Peptides for the prevention and treatment of fibrosis

Abstract

The present invention relates to a peptide for preventing or treating fibrosis, which has a property of inhibiting dissociation of TGF-beta 1 from LCC by binding with integrin, or a property of inhibiting the change from epithelial cells to mesenchymal cells and promoting the change from mesenchymal cells to epithelial cells, and a property of penetrating cells to bind with Smad2 and Smad3, and thus inhibiting phosphorylation thereof, and thus has an effect of preventing or treating fibrosis. The peptide according to the present invention exhibits a better therapeutic effect than nintedanib commercialized as a therapeutic agent for pulmonary fibrosis and liraglutide commercialized as a therapeutic agent for liver fibrosis, and thus can be used as a replacement therapeutic agent for these commercially available therapeutic agents or as a medicament for combination therapy with these therapeutic agents.

Description

[0001] This application is a divisional application of Chinese Patent Application No. 202280017052.2, filed on January 24, 2022, entitled "Peptide for the Prevention and Treatment of Fibrosis". Technical Field

[0002] This invention relates to a peptide for the prevention or treatment of fibrosis, and more specifically, to a peptide for the prevention or treatment of fibrosis that exhibits the effect of preventing or treating fibrosis by binding to integrin to inhibit the dissociation of TGF-β1 from LCC, or by inhibiting the transformation of epithelial cells to mesenchymal cells and promoting the transformation of mesenchymal cells to epithelial cells, and by binding to Smad2 and Smad3 through cell penetration to inhibit their phosphorylation. Background Technology

[0003] Fibrosis is caused by the excessive production and accumulation of collagen fibers in tissues, and is known to occur primarily in the lungs, liver, heart, and kidneys. As pulmonary fibrosis progresses, not only respiratory distress occurs, but also various complications arise. These may include systemic hypoxia due to reduced vital capacity and impaired oxygen diffusion; pulmonary hypertension due to compression of the pulmonary arteries and capillaries caused by fibrous tissue contraction; heart failure due to right ventricular congestion caused by impaired pulmonary circulation; and other complications such as thrombosis and pneumonia. Furthermore, inflammation and fibrosis may increase the risk of lung cancer. Pulmonary fibrosis can be caused by inhalation of toxic substances or persistent inflammation, but there is also idiopathic pulmonary fibrosis (IPF) with no identifiable cause. When idiopathic pulmonary fibrosis (IPF) occurs, the lung tissue thickens and hardens over time, making it unable to properly absorb oxygen and deliver it into the bloodstream. This eventually leads to permanent scarring and fibrosis of the lungs, resulting in difficulty breathing and reduced oxygen delivery to the body's major organs (Coward WR, The pathogenesis of idiopathic pulmonary fibrosis. Ther. Adv. Respir. Dis. 2010; 4:367-388).

[0004] Pulmonary fibrosis is a fatal disease with an average survival of 2 to 3 years after diagnosis, and there is still no way to restore lung tissue in advanced stages of fibrosis. Currently, steroids and immunosuppressants that suppress the inflammatory response are used as treatments for pulmonary fibrosis. However, fibrosis develops after the inflammatory response, and most patients only seek medical attention due to difficulty breathing. Therefore, treatment begins after the inflammatory phase has passed and the fibrotic phase has begun. Consequently, the fibrotic-suppressing effect of existing anti-inflammatory drugs is difficult to predict.

[0005] Meanwhile, in liver diseases, non-alcoholic steatohepatitis (NASH or non-alcoholic fatty liver disease (NAFLD)) is a type of fatty liver caused by non-alcoholic obesity, diabetes, hyperlipidemia, and medications. NAFLD progresses to cirrhosis and liver cancer due to inflammation and liver fibrosis caused by hepatocyte degeneration / necrosis resulting from fat accumulation in hepatocytes. Therefore, even in NAFLD, if fibrosis occurs, it is impossible to restore normal tissue. Besides the lungs and liver, fibrosis can also occur in the heart, kidneys, blood vessels, skin, and brain.

[0006] One of the currently available drugs for fibrosis is nintedanib (product name: Ofev). ® ), is a tyrosine kinase inhibitor targeting growth factor receptors involved in the mechanism of pulmonary fibrosis, specifically blocking platelet-derived growth factor receptor (PDGFR), fibroblast growth factor receptor (FGFR), and vascular endothelial growth factor receptor (VEGFR) (Wollin L, Mode of action of nintedanib in the treatment of idiopathic pulmonary fibrosis. Eur. Respir. J. 2015; 45:1434-1445; EP 1224170 B9). Additionally, pirfenidone is used to treat idiopathic pulmonary fibrosis, where fibrosis progresses in the lungs without a clear cause. This drug is a small molecule oral agent that inhibits collagen synthesis, reduces pro-fibrotic cytokines, and inhibits the proliferation of collagen-producing fibroblasts (US 8383150 B2).

[0007] Under normal conditions, collagen levels in tissues remain constant. However, in certain pathological conditions, this balance is disrupted, leading to excessive collagen production and accumulation in tissues, resulting in fibrosis. Inhibitors that reduce fibrosis primarily function by: 1) reducing fibrosis-related inflammatory responses, 2) inhibiting the activity of pro-fibrotic cytokines or growth factors, 3) inhibiting cell proliferation, and 4) reducing procollagen biosynthesis and processing. Therefore, these mechanisms of action are the main targets of fibrosis inhibitors. TGF-β (transforming growth factor-β) is known as a representative pro-fibrotic growth factor, and compounds that directly inhibit TGF-β are being used to develop fibrosis therapeutics. However, because TGF-β is involved in various physiological responses in the body, direct inhibition by targeting TGF-β or its receptors can have significant side effects. Therefore, a drug with a mechanism of indirect TGF-β inhibition is needed.

[0008] When fibrosis is viewed from a cellular perspective, TGF-β expression increases when normal epithelial cells are damaged by inflammation or some kind of stimulation, and the transformation of epithelial cells into mesenchymal cells (epithelial-mesenchymal transition, EMT) increases, leading to the transformation of epithelial cells into myofibroblasts. These myofibroblasts are known to lead to fibrosis by overproducing extracellular matrix proteins such as collagen (Rock JR, Multiple stromal populations contribute to pulmonary fibrosis without evidence for epithelial to mesenchymal transition. Proc. Natl. Acad. Sci. USA 2011; 108:E1475-E1483; Fernandez IE, The impact of TGF-β on lung fibrosis: from targeting to biomarkers. Proc. Am. Thorac. Soc. 2012; 9:111-116; Decaris ML, Proteomic analysis of altered extracellular matrix turnover in bleomycin-induced pulmonary fibrosis. Mol. Cell Proteomics 2014; 13:1741-1752). Therefore, it prevents the excessive transformation of epithelial cells into myofibroblasts, and conversely promotes the transformation of mesenchymal cells into normal epithelial cells (mesenchymal-epithelial transition, MET), i.e., cell reprogramming, thereby treating fibrosis.

[0009] Therefore, the inventors have determined that the peptide developed by the inventors does not directly inhibit TGF-β1, but binds to integrin present in lung fibroblasts, thereby preventing TGF-β1 from dissociating from the LCC (large latent complex). Thus, it inhibits fibrosis by inhibiting TGF-β1-induced profibrosis signal transduction. Furthermore, it has been determined that the peptide inhibits the conversion of epithelial cells to mesenchymal cells and simultaneously promotes the conversion of mesenchymal cells to epithelial cells, inducing cell reprogramming. It also inhibits fibrosis by binding to Smad2 and Smad3 through cell penetration and thereby inhibiting their phosphorylation. In addition, when the peptide is administered to animal models of pulmonary fibrosis or animal models of liver fibrosis induced by non-alcoholic steatohepatitis, the peptide effectively reduces fibrosis, thus completing the present invention.

[0010] The information described in the background section is for the purpose of improving the understanding of the background of the present invention and should not be construed as including information on related technologies known to those skilled in the art to which this invention pertains. Summary of the Invention

[0011] One object of the present invention is to provide a peptide that effectively prevents and treats fibrosis.

[0012] Another object of the present invention is to provide a pharmaceutical composition comprising the peptide for the prevention or treatment of fibrosis.

[0013] Another object of the present invention is to provide a method for preventing or treating fibrosis, including administering the peptide.

[0014] Another object of the present invention is to provide the use of peptides for the prevention or treatment of fibrosis.

[0015] Another object of the present invention is to provide the use of the peptide in the preparation of medicaments for the prevention or treatment of fibrosis.

[0016] To achieve the above objectives, the present invention provides a peptide for the prevention or treatment of fibrosis, the peptide being represented by the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2.

[0017] Furthermore, the present invention provides a pharmaceutical composition for the prevention or treatment of fibrosis, the pharmaceutical composition comprising, as an active ingredient, a peptide represented by the amino acid sequence of SEQ ID NO: 1 and / or a peptide represented by the amino acid sequence of SEQ ID NO: 2.

[0018] Furthermore, the present invention provides a method for preventing or treating fibrosis, the method comprising administering to a subject in need of it a peptide represented by the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2 or a composition comprising the peptide.

[0019] Furthermore, the present invention provides the use of a peptide represented by the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2, or a composition containing the peptide, for the prevention or treatment of fibrosis.

[0020] Furthermore, the present invention provides the use of a peptide represented by the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2, or a composition containing the peptide, in the preparation of a medicament for the prevention or treatment of fibrosis. Attached Figure Description

[0021] Figure 1 The illustration schematically demonstrates the effect of preventing or treating fibrosis by using a peptide according to the invention that binds to integrin to inhibit TGF-β1-induced profibrotic intracellular signal transduction.

[0022] Figure 2a The illustration schematically demonstrates the effect of preventing or treating fibrosis by using the peptides according to the invention to inhibit the transformation of epithelial cells into mesenchymal cells and to promote the transformation of mesenchymal cells into epithelial cells.

[0023] Figure 2b The illustration schematically demonstrates the effect of preventing or treating fibrosis by using a peptide according to the invention to inhibit profibrotic intracellular signal transduction, the peptide binding to Smad2 and Smad3 via cell penetration to inhibit their phosphorylation.

[0024] Figure 3 Results confirming the ability of the peptide of SEQ ID NO: 1 to bind to integrins β1, β3, αv, α4 and α5 are shown.

[0025] Figure 4 Results confirming the ability of the peptide of SEQ ID NO: 2 to bind to Smad2 or Smad3 are shown.

[0026] Figure 5 The results show the effect of the peptide of SEQ ID NO: 2 inhibiting the expression of pSmad2 and pSmad3.

[0027] Figure 6 Results confirming the effect of the peptide in SEQ ID NO: 1 or 2 on inhibiting the expression of COL1A2 and COL3A1 genes are shown.

[0028] Figure 7a The results show the observed increase in MET marker expression by the peptide of SEQ ID NO: 2.

[0029] Figure 7b The results show the observed inhibition of EMT marker expression by the peptide of SEQ ID NO: 2.

[0030] Figure 8a An experimental protocol for confirming the therapeutic effect of treating an animal model of pulmonary fibrosis with the peptide according to the invention is shown.

[0031] Figure 8b The results of changes in body weight observed when an animal model of pulmonary fibrosis was treated with the peptide according to the invention are shown.

[0032] Figure 8c Results confirming the effect of treating an animal model of pulmonary fibrosis with the peptide according to the invention on reducing fibrosis deposition are shown.

[0033] Figure 8d The results show the effect of fibrosis reduction by measuring the area of ​​fibrosis in an animal model of pulmonary fibrosis treated with the peptide according to the invention.

[0034] Figure 9a An experimental protocol for confirming the therapeutic effect of treating an animal model of liver fibrosis with the peptide according to the invention is shown.

[0035] Figure 9b The results of H&E staining are shown to confirm the therapeutic effect of treating an animal model of liver fibrosis with the peptide according to the invention.

[0036] Figure 9c The results of measuring NAFLD activity scores are shown to confirm the therapeutic effect of treating an animal model of liver fibrosis with the peptides according to the invention.

[0037] Figure 9d The results of Masson's trichrome staining are shown to confirm the therapeutic effect of treating an animal model of liver fibrosis with the peptide according to the invention.

[0038] Figure 9e The results of measuring the fibrosis area are shown to confirm the therapeutic effect of treating an animal model of liver fibrosis with the peptide according to the invention. Detailed Implementation

[0039] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. In general, the terminology used herein and the experimental methods described below are well-known and typical in the art.

[0040] TGF-β1 is known to be a protein that plays an important role in increasing fibrosis. In vivo, TGF-β1 binds to LLC (large latent complex) and exists in a latent form. LLC is composed of LAP (latency-associated peptide), LTBP1 (latent TGFβ-binding protein), and TGF-β1. When LAP in LLC binds to integrin receptors on the cell surface, latent TGF-β1 dissociates from LLC. This free TGF-β1 binds to TGFβ receptors, thereby inducing pro-fibrotic intracellular signal transduction. The peptide of SEQ ID NO: 1 binds to integrin to inhibit the LLC-integrin response, thereby preventing the release of TGF-β1. Ultimately, the expression of fibrosis-associated proteins induced by TGF-β1 is suppressed. Figure 1 ).

[0041] In fibrosis, normal epithelial cells are damaged, and TGF-β1 expression increases, thus increasing epithelial-mesenchymal transition (EMT), leading to the transformation of epithelial cells into myofibroblasts. These myofibroblasts are known to ultimately induce fibrosis by overproducing extracellular matrix proteins such as collagen. In this invention, the peptide of SEQ ID NO: 2 inhibits the excessive transformation of epithelial cells into mesenchymal cells, particularly myofibroblasts, and conversely promotes the transformation of mesenchymal cells into normal epithelial cells (mesenchymal-epithelial transition, MET), i.e., increases cell reprogramming, confirming that matrix protein deposition can be inhibited and fibrosis can be treated. Figure 2a Furthermore, the peptide of SEQ ID NO: 2 inhibits TGF-β1-induced profibrosis by suppressing the phosphorylation of Smad2 and Smad3, which are increased by TGF-β1. This peptide of SEQ ID NO: 2 can penetrate cells and bind to Smad2 and Smad3 proteins in the cell, thereby inhibiting their phosphorylation and thus suppressing TGF-β1-induced fibrosis. Figure 2b ).

[0042] Therefore, one aspect of the present invention relates to peptides for the prevention or treatment of fibrosis, which are represented by the amino acid sequence of SEQ ID NO:1 or SEQ ID NO:2 below.

[0043] SEQ ID NO: 1: YGLRSKSKKFRRPDIQYPDAT

[0044] SEQ ID NO: 2: GKCSTRGRKSSRRKK

[0045] In this invention, the peptide represented by the amino acid sequence of SEQ ID NO: 1 prevents TGF-β1 from dissociating from LLC (large latent complex) by binding to integrin, thereby inhibiting TGF-β1-induced profibrotic intracellular signal transduction and thus inhibiting fibrosis.

[0046] In this invention, the peptide represented by the amino acid sequence of SEQ ID NO: 2 inhibits epithelial-mesenchymal transition and also inhibits the phosphorylation of Smad2 and Smad3 by binding to them through cell penetration, thereby inhibiting fibrosis.

[0047] Another aspect of the present invention relates to a pharmaceutical composition for the prevention or treatment of fibrosis, the pharmaceutical composition comprising, as an active ingredient, a peptide represented by the amino acid sequence of SEQ ID NO: 1 and / or a peptide represented by the amino acid sequence of SEQ ID NO: 2.

[0048] In this invention, fibrosis is a disease caused by excessive collagen deposition and can be selected from, but is not limited to, the group consisting of pulmonary fibrosis, liver fibrosis, kidney fibrosis and cardiac fibrosis. The peptide and pharmaceutical compositions according to the invention can be used to prevent or treat all fibrotic diseases caused by excessive collagen deposition.

[0049] In this invention, pharmaceutical compositions can be formulated into any dosage form selected from the group consisting of: injections, oral formulations, patches, liquids, capsules, granules, tablets, powders, sprays, nasal administration formulations, inhalation sprays, ointments, gels, mucosal administration formulations, and suppositories, but are not limited thereto. These formulations can be prepared by methods typical of formulations used in the art or by methods disclosed in Remington's Pharmaceutical Science (latest edition), Mack Publishing Company, Easton PA, and can be prepared in various forms depending on the individual disease or ingredient. However, the above description is illustrative, and the formulations applicable to this invention are not limited thereto.

[0050] In this invention, the pharmaceutical composition may further comprise an acceptable adjuvant, which may be, for example, a carrier. Pharmaceutically acceptable carriers may include saline, sterile water, Ringer's solution, buffered saline, dextran solution, maltodextrin solution, glycerol, ethanol, and one or more mixtures thereof, as well as other typical additives that may be added if necessary, such as antioxidants, buffers, antibacterial agents, etc. Furthermore, diluents, dispersants, surfactants, binders, and lubricants may be added to prepare injectable formulations (such as aqueous solutions, suspensions, emulsions, etc.), formulations for nasal administration, inhalation sprays, pills, capsules, granules, or tablets. However, the above description is illustrative, and the adjuvants or carriers applicable to this invention are not limited thereto.

[0051] Depending on the desired method, the compositions of the present invention can be administered orally or parenterally (e.g., intravenously, subcutaneously, intraperitoneally, intranasally, intratracheally, or topically), and the dosage varies depending on the patient's weight, age, sex, health status, diet, time of administration, method of administration, excretion rate, and severity of disease. The peptide of SEQ ID NO: 1 or 2 of the present invention can be administered at a dose of about 1 μg / kg to 100 mg / kg daily, preferably 5 μg / kg to 50 mg / kg, and can be administered daily or 1-3 times weekly. However, the dosage and dosing interval are not limited thereto.

[0052] Here, the dosing regimen and dosage can vary depending on the individual patient's age, weight, and response. Those skilled in the art, taking these factors into account, can readily select an appropriate dosing regimen and dosage.

[0053] Another aspect of the invention relates to a method for preventing or treating fibrosis, the method comprising administering to a subject in need of the peptide represented by the amino acid sequence of SEQ ID NO: 1 or the amino acid sequence of SEQ ID NO: 2.

[0054] Another aspect of the invention relates to a method for preventing or treating fibrosis, the method comprising administering to a subject in need a composition comprising a peptide represented by the amino acid sequence of SEQ ID NO: 1 and / or a peptide represented by the amino acid sequence of SEQ ID NO: 2.

[0055] Another aspect of the invention relates to the use of a peptide represented by the amino acid sequence of SEQ ID NO: 1 or the amino acid sequence of SEQ ID NO: 2 for the prevention or treatment of fibrosis.

[0056] Another aspect of the invention relates to the use of a composition comprising a peptide represented by the amino acid sequence of SEQ ID NO: 1 and / or a peptide represented by the amino acid sequence of SEQ ID NO: 2 for the prevention or treatment of fibrosis.

[0057] Another aspect of the invention relates to the use of a peptide represented by the amino acid sequence of SEQ ID NO: 1 or the amino acid sequence of SEQ ID NO: 2 in the preparation of a medicament for the prevention or treatment of fibrosis.

[0058] Another aspect of the invention relates to the use of compositions comprising a peptide represented by the amino acid sequence of SEQ ID NO: 1 and / or a peptide represented by the amino acid sequence of SEQ ID NO: 2 in the preparation of a medicament for the prevention or treatment of fibrosis.

[0059] Since the above-mentioned "peptide or composition containing the peptide" is used in the prevention or treatment methods and uses of the present invention, further details thereof will be omitted.

[0060] In this invention, a specific amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2 is described; however, it will be apparent to those skilled in the art that, within the scope of the technical concept of this invention, an equivalent range of amino acid sequences in which the amino acid sequence of this invention is partially modified is interpreted as being within the scope of this invention.

[0061] The invention will be described in more detail below with reference to embodiments. These embodiments are for illustrative purposes only, and it will be apparent to those skilled in the art that the scope of the invention should not be construed as limited to these embodiments.

[0062] Example 1: Peptide Synthesis

[0063] The amino acids and reagents used for synthesis were purchased from GL Biochem and Sigma-Aldrich. Peptides were synthesized from the C-terminus using an F-moc solid-phase chemical synthesis method. Specifically, rink resin (0.075 mmol / g, 100-200 mesh, 1% DVB crosslinked) with Fmoc-(9-fluorenylmethoxycarbonyl) as the blocking group was used for synthesis. 50 mg of rink resin was placed in the synthesizer, swollen with DMF, and then a 20% piperidine / DMF solution was added to remove the Fmoc- group. Starting from the C-terminus, 0.5 M amino acid solution (solvent: dimethylformamide, DMF), 1.0 M MDIPEA (solvent: dimethylformamide & N-methylpyrrolidone, DMF & NMP), and 0.5 M HBTU (solvent: dimethylformamide, DMF) were added sequentially in amounts of 5, 10, and 5 equivalents, respectively, and the reaction was carried out under a nitrogen stream for 1 to 2 hours. Each time the deprotection and coupling steps are completed, the mixture is washed twice with DMF and isopropanol. After coupling the last amino acid, deprotection is performed to remove the Fmoc- group.

[0064] The synthesis was confirmed using the ninhydrin assay, and the tested and synthesized resin was dried with tetrahydrofuran (THF) or dichloromethane (DCM). Trifluoroacetic acid (TFA) cleavage cocktail reagent was then added in a volume of 20 ml relative to 1 g of resin, shaken for 3 hours, and then filtered to separate the cocktail reagent containing the dissolved resin and peptides. After removing the filtered solution using a rotary evaporator, cold diethyl ether was added, or excess cold diethyl ether was added directly to the TFA cocktail reagent solution containing the dissolved peptides, causing the peptides to crystallize into a solid phase, which was then separated by centrifugation. Here, the TFA cocktail reagent was completely removed by washing several times with diethyl ether and centrifugation. The peptides thus obtained were dissolved in distilled water and lyophilized.

[0065] The synthesized peptides were cleaved from the resin, washed, lyophilized, and then separated and purified by high-performance liquid chromatography (HPLC) (Shimadzu, Japan). A C14 filter with a diameter of 4.6 mm was used. 18 The column was analyzed as follows: 0.1% TFA / H₂O and 0.092% TFA / acetonitrile were flowed at a flow rate of 1 ml / min for 30 minutes, with a change of 0-60%. Here, the UV detector wavelength was set to 220 nm. Purification was performed using a 2.2 cm diameter column at a flow rate of 20 ml / min under the same solvent and detection wavelength conditions. The molecular weight of the purified peptide was determined by mass spectrometry.

[0066] The peptides of SEQ ID NO: 1 and SEQ ID NO: 2 were synthesized in the same manner as described above.

[0067] SEQ ID NO: 1: YGLRSKSKKFRRPDIQYPDAT

[0068] SEQ ID NO: 2: GKCSTRGRKSSRRKK

[0069] Example 2: Confirmation of the binding ability of the peptide of SEQ ID NO: 1 to integrin using immunoprecipitation

[0070] Human lung fibroblasts derived from patients with idiopathic pulmonary fibrosis (IPF) (disease-related lung fibroblasts from IPF patients, DHLF-IPF, Lonza, #CC-7231) were cultured in complete growth medium (fibroblast growth basal medium supplemented with hFGF-B, insulin, GA-1000 and 2% FBS-2, Lonza #CC-3132). They were then treated with TGF-β1 (R&Dsystems, #240-B-002) for 3 × 10⁶ cells / day. 6 Before DHLF-IPF, the culture medium was replaced with Opti-MEM (Gibco, #31985-070) and then cultured for 24 hours. After 24 hours, the culture medium was replaced with the existing growth medium, and the cells were treated with 10 ng / ml TGF-β1 (R&D systems, #240-B-002) for 24 hours. After 24 hours, the cells were treated with 100 µM of the biotin-bound peptide of SEQ ID NO:1 (Biotin-Seq 1) for 1 hour. Cells treated alone with 100 µM of the biotin-bound peptide of SEQ ID NO:1 (Biotin-Seq 1) were also included in this group.

[0071] Cells treated in this manner were washed with ice-cold PBS, and 500 μl of RIPA buffer (pH 8.0, 10 mM Tris-HCl, 1 mM EDTA, 0.5 mM EGTA, 1% Triton X-100, 0.1% sodium deoxycholate, 0.1% SDS, 140 mM NaCl, a complete protease inhibitor cocktail (Sigma-Aldrich), and a phosphatase inhibitor cocktail (Sigma-Aldrich)) was added, followed by cell lysis for 20 minutes. The cells were then centrifuged at 13000 g for 25 minutes, and the cell lysate was reacted with anti-biotin antibody (1:100 dilution; Abcam, ab53494) at 4°C overnight using a vertical rocker. 20 μl of protein-A agarose beads (Santa Cruz Biotechnology, sc-2003) were added, and the reaction was continued for another 3 hours. The cells were briefly centrifuged at 13000 g, the supernatant was discarded, and the beads were washed with PBS. Proteins bound to the beads were separated by adding 5x SDS sample buffer, followed by boiling, and then SDS-PAGE and Western blotting. Immunoblotting was performed using antibodies against integrin β1, β3, αv, α4, α5 (Santa Cruz Biotechnology, sc-374429, sc-46655, sc-9969, sc-365209, sc-376199) and IgG (Cell Signaling Technology). Protein levels are expressed as total lysate (input) and immunoprecipitated protein (IP).

[0072] As a result, the peptide of SEQ ID NO: 1 (Biotin-Seq 1) exhibited the ability to selectively bind to integrin β1, β3, and αv, and this binding ability was further increased when treated with both the peptide of SEQ ID NO: 1 and TGF-β1. Figure 3 This means that the ability of the peptide of SEQ ID NO: 1 to selectively bind to integrins β1, β3 and αv is further increased under pathological conditions of high TGF-β1.

[0073] Example 3: Confirmation of the binding affinity of the peptide of SEQ ID NO: 2 to Smad2 and Smad3 using immunoprecipitation

[0074] LX-2 (human hepatic stellate cell line, Millipore #SCC064) were cultured in a medium containing 5% DMEM high glucose (Gibco, 11965092), 10% FBS (Gibco, 16000044) and 1% antibiotic-antifungal agent (Gibco, 15240062).

[0075] 3 × 10⁻⁶ cells were treated with 100 µM of the biotin-binding peptide SEQ ID NO:2 (Biotin-Seq 2). 6 LX-2 cells were lysed for 1 hour. The cells were washed with ice-cold PBS, and 500 μl of RIPA buffer (pH 8.0, 10 mM Tris-HCl, 1 mM EDTA, 0.5 mM EGTA, 1% Triton X-100, 0.1% sodium deoxycholate, 0.1% SDS, 140 mM NaCl, a complete protease inhibitor cocktail (Sigma-Aldrich), and a phosphatase inhibitor cocktail (Sigma-Aldrich)) was added, followed by cell lysis for 20 minutes. The cells were then centrifuged at 13000 g for 25 minutes, and the cell lysate was reacted overnight at 4°C with anti-biotin antibody (1:100 dilution; OriGene) using a vertical shaker. 20 μl of protein-A agarose beads (Santa Cruz Biotechnology, sc-2003) were added, and the reaction was continued for another 3 hours. The cells were briefly centrifuged at 13000 g, the supernatant was discarded, and the beads were washed with PBS. Proteins bound to the beads were separated by adding 5x SDS sample buffer, followed by boiling, and then performing SDS-PAGE and Western blotting. Immunoblotting was performed using antibodies against Smad2 (Cell Signaling, #5339) and Smad3 (Cell Signaling, #9513). Protein levels are expressed as total lysate (input) and immunoprecipitated protein (IP).

[0076] As a result, the peptide of SEQ ID NO: 2 (Biotin-Seq 2) showed the ability to bind to both Smad2 and Smad3, especially with a higher affinity for Smad3. Figure 4 This indicates that the peptide in SEQ ID NO: 2 acts on the TGF-β1 sub-signals Smad2 and Smad3.

[0077] Example 4: Confirmation of the inhibition of Smad2 and Smad3 phosphorylation by the peptide of SEQ ID NO: 2

[0078] LX-2 (human hepatic stellate cell line, Millipore #SCC064) cells were cultured in a medium containing 5% DMEM high glucose (Gibco, 11965092), 10% FBS (Gibco, 16000044), and 1% antibiotic-antifungal agent (Gibco, 15240062). Three × 10⁶ cells were treated with 10 ng / ml TGF-β1. 6 LX-2 cells were cultured for 24 hours. Cells were treated in fresh culture medium with 10, 100, or 200 μM of the peptide of SEQ ID NO: 2 or 10 μM of SIS3 (Smad3 inhibitor, Sigma-Aldrich, catalog #566405) and cultured for 24 hours.

[0079] Proteins were lysed using RIPA lysis buffer (25 mM Tris HCl pH 7.6, 150 mM NaCl, 1% NP-40, 1% sodium deoxycholate, 0.1% SDS) containing protease and phosphatase inhibitors. Proteins were quantified by BCA protein assay, and the expression levels of pSmad2, Smad2, pSmad3, Smad3, TGFβ receptor 1, and GAPDH proteins were determined by Western blotting. For Western blotting, equal amounts of sample and size markers were loaded onto an 8% SDS-PAGE gel, electrophoresed for approximately 2 hours, and then transferred to a nitrocellulose membrane. The transferred membrane was blocked with 5% skim milk for 1 hour and then reacted with primary antibody at a 1:1000 ratio overnight. The primary antibodies used were pSmad2 (Cell Signaling, #3108), Smad2 (Cell Signaling, #5339), pSmad3 (Abcam, ab52903), Smad3 (Cell Signaling, #9513), TGFβ receptor 1 (Abcam, ab31013), and GAPDH (Cell Signaling, #2118S). Subsequently, the membrane was washed with TBST containing 0.1% Tween 20 and reacted with secondary antibodies conjugated with HRP at a ratio of 1:5000 for 1 hour, followed by chemiluminescence with ECL substrate.

[0080] As a result, pSmad3 expression completely disappeared at a peptide concentration of 10 μM (SEQ ID NO: 2), and pSmad2 expression decreased with increasing peptide concentration of SEQ ID NO: 2. Figure 5However, the expression level of the TGFβ receptor remained unchanged. This means that the peptide of SEQ ID NO: 2 binds to Smad2 and Smad3, thereby inhibiting their phosphorylation.

[0081] Example 5: Confirmation of inhibition of collagen gene expression in cells

[0082] Human lung fibroblasts (DHLF-IPF, Lonza, #CC-7231) derived from patients with idiopathic pulmonary fibrosis were cultured in complete growth medium (fibroblast growth basal medium-2 supplemented with hFGF-B, insulin, GA-1000, and 2% FBS, Lonza #CC-3132). Cells were seeded in 24-well plates and cultured for 24 hours in starvation medium (Dulbecco modified Eagle medium / F12, FBS-free, supplemented with 0.1 mM 2-phosphate-L-ascorbic acid). Recombinant human TGF-β1 protein (R&D Systems, #240-B-002) was diluted to 20 μg / ml in sterile 4 mM HCl containing 1 mg / ml bovine serum albumin. Cells were then cultured for 24 hours in complete growth medium containing 10 ng / ml TGF-β1. Cells were treated with SEQ ID NO: 1 (200 μM), SEQ ID NO: 2 (200 μM), or nintedanib (10 μM) in fresh culture medium and cultured for 24 hours. The peptide was dissolved in purified water, and nintedanib was dissolved in dimethyl sulfoxide (DMSO), setting the DMSO concentration in the culture medium to 1%. The culture medium was removed, followed by washing with DPBS (Welgene, LB001-02) and lysing the cells with 1 ml of Trizol reagent (Ambion, 15596). 250 μl of chloroform was added, the mixture was stirred, and centrifuged at 10,000 rpm for 15 minutes. After removing the aqueous layer with a pipette, 550 μl of isopropanol (Sigma, I9516) was added and gently shaken. After centrifugation at 15,000 rpm for 25 minutes, the isopropanol was removed, and 1 ml of 75% ethanol was added. After removing the ethanol and drying in air, the precipitate was dissolved in DEPC water (Invitrogen, AM9915G). The RNA concentration was measured using a Nano Drop (Thermo Scientific, NanoDrop One, AZY1913798).

[0083] cDNA was synthesized from the extracted RNA using the Maxima First-Strand cDNA Synthesis Kit (Thermo Fisher, K1641), according to the manufacturer's instructions. Furthermore, real-time PCR was performed using a QuantStudio 3 (Applied Biosystems, A28132), also according to the manufacturer's instructions. PCR amplification was performed by repeating 50 cycles at 95°C for 30 seconds, 60°C for 30 seconds, and 72°C for 30 seconds. The primer sequences used are shown in Table 1 below.

[0084] [Table 1] Primer sequences

[0085]

[0086] As a result, treatment with TGF-β1 increased the expression of COL1A2 and COL3A1 genes, but when cells were treated with TGF-β1 and then with the peptides of SEQ ID NO: 1 or SEQ ID NO: 2, the expression of each collagen gene was significantly reduced (*p<0.05), confirming that the peptides of the present invention are more effective than nintedanib. Figure 6 ).

[0087] Example 6: Confirmation of the cell reprogramming effect

[0088] LX-2 (human hepatic stellate cell line, Millipore #SCC064) was cultured in a medium containing 5% DMEM high glucose (Gibco, 11965092), 10% FBS (Gibco, 16000044), and 1% antibiotic-antifungal agent (Gibco, 15240062) at a concentration of 1×10⁻⁶. 6 Inoculated into each well of a 6-well plate (Nunc, 140675) and incubated. In Opti-MEM... TM (Gibco, 31985-070) After 6 hours of starvation, fresh Opti-MEM was used TMThe culture medium was replaced, and cells were treated with 10 ng / ml TGF-β1 and 100 μM of the peptide of SEQ ID NO: 2 for 24 hours. The culture medium was removed, followed by washing with DPBS (Welgene, LB001-02) and lysing the cells with 1 ml of Trizol reagent (Ambion, 15596). 250 μl of chloroform was added, the tube was stirred, and centrifuged at 10000 rpm for 15 minutes. After removing the aqueous layer with a pipette, 550 μl of isopropanol (Sigma, I9516) was added and gently shaken. After centrifugation at 15000 rpm for 25 minutes, the isopropanol was removed, and 1 ml of 75% ethanol was added. After removing the ethanol and air-drying, the precipitate was dissolved in DEPC water (Invitrogen, AM9915G). RNA concentration was measured using a Nano Drop (Thermo Scientific, NanoDrop One, AZY1913798).

[0089] cDNA was synthesized from the extracted RNA using the Maxima First-Strand cDNA Synthesis Kit (Thermo Fisher, K1641), according to the manufacturer's instructions. Furthermore, real-time PCR was performed using a QuantStudio 3 (Applied Biosystems, A28132), also according to the manufacturer's instructions. PCR amplification was performed by repeating 40 cycles at 95°C for 15 seconds, 60°C for 50 seconds, and 72°C for 15 seconds. The primer sequences used are shown in Table 2 below.

[0090] [Table 2] Primer sequences

[0091]

[0092] As a result, the peptide of SEQ ID NO: 2 increased the gene expression of E-cadherin and EpCAM, which are markers of MET, and decreased the gene expression of N-cadherin, Twist1, and Snail1, which are markers of EMT. Conversely, TGF-β1 decreased the expression of MET markers and increased the expression of EMT markers (Figure 7). Therefore, the peptide of SEQ ID NO: 2 was found to increase MET and inhibit EMT.

[0093] Example 7: Confirmation of the therapeutic effect in an animal model of bleomycin-induced lung disease

[0094] 7-1: Animal testing methods

[0095] To confirm the therapeutic effect of the peptides according to the invention on fibrosis at the animal level, an animal model of bleomycin-induced lung disease was used. C57BL / 6J mice (male, 7 weeks old) were purchased from Charles River. Mice were anesthetized by intraperitoneal administration of a mixture of Imalgene (ketamine, 100 mg / kg) and Rompun (xylazine, 20 mg / kg), and intratracheally injected with a total of 50 μl of bleomycin sulfate (1.45 mg / kg, MBcell, CA, USA), while the control group received intratracheal saline (day 0). On day 7, the mice were divided into groups of eight. They were administered subcutaneous injections of PBS (NT), SEQ ID NO: 1 (30 mg / kg), and SEQ ID NO: 2 (30 mg / kg) three times weekly, and nintedanib (fluorochem Ref = 050741, 100 mg / kg) orally daily. Each of the peptides in SEQ ID NO: 1 and SEQ ID NO: 2 was dissolved in water for injection before administration, and nintedanib was administered after being mixed with water containing 0.5% hydroxyethyl cellulose (Sigma; batch BCBV5033) and 0.01% Tween 80 (Sigma; batch BCBR6211V). Weight was measured every two days during the 21-day administration period, and other clinical symptoms were monitored. The experimental protocol is as follows: Figure 8a As shown in the image.

[0096] 7-2: Observation of weight changes

[0097] Based on measurements of weight changes from day 0 to day 22, weight continued to decrease until day 7 after administration of bleomycin, but increased after treatment with the peptide and nintedanib according to the invention. Figure 8b Furthermore, no weight loss was observed due to the toxicity of the peptides and nintedanib according to the invention.

[0098] 7-3: Observations on fiber deposition

[0099] Mice were euthanized with isoflurane on day 22, and lung tissue was excised for histological analysis. Paraffin was introduced into the lungs, and 5 μm paraffin sections were prepared. Sirius red and hematoxylin / fluorescein staining were performed to assess the degree of pulmonary fibrosis and inflammation, and the degree of inflammation and fibrosis was observed under a microscope. Therefore, in the bleomycin-induced lung disease group treated with PBS (NT), extensive fibrosis deposition was observed in the lungs, while in the nintedanib group, although some therapeutic effect was observed, extensive fibrosis deposition was still observed. However, compared with the NT group and the nintedanib group, the groups treated with the peptide of SEQ ID NO: 1 and the groups treated with the peptide of SEQ ID NO: 2 were confirmed to have significantly reduced fibrosis. Figure 8c ).

[0100] 7-4: Measurement of fibrous area

[0101] To quantify changes in fibrosis, the area of ​​red (fibrotic) areas in samples stained with Sirius red was quantified. The results showed that, compared to the NT group and the nintedanib treatment group, the groups treated with the peptides of SEQ ID NO: 1 and SEQ ID NO: 2 exhibited a significant reduction in fibrotic area. Figure 8d (P<0.05).

[0102] Example 8: Confirmation of efficacy in a methionine-choline-deficient mouse model

[0103] 8-1: Animal testing methods

[0104] To further confirm the therapeutic effect of the peptides according to the invention on fibrosis at the animal level, hepatitis and liver fibrosis were induced in mice (methionine-choline deficient diet mouse models) by administering a methionine-choline deficient (MCD) diet. Six-week-old male C57BL / 6J mice were used and randomly divided into a normal diet group, an untreated group fed an MCD diet, a group injected with peptides SEQ ID NO: 1 (20 mg / kg) and SEQ ID NO: 2 (20 mg / kg), and a positive control group injected subcutaneously with liraglutide (0.2 mg / kg), wherein liraglutide is a glucagon-like peptide-1 receptor agonist used as a hepatitis treatment agent. After 10 weeks of autonomous MCD feeding, eight mice were assigned to each group and administered the treatment. The peptides and liraglutide were administered once daily for a total of 12 times, with 3-day intervals. Figure 9a ).

[0105] 8-2: Measurement of the degree of liver inflammation

[0106] For all animals, body weight was measured immediately before administration and twice weekly throughout the trial. After the last administration, animals were anesthetized by intraperitoneal injection of 300 μl of Avertin. Blood was collected from the abdominal vena cava via laparotomy, from which the liver and spleen were removed, and the weight of the removed organs was measured. Tissues from all animals undergoing necropsy were fixed in neutral buffered formalin solution, and paraffin blocks were prepared and sectioned by tissue processing. The resulting tissue sections were then stained with H&E. H&E-stained specimens were scored for fatty degeneration, ballooning degeneration, and lobular inflammation (Table 3), and the degree of liver inflammation was measured (NAFLD activity score).

[0107] [Table 3] NAFLD Activity Score

[0108]

[0109] The result is as follows Figure 9b and Figure 9c As shown in the figure. Compared with the NT group or the positive control GLP-1 agonist group, both groups treated with peptides SEQ ID NO: 1 and SEQ ID NO: 2 showed a significant reduction in NAFLD activity scores.

[0110] 8-3: Measurement of fibrous area

[0111] Tissue sections prepared in the same manner as in Examples 8-2 were stained with Masson's trichrome, and 100x magnified images were obtained at five randomly designated sites on all Masson's trichrome stained slides (300MI CMOS camera, Aptina) using an optical microscope (BX51, Olympus, GER). The blue-stained area (Image-Pro Plus, MediaCybernetics Inc., UK) was measured and analyzed as a percentage (Masson's trichrome positive area (%)).

[0112] As a result, Figure 9d and 9e As shown, compared with the NT group or the positive control GLP-1 agonist group, the two groups that were administered peptides of SEQ ID NO: 1 and SEQ ID NO: 2 were found to have a significant reduction in fibrosis area.

[0113] Industrial applicability

[0114] According to the present invention, compared with commercially available nintedanib as a treatment agent for pulmonary fibrosis and commercially available liraglutide as a treatment agent for liver fibrosis, the peptide represented by the amino acid sequence of SEQ ID NO: 1 and the peptide represented by the amino acid sequence of SEQ ID NO: 2 can exhibit superior therapeutic effects, and therefore can replace these commercially available treatment agents and can be used in combination with these treatment agents.

[0115] The specific aspects of the invention have been described in detail above. It will be apparent to those skilled in the art that these specific descriptions are merely preferred embodiments, and the scope of the invention is not limited thereto. Therefore, the essential scope of the invention is defined by the appended claims and their equivalents.

[0116] Sequence List Free Text

[0117] Please attach the electronic file.

[0118] This disclosure relates to the following implementation plan.

[0119] 1. A peptide for the prevention and treatment of fibrosis, represented by the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2.

[0120] 2. The peptide according to embodiment 1, wherein the peptide represented by the amino acid sequence of SEQ ID NO: 1 inhibits the dissociation of TGF-β1 from the large latent complex (LLC) by binding to integrin.

[0121] 3. The peptide according to embodiment 1, wherein the peptide represented by the amino acid sequence of SEQ ID NO: 2 inhibits epithelial-mesenchymal transition, or inhibits their phosphorylation by binding to Smad2 and Smad3 through cell penetration.

[0122] 4. A pharmaceutical composition for the prevention or treatment of fibrosis, said pharmaceutical composition comprising, as an active ingredient, a peptide represented by the amino acid sequence of SEQ ID NO: 1 and / or a peptide represented by the amino acid sequence of SEQ ID NO: 2.

[0123] 5. The pharmaceutical composition according to embodiment 4, wherein the fibrosis is selected from the group consisting of pulmonary fibrosis, liver fibrosis, renal fibrosis and cardiac fibrosis.

[0124] 6. The pharmaceutical composition according to embodiment 4, wherein the pharmaceutical composition is formulated into any dosage form selected from the group consisting of: injections, oral preparations, patches, liquids, capsules, granules, tablets, powders, sprays, nasal administration preparations, inhalation sprays, ointments, gels, mucosal administration preparations, and suppositories.

[0125] 7. The pharmaceutical composition according to embodiment 4, wherein the pharmaceutical composition further comprises an acceptable adjuvant.

[0126] 8. The pharmaceutical composition according to embodiment 4, wherein the peptide is administered at a dose of 1 μg / kg to 100 mg / kg per day, and administered daily or 1-3 times per week.

Claims

1. Use of the peptide represented by the amino acid sequence of SEQ ID NO: 2 in the preparation of a medicament for the prevention or treatment of pulmonary fibrosis or liver fibrosis.

2. The use according to claim 1, wherein, The drug is formulated into any dosage form selected from the group consisting of: injections, oral preparations, patches, liquids, capsules, granules, tablets, powders, sprays, nasal preparations, inhaled sprays, ointments, gels, mucosal preparations, and suppositories.

3. The use according to claim 1, wherein, The pharmaceutical composition also contains an acceptable adjuvant.

4. The use according to claim 1, wherein, The peptide is administered at a dose of 1 μg / kg to 100 mg / kg per day, and may be administered daily or 1-3 times per week.

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