Peptide inhibitors and their use

Abstract

The present invention relates to a peptide inhibitor for inhibiting the activation of TGF-β1 by the involvement of TSP-1, or a pharmaceutically acceptable salt, solvate or prodrug thereof, and a pharmaceutical composition comprising the peptide or a pharmaceutically acceptable salt, solvate or prodrug thereof, and a method and a pharmaceutical composition for treating or preventing TGF-β1-related diseases, particularly fibrosis and solid tumors, by using the peptide inhibitor or a pharmaceutically acceptable salt, solvate or prodrug thereof.

Description

Technical Field

[0001] The present invention relates to a peptide inhibitor for inhibiting the activation of TGF-β1 by the involvement of TSP-1, or a pharmaceutically acceptable salt, solvate or prodrug thereof, and a pharmaceutical composition comprising the peptide or a pharmaceutically acceptable salt, solvate or prodrug thereof, and a method and a pharmaceutical composition for treating or preventing TGF-β1-related diseases, particularly fibrosis and solid tumors, by using the peptide inhibitor or a pharmaceutically acceptable salt, solvate or prodrug thereof.

Background Art

[0002] Fibrosis is a pathological condition in which fibrous connective tissue abnormally proliferates during the wound repair process to form tissue scars, and is characterized by excessive deposition of the extracellular matrix (ECM). Fibrosis leads to an irreversible decline in tissue and organ function, and in severe cases, affects the quality of life of patients and even endangers the patient's life. In addition, the formation of fibrosis contributes to tumorigenesis and distant metastasis, promotes the formation of an immunosuppressive tumor microenvironment, and results in immunotherapy resistance or immune non-responsiveness.

[0003] Transforming growth factor β (TGFβ) is a central regulatory molecule important for the mechanisms of fibrosis and the occurrence and progression of tumors. TGF-β and its signaling pathway have been proposed as therapeutic targets for the treatment of fibrotic diseases and tumors. However, TGF-β, a multifunctional cytokine, is also involved in various normal physiological processes, and as a result, the toxicity of drugs that directly target TGF-β and its signaling pathway is high. Currently, some of these drug development programs have been slowed down or even stopped due to severe drug-related adverse responses after entering the clinical stage.

[0004] TGF-β is synthesized and secreted as an inactive precursor structure, and its active fragment needs to undergo a series of activation events to become an active fragment before it can bind to its receptor and mediate downstream conversion pathways. The activation events of TGF-β are achieved by different protein molecules under different environments and conditions. Therefore, targeting specific proteins related to activation in disease states has become the latest research trend to break through the difficulties of TGF-β drug development.

[0005] Thrombospondin-1 (TSP-1) is a protein that has been clinically shown to be actively expressed only in fibrosis and tumor-related symptoms. TSP-1 has been shown to be involved in TGF-β activation events between chronic inflammation and fibrosis and is a major activator of TGF-β1 in vivo (Cell, Vol. 93, 1159-1170, June 26, 1998, Copyright (C) 1998 by Cell Press). Considering clinical applications, peptide drugs that target the activation of TGF-β can provide a safer strategy than other therapeutic anti-TGF-β1 molecules because they act at the site where TGF-β1 is activated, are easily metabolized, and have low antigenicity. Therefore, the present invention intends to design a polypeptide component that targets the domain of TSP-1 related to the activation of TGF-β to provide a new peptide drug for improving fibrosis and cancer.

Summary of the Invention

Problems to be Solved by the Invention

[0006] Through systematic research, the inventors unexpectedly discovered a group of short peptides consisting of 5 to 13 amino acids containing the amino acid motif (DXED), which are not only effective in inhibiting the interaction between the TSP-1 receptor and the TSP-1 / L-TGF-β1 complex, but also effective in blocking TSP-1-dependent L-TGF-β1 activation and subsequent TGF-β signaling. Based on this, the inventors established the inhibitory peptides of the present invention (hereinafter also referred to as "TGF-β1 activation inhibitory peptides"). As shown in the examples, the inhibitory peptides of the present invention exhibit inhibitory activity against TGF-β1 activation in both cyclic and linear forms; they show stronger inhibition of TGF-β1 activation compared to similar peptides (SEQ ID NO: 50) known to those skilled in the art, and the peptide length is shortened to achieve a higher cost-effectiveness. In some preferred embodiments, the inhibitory peptides of the present invention also exhibit better solubility, stability and / or bioavailability compared to known similar peptides.

[0007] In addition, the inhibitory peptides of the present invention also exhibit advantages in favorable drug safety. Studies on TSP-1 have shown that TSP-1 contains multiple functional domains, which bind independently to different cell surface receptors and are involved in various physiological processes such as hemostasis, cell adhesion, cell migration, apoptosis and anti-angiogenesis. Due to the low molecular weight, high activity and design for specific domains on TSP-1 related to the activation of TGF-β1 of the inhibitory peptides of the present invention, interference with other functional domains of TSP-1 and related physiological functions can be effectively reduced, protecting the specificity and safety of drug action.

Means for Solving the Problems

[0008] Therefore, in a first aspect, the present invention provides a TGF-β1 activation inhibitory peptide, or a pharmaceutically acceptable salt, solvate or prodrug thereof. The TGF-β1 activation inhibitory peptide according to the present invention can specifically block TGF-β1 activation involving TSP-1, and has the following general formula (I): R1 -X 1 -X 2 -X 3 -X 4 -X 5 -X 6 -X 7 -R 2 (I), (wherein, R 1 is acetyl or absent; R 2 is amino or absent; X 1 is absent or selected from T and S; X 2 is selected from natural amino acids or non-natural amino acids; preferably selected from polar side chain amino acids such as uncharged polar side chain amino acids S, C, G, N, Q, T, Y, or negatively charged polar side chain amino acids D and E; more preferably selected from amino acids N, C, Q, S, T, E, and D; even more preferably selected from amino acids Q, C, N, E, and S; X 3 is D; X 4 is selected from small side chain amino acids such as A, C, G, P, S, T, and V; more preferably selected from amino acids A, P, and S; even more preferably selected from amino acids A and P; X 5 is E; X 6 is D; X 7 is absent or starting from the amino terminus to the carboxyl terminus, Z 1 Z 2 Z 3 SZ 4 is either a sequence of 1, 2, 3, 4, 5, 6, or 7 amino acids continuously selected from the amino acid sequence of LQ; Z 1 is an amino acid of N, Q, P, A, L, V, M, or I; preferably N, P, or L; most preferably N or P; Z 2is an amino acid of T, S, V, C, A, K or R, preferably T, C or K, more preferably, Z 2 When Z is an amino acid of C or K, 2 Z is amino acid X 2 or X 3 forms a covalent bond with the side chain of, Z 3 is an amino acid of A, V, L or I, preferably A or V, Z 4 is an amino acid of F, Y, H or W, preferably F or H; Optionally, when X 4 is A, 0 to 1 small side chain amino acids such as P or G, preferably P, are inserted between X 3 and X 4 ; Optionally, when X 7 consists of 1 to 5 amino acids, the peptide has 0 to 2 additional amino acid residues added after X 7 , such as 1 to 2 amino acid residues selected from amino acid residues of Q, E, D and N being added, or 1 amino acid residue selected from C or K being added), and has an amino acid sequence.

[0009] In some embodiments, in the peptide of the present invention, X 1 is absent or selected from T and S; X 2 is selected from amino acids Q, N, C, S, T, E and D; X 3 is D; X 4 is selected from A, C, G, P, S, T and V; X 5 is E; X 6 is D; X 7 is absent or starting from the amino terminus to the carboxyl terminus, Z 1 Z 2 Z 3 SZ 4It is any one of sequences of 1, 2, 3, 4, 5, 6, or 7 amino acids consecutively selected from the amino acid sequence of LQ; Z 1 is N, P, or L; Z 2 is T, C, or K; Z 3 is A or V; Z 4 is F, Y, H, or W.

[0010] In some embodiments, in the peptide of the present invention, X 1 is selected from T and S; X 2 is selected from Q and N; X 3 is D; X 4 is selected from A and P; X 5 is E; X 6 is D.

[0011] In some embodiments, X 7 is absent. In some other embodiments, X 7 is Z 1 Z 1 Z 2 Z 1 Z 2 Z 3 Z 1 Z 2 Z 3 S, Z 1 Z 2 Z 3 SZ 4 Z 1 Z 2 Z 3 SZ 4 L or Z 1 Z 2 Z 3 SZ 4 consisting of LQ, preferably X 7 is Z 1 Z 2 Z 3 Z1 Z 2 Z 3 S or Z 1 Z 2 Z 3 SZ 4 and consists of Preferably Z 1 is N, P or L, preferably N or P; Z 2 is T, K or C; Z 3 is A or V, preferably A; Z 4 is F, Y, H or W, preferably F or H.

[0012] The peptide of the present invention may be a linear or cyclic peptide. In a second aspect, the present invention also provides the therapeutic and / or prophylactic use of the inhibitory peptide of the present invention or a pharmaceutically acceptable salt, solvate or prodrug thereof in a disease caused by or associated with TGF-β1 activation involving TSP-1 (i.e., a "TGF-β1-related disease"). The present invention also provides the use of the inhibitory peptide of the present invention or a pharmaceutically acceptable salt, solvate or prodrug thereof in the manufacture of a medicament for preventing or treating a fibrotic disorder. The fibrotic disorder is characterized by TGF-β1 activation involving TSP-1 and exhibits excessive deposition of the extracellular matrix (ECM), but is not limited to the site of the disease or the conventional disease classification. The present invention also provides the use of the inhibitory peptide of the present invention in the manufacture of a medicament for preventing or treating cancer associated with TGF-β1 activation. BRIEF DESCRIPTION OF THE DRAWINGS

[0013]

Figure 1

Figure 2

Figure 3

Modes for Carrying Out the Invention

[0014] Definitions For the present invention to be more easily understood, certain scientific and technical terms are specifically defined below. Unless otherwise explicitly defined herein, the scientific and technical terms used herein have the meanings generally understood by those skilled in the art to which the present invention pertains. The abbreviated forms of amino acid residues are the standard three-letter and / or one-letter codes used in the art to refer to the 20 commonly used amino acids. Unless otherwise indicated, amino acid sequences are written from left to right in the amino- to carboxyl direction.

[0015] Unless otherwise clearly indicated in the context, the singular forms used herein, including in the claims, include their corresponding plural forms. The term "about" refers to a value that is within the acceptable error range of a specific value determined by those skilled in the art, and the error range is determined by the measurement means used for measuring or determining the value, that is, the limits of the measurement system. For example, "about" can mean within 1 or greater than 1 standard deviation according to the practice in the art. Alternatively, "about" can refer to a range of up to 5%, 10% or 20% (i.e., ±5%, ±10% or ±20%).

[0016] The term "and / or" should be understood to mean any one of the alternatives or any two or more of the alternatives when used with respect to two or more alternatives. As used herein, the terms "comprising" or "including" are intended to include the recited elements, integers or steps, but not to exclude any other elements, integers or steps. When used herein, the terms "comprising" or "including" also embrace the recited elements, integers or steps "consisting of", unless otherwise indicated. For example, when referring to "comprising" a particular sequence, it is also intended to encompass peptides or polypeptides consisting of the particular sequence.

[0017] As used herein, the terms "peptide" and "polypeptide" are used interchangeably to refer to an amino acid sequence having a length of 2 to 100 amino acids, wherein the amino acids are linked by peptide bonds. The amino acids can be naturally occurring and non-naturally occurring.

[0018] As used herein, the term "conservative amino acid substitution" or "conservative amino acid replacement" means an amino acid substitution that does not adversely affect or modify the biological function of a polypeptide containing the amino acid sequence. Typically, a conservative amino acid substitution refers to the substitution of one amino acid for another amino acid having similar chemical properties (e.g., charge or hydrophobicity). Tables of conservative substitutions for functionally equivalent amino acids are well known in the art. In the present invention, conservative substitution residues can be obtained from the following table of conservative substitutions, and in particular, those in the following table are preferred conservative amino acid substitution residues.

[0019]

Table 1

[0020] Detailed Description In this study, the inventors surprisingly discovered that a short peptide having a motif (DXED) rich in negatively charged amino acids can effectively initiate an inhibitory effect on TSP-1-dependent TGF-β1 activation. Through analyses such as structure-activity correlation and amino acid substitution, the inventors further identified the essential amino acids and possible substitution sites within the peptide sequence; and by peptide cyclization and / or backbone modification, they optimized the structure, thereby establishing a series of novel inhibitory peptides against TGF-β1 activation with improved bioactivity and / or physicochemical properties.

[0021] The inhibitory peptide of the present invention In one aspect, the present invention thus provides a TGF-β1 activation inhibitory peptide or a pharmaceutically acceptable salt, solvate or prodrug thereof. The peptide of the present invention contains a negatively charged core motif (DXED) and has a length of 5 to 13 amino acids. Preferably, the peptide of the present invention consists of 7 to 11 amino acids. In some embodiments, the peptide of the present invention is a linear or cyclic pentapeptide or hexapeptide, such as a linear or head-to-tail cyclized hexapeptide. In some embodiments, the peptide of the present invention is a cyclic heptapeptide, such as a linear or head-to-tail cyclized heptapeptide. In some other embodiments, the peptide of the present invention is a linear or cyclic octapeptide, particularly an octapeptide cyclized by side chains, preferably by disulfide bonds. In still other embodiments, the peptide of the present invention is a linear or cyclic nonapeptide, particularly a linear nonapeptide. In still other embodiments, the peptide of the present invention is a linear or cyclic decapeptide, such as a head-to-tail cyclized decapeptide. In still other embodiments, the peptide of the present invention is a linear or cyclic undecapeptide, dodecapeptide or tridecapeptide, preferably a linear undecapeptide.

[0022] In some embodiments, when the peptide of the present invention is compared with the sequence of TQDAEDNTVSFLQ (SEQ ID NO: 51) for maximum alignment, there are 0 to 5 amino acid differences, for example 0 to 2 amino acid differences, between the full-length peptide of the present invention and the corresponding aligned portion of SEQ ID NO: 51. Amino acid differences include amino acid substitutions, additions, and deletions. For example, the peptide of the present invention may be identical to SEQ ID NO: 51 in the corresponding portion, or have 1 or 2 amino acid substitutions, or have an addition of 1 to 2 amino acids at the N-terminus or C-terminus, particularly at the C-terminus. The amino acids used for substitution can be naturally occurring or non-naturally occurring amino acids. In some embodiments, the peptide of the present invention includes a proline substitution preferably located at the position of the second residue of the core sequence DXED; or at the position of the first residue directly adjacent to the C-terminus of the core sequence. In some embodiments, the total net charge of the peptide of the present invention is negative, for example -1, -2, -3, or -4, preferably -2 or -3.

[0023] In some embodiments, the peptide includes a core amino acid sequence selected from DAED or DPED. In other embodiments, the peptide further includes an N or P residue located at the C-terminus of the core sequence, and thus the peptide includes an amino acid sequence selected from DAEDN; DPEDN; or DAEDP.

[0024] In some embodiments, the peptide of the present invention is a cyclic peptide. In some embodiments, the peptide of the present invention is cyclized by a covalent bond between the first amino acid and the last amino acid. In some other embodiments, the peptide of the present invention is cyclized by a lactam bond or a disulfide bond formed between the side chains of two amino acids. For this purpose, analogs with appropriate amino acids or appropriate side chain groups may be placed at appropriate positions within the peptide of the present invention to contribute to the formation of molecular lactam bonds or disulfide bonds within the peptide. In some embodiments, the lactam is formed by coupling the side chain amino functional group of the amino acid residue located at the C-terminus of the core motif (DXED) of the peptide of the present invention to the carboxylic acid group of the amino acid at the N-terminus of the core motif (DXED). In some embodiments, the disulfide bond is formed by oxidatively coupling the cysteine located at the C-terminus of the core motif (DXED) to the cysteine located at the N-terminus of the core motif (DXED). In some embodiments of the lactam cyclic peptide, the lactam bond is formed between the side chain carboxyl group of the first aspartic acid (D) of the core motif or the aspartic acid amino acid (D) or glutamic acid (E) located at the N-terminus of the core motif and the side chain amino group of the lysine (K) located at the C-terminus of the core motif, and the two amino acids for cyclization are separated by four or more amino acids, for example, 4, 5 or 6 amino acids apart, and preferably, the lysine residue for cyclization is the last residue at the C-terminus of the peptide. In some embodiments of the disulfide-cyclized peptide, the disulfide bond is formed between the cysteine, which is the first amino acid directly adjacent to the N-terminus of the core motif (DXED), and the cysteine located at the C-terminus of the core motif (DXED), and preferably, the two amino acids for cyclization are separated by five or more amino acids, preferably 5 or 6 amino acids apart, and more preferably, the cysteine at the C-terminus for cyclization is the last residue at the C-terminus of the peptide.In some embodiments, the peptide of the present invention is preferably a disulfide-cyclized peptide, preferably consisting of 8 amino acids, and more preferably, the N-terminal amino group of the cyclic peptide is acetylated and / or the C-terminal carboxyl group is amidated.

[0025] The peptide of the present invention may include chemical modifications, such as N-terminal acetylation, C-terminal amidation, PEG modification, lipid modification, D-amino acid substitution, or non-naturally occurring amino acid substitution. In some embodiments, the peptide of the present invention is modified by covalent attachment to a molecule such that the ability of the peptide to inhibit TGF-β1 activation, including, for example, glycosylation, acetylation, PEGylation, phosphorylation, amidation, or derivatization using known protecting / blocking groups, can be maintained. In one embodiment, the modification is N-terminal acylation (especially acetylation). In one embodiment, the modification is C-terminal amidation. Preferably, the peptide of the present invention is an N-terminal acylated (especially acetylated) linear peptide or a side-chain cyclized peptide, and more preferably the peptide also has C-terminal amidation. In some embodiments, the peptide can be linked to a biomolecule or a material for binding, labeling, or identification.

[0026] The TGF-β1 activation inhibitory peptide according to the present invention can inhibit TSP-1-dependent TGF-β1 activation and preferably has the following properties: - Reducing the amount of active TGF-β1 in fibrotic tissue; - Inhibiting TGF-β1-mediated downstream signaling; - Decreasing collagen deposition in fibrotic tissue; - Inhibiting inflammatory and / or fibrotic lesions mediated by TGF-β1; - Inhibiting the expression of extracellular matrix-related genes stimulated by TGF-β1; - Inhibiting the migration of tumor cells; - Preventing or treating TGF-β1-related diseases, especially fibrosis or cancer It has at least one of them. A person skilled in the art can determine the above characteristics of the peptide of the present invention according to methods known to those skilled in the art or described in the examples.

[0027] In some embodiments, the peptide of the present invention comprises or consists of an amino acid sequence corresponding to one of SEQ ID NOs: 1 to 49. In some embodiments, the peptide of the present invention comprises or consists of one of SEQ ID NOs: 1 to 49. In some embodiments, the peptide of the present invention comprises or consists of an amino acid sequence corresponding to one of SEQ ID NOs: 6, 14 to 15, 22 to 26, 33, 36, 40, 43 to 46, and 48 to 49. In some embodiments, the peptide of the present invention comprises or consists of one of SEQ ID NOs: 6, 14 to 15, 22 to 26, 33, 36, 40, 43 to 46, and 48 to 49. Preferably, the peptide of the present invention blocks the binding of TSP-1 to its receptor in an assay such as Example 2. In some embodiments, the peptide of the present invention exhibits a blocking rate of 15%, 20%, 25%, 30%, 35%, 40% or higher in an assay such as Example 2.

[0028] In some embodiments, the peptide of the present invention comprises or consists of an amino acid sequence corresponding to one of SEQ ID NOs: 3 to 6, 14, 17, 22, 24 to 26, 31, 33, 36, 40 to 47. In some embodiments, the peptide of the present invention comprises or consists of one of SEQ ID NOs: 3 to 6, 14, 17, 22, 24 to 26, 31, 33, 36, 40 to 47. In some embodiments, the peptide of the present invention comprises or consists of an amino acid sequence corresponding to one of SEQ ID NOs: 3, 6, 14, 22, 24 to 26, 33, 41 to 46. In some embodiments, the peptide of the present invention comprises or consists of one of SEQ ID NOs: 3, 6, 14, 22, 24 to 26, 33, 41 to 46. Preferably, the peptide of the present invention inhibits TSP-1-dependent TGF-β1 activation in an assay such as Example 3, and preferably reduces the activation of TGF-β1 by 25%, 30%, 35% or more compared to a negative control without adding the polypeptide.

[0029] In some embodiments, the peptide of the present invention comprises or consists of an amino acid sequence corresponding to one of SEQ ID NOs: 6, 22, 25-26, 33, 40, 43, 44, 46. In some embodiments, the peptide of the present invention comprises or consists of one of SEQ ID NOs: 6, 22, 25-26, 33, 40, 43, 44, 46. In some embodiments, the peptide of the present invention comprises or consists of SEQ ID NO: 6. In some embodiments, the peptide of the present invention comprises or consists of SEQ ID NO: 22. In some embodiments, the peptide of the present invention comprises or consists of SEQ ID NO: 25. In some embodiments, the peptide of the present invention comprises or consists of SEQ ID NO: 26. In some embodiments, the peptide of the present invention comprises or consists of SEQ ID NO: 33. In some embodiments, the peptide of the present invention comprises or consists of SEQ ID NO: 43.

[0030] The following are some embodiments of the TGF-β1 activation inhibitory peptide of the present invention. 1. Formula (I): R 1 -X 1 -X 2 -X 3 -X 4 -X 5 -X 6 -X 7 -R 2 (I), (wherein, R 1 is acetyl or absent; X 1 is absent or is a naturally occurring or non-naturally occurring amino acid residue; X 2 and X 4 each is independently a naturally occurring or non-naturally occurring amino acid residue; X 3 is D; X 5 is E; X 6 is D; X 7is a sequence consisting of 0 to 7 amino acids; R 2 is an amino acid or absent), a peptide having an amino acid sequence or a pharmaceutically acceptable salt, solvate or prodrug thereof.

[0031] 2.X 1 is absent or selected from homoserine, allothreonine, T and S; X 2 is selected from naturally occurring amino acids or non-naturally occurring amino acids; preferably, selected from polar side chain amino acids such as uncharged polar side chain amino acids S, C, G, N, Q, T, Y or negatively charged polar side chain amino acids D and E; more preferably, selected from amino acids N, C, Q, S, T, E and D; more preferably, selected from amino acids Q, C, N, E and S; X 3 is D; X 4 is selected from minor side chain amino acids such as A, C, G, P, S, T and V, more preferably selected from amino acids A, P and S, more preferably selected from amino acids A and P; X 5 is E; X 6 is D; X 7 is absent or starting from the amino terminus to the carboxyl terminus, Z 1 Z 2 Z 3 SZ 4 is either a sequence of 1, 2, 3, 4, 5, 6 or 7 amino acids continuously selected from the amino acid sequences of LQ; Z 1 Z 2 Z 3 Z 4 are each independently selected from naturally occurring amino acids and non-naturally occurring amino acids, preferably: Z 1 is amino acid N, Q, P, A, L, V, M or I, preferably N, P or L, most preferably N or P; Z2 is amino acid T, S, V, C, A, K or R, preferably T, C or K, more preferably Z 2 When Z is amino acid C or K 2 is amino acid X 2 or X 3 forms a covalent bond with the side chain of Z 3 is amino acid A, V, L or I, preferably A or V Z 4 is amino acid F, Y, H or W, preferably F or H; Optionally, when X 4 is A, 0 to 1 small side chain amino acids such as P or G, preferably P, are inserted between X 3 and X 4 ; Optionally, when X 7 consists of 1 to 5 amino acids, the peptide has 0 to 2 additional amino acid residues added after X, such as 1 to 2 amino acid residues independently selected from Q, E, D and N, or 1 amino acid residue selected from C or K, the peptide or a pharmaceutically acceptable salt, solvate or prodrug thereof according to Embodiment 1. 7 The peptide or a pharmaceutically acceptable salt, solvate or prodrug thereof according to Embodiment 1, which has 0 to 2 additional amino acid residues added after X

[0032] 3. X 1 is absent, the peptide or a pharmaceutically acceptable salt, solvate or prodrug thereof according to Embodiments 1 to 2. 4. X 1 is T or S, the peptide or a pharmaceutically acceptable salt, solvate or prodrug thereof according to Embodiments 1 to 2.

[0033] 5. X 1 is T, the peptide or a pharmaceutically acceptable salt, solvate or prodrug thereof according to Embodiments 1 to 2. 6. X 2 is selected from polar side chain amino acids, the peptide or a pharmaceutically acceptable salt, solvate or prodrug thereof according to Embodiments 1 to 5.

[0034] 7. X 2 is the peptide according to Embodiments 1 to 5, or a pharmaceutically acceptable salt, solvate or prodrug thereof, which is selected from the uncharged polar side chain amino acids S, C, G, N, Q, T and Y.

[0035] 8. X 2 is the peptide according to Embodiments 1 to 5, or a pharmaceutically acceptable salt, solvate or prodrug thereof, which is selected from the negatively charged polar side chain amino acids D and E. 9. X 2 is the peptide according to Embodiments 1 to 5, or a pharmaceutically acceptable salt, solvate or prodrug thereof, which is selected from the amino acids N, C, homocysteine, Q, S, homoserine, T, allothreonine, E and D.

[0036] 10. X 2 is the peptide according to Embodiments 1 to 5, or a pharmaceutically acceptable salt, solvate or prodrug thereof, which is selected from the amino acids Q, C, N, E or S. 11. X 2 is the peptide according to Embodiments 1 to 5, or a pharmaceutically acceptable salt, solvate or prodrug thereof, which is Q.

[0037] 12. X 2 is the peptide according to Embodiments 1 to 5, or a pharmaceutically acceptable salt, solvate or prodrug thereof, which is C. 13. X 2 The side chain of 7 forms a covalent bond with the side chain of the amino acid contained in X 2 Preferably, both X 2 and Z

[0038] 14. X 4is a peptide or a pharmaceutically acceptable salt, solvate or prodrug thereof according to Embodiments 1 to 13, which is a small side-chain amino acid, for example, A, C, G, P, S, T or V.

[0039] 15.X 4 is a peptide or a pharmaceutically acceptable salt, solvate or prodrug thereof according to Embodiments 1 to 13, which is selected from amino acids A, P and S. 16.X 4 is a peptide or a pharmaceutically acceptable salt, solvate or prodrug thereof according to Embodiments 1 to 13, which is A.

[0040] 17.X 4 is a peptide or a pharmaceutically acceptable salt, solvate or prodrug thereof according to Embodiments 1 to 13, which is P. 18.X 7 is a peptide or a pharmaceutically acceptable salt, solvate or prodrug thereof according to Embodiments 1 to 17, which is absent.

[0041] 19.X 7 is Z 1 Z 2 Z 3 SZ 4 is a sequence consisting of 1, 2, 3, 4, 5, 6 or 7 amino acids consecutive from the amino terminus to the carboxyl terminus in the amino acid sequence of LQ, which is a peptide or a pharmaceutically acceptable salt, solvate or prodrug thereof according to Embodiments 1 to 17.

[0042] 20.X 7 is Z 1 Z 2 Z 3 SZ 4 is a sequence consisting of 1, 2, 3, 4 or 5 amino acids consecutive from the amino terminus to the carboxyl terminus in the amino acid sequence of, which is a peptide or a pharmaceutically acceptable salt, solvate or prodrug thereof according to Embodiments 1 to 17.

[0043] 21.X 7 is Z 1The peptide according to any one of Embodiments 1 to 17, or a pharmaceutically acceptable salt, solvate or prodrug thereof. 22.X 7 is Z 1 Z 2 The peptide according to any one of Embodiments 1 to 17, or a pharmaceutically acceptable salt, solvate or prodrug thereof.

[0044] 23.X 7 is Z 1 Z 2 Z 3 The peptide according to any one of Embodiments 1 to 17, or a pharmaceutically acceptable salt, solvate or prodrug thereof. 24. The peptide according to any one of Embodiments 1 to 17, or a pharmaceutically acceptable salt, solvate or prodrug thereof, wherein X 7 is Z 1 Z 2 Z 3 A peptide consisting of S.

[0045] 25. The peptide according to any one of Embodiments 1 to 17, or a pharmaceutically acceptable salt, solvate or prodrug thereof, wherein X 7 is Z 1 Z 2 Z 3 SZ 4 A peptide consisting of. 26. The peptide according to any one of Embodiments 1 to 17, or a pharmaceutically acceptable salt, solvate or prodrug thereof, wherein X 7 is Z 1 Z 2 Z 3 SZ 4 A peptide consisting of L.

[0046] 27.X 7 is Z 1 Z 2 Z 3 SZ 4 The peptide according to any one of Embodiments 1 to 17, or a pharmaceutically acceptable salt, solvate or prodrug thereof, consisting of LQ. 28.Z 1The peptide or a pharmaceutically acceptable salt, solvate or prodrug thereof according to Embodiments 1 to 27, wherein is N, Q, P, A, N-methylalanine, L, norleucine, V, M, N-methylmethionine or I.

[0047] 29.Z 1 The peptide or a pharmaceutically acceptable salt, solvate or prodrug thereof according to Embodiments 1 to 27, wherein is N. 30.Z 1 The peptide or a pharmaceutically acceptable salt, solvate or prodrug thereof according to Embodiments 1 to 27, wherein is P.

[0048] 31.Z 1 The peptide or a pharmaceutically acceptable salt, solvate or prodrug thereof according to Embodiments 1 to 27, wherein is L. 32.Z 2 The peptide or a pharmaceutically acceptable salt, solvate or prodrug thereof according to Embodiments 1 to 31, wherein is T, allothreonine, S, homoserine, V, C, homocysteine, A, N-methylalanine, K or R.

[0049] 33.Z 2 The peptide or a pharmaceutically acceptable salt, solvate or prodrug thereof according to Embodiments 1 to 31, wherein is T. 34.Z 2 is C, and is covalently linked to X by a side chain 2 The peptide or a pharmaceutically acceptable salt, solvate or prodrug thereof according to Embodiments 1 to 31.

[0050] 35.Z 2 is K, and is covalently linked to X by a side chain 3 The peptide or a pharmaceutically acceptable salt, solvate or prodrug thereof according to Embodiments 1 to 31. 36.Z 3The peptide or a pharmaceutically acceptable salt, solvate or prodrug thereof according to Embodiments 1 to 35, wherein is A, N-methylalanine, V, L, norleucine or I.

[0051] 37. Particularly when Z 1 is N or P, Z 3 The peptide or a pharmaceutically acceptable salt, solvate or prodrug thereof according to Embodiments 1 to 35, wherein is A. 38. Z 3 The peptide or a pharmaceutically acceptable salt, solvate or prodrug thereof according to Embodiments 1 to 35, wherein is V.

[0052] 39. Z 4 The peptide or a pharmaceutically acceptable salt, solvate or prodrug thereof according to Embodiments 1 to 38, wherein is an aromatic amino acid. 40. Z 4 The peptide or a pharmaceutically acceptable salt, solvate or prodrug thereof according to Embodiments 1 to 38, wherein is F, N-methylphenylalanine, homophenylalanine, Y, H or W.

[0053] 41. Z 4 The peptide or a pharmaceutically acceptable salt, solvate or prodrug thereof according to Embodiments 1 to 38, wherein is F or H. 42. Z 4 The peptide or a pharmaceutically acceptable salt, solvate or prodrug thereof according to Embodiments 1 to 38, wherein is H.

[0054] 43. X 7 is Z 1 consists of; or Z 1 Z 2 consists of; or Z 1 Z 2 Z 3 consists of; or Z 1 Z 2 Z 3 consists of S; or Z 1 Z 2 Z 3 SZ4 The peptide according to any one of Embodiments 1 to 42, or a pharmaceutically acceptable salt, solvate or prodrug thereof, which consists of

[0055] 44.X 7 is an amino acid sequence selected from: N; P; L; NTA; NTV; PTA; NTAS; PTAS; NTVSF; NTASF; NTASH; NTVSFLQ; the peptide according to any one of Embodiments 1 to 43, or a pharmaceutically acceptable salt, solvate or prodrug thereof.

[0056] 45. The peptide has 0 to 2 additional amino acid residues appended after X 7 ; the peptide according to any one of Embodiments 1 to 44, or a pharmaceutically acceptable salt, solvate or prodrug thereof.

[0057] 46. The peptide contains an amino acid sequence selected from DAED; DPED; DAEDN; DPEDN; or DAEDP; the peptide according to any one of Embodiments 1 to 45, or a pharmaceutically acceptable salt, solvate or prodrug thereof.

[0058] 47.X 1 is T, and X 2 is Q; the peptide according to any one of Embodiments 1 to 46, or a pharmaceutically acceptable salt, solvate or prodrug thereof. 48.R 1 is acetyl; the peptide according to any one of Embodiments 1 to 47, or a pharmaceutically acceptable salt, solvate or prodrug thereof.

[0059] 49.R 2 is amino; the peptide according to any one of Embodiments 1 to 48, or a pharmaceutically acceptable salt, solvate or prodrug thereof. 50. The following general formula (II): R 1 -X 1 -X 2 -D-X 4 -E-D-X 7 -R 2 (II), (wherein, R 1 is acetyl or absent; R 2 is amino or absent; X 1 is T or S; X 2 is selected from polar side chain amino acids; X 4 is selected from amino acids A or P; X 7 is Z 1 TZ 3 SZ 4 is a sequence consisting of 1, 2, 3, 4 or 5 amino acids consecutive from the amino terminus to the carboxyl terminus in the amino acid sequence of Z 1 is N, P or L; Z 3 is A, V, L or I, Z 4 is F, Y, H or W, preferably F or H) a peptide having the amino acid sequence, or a pharmaceutically acceptable salt, solvate or prodrug thereof.

[0060] 51. X 2 is selected from amino acids Q, N, C, S, T, E and D; preferably selected from Q, N, E or S; more preferably, X 2 is Q or N, the peptide according to embodiment 50 or a pharmaceutically acceptable salt, solvate or prodrug thereof.

[0061] 52. X 2 is Q, the peptide according to embodiment 50 or a pharmaceutically acceptable salt, solvate or prodrug thereof. 53. Z 1 is N, and Z 3 is A or V; or Z 1 is P, and Z 3 is A, the peptide according to embodiments 50 to 52 or a pharmaceutically acceptable salt, solvate or prodrug thereof.

[0062] 54.Z 4 The peptide or a pharmaceutically acceptable salt, solvate or prodrug thereof according to Embodiments 50 to 53, wherein it is F or H. 55. The following general formula (III): R 1 -X 1 -cyclo(-C-D-X 4 -E-D-Z 1 -C)-X 8 -R 2 (III) (In the formula, R 1 is acetyl or absent; R 2 is amino or absent; X 1 is T or S; X 4 is selected from minor side chain amino acids such as A, C, G, P, S, T and V, more preferably selected from amino acids A, P and S, and even more preferably selected from amino acids A and P; Z 1 is a naturally occurring amino acid or a non-naturally occurring amino acid; X 8 is absent or is an array consisting of 1 to 5 amino acids) a peptide having an amino acid sequence or a pharmaceutically acceptable salt, solvate or prodrug thereof.

[0063] 56. X 4 The peptide or a pharmaceutically acceptable salt, solvate or prodrug thereof according to Embodiment 55, wherein it is A, C, G, P, S, T or V. 57. X 4 The peptide or a pharmaceutically acceptable salt, solvate or prodrug thereof according to Embodiment 55, wherein it is A.

[0064] 58. X 4 The peptide or a pharmaceutically acceptable salt, solvate or prodrug thereof according to Embodiment 55, wherein it is P. 59. Z1 The peptide or a pharmaceutically acceptable salt, solvate or prodrug thereof according to Embodiments 55 to 58, wherein is a polar uncharged amino acid or a nonpolar amino acid.

[0065] 60.Z 1 The peptide or a pharmaceutically acceptable salt, solvate or prodrug thereof according to Embodiments 55 to 58, wherein is N, Q, P, A, L, V, M or I. 61.Z 1 The peptide or a pharmaceutically acceptable salt, solvate or prodrug thereof according to Embodiments 55 to 58, wherein is N.

[0066] 62.X 8 The peptide or a pharmaceutically acceptable salt, solvate or prodrug thereof according to Embodiments 55 to 61, wherein does not exist. 63.X 8 wherein is Z 3 SZ 4 A peptide consisting of 1, 2, 3, 4 or 5 amino acids consecutive from the amino terminus to the carboxyl terminus in the amino acid sequence of LQ, and 3 wherein is A, V, L or I, and 4 The peptide or a pharmaceutically acceptable salt, solvate or prodrug thereof according to Embodiments 55 to 61, wherein is F, Y, H or W, preferably F or H.

[0067] 64.Z 3 wherein is A or V, and 4 The peptide or a pharmaceutically acceptable salt, solvate or prodrug thereof according to Embodiment 63, wherein is F or H. The peptide or a pharmaceutically acceptable salt, solvate or prodrug thereof according to Embodiments 55 to 64, wherein the peptide consists of 5 to 13 amino acids, preferably 7, 8, 9, 10 or 11 amino acids.

[0068] 66. The peptide consists of 5 to 13 amino acids, preferably 7, 8, 9, 10 or 11 amino acids, and is the peptide or a pharmaceutically acceptable salt, solvate or prodrug thereof described in Embodiments 1 to 54.

[0069] 67. The peptide is a linear peptide, and is the peptide or a pharmaceutically acceptable salt, solvate or prodrug thereof described in Embodiment 66. 68. The peptide is a cyclic peptide, and preferably the cyclized region of the peptide contains the core motif (DXED), and is the peptide or a pharmaceutically acceptable salt, solvate or prodrug thereof described in Embodiment 66.

[0070] 69. The peptide is head-to-tail cyclized or side-chain-to-side-chain cyclized, and is the peptide or a pharmaceutically acceptable salt, solvate or prodrug thereof described in Embodiment 68.

[0071] 70. The peptide is head-to-tail cyclized, and is the peptide or a pharmaceutically acceptable salt, solvate or prodrug thereof described in Embodiment 68. 71. The peptide is side-chain-to-side-chain cyclized, and is the peptide or a pharmaceutically acceptable salt, solvate or prodrug thereof described in Embodiment 68.

[0072] 72. The peptide is cyclized by coupling the side chains of the amino acids at positions 3 and 8 or by coupling the side chains of the amino acids at positions 2 and 8, and is the peptide or a pharmaceutically acceptable salt, solvate or prodrug thereof described in Embodiment 68.

[0073] 73. The peptide is cyclized by a lactam bond formed between amino acid side chains, and is the peptide or a pharmaceutically acceptable salt, solvate or prodrug thereof described in Embodiments 71 to 72.

[0074] 74. The peptide according to embodiments 71 to 72, or a pharmaceutically acceptable salt, solvate or prodrug thereof, wherein the peptide is cyclized by a disulfide bond formed between amino acid side chains.

[0075] 75. The peptide is cyclized by a side chain, X 3 is D, and Z 2 is K, and the peptide is cyclized by a lactam bond formed between the side chains of X 3 and Z 2 ; or X 2 is C, and Z 2 is C, and the peptide is cyclized by a disulfide bond formed between the side chains of X 2 and Z 2 ; Preferably, when Z 2 is amino acid C or K, X 7 is Z 1 Z 2 consists of, and X 1 is T or S. The peptide according to embodiments 71 to 74, or a pharmaceutically acceptable salt, solvate or prodrug thereof.

[0076] 76. The peptide has an amino acid sequence corresponding to any one selected from SEQ ID NOs: 1 to 49, or contains or consists of any one selected from SEQ ID NOs: 1 to 49. The peptide, or a pharmaceutically acceptable salt, solvate or prodrug thereof.

[0077] 77. The peptide has an amino acid sequence corresponding to any one selected from SEQ ID NOs: 6, 22, 25, 26, 33, 40, 43, 44 and 46, or contains or consists of any one selected from SEQ ID NOs: 6, 22, 25, 26, 33, 40, 43, 44 and 46. The peptide, or a pharmaceutically acceptable salt, solvate or prodrug thereof.

[0078] 78. A peptide or a pharmaceutically acceptable salt, solvate or prodrug thereof, wherein the peptide comprises or consists of SEQ ID NO: 6. 79. A peptide or a pharmaceutically acceptable salt, solvate or prodrug thereof, wherein the peptide comprises or consists of SEQ ID NO: 22.

[0079] 80. A peptide or a pharmaceutically acceptable salt, solvate or prodrug thereof, wherein the peptide comprises or consists of SEQ ID NO: 25. 81. A peptide or a pharmaceutically acceptable salt, solvate or prodrug thereof, wherein the peptide comprises or consists of SEQ ID NO: 26.

[0080] 82. A peptide or a pharmaceutically acceptable salt, solvate or prodrug thereof, wherein the peptide comprises or consists of SEQ ID NO: 33. 83. A peptide or a pharmaceutically acceptable salt, solvate or prodrug thereof, wherein the peptide comprises or consists of SEQ ID NO: 40.

[0081] 84. A peptide or a pharmaceutically acceptable salt, solvate or prodrug thereof, wherein the peptide comprises or consists of SEQ ID NO: 43. 85. A peptide or a pharmaceutically acceptable salt, solvate or prodrug thereof, wherein the peptide comprises or consists of SEQ ID NO: 44.

[0082] 86. A peptide or a pharmaceutically acceptable salt, solvate or prodrug thereof, wherein the peptide comprises or consists of SEQ ID NO: 46. 87. The peptide has 1, 2, 3, 4, or 5 amino acid differences, preferably 1 or 2 amino acid substitutions, deletions, or additions, compared to the peptide according to one of Embodiments 76 to 86, and preferably, for example, in the case of conservative amino acid substitutions, the substitution occurs at an amino acid residue other than the core motif (DXED), a peptide or a pharmaceutically acceptable salt, solvate, or prodrug thereof.

[0083] 88. The peptide is chemically modified, the peptide according to Embodiments 1 to 87 or a pharmaceutically acceptable salt, solvate, or prodrug thereof. 89. The peptide contains PEG modification, the peptide according to Embodiments 1 to 88 or a pharmaceutically acceptable salt, solvate, or prodrug thereof.

[0084] 90. The peptide contains lipid modification, the peptide according to Embodiments 1 to 89 or a pharmaceutically acceptable salt, solvate, or prodrug thereof. 91. The peptide contains D - type amino acid substitution and / or non - naturally occurring amino acid substitution, the peptide according to Embodiments 1 to 90 or a pharmaceutically acceptable salt, solvate, or prodrug thereof.

[0085] Method for preparing the inhibitory peptide of the present invention In one aspect, the present invention provides a method for preparing the inhibitory peptide of the present invention. The preparation of the peptide of the present invention can be carried out using chemical synthesis methods, fermentation, or genetic recombination techniques.

[0086] Methods for chemically synthesizing peptides are well - known in the art, and there are solid - phase synthesis and liquid - phase synthesis techniques. The desired peptide chain can be synthesized using the solid - phase polypeptide synthesis (SPPS) technique developed by Merrifield. To perform the synthesis, an automated peptide synthesizer can also be used. The subsequent purification of the SPPS peptide product is simpler and easier than that of peptides synthesized recombinantly. Therefore, it is preferable to apply the SPPS technique to synthesize the short peptides of the present invention.

[0087] Generally, the SPPS technique consists of cycles of coupling and deprotection. In the coupling step in the Fmoc synthesis strategy, the Fmoc-protected amino acid (Fmoc-AA-OH) is coupled with the solid polymer resin by applying a condensing agent such as HBTU / HATU / DIC. In the deprotection step, the protecting group Fmoc is removed from the amino acid by a deprotecting agent such as piperidine, releasing the amino group and then proceeding to the next coupling and deprotection cycle. The entire process can be carried out in a screening reactor until the final peptide is synthesized and cleaved from the resin. Thereafter, the synthesized peptide product can be purified and detected by high performance liquid chromatography.

[0088] Various resins such as 4-methylbenzhydrylamine (HMBA) resin, Wang resin, 2-chlorotrityl chloride (CTC) resin and Merrifield resin can be used for the solid-phase synthesis of peptides. In addition, various functional resins have been developed by coupling the resin to different linkers to enable peptide cyclization within the solid phase.

[0089] In one embodiment, the present invention provides a method for preparing a peptide of the present invention, comprising: (1) producing a linear peptide having a defined sequence by successively and continuously linking amino acids one by one in the presence of a condensing agent and under alkaline conditions, using, for example, 4-methylbenzhydrylamine (HMBA) resin as a starting material, Fmoc-protected amino acids as monomers, and a hexahydropyridine / DMF solution as a deprotecting reagent; (2) optionally, cyclizing the linear peptide obtained in step (1); and (3) purifying the peptide product obtained in step (1) or (2).

[0090] In another embodiment, the present invention provides a method for preparing a peptide of the present invention, comprising: (1) recombinantly expressing a linear peptide having a defined sequence; (2) optionally, cyclizing the linear peptide obtained in step (1); and (3) purifying the peptide product obtained in step (1) or (2). In some aspects, the present invention also provides a nucleic acid encoding the amino acid sequence of the peptide of the present invention, a vector (e.g., an expression vector), and a host cell containing the nucleic acid.

[0091] Peptide cyclization is a common peptide modification technique that includes various strategies such as head-to-tail cyclization, side-chain-to-side-chain cyclization, and backbone-to-side-chain cyclization. Typically, a single linear peptide is overly flexible when not linked to other peptides. In contrast, cyclization can promote the formation of secondary structure within the peptide by constituting intramolecular interactions, thereby improving the stability of the peptide.

[0092] In one embodiment according to the present invention, the peptide of the present invention forms a monocyclic peptide by head-to-tail cyclization. In another embodiment according to the present invention, the peptide of the present invention forms a lactam bridge between the side chain of glutamic acid (E) or aspartic acid (D) and lysine (K) by side chain cyclization. In another embodiment according to the present invention, the peptide of the present invention forms a disulfide bond between the side chains of two cysteine (C) residues by side chain cyclization. In an embodiment of the cyclic peptide of the present invention containing a lactam bridge, preferably, the lysine amino acid residue is located at the C-terminus of the core sequence (DXED) of the present invention and forms a lactam bridge with the first residue aspartic acid D of the core sequence (DXED); more preferably, the K residue is at least 4 residues, for example, 4, 5 or 6 residues away from the D residue, and preferably, the lysine residue is the last residue at the C-terminus of the peptide of the present invention. In an embodiment of the cyclic peptide of the present invention containing a disulfide bond, preferably, the two cysteine residues for cyclization are located at the N-terminus and C-terminus of the core sequence (DXED) respectively, and more preferably, the two cysteine residues are at least 5 residues, for example, 5, 6, 7, 8 residues apart. In a preferred embodiment, the amino acid at the 3rd position of the peptide of the present invention is D, the amino acid at the 8th position is K, and the peptide of the present invention is cyclized by the formation of a lactam bond through their side chains. In another preferred embodiment, the amino acid at the 2nd position of the peptide of the present invention is C, the amino acid at the 8th position is C, and the peptide of the present invention is cyclized by the formation of a disulfide bond through their side chains.

[0093] After the peptide is synthesized, it can be modified with or without using pharmaceutical chemistry techniques. In some cases, modifying the peptide can be advantageous, for example, by mimicking, stabilizing or constructing a more appropriate secondary structure to improve the biological activity of the peptide drug and / or to improve the selectivity, stability and solubility of the peptide drug. Peptide modification may be an amino acid substitution or residue modification of a non-essential amino acid and / or a modification at the N-terminus and / or C-terminus of the peptide, such as N-terminal acetylation and C-terminal amidation. The modification may also be a substitution of an important amino acid residue that affects the biological activity of the peptide to explore changes in activity.

[0094] In some cases, the peptide may contain non-naturally occurring amino acids. Optionally, the peptide may contain 1, 2, 3, 4, 5 or 6 or more non-naturally occurring amino acids. Alternatively, each of the amino acids contained in the peptide may be independently selected from naturally occurring amino acids.

[0095] Chemically synthesized peptides often possess free amino groups and free carboxyl groups. In some cases, the ends of the peptide can be blocked, i.e., N-terminal acetylation and C-terminal amidation, to create a synthetic peptide that more closely mimics a natural protein and improve the stability of the peptide.

[0096] Thus, in some embodiments, the peptides of the present invention may include D-amino acids, non-naturally occurring amino acids, amino acid analogs and / or group substitutions and modifications; or may be linked to a polymer or drug carrier.

[0097] Pharmaceutical composition In one aspect, the present invention also provides a pharmaceutical composition comprising the peptide of the present invention or a pharmaceutically acceptable salt, solvate or prodrug thereof. As is well understood in the art, the pharmaceutical composition may further optionally comprise suitable pharmaceutical adjuvants, pharmaceutical carriers, pharmaceutical excipients, including buffers.

[0098] Routes of administration for peptide delivery include, but are not limited to, subcutaneous administration, intramuscular administration, intravenous administration, mucosal administration (e.g., nasal administration, pulmonary mucosal administration, sublingual administration), oral administration (e.g., with the addition of gastroenteric proosmotic agents or carriers), and transdermal administration. The peptide can be formulated into injections, for example, any formulation suitable for administration such as intravenous injection or infusion, lyophilized powder, etc. Auxiliary materials for use in different dosage forms are known in the art.

[0099] As is well understood in the art, the pharmaceutical composition may further comprise another therapeutic agent beneficial for the particular disease being treated. Thus, in some embodiments, the present invention also provides a pharmaceutical composition comprising the peptide of the present invention or a pharmaceutically acceptable salt, solvate or prodrug thereof, and further comprising another therapeutic agent beneficial for the particular disease being treated. In such embodiments, the peptide of the present invention may be included in the same or a different composition as the other therapeutic agent; it may be administered in parallel with, sequentially or in any order with, and using any dosing regimen, as the other therapeutic agent.

[0100] In some embodiments, the pharmaceutical composition of the present invention particularly comprises the peptide of the present invention or a pharmaceutically acceptable salt thereof, particularly the peptide of the present invention or a pharmaceutically acceptable salt thereof according to one of the preceding embodiments 76 - 86, particularly the peptide of the present invention or a pharmaceutically acceptable salt thereof according to one of the preceding embodiments 78 - 86.

[0101] Methods of treatment and uses In one aspect, the present invention provides a peptide of the present invention or a pharmaceutically acceptable salt, solvate or prodrug thereof, or a pharmaceutical composition of the present invention for use as a therapy for preventing or treating TGF-β related diseases in a subject, as well as a method for treating and preventing said diseases.

[0102] As used herein, the term "TGF-β related disease" refers to diseases and disorders associated with and / or caused by TGF-β1 activation or pathological increase in TGF-β1 activity involving TSP-1, including but not limited to fibrosis, chronic inflammation and cancer. TGF-β related diseases would benefit from the inhibitory effect on TSP-1 related TGF-β1 activation.

[0103] As used herein, the term "subject" or "patient" or "individual" includes any human or non-human animal. The term "non-human animal" includes all vertebrates such as mammals and non-mammals, non-human primates, sheep, dogs, cats, horses, cows, chickens, amphibians, reptiles, etc. Preferably, the subject according to the present invention is a human.

[0104] As used herein, the term "effective amount" or "effective dosage" refers to an amount of a peptide of the present invention, or a pharmaceutically acceptable salt, solvate or prodrug thereof, which, when administered to a cell, tissue or subject alone or in combination with other therapeutic agents, is effective to prevent or alleviate the symptoms of one or more diseases or disorders, or the progression of a disease or disorder. An effective dosage also refers to an amount sufficient to improve symptoms, such as an amount sufficient to treat, cure, prevent or ameliorate a related medical condition, or to increase the rate of treating, curing, preventing or ameliorating the condition. When the active ingredient is administered alone to an individual, an effective dosage refers only to that ingredient. When administered in combination, an effective amount refers to the combined amount of the active ingredients that produces a therapeutic or preventive effect, whether administered concurrently, sequentially or simultaneously. In some embodiments, the effective amount will result in at least a 10%, typically at least a 20%, preferably at least about 30%, more preferably at least 40%, and most preferably at least 50% improvement in a diagnostic criterion or parameter.

[0105] In some embodiments described herein, "treatment" of a disease or disorder means ameliorating the disease or disorder (i.e., slowing, inhibiting or reducing at least one of the progression of the disease or the clinical symptoms). In some other embodiments, "treatment" refers to reducing or ameliorating at least one physical parameter, including physiological parameters that may not be distinguishable in a patient. In some other embodiments, "treatment" refers to physically improving (e.g., stabilizing a distinguishable symptom), physiologically improving (e.g., stabilizing a physical parameter), or both, a disease or disorder. Unless specifically and explicitly stated otherwise herein, methods for assessing the treatment and / or prevention of a disease are generally known in the art.

[0106] In a further embodiment according to the present invention, "prevention" of a disease or disorder includes inhibition of the onset or progression of the disease or disorder or of specific symptoms of the disease or disorder. Typically, in the context of fibrosis or cancer, the term "prevention" refers to administering a drug, particularly to a subject at risk of developing the disease, before the signs or symptoms of fibrosis or cancer occur.

[0107] Accordingly, in some embodiments, the present invention provides a method for preventing or treating a TGF-β related disease, the method comprising administering to a subject in need thereof an effective amount of a peptide according to the present invention or a pharmaceutically acceptable salt, solvate or prodrug thereof or a pharmaceutical composition according to the present invention. Optionally, the method further comprises administering to the subject an effective amount of a second therapeutic agent such as a chemotherapeutic agent or an immunotherapeutic agent. Preferably, the TGF-β related disease treated by the method of the present invention has macrophage infiltration at the lesion site. In some embodiments, the TGF-β related disease is associated with tissue damage, inflammation or fibrosis. In some embodiments, the TGF-β related disease is related to the metastasis of tumor cells.

[0108] In some embodiments of the preventive or therapeutic method of the present invention, a peptide according to the present invention or a pharmaceutically acceptable salt thereof, particularly a peptide according to the present invention or a pharmaceutically acceptable salt thereof of one of the preceding embodiments 76 - 86, particularly a peptide according to the present invention or a pharmaceutically acceptable salt thereof of one of the preceding embodiments 78 - 86, may be particularly used.

[0109] Treatment of fibrotic disorders In some embodiments, the disorder associated with a pathological increase in TGF-β activity is fibrosis. In one embodiment, the present invention provides a peptide according to the present invention or a pharmaceutically acceptable salt, solvate or prodrug thereof or a pharmaceutical composition according to the present invention for use as a therapy for preventing or treating a fibrotic disorder in a subject, as well as corresponding therapeutic and prophylactic methods.

[0110] A wide range of diseases are now recognized to exhibit fibrotic characteristics. These diseases are classified based on the tissues and organs affected by fibrosis, such as the lung, liver, kidney, bone marrow, and other tissues, or by systemic conditions, such as chronic inflammatory diseases, tumor diseases, and immunodeficiencies. The presence of fibrosis as a pathological feature has become an important factor in redefining and further naming the diagnosis and treatment of fibrotic disorders, going beyond the limitations of conventional classifications and findings based on disease location.

[0111] Accordingly, as used herein, "fibrotic disorder" refers to a disease characterized by a fibrotic pathological phenotype and is not limited to the specific site where the disease develops or to the conventional classification of the disease. Examples of fibrotic disorders that can be treated by the methods of the present invention include, but are not limited to, fibrosis of various tissues and organs, such as pulmonary fibrosis, hepatic fibrosis, renal fibrosis; as well as chronic inflammatory, tumor, and immune disorders.

[0112] Accordingly, in some embodiments, the present invention provides a method for preventing or treating a fibrotic disorder in a subject, the method comprising administering to a subject in need thereof a prophylactically or therapeutically effective amount of a peptide according to the present invention or a pharmaceutically acceptable salt, solvate, or prodrug thereof or a pharmaceutical composition according to the present invention. In some embodiments, the fibrotic disorder is hepatic fibrosis, pulmonary fibrosis, renal fibrosis, myelofibrosis, dermal fibrosis, or cardiac fibrosis.

[0113] In some embodiments, the fibrotic disorder is pulmonary fibrosis, such as idiopathic pulmonary fibrosis (IPF). In some embodiments, the method comprises administering the peptide of the present invention to the subject by inhalation, for example, as a nebulized formulation.

[0114] The therapeutic uses and methods described herein may also include co-formulating and / or co-administering the peptide of the present invention or a pharmaceutically acceptable salt, solvate, or prodrug thereof or a pharmaceutical composition with another therapeutically effective agent for preventing and / or treating a pathological fibrotic disease.

[0115] Treatment of tumors In some embodiments, the disorder associated with a pathological increase in TGF-β activity is cancer. In one embodiment, the present invention provides a peptide of the present invention or a pharmaceutically acceptable salt, solvate or prodrug thereof or a pharmaceutical composition for use as a therapy for preventing or treating cancer in a subject, as well as corresponding treatment and prevention methods. In some embodiments, the cancer is a solid tumor, preferably selected from: lung cancer, liver cancer, breast cancer, uterine cancer, prostate cancer, pancreatic cancer, colon cancer, skin cancer, central nervous system cancer, fibromyoma, fibroma, fibroadenoma and fibrosarcoma. In some embodiments, the cancer is sarcoma, pancreatic cancer, glioblastoma, head and neck cancer, melanoma, breast cancer or colorectal cancer. In some embodiments, the cancer is selected from squamous cell carcinoma, epidermoid carcinoma, urothelial carcinoma, adenocarcinoma, adrenocortical carcinoma, basal cell carcinoma, ductal carcinoma in situ (DCIS), invasive pancreatic ductal carcinoma, thymic carcinoma and renal cell carcinoma.

[0116] In some embodiments, the method comprises administering to the subject the peptide of the present invention or a pharmaceutically acceptable salt, solvate or prodrug thereof or a pharmaceutical composition orally, intravenously, intratumorally, transdermally, subcutaneously or topically. In some embodiments, the method comprises contacting a cancerous tissue of the subject (e.g., cancerous skin tissue) with a formulation comprising a therapeutically effective amount of the peptide of the present invention or a pharmaceutically acceptable salt, solvate or prodrug thereof or a pharmaceutical composition for a time sufficient to treat the cancer.

[0117] The therapeutic uses and methods described herein may also include co-formulating and / or co-administering the peptide of the present invention or a pharmaceutically acceptable salt, solvate or prodrug thereof or a pharmaceutical composition with another therapeutically effective agent for preventing and / or treating cancer.

[0118] Other uses In some aspects, the present invention: - blocking the binding of CD36 to TSP-1; - inhibiting TSP-1-dependent activation of TGF-β1; - reducing the amount of active TGF-β1 in fibrotic tissue; - inhibiting downstream signaling mediated by TGF-β1; - reducing collagen deposition in fibrotic tissue; - inhibiting inflammatory and / or fibrotic lesions mediated by TGF-β1; - inhibiting the expression of extracellular matrix-related genes stimulated by TGF-β1; - inhibiting the migration of tumor cells; - for preventing or treating TGF-β1-related diseases, especially fibrosis or cancer; alternatively, in the manufacture of a medicament for the above uses, also provided is the use of the peptide of the present invention or a pharmaceutically acceptable salt, solvate or prodrug thereof.

[0119] The above uses may be in vitro or in vivo. In a preferred embodiment, the use is an in vivo use for the treatment or prevention of a disease. The method for in vivo use comprises administering a therapeutically effective amount of the peptide of the present invention or a pharmaceutically acceptable salt, solvate or prodrug thereof, optionally in combination with another therapeutic agent.

[0120] In the prevention or treatment of a disease, the appropriate dosage of the peptide of the present invention or a pharmaceutically acceptable salt, solvate or prodrug thereof (when used alone or in combination with one or more other therapeutic agents) will depend on the type of disease being treated, the type of particular drug, the severity and course of the disease, whether the administration is for prophylactic or therapeutic purposes, the treatment history, the clinical history of the patient and the response to the dosage, as well as the judgment of the attending physician. The drug can be appropriately administered to the patient as a single treatment or in multiple treatments.

[0121] In a further aspect, the present invention also provides the use of the peptide of the present invention or a pharmaceutically acceptable salt, solvate or prodrug thereof for the manufacture of a medicament for use in the aforementioned method (for example, for therapeutic use).

[0122] The following examples are provided to assist in understanding the present invention. However, it should be understood that these examples are not intended to limit the scope of protection of the present invention in any way and should not be construed as constituting the present invention.

[0123] In the present invention, when referring to the amino acid residues herein, the following amino acid codes including the three-letter and one-letter codes are applicable.

[0124]

Table 2

Examples

[0125] Example 1 Polypeptide Preparation The polypeptide used in the examples of the present invention was prepared by Fmoc (9-fluorenylmethoxycarbonyl) solid-phase synthesis. Using the carboxy-terminal amino acid of the linear peptide as the starting point of the synthesis, Rink Amide MBHA Resin (Gill Biochemistry, 49101) was first immersed in a DCM solution for 30 minutes in a polypeptide solid-phase synthesis reaction column to activate it, then pump-dried, and a 20% hexahydropyridine + 80% DMF solution was added under nitrogen gas with stirring and shaking for 30 minutes to carry out a deprotection reaction. The synthesis reaction was carried out by supplying Fmoc-protected amino acids, a condensing agent and an organic base. After the reaction, the ninhydrin colorimetric method was performed to detect whether the reaction was complete. According to the amino acid sequence (from the carboxyl terminus to the amino terminus) of each peptide, the above supply, reaction and detection steps were repeated until the last amino acid, and then the desired peptide was cleaved from the resin to obtain a crude product.

[0126] The cyclic peptides were prepared as follows: (1) Disulfide cyclization: The linear polypeptide synthesized as described above and cleaved from the resin was cyclized in an aqueous solution (peptide concentration 1 g / L) at pH = 7.5 - 8 with stirring for more than 12 hours.

[0127] (2) Side-chain cyclization: During the synthesis of the above-mentioned linear polypeptide, special side-chain protecting groups such as D(oall) and K(alloc) were selected to protect the side-chain carboxyl group of D and the side-chain amino group of K. At the end of the synthesis of the linear polypeptide sequence, -oall / -alloc was removed from the resin, and cyclization was carried out using the corresponding condensing agent.

[0128] (3) Head-to-tail cyclization: The linear polypeptide synthesized and cleaved from the resin as described above was prepared into a fully protected polypeptide fragment, and the corresponding condensing agent in DMF or other solvents was added for cyclization. At the end of the reaction, the product was transferred from water, dried, and the protecting groups on the cyclic peptide were removed to obtain the deprotected head-to-tail cyclized polypeptide as a crude product.

[0129] The synthesized linear and cyclic crude peptides were separated and purified by HPLC on a C18 column using 0.1% TFA / acetonitrile solution as eluent A and 0.1% TFA / aqueous solution as eluent B. Fractions from the main peak were collected, the product was detected by mass spectrometry, and after confirming that it had a molecular weight consistent with the theoretical value, it was lyophilized to obtain the purified peptide. The sequences of the synthesized polypeptides are shown in Table 1. The peptides represented by each sequence number therein consist of the "sequence" and "modification" provided in the description of the sequence.

[0130]

Table 3

[0131] Example 2 In vitro target blocking activity assay of polypeptidesThe ELISA assay was performed to detect the ability of the polypeptide to block the binding of TSP-1 protein to CD36, a TSP-1 receptor protein. TSP-1 protein (novoprotein, catalog number CU45) was diluted to 2 μg / mL using 100 mM carbonate buffer and added to the sample wells of a 96-well ELISA plate (Thermo, catalog number 437111) (100 μL / well), and incubated overnight at 4°C; after washing the plate twice with 1xPBST washing solution, 200 μL of 1.5% BSA (Sigma, catalog number B2064) was added to each well and incubated at 37°C for 2 hours; after washing the plate three times with 1xPBST washing solution, the analyte [100 μL / well, three replicate wells; in the control group, 0.25 μg / mL CD36 protein with Fc tag (R&D Systems, catalog number 1955-CD-050); in the polypeptide treatment group, a mixture containing CD36 protein with Fc tag and polypeptide at final concentrations of 0.25 μg / mL and 100 μM, respectively] was added and incubated at 37°C for 3 hours. After washing the plate five times with 1xPBST washing solution, a 1:500 dilution solution of FITC-labeled anti-Fc tag antibody (Beyotime, catalog number A0556) was added and incubated at 37°C for 1 hour; after washing the plate five times with 1xPBST washing solution, the intensity of the FITC fluorescence signal in each sample well was detected using a multifunctional ELISA instrument (TECAN, model: Infinite 200 Pro). The ability of the polypeptide to block the binding of CD36 to TSP-1 protein (Table 2-1) was evaluated by calculating the ratio of the fluorescence signal of the polypeptide treatment group to the fluorescence signal of the control group, which enables the determination of the blocking rate of the polypeptide that inhibits the binding of CD36 to TSP-1 protein, i.e., (fluorescence signal of the control group - fluorescence signal of the polypeptide treatment group) / fluorescence signal of the control group × 100%.Based on the results of the blocking rate obtained at a single concentration and the molecular characteristics of the polypeptide, the preferred polypeptide molecules were assayed for concentration dependence (0 μM, 0.5 μM, 1 μM, 5 μM, 10 μM, and 100 μM), and the IC of the polypeptide for blocking the binding of CD36 to the TSP-1 protein. 50 The value was determined to further evaluate the ability of the preferred polypeptide molecules to block the binding of CD36 to TSP-1 (Table 2-2). The assay results showed that all of the polypeptides of the present invention inhibited the binding of CD36 to TSP-1.

[0132]

Table 4

[0133]

Table 5

[0134] Example 3 Inhibitory assay of polypeptide against TSP-1-dependent TGF-β activation in a cell-based model In this example, the inhibitory effect of the polypeptide against TSP-1-dependent TGF-β activation was detected using a model based on high TSP-1-expressing cells. In the assay, THP-1 cells (American Type Culture Collection, ATCC, TIB-20) were induced by phorbol ester (phorbol 12-myristate 13-acetate, PMA) (Sigma, catalog number P1585) and differentiated into macrophage-like cells with a unique macrophage expression profile. In the cell-based model, the expression of the TSP-1 protein was significantly increased, and L-TGF-β was secreted; in the absence of factors that prevent the activation of L-TGF-β, the fibrinolytic enzyme secreted by the cells recognized and cleaved the TSP-1 / L-TGF-β complex bound to the CD36 receptor on the cell surface, resulting in an increase in the production of active TGF-β.

[0135] The experiment was conducted as follows: THP-1 cells were seeded in a 96-well plate, treated with 300 nM PMA to differentiate and adhere to form macrophage-like cells. 100 μM of the polypeptide was added [in the negative control group, an equal volume of PBS (the solvent of the polypeptide) was added, and in the positive control group, the polypeptide shown in SEQ ID NO: 50 was administered at the same concentration]. The cells were treated for 24 hours, and then the 293 reporter cell line, 293-TGFβRes cell line (novoprotein, catalog number XCC03-1) was added to detect TGF-β activity, and they were co-cultured for 24 hours. Bio-Lite Luciferase Assay lysate (Vazyme, catalog number DD1201-01) containing a fluorescent substrate was added, and the fluorescence intensity was detected using a multifunctional microplate reader (TECAN, model Infinite 200 Pro). The inhibitory effect of the polypeptide on the activation of TGF-β was evaluated by calculating the ratio of the fluorescence signal of the polypeptide-treated group to the fluorescence signal of the negative control group. The experimental results are shown in Table 3. In this cell-based model, the polypeptide exhibited varying degrees of inhibition against the activation of TGF-β, indicating that the polypeptide of the present invention inhibits the activation of TSP-1-dependent TGF-β.

[0136]

Table 6

[0137] Example 4 Inhibitory assay of polypeptide on active TGFβ levels in a mouse model of fibrotic disease This example was to assess the inhibitory effect of the polypeptide on the active TGF-β levels during the progression of fibrotic disease. In this example, a mouse model of pulmonary fibrosis induced by bleomycin (BLM) was used. The BLM animal model offers advantages including mimicking the pathological features of the primary disease closely, ease of use, and good reproducibility. The animal model is the most representative model for studying the mechanism of fibrosis and is recommended for the assessment of drug efficacy.

[0138] In this example, male C57 / 6J mice, 6 - 8 weeks old and weighing 18 - 20 g, were used as the research subjects. The experimental animals were randomized into groups of 8 animals per group. In this model, the expression of profibrotic factors was detected at the end point of the experiment (day 8). In this example, an aerosol intratracheal quantitative dosing applicator (Yuyan, model YAN30012) was used for intratracheal administration in the modeling and drug delivery process. The dosing was carried out as follows (An Official American Thoracic Society Workshop Report: Use of Animal Model for the Preclinical Assessment of Potential Therapies for Pulmonary Fibrosis, Am J Respir Cell Mol Biol Vol. 56, No. 5, pp. 667 - 679, May 2017). The experimental animals were anesthetized with aerosolized isoflurane (RWD, lot number 22041701) using a small animal anesthetic machine (RWD, model R500IE). The animals in an appropriate anesthetic state were placed supine on the operating table with their heads up and their bodies tilted (about 60°) and fixed with their incisors. The operator gently lifted the tongue using forceps for holding tissue pieces in the right hand and pushed it outward and upward to open the animal's lower jaw. Holding a laryngoscope (Yuyan, model SR310 - RW) in the left hand, the operator slid the blade in the shape of its anterior lobe along the midline of the tongue into the oral cavity until it reached the glottis, and gently pressed the body and the base of the tongue upward. The injection needle of the drug applicator was then inserted through the mouth into the glottis together with the laryngoscope, and 50 μL of the liquid was quickly injected. On the day of modeling (day 1), the experimental animals were randomly assigned to groups, and a bleomycin solution (HanHui Pharmaceutical, batch number 20080511) at a dose of 0.7 USP / kg was administered via the tracheal route to induce the model. On day 7, each peptide treatment group was administered a single intratracheal dose of 50 μL of the peptide drug (a dosing amount of 3 mg / kg), and the model group was given an equal volume of solvent (physiological saline, Kelong, batch number L221062501) as a control.At 24 hours after drug administration, the animals were dissected to collect lung tissues, and the tissues were pulverized in liquid nitrogen to extract total proteins from the lung tissues. The active TGF-β content in the lung tissues of mice from each group was measured using a TGF-β ELISA kit (BOSTER, catalog number EK0515). The ratio of the active TGF-β content between the peptide treatment group and the model group was calculated to evaluate the effect of the peptide on the active TGF-β level. The results are shown in Table 4. In the fibrotic disease model, the peptide treatment group showed various degrees of decrease in the active TGF-β level compared with the model group, indicating that the peptide of the present invention has an inhibitory effect on the increase in the active TGF-β level induced by lung injury during the progression of fibrosis.

[0139]

Table 7

[0140] Example 5 Inhibitory assay of polypeptide on the signal transduction pathway downstream of TGFβ in a mouse model of fibrotic disease This example was intended to evaluate the inhibitory effect of the polypeptide on the signal transduction pathway downstream of TGF-β during the progression of fibrosis. TGF-β intracellular signal transduction utilizes phosphorylation events of Smad family proteins. An increase in the phosphorylation level of Smad2 / 3 is an important indicator that signal transduction has occurred in the TGF-β signal transduction pathway.

[0141] In this example, male C57 / 6J mice aged 6 - 8 weeks with a body weight of 18 - 20 g were used as the research subjects. The experimental animals were randomized into groups of 8 animals per group. In this example, as described in Example 4, intratracheal administration was performed using an aerosol intratracheal metered - dose applicator both during modeling and the drug delivery process. On the day of modeling (day 1), the experimental animals were randomly assigned to groups, and a dose of 0.7 USP / kg of bleomycin solution (HanHui Pharmaceutical, batch number 20080511) was administered via the tracheal route to induce the model. On day 7, each peptide - treated group was administered a single intratracheal dose of the peptide drug at a dosage of 3 mg / kg, and the BLM model group was given an equal volume of solvent (physiological saline) as a control. The animals were dissected 24 hours after drug administration, lung tissues were collected, and frozen in liquid nitrogen for future use. Total protein from the lung tissues was extracted after grinding the tissues in liquid nitrogen, and the total P - Smad2 / 3 content in the tissues was measured by using a P - Smad2 / 3 ELISA kit (CST, catalog number 12001C). The ratio of the P - Smad2 / 3 content between the peptide - treated group and the model group was calculated to evaluate the effect of the peptide on TGFβ signaling. The results are shown in Table 5. In the fibrotic disease model, the peptide - treated groups showed varying degrees of decrease in the P - Smad2 / 3 protein level compared with the model group, indicating that the peptides of the present invention have varying degrees of inhibitory effects on the signal transduction of the TGFβ signaling pathway during the progression of fibrosis.

[0142]

Table 8

[0143] Example 6 Inhibitory assay of polypeptide on collagen deposition in a mouse model of fibrotic disease This example was to evaluate the inhibitory effect of the polypeptide on collagen deposition in fibrotic tissue. In this example, male C57 / 6J mice, 6-8 weeks old and weighing 18-20 g, were used as the research subjects. The experimental animals were randomized into groups of 8 animals per group. On the day of modeling (day 1), a bleomycin solution at a dose of 0.7 USP / kg was administered via the tracheal route to induce the model. Treatment dosing started on day 8. In each peptide treatment group, the peptide drug was administered intratracheally at a dosing amount of 3 mg / kg, twice a week for a total of 4 doses. In the BLM model group, an equal volume of solvent (physiological saline) was administered as a control. The administration procedure followed the procedure described in Example 4. Day 21 was set as the experimental endpoint for evaluating the degree of fibrosis in the model.

[0144] On the 21st day, the animals were dissected to collect lung tissues. After pulverizing the lung tissues in liquid nitrogen, hydroxyproline (HYP) was extracted from the tissues using acid hydrolysis. HYP is a unique amino acid found in collagen, and the determination of HYP content is an important indicator reflecting the metabolism of collagen tissue and the degree of fibrosis. 1 mL of 6N HCl was added per 100 mg of tissue, mixed well, and then hydrolyzed at 121 °C for 6 hours. The hydrolyzate was centrifuged, and the obtained supernatant was used for the determination of HYP content together with the gradient of HYP standard solutions (Sigma, catalog number V900395-25G) with different concentrations. 10 μL of the hydrolyzate supernatant was added to each well of a 96-well plate and dried in an oven at 65 °C. 100 μL of 1.4% chloramine T oxidation solution (Sigma, catalog number 23270-50G) was added per well and incubated at room temperature for 15 minutes. 100 μL of Ehrlich chromogenic developing solution (Solarbio, catalog number G1290) was added per well and incubated at 65 °C for 30 minutes. The absorbance of each sample well was measured at 560 nm (OD560) using a multifunctional microplate reader (TECAN, model Infinite 200 Pro). The total HYP content in the lung tissues of each experimental group was calculated, and the percentage decrease in HYP content in the peptide treatment group compared to the model group was determined to evaluate the inhibitory effect of the peptide molecule on collagen deposition in the fibrotic site. The results are shown in Table 6. In the fibrotic disease model, the peptide treatment group showed various degrees of decrease in HYP content compared to the model group, indicating that the peptide of the present invention has various degrees of inhibitory effects on collagen deposition in fibrotic tissues.

[0145]

Table 9

[0146] Example 7 Inhibitory assay of polypeptide on inflammatory and fibrotic pathology in a mouse model of fibrotic disease This example was to evaluate the inhibitory effect of the polypeptide on fibrotic pathology. Male C57 / 6J mice, 6 - 8 weeks old and weighing 18 - 20 g, were used as the research subjects. The experimental animals were randomized into groups of 6 animals per group. On the day of modeling (day 1), a single dose of 0.7 USP / kg of BLM solution was administered via the tracheal route to the BLM group and each peptide treatment group to induce the model. An equal volume of physiological saline was administered to the negative control group. Treatment dosing started on day 8. The peptide drug was administered intratracheally at a dosing amount of 3 mg / kg to each peptide treatment group, and the peptide of SEQ ID NO: 50 was administered at the same dosing amount to the positive control group. The dosing frequency was 2 times a week for a total of 4 doses. An equal volume of solvent (physiological saline) was administered as a control to the BLM model group and the negative control group. The procedure followed the procedure described in Example 4.

[0147] On the 21st day, the lung tissues were dissected, washed with physiological saline to remove blood stains, and dried with gauze. Subsequently, histopathological analysis was performed on the dissected lung tissues. The lung tissues were perfused with 0.5 mL of paraformaldehyde through the trachea and placed in 4 mL of paraformaldehyde. A small amount of gauze was added to ensure that the lung tissues were completely impregnated with the fixing solution. The tissues were fixed for over 24 hours. One lobe of the left lung and four lobes of the right lung were trimmed against the maximum coronal plane, placed in embedding cassettes, dehydrated overnight, and then paraffin-embedded to prepare 3-μm sections. Using an automatic staining machine (Leica, model ST5010), hematoxylin (Herst, lot number 201905)-eosin (Chron, lot number 2018070301) (HE) staining and Masson's trichrome staining (Celestine blue, Chron, lot number MKCH8129; aniline blue, Yuanye, lot number H70J11S115483; ponceau, Sigma, lot number SHBM2047) were performed. After the sections were mounted, they were scanned using a digital panoramic scanner (Zhiyue, model WS-10). For each lung lobe, ≤10 fields of view were captured at a magnification of 20x. The severity of inflammation and the severity of fibrosis were evaluated using HE staining and Masson's trichrome staining respectively to assess the inhibitory effect of the polypeptide on inflammatory infiltration and fibrotic deposition at the pathological fibrotic sites. The results are shown in Figures 1 and 2.

[0148] Figure 1 shows a HE-stained pathological section (magnification 20×). In the lung tissues of the negative control group, the alveolar cavities had a vacuolar-like and thin-walled structure. The BLM model group showed significant infiltration of inflammatory cells and fibroblasts in the interstitium and alveoli, accompanied by marked thickening of the alveolar septum and compression and deformation of the alveolar structure. All peptide treatment groups showed varying degrees of reduction in inflammatory cells and reduction in the thickened alveolar septum region.

[0149] Figure 2 shows a pathological section stained with Masson (magnification 20×). In the lung tissue of the negative control group mice, the collagen layer of the bronchial wall was relatively thin, and a small number of long, strip-shaped collagen fibers were observed to be distributed among the alveoli. In the BLM model group, the alveolar septum was expanded with significant deposition of collagen fibers, and fibrous masses were formed in some regions. In each peptide treatment group, only a small amount of alveolar septum was expanded, and the area of collagen fiber deposition showed varying degrees of reduction compared with the BLM model group.

[0150] The results of the inflammatory and fibrosis scores are shown in Table 7. The inflammatory and fibrosis scores were significantly higher in the BLM model group compared with the negative control group. In each polypeptide administration group, the inflammatory and fibrosis scores showed varying degrees of reduction compared with the model group.

[0151]

Table 10

[0152] Example 8 Inhibitory assay of polypeptide on collagen deposition in a rat fibrotic disease model This example was to evaluate the inhibitory effect of a polypeptide on collagen deposition at the pathological site of pulmonary fibrosis. Male SD rats, 6 - 8 weeks old and weighing 200 - 220 g, were used as the research subjects. The experimental animals were randomized into groups of 8 animals per group. On the day of modeling (day 1), a single dose of 1.8 USP / kg bleomycin solution was administered via the tracheal route to the model group and each peptide treatment group to induce the model. Starting from day 1, each peptide treatment group received intratracheal administration of the peptide at a dosage of 2 mg / kg, twice a week for a total of 4 times. The model group was administered an equal volume of solvent (physiological saline), 100 μL, as a control. The procedure followed the procedure described in Example 4. Day 15 was set as the experimental endpoint for evaluating the degree of fibrosis in the model.

[0153] On the 15th day, the animals were dissected and lung tissues were collected. After pulverizing the rat lung tissues in liquid nitrogen, total hydroxyproline (HYP) was extracted from the tissues using acid hydrolysis, and the HYP content was determined by chloramine T oxidation colorimetric analysis. 1 mL of 6N HCl was added per 100 mg of tissue, mixed well, and then hydrolyzed at 121 °C for 6 hours. The hydrolyzate was centrifuged, and the obtained supernatant was used for the determination of the HYP content. In a 96-well plate, 10 μL per well of the hydrolyzate supernatant or a gradient of different concentrations of HYP standard solution (Sigma, catalog number V900395-25G) was added and dried in an oven at 65 °C. 100 μL per well of 1.4% chloramine T oxidation solution (Sigma, catalog number 23270-50G) was added and incubated at room temperature for 15 minutes. 100 μL per well of Ehrlich chromogenic developing solution (Solarbio, catalog number G1290) was added and incubated at 65 °C for 30 minutes. The absorbance of each sample well was measured at 560 nm (OD560) using a multifunctional microplate reader (TECAN, model Infinite 200 Pro). The total HYP content in the lung tissues of each experimental group was calculated, and the percentage decrease in the HYP content in the peptide treatment group compared to the model group was determined to evaluate the inhibitory effect of the peptide molecule on collagen deposition at the fibrotic site. The results are shown in Table 8. In the fibrotic disease model, the peptide treatment group showed various degrees of decrease in the HYP content compared to the model group, indicating that the peptide of the present invention has various degrees of inhibitory effects on collagen deposition in fibrotic tissues.

[0154]

Table 11

[0155] Example 9 Inhibitory assay of polypeptide on fibrotic pathology in a rat model of fibrotic disease This example was to evaluate the inhibitory effect of polypeptide administration on the expression level of profibrotic factors in pathological fibrotic sites. Male SD rats, 6 - 8 weeks old and weighing 200 - 220 g, were used as the research subjects. The experimental animals were randomized into groups of 6 animals per group. On the day of modeling (day 1), a single dose of 1.8 USP / kg of bleomycin solution was administered via the tracheal route to the BLM model group and each peptide treatment group to induce the model. The negative control group was given an equal volume of physiological saline. Starting from day 1, each peptide treatment group received intratracheal administration of the peptide at a dosage of 2 mg / kg, twice a week for a total of 4 times. The BLM model group and the negative control group were administered an equal volume of solvent (physiological saline) as a control at a volume of 100 μL. The procedure followed the procedure described in Example 4.

[0156] On day 15, the lung tissue was dissected, washed with physiological saline to remove blood streaks, dried with gauze, and weighed. The lung index was calculated [lung index = (wet lung weight / body weight) × 1000. This index reflects the general symptoms of lung tissue including edema, inflammation, and fibrosis]. Histopathological analysis was performed on the dissected lung tissue. The lung tissue was perfused with 5 mL of paraformaldehyde through the trachea and placed in 40 mL of paraformaldehyde. A small amount of gauze was added to ensure that the lung tissue was completely impregnated with the fixing solution. The tissue was fixed for more than 24 hours. One lobe of the left lung and four lobes of the right lung were trimmed with respect to the maximum coronal plane, and the left and right lungs were individually placed in two embedding cassettes and then paraffin-embedded to prepare 3-μm sections. Masson trichrome staining was performed as described in Example 7. After digital scanning, for each lung lobe, ≤10 fields were captured at a magnification of 10x. The severity score of fibrosis was evaluated to assess the inhibitory effect of the polypeptide on fibrotic deposition in the pathological fibrotic site.

[0157] The results are shown in Figure 3 (magnification 10×). In the negative control group, the collagen layer of the bronchial wall in rat lung tissue was relatively thin, and a small number of long and narrow, band-shaped collagen fibers were observed to be distributed among the alveoli. In the BLM model group, the alveolar septum in the lung tissue was expanded with significant deposition of collagen fibers, and fibrous masses were formed in some areas. In each peptide treatment group, a small number of alveolar septa were expanded, and the areas of collagen fiber deposition showed varying degrees of reduction compared with the BLM model group.

[0158] The results of the fibrosis score are shown in Table 9. In the BLM model group, the lung index and the severity score of fibrosis were significantly increased compared with the negative control group. In each peptide treatment group, the lung index and the severity score of fibrosis showed varying degrees of reduction compared with the model group.

[0159]

Table 12

[0160] Example 10 Inhibitory assay of polypeptide on tumor cell migration ability TGF-β plays two roles in the tumor microenvironment (TME). In the early tumor stage, the TGF-β signaling pathway induces apoptosis and suppresses the proliferation of tumor cells. In the advanced tumor stage, TGF-β functions as an immunosuppressive cytokine, inhibits the immune response, and promotes tumor progression by regulating the motility and metastasis of tumor cells, etc.

[0161] This example was to evaluate the effect of the polypeptide on the migration ability of different tumor cells. The experiment was carried out using a Transwell nested chamber (membrane pore size 8 μm). THP-1 cells were seeded in the lower chamber and treated with 300 nM PMA (Sigma, catalog number P1585) to differentiate and adhere to the chamber wall, and then treated with 100 μM polypeptide for 48 hours (in the negative control, an equal volume of solvent was added). In the upper chamber, tumor cells, human lung adenocarcinoma A549 cells (American Type Culture Collection, ATCC, CCL-18) were seeded and co-cultured for 24 hours; or human liver cancer HepG2 / C3A cells (ATCC, CRL-10741) were seeded and co-cultured for 24 hours; or human breast cancer MCF7 cells (ATCC, HTB-22) were seeded and co-cultured for 48 hours. Then, the upper chamber was removed, fixed with 70% ethanol for 10 minutes, washed, and then stained with Giemsa staining solution (Beyotime, catalog number C0131) for 45 minutes. After washing 2 - 3 times with twice the volume of distilled water, the non-migrating cells were wiped off from the upper layer using a cotton swab. After drying the chamber, it was observed under a microscope, and 5 different fields of view with a magnification of 100× were randomly selected for counting and statistics. The results are shown in Table 10. The polypeptide had a significant inhibitory effect on the migration ability of human lung adenocarcinoma cells, human liver cancer cells, and human breast cancer cells.

[0162]

Table 13

[0163] Example 11 Inhibition of the polypeptide on the expression of ECM-related genes in a cell-based model of liver fibrosis This example was to assess the inhibitory effect of a polypeptide on the expression of extracellular matrix (ECM)-related genes in a cell model of liver fibrosis. The experiment utilized human hepatic stellate cells (LX-2), which are the main source of myofibroblasts in the liver. When activated, hepatic stellate cells secrete ECM and transform into myofibroblasts that play a direct role in the development of liver fibrosis. Transient stimulation of LX-2 cells with TGF-β1 can initiate the positive feedback regulation of TSP-1-TGF-β1 activation, which continuously enhances the characteristics of myofibroblasts, including ECM production.

[0164] This experiment was conducted as follows. LX-2 cells (Procell, CL-0560) were stimulated with 20 ng / mL TGF-β1 (Genscript, catalog number Z03411) for 24 hours, then 100 μM of the polypeptide was added and cultured for 48 hours. An equal volume of solvent was added to the model group, and LX-2 cells without any treatment were used as the blank control. Subsequently, the LX-2 cells were collected, and total RNA for RT-qPCR was extracted to detect the expression of ECM-related genes, EDA-fibronectin (fibronectin, FN) and collagen I (COL1). The assay results are shown in Table 11. The polypeptide was able to reduce the expression of FN and COL1 genes in LX-2 and inhibit ECM production.

[0165]

Table 14

[0166] Summary of the Sequence Listing This application is accompanied by a sequence listing. The following table provides information regarding each peptide represented by the sequence numbers in the sequence listing, where Ac represents an acetyl group and NH2 represents an amino group.

[0167]

Table 15

Claims

1. Formula (I), R １ -X １ -X ２ -X ３ -X ４ -X ５ -X ６ -X ７ -R ２ (I) (wherein, R １ is acetyl or absent; R ２ is amino or absent; X １ is absent or is selected from T and S; X ２ is selected from naturally occurring amino acids or non-naturally occurring amino acids; preferably selected from polar side chain amino acids such as uncharged polar side chain amino acids S, C, G, N, Q, T, Y, or negatively charged polar side chain amino acids D and E; more preferably selected from amino acids N, C, Q, S, T, E and D; even more preferably selected from amino acids Q, C, N, E and S; X ３ is D; X ４ is selected from small side-chain amino acids such as A, C, G, P, S, T, and V, more preferably selected from amino acids A, P, and S, and even more preferably selected from amino acids A and P; X ５ is E; X ６ is D; X ７ is absent or starting from the amino terminus to the carboxyl terminus, Z １ Z ２ Z ３ SZ ４ is either a sequence of 1, 2, 3, 4, 5, 6 or 7 amino acids consecutively selected from the amino acid sequence of LQ; Z １ is an amino acid of N, Q, P, A, L, V, M or I, preferably N, P or L, most preferably N or P; Z ２ is an amino acid of T, S, V, C, A, K or R, preferably T, C or K, more preferably, Z ２ is an amino acid of C or K, Z ２ is amino acid X ２ or X ３ forms a covalent bond with the side chain of, Z ３ is an amino acid of A, V, L or I, preferably A or V, Z ４ is an amino acid of F, Y, H or W, preferably F or H; Optionally, X ４ When is A, 0 to 1 small side chain amino acids such as P or G, preferably P, are inserted between X ３ and X ４ ; Optionally, X ７ When consisting of 1 to 5 amino acids, 1 to 2 amino acid residues selected from the amino acid residues of Q, E, D and N are added to the peptide, or 1 amino acid residue selected from C or K is added, etc., X ７ (having 0 to 2 additional amino acid residues added after X) A peptide having an amino acid sequence or a pharmaceutically acceptable salt, solvate or prodrug thereof.

2. X １ is absent or is selected from T and S; X ２ is selected from amino acids Q, N, C, S, T, E, and D; X ３ is D; X ４ is selected from A, C, G, P, S, T, and V; X ５ is E; X ６ is D; X ７ is absent or starting from the amino terminus to the carboxyl terminus, Z １ Z ２ Z ３ SZ ４ is either a sequence of 1, 2, 3, 4, 5, 6 or 7 amino acids consecutively selected from the amino acid sequence of LQ; Z １ is N, Q, P, A, L, V, M or I; Z ２ is T, K or C; Z ３ is A or V; Z ４ wherein Z is F, Y, H or W The peptide according to Claim 1, or a pharmaceutically acceptable salt, solvate or prodrug thereof.

3. X １ is selected from T and S; X ２ is selected from Q, N, E, S, and C; X ３ is D; X ４ is selected from A and P; X ５ is E; X ６ is D The peptide according to Claim 1 or 2, or a pharmaceutically acceptable salt, solvate or prodrug thereof.

4. The peptide is - DAED; - DPED; - DAEDN; - DPEDN; or - DAEDP The peptide according to any one of Claims 1 to 3, or a pharmaceutically acceptable salt, solvate or prodrug thereof, comprising an amino acid sequence selected from.

5. X ７ is Z １ Z １ Z ２ Z １ Z ２ Z ３ Z １ Z ２ Z ３ S, Z １ Z ２ Z ３ SZ ４ Z １ Z ２ Z ３ SZ ４ L or Z １ Z ２ Z ３ SZ ４ consists of LQ, and preferably, X ７ is Z １ Z ２ Z ３ Z １ Z ２ Z ３ S or Z １ Z ２ Z ３ SZ ４ consists of Z １ is N, P, or L; Z ２ is T, K or C; Z ３ is A or V; Z ４ wherein Z is F, Y, H or W The peptide according to any one of Claims 1 to 4, or a pharmaceutically acceptable salt, solvate or prodrug thereof.

6. Z １ is N or P; Z ２ is T, K or C; Z ３ is A or V, preferably A; Z ４ is F or H, The peptide according to Claim 5, or a pharmaceutically acceptable salt, solvate or prodrug thereof.

7. X ７ The peptide or a pharmaceutically acceptable salt, solvate or prodrug thereof according to any one of claims 1 to 6, wherein ７ is an amino acid sequence selected from N; P; L; NTA; NTV; PTA; NTAS; PTAS; NTVSF; NTASF; NTASH; and NTVSFLQ.

8. X １ The peptide or a pharmaceutically acceptable salt, solvate or prodrug thereof according to any one of claims 1 to 7, wherein X is T.

9. X ４ The peptide or a pharmaceutically acceptable salt, solvate or prodrug thereof according to any one of claims 1 to 8, wherein X is A.

10. X ４ The peptide according to any one of claims 1 to 8, or a pharmaceutically acceptable salt, solvate or prodrug thereof, wherein X is P.

11. Z １ The peptide or a pharmaceutically acceptable salt, solvate or prodrug thereof according to any one of claims 1 to 10, wherein Z is N.

12. Z １ The peptide or a pharmaceutically acceptable salt, solvate or prodrug thereof according to any one of claims 1 to 10, wherein Z is P.

13. Z ３ The peptide or a pharmaceutically acceptable salt, solvate or prodrug thereof according to any one of claims 1 to 12, wherein Z is A.

14. X ２ The peptide according to any one of claims 1 to 13, or a pharmaceutically acceptable salt, solvate or prodrug thereof, wherein X is Q.

15. Z ２ The peptide or a pharmaceutically acceptable salt, solvate or prodrug thereof according to any one of claims 1 to 14, wherein Z is T.

16. X ２ is C, and Z ２ is C, and the side chains of amino acids X ２ and Z ２ are covalently linked to form a disulfide, the peptide according to any one of claims 1 to 13, or a pharmaceutically acceptable salt, solvate or prodrug thereof.

17. X ３ is D, and Z ２ is K, and the side chains of amino acids X ３ and Z ２ are covalently linked to form a lactam bond, the peptide according to any one of claims 1 to 13 or a pharmaceutically acceptable salt, solvate or prodrug thereof.

18. R １ The peptide according to any one of claims 1 to 17, or a pharmaceutically acceptable salt, solvate or prodrug thereof, wherein R is acetyl.

19. R ２ The peptide or a pharmaceutically acceptable salt, solvate or prodrug thereof according to any one of claims 1 to 18, wherein R is amino.

20. The peptide consists of 5 to 13 amino acids, preferably 7, 8, 9, 10 or 11 amino acids, the peptide according to any one of Claims 1 to 19, or a pharmaceutically acceptable salt, solvate or prodrug thereof.

21. The peptide is a linear peptide or a cyclic peptide, the peptide according to any one of Claims 1 to 20, or a pharmaceutically acceptable salt, solvate or prodrug thereof.

22. The peptide is a cyclic peptide, preferably, the cyclic peptide is head-to-tail cyclized, D-K side chain cyclized, or disulfide cyclized, The cyclized region of the peptide contains a core motif (DXED), Preferably, the peptide is cyclized by coupling of the side chains of the amino acids at positions 3 and 8; or by coupling of the side chains of the amino acids at positions 2 and 8, or Preferably, X ３ is D, Z ２ is K, and the peptide is cyclized by a lactam bond formed between the side chains of X ３ and Z ２ ; or Preferably, X ２ is C, Z ２ is C, and the peptide is cyclized by a disulfide bond formed between the side chains of X ２ and Z ２ ; More preferably, when Z ２ is an amino acid of C or K, X ７ is Z １ Z ２ consists of, and X １ is T or S The peptide according to any one of Claims 1 to 21, or a pharmaceutically acceptable salt, solvate or prodrug thereof.

23. The peptide according to any one of claims 1 to 22, or a pharmaceutically acceptable salt, solvate or prodrug thereof, wherein the peptide is chemically modified, for example, PEG-modified, lipid-modified, replaced D-type amino acid, or replaced non-naturally occurring amino acid.

24. The peptide according to any one of claims 1 to 22, or a pharmaceutically acceptable salt, solvate or prodrug thereof, wherein the peptide is selected from any one of SEQ ID NOs: 1 to 49, or is different therefrom by substitution, deletion or addition of 1 or 2 amino acids in the amino acid sequence, preferably, the substitution, deletion and addition occur at amino acid residues other than those of the core motif (DXED), for example, conservative amino acid substitution.

25. The peptide according to any one of SEQ ID NOs: 6, 22, 25, 26, 33, 40, 43, 44 and 46, or a pharmaceutically acceptable salt, solvate or prodrug thereof.

26. A method for preparing the peptide according to any one of claims 1 to 25, or a pharmaceutically acceptable salt, solvate or prodrug thereof, preferably, the method includes a step of synthesizing the peptide using solid-phase synthesis.

27. A pharmaceutical composition comprising the peptide according to any one of claims 1 to 25, or a pharmaceutically acceptable salt, solvate or prodrug thereof, and a pharmaceutically acceptable carrier.

28. A method for preventing or treating TGF-β related diseases, comprising administering a prophylactically or therapeutically effective amount of the peptide according to any one of claims 1 to 25, or a pharmaceutically acceptable salt, solvate or prodrug thereof, or the pharmaceutical composition according to claim 27, to a subject in need thereof; Optionally, further comprising a step of administering an effective amount of a second therapeutic agent to the subject; Preferably, the disease has macrophage infiltration at the site of the lesion, or is related to tissue damage, inflammation or fibrosis.

29. The method according to claim 28, wherein the TGF-β related disease is a fibrotic disorder, preferably selected from pulmonary fibrosis (for example, IPF), liver fibrosis, kidney fibrosis, myelofibrosis, myocardial fibrosis and / or dermal fibrosis.

30. The method according to claim 28, wherein the TGF-β related disease is a solid tumor, preferably selected from lung cancer, liver cancer, breast cancer, uterine cancer, prostate cancer, pancreatic cancer, colon cancer, skin cancer, central nervous system cancer, fibromyoma, fibroma, fibroadenoma and fibrosarcoma.

31. Use of a peptide according to any one of claims 1 to 25 or a pharmaceutically acceptable salt, solvate or prodrug thereof, in vitro or in vivo: - Blocking the binding of CD36 to TSP-1; - Inhibiting TSP-1-dependent TGF-β1 activation; - Reducing the amount of active TGF-β1 in fibrotic tissue; - Inhibiting TGF-β1-mediated downstream signaling; - Reducing collagen deposition in fibrotic tissue; - Inhibiting inflammatory and / or fibrotic lesions mediated by TGF-β1; - Inhibiting the expression of extracellular matrix-related genes stimulated by TGF-β1; - Inhibiting the migration of tumor cells; - Preventing or treating TGF-β1-related diseases, particularly fibrosis or cancer; or Use in the manufacture of a medicament for the above use.

Images