be intravenous, intramuscular, intraarterial, intramedullary, intradural, intracardiac, transdermal, subcutaneous, intraperitoneal, intranasal, intestinal, topical, sublingual, and rectal administration, but is not limited thereto.

[0098] The pharmaceutical composition of the present invention can be formulated into a preparation for oral administration or parenteral administration according to the administration route as described above. In the case of formulation, it can be prepared by using more than one buffer (e.g., saline solution or phosphate buffered saline (PBS)), carbohydrates (e.g., glucose, mannose, sucrose or dextran, etc.), antioxidants, antibacterial agents, chelating agents (e.g., ethylenediaminetetraacetic acid (EDTA) or glutathione), fillers, extenders, adhesives, adjuvants (e.g., aluminum hydroxide), suspending agents, thickeners, wetting agents, disintegrating agents, surfactants, diluents or excipients.

[0099] Solid preparations for oral administration include tablets, pills, powders, granules, liquids, gels, syrups, slurries, suspensions or capsules, etc. Such solid preparations can be prepared by mixing at least one excipient with the pharmaceutical composition of the present invention, and examples of the above excipients include starch (including corn starch, wheat starch, rice starch and potato starch, etc.), calcium carbonate, sucrose, lactose, glucose, sorbitol, mannitol, xylitol, erythritol maltitol, cellulose, methylcellulose, sodium carboxymethylcellulose, hydroxypropyl methylcellulose or gelatin, etc. For example, after the active ingredient is mixed with the solid excipient and crushed, a suitable auxiliary agent is added thereto and processed into a granular mixture, thereby obtaining a tablet or a sugar-coated tablet.

[0100] In addition to simple excipients, lubricants such as magnesium stearate and talc can also be used. Liquid preparations for oral administration correspond to suspensions, internal liquids, emulsions, syrups, etc., and in addition to the commonly used simple diluents such as water or liquid paraffin, they can also include several excipients such as wetting agents, sweeteners, aromatics or preservatives.

[0101] Furthermore, depending on the situation, cross-linked polyvinyl pyrrolidone, agar, alginic acid or sodium alginate may be added as a disintegrator, and anticoagulants, lubricants, wetting agents, flavors, emulsifiers and preservatives may also be added.

[0102] In the case of parenteral administration, the pharmaceutical composition of the present invention can be formulated into an injection form, a transdermal agent and an intranasal inhalation agent together with a suitable carrier for parenteral administration by methods known in the art. The above-mentioned injection must be sterilized and needs to be protected from contamination by microorganisms such as bacteria and fungi. Examples of suitable carriers for injections may include water, ethanol, polyols (such as glycerol, propylene glycol, liquid polyethylene glycol, etc.), mixtures thereof and / or solvents or dispersants including vegetable oils, but are not limited thereto. More preferably, Hanks solution, Ringer's solution, phosphate buffered saline (PBS) containing triethanolamine or sterile water for injection, or isotonic solution (such as 10% ethanol, 40% propylene glycol or 5% glucose) can be used as a suitable carrier. In order to protect the above-mentioned injection from microbial contamination, the injection may also contain various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, sorbic acid and thimerosal, etc. Also, in most cases, the above injections may also contain isotonic agents such as sugars or sodium chloride.

[0103] Forms of transdermal preparations include ointments, creams, lotions, gels, external solutions, plasters, coatings and aerosols, etc. The above-mentioned "transdermal administration" refers to the topical administration of a pharmaceutical composition to the skin to deliver an effective amount of active ingredients contained in the pharmaceutical composition into the skin.

[0104] In the case of inhalants, the compound used according to the present invention can be conveniently delivered from a pressurized package or atomizer using a suitable propellant such as dichlorofluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas in the form of an aerosol spray. In the case of a pressurized aerosol, the dosing unit can be determined by providing a valve that delivers the measured amount. For example, gelatin capsules and cartridges used in inhalers or insufflators can be formulated into a powder mixture containing the compound and a suitable powder material such as lactose or starch. The dosage form for parenteral administration is described in a formulary (Remington's Pharmaceutical Science, 15th Edition, 1975. Mack Publishing Company, Easton, Pennsylvania 18042, Chapter 87: Blaug, Seymour) that is generally known in pharmaceutical chemistry.

[0105] When the pharmaceutical composition of the present invention includes an effective amount of CHP, a preferred effect of preventing, improving or treating fibrosis can be provided. In the present application, the term "effective amount" refers to an amount showing more responses compared to a control group, preferably an amount sufficient to prevent, improve or treat fibrosis. The pharmaceutical composition of the present invention may contain 0.01% to 99.9% CHP, with the remainder being a pharmaceutically acceptable carrier. The effective amount of CHP contained in the pharmaceutical composition of the present invention may vary depending on the form in which the composition is commercialized.

[0106] The total effective amount of the pharmaceutical composition of the present invention can be administered to the patient in a single dose, or in multiple doses by a fractionated treatment protocol for long-term administration. The pharmaceutical composition of the present invention can change the content of the active ingredient according to the degree of the disease. For example, based on CHP, 0.001 mg to 100 mg is administered per 1 kg of body weight per day, and it is administered once or more a day. More preferably, 0.01 mg to 10 mg is administered per 1 kg of body weight per day, and it is administered once or more a day. However, the dosage of the above-mentioned CHP considers various factors such as the route of administration and the number of treatments of the pharmaceutical composition, the patient's age, weight, health status, gender, severity of the disease, diet and excretion rate to determine the effective dose for the patient. Therefore, in view of this, as long as a person of ordinary skill in the art can determine the above-mentioned CHP for the prevention, treatment or improvement of fibrosis for a specific purpose. The pharmaceutical composition of the present invention is not particularly limited to its dosage form, route of administration and method of administration as long as it shows the effect of the present invention.

[0107] The pharmaceutical composition for preventing or treating fibrosis of the present invention can be used alone or in combination with a method using surgery, radiotherapy, hormone therapy, chemotherapy or a biological response controller.

[0108] The pharmaceutical composition for preventing or treating fibrosis of the present invention can also be provided in the form of an external preparation containing CHP, etc. In this aspect, the composition of the present invention can be a quasi-drug composition for preventing or improving fibrosis and a quasi-drug containing the above composition.

[0109] The above-mentioned topical preparation can be directly applied to the skin or oral cavity. In the case of using the pharmaceutical composition for preventing or treating fibrosis of the present invention as a topical preparation, it can also include an adjuvant commonly used in the field of dermatology, such as a fatty substance, an organic solvent, a solubilizing agent, a concentrator, a gelling agent, a softener, an antioxidant, a suspending agent, a stabilizer, a foaming agent (forming agent), a fragrance, a surfactant, water, an ionic emulsifier, a nonionic emulsifier, a filler, a sequestrant, a chelating agent, a preservative, a vitamin, a retarder, a wetting agent, an essential oil, a dye, a pigment, a hydrophilic active agent, a lipophilic active agent or a lipid vesicle, etc., which are commonly used in any component of a skin topical preparation. In addition, the above-mentioned components can be introduced in an amount commonly used in the field of dermatology.

[0110] When the composition of the present invention is provided as an external preparation, it can be in the form of a liquid preparation, an ointment, a patch, a gel, a cream or an aerosol, but is not limited thereto. According to one embodiment of the present invention, the quasi-drug of the present invention can be an oral care product including toothpaste, a mouthwash and an oral spray, an ointment, a facial mask, a mud compress, a patch and a transdermal absorbent, etc.

[0111] When the composition of the present invention is used as a quasi-drug composition, CHP may be added directly or used appropriately with other quasi-drug components according to a conventional method. The amount of the active ingredient mixed may be appropriately determined according to the purpose of use (prevention, health care or treatment).

[0112] The contents of the pharmaceutical composition of the present invention can be applied to the quasi-drug composition and quasi-drug of the present invention.

[0113] In the present invention, the term "health functional food" means "functional food" and "health food".

[0114] As a specific example of such functional food, by using the above composition, it is possible to prepare a processed food which utilizes the properties of agricultural products, livestock products, or marine products and has good storage properties.

[0115] The health functional food composition of the present invention can also be prepared in the form of nutritional supplements, food additives, feeds, etc., targeting animals including humans or livestock.

[0116] The type of the above-mentioned food composition can be prepared into various forms according to conventional methods well known in the art. It can be prepared by adding CHP to general food, which includes beverages (including alcoholic beverages), fruits and processed foods thereof (such as canned fruit, bottled food, jam, jam with skin, etc.), fish, meat and processed products thereof (such as ham, sausage, corned beef, etc.), bread, noodles (such as udon noodles, buckwheat noodles, ramen, pasta, macaroni, etc.), fruit juice, various beverages, biscuits, syrup, dairy products (such as butter, milk cool, etc.), edible vegetable oils, margarine, vegetable protein, steamed food, frozen food and various seasonings (such as soybean paste, soy sauce, sauce, etc.), but is not limited thereto.

[0117] Also, nutritional supplements may be prepared by adding CHP to capsules, tablets, pills, etc., but are not limited thereto.

[0118] In the case where the food composition of the present invention is used as a health drink composition, the above-mentioned health drink composition may contain some flavorings or natural carbohydrates such as those in ordinary beverages as additional ingredients. The above-mentioned carbohydrates may be: monosaccharides such as glucose and fructose; disaccharides such as maltose and sucrose; polysaccharides such as dextrin and cyclodextrin; sugar alcohols such as xylitol, sorbitol and erythritol. Sweeteners may use: natural sweeteners such as teff, stevia extract; and synthetic sweeteners such as saccharin and aspartame. The proportion of the above-mentioned natural carbohydrates is generally about 0.01g to 0.04g per 100mL of the composition of the present invention, preferably about 0.02g to 0.03g.

[0119] In addition to the above, the health functional food of the present invention may contain various nutrients, vitamins, electrolytes, flavoring agents, colorants, pectin acid and its salts, alginic acid and its salts, organic acids, protective colloid thickeners, pH value regulators, stabilizers, preservatives, glycerol, alcohols or carbonating agents, etc. In addition, the health food of the present invention may contain pulp used to make natural fruit juice, fruit juice drinks and vegetable drinks. Such ingredients can be used alone or in combination. The proportion of such additives is not very important, but is usually selected from the range of 0.01 to 0.1 parts by weight relative to 100 parts by weight of the composition of the present invention.

[0120] Furthermore, the present invention relates to a method for preventing or treating fibrosis, comprising the step of administering an effective amount of CHP to an individual in need thereof.

[0121] In the methods of the present invention, the term "subject" includes any animal (e.g., humans, horses, pigs, rabbits, dogs, sheep, goats, non-human primates, cattle, cats, guinea pigs, or rodents), but is not limited thereto. This term does not indicate a particular age or gender. Thus, female / female, male / male, adult / adult and newborn subjects and embryos are included. Patient refers to a subject suffering from a disease or disorder. The term "patient" includes humans and veterinary subjects.

[0122] In the method of the present invention, the effect of CHP and the description of its administration route, administration frequency, dosage, etc. are the same as those described above, and therefore, the description thereof will be omitted.

[0123] Furthermore, the present invention provides the use of CHP in the preparation of a pharmaceutical composition for preventing or treating fibrosis.

[0124] Hereinafter, the present invention will be described in more detail by way of examples. However, the present invention may be implemented in various variations and may have various embodiments. The specific embodiments and descriptions described below are only to help understand the present invention, and the present invention is not limited to the specific disclosed embodiments. It should be understood that the scope of the present invention includes all variations, equivalent technical solutions or alternative technical solutions within the scope of the present invention's ideas and techniques.

[0125] Embodiments of the present invention

[0126] Preparation Example

[0127] CHP used in the following examples was purchased from BACHEM, Switzerland, and used.

[0128] Example 1

[0129] Confirmation of the antifibrotic effect in human proximal tubular epithelial cells treated with CHP

[0130] 1-1. Isolation and culture of human proximal tubular epithelial cells

[0131] like Figure 1 As shown, glomerular and tubulointerstitial tissues were isolated from normal adult kidney tissues and cultured in primary culture, thereby isolating and culturing pure human glomerular endothelial cells and human proximal tubular epithelial cells that can be used in cell experiments.

[0132] 1-2. Observation of cytological morphology after CHP treatment in a human proximal tubular epithelial cell fibrosis model

[0133] In the human proximal tubular epithelial cells isolated and cultured in the above Example 1-1, recombinant transforming growth factor β (2 ng / ml) was treated to induce cell fibrosis, thereby causing cytoskeletal remodeling and morphological changes (becoming elongated and losing structure), thereby confirming that fibrosis was appropriately induced ( Figure 2 ).

[0134] After that, when treated with various concentrations of CHP (4 μg / ml, 20 μg / ml, and 100 μg / ml), fibrosis was confirmed to be improved by cell morphology ( Figure 2 ).

[0135] 1-3. Expression of E-cadherin, fibroblasts and fibroblasts in human proximal tubular epithelial cells after CHP treatment in a fibrosis model Zonulin and pSTAT3 protein expression

[0136] Under the same conditions as in Example 1-2, after administration of CHP, the protein expression levels of E-cadherin, a junction marker, fibronectin, a fibrosis marker, and pSTAT3, a fibrosis transcription factor, were confirmed by Western blotting.

[0137] Results, such as Figure 3 As shown, it was confirmed that the protein expression of E-cadherin was restored and the protein expressions of fibronectin and pSTAT3 were reduced with the administration of CHP.

[0138] Example 2

[0139] Confirmation of the antifibrotic effect of CHP in human glomerular endothelial cells

[0140] Observation of cell morphology after CHP treatment in a human-derived glomerular endothelial cell fibrosis model

[0141] Similar to the human proximal tubular epithelial cell fibrosis model of Example 1-1 above, transforming growth factor β (2 ng / ml) was treated to induce cell fibrosis in glomerular endothelial cells, thereby causing cytoskeletal remodeling and morphological changes, thereby confirming that fibrosis was appropriately induced ( Figure 4 ).

[0142] After that, when treated with various concentrations of CHP (40 μg / ml and 100 μg / ml), fibrosis was confirmed to be improved by cell morphology ( Figure 4 ).

[0143] The above results confirm that CHP has an excellent therapeutic effect on renal fibrosis.

[0144] Example 3

[0145] Confirmation of antifibrotic effects in human hepatocytes treated with CHP

[0146] 3-1. Cultivation of human hepatocytes, induction of fibrosis and treatment of CHP

[0147] In order to culture the Huh7 hepatocyte line, DMEM medium and fetal bovine serum (FBS) were purchased from Hyclone Laboratories Inc., and transforming growth factor β for inducing fibrosis was purchased from R&D systems, Inc., USA. The cells were cultured in an incubator at 37°C and 5% CO2 using DMEM medium supplemented with 10% fetal bovine serum, and the transforming growth factor β was treated to a concentration of 2 ng / ml. At the same time, CHP was treated to a concentration of 0 ng / ml, 62.5 ng / ml, 125 ng / ml, and 250 ng / ml, and the cells were collected 48 hours after the treatment.

[0148] 3-2. Confirmation of fibrosis marker fibronectin expression after CHP treatment in a human hepatocyte fibrosis model

[0149] In order to analyze the protein expression of fibronectin, a fibrogenic protein, RIPA lysis buffer and Halt TM Protease and phosphatase inhibitor cocktails were used. For protein quantification, the BCA protein quantification kit from Thermo Fisher Scientific was purchased. For western blotting, the Bolt TM Protein gel electrophoresis system and wet transfer system from Bio-Rad Laboratories, Inc., USA. Antibodies against fibronectin, a marker of liver fibrosis, and GAPDH, a loading control, were purchased from Abcam, USA. 100ul of RIPA buffer supplemented with protease and phosphatase inhibitors was added to the recovered Huh7 cells, and the cells were lysed by pipetting. After placing on ice for 10 minutes, centrifuge at 15,000 rpm for 10 minutes at 4°C. The supernatant was recovered and the protein concentration was determined by the BCA quantitative method, and the protein concentration was determined by Bolt TMThe protein gel electrophoresis system separated the same amount of protein in the sample and transferred it to a nitrocellulose membrane. The membrane was blocked with 5% skim milk for 1 hour at room temperature and then reacted with fibronectin and GAPDH antibodies as primary antibodies at 4°C overnight. The membrane was washed with TBST 3 times for 10 minutes each time, and then reacted with the secondary antibody for 1 hour at room temperature. The membrane was washed with TBST for 3 minutes, 10 minutes each time, and reacted with ECL to determine the expression level. The size of the bands shown was quantified using the ImageJ program and corrected by dividing the fibronectin band size value by the GAPDH band size value. The statistical significance was analyzed by the Student's t-test statistical method compared with the control group. #p<0.05 (negative control group: transforming growth factor β-treated control group).

[0150] Experimental results, such as Figure 5a and Figure 5b As shown, the following tendency was confirmed, that is, when transforming growth factor β and 250 ng / ml CHP were treated, the expression level of fibronectin, which was increased by transforming growth factor β treatment, decreased. Therefore, it was confirmed that CHP can improve fibrosis in Huh7 cells, which is a liver cell line.

[0151] Example 4

[0152] Confirmation of antifibrotic effects in animal models of liver fibrosis by administration of CHP

[0153] Long-term high-fat diet intake is known as a risk factor for inducing liver fibrosis. Due to the insulin resistance and increased blood fatty acids, adipokines, and cytokines that occur when a fat diet is consumed for a long time, an inflammatory environment is induced in the liver tissue, and the interaction of immune cells accumulated in the liver tissue promotes fibrosis. In particular, interleukin-13 cytokines secreted by Th2CD4+T cells are produced in large quantities by macrophages, which is a key inducer of fibrosis. Signaling through transforming growth factor-β causes transformation of cells in liver tissue and promotes the progression of fibrosis (Rosselli, M et al., Current Pharmaceutical Design, 20(31), 5010-5024, 2014; Wu. D et al., Science, 332:243-7, 2011; J Investig Med, 60(8):1147-1150, 2012; Fichtner-Feigl S et al., Nat Med, 12:99-106, 2006; Lee CG et al., J Exp Med, 194:809-21, 2001).

[0154] When normal mice were fed a high-fat diet containing 60% fat for 10 to 16 weeks, liver fibrosis was observed and confirmed by the expression of fibronectin, a representative fibrosis marker protein (Kim, I. Het al., AGE, 38(4), 291-302, 2016; Chen, HJ, & Liu, J., Biomedicine & Pharmacotherapy, 97, 1386-1396, 2018.1-2).

[0155] 4-1. Design of experimental animals and induction of liver fibrosis

[0156] For the liver fibrosis experiment in animals, 12-week-old C57BL / 6 mice that had been fed a high-fat diet containing 60% fat for 6 weeks were purchased from Jackson, USA, and Research diets D12492, a high-fat diet containing 60% fat, was purchased from Saeronbio, South Korea, and the mice were fed for 17 weeks. The mice were allowed to eat and drink freely in an environment of 24±3°C. The C57BL / 6 mice that had been fed a high-fat diet for 6 weeks were weighed after adapting to the feeding environment for 1 week, and were evenly divided into 3 groups as shown in Table 1. CHP was administered once a day to each group at a concentration of 5 mg / kg or 35 mg / kg, and the administration was orally administered for a total of 16 weeks. For the control group, the same amount of distilled water was orally administered by the same method.

[0157] Table 1

[0158]

[0159] 4-2. Confirmation of the expression of fibrosis marker fibronectin in the animal model of liver fibrosis induced by CHP administration

[0160] Isoflurane required for anesthesia and dissection of mice was purchased from Hana Pharm, and the RC2 Rodent Circuit Controller Anesthesia System of VetEquip Inc. of the United States was prepared. Phosphate buffer was purchased from Hyclone, USA. In order to separate the liver tissue of mice, the mice were anesthetized by respiratory anesthesia using 3% to 3.5% isoflurane. After blood was collected from the heart of the anesthetized mouse, the liver tissue was immediately removed, washed with phosphate buffer, 50 mg of liver tissue was cut off and placed in 500 ul of RIPA buffer supplemented with protease and phosphatase inhibitors, and crushed using a T10 homogenizer from IKA, Germany. After placing on ice for 15 minutes, centrifuge at 15,000 rpm at a temperature of 4°C. In order to analyze the expression of fibronectin as a fibrosis protein, protein blotting was performed by the same method as in Example 3-2. Statistical significance was analyzed by Student's t-test compared with the HFD control group (*p<0.05, **p<0.01).

[0161] Experimental results, such as Figure 6a and Figure 6b As shown, it was confirmed that the expression level of fibronectin, a representative fibrosis marker, was significantly reduced in the CHP-administered group, and the effect was enhanced according to the concentration of CHP. This indicates that CHP improves liver fibrosis and is clear evidence that it can be used to treat liver fibrosis.

[0162] Example 5

[0163] Confirmation of antifibrotic effects in human lung cells treated with CHP

[0164] 5-1. Cultivation of human lung cells and treatment of CHP after induction of fibrosis

[0165] In order to culture the L-132 lung cell line, MEM medium and fetal bovine serum were purchased from Hyclone, USA, and transforming growth factor β for inducing fibrosis was purchased from R&D systems, USA. The cells were cultured in an incubator at 37°C and 5% CO2 using MEM medium supplemented with 10% fetal bovine serum, and the transforming growth factor β was treated to a concentration of 2 ng / ml, and CHP was treated to a concentration of 0 ng / ml, 62.5 ng / ml, or 125 ng / ml, and the cells were collected after 48 hours of treatment.

[0166] 5-2. Protein identification of fibrosis marker fibronectin after CHP treatment in a human lung cell fibrosis model Quality Expression

[0167] In order to analyze the expression of fibronectin as a fibrogenic protein, Western blotting was performed by the same method as in Example 3-2. Statistical significance was analyzed by Student's t-test statistical method with the control group (#p<0.05 (negative control group: transforming growth factor β-treated control group), **p<0.01 (transforming growth factor β-treated control group: CHP 125ng / ml treated group)).

[0168] Experimental results, such as Figure 7a and Figure 7b As shown, it was confirmed that when transforming growth factor β and 125 ng / ml CHP were treated, the expression of fibronectin, which was increased by transforming growth factor β treatment, was significantly reduced. The results that CHP improved fibrosis in L-132 cells, a lung cell line, confirmed that CHP has an excellent therapeutic effect on pulmonary fibrosis.

[0169] Example 6

[0170] Confirmation of antifibrotic effects in animal models of pulmonary fibrosis by administration of CHP

[0171] Due to long-term intake of a high-fat diet, pulmonary fibrosis occurs. This seems to be caused by increased inflammation levels caused by high fat intake. It is reported that when normal mice are fed a high-fat diet containing 60% fat for 15 weeks, pulmonary fibrosis occurs (Ge, XN et al., Experimental Lung Research, 39 (9), 365-378, 2016). Specifically, long-term intake of a high-fat diet induces obesity, and the adipose tissue and the immune cells gathered there secrete adipose factors and cytokines that interfere with the movement of immune cells from the bone marrow to the lungs, thereby causing a chronic inflammatory response (de Vries A et al., Clin Exp Allergy, 39: 731-739, 2009). In addition, interleukin-13 cytokines secreted by Th2CD4+T cells generate a large amount of transforming growth factor β, which is a key inducer of fibrosis, and cause the transformation of lung tissue and promote fibrosis through the signal transduction of transforming growth factor β (Fichtner-Feigl S et al., Nat Med, 12: 99-106, 2006; Lee CG et al., J Exp Med, 194: 809-21, 2001). Pulmonary fibrosis can be confirmed by the expression of transforming growth factor β gene, etc., which is increased relative to normal (Ge, XN et al., Experimental Lung Research, 39 (9), 365-378, 2016).

[0172] For the pulmonary fibrosis experiment in animals, a pulmonary fibrosis animal model was established by the same method as in Example 4-1.

[0173] 6-2. Confirmation of fibrosis markers, namely transforming growth factor-β and collagen, in an animal model of pulmonary fibrosis induced by CHP administration Pro3 gene expression

[0174] Isoflurane required for anesthesia and dissection of mice was purchased from Hana Pharm Co., Ltd., South Korea, and the RC2 Rodent Circuit Controller Anesthesia System of Vitrap Co., Ltd., USA was prepared. Phosphate buffer was purchased from Hyclone Co., Ltd., USA. In order to isolate the lung tissue of mice, the mice were anesthetized by respiratory anesthesia using 3% to 3.5% isoflurane. After blood was collected from the heart of the anesthetized mice, the lung tissue was immediately removed, 50 mg of the lung was cut and placed in 500 μL of NucleoZOL, and it was crushed using the T10 homogenizer of Aika Co., Ltd., Germany. Then, RNA was extracted according to the total RNA isolation protocol of NucleoZOL, and 1 μg of RNA was synthesized into cDNA by reverse transcription polymerase chain reaction using the iScript cDNA synthesis kit. The synthesized cDNA was analyzed by real-time polymerase chain reaction using iQ SYBR Green Supermix using the forward / reverse primer set corresponding to each gene. The expression value of each gene was corrected by dividing it by the expression value of GAPDH, which is a housekeeping gene. The primers used for real-time polymerase chain reaction had the base sequences shown in Table 2 and were synthesized and used by Bioneer Co., Ltd., Korea.

[0175] Table 2

[0176]

[0177] Statistical significance was analyzed using One-way ANOVA, and Dunnett's post hoc test was used to compare the significance with the control group (*p<0.05, ***p<0.001).

[0178] Experimental results, such as Figure 8 As shown, it was confirmed that the gene expressions of transforming growth factor β and collagen 3, which are major fibrosis markers shown in the lungs, were significantly reduced depending on the CHP administration concentration. This means that CHP improves pulmonary fibrosis and has an excellent therapeutic effect on the treatment of pulmonary fibrosis.

[0179] Example 7

[0180] Confirmation of antifibrotic effects in human skin cells treated with CHP

[0181] 7-1. Cultivation of human skin cells and treatment of CHP after induction of fibrosis

[0182] In order to culture the cell line derived from HS68 skin fibroblasts, DMEM medium and fetal bovine serum were purchased from Hyclone, USA, and transforming growth factor β for inducing fibrosis was purchased from R&D systems, USA. The cells were cultured in a 37°C, 5% CO2 incubator using DMEM medium supplemented with 10% fetal bovine serum, and the transforming growth factor β was treated to a concentration of 2 ng / ml, and CHP was treated to a concentration of 0 ng / ml, 62.5 ng / ml, 125 ng / ml, 250 ng / ml, or 500 ng / ml, and the cells were collected 48 hours after the treatment.

[0183] 7-2. Confirmation of fibrosis marker fibronectin expression after CHP treatment in a human skin cell fibrosis model

[0184] In order to analyze the expression of fibronectin as a fibrogenic protein, Western blotting was performed by the same method as in Example 3-2. The statistical significance was analyzed by Student's t-test statistical method with the control group (#p<0.05 (negative control group: transforming growth factor β-treated control group), *p<0.05 (transforming growth factor β-treated control group: CHP500ng / ml treated group)).

[0185] Experimental results, such as Figure 9a and Figure 9b As shown, it was confirmed that when transforming growth factor β and 500 ng / ml CHP were treated, the expression level of fibronectin, which is a fibrogenic protein, which increased with transforming growth factor β treatment, was significantly reduced. The results that CHP improved fibrosis in HS68 cells, a skin cell line, confirmed that CHP can be used to treat skin fibrosis.

[0186] Example 8

[0187] Confirmation of the antifibrotic effect in an animal model of cardiac fibrosis by administration of CHP

[0188] Well-known risk factors for cardiac fibrosis include poor eating habits, obesity, and high cholesterol levels. When a high-fat diet is consumed for a long time, obesity causes excessive blood volume, which overloads the heart and causes deformation of the heart's systolic and diastolic functions. At the same time, symptoms such as high blood sugar, insulin resistance, hyperlipidemia, and chronic inflammation play a decisive role in cardiac fibrosis and are well known (Kaltman AJ, Goldring RM., Am J Med, 60: 645-653, 1976; Xia Y et al., Histochem Cell Biol, 131: 471-481, 2009; Ulasova E et al., J Mol Cell Cardiol., 50: 147-156, 2011; Lo CS et al., J Cell Biochem., 103: 1999-2009, 2008; Cavalera, M et al., Translational Research, 164 (4), 323-335, 2014). In particular, the increase in cardiac mast cells and their degranulation caused by obesity provide fibrosis-inducing factors such as transforming growth factor-β to cardiac tissue and further promote cardiac fibrosis (Kong P, Christia P, Frangogiannis NG, Cell Mol Life Sci., 71: 549-574, 2014).

[0189] Furthermore, it has been reported that cardiac fibrosis occurs when normal mice are fed a high-fat diet for a long time. It has been reported that when normal mice are fed a high-fat diet containing 60% fat for 14 to 22 weeks, cardiac fibrosis is induced, and changes in the expression of transforming growth factor β, collagen genes, etc., which are increased compared with normal mice, can be confirmed (Li, W et al., Nutrition & Metabolism, 14(1); 68, 2017; Ternacle, J et al., European Heart Journal, 18(11), 1283-1291, 2017; Zhao, Y et al., Biochimica et Biophysica Acta (BBA)-Molecular Basis of Disease, 1863(8), 1991-2000, 2017; Xu, Z., & Kong, XQ, Biomedicine & Pharmacotherapy, 89, 991-1004, 2017).

[0190] 8-1. Design of experimental animals and induction of cardiac fibrosis

[0191] For the cardiac fibrosis experiment in animals, a cardiac fibrosis animal model was established by the same method as in Example 4-1.

[0192] 8-2. Confirmation of fibrosis markers in the animal model of cardiac fibrosis induced by CHP administration, namely transforming growth factor-β, fibroblast growth factor-β and Zonulin, collagen 1, collagen 2, collagen 3 and collagen 4 gene expression

[0193] Isoflurane required for anesthesia and dissection of mice was purchased from Hana Pharm, South Korea, and the RC2 Rodent Circuit Controller Anesthesia System of Vitpur, USA was prepared. Phosphate buffer was purchased from Hyclone, USA. In order to isolate the cardiac tissue of mice, the mice were anesthetized by respiratory anesthesia using 3% to 3.5% isoflurane. After blood was collected from the heart of the anesthetized mouse, the heart tissue was immediately removed, and 50 mg of the heart was cut off. RNA extraction and gene expression analysis were performed using the same method as Example 6-2. The primers used for real-time polymerase chain reaction were the base sequences shown in Table 3, which were synthesized and used by Bioneer, South Korea. The expression value of each gene was corrected by dividing it by the expression value of the housekeeping gene β-actin.

[0194] Table 3

[0195]

[0196] Statistical significance was analyzed using ANOVA statistical analysis and compared with the HFD control group by Dunnett's post hoc test (*p<0.05, **p<0.01, ****p<0.0001).

[0197] Experimental results, such as Fig.10 As shown, it was confirmed that the expression of fibronectin, transforming growth factor β, collagen 1, collagen 3, and collagen 4 genes shown as major fibrosis markers in the heart was significantly reduced by administering CHP. However, the effect of further reduction according to the concentration of CHP was not confirmed, which can be regarded as a sufficient effect of CHP at only 5 mg / kg concentration. This result that CHP inhibits the progression of cardiac fibrosis and improves cardiac fibrosis means that CHP can be used for the treatment of cardiac fibrosis.

[0198] Example 9

[0199] Confirmation of antifibrotic effect in animal model of adipose fibrosis by administration of CHP

[0200] The best known way to prevent fibrosis is to block the inflammatory response. In particular, adipose tissue secretes inflammatory substances to lead to a vicious cycle of inflammation-induced adipose fibrosis. It has been reported that when normal mice were fed a high-fat diet containing 60% fat for 16 to 24 weeks, adipose tissue fibrosis occurred (Hu, M et al., Evidence-Based Complementary and Alternative Medicine, 1-12, 2018; Kwon, EY, & Choi, MS, Nutrients, 10(10), 1415, 2018; Nakazeki, F et al., Scientific Reports, 8(1), 2018; Muniappan, L et al..Scientific Reports, 7(1), 2017; Lancha, A et al., PLoS ONE, 9(5), e98398, 2014; Velαzquez, KT et al., Physiological Reports, 5(18), e13412, 2017; Wang, L., Ye, X., Hua, Y., & Song, Y., Biomedicine & Pharmacotherapy, 105, 121-129, 2018). When a high-fat diet is consumed for a long time, if fat cells expand due to excess nutrition, neutral fat accumulation, adipocyte apoptosis, production of adipokines and cytokines, endoplasmic reticulum stress, adipose tissue hypoxia, etc. will occur, causing immune cells to infiltrate into adipose tissue and cause chronic inflammation (Schenk S et al., J Clin Invest, 118: 2992-3002, 2008; Sun K, Kusminski CM, Scherer PE, J Clin Invest, 121: 2094-101, 2011). The interaction between various inflammatory immune cells accumulated in adipose tissue and macrophages-adipocytes generates transforming growth factor-β, which is a key inducer of fibrosis. As a result, adipose tissue is deformed, thereby inducing adipose fibrosis (Lee CG et al., J Exp Med, 194: 809-21, 2001; Fichtner-Feigl S et al., Nat Med, 12: 99-106, 2006; Pessin, JE, & Kwon, H, Journal of Investigative Medicine, 60 (8), 1147-1150, 2012). The fibrosis of adipose tissue can be confirmed by the expression of representative fibrosis gene markers that are increased relative to normal adipose tissue.

[0201] 9-1. Design of experimental animals and induction of adipose fibrosis

[0202] For the adipose fibrosis experiment in animals, an adipose fibrosis animal model was established by the same method as in Example 4-1.

[0203] 9-2. Confirmation of fibrosis markers in the animal model of adipose fibrosis induced by CHP administration, namely transforming growth factor-β, fibroblast growth factor-β and Zonulin, collagen 3 and connective tissue growth factor gene expression

[0204] Isoflurane required for anesthesia and dissection of mice was purchased from Hana Pharm in South Korea, and the RC2 Rodent Circuit Controller Anesthesia System of Vitpur in the United States was prepared. Phosphate buffer was purchased from Hyclone in the United States. In order to isolate the adipose tissue of mice, the mice were anesthetized by respiratory anesthesia using 3% to 3.5% isoflurane. After blood was collected from the heart of the anesthetized mice, the epididymal adipose tissue (EAT) was immediately removed, and 100 mg of fat was cut off. RNA extraction and gene expression analysis were performed by the same method as Example 6-2. The primers used for real-time polymerase chain reaction have the base sequences shown in Table 4, which were synthesized and used by Bioneer. The expression value of each gene was corrected by dividing it by the expression value of GAPDH, which is a housekeeping gene.

[0205] Table 4

[0206]

[0207]

[0208] Statistical significance was analyzed using ANOVA statistical analysis and compared with the control group by Dunnett's post hoc test (*p<0.05, **p<0.01).

[0209] Experimental results, such as Fig.11 As shown, it was confirmed that the gene expressions of fibronectin, transforming growth factor β, collagen 3, and connective tissue growth factor, which are major fibrosis markers in fat, were significantly reduced by administration of CHP. This means that CHP improves fat fibrosis and can be confirmed to have an excellent therapeutic effect for the treatment of fat fibrosis. Sequence Listing <110> Nometa Pharmaceuticals Ltd. Seoul National University Hospital Seoul National University Industry-Academic Cooperation Group <120> Use for preventing, improving or treating fibrotic CHP <130> 1067299 <150> KR10-2019-0036269 <151> 2019-03-28 <160> 16 <170> PatentIn Version 3.2 <210> 1 <211> 20 <212> DNA <213> Artificial <220> <223> Transforming growth factor β forward primer <400> 1 cctgagtggc tgtcttttga 20 <210> 2 <211> twenty one <212> DNA <213> Artificial <220> <223> TGF-β reverse primer <400> 2 aatcgaaagc cctgtattcc g 21 <210> 3 <211> 20 <212> DNA <213> Artificial <220> <223> Collagen 3 forward primer <400> 3 agtcaaggag aaagtggtcg 20 <210> 4 <211> 19 <212> DNA <213> Artificial <220> <223> Collagen 3 reverse primer <400> 4 ccagggaaac ccatgacac 19 <210> 5 <211> 20 <212> DNA <213> Artificial <220> <223> GAPDH forward primer <400> 5 acactgagca agagagaggc 20 <210> 6 <211> 20 <212> DNA <213> Artificial <220> <223> GAPDH reverse primer <400> 6 tgggggtctg ggatggaaat 20 <210> 7 <211> 18 <212> DNA <213> Artificial <220> <223> Fibronectin forward primer <400> 7 ttggagagga gtgggagc 18 <210> 8 <211> 20 <212> DNA <213> Artificial <220> <223> Fibronectin reverse primer <400> 8 gaaatgacca ctgccaaagc 20 <210> 9 <211> 19 <212> DNA <213> Artificial <220> <223> Collagen 1 forward primer <400> 9 ctaacgtggt tcgtgaccg 19 <210> 10 <211> 20 <212> DNA <213> Artificial <220> <223> Collagen 1 reverse primer <400> 10 atccatcggt catgctctct 20 <210> 11 <211> 20 <212> DNA <213> Artificial <220> <223> Collagen 4 forward primer <400> 11 cggtacacag tcagaccatt 20 <210> 12 <211> 20 <212> DNA <213> Artificial <220> <223> Collagen 4 reverse primer <400> 12 catcacgaag gaatagccga 20 <210> 13 <211> 18 <212> DNA <213> Artificial <220> <223> β-actin forward primer <400> 13 gggaaggtga cagcattg 18 <210> 14 <211> twenty three <212> DNA <213> Artificial <220> <223> β-actin reverse primer <400> 14 atgaagtatt aaggcggaag att 23 <210> 15 <211> 20 <212> DNA <213> Artificial <220> <223> Connective tissue growth factor forward primer <400> 15 ctgtgcctgc cattacaact 20 <210> 16 <211> twenty one <212> DNA <213> Artificial <220> <223> Connective tissue growth factor reverse primer <400> 16 ccatgtctcc gtacatcttc c 21

Claims

1. Use of CHP or a pharmaceutically acceptable salt thereof in the preparation of a pharmaceutical composition for preventing or treating fibrosis, wherein the fibrosis is caused by dysregulated proliferation or activity of fibroblasts, abnormal accumulation of fibronectin, or pathological or excessive accumulation of collagen tissue, and the CHP exhibits anti-fibrotic activity, wherein the fibrosis is one or more selected from the group consisting of renal fibrosis, liver fibrosis, lung fibrosis, skin fibrosis, cardiac fibrosis, adipose fibrosis, and muscle fibrosis.

2. The use according to claim 1, wherein the fibrosis is idiopathic pulmonary fibrosis.

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