Composition for preventing or treating fibrotic diseases containing HAPLN1

Abstract

The present invention discloses a pharmaceutical composition for preventing or treating fibrotic diseases, which contains hyaluronan and proteoglycan link protein 1 (HAPLN1) or a gene encoding the same as an active ingredient, and a method for preventing or treating fibrotic diseases using the composition. According to the present invention, by preventing and suppressing fibrosis of cells or tissues, the occurrence or progression of various diseases induced by fibrosis can be fundamentally suppressed, and the diseases can be prevented or treated.

Description

[Technical Field]

[0001] The present invention relates to a composition for repairing various tissue damages caused by fibrosis and for preventing or treating fibrotic diseases, which comprises hyaluronan and proteoglycan link protein 1 (HAPLN1) or a gene encoding it as an active ingredient. Specifically, the present invention relates to a composition for repairing and regenerating tissue fibrosis and damage caused by fibrosis, which comprises HAPLN1 as an active ingredient, and also to a composition for repairing and recovering various tissue damages and functional loss caused by fibrosis, and for preventing or treating aging and degenerative diseases including fibrotic diseases.

[0002] Furthermore, the present invention discloses a composition for preventing or treating fibrotic diseases, and a method for preventing or inhibiting cellular fibrosis using the composition. According to the present invention, by preventing and inhibiting cellular fibrosis, it is possible to fundamentally inhibit the occurrence or progression of various diseases induced by the intervention of cellular fibrosis, as well as to treat the diseases. [Background technology]

[0003] The medical definition of "pulmonary fibrosis," which causes the lungs to harden like stone, is a disease in which healthy lung tissue turns into scar tissue due to some cause, causing the lung tissue to thicken and harden. There are clearly known cases, such as the humidifier disinfectant incident in South Korea, where the cause was a specific toxic chemical used to maintain the sterilizing effect of a humidifier, but in most cases, the cause is unknown and the disease that causes the lungs to harden is actually "idiopathic pulmonary fibrosis (IPF)." This disease is a type of interstitial lung disease (ILD) that exhibits symptoms of "usual interstitial pneumonia (UIP)," characterized histologically by inflammation and fibrosis of the lung parenchyma. Various risk factors are known, including aging, smoking, gastroesophageal reflux disease (GERD), environmental and occupational exposure, dusty work, radiation exposure, autoimmunity, drug toxicity, hypersensitivity pneumonitis, viral and pathogenic bacterial infections, and family history. However, despite recent advances in medical technology and the efforts of scientists, the etiology of this disease remains unclear. The average age of onset is 69 years, and it primarily occurs in older adults. Men are more than twice as likely to develop this disease than women, and the prognosis is poor. Idiopathic pulmonary fibrosis, a typical form of pulmonary fibrosis, has a patient rate of 1.7 per 100,000 people in Korea. It is a frightening disease, with its unknown cause making treatment difficult. The average survival time is 60 months, but 14% of patients experience exacerbations due to infection or other factors every year. If an exacerbation occurs, survival time drops sharply to 15 months.

[0004] In patients with pulmonary fibrosis, the alveolar walls become thickened with scar tissue, which gradually worsens and destroys the lung parenchyma. This makes it particularly difficult to inhale air, preventing patients from receiving enough oxygen to supply the blood. Ultimately, the alveoli are unable to perform their normal functions, resulting in shortness of breath, a persistent dry cough, fatigue, and characteristic club-like changes in the fingers and toes. The three main symptoms are coughing, exertional dyspnea, and crackling due to phlegm. The current pandemic, COVID-19, can also cause lung infection and inflammation, leading to pulmonary fibrosis during the recovery process.

[0005] The most well-known risk factor for fibrosis is advanced age. Researchers studying lung tissue lesions in particular have recently discovered that lung cells in patients with pulmonary fibrosis are unable to sustain division and growth, a sign of cellular senescence. The cellular senescence theory posits that aging in organisms, including humans, is due to the accumulation of senescent cells that are not physiologically beneficial but rather promote pathological conditions. Senescence is a phenomenon in which cells enter a series of processes called cell cycle arrest, preventing further cell division and production at a normal rate. A recent report systematically and effectively described the impact of cellular senescence on fibrosis in elderly pneumonia (Yanagi 2017, Int. J. Mol. Sci. 18, 503).

[0006] Intracellular DNA damage and chromosome "end segment wear," or telomere attrition, that occur with smoking or aging progress along with the phenomenon of immunosenescence that occurs with aging. Not only does this limit the ability to remove senescent cells from the body, but the cells also stop proliferating and fail to increase in number (cell growth arrest). Instead, they secrete inflammation- and senescence-inducing substances called SASP (senescence-associated secretory phenotype), such as interleukin-1 beta (IL-1β), which affect surrounding normal cells and turn them into senescent cells. As a result, the number of senescent cells gradually increases, secreting even more SASP, which leads to a pathological state accompanied by chronic low-grade inflammation. As a result, the tissue's regenerative and repair abilities naturally decrease, and eventually, normal lung tissue structure begins to be destroyed. In particular, in the case of elderly people, as various coexisting factors are added to their own, resistance to various external pathogenic attacks decreases (high vulnerability), and the fibrosis that initially developed further develops and worsens.

[0007] Specifically, the present inventors have focused on the fact that aging is a risk factor for pulmonary fibrosis, as mentioned above, and have been searching for "endogenous substances in the blood" that can prevent or treat the onset of this disease through qualitative and quantitative analysis using heterochronic parabiosis technology between young and old mice and proteomics technology. During this search, they have focused on the link protein between hyaluronic acid (HA) and proteoglycan (HAPLN1: hyaluronan and proteoglycan link protein 1) as the most likely substance. According to various literature, the HAPLN1 protein is one of the constituent proteins in the extracellular matrix (ECM) first discovered in vertebrate cartilage, and is reported to play a role not only in stabilizing the structural aggregates of hyaluronic acid and proteoglycans, but also in creating a bottle-brush shape by tightly packing the spaces between the proteoglycans. The present inventors have demonstrated that the recombinant human HAPLN1 (rhHAPLN1) protein, via its hinge-like link function, can actually inhibit the degradation of its partner molecules, high molecular weight HA (HMWHA) and aggrecan (a type of proteoglycan), thereby reducing the production of low molecular weight HA (LMWHA) or 32-mer peptides as damage-associated molecular patterns (DAMPs). Myofibroblasts are the primary cells responsible for the excessive formation of collagen-like fibrous tissues in fibrotic diseases. These myofibroblasts characteristically express α-smooth muscle actin (αSMA), and α-SMA expression is therefore an important indicator of myofibroblast expression. In fact, α-SMA-expressing myofibroblasts are elevated in various fibrotic diseases, such as idiopathic pulmonary fibrosis and renal fibrosis, and increased αSMA expression has also been reported in in vivo fibrosis models. These myofibroblasts are primarily derived from fibroblasts, but have also been reported to be derived from endothelial cells, epithelial cells, and stem cells (The American Journal of Pathology, 2007, 170 (6): 1807-1816). In the case of liver fibrosis, hepatic stellate cells are activated and play a role in excessively forming fibrous tissues such as collagen. αSMA expression also increases during hepatic stellate cell activation, which is used as an indicator of hepatic stellate cell activation (Cells 2019, 8 (11), 1419).

[0008] The most well-known factor inducing myofibroblast differentiation (or hepatic stellate cell activation) is TGF-β1, but mechanical stress in a stiff environment has also been reported as a factor in myofibroblast differentiation (The American Journal of Pathology, 2007 170 (6): 1807-1816). Therefore, in vitro fibrosis models have been used to induce αSMA expression in various cells using TGF-β1, and the ability to reduce induced αSMA expression has been determined to have anti-fibrotic efficacy (Molecular Medicine 2021 27: 22).

[0009] To date, various animal models have been established to demonstrate the efficacy of drugs against pulmonary fibrosis. The most widely accepted and used model is the inhaled bleomycin (BL)-induced pulmonary fibrosis (BIPF) mouse model. Originally, bleomycin was used as an anticancer drug and for the treatment of warts. In 1998, a three-dose bleomycin (BL)-hamster model was established by Professor Giri's research team at the University of California, Davis, College of Veterinary Medicine (Iyer 1998). In 2008, Oku et al. established a mouse model in which bleomycin was administered intravenously once daily for five consecutive days (Oku 2008, European Journal of Pharmacology 590; 400-408). Recently, Song et al. (EXPERIMENTAL AND THERAPEUTIC MEDICINE 16: 1800-1806) (2018) established a rat model in which bleomycin was administered via the bronchial route once a day, followed by oral administration of the approved drug pirfenidone (PFD) 14 or 28 times a day, starting the day after the administration. This model successfully induced pulmonary fibrosis, and the researchers investigated the efficacy of the drug and some of its mechanisms. They reported that the levels of periostin and TGF-β1, which had increased with bleomycin, decreased in the 14- and 28-day groups (Song 2018).

[0010] In fact, only two IPF treatments utilizing such mouse models have been approved for sale. One of them, pirfenidone (PFD, trade name Pirespa), was first approved by Shionogi & Co., Ltd. in Japan in 2008, followed by the European Union in 2011, Canada in 2012, and the United States in 2014. The other, nintedanib, trade names Ofev and Vargatef, is a pulmonary fibrosis treatment recently approved in the United States in March 2020. According to Oku et al. (Oku 2008), who developed pirfenidone (PFD), when mice were given intravenous injections of bleomycin five times daily, the inflammatory response reached its peak around the 10th day after the start of administration, after which it gradually decreased, and from around the 8th to 9th day after the start of administration, fibrosis gradually progressed.

[0011] More specifically, to investigate the efficacy of the candidate drug pirfenidone against pulmonary fibrosis, the team administered it three times daily for 10 or 28 days, starting on the day immediately following bleomycin administration (day 0). Oku et al. found that concurrent treatment with prednisolone, an anti-inflammatory corticosteroid, reduced inflammation-related biomarker levels but not TGF-β1 levels, a measure of anti-fibrotic efficacy. Therefore, the team concluded that the role of inflammation in the fibrotic process is not only inconclusive, but also that an early inflammatory response may be initiated immediately after bleomycin administration, and that early drug administration may slow or attenuate the gradual progression of the fibrotic process.

[0012] In addition, du Bois et al. reached a similar conclusion in a paper published in 2010, two years after Oku et al.'s publication (du Bois, RM Strategies for treating idiopathic pulmonary fibrosis, Nature Rev. Drug Discov, 2010, 9, 129-140).

[0013] Based on such literature evidence, the inventors have discovered, using the TGFβ1-induced fibrotic disease cell model and the bleomycin-induced pulmonary fibrosis mouse model (BIPF) as described above, that the HAPLN1 protein composition exhibits excellent efficacy in preventing and treating fibrosis in various tissues even at very low concentrations, thereby completing the present invention.

[0014] Regarding HAPLN1, for example, US 2013 / 0052198 discloses HAPLN1 polypeptide as one of a wide range of individual factors secreted by marrow stromal cells (MSCs), suggesting that its administration to individuals with inflammatory diseases may attenuate the characteristics of the disease, but makes no mention of its ability to prevent or treat fibrosis in various tissues.

[0015] If it were possible to directly prevent or suppress abnormally excessive fibrosis in cells within the body, this would be an effective countermeasure against various diseases in which fibrosis itself acts as a cause of disease or in which the fibrotic effect accelerates the progression of an already occurring disease, and there is a constant demand for such a measure. Summary of the Invention [Problem to be solved by the invention]

[0016] The present invention provides a composition for preventing and treating fibrotic diseases, which contains hyaluronan and proteoglycan link protein 1 (HAPLN1) or a gene encoding it as an active ingredient, and a method for preventing and treating fibrotic diseases using the same.

[0017] Furthermore, by providing a reagent composition containing hyaluronan and proteoglycan link protein 1 (HAPLN1) or the gene encoding it as an active ingredient, it can be used in research on the aforementioned diseases, contributing to elucidating the mechanisms of the diseases and developing preventive or therapeutic compositions for related diseases. [Means for solving the problem]

[0018] In order to solve the above problems, the present invention provides the following aspects of the invention.

[0019] [1] One aspect of the present invention relates to a pharmaceutical composition for the prevention or treatment of fibrotic diseases, which comprises, as an active ingredient, hyaluronan and proteoglycan link protein 1 (HAPLN1) or a gene encoding it. [2] Another aspect of the present invention relates to a composition, wherein the protein of the composition has 80% or more sequence identity with the amino acid sequence of SEQ ID NO:1. [3] Another aspect of the present invention relates to a composition, wherein the nucleic acid associated with the gene in the composition is contained in an expression vector. [4] Another aspect of the present invention relates to a composition in which the lesion of the fibrotic disease is selected from the group consisting of skin, liver, intestine, heart, lung, and kidney. [5] Another aspect of the present invention relates to a composition in which the fibrotic disease lesion is selected from the group consisting of skin fibroblasts, hepatic stellate cells, colonic fibroblasts, cardiac peripheral blood endothelial cells, pulmonary fibroblasts, kidney renal tubule cells, and kidney proximal tubule epithelial cells. [6] Another aspect of the present invention relates to a composition, wherein the fibrotic disease is ischemic fibrosis. [7] Another aspect of the present invention relates to the composition, wherein the single dose of the composition is 0.1 ng / ml to 500 ng / ml. [8] Another aspect of the present invention relates to a composition that, when administered in vivo, is administered at a dose of 0.001 to 5 mg / kgBW of rhHAPLN1 protein. [9] Another aspect of the present invention relates to a composition for preventing or suppressing cellular fibrosis, wherein the composition is contained as a main active ingredient or a supplementary active ingredient.

[10] Yet another aspect of the present invention relates to a method for preventing or inhibiting cellular fibrosis by treating cells with a composition containing, as an active ingredient, hyaluronan and proteoglycan link protein 1 (HAPLN1) or the gene encoding it.

[11] Furthermore, a further aspect of the present invention relates to a kit for preventing or suppressing cellular fibrosis, comprising a composition according to any one of items [1] to [9] and instructions for treatment according to the method of item

[10] .

[12] Furthermore, a further aspect of the present invention relates to an experimental reagent composition for preventing or inhibiting cell fibrosis, comprising a composition according to any one of items [1] to [9]. [Effects of the Invention]

[0020] As described above, the compositions and methods of the present invention prevent and inhibit cell fibrosis, thereby fundamentally inhibiting the onset or progression of various diseases caused by the intervention of cell fibrosis, dramatically increasing the possibility of preventing or treating such diseases. That is, they can prevent or treat fibrosis that occurs in various tissues due to a decrease in tissue regeneration and repair ability caused by aging and environmental factors such as smoking.

[0021] The composition of the present invention has fewer side effects than existing drugs for treating fibrotic diseases, and can safely and effectively treat fibrotic diseases, which are increasing every year with the global aging trend.

[0022] Furthermore, the present invention can also provide a method for preventing or treating diseases caused by fibrosis by utilizing the above-mentioned composition of the present invention.

[0023] Furthermore, the reagent composition of the present invention can be used in research into various diseases associated with cell fibrosis, and can be useful in elucidating the mechanisms of these diseases and developing preventive or therapeutic compositions for these diseases. [Brief explanation of the drawings]

[0024] [Figure 1A] 1 is a photograph of protein bands in a Western blot showing the anti-fibrotic effect of the composition of the present invention on skin fibrosis using normal human dermal fibroblasts (NHDFs) as a sample. The expression levels of αSMA and GAPDH proteins at various concentrations of the composition of the present invention and pirfenidone are shown as the intensity of the Western blot bands. [Figure 1B] 1B is a graph showing the relative αSMA levels at various concentrations of the composition of the present invention and pirfenidone by quantifying the intensity of the Western blot bands in FIG. 1A. [Figure 2A]FIG. 1 is a diagram showing an experimental design for evaluating the anti-fibrotic effect of the composition of the present invention on liver fibrosis using human hepatic stellate cells (HHSCs) as a sample. [Figure 2B] 2B is a photograph of a Western blot band showing the expression levels of αSMA and GAPDH proteins at various concentrations of the composition of the present invention and pirfenidone according to the experimental design of FIG. 2A. [Figure 2C] 2B is a graph showing the relative αSMA levels at various concentrations of the composition of the present invention and pirfenidone, quantifying the intensity of the Western blot bands in FIG. 2A. [Figure 3A] FIG. 1 is a diagram showing an experimental design for evaluating the anti-fibrotic effect of the composition of the present invention on intestinal fibrosis using a human colon fibroblast cell line (CCD-18Co) as a sample. [Figure 3B] 3B is a photograph of a Western blot band showing the expression levels of αSMA and GAPDH proteins at various concentrations of the composition of the present invention and pirfenidone according to the experimental design of FIG. 3A. [Figure 3C] 3C is a graph showing the relative αSMA levels at various concentrations of the composition of the present invention and pirfenidone, quantifying the intensity of the Western blot bands in FIG. 3B. [Figure 4A] FIG. 1 is a diagram showing an experimental design for evaluating the anti-fibrotic effect of the composition of the present invention on cardiac fibrosis using human cardiac microvascular endothelial cells (HCMEC) as a sample. [Figure 4B] 4B is a photograph of a Western blot band showing the expression levels of αSMA and GAPDH proteins at various concentrations of the composition of the present invention and pirfenidone according to the experimental design of FIG. 4A. [Figure 4C]4C is a graph showing the relative αSMA levels at various concentrations of the composition of the present invention and pirfenidone, quantifying the intensity of the Western blot bands in FIG. 4B. [Figure 5A] 1 is a photograph of a Western blot band showing the antifibrotic effect of the composition of the present invention on skin fibrosis using normal human lung fibroblasts (NHLFs) as a sample. The expression levels of αSMA and GAPDH proteins at various concentrations of the composition of the present invention and pirfenidone are shown as the intensity of the Western blot band. [Figure 5B] 5B is a graph showing the relative αSMA levels at various concentrations of the composition of the present invention and pirfenidone, quantifying the intensity of the Western blot bands in FIG. 5A. [Figure 6A] FIG. 1 is a diagram showing an experimental design for evaluating the fibrosis-preventing effect of the composition of the present invention on renal fibrosis using a human kidney tubular cell (HK-2: human kidney 2) epithelial cell line as a sample. [Figure 6B] 6B is a photograph of a Western blot band showing the expression levels of αSMA and GAPDH proteins at various concentrations of the composition of the present invention and pirfenidone according to the experimental design of FIG. 6A. [Figure 6C] 6C is a graph showing the relative αSMA levels at various concentrations of the composition of the present invention and pirfenidone, quantifying the intensity of the Western blot bands in FIG. 6B. [Figure 7A] FIG. 1 is a diagram showing an experimental design for evaluating the anti-fibrotic effect of the composition of the present invention on renal fibrosis using a human kidney tubular cell (HK-2: human kidney 2) epithelial cell line as a sample. [Figure 7B] 7B is a photograph of a Western blot band showing the expression levels of αSMA and GAPDH proteins at various concentrations of the composition of the present invention and pirfenidone according to the experimental design of FIG. 7A. [Figure 7C]7C is a graph showing the relative αSMA levels at various concentrations of the composition of the present invention and pirfenidone, quantifying the intensity of the Western blot bands in FIG. 7B. [Figure 8A] This is an experimental design diagram for evaluating the efficacy of rhHAPLN1 in TGFβ1-induced fibrotic morphological changes in human kidney 2 (HK-2) epithelial cell line. [Figure 8B] 8B is a micrograph showing changes in cell morphology at various concentrations of the composition of the present invention and pirfenidone according to the experimental design of FIG. 8A. [Figure 9A] 1 is a photograph of a Western blot band showing the anti-fibrotic effect of the composition of the present invention on senescence-induced fibrosis in renal proximal tubule epithelial cells (RPTECs). The expression levels of α-SMA and GAPDH proteins at various concentrations of the composition of the present invention are shown as the intensity of the Western blot band for cells cultured for 11 passages. [Figure 9B] 9B is a graph showing the relative αSMA levels quantified by quantifying the intensity of the Western blot bands shown in FIG. 9A and varying concentrations of the composition of the present invention. [Figure 10A] At the 4th and 11th passages, cell nuclei were stained with 4',6-diamidino-2-phenylindole (DAPI) to observe the cell nuclei. The photographs show the level of intracellular αSMA expression by fluorescence staining using an αSMA antibody (ab7817 (Abcam)) diluted 1:1000 in 1X PBST (1X PBS with 0.1% Triton X-100, 1% BSA). [Figure 10B] This is a photograph showing the relative cellular αSMA levels by fluorescence after treatment with various concentrations of the composition of the present invention and pirfenidone at the 4th passage as a control group, and at the 11th passage when senescence was induced. [Figure 11A]This is an experiment to confirm the effect of rhHAPLN1 on anti-fibrotic markers using the BioMAP Fibrosis panel, and is a graph showing the significant reducing effect of the composition of the present invention (rhHAPLN1) on αSMA and collagen I at each concentration in the SAEMyoF system, a lung fibrosis disease model, as a relative fold change compared to the vehicle control group. [Figure 11B] This is an experiment to confirm the effect of rhHAPLN1 on anti-fibrotic markers using the BioMAP Fibrosis panel, and is a graph showing the significant reducing effect of the composition of the present invention (rhHAPLN1) on collagen I at each concentration in the REMyoF system, a kidney fibrosis disease model, as a relative fold increase compared to the vehicle control group. [Figure 11C] This is an experiment to confirm the effect of rhHAPLN1 on anti-fibrotic markers using the BioMAP Fibrosis panel, and is a graph showing the significant reducing effect of the composition of the present invention (rhHAPLN1) on collagen IV in myofibroblasts at each concentration, expressed as a relative fold increase compared to the vehicle control group. [Figure 12A] FIG. 1 is a diagram illustrating an outline of an experiment for evaluating the anti-fibrotic efficacy (ability to prevent fibrotic diseases) of the composition of the present invention using a mouse bleomycin-induced pulmonary fibrosis (BIPF) model. [Figure 12B]In an experiment using a mouse bleomycin-induced pulmonary fibrosis model (Figure 12A), mice were divided into four groups (mouse 1 to mouse 4). The normal group was treated with PBS, the control group received no treatment except for PBS, and the control group was treated with 0.0005% (w / w) of the composition of the present invention (rhHAPLN1) or 0.0015% (w / w) of the composition of the present invention (rhHAPLN1). Lung tissue was removed from four mice per group, and the left large lung lobe was cut in half horizontally, and the upper part was fixed in formalin and then stained with hematoxylin and eosin (H&E). [Figure 12C] The middle part of the lung tissue of the largest lobe on the left side of the mouse was cut, and three tissue slides were prepared per mouse. Three areas on each slide were randomly photographed, and for a total of nine sections, the stained red areas were separated from the unstained white areas. The areas of the stained red areas were measured using Image J software, and the obtained values ​​were averaged to obtain the average value per mouse, and the statistical significance was confirmed. [Figure 12D] This is a photograph of the Ashcroft score, which serves as an official guideline for easily measuring the severity of pulmonary fibrosis with the naked eye (Ashcroft et al 1988, J Clin Pathol 41:467-470). [Figure 12E] This is a description of the Ashcroft score, which serves as a formal guide to easily measure the severity of pulmonary fibrosis by visual inspection (Ashcroft et al 1988, J Clin Pathol 41:467-470). [Figure 12F] 13A and 13B are graphs related to the experimental results showing the severity of pulmonary fibrosis in terms of Ashcroft scores. [Figure 13A] FIG. 1 is a diagram illustrating an outline of an experiment for evaluating the anti-fibrotic efficacy (ability to treat fibrotic diseases) of the composition of the present invention using a mouse bleomycin-induced pulmonary fibrosis (BIPF) model. [Figure 13B] In an experiment using a mouse bleomycin-induced pulmonary fibrosis model (Figure 13A), mice were divided into four groups (normal group, control group, 0.00075% (w / w) rhHAPLN1, 0.0015% (w / w) rhHAPLN1, 0.003% (w / w) rhHAPLN1) with four mice per group. After the experiment, lung tissue from four mice per group was removed, and the left large lung lobe was cut in half horizontally, and the upper part was fixed in formalin and then stained with hematoxylin and eosin (H&E). [Figure 13C] The middle part of the lung tissue of the largest lobe on the left side of the mouse was cut, and three tissue slides were prepared per mouse. Three areas on each slide were randomly photographed, and for a total of nine sections, the stained red areas were separated from the unstained white areas. The areas of the stained red areas were measured using Image J software, and the obtained values ​​were averaged to obtain the average value per mouse, and the statistical significance was confirmed. [Figure 13D] The middle part of the lung tissue of the largest lobe on the left side of the mouse was cut, and three tissue slides were prepared per mouse. Three areas on each slide were randomly photographed, and for a total of nine sections, the stained red areas were separated from the unstained white areas. The areas of the stained red areas were measured using Image J software, and the obtained values ​​were averaged to obtain the average value per mouse, and the statistical significance was confirmed. [Figure 13E] 13C and shows a graph of the experimental results indicating the severity of pulmonary fibrosis as Ashcroft scores. [Figure 14A] FIG. 1 is a diagram showing an experimental design for an animal model experiment on induced renal fibrosis, which utilizes ischemia / reperfusion to evaluate the anti-fibrotic effect of the composition of the present invention on renal fibrosis. [Figure 14B]Figure 14A is a photograph of protein bands from a Western blot showing the level of αSMA protein expression following administration of the composition of the present invention and pirfenidone, according to the experimental design (SV: sham-vehicle, SB: sham-rhHAPLN1-dissolving solution, sham-rhHAPLN1 dose B, a group used to confirm the effects of treating the sham control group with rhHAPLN1, IRV: IR-vehicle, IRP: IR-pirfenidone, IRA: IR-rhHAPLN1 dose A, IRB: IR-rhHAPLN1 dose B, IRC: IR-rhHAPLN1 dose C). [Figure 14C] FIG. 14C is a graph showing the fold change in the expression level of αSMA relative to IRV at various concentrations of pirfenidone and the composition of the present invention, based on the numerical values ​​of the intensity of the Western blot bands in FIG. 14B. [Figure 14D] 1 is a photograph showing the level of collagen expression in an acute kidney injury induced model at various concentrations of pirfenidone and the composition of the present invention, stained with Sirius Red and PAS. Purple indicates collagen expression. [Figure 14E] In an acute kidney injury induced model, to quantify the level of collagen expression at various concentrations of pirfenidone and the composition of the present invention, two locations in the outer medulla of the kidney were randomly imaged using histological staining, and the photograph shows only the collagen-positive areas. [Figure 14F] This is a graph showing the collagen-positive areas in Figure 14E, which were marked and the collagen area (%) was quantified using i-solution software (IMT). [Figure 14G]1 is a graph showing the creatinine clearance rate measured 21 days after ischemia / reperfusion using various concentrations of pirfenidone and the composition of the present invention in an acute kidney injury induced model. DETAILED DESCRIPTION OF THE INVENTION

[0025] First, various terms used in this specification may be defined as follows.

[0026] In the present specification, "rhHAPLN1" is an abbreviation for recombinant human HAPLN1 and refers to recombinant human HAPLN1.

[0027] In the present specification, the term "fibroblast" refers to a cell that produces connective tissue such as collagen fibers and is involved in maintaining tissue structure, but is not an epithelial, blood vessel, lymphatic vessel, or inflammatory cell.

[0028] In the present specification, the term "myofibroblast" refers to a fibroblast that is activated by physical injury or inflammation, expresses alpha-smooth muscle actin (α-SMA), and has contractile function similar to that of smooth muscle cells.

[0029] In the present specification, "αSMA" or "α-SMA (alpha-smooth muscle actin)" is a protein expressed in pathological vascular smooth muscle cells and stromal fibroblasts, and is a major marker of tissue fibrosis. High expression is observed in kidneys showing signs of renal fibrosis, and such high expression level or activity indicates high myofibroblast activity, which is closely related to the degree of interstitial fibrosis. Furthermore, SMA expression in patients is closely related to a decrease in the renal creatine clearance rate, and therefore functions as a marker for confirming that creatine, a waste product, is not being eliminated from the body.

[0030] In the present specification, "transforming growth factor beta 1 (TGFβ1 or TGF-β1)" refers to a cytokine used in various embodiments of the present invention to transform fibroblasts into myofibroblasts, and the transformation dose varies depending on the cell type. In general, the transformation of fibroblasts into myofibroblasts involves various physiologically active substances such as cytokines, chemokines, growth factors, and hormones.

[0031] In the present specification, "pirfenidone" is an antifibrotic drug approved for the treatment of pulmonary fibrosis and currently being used in clinical trials for the treatment of renal fibrosis. It was used as a control substance in the experiments of the present invention. Effective doses may vary depending on the cell type, but relevant literature reports that pirfenidone is effective at concentrations of 100 μg / mL or higher.

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

[0033] One aspect of the present invention relates to an antifibrotic composition containing hyaluronan and proteoglycan link protein 1 (HAPLN1) or its encoding gene as an active ingredient, which can function as a pharmaceutical composition for the prevention or treatment of fibrotic diseases.

[0034] According to one embodiment of the present invention, the composition of the present invention is also a recombinant human HAPLN1 protein represented by SEQ ID NO: 1. In relation thereto, the HAPLN1 protein of the present invention is also a protein having 80%, preferably 85%, more preferably 90%, even more preferably 95%, and most preferably 100% sequence identity to the amino acid sequence of SEQ ID NO: 1, as long as it maintains its anti-fibrotic function.

[0035] According to one embodiment of the present invention, the nucleic acid related to the gene encoding the HAPLN1 protein in the composition of the present invention is contained in an expression vector and is also contained in the composition.

[0036] In addition, in the present invention, the cells on which the composition of the present invention acts are not particularly limited, but preferably include various types of fibroblasts, fibroblasts, endothelial cells, and epithelial cells, more specifically, skin fibroblasts, hepatic stellate cells, human colon cells, peripheral blood endothelial cells, pulmonary fibroblasts, renal tubule cells, and proximal tubule epithelial cells, and include cells in various contexts such as in vivo, in vitro, ex vivo, and in situ. In addition, the organs to which the composition of the present invention can be applied are not particularly limited as long as they are organs in which fibrosis can be induced, but preferably include lungs, kidneys, skin, liver, intestines, and heart.

[0037] According to one embodiment of the present invention, the composition of the present invention is administered at a single dose to cells of 0.1 ng / ml to 500 ng / ml, preferably 1 ng / ml to 300 ng / ml, more preferably 3.0 ng / ml to 50 ng / ml, and even more preferably 10 ng / ml to 13 ng / ml, although depending on the application site or circumstances, it may be more specifically 3, 5, 11, 30, 50, or 100 ng / ml. Alternatively, according to one embodiment of the present invention, the composition of the present invention is administered at a single dose to cells of 0.00001 to 0.1% (w / w), preferably 0.0001 to 0.05% (w / w), more preferably 0.0003 to 0.03% (w / w), and even more preferably 0.0005 to 0.015% (w / w).

[0038] In one embodiment of the present invention, the composition of the present invention is contained as a main active ingredient or auxiliary active ingredient in a composition for preventing or suppressing fibrotic action of cells.

[0039] Furthermore, according to one aspect of the present invention, the present invention relates to a method for preventing or suppressing cellular fibrosis by treating cells with a composition containing, as an active ingredient, hyaluronan and proteoglycan link protein 1 (HAPLN1) or the gene encoding it.

[0040] Another aspect of the present invention relates to a kit for preventing or suppressing cell fibrosis, which kit comprises the composition of the present invention and a treatment guideline.

[0041] Another aspect of the present invention relates to a reagent composition for experiments related to the prevention or inhibition of cell fibrosis, which comprises the composition of the present invention.

[0042] Furthermore, the present invention relates to the use of the composition of the present invention for the production of a pharmaceutical composition for the prevention or treatment of diseases induced by cell fibrosis.

[0043] In certain embodiments, the pharmaceutical composition generally also comprises a molecularly and pharmaceutically acceptable carrier. As used herein, the term "pharmaceutically acceptable carrier" includes saline, solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonicity agents, and absorption delaying agents, etc., that are compatible with pharmaceutical administration. Supplementary active compounds may also be included in the composition. In other words, the pharmaceutical composition of the present invention may further comprise a pharmaceutical additive selected from the group consisting of pharmaceutically acceptable carriers, diluents, binders, disintegrants, lubricants, and any combination thereof.

[0044] The pharmaceutical composition may be formulated to be compatible with the intended route of administration. Preferably, the composition of the present invention is in a dosage form selected from the group consisting of eye drops, ointments, tablets, pills, capsules, lozenges, inhalants, injections, patches, and suppositories.

[0045] Examples of routes of administration include parenteral, e.g., intravenous, intradermal, subcutaneous, oral (e.g., inhalation), transdermal (topical), transmucosal, and rectal administration. Parenteral administration is preferred.

[0046] Solutions or suspensions used for parenteral, intradermal, or subcutaneous application may also include the following components: a sterile diluent such as water for injection, saline, fixed oils, polyethylene glycol, glycerin, propylene glycol, or other synthetic solvents; an antibacterial agent such as benzyl alcohol or methylparaben; an antioxidant such as ascorbic acid or sodium bisulfite; a chelating agent such as ethethylenediaminetetraacetic acid; a buffer such as acetate, citrate, or phosphate; and a tonicity adjusting agent such as sodium chloride or dextrose. The pH may be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide. Parenteral preparations may be enclosed in ampoules, single-use syringes, or multiple-dose vials made of glass or plastic.

[0047] Pharmaceutical compositions suitable for injection include sterile aqueous solutions (where water soluble), or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. Suitable carriers for intravenous administration include physiological saline, bacteriostatic water, or phosphate-buffered saline (PBS). In all cases, the composition must be sterile and sufficiently fluid for easy syringability. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can also be a solvent or dispersion medium containing, for example, water, ethanol, polyol (e.g., glycerol, propylene glycol, and liquid polyethylene glycol), and suitable mixtures thereof. Proper fluidity can be maintained, for example, by the use of a coating such as lecithin, the maintenance of the required particle size in the case of dispersion, and the use of surfactants. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, and thimerosal. In many cases, it will be desirable to include isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, sodium chloride in the composition. Prolonged absorption of the injectable compositions can be achieved by including in the composition agents delaying absorption, for example, aluminum monostearate and gelatin.

[0048] Sterile injectable solutions can be prepared by incorporating the active compound in the required amount in a suitable solvent with one or a combination of the above-listed ingredients, as needed, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle containing a basic dispersion medium and the other required ingredients listed above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred preparation method is vacuum drying and freeze-drying, which produces a powder of the active ingredient and any additional desired ingredients from a previously sterile-filtered solution.

[0049] Oral compositions generally contain an inert diluent or an edible carrier. For oral therapeutic administration, the active compound may be incorporated with an excipient and used in the form of tablets, troches, or capsules, such as gelatin capsules. Oral compositions may also be prepared using a fluid carrier for use as a mouthwash. Pharmaceutically suitable binders and / or adjuvants may also be included as part of the composition. Tablets, pills, capsules, troches, etc. may contain any of the following ingredients, or compounds of a similar nature: binders such as microcrystalline cellulose, tragacanth gum, or gelatin; disintegrating agents such as starch or lactose, disintegrating agents such as alginic acid, Primogel, or corn starch; lubricants such as magnesium stearate or sterols; glidants such as colloidal silicon dioxide; sweeteners such as sucrose or saccharin; or flavoring agents such as peppermint, methyl salicylate, or orange flavoring.

[0050] For administration by inhalation, the compounds are delivered in the form of an aerosol spray from pressured container or dispenser which contains a suitable propellant, eg, a gas such as carbon dioxide, or a nebulizer.

[0051] Systemic administration can also be achieved by transmucosal or transdermal means. For transmucosal or transdermal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art and include, for example, those for transmucosal administration, detergents, bile salts, and fusidic acid derivatives. Transmucosal administration can also be achieved using nasal sprays or suppositories. For transdermal administration, the active compound is formulated into ointments, salves, gels, or creams, as generally known in the art.

[0052] The compounds can also be prepared in the form of suppositories (eg, with common suppository bases such as cocoa butter and other glycerides) or retention enemas for rectal delivery.

[0053] The data obtained from cell culture assays and animal studies can be used to formulate various dosages for use in humans. The dosage of such compounds is preferably within the ED range with little or no toxicity. 50 The dosage will vary within this range depending upon the dosage form employed and the route of administration utilized.

[0054] Therapeutically effective amounts (i.e., effective dosages) of the compositions of the present invention will vary depending on the circumstances of the selected patient. For example, a single dose ranging from about 1 pg to 1,000 mg may be administered; in some embodiments, 10, 30, 100, or 1,000 pg, 10, 30, 100, or 1,000 ng, 10, 30, 100, or 1,000 μg, or 10, 30, 100, or 1,000 mg may be administered.

[0055] Furthermore, according to one embodiment of the present invention, when the composition of the present invention is administered directly to a living body in vivo, the dose of rhHAPLN1 protein is 0.001 to 10 mg / kg / BW, preferably 0.004 to 5 mg / kg / BW, more preferably 0.1 to 1.0 mg / kg / BW, and even more preferably 0.15 to 0.5 mg / kg / BW.

[0056] Alternatively, in some embodiments, a pharmaceutical composition may be administered at a concentration of 1 ng / ml to 100 μg / ml, preferably 3 ng / ml to 50 μg / ml, more preferably 5 ng / ml to 500 ng / ml, and particularly preferably 10 ng / ml to 200 ng / ml. The pharmaceutical composition may be administered from once or more times daily to once or more times weekly, including once every other day. A skilled artisan will recognize that certain factors may influence the dosage and timing required to effectively treat an individual, including, but not limited to, the severity of the disease or disorder, previous treatments, the individual's general health and / or age, and other pre-existing conditions. Furthermore, treating an individual with a therapeutically effective amount of a molecule of the invention can include a single treatment or, preferably, a series of treatments.

[0057] The composition of the present invention may be administered at a dosage of 5 mg / kg / week to 500 mg / kg / week, for example, 5 mg / kg / week, 10 mg / kg / week, 15 mg / kg / week, 20 mg / kg / week, 25 mg / kg / week, 30 mg / kg / week, 35 mg / kg / week, 40 mg / kg / week, 45 mg / kg / week, 50 mg / kg / week, 55 mg / kg / week, 60 mg / kg / week, 65 mg / kg / week, 70 mg / kg / week, 75 mg / kg / week, 80 mg / kg / week, 85 mg / kg / week, 90 mg / kg / week, 95 mg / kg / week, 100 mg / kg / week, 100 mg / kg / week, 15 mg / kg / week, 20 mg / kg / week, 25 mg / kg / week, 30 mg 100 mg / kg / week, 70 mg / kg / week, 75 mg / kg / week, 80 mg / kg / week, 85 mg / kg / week, 90 mg / kg / week, 95 mg / kg / week, 100 mg / kg / week, 150 mg / kg / week, 200 mg / kg / week, 250 mg / kg / week, 300 mg / kg / week, 350 mg / kg / week, 400 mg / kg / week, 450 mg / kg / week, and 500 mg / kg / week. In specific embodiments, the dosage of a dual acting molecule according to the invention is in the range of 10 mg / kg / week to 200 mg / kg / week, 20 mg / kg / week to 150 mg / kg / week, or 25 mg / kg / week to 100 mg / kg / week. In certain embodiments, the compositions of the invention are administered once per week for 2 weeks to 6 months, e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 26 weeks, 6 months, 8 months, 10 months, or a year or more. In certain embodiments, the compositions of the invention are administered twice per week. In other embodiments, the compositions of the invention are administered every other week.

[0058] The compositions of the present invention can also be formulated into pharmaceutical compositions containing a pharmacologically effective amount of a HAPLN1 protein molecule and a pharmaceutically acceptable carrier. A pharmacologically or therapeutically effective amount refers to an amount effective to produce an intended pharmacological, therapeutic, or preventive result. The phrases "pharmacologically effective amount" and "therapeutically effective amount," or simply "effective amount," refer to an amount of a dual-acting molecule effective to produce an intended pharmacological, therapeutic, or preventive result. For example, if a given clinical treatment is considered effective when a measurable parameter associated with a disease or disorder is reduced by 20% or more, then a therapeutically effective amount of a drug for treating that disease or disorder is the amount necessary to reduce that parameter by at least 20%.

[0059] Properly formulated pharmaceutical compositions of the present invention may be administered by any means known in the art, such as parenteral routes, including intravenous, intramuscular, intraperitoneal, subcutaneous, transdermal, respiratory (aerosol), rectal, vaginal, and topical (including buccal and sublingual) administration. In some embodiments, the pharmaceutical compositions are administered by intravenous or parenteral infusion or injection.

[0060] Generally, suitable dosage units of a molecule range from 0.001 to 0.25 mg per kg of recipient body weight per day, from 0.01 to 20 mg per kg of body weight per day, from 0.01 to 10 mg per kg of body weight per day, from 0.10 to 5 mg per kg of body weight per day, or from 0.1 to 2.5 mg per kg of body weight per day. Pharmaceutical compositions containing a molecule can be administered once daily. However, therapeutic agents can also be administered in dosage units containing 2, 3, 4, 5, 6, or more subdoses administered at appropriate intervals throughout the day. Dosage units can also be compounded into a single dose over several days, for example, using a conventional sustained-release dosage form that provides sustained and consistent release of the molecule over a period of several days. Such sustained-release dosage forms are well known in the art. In this embodiment, the dosage unit contains a multiple appropriate for the daily dosage.

[0061] The pharmaceutical compositions may also be included in a kit, container, pack, or dispenser together with instructions for administration.

[0062] As used herein, "treatment" or "treating" is defined as the application or administration of a therapeutic agent (e.g., a molecule of the invention) to a patient, or the application or administration of a therapeutic agent to an isolated tissue or cell line, with the intent to cure, heal, alleviate, relieve, alter, relieve, ameliorate, improve or affect a disease or disorder, a symptom of a disease or disorder, or a predisposition to a disease or disorder.

[0063] Furthermore, one aspect of the present invention relates to the use of a composition containing hyaluronan and proteoglycan link protein 1 (HAPLN1) as an active ingredient for preventing or suppressing cellular fibrosis. [Example]

[0064] The present invention will be described in more detail below through examples. However, the following examples are for illustrative purposes only and are not intended to limit the scope of the present invention.

[0065] [Manufacturing Example 1] Production of hyaluronan and proteoglycan link protein 1 (HAPLN1) A vector containing a polynucleotide encoding the human HAPLN1 protein of amino acid sequence SEQ ID NO: 1 was inserted into CHO-K1 cells to generate a CHO-K1 cell line producing recombinant human HAPLN1 protein.

[0066] The amino acid sequence of SEQ ID NO: 1 is as follows:

[0067]

number

[0068] A cell line with excellent protein production and quality was selected as an MCB (master cell bank). The MCB was subcultured to obtain 0.40±0.05×10 6 The cells were inoculated into a Thermo Hyperforma SUB 250L bioreactor at a concentration of 1000 cells / mL and cultured in a fed-batch manner. TM The medium was composed of 0.5846 g glutamine, 10.00 g HT Supplement (Thermo Fisher Scientific), 4.29 g 10 N NaOH, and 1.80 g NaHCO3. The culture temperature was set at 36.5°C, the dissolved oxygen (DO) was set at 40.0%, and the pH was set at 7.00 ± 0.20. 1 M sodium carbonate monohydrate was used as a pH adjusting solution. 181.04 g HyClone was used as the feeding medium (FM). TM Cell Boost 7a + 12.28g FM020a with 10N NaOH, and 94.60g HyClone TM Cell Boost 7b + 105.93g FM020b of 10N NaOH was used.

[0069] Recombinant human HAPLN1 (rhHAPLN1) protein was isolated and purified from the cells cultured as described above through processes such as harvest and clarification, ultrafiltration / diafiltration 1 (UF / DF1), anion exchange chromatography, S / D (solvent / detergent) viral inactivation, cation exchange chromatography, mixed-mode chromatography (MMC), hydrophobic interaction chromatography, ultrafiltration / diafiltration 2 (UF / DF2), and intermediate depth filtration (Int.DF). Such compositions of the present invention were used in the following examples.

[0070] [Manufacturing Example 2] Production of the composition of the present invention containing HAPLN1 protein The HAPLN1 protein purified in Preparation Example 1 was stabilized in 20 mM acetate buffer, 8% (w / v) sucrose, and 0.04% (w / v) PS80 (pH 5.0) to prepare a composition of the present invention containing the HAPLN1 protein itself as the main active ingredient.

[0071] [Manufacturing Example 3] Preparation of the composition of the present invention comprising an expression vector containing a gene encoding the HAPLN1 protein A composition containing an expression vector carrying a gene encoding the HAPLN1 protein was prepared using the jetPRIME Transfection Reagent Kit (Product 14-15 (PolyPlus)). More specifically, a plasmid vector designed to express human HAPLN1 (hereinafter referred to as "Human HAPLN1 ORF Clone") was prepared using Product RC209274 (OriGene). This vector can express the gene it transports in the nucleus of host cells.

[0072] For reference, the jetPRIME Transfection Reagent Kit consists of jetPRIME buffer solution and jetPRIME Transfection reagent. The jetPRIME buffer solution is used to dilute the plasmid vector to be delivered to the host cell, and the jetPRIME Transfection reagent captures the plasmid vector in the form of a vesicle (liposome) so that it can penetrate into the host cell.

[0073] The Human HAPLN1 ORF Clone was diluted with jetPRIME buffer to prepare a composition of the present invention containing an expression vector harboring a gene encoding the HAPLN1 protein.

[0074] JetPRIME Transfection Reagent was added to the cells, and the mixture was left for 10 minutes to allow the plasmid vector to be trapped in vesicles (liposomes), which were then dropped onto the cells in culture. After culturing the cells for over 48 hours, increased expression of the human HAPLN1 gene was confirmed by real-time qPCR, and increased expression of human HAPLN1 protein was confirmed by Western blot.

[0075] [Example 1] Evaluation of the anti-fibrotic efficacy of the composition of the present invention on human skin fibroblasts Normal human dermal fibroblasts (NHDF) were used as samples to examine the anti-fibrotic effect of the composition of the present invention on skin fibrosis.

[0076] 1. Experimental Method (1) Seeding of normal human dermal fibroblasts (NHDF) into a 6-well plate (1.0x10 5The cells were then cultured in Fibroblast Growth Medium-2 (FBM-2) (CC-3131 (Lonza)) medium at 37°C and 5% CO2 for 24 hours. (2) After replacing the medium with serum-free medium, the cells were incubated for 24 hours. (3) In contrast, under the conditions of treatment with TGFβ1 (10 ng / ml) to induce fibrosis, each well of the plate was treated with the composition of the present invention based on Preparation Example 2 but with rhHAPLN1 protein contents of 0, 3, 10, 30, and 100 ng / ml, and with 100 μg / mL and 200 μg / mL of the commercially available anti-fibrotic agent pirfenidone (Selleckchem), and then incubated for 24 hours. (4) After that, each sample was washed twice with phosphate buffered saline (PBS, pH 7.2), and the expression level of αSMA, a marker for myofibroblasts, was confirmed by Western blotting.

[0077] 2.Results In Figure 1A, the Western blot band photograph confirmed that the composition of the present invention exhibits a far superior anti-fibrotic effect compared to pirfenidone, based on the intensity and thickness of the αSMA protein band relative to the degree of fibrosis in normal human skin fibroblasts.

[0078] Furthermore, Figure 1B shows that after fibrosis induction with TGF1β (10 ng / ml), treatment with the composition of the present invention resulted in significantly lower αSMA expression levels across the entire rhHAPLN1 protein content range compared to treatment with a composition lacking rhHAPLN1 protein, demonstrating anti-fibrotic efficacy. In particular, the sample with an rhHAPLN1 protein content of 100 ng / ml of the composition of the present invention exhibited the most excellent anti-fibrotic efficacy, demonstrating 25% greater anti-fibrotic efficacy than pirfenidone at 100 μg / mL (100,000 ng / mL), despite being 1,000-fold lower. Because pirfenidone at 200 μg / mL exhibited higher αSMA expression levels than at 100 μg / mL, it is possible to infer that its anti-fibrotic efficacy declines at higher concentrations, with 100 μg / mL being the optimal anti-fibrotic concentration. If this is the case, it can be seen that the composition of the present invention has far superior antifibrotic efficacy compared to pirfenidone, a commercially available antifibrotic agent.

[0079] [Example 2] Evaluation of the anti-fibrotic efficacy of the composition of the present invention on human hepatic stellate cells Using human hepatic stellate cells (HHSCs) as samples, the anti-fibrotic effect of the composition of the present invention on liver fibrosis was examined.

[0080] 1. Experimental Method (1) According to the experimental design shown in Figure 2A, primary human hepatic stellate cells (HHSCs) (iXCells Biotechnologies, 10HU-210) were seeded into a 6-well plate (1.5x10 5 The cells were then cultured in Stellate Cell Growth Medium (iXCells Biotechnologies, MD-0014) and incubated at 37°C in a 5% CO2 incubator for 24 hours. (2) After replacing the medium with serum-free DMEM, the cells were incubated for 24 hours. (3) In contrast, the cells were treated with TGFβ1 (2 ng / ml) to induce fibrosis, followed by incubation for 24 hours. (4) Then, each well of the plate was treated with the composition of the present invention at a content of 0, 3, 10, 30, and 100 ng / ml of rhHAPLN1 protein, and 100 μg / mL and 200 μg / mL of pirfenidone, respectively, and then incubated for 24 hours. (5) Then, each sample was treated with a cell lysis buffer and then harvested. (6) Western blotting was performed to confirm the protein expression levels of αSMA and GAPDH using anti-αSMAA antibody (ab7817, Abcam) and anti-GAPDH antibody (sc-32233, Santa Cruz) as primary antibodies, and HRP-conjugated anti-mouse IgG antibody (#7076, California State University) as secondary antibody.

[0081] 2.Results In Figure 2B, the Western blot band photographs confirmed that the composition of the present invention exhibited a much superior anti-fibrotic effect compared to pirfenidone across the entire range of rhHAPLN1 protein content, based on the intensity and thickness of the αSMA protein band in relation to the degree of fibrosis of human hepatic stellate cells.

[0082] Furthermore, Figure 2C shows that after fibrosis induction with TGFβ1 (2 ng / ml), treatment with the composition of the present invention resulted in significantly lower expression levels of αSMA, a marker of hepatic stellate cell activity, across the entire range of rhHAPLN1 protein content compared to treatment with a composition lacking rhHAPLN1 protein, demonstrating anti-fibrotic efficacy. In particular, the sample with an rhHAPLN1 protein content of 3 ng / ml of the composition of the present invention exhibited the most excellent anti-fibrotic efficacy, demonstrating a much higher anti-fibrotic efficacy than not only pirfenidone 100 μg / mL (100,000 ng / ml) but also the even higher dose of pirfenidone 200 μg / mL (200,000 ng / ml), despite being 3 / 200,000th of the original concentration.

[0083] [Example 3] Evaluation of the antifibrotic efficacy of the composition of the present invention on a human colonic fibroblast cell line Using a human colon fibroblast cell line (CCD-18Co) as a sample, the anti-fibrotic effect of the composition of the present invention on intestinal fibrosis was examined.

[0084] 1. Experimental Method (1) According to the experimental design shown in Figure 3A, human colon fibroblast cell line (CCD-18Co) (CRL-1459 (ATCC)) was seeded into a 6-well plate (1.5 × 105 cells / well) and incubated in EMEM (Eagle's Minimum Essential Medium) (ATCC) medium containing 10% fetal bovine serum (FBS) at 37°C in a 5% CO2 incubator for 24 hours. (2) After replacing the medium with serum-free EMEM, the cells were incubated for 24 hours. (3) In contrast, the cells were treated with TGF-β (10 ng / ml) to induce fibrosis, and then incubated for 24 hours. (4) Then, each well of the plate was treated with the composition of the present invention at a content of 0, 3, 10, 30, and 100 ng / ml of rhHAPLN1 protein, and 100 μg / mL and 200 μg / mL of pirfenidone, respectively, and then incubated for 24 hours. (5) Then, each sample was treated with a cell lysis buffer and then collected. (6) Western blot analysis was performed using anti-αSMA antibody (ab7817, Abcam) and anti-GAPDH antibody (sc-32233, Santa Cruz) as primary antibodies, and HRP-conjugated anti-mouse IgG antibody (#7076, California State University) as secondary antibody to confirm the protein expression levels of αSMA and GAPDH.

[0085] 2.Results In Figure 3B, the Western blot band photographs confirmed that the composition of the present invention exhibited a significantly superior anti-fibrotic effect compared to pirfenidone across the entire range of rhHAPLN1 protein content, based on the intensity and thickness of the αSMA protein band in relation to the degree of fibrosis of human colonic fibroblasts.

[0086] Furthermore, Figure 3C shows that after fibrosis was induced with TGFβ1 (10 ng / ml), treatment with the composition of the present invention resulted in significantly lower expression levels of αSMA, a myofibroblast marker, across the entire range of rhHAPLN1 protein content compared to treatment with a composition not containing rhHAPLN1 protein, demonstrating anti-fibrotic efficacy.For reference, in intestinal fibrosis, myofibroblasts undergo transition into myofibroblasts and produce excessive fibrous tissue.

[0087] In particular, among the compositions of the present invention, the sample with an rhHAPLN1 protein content of 10 ng / ml showed the most excellent anti-fibrotic efficacy. Despite being 10,000-fold lower than pirfenidone at 100 μg / mL (100,000 ng / ml), the level of αSMA induced by TGFβ1 was calculated to be inhibited by 71.6% ((3.82-1.8) / (3.82-1)*100) for rhHAPLN1 and 10.6% ((3.82-3.52) / (3.82-1)*100) for pirfenidone. This indicates that the anti-fibrotic efficacy is approximately 7-fold higher than that of pirfenidone at 200 μg / mL (200,000 ng / ml), and is more than twice as effective, despite being 20,000-fold lower.

[0088] Since pirfenidone exhibits higher αSMA expression levels at 200 μg / mL than at 100 μg / mL, it can be inferred that the antifibrotic efficacy of pirfenidone decreases at higher concentrations, and that 100 μg / mL is the optimal antifibrotic concentration of pirfenidone. If this is the case, it can be seen that the composition of the present invention has significantly superior antifibrotic efficacy compared to pirfenidone, a commercially available antifibrotic agent.

[0089] [Example 4] Evaluation of the anti-fibrotic efficacy of the composition of the present invention on human peripheral blood endothelial cells Human cardiac microvascular endothelial cells (HCMEC) were used as samples to examine the anti-fibrotic effect of the composition of the present invention on cardiac fibrosis.

[0090] 1. Experimental Method (1) According to the experimental design shown in Figure 4A, primary human cardiac microvascular endothelial cells (HCMEC) (#6000 (ScienCell)) were seeded into a 6-well plate (5.7 x 10 4The cells were then cultured on endothelial cell medium (ScienCell) in an incubator at 37°C and 5% CO2 for 24 hours. (2) In contrast, the cells were treated with TGFβ1 (5 ng / ml) to induce fibrosis, and then incubated for 3 days. (3) Then, each well of the plate was treated with the composition of the present invention at a content of 0, 3, 10, 30, and 100 ng / ml of rhHAPLN1 protein, and 100 μg / mL and 200 μg / mL of pirfenidone, respectively, and then incubated for 2 days. (4) Then, each sample was treated with a cell lysis buffer and then harvested. (5) Western blotting was performed to confirm the protein expression levels of αSMA and GAPDH using anti-αSMAA antibody (ab7817, Abcam) and anti-GAPDH antibody (sc-32233, Santa Cruz) as primary antibodies, and HRP-conjugated anti-mouse IgG antibody (#7076, California State University) as secondary antibody.

[0091] 2.Results In Figure 4B, the Western blot band photographs confirmed that the composition of the present invention exhibits a far superior anti-fibrotic effect compared to pirfenidone across the entire range of rhHAPLN1 protein content, based on the intensity and thickness of the αSMA protein band, which indicates the degree of fibrosis of human cardiac peripheral blood endothelial cells.

[0092] Furthermore, Figure 4C shows that after fibrosis was induced with TGFβ1 (5 ng / ml), treatment with the composition of the present invention resulted in significantly lower expression levels of αSMA, a myofibroblast marker, across the entire range of rhHAPLN1 protein content, compared to treatment with a composition not containing rhHAPLN1 protein, demonstrating anti-fibrotic efficacy.For reference, in cardiac fibrosis, endothelial cells are transformed into myofibroblasts and produce excessive fibrous tissue.

[0093] In particular, among the compositions of the present invention, the sample with an rhHAPLN1 protein content of 3 ng / ml exhibited the most excellent anti-fibrotic efficacy. Although this is a concentration that is 3 / 100,000th of that of pirfenidone at 100 μg / mL (100,000 ng / ml), it exhibited anti-fibrotic efficacy not exhibited by pirfenidone, and although this is a concentration that is 3 / 200,000th of that of pirfenidone at 200 μg / mL (200,000 ng / ml), it exhibited anti-fibrotic efficacy not exhibited by pirfenidone.

[0094] Regardless of the concentration of pirfenidone, the expression level of αSMA was higher than that of a composition not containing rhHAPLN1 protein, indicating that its anti-fibrotic effect in peripheral cardiac endothelial cells was further reduced. If this is the case, it suggests that the composition of the present invention may be an almost unique anti-fibrotic agent, at least in peripheral cardiac endothelial cells.

[0095] [Example 5] Evaluation of the anti-fibrotic efficacy of the composition of the present invention on normal human lung fibroblasts Normal human lung fibroblasts (NHLF) were used as samples to examine the anti-fibrotic effect of the composition of the present invention on pulmonary fibrosis.

[0096] 1. Experimental Method (1) Normal human lung fibroblasts (NHLFs) cultured for 9 passages were seeded into a 6-well plate (1.0x10 5 The cells were then cultured in Fibroblast Growth Medium-2 (FBM-2) (CC-3131 (Lonza)) medium at 37°C and 5% CO2 for 24 hours. (2) After replacing the medium with serum-free medium, the cells were incubated for 24 hours. (3) In contrast, under the condition of treatment with TGFβ1 (10 ng / ml) to induce fibrosis, each well of the plate was treated with the composition of the present invention at rhHAPLN1 protein contents of 0, 3.1, 12.5, 25, and 100 ng / ml, and pirfenidone (Selleckchem) at 100 μg / mL and 200 μg / mL, respectively, and then incubated for 24 hours. (4) After that, each sample was washed twice with phosphate-buffered saline (PBS (pH 7.2)), and the expression level of αSMA, a marker for myofibroblasts, was confirmed by Western blotting.

[0097] 2.Results In Figure 5A, the Western blot band photographs confirmed that the composition of the present invention exhibited a far superior anti-fibrotic effect compared to pirfenidone across the entire range of rhHAPLN1 protein content, based on the intensity and thickness of the αSMA protein band relative to the degree of fibrosis in normal human lung fibroblasts.

[0098] Furthermore, in Figure 5B, after inducing fibrosis with TGFβ1 (10 ng / ml), treatment with the composition of the present invention showed significantly lower expression levels of αSMA, a myofibroblast marker, across the entire range of rhHAPLN1 protein content, compared to treatment with a composition not containing rhHAPLN1 protein, demonstrating anti-fibrotic efficacy.

[0099] In particular, among the compositions of the present invention, the sample with an rhHAPLN1 protein content of 12.5 ng / ml exhibited the most excellent anti-fibrotic efficacy. Despite having a content 10,000 times lower than that of pirfenidone at 100 μg / mL (100,000 ng / ml), the anti-fibrotic efficacy was approximately twice as high, and similarly, it was approximately twice as high as that of pirfenidone at 200 μg / mL (200,000 ng / ml).

[0100] Regardless of the concentration, pirfenidone treatment showed almost no difference in αSMA expression levels compared to treatment with a composition not containing rhHAPLN1 protein, indicating that it has no antifibrotic effect, at least in lung fibroblasts. If this is the case, it suggests that the composition of the present invention may be an almost unique antifibrotic formulation in lung fibroblasts.

[0101] [Example 6] Evaluation of the fibrosis prevention efficacy of the composition of the present invention in human kidney renal tubular cells Using a human kidney tubular cell (HK-2: human kidney 2) epithelial cell line as a sample, the fibrosis preventive effect of the composition of the present invention on kidney fibrosis was examined.

[0102] 1. Experimental Method (1) According to the experimental design in Figure 6A, human kidney tubular cells (HK-2) were seeded into a 6-well plate (1.0x10 5 The cells were then cultured in RPMI 1640 Medium (11875-093 (Gibco)) containing 10% FBS and incubated in an incubator at 37°C and 5% CO2 for 24 hours. (2) After washing with PBS several times, each well of the plate was simultaneously treated with TGFβ1 (5 ng / ml), the composition of the present invention (rhHAPLN1 protein content: 0, 5, 10, 20, and 50 ng / ml), and pirfenidone 0.2 mg / mL (200 μg / mL), followed by incubation for 24 hours. (3) After that, each sample was washed several times with ice-cold PBS, treated with RIPA (radioimmunoprecipitation assay) Lysis and Extraction Buffer (Thermo Scientific) containing protease / phosphatase inhibitors, scraped with a scraper to prepare the sample, and then transferred to an E-tube. (4) After centrifugation (12,000 rpm, 20 minutes, 4°C), the supernatant was collected and subjected to a BCA (bicinchoninic acid) assay to obtain a protein quantification value. (5) After the obtained quantitative values ​​were determined, the samples were cooked at 100°C for 3 minutes and then stored in a refrigerator at -20°C. (6) 4–15% precast acrylamide PAGE gels were used, and 10 μg of protein was loaded per lane and run (80 V: 20 min, 120 V: 1 hour 30 min). (7) Changed to 100V: 1 hour 30 minutes and blocked with a smart blocker for about 5 minutes. (8) The primary antibody was diluted 1:1,000 in 1% BSA-TBST (bovine serum albumin-Tris-buffered saline & polysorbate 20), dispensed, and incubated overnight at 4°C. (9) The next day, the plate was washed three times with TBST for 10 minutes each. (10) The secondary antibody was diluted 1:2,000 in 1% BSA-TBST and dispensed, followed by shaking at room temperature for approximately 1 hour. (11) After further washing with TBST three times for 10 min each, the plate was detected using ChemiDoc.

[0103] 2.Results In Figure 6B, the Western blot band photograph confirmed that the composition of the present invention exhibits superior fibrosis prevention effects compared to pirfenidone at a specific rhHAPLN1 protein content range (rhHAPLN1 protein content 50 ng / ml), based on the concentration and thickness of the αSMA protein band in relation to the degree of fibrosis of human kidney renal tubule cells.

[0104] Furthermore, Figure 6C shows that after inducing fibrosis with TGF-β (5 ng / ml), treatment with the composition of the present invention containing 50 ng / ml of rhHAPLN1 protein exhibited superior fibrosis prevention efficacy compared to treatment with a composition not containing rhHAPLN1 protein. This result indicates that despite the concentration being 1 / 4,000 of that of pirfenidone at 200 μg / mL (200,000 ng / ml), the fibrosis prevention efficacy was much higher.

[0105] [Example 7] Evaluation of the anti-fibrotic efficacy of the composition of the present invention on human kidney tubular cells The anti-fibrotic effect of the composition of the present invention on renal fibrosis was examined using an epithelial cell line of human kidney tubular cells (HK-2: human kidney 2) as a sample.

[0106] 1. Experimental Method (1) According to the experimental design in Figure 7A, human kidney tubular cells (HK-2) were seeded into a 60-well plate (2.8 x 10 5 The cells were then cultured in RPMI 1640 Medium (11875-093 (Gibco)) containing 10% FBS and incubated in an incubator at 37°C and 5% CO2 for 24 hours. (2) After that, the cells were washed several times with PBS, and each well of the plate was treated with TGFβ1 (5 ng / ml) and incubated for 24 hours. (3) Subsequently, the cells were treated with the composition of the present invention (rhHAPLN1 protein content: 0, 5, 10, 20, and 50 ng / ml) and pirfenidone at 0.2 mg / mL (200 μg / mL), respectively, and then incubated for 48 hours. (4) After that, each sample was washed several times with ice-cold PBS, treated with RIPA (radioimmunoprecipitation assay) Lysis and Extraction Buffer (Thermo Scientific) containing protease / phosphatase inhibitors, scraped with a scraper to prepare the sample, and then transferred to an E-tube. (5) After centrifugation (12,000 rpm, 20 minutes, 4°C), the supernatant was collected and subjected to a BCA assay to obtain a quantitative protein value. (6) After the obtained quantitative values ​​were determined, the samples were cooked at 100°C for 3 minutes and then stored in a refrigerator at -20°C. (7) 8% precast acrylamide PAGE gels were used, and 10 μg of protein was loaded per lane and run (80 V: 20 min, 120 V: 1 h 30 min). (8) Changed to 100V: 1 hour 30 minutes and blocked with a smart blocker for about 5 minutes. (9) The primary antibody was diluted 1:1,000 in 1% BSA-TBST, dispensed, and reacted overnight at 4°C. (10) The next day, the plate was washed three times with TBST for 10 minutes each. (10) The secondary antibody was diluted 1:2,000 in 1% BSA-TBST and dispensed into aliquots, followed by shaking at room temperature for approximately 1 hour. (11) After further washing with TBST three times for 10 min each, the plate was detected using ChemiDoc.

[0107] 2.Results In Figure 7B, the Western blot band photograph confirmed that the composition of the present invention exhibits anti-fibrotic activity in a specific range of rhHAPLN1 protein content (rhHAPLN1 protein content of 10 to 50 ng / ml), based on the concentration and thickness of the αSMA protein band, which indicates the degree of fibrosis of human kidney renal tubule cells.

[0108] 7C shows that after fibrosis induction with TGF-β (10 ng / ml), treatment with the composition of the present invention at 10 to 50 ng / ml of rhHAPLN1 protein, especially treatment with 50 ng / ml of rhHAPLN1 protein, showed superior anti-fibrotic efficacy compared to treatment with a composition not containing rhHAPLN1 protein. It is quite noteworthy that treatment with 50 ng / ml of the composition of the present invention showed almost the same anti-fibrotic efficacy as pirfenidone, despite being a quarter of the dose of 0.2 mg / ml, i.e., 200 ng / ml, of pirfenidone.

[0109] [Example 8] Observation of cell morphological changes due to the anti-fibrotic effect of the composition of the present invention on human kidney tubular cells Using a human kidney tubular cell (HK-2: human kidney 2) epithelial cell line as a sample, the anti-fibrotic effect of the composition of the present invention on renal fibrosis was verified by observing changes in cell morphology.

[0110] 1. Experimental Method (1) According to the experimental design of Figure 8A, human kidney tubular cells (HK-2) were seeded into a 6-well plate (3.0x10 5 The cells were then cultured in RPMI 1640 Medium (11875-093 (Gibco)) containing 10% FBS and incubated in an incubator at 37°C and 5% CO2 for 24 hours. (2) After washing with PBS several times, each well of the plate was treated with TGFβ1 (10 ng / ml) and incubated for 24 hours. (3) Subsequently, the cells were treated with the composition of the present invention (rhHAPLN1 protein content: 0, 5, 10, 20, 50, and 100 ng / ml) and pirfenidone at 0.2 mg / mL (200 μg / mL), respectively, and then incubated for 24 hours. (4) Then, each well containing a sample was washed twice with 1X PBS, and then treated with 4% paraformaldehyde and fixed for 1 hour. (5) After washing twice with 1X PBS, the appearance and morphology of the cells were observed under a microscope.

[0111] 2.Results In Figure 8B, cell morphology varied depending on the treatment. In the control group (normal cells without any treatment), the cells were round, but treatment with 10 ng / ml TGFβ1 confirmed that the round cell morphology changed to a pointed shape due to fibrosis.

[0112] However, by adding the compositions of the present invention having various rhHAPLN1 protein contents, the morphology of the cells changed to a rounder form, as in the case of pirfenidone.

[0113] This proves that the composition of the present invention reliably exhibits excellent anti-fibrotic efficacy on cells.

[0114] [Example 9] Evaluation of the anti-fibrotic efficacy of the composition of the present invention on renal proximal tubular epithelial cells 1 Using primary epithelial cells of renal proximal tubule epithelial cells (RPTEC), the anti-fibrotic effect of the composition of the present invention on aging-induced renal fibrosis was examined.

[0115] 1. Experimental Method (1) Renal proximal tubular epithelial cells (RPTECs) from the 3rd and 10th passages were seeded onto a 60mm diameter culture dish (5.0x10 5 The cells were cultured in complete Renal Epithelial Cell Medium (ATCC PCS-400-030 + ATCC PCS-400-040) in an incubator at 37°C and 5% CO2 for 24 hours. (2) Subsequently, the cells were treated with the compositions of the present invention (rhHAPLN1 protein contents of 0, 10, and 100 ng / ml), and then further cultured for 72 hours. (4) After that, the wells were washed three times with ice-cold PBS, and then treated with 300 μl of RIPA Lysis and Extraction Buffer containing protease inhibitors and phosphatase inhibitors per well. The cells were then scraped with a scraper to recover the cells, which were then transferred to an E-tube. (5) After centrifugation (12,000 rpm, 20 minutes, 4°C), the supernatant was collected and subjected to a BCA assay to obtain a protein quantification value. (6) 4–15% precast acrylamide PAGE gels were used, and 20 μg of protein was loaded per lane and run (80 V: 20 min, 120 V: 1 h 30 min). (7) Western blot analysis was performed using anti-αSMAA antibody (ab7817, Abcam) and anti-GAPDHA antibody (sc-32233, Santa Cruz) as primary antibodies, and HRP-conjugated anti-mouse IgG antibody (#7076, CST) as secondary antibody.

[0116] 2.Results As can be seen from Figure 9A, as the cells aged, i.e., as the number of subcultures increased from 4 to 11, the expression level of αSMA protein increased, as can be seen from the increased density and thickness of the protein band in the Western blot band photograph.

[0117] 9B shows that in senescent cells (P11), treatment with the composition of the present invention at a concentration of 100 ng / ml of rhHAPLN1 protein reduced the expression level of αSMA protein by approximately one-third compared to treatment with a composition not containing rhHAPLN1 protein, demonstrating excellent anti-fibrotic efficacy.

[0118] [Example 10] Evaluation of the anti-fibrotic efficacy of the composition of the present invention on renal proximal tubular epithelial cells 2 Using primary epithelial cells of renal proximal tubule epithelial cells (RPTEC), the anti-fibrotic effect of the composition of the present invention on aging-induced renal fibrosis was examined.

[0119] 1. Experimental Method (1) Renal proximal tubular epithelial cells (RPTECs) from passages 3 and 10 were seeded into 12-well plates (1.5x10 5 The cells were cultured in complete Renal Epithelial Cell Medium (ATCC PCS-400-030 + ATCC PCS-400-040) in an incubator at 37°C and 5% CO2 for 24 hours. (2) Subsequently, the cells were treated with the composition of the present invention (rhHAPLN1 protein content: 0, 10, 20, 50, and 100 ng / ml) and pirfenidone at 0.2 mg / mL (200 μg / mL), and then cultured for 72 hours. (3) Then, each well was washed twice with PBS, and then treated with 4% paraformaldehyde for fixation for 1 hour. (4) After washing four times with 1x PBS, the sections were treated with 1x PBST (1x PBS + 0.1% Triton X-100) containing 1% BSA and blocked for 2 hours. (5) The primary antibody against αSMA (Abcamab7817) was diluted 1:200 in 1X PBSTdp and incubated overnight at 4°C. (6) The next day, the cells were washed four times with 1X PBST. (7) Secondary antibody (anti-mouse IgG Alexa Fluor TM After treatment with 4881:500 (A28175 (Invitrogen)) and DAPI 1:2,000 (D1306 (Invitrogen)), the sections were incubated at room temperature for 1 hour. (8) Then, 50 μl of Vectashield (H-1000 (Vectorlabs)) was dropped onto the cells, which were then covered with a coverslip and photographed under a fluorescence microscope.

[0120] 2.Results As can be seen from Figure 10A, as the cells aged, i.e., as the number of subcultures increased from 4 to 11, the expression level of αSMA protein increased, i.e., fibrosis progressed, as can be seen from the increase in bright fluorescent areas in the right-hand photograph of subculture 11.

[0121] 10B, in senescent cells (cultured for 11 passages), treatment with the composition of the present invention revealed an increased rhHAPLN1 protein content compared to treatment with a composition not containing rhHAPLN1 protein, and in particular, at rhHAPLN1 protein concentrations of 50 and 100 ng / mL, fewer fluorescently stained areas were observed, similar to treatment with pirfenidone. Considering that the pirfenidone content is 0.2 mg / mL (200,000 ng / mL), and the rhHAPLN1 protein content in the composition of the present invention is only 1 / 4,000 to 1 / 2,000 of that of pirfenidone, it can be seen that the composition of the present invention exhibits superior antifibrotic efficacy compared to conventional antifibrotic preparations.

[0122] [Example 11] Evaluation of the antifibrotic efficacy of the composition of the present invention using various antifibrotic markers The BioMAP Fibrosis Panel (provided by Eurofins discovery) was used to examine the expression levels of various fibrosis markers, thereby verifying the anti-fibrotic effect of the composition of the present invention on pulmonary fibrosis and renal fibrosis.

[0123] The BioMAP Fibrosis panel consists of three models: the SAEMyoF system (co-culturing small airway epithelial cells and adult fibroblasts), the REMyoF system (co-culture of renal proximal tubule epithelial cells and adult fibroblasts), and the MyoF system (differentiated lung myofibroblasts). The SAEMyoF system represents interstitial lung disease (IDL), including pulmonary fibrotic diseases such as idiopathic pulmonary fibrosis (IPF). The REMyoF system represents kidney fibrosis associated with end-stage renal failure. However, the MyoF system was used to investigate the effects of fibrosis on myofibroblasts only.

[0124] 1. Experimental Method (1) Each primary cell line was prepared by pooling cells derived from various donors (n = 3–6). The cells were first cultured in a 96-well plate until they reached confluence, after which peripheral blood mononuclear cells (PBMCs) were added. (2) Subsequently, the cells were treated with the compositions of the present invention (rhHAPLN1 protein contents of 0, 3.7, 11, and 33 ng / ml) at different concentrations, and then cultured for 1 hour. (3) Then, the cells were treated with TNFα and TGFβ1 and incubated for 48 hours. (4) Direct ELISA (enzyme-linked immunosorbent assay) was performed to examine the expression levels of various biomarkers.

[0125] 2.Results Each quantitative value was compared with the vehicle control group using the relative fold change. Statistical analysis was performed using an unpaired t-test (significant differences vs. vehicle control) (*p<0.05, **p<0.01, and ***p<0.001).

[0126] Experimental results confirmed that the composition of the present invention (rhHAPLN1) significantly reduced αSMA and collagen I expression in the SAEMyoF system, a pulmonary fibrosis disease model (Figure 11A). Specifically, in the case of αSMA, treatment with the composition of the present invention consistently reduced αSMA expression by 0.9-fold or less compared to treatment with a composition lacking rhHAPLN1 protein, regardless of the rhHAPLN1 protein content in the composition. Furthermore, in the case of collagen I, treatment with the composition of the present invention consistently reduced collagen I expression by a significant amount compared to treatment with a composition lacking rhHAPLN1 protein, regardless of the rhHAPLN1 protein content in the composition. In particular, when the rhHAPLN1 protein content in the composition was 11 ng / ml, collagen I expression was reduced by 0.8-fold or less.

[0127] Furthermore, in the REMyoF system, a kidney fibrosis disease model, we confirmed that the composition of the present invention (rhHAPLN1) significantly reduced collagen I expression (Figure 11B). That is, compared to treatment with a composition not containing rhHAPLN1 protein, treatment with the composition of the present invention reduced collagen I expression by 0.9-fold or less, regardless of the rhHAPLN1 protein content in the composition. In particular, when the rhHAPLN1 protein content in the composition was 11 ng / ml, collagen I expression was reduced by almost 0.7-fold, suggesting that this concentration is the optimal concentration for exerting anti-fibrotic effects in the kidney.

[0128] Furthermore, in the MyoF system involving myofibroblasts, we confirmed that the composition of the present invention (rhHAPLN1) significantly reduced collagen IV (Figure 11C). That is, compared to treatment with a composition not containing rhHAPLN1 protein, treatment with the composition of the present invention reduced collagen I expression by 0.8-fold or less, in proportion to the rhHAPLN1 protein content in the composition. Therefore, when the rhHAPLN1 protein content in the composition was 33 ng / ml, collagen IV expression was reduced by 0.7-fold.

[0129] [Example 12] Evaluation of the anti-fibrotic efficacy (ability to prevent fibrotic diseases) of the composition of the present invention using an animal model of pulmonary fibrosis An experiment was conducted in which the composition of the present invention was repeatedly administered by inhalation to animals with induced pulmonary fibrosis, to verify its effectiveness in preventing bleomycin-induced pulmonary fibrosis (BILP).

[0130] 1. Preparation and care of experimental animals The experimental animals were 8-week-old female C57BL / 6N (KBSI, Daejeon, Republic of Korea) mice. They were divided into a normal control group (PBS) and two treatment groups (the composition of the present invention containing rhHAPLN1 protein). Water was changed every two days and was available ad libitum. Mice were housed in cages of four. The temperature in the room was maintained at 21-24°C, the humidity was maintained at 40-60%, and the day-night cycle was 12 hours.

[0131] 2. Lung tissue staining and microscopic observation To construct the bleomycin-induced pulmonary fibrosis (PF) model, 8-week-old female C57BL / 6N mice were divided into four groups (normal, control, 0.0005% (w / w) rhHAPLN1, and 0.0015% (w / w) rhHAPLN1) with four mice per group. The IPF disease induction method was as described in Liu et al. (Methods in Molecular Biology 2017, DOI: 10, 1007 / 978-1-4939-7113-8_2) (see Figure 12A). To induce idiopathic pulmonary fibrosis (IPF), bleomycin (bleomycin sulfate from Streptomyces verticillus) (B8416, Sigma-Aldrich Co.) was administered orally via a single, careful intratracheal instillation at a concentration of 2 U / kg using a 1 ml syringe and a 0.9 × 50 mm, 20-gauge oral needle. After careful intratracheal instillation, 10 ml of fresh phosphate-buffered saline (1× PBS, pH 7.4, Gibco Co.) was added to a nebulizer, allowing the nebulizer to generate aerosols that could be inhaled freely.

[0132] Starting one day after bleomycin administration, the control group received a mixture of 50 μl of a stock solution composed of 20 mM Tris-HCl, 0.5 M NaCl (pH 8.0), and 50% glycerol and 10 ml of PBS (1X, pH 7.4 (Gibco Co.)). The two groups receiving the composition of the present invention received 50 μl of rhHAPLN1 stock solution, adjusted to rhHAPLN1 weight concentrations of 0.0005% (w / w), i.e., 5,000 ng / mL, and 0.0015% (w / w), i.e., 15,000 ng / mL, which were then mixed with 10 ml of PBS (1X, pH 7.4 (Gibco Co.)). The mixture was administered to each group once daily for one hour via nebulization using the aforementioned nebulizer. The weight of each experimental animal was measured every two days to confirm that there was no change in weight, and a Mass Dosing System and Aerosol Chamber (Data Science International Co.) were used to generate smoke.

[0133] The total experimental period was 12 days, and the recombinant HAPLN1 protein at each concentration was administered 10 times for 10 days, once a day for 1 hour, and then the mice were anesthetized on the 11th day and dissected.

[0134] 3.Results 1) Photographic comparison of lung tissue 12B is a photograph of the lung tissue after removal, in which the left large lobe was cut in half transversely, the upper part fixed in formalin, and stained with hematoxylin and eosin (H&E). From the top of the photograph, it can be seen that compared to the control group, administration of the composition of the present invention, especially at a weight ratio concentration of 0.0015% (w / w) of rhHAPLN1, showed a morphology significantly similar to that of the normal group.

[0135] 2) Numerical values ​​of the preventive efficacy of the composition of the present invention Next, the middle portion of the left large lung lobe of each mouse was cut, and three histological slides were prepared per mouse. Three randomly selected areas on each slide were photographed, and the nine sections were divided into red and white areas. The areas of the red stained areas were measured using Image J software, and the average values ​​were calculated to obtain the mean value per mouse. The red areas indicate areas with advanced fibrosis.

[0136] The remaining three mice were also measured using the same method to obtain mean values, which were then re-averaged to finally obtain the mean area reflecting fibrosis and its deviation. The P value for significance between the normal group not treated with bleomycin and the control group was 0.00038 (***P<0.001), and the P value for significance between the group not treated with the composition of the present invention (rhHAPLN1) and the 0.0015% (w / w) rhHAPLN1-treated group was 0.00355 (**P<0.01), confirming statistical significance.

[0137] 12C, which shows these average values ​​in a graph, reveals that when the composition of the present invention was administered, the average area of ​​the red portion was reduced compared to the control group, particularly when rhHAPLN1 was administered at a weight ratio concentration of 0.0015% (w / w), so that the average area was significantly closer to that of the normal group. This confirms that the composition of the present invention exhibits a preventive effect against pulmonary fibrosis in vivo.

[0138] 3) Preventive efficacy of the composition of the present invention based on Ashcroft score Figures 12D and 12E are photographs and captions illustrating a formula that allows for a simple visual assessment of the severity of pulmonary fibrosis (Ashcroft et al 1988, J Clin Pathol 41: 467-470).

[0139] Fibrosis can be graded using the Ashcroft score as follows: Score 0: Thin, small fibers are observed in some alveolar walls, but no obvious fibrous structure or burden is seen (normal lung). Score 1: Isolated soft fibrotic changes (septal thickness less than or equal to three times normal); alveoli partially enlarged and sparse, but no fibrous masses (masses) are present. Score 2: Clear fibrotic changes (septal thickness more than three times normal); alveoli are partially enlarged and sparse, but no fibrous masses are observed. Score 3: In the entire microscopic area, mainly continuous fibrous septa were observed (septal thickness was more than three times normal), and the alveoli were partially enlarged and sparse, but no fibrous masses were present. Score 4: Architectural change; single fibrous mass less than or equal to 10% of the microscopic area. Score 5: structural changes; merged fibrous mass (greater than 10% and less than 50% of the microscopic area or similar); lung architecture severely damaged but still retaining its morphology. Score 6: Architectural changes; little morphology present. Continuous fibrous mass (greater than 50% of the microscopic area); poorly preserved lung architecture. Score 7: Alveolar structure is absent. The alveoli are mostly obliterated by fibrous masses, but up to five air bubbles still remain. Score 8: Alveolar structures are absent. Under microscopic observation, the alveoli are completely obliterated by fibrous masses.

[0140] Based on the above evaluation criteria, three observers individually judged and scored the same data under conditions that were not influenced by the evaluations of others. The results were then combined to determine a final average score. The results of each evaluator were very similar, and the result of the representative observer is shown in Figure 12F.

[0141] In conclusion, the normal group had an Ashcroft score of 0, the control group had an Ashcroft score of 6.25, the group administered Composition 1 of the present invention (rhHAPLN 10.0005%) had an Ashcroft score of 5.25, and the group administered Composition 2 of the present invention (rhHAPLN 10.0015%) had an Ashcroft score of 0.5, demonstrating statistical significance between the normal group and the control group and between the control group and the group administered Composition 2 of the present invention.

[0142] [Example 13] Evaluation of the anti-fibrotic efficacy (ability to treat fibrotic diseases) of the composition of the present invention using an animal model of pulmonary fibrosis An experiment was conducted to verify the effectiveness of the composition of the present invention in improving bleomycin-induced pulmonary fibrosis (BILP) that had already occurred by repeatedly inhaling the composition of the present invention to animals with pulmonary fibrosis. This experiment was basically similar to Example 12, but bleomycin-induced pulmonary fibrosis (BILP) was fully developed 7 days after bleomycin treatment, and the experimental results presented here demonstrate that the composition of the present invention has the ability to treat fibrotic diseases.

[0143] 1. Preparation and care of experimental animals This is the same as in Example 12.

[0144] 2. Staining and Microscopic Observation of Lung Tissue To establish the bleomycin-induced pulmonary fibrosis (PF) model, 8- to 10-week-old female C57BL / 6N mice were divided into four groups (normal group, PBS control group, 0.00075% (w / w) rhHAPLN1 group, and 0.0015% (w / w) rhHAPLN1 group) with four mice per group. The IPF disease induction method was as described in Liu et al. (Methods in Molecular Biology 2017, DOI: 10, 1007 / 978-1-4939-7113-8_2) (see Figure 13A). To induce idiopathic pulmonary fibrosis (IPF), bleomycin sulfate from Streptomyces verticillus (B8416, Sigma-Aldrich Co.) was administered intraorally at a dose of 50 U / kg (20 μl per mouse) using a 1 ml syringe and a 0.9 × 50 mm, 20-gauge oral needle. After anesthesia, the mice were carefully administered bleomycin intratracheally. After instillation, 10 ml of fresh phosphate-buffered saline (1× PBS, pH 7.4, Gibco Co.) was added to a nebulizer, and the nebulized aerosol was allowed to inhale freely.

[0145] From 8 days after bleomycin administration, the control group received a mixture of 50 μl of a stock solution composed of 20 mM Tris-HCl, 0.5 M NaCl (pH 8.0), and 50% glycerol and 10 ml of PBS (1X, pH 7.4 (Gibco Co.)). For the two groups receiving the composition of the present invention, a stock solution of rhHAPLN1 (20 mM acetate (pH 5.0), 8.0 M sucrose, 0.04% PS80) was diluted with phosphate-buffered saline (PBS) to adjust the weight ratio of rhHAPLN1 to 0.00075% (w / w), i.e., 7,500 ng / mL, and 0.0015% (w / w), i.e., 15,000 ng / mL, in 50 μl of the solution. The mixture was then mixed with 10 ml of PBS (1X, pH 7.4 (Gibco Co.)) and administered to each group via nebulization (10 ml / hour / 14 times / day) once daily for 1 hour. The weight of each experimental animal was measured every two days to ensure no weight change occurred. A mass dosing system and an aerosol chamber (Data Science International Co.) were used to generate the aerosol. The total experimental period was 23 days, and the actual recombinant human HAPLN1 protein at each concentration was administered at 10 ml per day for 1 hour, a total of 14 times for 14 days. The mice were anesthetized the day after the final exposure and then dissected.

[0146] 3.Results 1) Photographic comparison of lung tissue 13B is a photograph of the lung tissue after removal, in which the left large lobe was cut in half transversely, the upper part fixed in formalin, and stained with hematoxylin and eosin (H&E). From the top of the photograph, it can be seen that compared to the control group, administration of the composition of the present invention, particularly at a weight ratio concentration of rhHAPLN1 of 0.0015% (w / w), showed a morphology that was quite similar to that of the normal group.

[0147] 2) Therapeutic efficacy of the composition of the present invention expressed in numerical values Next, the middle portion of the left large lung lobe of each mouse was cut, and three histological slides were prepared per mouse. Three randomly selected areas on each slide were photographed, and the nine sections were divided into red and white areas. The areas of the red stained areas were measured using Image J software, and the average values ​​were calculated to obtain the mean value per mouse. The red areas indicate areas with advanced fibrosis.

[0148] The remaining three mice were also measured using the same method to obtain mean values, which were then re-averaged to finally obtain the mean area and deviation reflecting fibrosis. The P value for significance between the normal group not treated with bleomycin and the control group was 0.00038 (***P<0.001), and the P value for significance between the group not treated with the composition of the present invention (rhHAPLN1) and the 0.0015% (w / w) rhHAPLN1-treated group was 0.00355 (**P<0.01), confirming statistical significance.

[0149] Figure 13C, which shows these average values, reveals that when the composition of the present invention was administered, the average red area was reduced by 51% compared to the control group, particularly when rhHAPLN1 was administered at a weight ratio of 0.0015% (w / w). This confirms that the composition of the present invention exhibits a significant therapeutic effect on pulmonary fibrosis in vivo. This is further illustrated in Figure 13D. ***p<0.001, calculated by Student's t-test.

[0150] 3) Therapeutic efficacy of the composition of the present invention according to Ashcroft score Based on the evaluation criteria described in Example 12, three observers individually judged and scored the data under conditions unaffected by the evaluations of others, and then combined the results to determine a final average score. The results of each observer were very similar, and the result of the representative observer is shown in Figure 13E. This again confirmed that the composition of the present invention not only has a preventive effect but also an excellent therapeutic effect on pulmonary fibrosis in vivo.

[0151] [Example 14] Evaluation of the anti-fibrotic efficacy of the composition of the present invention using an animal model of induced renal fibrosis An experiment was conducted to verify the therapeutic effect of repeated administration of the composition of the present invention to animals with acute renal fibrosis.

[0152] 1. Construction of Acute Kidney Injury Induction Model According to the experimental design shown in Figure 14A, 8-week-old C57BL / 6 male mice underwent flank incision under pentobarbital anesthesia (50 mg / kg BW, i.p.). To induce ischemia, the kidneys were exposed and bilateral renal pedicles were occluded using non-traumatic microaneurysm clamps (Roboz Surgical Instruments) to occlude blood flow for 25 minutes. The clamps were then removed and the incision site was sutured. A control operation (sham operation) was performed identically to the ischemia / reperfusion experiment, except for the occlusion of blood flow.

[0153] One day after ischemia, blood urea nitrogen (BUN) was measured, and only mice with a BUN level of approximately 98 mg / dL or higher (hereinafter referred to as 100 mg / dL) were grouped into the experimental group for the experiment. Starting from day 7 after surgery, the experimental group received daily administration of various drugs, including the composition of the present invention. Kidneys were removed on day 14 and used for histological and biochemical analysis. Drug concentrations are shown in Table 1, and details of the experimental groups are shown in Table 2.

[0154] [Table 1]

[0155] [Table 2]

[0156] On the 7th day after drug administration, blood was taken to measure the blood urea nitrogen (BUN) concentration, and on the 14th day, blood and urine were taken to measure the creatinine concentration in the blood and urine, and the blood urea nitrogen (BUN) concentration in the blood. The creatinine clearance rate was also calculated.

[0157] 2. Results derivation experiment A. Evaluation of the efficacy of the composition of the present invention in reducing αSMA (1) Experimental method For biochemical analysis (evaluation of fibrosis index expression), Western blotting was performed using antibodies against αSMA to examine the expression level in kidney tissue.

[0158] To confirm the expression of αSMA in kidney tissues from control (sham-operated) and ischemia / reperfusion (IR) mice, samples from each experimental group were loaded onto a single gel and subjected to Western blotting to measure the density of each band. Ponceau and GADPH were used as equal loading markers (Figure 14B). Here, SV: sham-vehicle, SB: sham-rhHAPLN1-dissolving solution, IRV: IR-vehicle, IRP: IR-pirfenidone, IRA: IR-inventive composition (rhHAPLN1) A, IRB: IR-inventive composition (rhHAPLN1) B, and IRC: IR-inventive composition (rhHAPLN1) C are shown, respectively. Further details are shown in Tables 1 and 2. More specifically, the Sham-Vehicle group was a sham control group that received IP injection of rhHAPLN1-free buffer diluted with PBS. The Sham-rhHAPLN1-dissolving solution group was a group that received IP injection of rhHAPLN1 concentration B (0.02 mg / kg) diluted to confirm the effect of rhHAPLN1 on normal conditions. The IR-Vehicle group was a group treated with rhHAPLN1-free buffer in an I / R fibrosis induction model. The IR-Pirfenidone group received oral administration of 300 mg / kg of pirfenidone after I / R fibrosis induction.

[0159] In addition, the fold change in expression level relative to IRV in relation to αSMA expression was shown graphically (FIG. 14C).

[0160] (2) Results In the kidney, the expression of αSMA, an indicator of fibrosis, was significantly higher in patients with induced ischemia / reperfusion than in those with sham-operation (SV and SB).

[0161] However, the expression of αSMA in the groups administered with the composition of the present invention (rhHAPLN1) (IRA and IRB) was lower than that in the control drug-administered group (IRV), at a level similar to that of pirfenidone. Here, since the concentration of IRC was extremely low, it appears to be expressed at a level comparable to that of IRV.

[0162] B. Evaluation of the efficacy of the composition of the present invention for collagen reduction (1) Experimental method As part of the histological analysis of fibrosis, paraffin-fixed kidneys were sectioned at 3 μm, and the slide sections were stained with Sirius Red / PAS to evaluate collagen expression and tissue fibrotic lesions ( FIG. 14D ). Here, SV: sham-vehicle, SB: sham-rhHAPLN1-dissolving solution, IRV: IR-vehicle, IRP: IR-pirfenidone, IRA: IR-composition of the present invention (rhHAPLN1) at a concentration of 0.1 mg / kg, IRB: IR-composition of the present invention (rhHAPLN1) at a concentration of 0.02 mg / kg, and IRC: IR-composition of the present invention (rhHAPLN1) at a concentration of 0.004 mg / kg.

[0163] (2) Results As can be seen from Figure 14D, the group of kidneys exposed to ischemia / reperfusion (IRV) showed significantly more collagen-positive areas (red) in the renal interstitium (between the renal tubules) than the control surgery groups (SV and SB). To further objectively confirm the anti-fibrotic efficacy of the present invention related to collagen reduction, the histological staining photographs in Figure 14D were blindly examined by a total of three researchers. All three researchers confirmed that collagen expression was weaker in the experimental group IRP treated with pirfenidone and the groups treated with the composition of the present invention (IRA and IRB) than in the IRV group.

[0164] To quantify collagen expression in the tissues, two randomly selected areas of the outer medulla were imaged in the tissue staining photograph shown in Figure 14D, and collagen-positive areas were marked. The red color indicates Sirus red-positive signals (Figure 14E).

[0165] Based on this, the results were quantified using i-Solution software (IMT), and as seen under a microscope in Figure 14D, collagen-positive areas were weaker in the IRP, IRA, and IRB groups compared to the IRV group (Figure 14F). This indicates that the composition of the present invention (rhHAPLN1) reduced collagen expression to a similar extent as pirfenidone, indicating that it has anti-fibrotic efficacy in vivo.

[0166] C. Efficacy Evaluation of the Composition of the Present Invention on Creatinine Scavenging Rate (1) Experimental method For renal function analysis, blood was collected using heparinized glass capillary tubes via the retro-orbital venous plexus, and urine was obtained using metabolic cages. Blood urea nitrogen and plasma creatinine (PCr) concentrations were measured using a Vitros 250 (Johnson & Johnson).

[0167] Creatinine clearance is a method of estimating glomerular filtration rate using the amount of creatinine removed by the kidney.

[0168]

number

[0169] In this experiment, the values ​​were measured 21 days after ischemia / reperfusion (*p<0.05, N=4-6); *In the IR experimental group, only animals with a blood urea nitrogen (BUN) concentration of 100 mg / dL or higher on the first day after surgery, indicating a similar level of injury, were used.

[0170] (2) Results As can be seen from Figure 14G, the kidney group exposed to ischemia / reperfusion (IRV) showed a significantly reduced creatinine clearance rate, i.e., the amount of creatinine removed from the blood by normal kidney function, compared to the control groups (SV and SB) that did not experience ischemia / reperfusion.

[0171] However, in the groups treated with the composition of the present invention (IRA, IRB, and IRC), the creatinine clearance rate increased. In particular, the creatinine clearance rate in the IRA group (administered with 0.1 mg / kg / BW of rhHAPLN1), was significantly higher than that in the rhHAPLN1 buffer (IRV) group, which contains only PBS buffer without rhHAPLN1 protein, and the pirfenidone-administered group (IRP). In particular, the CrCl in the IRA group was statistically significantly higher than that in the IRV group. This indicates that administration of the composition of the present invention significantly alleviates the decline in renal function induced by ischemia / reperfusion injury.

[0172] [Sequence list free text] SEQ ID NO:1:

[0173]

number

Claims

1. A pharmaceutical composition for the prevention or treatment of a fibrotic disease, comprising hyaluronan and proteoglycan link protein 1 (HAPLN1) or a gene encoding it as an active ingredient, wherein the protein has 90% or more sequence identity with the amino acid sequence of SEQ ID NO: 1, and the lesion of the fibrotic disease is selected from the group consisting of skin, liver, intestine, heart, lung, and kidney.

2. The composition of claim 1 , wherein the nucleic acid encoding the gene is contained within an expression vector.

3. 2. The composition of claim 1, wherein the fibrotic disease lesions are selected from the group consisting of skin fibroblasts, hepatic stellate cells, colonic fibroblasts, cardiac peripheral blood endothelial cells, lung fibroblasts, kidney renal tubule cells, and kidney proximal tubule epithelial cells.

4. The composition of claim 1 , wherein the fibrotic disease is ischemic fibrosis.

5. 10. The composition of claim 1, wherein the single dose of the composition is from 0.1 ng / ml to 500 ng / ml.

6. The composition of claim 1, wherein the composition is administered in vivo at a dose of 0.001 to 5 mg / kg BK of rhHAPLN1 protein.

7. The composition of claim 1, wherein the composition is included as a main or auxiliary active ingredient of a composition for preventing or inhibiting cellular fibrosis.

8. A kit for preventing or suppressing cell fibrosis, comprising the composition according to any one of claims 1 to 7 and instructions for treatment.

9. A composition for preventing or suppressing cell fibrosis, which is used as an experimental reagent, comprising the composition according to any one of claims 1 to 7.

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