β-1 integrin agonist peptide and therapeutic use thereof

Abstract

The present invention relates to a β-1 integrin subunit agonist peptide and, more specifically, to a β-1 integrin subunit agonist peptide that binds to a β-1 integrin subunit to increase focal adhesion kinase (FAK) and AKT, which are intracellular downstream signals. The peptide then increases a regeneration signal to enhance the differentiation of tight junction proteins required for regeneration, thereby exhibiting the effect of preventing or treating diseases caused by fibrosis. The peptide according to the present invention can be used as a preventive or therapeutic agent for diseases caused by fibrosis, and can be used in combination with existing therapeutic agents.

Description

Beta-1 Integrin Agonist Peptide and Its Therapeutic Uses

[0001] The present invention relates to a β-1 integrin subunit agonist peptide, and more specifically, to a peptide capable of regenerating a damaged extracellular matrix (ECM) by binding to a β-1 integrin subunit. Specifically, the peptide according to the present invention is useful for preventing or treating diseases characterized by abnormal remodeling of the extracellular matrix, such as fibrosis, by increasing Focal adhesion kinase (FAK) and Protein kinase B (AKT), which are intracellular downstream signals of the β-1 integrin subunit, and then increasing regeneration signals to increase the differentiation of tight junction proteins necessary for regeneration.

[0002] When tissue damage is caused by inflammation or infection, the damaged tissue is repaired through a process of homeostatic tissue remodeling involving the formation of a new extracellular matrix (ECM) along with apoptosis or the removal of damaged cells. However, if the ECM accumulates excessively beyond normal levels during this remodeling process, fibrosis characterized by structural and functional abnormalities of the tissue may be induced. Such fibrosis is known to occur in various organs, including the lungs, intestines, liver, heart, kidneys, skin, and eyes.

[0003] As an example of a fibrotic disease, in the case of inflammatory bowel disease (IBD), anti-inflammatory agents are used in the early stages; however, it is known that intestinal fibrosis develops as the disease progresses in patients who do not respond sufficiently to such treatment. In particular, in patients who do not respond to anti-inflammatory therapy, intestinal strictures inevitably occur as a result of a prolonged inflammatory response. Such intestinal fibrosis and strictures have been reported as major pathological features that worsen the prognosis of IBD (e.g., Rieder, F. et al., Management, and Treatment of Fibrosis in Patients With Inflammatory Bowel Diseases. Gastroenterology 152, 340-350 e346 (2017)).

[0004] Various fibrotic diseases, such as idiopathic pulmonary fibrosis (IPF), non-alcoholic steatohepatitis (NASH), chronic kidney disease, and fibrosis associated with myocardial infarction, are known to begin with an inflammatory response and progress to gradual fibrosis as the disease becomes chronic. This progression of fibrosis alters the normal structure of each organ, which can result in the loss of the organ's inherent function. For example, in the case of idiopathic pulmonary fibrosis, the accumulation and contraction of fibrous tissue can compress the pulmonary arteries and / or capillaries, leading to pulmonary hypertension and impaired pulmonary circulation; this can cause blood to stagnate in the right ventricle, potentially inducing heart failure. In addition, complications such as thrombosis and pneumonia may occur in patients with idiopathic pulmonary fibrosis, and it has been reported that the risk of developing lung cancer increases due to persistent inflammatory responses and fibrosis (e.g., Coward WR, The pathogenesis of idiopathic pulmonary fibrosis. Ther Adv Respir Dis 2010; 4:367-388).

[0005] One of the currently used treatments for fibrosis is nintedanib (product name: Ofev®), which is a tyrosine kinase inhibitor that targets growth factor receptors involved in the pathogenesis of pulmonary fibrosis. Specifically, nintedanib has been reported to exhibit antifibrotic effects by 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).

[0006] In addition, pirfenidone is a small molecule oral agent used to treat idiopathic pulmonary fibrosis, a condition in which fibrosis progresses in the lungs without a clear cause. It is known to inhibit collagen synthesis, reduce the expression of profibrotic cytokines that promote fibrosis, and suppress the proliferation of collagen-producing fibroblasts (U.S. Patent No. 8,383,150).

[0007] Meanwhile, as an approach to treating fibrosis, compounds that directly inhibit transforming growth factor-β (TGF-β), which promotes fibrosis, have been studied; however, since TGF-β is a signaling molecule involved in various physiological responses in vivo, such as cell proliferation, differentiation, and immune regulation, it is recognized that directly inhibiting TGF-β or TGF-β receptors may lead to a high likelihood of systemic side effects.

[0008] In addition, fibrosis treatments that inhibit αvβ-type integrins or αvβ1 integrins are being studied in clinical trials; for example, an approach to reduce fibrosis in lung tissue by dually inhibiting αvβ and αvβ1 has been reported (Martin L. Decaris et al., Dual inhibition of αvβ and αvβ1 reduces fibrogenesis in lung tissue explants from patients with IPF, Respiratory Research 22:265 (2021)). Furthermore, a treatment that inhibits T lymphocyte migration to inflammatory bowel tissue using antibodies against α4β integrins has been approved as a treatment for ulcerative colitis and Crohn's disease (Feagan, BG et al., Vedolizumab as induction and maintenance therapy for ulcerative colitis, N Engl J Med 369:699-710 (2013)).

[0009] However, these existing therapeutic approaches primarily focus on suppressing inflammatory responses or the progression of fibrosis, and are difficult to regard as treatments that fundamentally induce structural recovery or functional regeneration of already damaged tissues.

[0010] The development of fibrosis can be suppressed by promoting the normal regeneration process of damaged tissue. Generally, when inflammation occurs, immune cells migrate to the extracellular matrix (ECM) located beneath the epithelial layer, and the structure of the ECM is partially destroyed by various chemokines secreted by these immune cells. As a result, collagen and / or other ECM proteins are degraded and released into the bloodstream, and it is understood that the exposure of β-1 integrin subunits increases in the damaged tissue area.

[0011] Epithelial cells express various α / β integrin heterodimers, and through these integrins, they play a role in maintaining the structural stability of the tissue by binding to ECM proteins and attaching to the basement membrane. The expression and distribution of these integrins have been reported to be closely associated with the maintenance and regeneration of epithelial tissue (e.g., Fujimoto, K. et al., Identification and isolation of candidate human colonic clonogenic cells based on cell surface integrin expression, Gastroenterology 123, 1941-1948 (2002); Beaulieu, JF et al., Immunolocalization of extracellular matrix components during organogenesis in the human small intestine, Anat Embryol (Berl) 183, 363-369 (1991)).

[0012] In particular, β-1 integrin subunits expressed in epithelial cells and the extracellular matrix are recognized as important factors that regulate the proliferation and migration of epithelial cells and are involved in the regeneration process of the damaged extracellular matrix (ECM).

[0013] β-1 integrins are known to detect a decrease in mechanical tension at the cell membrane level when the ECM is disrupted or damaged, thereby activating signaling axes such as focal adhesion kinase (FAK), Src, and AKT. This initial signaling activation induces the reorganization of the cytoskeleton and the reconstruction of adhesion complexes, providing a foundational environment for epithelial cells to migrate to the site of damage and reattach.

[0014] Subsequently, continuous β-1 integrin-FAK-AKT signaling stimulates cell cycle entry and cell survival pathways to promote epithelial cell proliferation, and it has been reported that during this proliferation phase, the expression of genes encoding ECM constituent proteins such as collagen, laminin, and fibronectin tends to increase concomitantly. Accordingly, the newly synthesized ECM reconstructs the basement membrane structure and restores the stability of cell adhesion, and the restored ECM can form a positive feedback loop in the tissue regeneration process by further enhancing the binding and / or signal activation of β-1 integrin.

[0015] From this perspective, epithelial cell proliferation functions as one of the important contributing factors to ECM regeneration, and β-1 integrin can be understood as a key regulatory factor mediating the continuous mechanistic flow between the two processes.

[0016] Meanwhile, the contents described in the background section are merely explanations to aid in understanding the background of the present invention and should not be interpreted as constituting prior art known to those skilled in the art to which the present invention belongs.

[0017] Prior art literature

[0018] Patent documents

[0019] (Patent Document 1) U.S. Patent No. 8,383,150

[0020] Non-patent literature

[0021] (Non-patent Document 1) Rieder, F. et al., Management, and Treatment of Fibrosis in Patients With Inflammatory Bowel Diseases. Gastroenterology 152, 340-350 e346 (2017).

[0022] (비특허문헌 2) Coward WR, The pathogenesis of idiopathic pulmonary fibrosis. Ther Adv Respir Dis 2010; 4:367-388.

[0023] (비특허문헌 3) Wollin L, Mode of action of nintedanib in the treatment of idiopathic pulmonary fibrosis. Eur Respir J 2015; 45:1434-1445.

[0024] (비특허문헌 3) 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.

[0025] (비특허문헌 4) Martin L. Decaris et al., Dual inhibition of αvβ6 and αvβ1 reduces fibrogenesis in lung tissue explants from patients with IPF. Respiratory Research volume 22, Article number: 265 (2021).

[0026] (비특허문헌 5) Feagan, B. G. et al. Vedolizumab as induction and maintenance therapy for ulcerative colitis. N Engl J Med 369, 699-710 (2013).

[0027] (Non-patent Document 6) Hossein-Ardeschir Ghofrani et al., Mechanisms and treatment of pulmonary arterial hypertension, Nature Reviews Cardiology volume 22, pages 105-120 (2025).

[0028] (Non-patent Document 7) Fujimoto, K. et al., Identification and isolation of candidate human colonic clonogenic cells based on cell surface integrin expression. Gastroenterology 123, 1941-1948 (2002).

[0029] (Non-patent document 8) Beaulieu, JF et al., Immunolocalization of extracellular matrix components during organogenesis in the human small intestine. Anat Embryol (Berl) 183, 363-369 (1991).

[0030] The objective of the present invention is to provide a therapeutically useful beta-1 integrin subunit agonist peptide.

[0031] Another objective of the present invention is to provide a pharmaceutical composition for the prevention or treatment of diseases characterized by abnormal remodeling of the extracellular matrix, such as fibrosis, comprising the above peptide.

[0032] Another objective of the present invention is to provide a method for preventing or treating a disease characterized by abnormal remodeling of the extracellular matrix, e.g., fibrosis, comprising the step of administering the peptide.

[0033] Conventionally, strategies for treating integrin-mediated fibrosis involved inhibiting fibrosis by suppressing the beta-1 integrin subunit; however, the inventors of the present invention sought to treat fibrosis by utilizing an agonist of the beta-1 integrin subunit to enhance the normal regeneration process of damaged tissue. This is a concept contrary to previously attempted treatment strategies.

[0034] The inventors intended to develop a peptide that acts as an integrin agonist to regenerate damaged epithelial cells and the ECM. The peptide of the present invention binds to the β-1 integrin subunit and increases the intracellular downstream signals Focal adhesion kinase (FAK) and Protein kinase B (AKT). Subsequently, it was confirmed that it increases the differentiation of tight junction proteins necessary for regeneration. The present invention was completed by confirming that when the peptide is administered to animal models of inflammatory bowel disease, pulmonary fibrosis, etc., it exhibits effects such as the treatment of inflammatory bowel disease, regeneration of intestinal tissue, and improvement of pulmonary fibrosis.

[0035] One aspect of the present invention provides a β-1 integrin subunit agonist peptide of a novel amino acid sequence.

[0036] The numbered items below exemplify some of the embodiments described in this specification.

[0037] 1. A β-1 integrin subunit agonist peptide comprising the amino acid sequence of the following chemical formula or an amino acid sequence that is at least 85% identical thereto.

[0038] GLX1SX2X3X4X5FX6X7PDIQX8PDA

[0039] In the above formula, X1 is arginine or glycine, X3 is serine, glutamic acid or lysine, X8 is tyrosine or phenylalanine, and X2, X4, X5, X6 and X7 are each independently cationic amino acids selected from the group consisting of arginine, histidine and lysine.

[0040] 2. A peptide comprising the amino acid sequence of SEQ ID NO. 1 or an amino acid sequence comprising one or two amino acid substitutions compared therewith, in the first embodiment above.

[0041] 3. A peptide comprising the amino acid sequence of SEQ ID NO. 2 or an amino acid sequence comprising one or two amino acid substitutions compared therewith, in the first embodiment above.

[0042] 4. A peptide comprising the amino acid sequence of SEQ ID NO. 3 or an amino acid sequence comprising one or two amino acid substitutions compared therewith, in the first embodiment above.

[0043] 5. A peptide in which, in any one of the first to fourth embodiments above, the β-1 integrin subunit forms a complex with an α-1, α-2, α-3, α-5, α-6, α-v, α-7, α-8, α-10, or α-11 integrin subunit.

[0044] 6. A peptide that selectively binds to a β-1 integrin subunit compared to a β-3, β-4, β-5, β-6, β-7, α-3, α-4, α-5, or α-v integrin subunit in any one of the above first to fifth embodiments.

[0045] 7. A peptide that, in any one of the first to sixth embodiments above, does not bind to a β-1 integrin subunit present in immune cells but binds to a β-1 integrin subunit present in epithelial cells.

[0046] 8. A peptide that can be used to regenerate damaged extracellular matrix and damaged epithelial tissue in any one of the first to seventh embodiments above.

[0047] 9. A peptide in any one of the first to eighth embodiments, wherein the damaged extracellular matrix is ​​associated with damaged epithelial tissue of an organ selected from the group consisting of lungs, liver, heart, blood vessels, kidneys, eyes, skin, and intestines.

[0048] One aspect of the present invention provides a pharmaceutical composition comprising a β-1 integrin subunit agonist peptide as described herein.

[0049] 10. A pharmaceutical composition for use in the prevention or treatment of a disease characterized by abnormal remodeling of the extracellular matrix, comprising a peptide according to any one of the first to ninth embodiments above.

[0050] 11. A pharmaceutical composition for use in the prevention or treatment of fibrosis in the above 10th embodiment.

[0051] 12. A pharmaceutical composition in which, in the above 11th embodiment, the fibrosis is selected from the group consisting of pulmonary fibrosis, hepatic fibrosis, renal fibrosis, and cardiac fibrosis.

[0052] 13. A pharmaceutical composition for use in the treatment of inflammatory bowel disease in the above 10th embodiment.

[0053] 14. A pharmaceutical composition according to the 13th embodiment, wherein the inflammatory bowel disease is Crohn's disease or ulcerative colitis.

[0054] 15. A pharmaceutical composition for use in the prevention or treatment of cancer or autoimmune disease in the above 10th embodiment.

[0055] 16. A pharmaceutical composition in which the peptide is administered at intervals of at least 72 hours in the above 10th to 15th embodiments.

[0056] The β-1 integrin subunit agonist peptide according to the present invention can bind to a β-1 integrin subunit and increase intracellular downstream signals, Focal adhesion kinase (FAK) and AKT. Subsequently, by increasing regeneration signals and increasing the differentiation of tight junction proteins necessary for regeneration, it can be used as a preventive or therapeutic agent for diseases caused by fibrosis, and may also be used in combination with existing therapeutic agents.

[0057] Figure 1 is the result of confirming the binding of the peptide of SEQ ID NO. 1 to integrin subunits present in lung tissue-derived LL-29 and human T immune cells, specifically HuT-78 cells, by immunoprecipitation.

[0058] Figure 2 is a three-dimensional image of the peptide of SEQ ID NO. 1 bound to an integrin complex of an α-v subunit and a β-1 subunit, showing that anionic amino acids are concentrated and distributed at the integrin site that binds to the peptide. The figure on the right shows amino acid residues of the β-1 integrin subunit located within 5 Å of the peptide.

[0059] Figure 3 shows a comparison of the change in force over time measured by Steered Molecular Dynamics (SMD) simulation when different peptide sequences according to the present invention bind to an integrin α-v / β-1 complex.

[0060] Figures 4 and 5 respectively show the results of comparing the relative expression levels of Col1a1 mRNA and FN1 mRNA induced by bleomycin (BLM) treatment in A549 cells when different peptide sequences according to the present invention were treated together, analyzed by quantitative real-time reverse transcription-polymerase chain reaction (qRT-PCR). Figure 4 shows the results of measuring Col1a1 mRNA expression, and Figure 5 shows the results of measuring FN1 mRNA expression.

[0061] Figure 6 is an immunohistochemistry result observing the colocalization of the β-1 integrin subunit and the peptide of SEQ ID NO. 1 expressed in the intestinal tissue of mice induced with colitis by DSS. Green represents the β-1 integrin subunit, red represents the peptide labeled with Cy5.5, and blue represents the nucleus. This demonstrates that the peptide according to the present invention specifically binds to the β-1 integrin subunit in individuals with inflammatory bowel disease. Peptide Sq1 refers to the peptide of SEQ ID NO. 1, and this is also the case in other figures.

[0062] Figure 7 shows the results of observing intracellular signal transduction via the integrin of the peptide of SEQ ID NO. 1. Figure 7A is a Western blot result observing the phosphorylation of integrin downstream signals FAK, AKT, and mTorr when the peptide, the FAK inhibitor PF-573228, and the peptide and FAK inhibitor were treated simultaneously. Figure 7B shows the results of quantifying the intensity of the Western blot bands using Image J. This demonstrates that the peptide according to the present invention activates downstream signal transduction of the beta-1 integrin subunit. ("PF" indicates PF-573228.)

[0063] Figures 8 and 9 show the results of measuring the phosphorylation of FAK and AKT among the integrin downstream signals by the peptide of SEQ ID NO. 1 over time. Figure 8 shows the results of measuring the phosphorylation retention time of FAK and AKT by Western blot. Figure 9 shows the results of measuring the intensity of the Western blot bands using Image J. This demonstrates that the peptide according to the present invention activates downstream signal transduction of the beta-1 integrin subunit.

[0064] Figures 10 and 11 show the results of measuring the expression of E-cadherin, Occludin, and ZO-1 over time to evaluate the expression of tight junction proteins, which are the final step of integrin downstream signaling by the peptide of SEQ ID NO. 1. Figure 10 shows the results of measuring the time during which the expression of tight junction proteins is maintained by the peptide of SEQ ID NO. 1 using Western blot. Figure 11 shows the results of measuring the intensity of the Western blot bands using Image J. In an inflammatory bowel disease model, the peptide according to the present invention activates downstream signaling of the beta-1 integrin subunit and increases the differentiation of tight junction proteins necessary for regeneration.

[0065] Figures 12 to 15 show the results demonstrating the therapeutic effect of the peptide of SEQ ID NO. 1 in mice induced with inflammatory bowel disease via DSS. Figure 12 shows the experimental protocol for inducing an animal model of chronic inflammatory bowel disease. Figure 13 shows the results of measuring the intestinal length of each treatment group after the sacrifice of mice. Figure 14 shows the results of observing the extracted intestinal tissue under a microscope after HE staining. Figure 15 shows the results of measuring the concentration of calprotectin present in stool and plasma, which are biomarkers for evaluating the degree of treatment for inflammatory bowel disease. This demonstrates the excellent therapeutic effect of the peptide according to the present invention on inflammatory bowel disease.

[0066] Figure 16 shows the results of an immunohistochemistry observation of the colocalization of the β-1 integrin subunit expressed in the lung tissue of mice induced with bleomycin-induced pulmonary fibrosis and the peptide of SEQ ID NO. 1. Green represents the β-1 integrin subunit, red represents the peptide labeled with Cy5.5, and blue represents the nucleus. This demonstrates that the peptide according to the present invention specifically binds to the β-1 integrin subunit in individuals with pulmonary fibrosis.

[0067] Figures 17 to 20 show the results demonstrating the therapeutic effect of the peptide of SEQ ID NO. 1 in mice induced with bleomycin-induced pulmonary fibrosis. Figure 17 shows the experimental protocol for inducing an animal model of pulmonary fibrosis. Figure 18 shows the results of observing lung tissue under a microscope after staining the collagen-deposited areas with Sirius red. Figure 19 shows the results of measuring the collagen-stained area and Ashcroft score to assess the improvement in fibrous deposition following peptide treatment in an animal model of pulmonary fibrosis. Figure 20 shows the results of confirming the expression of Claudin-3, a tight junction, through immunohistochemistry to observe lung tissue regeneration. This demonstrates the excellent therapeutic effect of the peptide according to the present invention on pulmonary fibrosis.

[0068] Unless otherwise specifically defined, the technical and scientific terms used in this specification have the meaning generally understood by those skilled in the art to which the present invention pertains.

[0069] The embodiments described in this specification and the configurations illustrated in the drawings are merely specific examples to aid in understanding the invention and do not limit the technical scope of the invention. At the time of filing this application, various modifications, equivalents, and applications performing substantially the same functions are possible, and a person skilled in the art will understand that such modifications and applications are also included within the scope of the invention.

[0070] The embodiments, features, components, and technical means described herein may be implemented independently or in any combination, unless otherwise explicitly stated to be exclusive or mutually excluded. In particular, technical features described in different paragraphs, items, or embodiments herein may be combined or optionally combined to the extent that there is no technical contradiction, as understood by a person skilled in the art. Such combinations should be understood to be included within the technical spirit and scope of the invention.

[0071] Accordingly, even if a component or feature described in a specific embodiment or paragraph is not explicitly described together with another embodiment or paragraph, it should be interpreted as being directly and clearly derivable from the disclosure of the present invention where it is recognized as technically reasonable by a person skilled in the art.

[0072] definition

[0073] Expressions used in the singular form in this specification are used to include the plural unless the context clearly indicates otherwise. Additionally, the expression “or” is interpreted to include the meaning of “and / or” unless otherwise specified in the context.

[0074] As used herein, the term “comprising” should be understood as an open expression that essentially includes the described components, components, steps, etc., unless specifically stated otherwise, without excluding the existence of additional components, components, steps, etc. Accordingly, the term “comprising” is interpreted as a concept that includes the more restrictive “consisting of” or “consisting essentially of.”

[0075] In this specification, the term “sequence” may be interpreted, depending on the context, as a nucleic acid (or polynucleotide) molecule or a protein (or polypeptide) molecule having a given sequence.

[0076] The term "peptide" as used in this specification means an amino acid sequence in which two or more amino acids are linked by peptide bonds.

[0077] As used in this specification, the term “anionic amino acid” refers to an amino acid having a side chain that carries a negative charge under physiological conditions (pH approximately 7.4) and includes glutamic acid (Glu, E) and aspartic acid (Asp, D). On the other hand, the term “cationic amino acid” refers to an amino acid having a side chain that carries a positive charge under physiological conditions (pH approximately 7.4) and includes lysine (Lys, K), arginine (Arg, R), histidine (His, H), etc.

[0078] In this specification, the term “other amino acid” means an amino acid selected from alanine, isoleucine, leucine, methionine, phenylalanine, proline, tryptophan, valine, aspartic acid, cysteine, glutamine, glycine, serine, threonine, tyrosine, aspartic acid, glutamic acid, arginine, histidine, lysine, and all known variants of said amino acids, excluding the amino acid that the wild-type protein has at the original mutation site, unless otherwise indicated.

[0079] As used herein, the terms “sequence identity” or “sequence homology” refer to a value representing the number of residues present at the same position as a percentage of the total length of the sequences when two amino acid sequences or nucleic acid sequences are aligned. When a specific sequence is described in this specification as having “at least X% sequence identity,” X may be, for example, 85%, 90%, 95%, 98%, or 99%. Sequence identity is typically calculated using BLAST (Basic Local Alignment Search Tool), ClustalW, EMBOSS, or other known sequence alignment algorithms, based on default parameters. For example, when aligning amino acid sequences using BLASTP, the identity value calculated using a BLOSUM62 matrix and a gap penalty as default values ​​may be used as the basis. Additionally, in this specification, “sequence identity” or “sequence homology” may include values ​​calculated according to global alignment or local alignment optimized by considering insertions, deletions, substitutions, etc., during sequence alignment, and is also used as a criterion to describe the scope of functional equivalents capable of maintaining the technical effects of the invention.

[0080] As used herein, the term “conservative amino acid substitution” refers to a substitution between amino acids with similar physicochemical properties (e.g., charge, size, hydrophobicity, polarity, etc.), and includes a substitution in which the structural stability or biological function of the protein can be substantially maintained.

[0081] For example, the following substitutions within an amino acid group may be considered conservative substitutions:

[0082] Hydrophobic amino acid group: Ala, Val, Leu, Ile, Met

[0083] Polar non-charged amino acid groups: Ser, Thr, Gln, Asn

[0084] Acidic amino acid group: Asp, Glu

[0085] Basic amino acid group: Lys, Arg, His

[0086] Aromatic amino acid group: Phe, Tyr, Trp

[0087] In this specification, the expression “having one or more conservative amino acid substitutions” includes the fact that even if the original amino acid is substituted with a physicochemically similar amino acid as described above, the function of the peptide (e.g., β-1 integrin agonist activity) is substantially maintained.

[0088] In this specification, “selectively binds” means that a specific ligand, protein, receptor, or target sequence, etc. binds with significantly higher binding affinity or specificity compared to other non-target substances. Herein, “significantly higher” includes cases where, under normal test conditions, a difference in relative binding strength is shown at a level or statistically significant or at a level or commonly recognized by researchers / in the technical field compared to the non-target.

[0089] In addition, “selective binding” does not require absolute exclusivity and applies when binding to the target is dominant or enhanced to a functionally meaningful level, even if some non-specific binding to a non-target exists. If necessary, selective binding may be defined by differences in binding parameters such as Kd, IC50, EC50, kon / koff, or differences in functional activation (such as increased signaling strength).

[0090] The peptide of the present invention may exhibit a relatively high binding affinity and / or functional activation ability for β1-based integrin complexes that are uniquely or predominantly expressed in epithelial cells, compared to the extent of substantially binding to integrins that are mainly expressed in immune cells. That is, the peptide of the present invention is characterized by exhibiting significantly high selectivity and / or activation specificity for epithelial cell integrins while minimizing non-specific binding to non-target immune cell integrins.

[0091] As used herein, the term “integrin” refers to a functional protein that is a transmembrane heterodimer receptor composed of an α (alpha) subunit and a β (beta) subunit, and converts binding with an extracellular ligand into intracellular signaling. Integrins play a key role in physiological and pathological processes such as cell adhesion, cell-cell / cell-extracellular matrix (ECM) interactions, cell migration, and the regulation of cell survival and / or proliferation signals, and mediate outside-in and / or inside-out signaling through conformational activation upon ligand binding.

[0092] In this specification, “integrin” is not limited to specific α / β combinations and includes all functional integrin complexes formed by commonly known subunit combinations such as α1-α11, α-v, β1-β8, etc. In particular, β1 integrins (α5β1, α4β1, etc.) can selectively bind to extracellular ligands (fibronectin, laminin, etc.) or agonist peptides of the present invention to exhibit functions such as regulating cell adhesion, activating signal transduction, tissue regeneration, or regulating antitumor activity.

[0093] As used herein, the terms “β1 integrin (Integrin β1)” or “β1 integrin subunit” refer to receptors containing a β1 subunit among integrin α / β heteromers, and include all functional complexes formed by binding with α subunits such as α1-α11 or αV. β1 integrin has a unique extracellular structure containing an I-like domain and a metal ion-dependent adhesion site (MIDAS), and exhibits ligand binding specificity optimized for binding to various extracellular matrix (ECM) components such as fibronectin, laminin, and collagen. Two NPXY motifs are present in the cytoplasmic tail of β1, which mediate the stabilization of the extended-open structure of the integrin and / or signal transduction activation through binding with talin, kindlin, etc.

[0094] β1 integrins induce outside-in signaling, including FAK, Src, ILK, PI3K / AKT, and MAPK pathways, upon binding to the ECM, and inside-out activation is regulated by the binding of talin or kindlin; through this, they perform various physiological and pathological functions such as cell adhesion, migration, survival, anti-apoptotic response, and / or tissue regeneration. Furthermore, β1 integrins have the characteristic of having a low energy barrier required for structural conversion between inactive and active forms, allowing them to be easily converted to the active form with only relatively small structural changes or ligand binding.

[0095] In this specification, β1 integrins include all β1-based integrin complexes having the above structural and functional properties, and in particular, refer to receptors to which the agonist peptide of the present invention binds to enable activation or functional regulation.

[0096] The β-1 integrin subunit combines with various α subunits such as α1, α2, α3, α5, α6, αv, α7, α8, α10, and α11 to form dimers such as α1β1, α2β1, α3β1, α5β1, α6β1, αvβ1, α7β1, α8β1, α10β1, and α11β1, and performs signal transduction specific to tissue and cell types.

[0097] The term “agonist” as used in the present invention, also known as an agent or agonist, refers to a molecule such as a compound, drug, enzyme activator, or hormone that enhances the activity of a target receptor or the activity of another molecule. In the present invention, a beta-1 agonist peptide refers to a peptide capable of specifically binding to a beta-1 integrin subunit and activating a downstream signal of the beta-1 integrin subunit (e.g., FAK-AKT-mTOR pathway), and any specific sequence, form, origin, or synthesis method of such a peptide is not limited. Whether a peptide is a beta-1 integrin subunit agonist can be identified by whether any component constituting a beta-1 integrin subunit downstream signaling pathway, such as the FAK-AKT-mROR pathway, is activated or by the functional changes caused by such downstream signaling pathways (e.g., increased differentiation of tight junction proteins required for regeneration), and a person skilled in the art of biotechnology is familiar with biochemical techniques that can confirm such activation or changes.

[0098] The term “complex” as used in this specification refers to a structure formed by two or more molecules (e.g., peptides, proteins, antibodies, antigen-binding fragments, nucleic acids, ligands, or receptors, etc.) being joined together by covalent or non-covalent interactions, and such joining may be direct or indirect through mediating molecules.

[0099] As used herein, the term “immune cell” refers to a cell involved in innate or adaptive immune responses, and includes all cells originating from or acting in blood and / or lymphatic tissues, such as T cells, B cells, NK cells, macrophages, dendritic cells, neutrophils, and monocytes. Immune cells primarily express integrins specialized for immune responses, such as α4β1, αLβ2 (LFA-1), and αMβ2 (Mac-1), and correspond to the non-target cell group of the peptides mentioned herein.

[0100] As used in this specification, the term “epithelial cell” refers to a cell constituting epithelial tissue, such as skin, mucous membranes, organ surfaces, and the inner surface of tubular structures, which is involved in barrier function and / or tissue structure maintenance. Epithelial cells richly express β1-based integrin complexes (α5β1, α3β1, α2β1, etc.) and include a group of target cells to which the peptide of the present invention selectively binds and which act functionally.

[0101] As used herein, the term “ECM (Extracellular Matrix)” refers to a complex matrix comprising fibrous proteins (fibronectin, collagen, laminin, etc.), polysaccharides, and proteoglycans that exist outside the cell and are involved in the structural support of tissues and / or the regulation of cell behavior. The ECM can act as a natural ligand for integrins and includes environmental components that interact, in particular with the β1 integrin family, to regulate cell adhesion, migration, and survival signaling.

[0102] In this specification, “ECM regeneration” refers to the process of restoring the structure and function of a damaged extracellular matrix, and, while not necessarily limited thereto, may generally include the following steps.

[0103] (1) Reduction and / or structural breakdown of damaged ECM proteins

[0104] (2) Increased accessibility to β-1 integrin and / or change in active state due to ECM disruption

[0105] (3) Activation of β-1 integrin downstream signals (FAK / AKT, etc.)

[0106] (4) Increased expression of tight junction proteins and / or restoration of epithelial cell barrier function

[0107] (5) Remodeling of normal tissue structure

[0108] The peptide of the present invention can contribute to the regeneration of the ECM of damaged tissue by enhancing at least some of the above steps, particularly (3) and / or (4).

[0109] In this specification, the terms “damaged” or “damaged” mean a state in which the structural and functional integrity of a tissue or extracellular matrix (ECM) is partially or entirely impaired by external stimuli (physical, chemical, or biological factors) or internal pathological conditions. Such damage may include disruption or reduction of ECM proteins, dissociation of junctions between epithelial cells, reduced cell adhesion, tissue breakdown due to inflammatory responses, and loss of function due to changes in the microenvironment. Damage does not require complete destruction and includes any level of defect in which the normal physiological function of the tissue is impaired or destabilized.

[0110] As used herein, the term “regeneration” refers to the process by which damaged tissue or ECM recovers its original structural and functional state or is restored to a substantially similar level. This may include the resynthesis and / or rearrangement of ECM constituent proteins, the recovery of cell adhesion and / or tissue binding structure, the migration, differentiation, and proliferation of epithelial cells, and the recovery of tissue stability and / or homeostasis. Regeneration includes not only physiological recovery but also artificial and therapeutic recovery processes promoted by the action of the peptide according to the present invention.

[0111] Although the various diseases mentioned in this invention may differ in their causes or tissue characteristics, they are known to commonly involve pathological conditions in which the regeneration and rearrangement processes of the ECM accompanying inflammation or tissue damage are not maintained normally. In such situations, the balance between the synthesis and degradation of the ECM is disrupted, or constituent proteins such as collagen and / or fibronectin are excessively accumulated or reduced, which may lead to functional changes such as changes in tissue stiffness, weakening of the epithelial-basement membrane junction, and inhibition of cell adhesion, migration, and / or signal transduction.

[0112] In this specification, the term "extracellular matrix (ECM) remodeling-related pathological condition" refers to a condition in which one or more of the processes of synthesis, degradation, rearrangement, or accumulation of the extracellular matrix are regulated outside the normal physiological range, and includes a pathological condition in which the structural stability, mechanical properties, and / or functional homeostasis of the tissue are impaired as a result. In such pathological conditions, excessive accumulation or abnormal degradation of ECM components such as collagen, elastin, fibronectin, and laminin may occur, along with abnormalities in the interaction between epithelial cells and the ECM, changes in cell adhesion signaling, or a decline in tissue barrier function.

[0113] Examples of pathological conditions related to ECM remodeling include fibrosis, inflammatory bowel disease, cancer, and autoimmune diseases. For instance, in fibrosis, chronic inflammation or repetitive tissue damage can induce an excessive accumulation of ECM components, leading to tissue stiffness and functional decline. In the case of inflammatory bowel disease, imbalances in ECM reorganization and damage to the epithelial barrier, along with persistent inflammatory responses in the intestinal mucosa, may be involved in the pathophysiology. Furthermore, in cancer, abnormal reorganization of the ECM within the tumor microenvironment can influence tumor growth, invasion, or metastasis, while in autoimmune diseases, structural changes in the ECM accompanying chronic inflammatory responses may be associated with tissue damage and functional abnormalities.

[0114] Such abnormalities in ECM remodeling have been reported in various diseases, including fibrotic diseases such as pulmonary fibrosis, intestinal tissue changes observed in inflammatory bowel disease, some autoimmune diseases, chronic wounds, and ECM reorganization in the tumor microenvironment of solid tumors.

[0115] As used herein, the term “fibrosis” refers to pathological tissue changes in which excessive activation of fibroblasts and / or myofibroblasts is induced by stimuli such as tissue damage or chronic inflammation, resulting in the abnormal accumulation of ECM proteins such as collagen, fibronectin, and laminin. Such fibrosis may include increased ECM stiffness, decreased tissue flexibility, abnormalities in cell migration or adhesion, and disruption of normal tissue structure; examples include pulmonary fibrosis, intestinal fibrosis in inflammatory bowel disease, and fibrotic changes associated with certain autoimmune diseases or cancer.

[0116] As used herein, the term “Inflammatory Bowel Disease (IBD)” refers to a group of diseases accompanied by chronic and recurrent inflammatory responses in the intestinal tract, damage to the epithelial barrier, structural changes in the ECM, and / or immune abnormalities. Examples may include Crohn's disease and ulcerative colitis, which are commonly characterized by chronic inflammation, damage to epithelial cells, impaired barrier function, and / or abnormalities in ECM remodeling.

[0117] In this specification, the term “cancer” refers to a group of diseases in which the regulation of cell proliferation is lost and incomplete differentiation and invasiveness increase, forming an abnormal proliferative mass (tumor) within a tissue or systemically. Cancer may include solid tumors and hematologic malignancies, and may commonly be accompanied by abnormal reorganization of the extracellular matrix (ECM), changes in the tumor microenvironment, distortion of cell migration and adhesion, and / or abnormal activation of integrin signaling.

[0118] In this specification, the term “autoimmune disease” refers to a group of diseases in which inflammation, tissue damage, and functional decline are induced as the immune system mistakes its own tissues or antigens for foreign antigens and attacks them. This may include various diseases such as rheumatoid arthritis, systemic lupus erythematosus (SLE), ankylosing spondylitis, psoriatic arthritis, and autoimmune skin diseases, and these diseases may be accompanied by pathological conditions such as epithelial barrier dysfunction, tissue ECM deformation, and chronic inflammatory response.

[0119] In this specification, the term “prevention” means any act or measure intended to suppress or delay the occurrence of a specific disease or symptom. Prevention includes preemptive measures taken before the disease manifests, or acts to prevent clinical deterioration or progression even if initial changes in the disease are present, and may be achieved through the biological action or mechanistic effect of the target substance.

[0120] In this specification, the terms “treatment” or “therapy” mean any medical or pharmacological measure intended to alleviate, relieve, stabilize, inhibit the progression of, or substantially improve an existing disease or symptom. This includes the alleviation of symptoms, restoration of tissue function, inhibition of pathophysiological changes, promotion of ECM regeneration, etc., and the degree of treatment includes both partial and complete improvement.

[0121] As used herein, the term “pharmaceuticalally acceptable carrier” means a substance that enables a pharmaceutical composition containing an active ingredient to be safely administered in vivo and provides physical and chemical stability, solubility, or suitability for administration of the active ingredient. Such a carrier may include water-soluble or water-insoluble media depending on the route of administration and may be selected within a range that does not substantially affect the pharmacological activity of the active ingredient.

[0122] As used herein, the term “pharmaceuticalally acceptable diluent” refers to a substance used to adjust the concentration of an active ingredient or to increase the volume or weight of a formulation, and which serves to improve the ease of administration, quantification, or uniformity of a pharmaceutical composition. The diluent may be selected within a range that is chemically stable with respect to the active ingredient and does not adversely affect the safety and efficacy of the pharmaceutical composition.

[0123] As used herein, the term “pharmaceuticalally acceptable excipient” means an auxiliary component used to improve the stability, release characteristics, bioavailability, or physical properties of an active ingredient during the preparation, storage, or administration of a pharmaceutical composition. Such excipients may include, but are not limited to, stabilizers, buffers, isotonic agents, preservatives, antioxidants, binders, disintegrants, or coating agents, for example.

[0124] 1. β-1 integrin agonist peptide

[0125] One aspect of the present invention provides a β-1 integrin agonist peptide of a novel amino acid sequence.

[0126] In one embodiment, the β-1 integrin may form a complex with an α-1, α-2, α-3, α-5, α-6, α-v, α-7, α-8, α-10, or α-11 integrin subunit.

[0127] The β-1 integrin agonist peptide according to the present invention can promote the activity of an integrin receptor containing β-1 integrin by binding to or interacting with a β-1 integrin subunit. It is known that β-1 integrin functions by forming dimeric complexes with various α-integrin subunits, and the agonist peptide of the present invention can act in a manner that induces the formation or stabilization of such β-1 integrin / α-integrin complexes or increases the active state of said complexes.

[0128] In one embodiment, the agonist peptide of the present invention may bind to a complex formed by a β-1 integrin with an α-1, α-2, α-3, α-5, α-6, α-v, α-7, α-8, α-10, or α-11 integrin subunit, thereby activating integrin-mediated signals associated with cell adhesion, cell signaling, cell survival, migration, or differentiation. Such activity may be identified, for example, by increased binding ability to extracellular matrix proteins, activation of downstream signaling pathways, or changes in cell function, but is not limited thereto.

[0129] In one embodiment, the agonist peptide of the present invention may include the amino acid sequence of the following chemical formula 1.

[0130] [Chemical Formula 1]

[0131] GLX1SX2X3X4X5FX6X7PDIQX8PDA

[0132] In the above formula, X1 is arginine or glycine.

[0133] In the above formula, X3 is serine, glutamic acid, or lysine.

[0134] In the above formula, X8 is tyrosine or phenylalanine.

[0135] In the above formula, X2, X4, X5, X6, and X7 are each independently cationic amino acids selected from the group consisting of arginine, histidine, and lysine.

[0136] The cationic amino acid residues included in the above chemical formula 1 can enhance binding affinity through electrical interaction with the negatively charged region on the surface of the integrin protein, and proline and aromatic amino acid residues can contribute to the stabilization of the peptide's stereochemical structure or binding specificity.

[0137] To understand the interactions between the peptide of the present invention and various α / β integrin complexes, homologous modeling using SWISS-MODEL, electric potential calculation (APBS), molecular docking (Pharmulator™), and molecular dynamics (MD) simulation (NAMD, CHARMM36 force field) were performed.

[0138] According to the results of molecular docking and molecular dynamics (MD) simulations, the agonist peptide of the present invention was observed to bind relatively stably to the β-1 integrin complex. When comprehensively considering the docking score, binding posture retention time, and whether dissociation occurred during the MD simulation, the agonist peptide of the present invention showed a tendency to remain in a region of concentrated negative charge near the β domain, which suggests the possibility that the corresponding site is involved in the binding.

[0139] In addition, as a result of electropotential analysis (APBS) and surface charge distribution evaluation, the ligand binding sites of β-1 integrins tended to contain negative charge clusters with Asp and Glu residues arranged in succession. On the other hand, the peptide of the present invention was analyzed to form patches of concentrated positive charge through the repeated arrangement of Lys, Arg, and His residues, suggesting that electrostatic complementarity between these charge patterns may contribute to binding stability. Molecular dynamics simulations also evaluated that these charge interactions may contribute to maintaining the binding posture to some extent.

[0140] Furthermore, when hypothetical analogs with different cationic amino acids introduced at the estimated positive charge patch positions were subjected to docking analysis under the same conditions, cases were observed where the binding energy and binding mode did not change significantly. This suggests that the continuous formation of positive charge patches may play a more important role in interactions with β-1 integrin than the individual structure of specific amino acid residues. Additionally, when docking analysis was performed on derivatives in which specific amino acids were substituted at positions X1, X2, and X3 in the amino acid sequence of Formula 1, a tendency was observed in some variants to maintain or relatively increase the binding affinity with β-1 integrin.

[0141] The β-1 integrin agonist peptide according to the present invention exhibited a pattern in which actual binding was observed in a system containing β-1 integrin, and it is understood that this binding characteristic is generally consistent with the results of the structure-based analysis described above. That is, the peptide according to the present invention includes structural features having a charge distribution complementary to a region of concentrated negative charge located near the β domain of the β-1 integrin complex, and binding stability with β-1 integrin can be ensured through this electrical and spatial complementarity.

[0142] In one embodiment, the agonist peptide of the present invention may include an analog having an amino acid sequence that is 85% or more identical to the amino acid sequence of Formula 1.

[0143] In one embodiment, the agonist peptide of the present invention may include an analog having an amino acid sequence that is 90% or more identical to the amino acid sequence of Formula 1.

[0144] In one embodiment, the agonist peptide of the present invention may include an analog having an amino acid sequence that is 95% or more identical to the amino acid sequence of Formula 1.

[0145] In one embodiment, the agonist peptide of the present invention may include an analog having an amino acid sequence that is 85% or more identical to the amino acid sequence of SEQ ID NO. 1.

[0146] In one embodiment, the agonist peptide of the present invention may include an analog having an amino acid sequence that is 90% or more identical to the amino acid sequence of SEQ ID NO. 1.

[0147] In one embodiment, the agonist peptide of the present invention may include an analog having an amino acid sequence that is 95% or more identical to the amino acid sequence of SEQ ID NO. 1.

[0148] In one embodiment, the agonist peptide of the present invention may include an analog having an amino acid sequence that is 85% or more identical to the amino acid sequence of SEQ ID NO. 2.

[0149] In one embodiment, the agonist peptide of the present invention may include an analog having an amino acid sequence that is 90% or more identical to the amino acid sequence of SEQ ID NO. 2.

[0150] In one embodiment, the agonist peptide of the present invention may include an analog having an amino acid sequence that is 95% or more identical to the amino acid sequence of SEQ ID NO. 2.

[0151] In one embodiment, the agonist peptide of the present invention may include an analog having an amino acid sequence that is 85% or more identical to the amino acid sequence of SEQ ID NO. 3.

[0152] In one embodiment, the agonist peptide of the present invention may include an analog having an amino acid sequence that is 90% or more identical to the amino acid sequence of SEQ ID NO. 3.

[0153] In one embodiment, the agonist peptide of the present invention may include an analog having an amino acid sequence that is 95% or more identical to the amino acid sequence of SEQ ID NO. 3.

[0154] The analogs may include amino acid substitutions, deletions, or additions compared to the amino acid sequence of Formula 1, but include functional equivalents that substantially retain β-1 integrin agonist activity. The functional equivalents include analogs that retain high-affinity binding to β-1 integrin and associated downstream signaling activation. Here, “downstream signaling activation” refers to a signaling process that proceeds sequentially at the membrane and cytoplasmic levels following ligand binding to β-1 integrin, comprising a series of molecular steps starting with an increase in FAK phosphorylation and leading to AKT and / or mTOR activation. Such downstream signaling activation can be confirmed by conventional biochemical methods.

[0155] When the agonist peptide of the present invention binds to a β-1 integrin subunit, downstream signaling activation is promoted, which increases the phosphorylation of FAK. Subsequently, the activation of AKT and mTOR proceeds sequentially, resulting in increased expression of tight junction-related proteins such as E-cadherin, ZO-1, and Occludin. This signaling process induces epithelial barrier restitution, which is a key component of the ECM repair process, and helps the damaged epithelial layer recover rapidly.

[0156] In one embodiment, when the agonist peptide of the present invention is an analog of the peptide of Formula 1, it may include one to five amino acid substitutions compared to the amino acid sequence of Formula 1.

[0157] In one embodiment, when the agonist peptide of the present invention is an analog of the peptide of Formula 1, it may include one or two amino acid substitutions compared to the amino acid sequence of Formula 1.

[0158] The above amino acid substitution means that one or more amino acids included in the amino acid sequence of Formula 1 are replaced with other amino acids, and said substitution may be performed within a range that substantially maintains the structural stability of the peptide or β-1 integrin agonist activity.

[0159] In one embodiment, when the agonist peptide of the present invention is an analogue of the peptide of SEQ ID NO. 1, it may include one to five amino acid substitutions compared to the amino acid sequence of SEQ ID NO. 1.

[0160] In one embodiment, when the agonist peptide of the present invention is an analogue of the peptide of SEQ ID NO. 1, it may include one or two amino acid substitutions compared to the amino acid sequence of SEQ ID NO. 1.

[0161] The above amino acid substitution means that one or more amino acids included in the amino acid sequence of SEQ ID NO. 1 are replaced with other amino acids, and said substitution may be performed within a range that substantially maintains the structural stability of the peptide or β-1 integrin agonist activity.

[0162] In one embodiment, when the agonist peptide of the present invention is an analogue of the peptide of SEQ ID NO. 2, it may include one to five amino acid substitutions compared to the amino acid sequence of SEQ ID NO. 2.

[0163] In one embodiment, when the agonist peptide of the present invention is an analogue of the peptide of SEQ ID NO. 2, it may include one or two amino acid substitutions compared to the amino acid sequence of SEQ ID NO. 2.

[0164] The above amino acid substitution means that one or more amino acids included in the amino acid sequence of SEQ ID NO. 2 are replaced with other amino acids, and said substitution may be performed within a range that substantially maintains the structural stability of the peptide or β-1 integrin agonist activity.

[0165] In one embodiment, when the agonist peptide of the present invention is an analogue of the peptide of SEQ ID NO. 3, it may include 1 to 5 amino acid substitutions compared to the amino acid sequence of SEQ ID NO. 3.

[0166] In one embodiment, when the agonist peptide of the present invention is an analogue of the peptide of SEQ ID NO. 3, it may include one or two amino acid substitutions compared to the amino acid sequence of SEQ ID NO. 3.

[0167] The above amino acid substitution means that one or more amino acids included in the amino acid sequence of SEQ ID NO. 3 are replaced with other amino acids, and said substitution may be performed within a range that substantially maintains the structural stability of the peptide or β-1 integrin agonist activity.

[0168] The above amino acid substitution may be, for example, a conservative substitution.

[0169] Whether a conservative amino acid substitution maintains protein function can generally be determined through integrin binding analysis, cell-based activity evaluation, receptor signaling analysis, or similar biological function evaluation experiments. Such conservative substitutions are substitutions designed to maintain the structural stability and function of a protein; they represent an example of a variant that a person skilled in the art can derive through ordinary experimental and predictive methods, and are recognized as a substantial variation of the technical concept of the embodiments described in the specification. Therefore, where the specification specifies that it includes “conservative amino acid substitutions,” this is interpreted to include substitutions with structurally similar and functionally equivalent amino acids.

[0170] In one embodiment, the agonist peptide of the present invention may selectively bind to a β-1 integrin subunit compared to a β-3, β-4, β-5, β-6, β-7, α-3, α-4, α-5, or α-v integrin subunit.

[0171] This selectivity of the agonist peptide of the present invention can provide a technical effect of reducing the possibility of non-specific signal transduction or undesirable biological reactions by minimizing the activation of non-target integrin subunits.

[0172] In one embodiment, the agonist peptide of the present invention may not bind to β-1 integrin subunits present in immune cells, but may bind to β-1 integrin subunits present in epithelial cells.

[0173] Here, the expression “does not bind to β-1 integrin subunits present in immune cells” should not be interpreted as being limited to the meaning that the agonist peptide of the present invention does not form any physical interaction at all with β-1 integrin subunits expressed in immune cells. This means that the binding affinity, binding persistence, and / or functional activation ability for β-1 integrin subunits present in immune cells are substantially low or negligible compared to β-1-based integrin complexes present in epithelial cells. Such relative binding characteristics and functional activation can be easily determined by a person skilled in the art through known binding analysis techniques, cell-based functional analysis, or similar conventional experimental methods.

[0174] The binding characteristics described above mean that even if the same β-1 integrin subunit is formed, it can be selectively recognized due to differences in the composition of the integrin complex formed according to the cell type, its distribution on the cell membrane, the surrounding microenvironment, or its three-dimensional arrangement. Accordingly, the agonist peptide of the present invention can selectively activate only β-1 integrin-mediated signals related to the functional recovery or regeneration of epithelial cells without causing the activation of immune cells, the induction of inflammatory responses, or immune-related side effects.

[0175] In one embodiment, the agonist peptide of the present invention can be used to regenerate damaged extracellular matrix and damaged epithelial tissue.

[0176] The agonist peptide of the present invention can promote the adhesion, survival, proliferation, or migration of epithelial cells by activating β-1 integrin-mediated signaling in a damaged extracellular matrix environment, thereby inducing structural and functional regeneration of damaged epithelial tissue. In particular, normal integrin signaling is often degraded or disrupted in a damaged extracellular matrix, and the agonist peptide of the present invention can act in a manner that complements or enhances β-1 integrin signaling in such an environment.

[0177] In one embodiment, the damaged extracellular matrix is ​​associated with damaged epithelial tissue of an organ selected from the group consisting of the lung, liver, heart, blood vessels, kidney, eye, skin, and intestine. Here, the expression “associated with damaged epithelial tissue” means that structural deformation, degradation, or reconstruction of the extracellular matrix is ​​spatially and functionally linked to damage, inflammation, functional decline, or regeneration processes of the said epithelial tissue.

[0178] Damage to epithelial tissue in the above organs can be caused by inflammation, fibrosis, trauma, ischemia, infection, or degenerative changes, and such damage is often accompanied by structural deformation or functional abnormalities of the extracellular matrix. The agonist peptide of the present invention can be applied to the damaged extracellular matrix and epithelial tissue environment occurring in these various organs to promote the regeneration and functional recovery of epithelial tissue through β-1 integrin-mediated signaling.

[0179] The agonist peptide of the present invention enables a novel therapeutic approach that promotes the regeneration of damaged ECM and epithelial tissue itself, rather than being limited to simple anti-inflammatory or anti-fibrotic effects, by inducing a series of biological changes including increased β-1 integrin exposure, activation of FAK / AKT / mTOR pathways, increased expression of tight junction proteins, and restoration of the epithelial barrier and / or ECM structure. This mechanism is understood to be able to aid in the recovery of epithelial cells and the reorganization of the ECM in situations where the balance of ECM synthesis and degradation is disrupted or the basement membrane is weakened due to inflammation or tissue damage.

[0180] Although not bound by any specific theory, the β-1 integrin agonist peptide of the present invention is understood to be capable of being used for the prevention or treatment of various diseases or conditions requiring the regeneration or functional recovery of damaged epithelial tissue, based on this mechanism of action.

[0181] The agonist peptide of the present invention may be provided in the form of a pharmaceutical composition alone or together with a pharmaceutically acceptable carrier, and may be administered topically, systemically, or in a form included in a delivery vehicle for targeting specific tissues. Such uses are based on, but are not limited to, the selective activation of β-1 integrin provided by the technical concept of the present invention.

[0182] The agonist peptide according to the present invention can be easily manufactured and practiced by a person skilled in the art without requiring excessive experimentation or creative effort, based on the description in this specification and general skills and knowledge known in the art. The agonist peptide of the present invention can be manufactured through solid-phase peptide synthesis, solution-phase synthesis, or similar known peptide synthesis methods, and techniques such as amino acid substitution, modification, or the use of protecting groups can also be implemented through methods widely known to a person skilled in the art.

[0183] The β-1 integrin binding characteristics, selectivity, or biological activity of the agonist peptide according to the present invention can be confirmed through binding analysis, cell-based activity evaluation, or similar biological experiments commonly used in the art, and such evaluation methods are widely known to those skilled in the art.

[0184] 2. Medical Use

[0185] The agonist peptide according to the present invention can be used for the prevention or treatment of diseases characterized by abnormal remodeling of the extracellular matrix.

[0186] In one embodiment, the agonist peptide of the present invention may be used for the prevention or treatment of fibrosis.

[0187] The above fibrosis may include diseases characterized by the deposition of excessive extracellular matrix, abnormal activation of fibroblasts, and destruction of normal tissue structures. In such fibrosis, β-1 integrin-mediated signaling is known to be involved in extracellular matrix-cell interactions, fibroblast activation, and increased tissue stiffness. The agonist peptide of the present invention can contribute to alleviating abnormal extracellular matrix accumulation or promoting the reconstruction of damaged tissue by regulating β-1 integrin signaling.

[0188] For example, the above fibrosis is pulmonary fibrosis.

[0189] For example, the above fibrosis is hepatic fibrosis.

[0190] For example, the above fibrosis is cardiac fibrosis.

[0191] However, it is not limited to this and may include fibrous diseases occurring in the kidneys, skin, or other organs.

[0192] Inflammatory bowel disease can be characterized by a chronic inflammatory response, damage to the intestinal epithelial barrier, changes in the structure of the extracellular matrix, and a decrease in tissue regenerative capacity. In these diseases, β-1 integrin is known to play an important role in the adhesion, migration, and regeneration of intestinal epithelial cells. The agonist peptide of the present invention can promote the recovery of damaged intestinal epithelium and contribute to the improvement of barrier function by activating β-1 integrin-mediated signaling.

[0193] For example, the inflammatory bowel disease mentioned above may be Crohn's disease.

[0194] For example, the above inflammatory bowel disease may be ulcerative colitis.

[0195] However, it is not limited to this and may include chronic enteritis or other diseases involving damage to the intestinal epithelium.

[0196] In one embodiment, the agonist peptide of the present invention may be used for the prevention or treatment of cancer or autoimmune diseases.

[0197] In cancer and autoimmune diseases, the reorganization of the extracellular matrix, changes in cell motility, and abnormalities in integrin-mediated signaling are often observed. In particular, β-1 integrin is known to be involved in the interaction between cancer cells and the extracellular matrix, as well as cancer cell survival and invasion, in the tumor microenvironment, or to influence the interaction between immune cells and tissue cells in autoimmune diseases. The agonist peptide of the present invention can be used to improve the pathological microenvironment or alleviate tissue damage by regulating these β-1 integrin-mediated signals.

[0198] For example, the above cancer may include epithelial-derived cancer, cancer accompanied by a fibrous stroma, or cancer where the reorganization of the tumor microenvironment is important for therapeutic effect.

[0199] For example, the above-mentioned autoimmune disease may include, but is not limited to, autoimmune inflammatory diseases accompanied by tissue damage and abnormal changes in the extracellular matrix.

[0200] 3. Pharmaceutical composition

[0201] One aspect of the present invention provides a pharmaceutical composition comprising an agonist peptide as described herein. The pharmaceutical composition may be used for medical uses as described herein.

[0202] The pharmaceutical composition of the present invention comprises a β-1 integrin agonist peptide as described herein as an active ingredient and may further comprise a pharmaceutically acceptable carrier, diluent, or excipient. The pharmaceutical composition may be formulated to improve the stability, bioavailability, or tissue reachability of the agonist peptide.

[0203] In one embodiment, the pharmaceutically acceptable carrier may be physiological saline, a buffer solution, a glucose solution, Ringer's solution, or a similar aqueous solution, and the excipient may include, but is not limited to, a stabilizer, an isotonic agent, a preservative, a buffer, or an antioxidant.

[0204] The pharmaceutical composition of the present invention can be prepared in various formulations depending on the route of administration. For example, the pharmaceutical composition may be provided as an injectable formulation, a topical formulation, an inhaled formulation, an oral formulation, or a mucosal formulation, and may be implemented as a sustained-release, sustained-release, or targeted-delivery formulation as needed.

[0205] In one embodiment, the pharmaceutical composition of the present invention may be designed to be selectively delivered to damaged epithelial tissue or a site where extracellular matrix is ​​present, and for this purpose may be included in liposomes, nanoparticles, polymer carriers, or other drug delivery systems. Such formulations may help the agonist peptide effectively activate β-1 integrin-mediated signaling and contribute to minimizing action in non-target tissues.

[0206] In one embodiment, the pharmaceutical composition of the present invention may contain a β-1 integrin agonist peptide as described herein in a therapeutically effective amount.

[0207] Here, “therapeutically effective content” means a content of the pharmaceutical composition of the present invention containing a sufficient amount of β-1 integrin agonist peptide to induce one or more of the intended therapeutic effects in a subject, such as promoting the regeneration of damaged epithelial tissue, regulating the remodeling of the extracellular matrix, inhibiting the progression of fibrosis, or alleviating inflammation, on a unit dosage form or unit dose basis, and includes a content that exhibits said effects within an acceptable range of side effects.

[0208] In one embodiment, the unit content of the β-1 integrin agonist peptide included in the pharmaceutical composition of the present invention may be set considering the type of formulation, route of administration, release characteristics, frequency of administration, type and severity of the disease, or characteristics of the therapeutic agent administered in combination, and such differences or variations should be understood as being included within the scope of the technical concept of the present invention.

[0209] In one embodiment, the therapeutically effective content may be provided as an amount contained in a single formulation unit, or may be provided dispersed across multiple formulation units, and may be configured to cumulatively produce a therapeutic effect through repeated administration of the unit formulation.

[0210] The pharmaceutical composition according to the present invention can be easily prepared and practiced by a person skilled in the art without requiring excessive experimentation or creative effort, based on the description provided herein and general skills and knowledge known in the art. Pharmaceutically acceptable carriers, excipients, stabilizers, and delivery systems that may be used in the pharmaceutical composition of the present invention include materials and formulation techniques known in the art, and such differences or variations should be understood to be included within the scope of the technical concept of the present invention.

[0211] 4. Treatment methods

[0212] One aspect of the present invention provides a method comprising administering an agonist peptide as described herein to an individual requiring medical use as described herein.

[0213] A treatment method according to the present invention comprises administering a pharmaceutical composition comprising a β-1 integrin agonist peptide as described in this specification to an individual who has a disease or condition associated with abnormal remodeling of the extracellular matrix, damage to epithelial tissue, or abnormalities in β-1 integrin-mediated signaling.

[0214] In one embodiment, the individual may be a human and may include a non-human mammal as needed. The treatment method of the present invention may be performed for the purpose of preventing a disease, or for the purpose of alleviating symptoms of a disease that has already occurred, inhibiting progression, or restoring tissue function.

[0215] In one embodiment, the treatment method may include administering the β-1 integrin agonist peptide of the present invention in a therapeutically effective amount.

[0216] Here, “therapeutically effective amount” means an amount sufficient to induce one or more therapeutic effects in an individual administered with the agonist peptide, such as promoting the regeneration of damaged epithelial tissue, regulating abnormal remodeling of the extracellular matrix, normalizing β-1 integrin-mediated signaling, or alleviating symptoms of the disease, inhibiting progression, and / or restoring function, and includes an amount that exhibits such effects within an acceptable safety range.

[0217] The above therapeutically effective amount may vary depending on the route of administration, formulation, frequency of administration, type and severity of disease or condition, age, body weight, general condition, or characteristics of concomitantly administered therapeutic agents, and such differences or variations should be understood as being included within the scope of the technical concept of the present invention.

[0218] In addition, the above therapeutically effective amount may be provided as a single dose, or in the form of repeated doses or divided doses.

[0219] The agonist peptide of the present invention can promote the adhesion, survival, proliferation, or migration of epithelial cells by activating β-1 integrin-mediated signals in damaged epithelial tissue or sites where extracellular matrix is ​​present, and as a result, induce tissue regeneration or functional recovery.

[0220] In one embodiment, the treatment method may include administering the pharmaceutical composition of the present invention alone, and in another embodiment, may include administering it in combination with an anti-inflammatory agent, an antifibrotic agent, an immunomodulator, a growth factor, an extracellular matrix modulator, or an anticancer agent. The combination administration may be performed with the same formulation or different formulations, and may be carried out simultaneously, sequentially, or at intervals of time.

[0221] The administration route in the above treatment method may be appropriately selected depending on the type of disease and the treatment site, and may include, for example, local administration, systemic administration, mucosal administration, or delivery to target tissues. In addition, the dosage, frequency of administration, and duration of administration may vary depending on the severity of the disease, treatment response, patient condition, and whether combination therapy is used, and such differences or variations should be understood as being included within the scope of the technical concept of the present invention.

[0222] In one embodiment, the treatment method of the present invention may include repeatedly administering the pharmaceutical composition of the present invention at intervals of at least 72 hours.

[0223] The fact that the pharmaceutical composition of the present invention can be administered at intervals of at least 72 hours reflects that the β-1 integrin agonist peptide of the present invention can induce a relatively sustained biological effect. This administration interval may be set by considering the in vivo stability of the agonist peptide, the persistence of β-1 integrin-mediated signaling, the duration of signal transduction maintenance after receptor activation, or the temporal characteristics of the tissue regeneration process.

[0224] In addition, a minimum administration interval of 72 hours may have the advantage of improving patient compliance, ensuring safety during long-term administration, and facilitating coordination with combination therapy. However, the above administration interval is exemplary, and such an administration schedule should be understood as being included within the scope of the technical concept of the present invention.

[0225] Examples

[0226] Example 1: Peptide Synthesis

[0227] Amino acids and reagents required for synthesis were purchased from GL Biochem and Sigma-Aldrich. Peptides were synthesized from C-terminuses using an automated peptide synthesizer via Fmoc solid-phase peptide synthesis.

[0228] Specifically, a link resin (0.075 mmol / g, 100-200 mesh, 1% DVB crosslinking) having a Fmoc-(9-fluorenylmethoxycarbonyl) protecting group was used. After adding 50 mg of link resin to the synthesizer, the resin was swollen using dimethylformamide (DMF). Subsequently, a 20% piperidine / DMF solution was applied to remove the Fmoc protecting group. In sequence order from the C-terminus, 5, 10, and 5 equivalents of 0.5 M amino acid solution (solvent: DMF), 1.0 M DIPEA solution (solvents: DMF and N-methylpyrrolidone (NMP)), and 0.5 M HBTU solution (solvent: DMF), respectively, were added, and the coupling reaction was performed for 1–2 hours under a nitrogen stream. After each deprotection and coupling step, the body was washed twice with DMF and isopropanol, respectively. Even after the coupling of the final amino acid, a deprotection step was performed to remove residual Fmoc protecting groups.

[0229] The progress of synthesis was confirmed via the ninhydrin test. The synthesized resin was dried with tetrahydrofuran (THF) or dichloromethane (DCM), after which a trifluoroacetic acid (TFA) cleavage cocktail was added at a ratio of approximately 20 mL per 1 g of resin and stirred for about 3 hours. Subsequently, the resin and the cleavage solution containing dissolved peptides were separated by filtration. The solvent was removed from the filtered solution using a rotary evaporator, and then cold ether was added to precipitate the peptides. The resulting precipitate was recovered by centrifugation and repeatedly washed with ether to remove residual TFA. The obtained peptides were dissolved in distilled water and freeze-dried.

[0230] The freeze-dried peptides were separated and purified using high-performance liquid chromatography (HPLC). For the analytical HPLC, a 4.6 mm diameter C18 column was used to elute 0.1% TFA / water and 0.092% TFA / acetonitrile at a flow rate of 1 mL / min for 30 minutes with a gradient of 0–60%, and the detection wavelength was set to 220 nm. Purification was performed using a 2.2 cm diameter C18 column at a flow rate of 20 mL / min under the same solvent composition and detection conditions. The molecular weight of the purified peptides was confirmed by mass spectrometry. Peptides corresponding to the following sequence numbers were synthesized using the above method.

[0231] Sequence No. 1 GLRSKSKKFRRPDIQYPDA

[0232] Sequence No. 2 GLGSKEKKFKKPDIQFPDA

[0233] Sequence No. 3 GLGSKLKKFRHPDIQFPDA

[0234] Other peptides having the amino acid sequence of Formula 1 disclosed in this specification can also be synthesized in the same way as above.

[0235] Example 2: Confirmation of binding force to integrins using the principle of surface plasmon resonance

[0236] The binding characteristics of the peptide according to the present invention to integrin proteins were evaluated using surface plasmon resonance (SPR) analysis. SPR analysis was performed using a Biacore system.

[0237] Specifically, β-1 integrin protein (Thermo Fisher Scientific, Waltham, MA, USA) at a concentration of 5 μg / mL was prepared in sodium acetate buffer at pH 5.0 and immobilized on a dextran-coated sensor chip surface (CM5 chip). The immobilization of the integrin protein was performed using N-hydroxysuccinimide (NHS) and N-ethyl-N′-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDAC) as crosslinking agents. Unreacted active groups on the dextran surface were blocked with a 1 M ethanolamine solution at pH 8.5. 10 mM sodium acetate buffer was used as the immobilization buffer. HBS-EP buffer was used as the driving buffer, and a 50 mM sodium hydroxide (NaOH) solution was used during the regeneration step. To evaluate the binding kinetics of the peptides, the peptides of SEQ ID NO. 1, SEQ ID NO. 2, and SEQ ID NO. 3 were each injected into the sensor chip for 2 minutes at a flow rate of 30 μL / min. Subsequently, a dissociation phase was carried out for 4 minutes.

[0238] SPR analysis was performed using a Biacore T200 system (GE Healthcare Bio-Sciences AB, Uppsala, Sweden), and a CM5 chip surface without immobilized β-1 integrin protein was used as a reference. The binding rate constant (ka), dissociation rate constant (kd), and equilibrium dissociation constant (KD) were calculated by fitting a 1:1 binding model to data obtained by injecting each peptide solution at different concentrations seven times using BIAevaluation software.

[0239] The equilibrium dissociation constants (KD) for each sequence number are summarized in Table 1. As a result of the analysis, the KD value of sequence number 2 was relatively smaller than that of sequence number 1, showing a tendency for higher binding affinity for β-1 integrin, while the KD value of sequence number 3 was observed to be similar to that of sequence number 1.

[0240]

[0241] In addition, the binding affinity of the peptide of SEQ ID NO. 1 to various integrin proteins, including the β-1 integrin subunit (Thermo Fisher Scientific, Waltham, MA, USA), β-4 integrin subunit (Abcam, Cambridge, UK), β-5 integrin subunit (Proteintech, Rosemont, IL, USA), β-6 integrin subunit (LSBio, Seattle, WA, USA), β-7 integrin subunit (Origene, Rockville, MD, USA), and α-3, α-4, α-5, and α-v integrin subunits (Abcam or Santa Cruz Biotechnology), was measured using the same method as above. The results are as follows.

[0242]

[0243] Example 3: Confirmation of the binding affinity of the peptide of SEQ ID No. 1 to the integrin using immunoprecipitation

[0244] The binding affinity of the α and β subunits of the integrin and the peptide SEQ NO. 1 in LL-29 (ATCC, CCL-134) cells was measured by immunoprecipitation. The peptide SEQ NO. 1 labeled with 100 μM biotin was treated to LL-29 cells for 1 hour. Human T-lymphocyte cell lines (HuT-78, ATCC, TIB-161) were used to confirm the cell specificity of the peptide SEQ NO. 1. LL-29 and HuT-78 cells were cultured in 10 cm culture dishes, and after reaching 80-90% confluency, the cells were cultured for 2 hours in a DMEM environment containing 0.5% FBS to restrict nutrition. Biotin-conjugated peptide SEQ NO. 1 at concentrations of 100 μM and 200 μM was treated for 1 hour, respectively.

[0245] The treated cells were lysed in Pierce IP lysis buffer containing a protease inhibitor (Thermo Fisher Scientific, Waltham, MA, USA) on ice for 30 minutes. The protein lysate (2 mg / mL) was precipitated overnight with 2 μg of the integrin primary antibody and then conjugated to A / G plus agarose beads (Santa Cruz Biotechnology) for 3 hours. The beads were washed four times with DPBS (Dulbecco's DPBS), pelleted, and boiled in 2x concentration electrophoresis sample buffer. The immunoprecipitated material was separated using SDS-PAGE and transferred to a nitrocellulose membrane. Anti-GAPDH (input) and anti-IgG (IP) were used as reference markers. A primary antibody against integrin and a secondary antibody conjugated to HRP (Lifespan Technologies, Salt Lake City, UT, USA) were used. It was developed using a chemiluminescent reagent (West-Zol, Intron, Seoul, Korea) with a ChemiDoc imaging system (BioRad, Hercules, CA, USA).

[0246] As a result, as confirmed from Figure 1, the peptide of SEQ ID NO. 1 selectively bound to the β-1 integrin subunit among the integrins in LL-29 cells. However, it did not bind to the β-1 integrin subunit in HuT-78 cells. Therefore, it was found that the peptide of SEQ ID NO. 1 does not bind to the β-1 integrin subunit in immune cells, but binds to the β-1 integrin subunit present in intestinal epithelial cells.

[0247] Example 4: Analysis of Peptide Integrin Binding Characteristics Using Computer Modeling

[0248] Using computer-based structural modeling, the binding characteristics of the peptide according to the present invention to the integrin protein were analyzed.

[0249] SWISS-MODEL is a web-based protein homologous modeling service that includes the steps of structural template identification, alignment of target sequences with template structures, model construction, and model quality evaluation. Since integrins are transmembrane heterodimers composed of two subunits, α and β, the sequences of the α and β subunits were entered simultaneously using the "Add hetero target" option. After entering the sequences, appropriate templates were searched from the SWISS-MODEL Template Library (SMTL), which is derived from the Protein Data Bank (PDB) containing experimentally determined protein structures. The top 50 templates were selected based on sequence coverage and sequence similarity, and these templates were sorted according to their estimated quality based on the Global Model Quality Estimate (GMQE). Sequence similarity was calculated based on a normalized BLOSUM62 substitution matrix. The top-ranked templates were selected and used for final model construction. The constructed model underwent preprocessing steps such as bond order assignment, addition of hydrogen atoms, generation of disulfide bonds, and supplementation of missing side chains; subsequently, structural strain was relieved and atomic arrangements were fine-tuned through constrained minimization. Structural models of peptides binding to αβ integrins were generated using AlphaFold2. Among the generated peptide models, the one with a radius of gyration of 9.22 Å was selected for subsequent analysis. The Adaptive Poisson-Boltzmann Solver (APBS) was used to calculate the electrical properties of the peptide and integrin complexes and to interpret the continuous electrochemical equations.

[0250] To perform molecular docking to investigate the interaction between the peptide and the integrin, the integrin protein structure was prepared by adding missing hydrogens while keeping the binding order fixed, optimizing hydrogen bonding based on the protonation state at pH 7.0, and performing constraint minimization. All hydrogens were added to the ligand peptide, and a canonical charge (-1) was assigned based on the charge state under pH 7.0 conditions. Molecular docking was performed under default parameter conditions using the Pharmulator (massive docking module). After preparing the receptor by setting up a grid box containing the center of the active site, docking was performed by allowing flexible sampling of the ligand.

[0251] As a result, Figure 2 shows that in an integrin dimer in which an α-v subunit and a β-1 subunit are combined, the anions ASP244, LEU245, and SER247 residues, which are shown in blue and are present in the β-1 integrin subunit, are located close to and bind to the cationic amino acids (Arginine, Lysine, Histidine) that make up the peptide.

[0252] Poses with high docking scores were selected and used as initial structures for subsequent Steered Molecular Dynamics (SMD) simulations. SMD simulations were performed to evaluate the binding strength between the integrin α-5 and β-1 subunits and the peptide.

[0253] Figure 3 shows the force profiles calculated from SMD simulations for each peptide sequence. The peak force of peptide SEQ No. 1 was observed to be approximately 650 kJ / mol / nm. In comparison, the peak force of peptide SEQ No. 2 was measured to be approximately 1,100 kJ / mol / nm, representing an increase of about 69% compared to peptide SEQ No. 1. Additionally, the peak force of peptide SEQ No. 3 was observed to be approximately 1,000 kJ / mol / nm, showing an increase of about 54% compared to peptide SEQ No. 1. These results suggest that peptide SEQ No. 2 and peptide SEQ No. 3 are likely to form stronger interactions with complexes containing integrins α-5 and β-1 compared to peptide SEQ No. 1.

[0254] Example 5: Evaluation of Antifibrotic Effect

[0255] To evaluate the antifibrotic effect, human lung epithelial cell line A549 cells were used. A549 cells were purchased from the American Type Culture Collection (ATCC) and cultured in Dulbecco's Modified Eagle's Medium (DMEM) containing 10% fetal bovine serum (FBS) and 1× penicillin-streptomycin solution at 37°C under humid 5% CO2 conditions.

[0256] 1 x 10⁻⁶ A549 cells in DMEM containing 10% FBS 5After diluting to a concentration of cells / mL, the cells were seeded into a 6-well plate. When the cells reached approximately 60-70% confluence, the medium was replaced with 2 mL of serum-free DMEM prior to treatment and cultured for 24 hours. Subsequently, the cells were treated with bleomycin (BLM) at a concentration of 50 μg / mL and co-treated with the peptides of SEQ ID NO. 1, SEQ ID NO. 2, or SEQ ID NO. 3 at a concentration of 100 μM each, followed by 3 days of culture. A group treated with BLM alone was used as a control.

[0257] After treatment, total RNA was extracted from the cells using the TRIzol reagent according to the manufacturer's instructions. 1 μg of the isolated RNA was used for cDNA synthesis using the Maxima First Strand cDNA Synthesis Kit and the GeneAmp PCR System 9700. Quantitative real-time reverse transcription-polymerase chain reaction (qRT-PCR) was performed using the SYBR™ Green PCR Master Mix and the QuantStudio 3 Real-Time PCR System. The reaction conditions consisted of an initial denaturation at 95°C for 5 minutes, followed by 40 cycles of 30 seconds at 95°C, 40 seconds at 60°C, and 30 seconds at 72°C; the fluorescence signal was measured at each cycle to calculate the threshold cycle (Ct). The primer sequences used are shown in Table 3 (Sequence Nos. 5 to 10).

[0258]

[0259] The relative quantification of the qRT-PCR analysis results was evaluated by comparing the Ct values ​​of the test substance treatment group with the control group. The relative expression value was calculated according to the following formula.

[0260]

[0261] The untreated group was used as the control group, and GAPDH was used as the housekeeping gene.

[0262] As can be seen from the qRT-PCR analysis results in Figures 4 and 5, in the group treated with bleomycin (BLM) alone, a significant increase in mRNA expression of Col1a1 and Fibronectin (FN1) genes was observed compared to the untreated control group. On the other hand, in the group treated with the agonist peptide of the present invention in combination with BLM, this increase in fibrosis-related gene expression tended to be mitigated.

[0263] Specifically, a tendency for Col1a1 and FN1 expression to decrease was observed in the group treated with SEQ ID NO. 1 compared to the group treated with BLM alone, and the inhibitory effect on the expression of the corresponding genes was more pronounced in the groups treated with SEQ ID NO. 2 and SEQ ID NO. 3. In particular, the lowest relative mRNA expression levels of Col1a1 and FN1 were observed in the group treated with SEQ ID NO. 3, suggesting the possibility of more effectively reducing molecular indicators related to fibrosis induced by BLM.

[0264] These results show that the agonist peptide according to the present invention can regulate gene expression related to extracellular matrix components under conditions of fibrosis induction, and exhibits a trend consistent with the previously identified integrin binding characteristics and structure-based analysis results. Therefore, it suggests that the agonist peptide of the present invention can exhibit an antifibrotic effect by alleviating pathological gene expression associated with fibrosis.

[0265]

[0266] Example 6: Measurement of the co-localization of the peptide of SEQ No. 1 and the β-1 integrin subunit in intestinal tissue induced by inflammatory bowel disease

[0267] To confirm the binding of the peptide of SEQ NO. 1 to the β-1 integrin subunit in colon tissues induced with colitis, the peptide of SEQ NO. 1 labeled with Cy 5.5 or the mismatch peptide (SEQ NO. 4: KVIWVGAHAGKYAVSRPFT) was subcutaneously injected into mice with acutely induced colitis via DSS. C57BL / 6 mice consumed drinking water containing 3% (w / v) DSS (dextran sulfate sodium, 36-50 kDa, MP Biomedicals, Solon, OH, USA) for 6 days. Normal mice consumed plain water for 6 days. The animals were randomized into four groups as follows: (a) 30 mg / kg of the Cy 5.5-labeled peptide of SEQ NO. 1 to normal mice; (b) 30 mg / kg of the Cy 5.5-labeled peptide of SEQ NO. 1 to DSS-induced colitis mice; (c) 30 mg / kg Cy5.5-labeled mismatch peptide in normal mice; (d) 30 mg / kg Cy5.5-labeled mismatch peptide in DSS-induced colitis mice.

[0268] After 7 days, DSS-induced colitis mice or normal mice were administered a single subcutaneous injection of 30 mg / kg Cy5.5-labeled peptide of SEQ No. 1 or 30 mg / kg Cy5.5-labeled mismatch peptide, and sacrificed 1 hour later. Colon tissues were fixed overnight in 4% paraformede and washed three times with PBS. After perfusion with sucrose, the tissues were inserted into Tissue-tek OCT blocks (Sakura Finetec) while frozen. The tissues contained in the OCTs were cut into frozen sections, and the samples were blocked with 5% BSA in DPBS. Each diluted anti-β1 integrin subunit antibody, secondary antibody, and DAPI staining was performed. The stained tissues were stained using a confocal microscope (LSM 700, Zeiss), and the images were analyzed using Zen Black software (Carl Zeiss).

[0269] As a result, in Figure 6, β-1 integrin subunits (green) were observed more frequently in intestinal tissue with induced colitis than in normal intestinal tissue. This indicates that the exposure of β-1 integrin subunits increases as the intestinal tissue is damaged. Additionally, the peptide of SEQ ID NO. 1 (red) was observed to be distributed more frequently (yellow) at the same location as the β-1 integrin subunit in intestinal tissue with induced colitis than in β-1 integrin subunits in normal tissue. This indicates that the peptide of SEQ ID NO. 1 binds more frequently to the β-1 integrin subunits in the intestinal tissue with induced colitis.

[0270]

[0271] Example 7: Verification of signal transmission via integrin

[0272] To investigate the effect of the peptide of SEQ ID NO. 1 on the activation of the FAK-AKT-mTOR pathway, a downstream signal of integrins, FAK inhibitor (PF-573228) was administered to HT-29 cells for evaluation. HT-29 cells were cultured in 60 mm culture dishes at a density of 2.5 x 10⁵ cells / cm² and inhibited in growth by incubating in serum-free medium for 20 hours. After replacing the medium, 100 μM of the peptide of SEQ ID NO. 1, 10 μM of PF-573228, and 10 μM of PF-573228 and 100 μM of the peptide of SEQ ID NO. 1 were administered for 1 hour. 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 inhibitors and phosphatase inhibitors. Proteins were quantified using the BCA protein assay, and the expression levels of p-FAK, FAK, p-AKT, AKT, p-mTOR, mTOR, and GAPDH proteins were confirmed by Western blot. Equal amounts of the sample were loaded onto an 8% SDS-PAGE gel along with a size marker and subjected to electrophoresis for approximately 2 hours, after which the samples were transferred to a nitrocellulose membrane. The transferred membrane was blocked with 5% skim milk for 1 hour, and the primary antibody was incubated at a 1:1000 ratio overnight.Primary antibodies are anti-p-FAK(Y397) (3283S, Cell signaling), anti-total-FAK (13009S, Cell signaling), anti-p-AKT(S473) (9271S, Cell signaling), anti-total-AKT (9272S, Cell signaling), anti-p-mTOR(S2448) (5536S, Cell signaling), anti-total antibodies mTOR (2983S, Cell signaling) and anti-GAPDH (2118S, Cell signaling) were used. Afterward, the membrane was washed with TBST containing 0.1% tween-20, and HRP-attached secondary antibodies, anti-Rabbit (Bethyl, A120-101P) and anti-Mouse (Bethyl, A90-116P), were reacted at a ratio of 1:5000 for 1 hour, after which chemiluminescence was confirmed using an ECL substrate.

[0273] As a result, as shown in Figure 7, p-FAK, p-AKT, and p-mTOR increased due to the peptide of SEQ ID NO. 1, but decreased due to the FAK inhibitor. When the FAK inhibitor and the peptide of SEQ ID NO. 1 were treated simultaneously, p-FAK, p-AKT, and p-mTOR also decreased. It was found that when FAK is inhibited, its downstream signals, AKT and mTOR, also decrease. This implies that the peptide of SEQ ID NO. 1 transmits a signal into the cell through FAK, a downstream signal of integrin.

[0274] When the peptide of SEQ ID NO. 1 was treated once, the duration of intracellular signal transduction via integrins and regeneration markers was measured. A549 cells (ATCC) were placed in a 60 mm culture dish at a rate of 1 x 10⁶ 5The cells were cultured at a density of / cm². After 7 days, the cells were treated with 5 μg / ml bleomycin (BLM) or untreated, and then treated with 100 μM of the peptide of SEQ ID NO. 1 for up to 72 hours and cultured. The production of focal adhesion kinase (FAK) and protein kinase B (AKT), which are the cell's initial signaling pathways, and tight junction markers E-cadherin, ZO-1, and Occludin as regeneration markers were measured by Western blot. The antibodies used were anti-p-FAK(Y397) (3283S, Cell signaling), anti-total-FAK (13009S, Cell signaling), anti-p-AKT(S473) (9271S, Cell signaling), anti-total-AKT (9272S, Cell signaling), anti-Occludin (33-1500, Thermo), anti-ZO-1 (33-9100, Thermo), anti-E-cadherin (3195S, Cell signaling), anti-β1 integrin (14-0299-82, Thermo), anti-GAPDH (2118S, Cell signaling), and anti-beta-actin (3700S, Cell signaling). GAPDH and beta-actin were used as reference markers.

[0275] As a result, in Figures 8 and 9, A549 cells treated only with bleomycin (BLM) maintained a constant p-FAK activity of approximately 1.0 for 72 hours. In contrast, when the peptide of SEQ ID NO. 1 was treated together with bleomycin (BLM), the level of p-FAK increased for more than 50 hours. Additionally, the level of p-AKT was slightly higher for 48 hours compared to cells treated only with BLM.

[0276] In Figures 10 and 11, when only the peptide of SEQ ID NO. 1 was treated once, the production of E-Cadherin, ZO-1, and Occludin increased for 72 hours. In A549 cells treated only with bleomycin, the production of E-Cadherin, ZO-1, and Occludin did not increase. On the other hand, when the peptide of SEQ ID NO. 1 and bleomycin were treated together, the production of tight junction proteins E-Cadherin, ZO-1, and Occludin increased for 72 hours. Even when cell damage was induced by bleomycin, simultaneous treatment with the peptide of SEQ ID NO. 1 initially increased the phosphorylation of FAK and AKT, and subsequently increased the production of tight junction proteins that contribute to the regeneration and recovery of epithelial cells. Given that tight junction proteins increase for 72 hours after a single administration of the peptide of SEQ ID NO. 1, the administration interval of the peptide of SEQ ID NO. 1 in animals or humans can be determined to be at least 72 hours.

[0277]

[0278] Example 8: Confirmation of therapeutic effect in an animal model of chronic inflammatory bowel disease

[0279] The chronic colitis mouse model was constructed according to a protocol approved in accordance with the guidelines of the Institutional Animal Care and Use Committee (IACUC) of Chungbuk National University (Approval No. # CBNUA-2196-23-02). The animals used were 8-week-old C57BL / 6 mice purchased from Central Lab. Animal Inc. (Seoul, South Korea) and housed at the facility of the Department of Experimental Animal Resources at Chungbuk National University during the experiment. To induce chronic inflammatory bowel disease, C57BL / 6 mice were fed drinking water supplemented with 2% (w / v) DSS for 7 days, followed by plain water without DSS for 14 days, and this process was repeated a total of 3 times (Fig. 12). The animals were randomly assigned to the following 7 groups: (a) normal; (b) 2% DSS-induced colitis group (defect); (c) positive control group with 10 mg / kg tofacitinib + DSS (tofacitinib 10 mg / kg); (d) 30 mg / kg SEQ ID NO. 1 peptide + DSS group (SEQ ID NO. 1 peptide 30 mg / kg); (e) 60 mg / kg SEQ ID NO. 1 peptide + DSS group (SEQ ID NO. 1 peptide 60 mg / kg); (f) 90 mg / kg SEQ ID NO. 1 peptide + DSS group (NP-201 90 mg / kg); and (g) 120 mg / kg SEQ ID NO. 1 peptide + DSS group (SEQ ID NO. 1 peptide 120 mg / kg). Tofacitinib was administered orally, and SEQ ID NO. 1 peptide was injected subcutaneously once daily into DSS-induced mice from day 42 to day 62. To evaluate the therapeutic effect, intestinal length, histological observation of the colon, and calprotectin, a biomarker for assessing the degree of treatment in stool and blood, were analyzed by ELISA.

[0280] The colon was washed with phosphate-buffered saline (PBS) and rolled into a Swiss roll. The Swiss roll-shaped colon was fixed in 10% buffered formalin, while other sections were stored on dry ice for later analysis. Histology was performed on 5 μm thick sections embedded in paraffin by staining with hematoxylin and eosin.

[0281] To analyze biomarkers contained in blood and feces, blood and feces were collected upon mouse sacrifice. Blood was collected using an EDTA-coated collection tube (366643, BD Biosciences). The tubes were then centrifuged in a cold centrifuge at 1,500 xg for 10 minutes at 4°C. The separated plasma was mixed with a protease inhibitor (1861279, Thermo Fisher Scientific) and a phosphatase inhibitor cocktail 2 (P5726, Sigma Aldrich) according to the manufacturer's instructions. Blood proteins separated from the stomach were quantified using a BCA protein assay kit (23225, Thermo Fisher Scientific). Fecal pellets were collected, weighed, and frozen immediately. The night before analysis, the pellet was left at 4°C with 1 mL of stool extraction buffer (0.1 mol / L Tris, 0.15 mol / L NaCl, 1.0 mol / L urea, 10 mmol / L CaCl2, 0.1 mol / L citric acid, 5 g / L bovine serum albumin, 0.25 mmol / L thimerosal, pH 8; Hycult Biotech, Wayne, PA). The pellet was crushed using a shaker and ceramic beads, and the supernatant was collected. Dilution with the extraction buffer was calculated assuming a density of 1 mg / mL, and the results were calculated based on the weight of the stool. Calprotectin in isolated stool and plasma was analyzed as a biomarker for evaluating IBD treatment using an ELISA kit (ab263885, Abcam) according to the manufacturer's manual.

[0282] As a result, as shown in Figure 13, the length of the intestine increased as the dosage of the peptide of SEQ ID NO. 1 increased. The length of the large intestine in normal mice was approximately 95 mm, and the length of the large intestine in the defect group was approximately 70 mm. In the group treated with the peptide of SEQ ID NO. 1, it increased to approximately 85 mm or more. Tofacitinib (10 mg / kg) increased the length of the large intestine to approximately 80 mm, which was equivalent to the lowest dose of the peptide of SEQ ID NO. 1. There was no significant difference between the groups treated with the peptide of SEQ ID NO. 1, which demonstrates that it is effective even at a low dose of 30 mg / kg of the peptide of SEQ ID NO. 1.

[0283] Figure 14 shows the results of histological analysis of colon tissues from each treatment group. In the normal group, distinct intestinal crypts arranged with absorptive cells, goblet cells, and lamina propria were observed. In the defect group, the epithelial layer was destroyed, the intestinal crypts were damaged, and inflammatory cells were observed to have infiltrated the mucosal and submucosal layers. The tofacitinib group succeeded in increasing colon length to some extent but failed to achieve regeneration similar to normal colon structure. Conversely, the peptide of SEQ ID NO. 1 not only increased colon length but also demonstrated regeneration of colon structure. Distinct crypt formation was observed in the mucosal layer, and unique goblet cells and submucosal lamina propria were identified. Inflammatory cell infiltration was minimal, and the absorptive cells in the epithelial layer were arranged normally. Figure 15 shows the concentration of calprotectin present in stool and plasma. The concentrations of calprotectin in stool and plasma were increased in the defect group compared to the normal group. The concentration of calprotectin was significantly reduced in both the peptide of SEQ ID NO. 1 and the tofacitinib group, and the concentration of calprotectin was reduced to a similar level at all doses of the peptide of SEQ ID NO. 1.

[0284]

[0285] Example 9: Measurement of the co-localization of the peptide of SEQ ID No. 1 and the β-1 integrin subunit in lung tissue with pulmonary fibrosis

[0286] To confirm the binding of the peptide of SEQ ID NO. 1 and the β-1 integrin subunit in lung tissue induced with pulmonary fibrosis, the peptide of SEQ ID NO. 1 labeled with Cy 5.5 was subcutaneously injected into mice with bleomycin-induced pulmonary fibrosis. The method of inducing pulmonary fibrosis was the same as in Example 8. The administration groups were as follows: (a) 30 mg / kg of the peptide of SEQ ID NO. 1 labeled with Cy 5.5 to normal mice; (b) 30 mg / kg of the peptide of SEQ ID NO. 1 labeled with Cy 5.5 to mice with bleomycin-induced pulmonary fibrosis.

[0287] Bleomycin-induced pulmonary fibrosis mice or normal mice were administered a single subcutaneous injection of 30 mg / kg of the Cy5.5-labeled peptide of SEQ ID NO. 1, and sacrificed 1 hour later. Lung tissues were fixed overnight in 4% paraformede and washed three times with PBS. After perfusion with sucrose, the tissues were inserted into Tissue-tek OCT blocks (Sakura Finetec) while frozen. The tissues contained in the OCTs were cut into frozen sections, and the samples were blocked with 5% BSA in DPBS. Each diluted anti-β1 integrin subunit antibody, secondary antibody, and DAPI staining was performed. The stained tissues were stained using a confocal microscope (LSM 700, Zeiss), and the images were analyzed using Zen Black software (Carl Zeiss).

[0288] As a result, in Figure 16, β-1 integrin subunits (green) were observed more frequently in lung tissue with fibrosis than in normal lung tissue. This indicates that the exposure of β-1 integrin subunits increases as the lung tissue becomes damaged. Additionally, the peptide of SEQ ID NO. 1 (red) was observed to be distributed more frequently (yellow) at the same locations as β-1 integrin subunits in lung tissue with fibrosis than in normal tissue. This means that the peptide of SEQ ID NO. 1 binds more frequently to β-1 integrin subunits in lung tissue with pulmonary fibrosis.

[0289]

[0290] Example 10: Confirmation of therapeutic effect in an animal model of bleomycin-induced lung disease

[0291] To confirm the therapeutic effect of the peptide according to the present invention on fibrosis at the animal level, an animal model in which lung disease was induced with bleomycin was used. C57BL / 6J mice (male, 7 weeks) 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 bleomycin sulfate (1.45 mg / kg, MBcell, CA, USA) was injected once into the trachea with a total volume of 50 µl, while the control group was injected into the bronchi with saline (day 0). On day 7, the groups were separated into 8 mice per group. PBS (NT) and the peptide of SEQ No. 1 were administered subcutaneously at doses of 30, 60, and 90 mg / kg three times a week, while nintedanib (Fluorochem Ref = 050741, 100 mg / kg) was administered orally daily. The peptide of SEQ No. 1 was dissolved in sterile water for injection and administered, while nintedanib was mixed with water containing 0.5% hydroxyethylcellulose (Sigma; lot BCBV5033) and 0.01% Tween 80 (Sigma; lot BCBR6211V) and administered. During the 21 days of drug administration, body weight was measured every two days and other clinical symptoms were checked; the experimental protocol is shown in Fig. 17. Mice were sacrificed with Isoflurane on day 22, after which lung tissues were resected and histological analysis was performed. After injecting paraffin into the lungs and preparing 5 μm paraffin sections, Sirius Red staining and hematoxylin / phloxin staining were performed to evaluate the degree of pulmonary fibrosis and inflammation, and the degree of inflammation and fibrosis was observed under a microscope, as shown in Fig. 18.

[0292] As a result, in the group that induced lung disease with bleomycin and administered PBS (NT), significant deposition of fibrous tissue was observed in the lungs, and in the group administered nintedanib, although there was some healing effect, significant deposited fibrous tissue was still observed. However, in the group administered the peptide of SEQ ID NO. 1, it was confirmed that fibrosis was significantly reduced compared to the NT group and the group administered nintedanib.

[0293] To quantify the degree of fibrosis, the area of ​​red (fibrotic portion) was measured in samples stained with Sirius red. As a result, compared to NT in Figure 19, the group administered the peptide of SEQ ID NO. 1 at a dose of 30 mg / kg showed a significant decrease. Compared to the nintedanib treatment group, the average value was found to be lower in all groups administered the peptide of SEQ ID NO. 1.

[0294] To demonstrate the regenerative effect of the peptide of SEQ ID NO. 1, the expression of the tight junction protein Claudin-3 in the lung tissue of pulmonary fibrosis mice was measured by immunohistochemistry. Figure 20 shows the results of observing Claudin-3 expression by immunohistochemistry and measuring fluorescence intensity. The peptide of SEQ ID NO. 1 significantly increased the expression of Claudin-3 compared to NT starting at 30 mg / kg, and significantly increased it compared to nintedanib starting at 60 mg / kg. Therefore, it was found that the peptide of SEQ ID NO. 1 is effective not only in reducing fibrosis but also in regenerating damaged lung tissue.

Claims

1. A β-1 integrin subunit agonist peptide comprising the amino acid sequence of the following chemical formula or an amino acid sequence that is at least 85% identical thereto. GLX1SX2X3X4X5FX6X7PDIQX8PDA In the above formula, X1 is arginine or glycine, X3 is serine, glutamic acid or lysine, X8 is tyrosine or phenylalanine, and X2, X4, X5, X6 and X7 are each independently cationic amino acids selected from the group consisting of arginine, histidine and lysine.

2. The peptide according to claim 1, comprising the amino acid sequence of SEQ ID NO. 1 or an amino acid sequence comprising one or two amino acid substitutions compared therewith.

3. The peptide according to claim 1, comprising the amino acid sequence of SEQ ID NO. 2 or an amino acid sequence comprising one or two amino acid substitutions compared therewith.

4. The peptide according to claim 1, comprising the amino acid sequence of SEQ ID NO. 3 or an amino acid sequence comprising one or two amino acid substitutions compared therewith.

5. A peptide according to claim 1 or 2, wherein the β-1 integrin subunit forms a complex with an α-1, α-2, α-3, α-5, α-6, α-v, α-7, α-8, α-10, or α-11 integrin subunit.

6. A peptide according to claim 1 or 2 that selectively binds to a β-1 integrin subunit compared to a β-3, β-4, β-5, β-6, β-7, α-3, α-4, α-5, or α-v integrin subunit.

7. The peptide according to claim 1 or 2, which does not bind to a β-1 integrin subunit present in immune cells but binds to a β-1 integrin subunit present in epithelial cells.

8. A peptide that can be used to regenerate damaged extracellular matrix and damaged epithelial tissue in accordance with claim 1 or 2.

9. A peptide according to claim 1 or 2, wherein the damaged extracellular matrix is ​​associated with damaged epithelial tissue of an organ selected from the group consisting of lungs, liver, heart, blood vessels, kidneys, eyes, skin, and intestines.

10. A pharmaceutical composition for use in the prevention or treatment of a disease characterized by abnormal remodeling of the extracellular matrix, comprising a peptide according to claim 1 or 2.

11. A pharmaceutical composition for use in the prevention or treatment of fibrosis, according to claim 10.

12. A pharmaceutical composition according to claim 11, wherein the fibrosis is selected from the group consisting of pulmonary fibrosis, hepatic fibrosis, renal fibrosis, and cardiac fibrosis.

13. A pharmaceutical composition for use in the treatment of inflammatory bowel disease, according to claim 10.

14. A pharmaceutical composition according to claim 13, wherein the inflammatory bowel disease is Crohn's disease or ulcerative colitis.

15. A pharmaceutical composition for use in the prevention or treatment of cancer or autoimmune diseases, according to claim 10.

16. A pharmaceutical composition according to claim 11 or 13, wherein the peptide is administered at intervals of at least 72 hours.

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