Beta-1 integrin agonist peptide for treating vascular fibrosis diseases

Abstract

The present invention relates to a pharmaceutical composition to be used in the prevention or treatment of diseases involving vascular fibrosis. Specifically, the pharmaceutical composition according to the present invention contains a peptide that reduces fibrosis of perivascular tissue or the extracellular matrix constituting the vascular wall, and is useful for preventing or treating diseases involving vascular fibrosis, particularly pulmonary arterial hypertension.

Description

Beta-1 integrin agonist peptide for the treatment of vascular fibrosis

[0001] The present invention relates to a pharmaceutical composition for use in the prevention or treatment of diseases accompanied by vascular fibrosis. Specifically, the pharmaceutical composition according to the present invention comprises a peptide that reduces fibrosis of the extracellular matrix constituting the blood vessel wall or the perivascular tissue, and is useful for the prevention or treatment of diseases accompanied by vascular fibrosis, particularly pulmonary hypertension.

[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] Vascular fibrosis is a pathological process in which fibrous connective tissue accumulates within vascular tissue. It is a widespread pathological phenomenon that occurs during vascular remodeling in various cardiovascular diseases such as atherosclerosis, hypertension, diabetic vasculopathy, and heart failure. This fibrosis leads to stiffening of the blood vessel walls and endothelial dysfunction, causing hemodynamic imbalances and ultimately reducing vascular elasticity, which can result in serious vascular diseases.

[0004] The pulmonary artery is a blood vessel capable of withstanding high-intensity pressure and wall stress, and accommodating a large volume of blood. In pulmonary hypertension, peripheral pulmonary blood vessels undergo remodeling, causing the vessel walls to gradually thicken and the vessel diameter to decrease over time. These changes are driven by vasoconstriction, inflammation, thrombus formation, hypertrophy and proliferation of vascular smooth muscle, and fibrosis.

[0005] Currently, Renin-Angiotensin System (RAS) inhibitors, antioxidants, anti-inflammatory agents, and anti-fibrotic drugs are being studied as treatments for vascular fibrosis, but effective treatment strategies are still lacking. In particular, existing treatments fail to completely inhibit the progression of vascular fibrosis or raise concerns about side effects with long-term use, highlighting the need for the development of new therapies.

[0006] β-1 integrins are known to be involved in vascular cells recognizing changes in the mechanical environment caused by structural changes or rearrangements of the ECM. These changes may be associated with the clustering of integrins and the formation of adhesion complexes at the cell membrane level, which have been reported to be accompanied by the activation of signaling factors such as FAK, Src, and AKT (Hynes, RO, Cell, 2002).

[0007] Such early integrin-mediated signaling may be associated with changes in cytoskeletal composition and dynamic reorganization of adhesion complexes in vascular endothelial cells and / or vascular smooth muscle cells, which is understood to be linked to the process by which cells adapt to an altered ECM environment to maintain their adhesion state or change their alignment state (Humphrey et al., Nat Rev Mol Cell Biol, 2014).

[0008] In addition, β-1 integrin-mediated signaling is known to be associated with signaling pathways related to the survival of vascular endothelial cells or the proliferation, migration, and contractile characteristics of vascular smooth muscle cells, and the activation of these signaling pathways may be associated with changes in cell behavior observed during the vascular remodeling process.

[0009] Meanwhile, changes in the composition and distribution of ECM constituent proteins, such as collagen, elastin, and fibronectin, are generally observed during the vascular remodeling process, and these changes in the ECM environment can, in turn, affect integrin-mediated signaling. Accordingly, changes in the state of vascular cells and ECM reorganization are understood as interrelated phenomena, and β-1 integrin has been reported to function as one of the molecules mediating the interaction between the ECM and cells in this process (Hynes, RO, Cell, 2002; Humphrey et al., Nat Rev Mol Cell Biol, 2014).

[0010] 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.

[0011] Prior art literature

[0012] Non-patent literature

[0013] (Non-patent literature 1) Hynes, RO, Cell, 2002.

[0014] (Non-patent literature 2) Humphrey et al., Nat Rev Mol Cell Biol, 2014.

[0015] The objective of the present invention is to provide a therapeutically useful beta-1 integrin subunit agonist peptide.

[0016] The objective of the present invention is to provide a peptide that can be therapeutically utilized for diseases associated with vascular fibrosis, particularly pathological conditions associated with pulmonary arterial hypertension.

[0017] Another objective of the present invention is to provide a pharmaceutical composition that can be used for the prevention or treatment of diseases characterized by abnormal remodeling of the extracellular matrix containing the peptide, for example, pulmonary arterial hypertension.

[0018] 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., pulmonary hypertension, comprising the step of administering the peptide.

[0019] Conventionally, therapeutic strategies for hypertensive diseases associated with integrin-mediated signaling have proposed approaches aimed at regulating cellular responses related to vascular remodeling by inhibiting signaling pathways involved in vasoconstriction or increased vascular resistance, or by inhibiting specific integrin functions. However, these approaches have limitations in that they fail to adequately consider structural abnormalities of the vascular ECM and the complex changes in cellular signaling associated with them.

[0020] In contrast, the inventors of the present invention have focused on the potential to regulate cell signaling and cell responses under abnormal vascular ECM environments through an agonist approach that selectively activates β-1 integrin subunits.

[0021] Based on this concept, the inventors have developed a peptide acting as an integrin agonist that can be utilized for the prevention or treatment of hypertensive diseases associated with abnormal vascular ECM remodeling and the accompanying imbalance of β-1 integrin-mediated signaling. The peptide of the present invention can bind to β-1 integrin subunits and induce intracellular responses associated with downstream signaling pathways including Focal adhesion kinase (FAK) and Protein kinase B (AKT), and the regulation of such signaling can be associated with the functional state of vascular endothelial cells and vascular smooth muscle cells.

[0022] One aspect of the present invention provides a β-1 integrin subunit agonist peptide of a novel amino acid sequence.

[0023] The numbered items below exemplify some of the embodiments described in this specification.

[0024] 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.

[0025] GLX1SX2X3X4X5FX6X7PDIQX8PDA

[0026] 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.

[0027] 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.

[0028] 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.

[0029] 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.

[0030] 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.

[0031] 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.

[0032] 7. A peptide that, in any one of the first to sixth embodiments, does not bind to a β-1 integrin subunit present in immune cells but binds to a β-1 integrin subunit present in vascular endothelial cells or vascular smooth muscle cells.

[0033] 8. A peptide that can be used in any one of the first to seventh embodiments to regenerate a damaged or pathologically remodeled extracellular matrix, maintain the adhesion, survival, and function of vascular endothelial cells, and / or alleviate abnormal proliferation or stiffness of vascular smooth muscle cells.

[0034] 9. A peptide in any one of the first to eighth embodiments, wherein the damaged or pathologically remodeled extracellular matrix is ​​associated with vascular tissue or epithelial tissue adjacent to a blood vessel of an organ selected from the group consisting of lung, liver, heart, blood vessel, kidney, eye, skin, and intestine.

[0035] One aspect of the present invention provides a pharmaceutical composition comprising a β-1 integrin subunit agonist peptide as described herein.

[0036] 10. A pharmaceutical composition for use in the prevention or treatment of diseases accompanied by vascular fibrosis, comprising a β-1 integrin subunit agonist peptide having an amino acid sequence of the following chemical formula or an amino acid sequence that is 85% or more identical thereto.

[0037] GLX1SX2X3X4X5FX6X7PDIQX8PDA

[0038] 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.

[0039] 11. A pharmaceutical composition in which, in the above 10th embodiment, the agonist peptide comprises the amino acid sequence of SEQ ID NO. 1 or an amino acid sequence comprising one or two amino acid substitutions compared therewith.

[0040] 12. A pharmaceutical composition in which, in the above 10th embodiment, the agonist peptide comprises the amino acid sequence of SEQ ID NO. 2 or an amino acid sequence comprising one or two amino acid substitutions compared therewith.

[0041] 13. A pharmaceutical composition in which, in the above 10th embodiment, the agonist peptide comprises the amino acid sequence of SEQ ID NO. 3 or an amino acid sequence comprising one or two amino acid substitutions compared therewith.

[0042] 14. A pharmaceutical composition in which, in any one of the 10th to 13th 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.

[0043] 15. A pharmaceutical composition in which, in any one of the 10th to 14th embodiments above, the agonist peptide 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.

[0044] 16. A pharmaceutical composition in which, in any one of the 10th to 15th embodiments above, the agonist peptide does not bind to a β-1 integrin subunit present in immune cells, but binds to a β-1 integrin subunit present in vascular endothelial cells or vascular smooth muscle cells.

[0045] 17. A pharmaceutical composition in which, in any one of the 10th to 16th embodiments above, the agonist peptide can be used to regenerate a damaged or pathologically remodeled extracellular matrix, maintain the adhesion, survival, and function of vascular endothelial cells, and / or alleviate abnormal proliferation or stiffness of vascular smooth muscle cells.

[0046] 18. A pharmaceutical composition in which the damaged or pathologically remodeled extracellular matrix of the above 17th embodiment is associated with vascular tissue or epithelial tissue adjacent to blood vessels of an organ selected from the group consisting of lung, liver, heart, blood vessels, kidney, eye, skin, and intestine.

[0047] 19. A pharmaceutical composition in which, in any one of the 10th to 18th embodiments above, the disease is selected from the group consisting of hypertension, arteriosclerosis, diabetic vascular disease, myocardial fibrosis, coronary artery disease, peripheral artery disease, pulmonary hypertension, and heart failure.

[0048] 20. A pharmaceutical composition in which the disease is pulmonary arterial hypertension, according to the 19th embodiment above.

[0049] The peptide according to the present invention can influence phenomena related to structural changes and increased thickness of blood vessel walls in pathological processes associated with vascular fibrosis, and thereby contribute to alleviating physiological abnormalities associated with vascular remodeling. In addition, the peptide according to the present invention can influence pathological changes associated with fibrosis of surrounding lung tissues observed in association with vascular fibrosis, and thus has the potential to provide effects associated with tissue structure recovery or regeneration in a damaged tissue environment. Therefore, the peptide according to the present invention can be usefully utilized for the prevention or treatment of diseases associated with vascular fibrosis, particularly diseases characterized by pulmonary arterial hypertension, and may be used as a monotherapy or in combination with existing therapeutic agents.

[0050] Figure 1 is a three-dimensional structural model image showing the state in which the peptide of SEQ ID NO. 1 is bound to an integrin complex composed of an integrin α-v subunit and a β-1 subunit. The figure shows that negatively charged amino acid residues are distributed around the peptide binding site, and the figure on the right shows the amino acid residues of the β-1 integrin subunit located within about 5 Å of the peptide.

[0051] Figure 2 shows a comparison of the change in force over time measured through Steered Molecular Dynamics (SMD) simulation when different peptide sequences according to the present invention bind to an integrin α-v / β-1 complex.

[0052] Figures 3 and 4 show the results of analyzing the pattern of integrin-mediated intracellular signal transduction induced by the peptide of SEQ ID NO. 1. Figure 3 shows the results of Western blot analysis of the phosphorylation status of integrin downstream signaling factors FAK, AKT, and mTOR under the conditions of the peptide of SEQ ID NO. 1, the FAK inhibitor PF-573228, and their combined treatment. Figure 4 shows the results of quantifying the intensity of the Western blot bands using the ImageJ program. "PF" represents PF-573228.

[0053] Figures 5 and 6 show the results of analyzing the time-dependent changes in the integrin downstream signal following treatment with the peptide of SEQ ID NO. 1. Figure 5 shows the results of analyzing the phosphorylation maintenance pattern of FAK and AKT over time using Western blot, and Figure 6 shows the results of quantifying the intensity of the corresponding Western blot bands using ImageJ.

[0054] Figures 7 to 10 show the results of analyzing histological changes in pulmonary artery and lung tissues following treatment with the peptide of SEQ ID NO. 1 in a mouse model of pulmonary arterial hypertension induced with monocrotaline (MCT). Figure 7 shows the experimental protocol regarding the induction and treatment of the animal model of pulmonary arterial hypertension. Figure 8 shows the results of measuring the pulmonary artery wall thickness in the animal model of pulmonary arterial hypertension; Peptide Sq1 refers to the peptide of SEQ ID NO. 1, and sildenafil is a comparative drug known as a treatment for pulmonary arterial hypertension. Figure 9 shows the results of observing collagen deposition sites in lung tissue using Masson's trichrome staining, and Figure 10 shows the results of observing collagen deposition sites in lung tissue using Sirius red staining. Here, MCT refers to monocrotaline, and NT (No Treatment) refers to the control group that was not treated with the test substance.

[0055] Figures 11 to 13 show the results of ultrasound evaluation of right ventricular function following treatment with the peptide of SEQ ID NO. 1 in mice with pulmonary arterial hypertension induced by monocrotalin. Figure 11 shows a parasternal short-axis ultrasound image taken at the level of the middle papillary muscle. Figure 12 shows the results of measuring the Tricuspid Annular Plane Systolic Excursion (TAPSE), where the red and green dotted lines represent the baselines for systole and diastole, respectively. Figure 13 shows the results of measuring the Right Ventricular Fractional Area Change (RVFAC), Right Ventricular Free Wall Thickness (RVFWT), and Left Ventricular Stroke Volume (SV).

[0056] 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.

[0057] 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.

[0058] 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.

[0059] 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.

[0060] definition

[0061] 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.

[0062] 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."

[0063] 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.

[0064] 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.

[0065] 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.

[0066] In this specification, the term “other amino acid” means an amino acid selected from among 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.

[0067] As used herein, the terms “sequence identity” or “sequence homology” refer to a value expressed as a percentage of the total length of the sequences, representing the number of residues present at the same position 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.

[0068] 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.

[0069] For example, the following substitutions within an amino acid group may be considered conservative substitutions:

[0070] Hydrophobic amino acid group: Ala, Val, Leu, Ile, Met

[0071] Polar non-charged amino acid groups: Ser, Thr, Gln, Asn

[0072] Acidic amino acid group: Asp, Glu

[0073] Basic amino acid group: Lys, Arg, His

[0074] Aromatic amino acid group: Phe, Tyr, Trp

[0075] 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.

[0076] In this specification, the term "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, there is a difference in relative binding strength that is typically recognized by researchers / in the technical field or is statistically significant.

[0077] 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).

[0078] 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 vascular endothelial cells or vascular smooth muscle 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.

[0079] 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.

[0080] 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.

[0081] As used herein, the terms “β1 integrin (Integrin β1)” or “β1 integrin subunit” refer to a receptor containing a β1 subunit among integrin α / β heteromers and include all functional complexes formed by binding with an α subunit such as α1-α11 or αV. β1 integrin has a unique extracellular structure including 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.

[0082] 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.

[0083] 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.

[0084] 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.

[0085] The term "agonist" as used in the present invention refers to a molecule, such as a compound, drug, enzyme activator, or hormone, which 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.

[0086] The term “complex” as used in this specification means 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.

[0087] 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.

[0088] As used herein, the term "epithelial cell" refers to a cell constituting epithelial tissue, such as the 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 acts functionally. In this specification, "epithelial cell" includes cells that share functional and molecular characteristics such as adhesion to the ECM, β1 integrin-mediated signaling, and mechanotransduction, and may include vascular endothelial cells lining the lumen of blood vessels as a group of cells sharing these characteristics.

[0089] As used herein, the term "endothelial cell" refers to a monolayer of cells lining the lumen of blood vessels that are involved in maintaining vascular tone, blood flow responsiveness, vascular permeability, vascular homeostasis, and vascular structure. Endothelial cells are known to express various integrins, including β1 integrin-based complexes, and are involved in cell adhesion, survival-related signaling, and mechanotransduction through interactions with the extracellular matrix (ECM).

[0090] As used herein, the term "vascular smooth muscle cell (VSMC)" refers to a cell that constitutes the media of the blood vessel wall and is involved in vasoconstriction and relaxation, regulation of blood vessel diameter, and maintenance of vascular structural stability. Vascular smooth muscle cells express the β1 integrin family, which mediates interactions with the ECM, and these integrin-mediated signals have been reported to be associated with cell proliferation, migration, survival, and phenotypic switching.

[0091] As used herein, the term “ECM (Extracellular Matrix)” refers to a complex matrix containing fibrous proteins (fibronectin, collagen, laminin, etc.), polysaccharides, and proteoglycans that exist outside of cells 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.

[0092] In this specification, "ECM regeneration" refers to the process of restoring the structure and function of a damaged extracellular matrix (ECM), and, while not necessarily limited thereto, may generally include the following steps.

[0093] (1) Reduction and / or structural breakdown of damaged ECM proteins

[0094] (2) Increased accessibility to β-1 integrin and / or change in active state due to ECM disruption

[0095] (3) Activation of β-1 integrin downstream signals (FAK / AKT, etc.)

[0096] (4) Increased expression of tight junction proteins and / or restoration of epithelial cell barrier function

[0097] (5) Remodeling of normal tissue structure

[0098] The peptide of the present invention promotes ECM regeneration and recovery of vascular function by enhancing at least some of the above steps, particularly (3) and / or (4).

[0099] As used herein, the terms “damaged” or “damaged” mean a state in which the structural and / or 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, but is not limited to, the disruption, denaturation, or abnormal rearrangement of ECM proteins, reduced cell adhesion, reduced stability of vascular or tissue structures, and functional abnormalities resulting from increased mechanical stress. Here, “damage” does not require the complete destruction of the tissue or ECM and is interpreted as a concept encompassing various levels of structural or functional defects, including a state in which normal physiological functions are impaired or unstable.

[0100] As used herein, the term "regeneration" refers to the process by which damaged tissue or the 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 in various tissues, including vascular tissue; the reduction or reorganization of abnormally accumulated fibrous ECM; the restoration of cell adhesion and / or tissue binding structure; the recovery of migration, proliferation, and function of epithelial cells and / or vascular endothelial cells; the alleviation of abnormal proliferation or stiffness of vascular smooth muscle cells; and the restoration of tissue stability and / or homeostasis. "Regeneration" as used herein does not necessarily require complete morphological restoration prior to injury, but includes functional normalization that contributes to the substantial recovery of tissue function, improvement of vascular compliance, or inhibition of disease progression in conditions involving pathological ECM remodeling, such as vascular fibrosis. In addition, regeneration includes not only natural physiological recovery processes but also therapeutic and artificial recovery processes promoted or induced by the action of the β-1 integrin agonist peptide according to the present invention.

[0101] 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.

[0102] As used herein, the term “diseases accompanied by vascular fibrosis” refers to diseases or pathological conditions in which abnormal accumulation, rearrangement, or stiffening of the extracellular matrix (ECM) in vascular tissues is involved as a major feature of pathophysiology, resulting in a decline in vascular structural stability, vascular compliance, or vascular function. Such vascular fibrosis is often accompanied by excessive accumulation of ECM components, such as collagen, elastin, and fibronectin, in the intima, media, and / or adventitia of the blood vessel wall, changes in the arrangement of ECM fibers, increased mechanical stiffness, vascular lumen stenosis, or increased vascular resistance. Diseases accompanied by vascular fibrosis may also involve dysfunction of vascular endothelial cells and / or vascular smooth muscle cells, abnormal vascular remodeling, and disturbance of integrin-mediated signaling, including β-1 integrin.

[0103] As used herein, the term "hypertension" refers to a disease or condition characterized by a sustained elevation of blood pressure in the vascular system, and includes a group of diseases in which dysfunction of vascular endothelial cells and / or vascular smooth muscle cells and changes in extracellular matrix (ECM) remodeling are involved in the pathophysiology. Such hypertension may be accompanied by, for example, but is not limited to, one or more of the following: accumulation of collagen and elastin in the blood vessel walls, increased ECM stiffness, decreased vascular compliance, vascular lumen stenosis, and increased vascular resistance. Examples include pulmonary arterial hypertension, essential hypertension, secondary hypertension, and hypertensive vascular disease.

[0104] 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.

[0105] 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, and promotion of ECM regeneration, and the degree of treatment includes both partial and complete improvement.

[0106] 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.

[0107] 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.

[0108] 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.

[0109] 1. β-1 integrin agonist peptide

[0110] One aspect of the present invention provides a β-1 integrin agonist peptide of a novel amino acid sequence.

[0111] 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.

[0112] 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.

[0113] 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.

[0114] In one embodiment, the agonist peptide of the present invention may include the amino acid sequence of the following chemical formula 1.

[0115] [Chemical Formula 1]

[0116] GLX1SX2X3X4X5FX6X7PDIQX8PDA

[0117] In the above formula, X1 is arginine or glycine.

[0118] In the above formula, X3 is serine, glutamic acid, or lysine.

[0119] In the above formula, X8 is tyrosine or phenylalanine.

[0120] 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.

[0121] 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.

[0122] 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.

[0123] 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.

[0124] 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.

[0125] 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.

[0126] 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.

[0127] 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.

[0128] 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.

[0129] 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.

[0130] 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.

[0131] 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.

[0132] 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.

[0133] 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.

[0134] 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.

[0135] 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.

[0136] 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.

[0137] 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.

[0138] 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.

[0139] The analogs described above 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 verified by conventional biochemical methods.

[0140] When the agonist peptide of the present invention binds to a β-1 integrin subunit, changes in the distribution or arrangement of β-1 integrins in the cell membranes of vascular endothelial cells and / or vascular smooth muscle cells may be induced, thereby promoting β-1 integrin-mediated downstream signaling activation with increased FAK phosphorylation. In conjunction with this, activation of downstream signaling pathways including AKT and / or mTOR may occur, resulting in enhanced signaling related to cytoskeletal reorganization, stabilization of adhesion complexes, and maintenance of vascular cell survival or functional stability. It is understood that the regulation of such signaling can assist in the restoration of vascular structural stability and mechanical homeostasis by contributing to the normalization of vascular endothelial cell function, mitigation of abnormal proliferation or contractile responses of vascular smooth muscle cells, and interactions with the vascular ECM.

[0141] 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.

[0142] 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.

[0143] 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.

[0144] 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.

[0145] 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.

[0146] 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.

[0147] 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.

[0148] 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.

[0149] 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.

[0150] 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.

[0151] 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.

[0152] 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.

[0153] The above amino acid substitution may be, for example, a conservative substitution.

[0154] 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 "conservative amino acid substitution," this is interpreted to include substitutions with structurally similar and functionally equivalent amino acids.

[0155] 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.

[0156] 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.

[0157] In one embodiment, the agonist peptide of the present invention may not exhibit substantial binding or functional activation to β-1 integrin subunits present in immune cells, but may selectively bind to β-1 integrin subunits present in vascular endothelial cells and / or vascular smooth muscle cells.

[0158] 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 with β-1 integrins expressed in immune cells. This means that, compared to β-1-based integrin complexes present in vascular endothelial cells or vascular smooth muscle cells, the binding affinity, binding persistence, and / or functional activation ability for β-1 integrins present in immune cells is substantially low or negligible. 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.

[0159] The binding selectivity described above means that even the same β-1 integrin subunit can be selectively recognized due to differences in the composition of the integrin complex formed according to cell type, distribution on the cell membrane, local ECM microenvironment, or three-dimensional arrangement. Accordingly, the agonist peptide of the present invention can selectively regulate only β-1 integrin-mediated signals in vascular endothelial cells and vascular smooth muscle cells without causing unintentional activation of immune cells or immune-related side effects.

[0160] In one embodiment, the agonist peptide of the present invention may be used to regenerate damaged or pathologically remodeled vascular extracellular matrix (ECM), maintain the adhesion, survival, and function of vascular endothelial cells, and / or alleviate abnormal proliferation or stiffness of vascular smooth muscle cells.

[0161] In particular, in conditions accompanied by vascular fibrosis, normal integrin signaling is often inhibited or disrupted due to abnormal accumulation or stiffness of the ECM, and the agonist peptide of the present invention can promote the recovery of structural stability and functional compliance of the vascular ECM by supplementing or regulating β-1 integrin-mediated signaling in such environments.

[0162] In one embodiment, the damaged or pathologically remodeled extracellular matrix may be spatially and / or functionally associated with vascular tissue or epithelial tissue adjacent to blood vessels of an organ selected from the group consisting of lungs, liver, heart, blood vessels, kidneys, eyes, skin, and intestines. Herein, the expression “associated” means that structural deformation, accumulation, or rearrangement of the ECM is pathophysiologically linked to the deterioration of vascular tissue function, inflammatory response, progression of fibrosis, or regeneration process.

[0163] The agonist peptide of the present invention enables a therapeutic approach that promotes the functional recovery of abnormally remodeled vascular ECM and vascular tissue itself, rather than merely having anti-inflammatory or anti-fibrotic effects, by inducing a series of biological changes including increased β-1 integrin exposure, regulation of downstream signaling pathways including FAK, AKT and / or mTOR, stabilization of vascular cell adhesion and survival signals, and / or reorganization of ECM structure.

[0164] Although not bound by any specific theory, the β-1 integrin agonist peptide of the present invention, based on these functional characteristics, can be usefully employed in the prevention or treatment of various vascular diseases in which vascular fibrosis and associated vascular dysfunction act as important factors in pathophysiology.

[0165] 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.

[0166] 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.

[0167] 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.

[0168] 2. Medical Use

[0169] The agonist peptide of the present invention can be used for the prevention or treatment of diseases accompanied by vascular fibrosis.

[0170] Vascular fibrosis is a pathological condition characterized by the abnormal accumulation, rearrangement, or stiffening of the extracellular matrix (ECM) in the blood vessel walls, and is known as a major pathological factor leading to decreased vascular compliance, increased vascular resistance, and impaired vascular function. Such vascular fibrosis can be observed in pulmonary hypertension, systemic hypertension, ischemic vascular disease, diabetic vascular disease, and other diseases involving abnormal vascular remodeling.

[0171] Diseases accompanied by chronic vascular fibrosis may include, for example, pulmonary arterial hypertension, systemic hypertension, hypertensive vascular disease, ischemic vascular disease, diabetic vascular disease, and vascular stiffness associated with vascular aging, but are not limited thereto.

[0172] In this specification, the term "disease involving vascular fibrosis" does not necessarily require that vascular fibrosis be the sole cause of the disease, but is interpreted as encompassing all cases in which vascular fibrosis substantially contributes to the onset or progression of the disease in combination with inflammation, mechanical stress, metabolic abnormalities, or genetic factors.

[0173] The agonist peptide of the present invention can contribute to stabilizing signals related to the adhesion, survival, and maintenance of function of vascular endothelial cells and alleviating abnormal proliferation, migration, or contractile responses of vascular smooth muscle cells by activating or regulating β-1 integrin-mediated signaling in a pathologically remodeled vascular ECM environment. This action can alleviate excessive ECM accumulation or stiffness in the blood vessel wall and promote the restoration of vascular structural stability and mechanical homeostasis.

[0174] In particular, in cases where vascular fibrosis has progressed, normal integrin-mediated signaling is often inhibited or distorted due to abnormal accumulation of ECM and changes in mechanical properties, and the agonist peptide of the present invention can exhibit the effect of inhibiting or delaying the pathological progression of the vascular remodeling process by supplementing or normalizing β-1 integrin signaling in such an environment.

[0175] The agonist peptide of the present invention not only inhibits the progression of fibrosis but also provides a therapeutic approach that induces substantial recovery or improvement of vascular function by regulating the interaction between abnormally remodeled vascular ECM and vascular cells. Due to these characteristics, the agonist peptide of the present invention can be usefully applied to the prevention or treatment of various diseases that include vascular fibrosis as a key pathophysiological element.

[0176] In one embodiment, the agonist peptide of the present invention may be used for the prevention or treatment of a disease selected from the group consisting of hypertension, arteriosclerosis, diabetic vascular disease, myocardial fibrosis, coronary artery disease, peripheral artery disease, pulmonary hypertension, and heart failure.

[0177] In one embodiment, the disease is pulmonary arterial hypertension.

[0178] 3. Pharmaceutical composition

[0179] 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.

[0180] 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.

[0181] 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.

[0182] 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.

[0183] 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.

[0184] 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.

[0185] Herein, "therapeutically effective content" means a content of the pharmaceutical composition of the present invention containing a sufficient amount of β-1 integrin agonist peptide on a unit dosage form or unit dose basis to improve pathological conditions associated with diseases involving vascular fibrosis in a subject to administration. The therapeutic effects may include, for example, structural and / or functional recovery of pathologically remodeled vascular extracellular matrix, stabilization of adhesion, survival, and function of vascular endothelial cells and / or vascular smooth muscle cells, inhibition of progression of vascular wall fibrosis, alleviation of vascular stiffness, improvement of vascular compliance, or reduction of vascular resistance, and include a content that exhibits such effects within an acceptable range of side effects.

[0186] 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.

[0187] 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.

[0188] 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.

[0189] 4. Treatment methods

[0190] 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.

[0191] 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 vascular extracellular matrix, vascular tissue damage, or abnormalities in β-1 integrin-mediated signaling.

[0192] 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 its progression, or restoring the structure and function of blood vessels.

[0193] In one embodiment, the treatment method may include administering the β-1 integrin agonist peptide of the present invention in a therapeutically effective amount.

[0194] Here, "therapeutically effective amount" means an amount sufficient to induce one or more therapeutic effects among normalization of pathologically remodeled vascular extracellular matrix, stabilization of vascular endothelial cell and / or vascular smooth muscle cell function, inhibition of progression of vascular wall fibrosis, alleviation of vascular stiffness, improvement of vascular compliance, or reduction of vascular resistance in subjects administered with the agonist peptide, and includes an amount that exhibits these effects within an acceptable safety range.

[0195] 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.

[0196] In addition, the above therapeutically effective amount may be provided as a single dose, or in the form of repeated doses or divided doses.

[0197] The agonist peptide of the present invention can promote the adhesion, survival, maintenance of function, and signal homeostasis of vascular endothelial cells and / or vascular smooth muscle cells by activating β-1 integrin-mediated signaling in an environment where the vascular extracellular matrix is ​​damaged or pathologically remodeled, thereby inducing stabilization of vascular structure and functional recovery.

[0198] 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, a vasodilator, an immunomodulator, a growth factor, an extracellular matrix modulator, or an anticancer agent. Combined administration may be performed with the same formulation or different formulations, and may be carried out simultaneously, sequentially, or at regular intervals.

[0199] 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 methods targeting vascular tissue. 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.

[0200] 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.

[0201] 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 relatively sustained biological effects and signal activation in vascular tissue. These administration intervals 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 vascular remodeling and recovery process.

[0202] 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.

[0203] Example 1: Peptide Synthesis

[0204] 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.

[0205] 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.

[0206] 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.

[0207] 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.

[0208] Sequence No. 1 GLRSKSKKFRRPDIQYPDA

[0209] Sequence No. 2 GLGSKEKKFKKPDIQFPDA

[0210] Sequence No. 3 GLGSKLKKFRHPDIQFPDA

[0211] Other peptides having the amino acid sequence of Formula 1 disclosed in this specification can also be synthesized in the same way as above.

[0212] Example 2: Confirmation of binding force to integrins using the principle of surface plasmon resonance

[0213] 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.

[0214] 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.

[0215] 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.

[0216] 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.

[0217]

[0218] 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.

[0219]

[0220]

[0221] Example 3: Analysis of Peptide Integrin Binding Characteristics Using Computer Modeling

[0222] Using computer-based structural modeling, the binding characteristics of the peptide according to the present invention to the integrin protein were analyzed.

[0223] 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.

[0224] 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.

[0225] As a result, Figure 1 showed 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.

[0226] 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.

[0227] Figure 2 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.

[0228]

[0229] Example 4: Analysis of Peptide Integrin Binding Characteristics Using Computer Modeling

[0230] 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.

[0231] As a result, as shown in Figures 3 and 4, 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.

[0232] 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 were cultured in DMEM medium supplemented with 10% FBS and 1X penicillin-streptomycin solution under 37°C in a 5% CO2 humidified incubator. Cells were diluted to 1X10⁵ cells / ml with DMEM containing 10% FBS and seeded into 6-well plates. When the cells reached 60-70% confluence, the medium was replaced with 2 ml of serum-free DMEM for 24 hours prior to treatment. Subsequently, the cells were treated with 50 μg / ml BLM and 100 μM peptide of SEQ ID NO. 1 for 3 days, or treated with BLM alone. The production amounts of Focal adhesion kinase (FAK) and protein kinase B (AKT), which are the initial signaling pathways of cells, and tight junction markers such as E-cadherin, ZO-1, and Occludin 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.

[0233] As a result, in A549 cells treated with BLM alone as shown in Figures 5 and 6, pFAK activity remained nearly constant for 72 hours around a baseline level of approximately 1.0. In the case of pAKT, a slight increase was observed in the group treated with BLM alone. In A549 cells treated with BLM and 100 μM peptide SEQ NO. 1, pFAK and pAKT levels increased at 1 hour, and then decreased back to baseline levels after 1 hour without any change in total FAK or AKT protein levels. Additionally, peptide SEQ NO. 1 treated with BLM induces a long-lived, low-level pFAK signal that persists for up to 50 hours.

[0234]

[0235] Example 5: Evaluation of pulmonary artery thickness and lung tissue fibrosis in a monocrotalin-induced pulmonary hypertension mouse model

[0236] To evaluate the therapeutic effect of the peptide according to the present invention on pulmonary arterial hypertension, a mouse model of pulmonary arterial hypertension induced by monocrotaline was used. Male C57BL / 6J mice (15-17 weeks old) were purchased from Charles River.

[0237] Mice were anesthetized by intraperitoneal administration of a mixture of Imalgene (ketamine, 100 mg / kg) and Rompun (xylazine, 20 mg / kg), and pulmonary arterial hypertension was induced by subcutaneous injection of monocrotalin (60 mg / kg, Sigma-Aldrich) once a week for 4 weeks. Simultaneously with the administration of monocrotalin, the peptide of SEQ ID NO. 1 was administered subcutaneously daily at doses of 30, 60, 90, or 120 mg / kg. The control group included a group administered only PBS (NT) and a group administered daily subcutaneously of sildenafil (20 mg / kg, Glentham Life Sciences) as a comparator drug. The complete experimental protocol is shown in Figure 7.

[0238] During the drug administration period (28 days), body weight was measured at 2-day intervals and the presence of clinical symptoms was observed. On the 29th day, mice were sacrificed with isoflurane, and lung tissues were excised for histological analysis. Lung tissues were embedded in paraffin, and 5 μm thick sections were prepared. Masson's trichrome staining and Sirius red staining were performed to evaluate the degree of pulmonary fibrosis and inflammation.

[0239] Images were acquired at 100x magnification using an optical microscope (BX51, Olympus) (300MI CMOS camera, Aptina) for five randomly selected sites in each specimen, and the thickness and fibrotic area of ​​the pulmonary artery walls were quantitatively analyzed using Image-Pro Plus software (Media Cybernetics Inc.). The degree of fibrosis was evaluated as the percentage of Masson's trichrome positive area (Masson Trichrome positive area, %).

[0240] As a result, as shown in Figure 8, the thickness of the pulmonary artery wall decreased in both the peptide of SEQ ID NO. 1 and the sildenafil administration group. In addition, as shown in Figures 9 and 10, vascular wall fibrosis and fibrosis within the lung tissue were significantly reduced in the group administered the peptide of SEQ ID NO. 1. These results demonstrate that the peptide of SEQ ID NO. 1 effectively reduces pulmonary artery wall thickening and lung tissue fibrosis observed in pulmonary hypertension.

[0241]

[0242] Example 6: Cardiac ultrasound analysis in a monocrotalin-induced pulmonary hypertension mouse model

[0243] In the same animal model used in Example 5, right ventricular function before sacrifice was evaluated using echocardiography. Mice were anesthetized by inhaling isoflurane through a face mask, and then transthoracic echocardiography was performed using a Vega Preclinical Ultrasound System (Revvity) and a transducer for mice (MS-550D, 22-55 MHz).

[0244] Right Ventricular Fraction Area Change (RVFAC) was measured along the parasternal short-axis at the level of the middle papillary muscle (see Fig. 11). Right Ventricular Free Wall Thickness (RVFWT) was measured at the end of diastole using M-mode imaging at the level of the mitral valve along the parasternal short-axis or at the level of the right ventricular outflow duct along the parasternal long-axis.

[0245] Pulse-wave Doppler echocardiography was used to record pulmonary blood flow outflow at the aortic valve level along the short axis to measure Pulmonary Acceleration Time (PAT) and Pulmonary Ejection Time (PET). Tricuspid annular plane systolic excursion (TAPSE) was measured using 2D M-mode ultrasound in an apical four-chamber view, with the cursor positioned at the lateral tricuspid annulus near the free wall of the right ventricle. Additionally, stroke volume (SV) was measured in the left ventricle (LV).

[0246] Representative ultrasound images are shown in Fig. 12. The area indicated by the blue lines represents the right ventricular wall, and a larger gap between the lines indicates an increase in right ventricular wall thickness. The right ventricular wall thickness increased most significantly in the group administered only monocrotalin (No Treatment), while the right ventricular wall thickness decreased when the peptide of SEQ ID NO. 1 or sildenafil was administered (see Fig. 13).

[0247] The quantitative results of ejection fraction, right ventricular wall thickness, and left ventricular stroke volume are shown in Figure 13. The ejection fraction decreased in the monocrotalin-treated group (NT) compared to the normal group, but significantly increased in the group administered the peptide of SEQ ID NO. 1 and the sildenafil-treated group. The right ventricular wall thickness increased in the monocrotalin-treated group and significantly decreased in the group administered the peptide of SEQ ID NO. 1. On the other hand, no statistically significant decrease was observed in the sildenafil-treated group compared to the untreated group.

[0248] Left ventricular stroke volume decreased in the monocrotalin-treated group compared to the normal group, and significantly increased in the group administered 60 mg / kg or more of the peptide of SEQ ID NO. 1. No significant difference was observed in the sildenafil-treated group compared to the untreated group.

[0249] From the above results, it was confirmed that the peptide of SEQ ID NO. 1 effectively restores cardiac function degraded by monocrotalin by reducing pulmonary artery wall thickening and improving right ventricular function and left ventricular ejection function in a pulmonary arterial hypertension model.

Claims

1. A pharmaceutical composition for use in the prevention or treatment of diseases accompanied by vascular fibrosis, comprising a β-1 integrin subunit agonist peptide having an amino acid sequence of the following chemical formula or an amino acid sequence that is 85% or more 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. A pharmaceutical composition according to claim 1, wherein the agonist peptide comprises the amino acid sequence of SEQ ID NO. 1 or an amino acid sequence comprising one or two amino acid substitutions compared thereto.

3. A pharmaceutical composition according to claim 1, wherein the agonist peptide comprises the amino acid sequence of SEQ ID NO. 2 or an amino acid sequence comprising one or two amino acid substitutions compared thereto.

4. A pharmaceutical composition according to claim 1, wherein the agonist peptide comprises the amino acid sequence of SEQ ID NO. 3 or an amino acid sequence comprising one or two amino acid substitutions compared thereto.

5. A pharmaceutical composition 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 pharmaceutical composition according to claim 1 or 2, wherein the agonist peptide 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. A pharmaceutical composition according to claim 1 or 2, wherein the agonist peptide does not bind to β-1 integrin subunits present in immune cells, but binds to β-1 integrin subunits present in vascular endothelial cells or vascular smooth muscle cells.

8. A pharmaceutical composition according to claim 1 or 2, wherein the agonist peptide can be used to regenerate damaged or pathologically remodeled extracellular matrix, maintain the adhesion, survival, and function of vascular endothelial cells, and / or alleviate abnormal proliferation or stiffness of vascular smooth muscle cells.

9. A pharmaceutical composition according to claim 8, wherein the damaged or pathologically remodeled extracellular matrix is ​​associated with vascular tissue or epithelial tissue adjacent to blood vessels of an organ selected from the group consisting of lungs, liver, heart, blood vessels, kidneys, eyes, skin, and intestines.

10. A pharmaceutical composition according to claim 1 or 2, wherein the disease is selected from the group consisting of hypertension, arteriosclerosis, diabetic vascular disease, myocardial fibrosis, coronary artery disease, peripheral artery disease, pulmonary hypertension, and heart failure.

11. A pharmaceutical composition according to claim 10, wherein the disease is pulmonary arterial hypertension.

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