Peptide complexes having VEGFR-2 agonist activity

Abstract

Provided is a novel compound having VEGFR-2 agonist activity. A peptide complex or a pharmaceutically acceptable salt thereof, which comprises a first peptide having an amino acid sequence represented by X1-W-X2-X3-X4-X5-X6-X7-X8-Y-X9-X10-X11-C (SEQ ID NO: 1) and having VEGFR-2 agonist activity, or a pharmaceutically acceptable salt thereof, and which is characterized in that: the first peptide has an amino acid sequence represented by X1-W-X2-X3-X4-X5-X6-X7-X8-Y-X9-X10-X11-C (SEQ ID NO: 1); or comprising an amino acid sequence obtained by substitution, deletion, addition or insertion of 1-3 amino acids in the amino acid sequence represented by SEQ ID NO: 1.

Description

Technical Field

[0001] This invention relates to novel peptide complexes having VEGFR-2 agonist activity and cell culture compositions comprising such peptide complexes. Furthermore, this invention relates to novel peptide complexes having cell proliferation capacity derived from VEGFR-2 agonist activity and cell culture compositions comprising such peptide complexes. Background Technology

[0002] Angiogenesis is crucial in the normal physiological processes encompassing embryonic development, follicular development, and wound healing. Vascular endothelial growth factor (VEGF) is a key mediator of angiogenesis. VEGF's receptor, VEGFR, is a receptor-type tyrosine kinase, and three proteins, VEGFR-1 through VEGFR-3, exist. In particular, the binding of VEGF to VEGFR-2 induces receptor dimerization and autophosphorylation of tyrosine residues. This autophosphorylation triggers the activation of several signal transduction cascades, inducing endothelial cell differentiation, proliferation, migration, vascular permeability, and angiogenesis. Studies on the use of VEGFR-2 agonists for the treatment of trauma, liver dysfunction and liver disease, prevention of liver damage, and treatment of kidney disease have been reported (Patent Literature 1, 2, 3, 4). However, the VEGFR-2 agonists used in these studies are natural VEGF or its variants, and no chemically synthesized VEGFR-2 agonist peptides have been reported. Existing technical documents Patent documents

[0003] Patent Document 1: Japanese Patent Application Publication No. 2008-546707 Patent Document 2: Japanese Patent Application Publication No. 2010-159266 Patent Document 3: Japanese Patent 5111729 Patent Document 4: Japanese Patent Publication No. 2009-541207 Non-patent literature

[0004] Non-patent literature 1: Rom J Morphol Embryol 2018, 59, 455. Non-patent literature 2: Biomolecules 2020, 10, 1673. Non-patent literature 3: Biology Methods and Protocols, 2023, 8, bpac034. Non-patent literature 4: Scientific Reports 2020, 10, 17937. Non-patent literature 5: Scientific Reports 4: 6716 doi: 10.1038 / srep06716. Non-patent literature 6: Developmental Cell 2020, 54, 516. Non-patent literature 7: Cell Reports 2018, 23, 1620. Summary of the Invention The problem that the invention aims to solve

[0005] One aspect of the present invention provides novel compounds having VEGFR-2 agonist activity. Another aspect of the present invention also provides novel compounds having cell proliferation capacity derived from VEGFR-2 agonist activity. Methods for solving problems

[0006] Although not limited, the present invention includes the following methods. [1] A peptide complex or a pharmaceutically acceptable salt thereof, which is a peptide complex containing a first peptide and having VEGFR-2 agonist activity or a pharmaceutically acceptable salt thereof. The first peptide has X 1 -WX 2 -X 3 -X 4 -X 5 -X 6 -X 7 -X 8 -YX 9 -X 10 -X 11 The amino acid sequence shown in -C (Sequence Number 1), or a sequence consisting of 1 to 3 amino acids substituted, deleted, added, or inserted in the amino acid sequence shown in Sequence Number 1. X 1 For amino acids with chain alkyl groups that can be substituted by polar groups on their side chains or with aromatic rings that can be substituted, X 2 For amino acids with alkyl groups on their side chains that can be substituted, X 3 For amino acids with substituted chain alkyl groups in their side chains, X 4 For amino acids with alkyl groups on their side chains that can be substituted, X 5 For any amino acid, X 6 For amino acids with chain alkyl groups that can be substituted by polar groups on the side chain, or amino acids without side chains, X 7For amino acids with substituted chain alkyl groups in their side chains, X 8 For amino acids with substituted chain alkyl groups in their side chains, X 9 For amino acids with an aromatic ring in the side chain that can be substituted, X 10 For amino acids with alkyl groups on their side chains that can be substituted, X 11 It can be any amino acid. [2] The peptide complex or a pharmaceutically acceptable salt thereof according to [1], wherein, X 2 Amino acids having chain-like or cyclic alkyl groups in their side chains, or having chain-like alkyl groups that can be substituted with aromatic rings, X 3 For amino acids with chain-like alkyl groups on the side chain that can be replaced by polar groups, X 4 Amino acids having chain-like or cyclic alkyl groups in their side chains, or having chain-like alkyl groups that can be substituted by polar groups. X 7 For amino acids with chain alkyl groups on the side chain that can be substituted by polar groups or with chain alkyl groups that can be branched, X 8 For amino acids with chain alkyl groups on the side chain that can be substituted by polar groups or with chain alkyl groups that can be branched, X 10 Amino acids having chain-like or cyclic alkyl groups in their side chains, or having chain-like alkyl groups that can be substituted with aromatic or heterocyclic rings. [3] The peptide complex or a pharmaceutically acceptable salt thereof according to [2], wherein, X 1 The possible values ​​are W, Hcit, 4Py2NH2, 3Py6NH2, F4aao, or W7N. X 2 For V, Tbg, Chg, or Hty, X 3 For D, Q, or MetO2, X 4 For V, Tbg, alT, or Chg, X 5 For Q, Ahp, W, Hph, Cha, F4COO, or K, X 6 For D, G, or N, X 7 For D or V, X 8 For L, N, or Atb, X9 For F, Bph, Yph, F4G, or F4C, X 10 For V, Hph, Hty, Chg, or H4Py, X 11 The options are D, A, S, F, F4aao, Har, or Hyp. [4] The peptide complex or a pharmaceutically acceptable salt thereof as described in [1], wherein, X 2 For amino acids with chain-like or cyclic alkyl groups in their side chains, X 4 For amino acids with chain alkyl groups on the side chain that can be substituted by polar groups or with chain alkyl groups that can be branched, X 6 For amino acids with chain-like alkyl groups on the side chain that can be replaced by polar groups, X 7 For amino acids with chain-like alkyl groups on the side chain that can be replaced by polar groups, X 8 For amino acids with chain alkyl groups on the side chain that can be substituted by polar groups or with chain alkyl groups that can be branched, X 10 Amino acids having chain-like or cyclic alkyl groups in their side chains, or having chain-like alkyl groups that can be substituted with aromatic rings. [5] The peptide complex or a pharmaceutically acceptable salt thereof according to [4], wherein, X 1 For W or W7N, X 2 For V, Tbg, or Chg, X 3 D, X 4 For V or alT, X 5 For Ahp, Cha, or K, X 6 For D or N, X 7 D, X 8 For L, N, or Atb, X 9 For F or F4G, X 10 For V, Hty, or Chg, X 11 It can be D or S. [6] The peptide complex or a pharmaceutically acceptable salt thereof according to [1], wherein, The first peptide has the amino acid sequence shown in WWVDVQDDLYFVDC (Sequence Number 2), or is composed of an amino acid sequence in which 1 to 3 amino acids in the amino acid sequence shown in Sequence Number 2 are substituted, deleted, added or inserted. [7] The peptide complex or a pharmaceutically acceptable salt thereof according to [1], wherein, The first peptide has an amino acid sequence in which glycine has been added to the C-terminus. [8] The peptide complex or a pharmaceutically acceptable salt thereof according to [1], wherein, The first peptide is a peptide composed of any one of the amino acid sequences shown in sequence numbers 3 to 51. [9] The peptide complex or a pharmaceutically acceptable salt thereof according to [1], wherein, The first peptide is a cyclic peptide.

[10] The peptide complex or a pharmaceutically acceptable salt thereof according to [1], wherein, The first peptide is an N-terminal amino acid residue that is chloroacetylated, and has an intrapeptide cysteine ​​residue and a cyclic peptide in which the N-terminal amino acid residue is bound to the cysteine ​​residue.

[11] The peptide complex or a pharmaceutically acceptable salt thereof according to [1], wherein, The peptide complex consists of a first peptide, a second peptide, and a linker connecting the first peptide and the second peptide. The second peptide may be the same as or different from the first peptide, having the amino acid sequence shown in Sequence Number 1, or consisting of an amino acid sequence in which 1 to 3 amino acids in the amino acid sequence shown in Sequence Number 1 are substituted, deleted, added or inserted.

[12] The peptide complex or a pharmaceutically acceptable salt thereof according to

[11] , wherein, The homology between the first peptide and the second peptide is more than 90% and less than 100%.

[13] The peptide complex or a pharmaceutically acceptable salt thereof according to

[11] , wherein, The first peptide and the second peptide are peptides with the same sequence.

[14] The peptide complex or a pharmaceutically acceptable salt thereof according to

[11] , wherein, The sixth amino acid of the first peptide is linked to the sixth amino acid of the second peptide, or the C-terminus of the first peptide is linked to the C-terminus of the second peptide via the linker.

[15] The peptide complex or a pharmaceutically acceptable salt thereof according to

[11] , wherein, The linker is a PEG linker or a linker that has been added with 1 to 6 amino acids to a PEG linker.

[16] A composition comprising the peptide complex according to [1] or a pharmaceutically acceptable salt thereof and a carrier.

[17] A cell culture composition comprising a peptide complex according to [1] or a pharmaceutically acceptable salt thereof and a carrier, and for use in cell culture.

[18] A composition comprising the peptide complex according to [1] or a pharmaceutically acceptable salt thereof and a carrier, for medical, diagnostic or research use. The effects of the invention

[0007] According to the present invention, novel compounds possessing VEGFR-2 agonist activity can be provided. Furthermore, the present invention can further provide novel compounds possessing cell proliferation capacity derived from VEGFR-2 agonist activity. Attached Figure Description

[0008] Figure 1A and Figure 1B This indicates the results of the Human Phospho-RTK Array analysis using the peptide complex and VEGF165a of the present invention. Figure 1A An array map representing the target factor. Figure 1B This indicates the evaluation result. Detailed Implementation

[0009] This invention includes, without limitation, the following methods. In this specification, unless otherwise specified, the technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. The substances, materials, and examples disclosed herein are illustrative only and are not intended to be limiting. The phrase "in one method" in this specification is not limited to that method; that is, it is meant to be non-limiting.

[0010] The first invention of this specification relates to a peptide complex or a pharmaceutically acceptable salt thereof. The peptide complex or a pharmaceutically acceptable salt thereof contains a first peptide and has VEGFR-2 agonist activity. Furthermore, the peptide complex or a pharmaceutically acceptable salt thereof possesses cell proliferation capacity derived from VEGFR-2 agonist activity. "Pharmaceutically acceptable salt" means a pharmaceutically acceptable salt of the peptide complex. Pharmaceutically acceptable salts are described below. Hereinafter, for brevity, the peptide complex or a pharmaceutically acceptable salt thereof will sometimes be simply referred to as a peptide complex.

[0011] peptide complex In one embodiment, the peptide complex of the present invention comprises a first peptide and has VEGFR-2 agonist activity. In another embodiment, the peptide complex of the present invention has cell proliferation capacity derived from VEGFR-2 agonist activity. The peptide complex is a peptide comprising a first peptide and other peptides or compounds, a peptide-containing compound, or a pharmaceutically acceptable salt thereof. The peptide complex may comprise one or more (three or more or four) first peptides. The peptide complex may further comprise one or more partial peptides different from the first peptide. The peptide complex is preferably composed of the first peptide and partial peptides linked by linkers. The peptide complex may be a homomultimer comprising only peptides having the same amino acid sequence. The peptide complex may be a heteromultimer comprising peptides having different amino acid sequences. The peptide complex is preferably a homodimer comprising two first peptides having the same amino acid sequence linked by linkers. In a homodimer, the two peptide portions may have completely identical amino acid sequences or substantially identical amino acid sequences. The peptide complex has VEGFR-2 agonist activity, but further, the first peptide and partial peptides constituting the peptide complex may have VEGFR-2 agonist activity. As shown in the examples, the first peptide has a peptide complex structure formed by linkers, thereby exhibiting VEGFR-2 agonist activity.

[0012] In one embodiment, the peptide complex of the present invention may consist of a first peptide, a second peptide, and a linker connecting the first peptide and the second peptide. In this case, the second peptide is preferably the same as or different from the first peptide, having the amino acid sequence shown in Sequence Number 1, or consisting of an amino acid sequence in which 1 to 3 amino acids in the amino acid sequence shown in Sequence Number 1 are substituted, deleted, added, or inserted. Furthermore, the peptide complex preferably has a homology of 90% or more and 100% or less between the first peptide and the second peptide, more preferably the first peptide and the second peptide are substantially identical homodimers.

[0013] First peptide In one embodiment, the first peptide has X 1 -WX 2 -X 3 -X 4 -X 5 -X 6 -X 7 -X 8 -YX 9 -X 10 -X 11The amino acid sequence shown in -C (Sequence Number 1), or a sequence consisting of 1 to 3 (1, 2, or 3) amino acids substituted, deleted, added, or inserted in the amino acid sequence shown in Sequence Number 1. Among the substituted amino acids, a pharmaceutically acceptable salt of the amino acid is included. A preferred example of a peptide having a sequence consisting of amino acid sequences consisting of substituted, deleted, added, or inserted amino acids in the amino acid sequence shown in Sequence Number 1 is a peptide having an amino acid sequence with conserved amino acid substitutions.

[0014] Conservative amino acid substitution When substituting, deleting, adding, or inserting one, two, or three amino acid residues from a specific sequence, conserved amino acid substitution is preferred. "Conservative amino acid substitution" refers to substitution with a functionally equivalent or similar amino acid. Conservative amino acid substitution in a peptide causes a static change in the peptide's amino acid sequence. For example, one or more amino acids with the same polarity function equivalently and cause a static change in the peptide's amino acid sequence. Generally, substitutions within a certain group can be considered structurally and functionally conserved. However, it will be apparent to those skilled in the art that the role of a particular amino acid residue can be determined by its position in the three-dimensional structure of the molecule containing that amino acid. For example, cysteine ​​residues are less polar than their reduced (thiol) form and can be in their oxidized (disulfide) form. Long aliphatic portions of the arginine side chain can constitute structurally and functionally important features. Additionally, side chains containing aromatic rings (tryptophan, tyrosine, phenylalanine) can contribute to ion-aromatic interactions or cation-pi interactions. In this case, even if amino acids with these side chains are replaced with amino acids belonging to the acidic or nonpolar groups, the structure and function can remain conserved. Residues such as proline, glycine, and cysteine ​​(in disulfide form) may have a direct effect on the conformation of the main chain and cannot be repeatedly substituted without structural deformation. Conserved amino acid substitutions are shown below, including specific substitutions based on side chain similarity (Lehninger, Biochemistry, 2nd edition, revised 2nd edition, 1975, pp. 73-75, Worth Publisher, New York (1975)) and typical substitutions.

[0015] In addition, the preferred method of conservative amino acid substitution is, for example, as described below, in a group in which natural amino acids are classified based on the properties of their common side chains, to replace an amino acid with an amino acid belonging to the same group. Hydrophobic (also known as nonpolar) amino acids: Amino acids that exhibit hydrophobic (nonpolar) properties, including alanine (“Ala” or abbreviated as “A”), glycine (“Gly” or abbreviated as “G”), valine (“Val” or abbreviated as “V”), leucine (“Leu” or abbreviated as “L”), isoleucine (“Ile” or abbreviated as “I”), proline (“Pro” or abbreviated as “P”), phenylalanine (“Phe” or abbreviated as “F”), tryptophan (“Trp” or abbreviated as “W”), tyrosine (“Tyr” or abbreviated as “Y”), and methionine (“Met” or abbreviated as “M”). It should be noted that hydrophobic amino acids can be further classified into the following groups. Aliphatic amino acids: These are amino acids that have fatty acid or hydrogen in their side chain, including Ala, Gly, Val, Ile, and Leu. Aliphatic / branched-chain amino acids: These are amino acids with branched fatty acids in their side chains, including Val, Ile, and Leu. Aromatic amino acids: These are amino acids that have an aromatic ring in their side chain, including Trp, Tyr, and Phe. Hydrophilic (also known as polar) amino acids: Amino acids that exhibit hydrophilic (polar) properties include serine ("Ser" or abbreviated as "S"), threonine ("Thr" or abbreviated as "T"), cysteine ​​("Cys" or abbreviated as "C"), asparagine ("Asn" or abbreviated as "N"), glutamine ("Gln" or abbreviated as "Q"), aspartic acid ("Asp" or abbreviated as "D"), glutamic acid ("Glu" or abbreviated as "E"), lysine (also written as Lysine, "Lys" or abbreviated as "K"), arginine ("Arg" or abbreviated as "R"), and histidine ("His" or abbreviated as "H"). It should be noted that hydrophilic amino acids can be further classified into the following groups. Acidic amino acids: These are amino acids whose side chains exhibit acidity, including Asp and Glu. Basic amino acids: These are amino acids whose side chains exhibit basicity, including Lys, Arg, and His. Neutral amino acids: These are amino acids whose side chains exhibit neutrality, including Ser, Thr, Asn, Gln, and Cys. In addition, Gly and Pro can also be classified as "amino acids that affect the direction of the main chain", and amino acids containing sulfur molecules in the side chain, Cys and Met can also be classified as "sulfur-containing amino acids".

[0016] In this specification, "amino acid" includes not only naturally occurring amino acids but also non-natural amino acids. Among non-natural amino acids are N-alkyl amino acids, such as those formed by N-alkylation of naturally occurring amino acids as described above, and amino acids in which the nitrogen atoms forming peptide bonds are modified by branched or unbranched lower-order alkyl groups (e.g., C1-C5, preferably C1-C3, more preferably C1). Among N-alkyl amino acids, N-ethyl amino acids, N-butyl amino acids, or N-methyl amino acids are preferred, with N-methyl amino acids being more preferred. Furthermore, among non-natural amino acids are chemically modified amino acids such as D-type amino acids (also described as D-amino acids), β-amino acids, γ-amino acids, amino acid variants, and amino acid derivatives, as well as amino acids such as ortholeucine and ornithine that cannot serve as building blocks of proteins in living organisms. In addition, it also includes amino acids in which the side chains of natural amino acids have been further added with functional groups or the functional groups have been replaced with other functional groups (e.g., amino acids with substituted or added functional groups in the arylene, alkylene, or other parts of the side chain; amino acids in which the number of carbons in the arylene, alkylene, or alkyl groups of the side chain has been increased; amino acids with substituted aromatic rings in the side chain; and amino acids that have been heterocyclic or fused-ringed). It should be noted that by adding or substituting functional groups or other structures into the side chains of natural amino acids, properties different from those of the natural amino acids can be imparted. For example, A4p is alanine with a piperidine group added to its side chain, but because of this addition, it exhibits basic polarity, unlike alanine which belongs to the nonpolar amino acid group. That is, the group that classifies natural amino acids based on the properties of their common side chains may include non-natural amino acids that have the same side chain properties. For example, N-methylarginine (MeR), the N-methylated amino acid of arginine, which is a basic amino acid, is a non-natural amino acid, but it can be classified as a basic amino acid because it exhibits basicity. Thus, non-natural amino acids that exhibit the same side chain properties as an amino acid can also be included as objects of conserved amino acid substitution. Non-limitingly, non-natural amino acids include N-methyl amino acids, 4Py, alT, Cit, P4Sh, F4CON, F4COO, 3Py, HyP, SMe, A4paa, Atp, Hgl, KAc, Na1, W6N, W7N, PeG, etc. It should be noted that N-methyl amino acids can also be classified as N-alkyl amino acids, or according to the nature of the side chain of the un-N-methylated original amino acid.

[0017] peptide length In one embodiment, the first peptide, for example, includes addition amino acid residues in addition to the amino acid sequences described in sequence numbers 2 to 51, or the first to 14th amino acid sequences of these sequences. The addition amino acid residues may be included in a cyclic peptide, or may be added to the cyclic peptide in a chain-like manner. The number of peptides and the number of amide bonds (number of amino acids, length) at the peptide site are not particularly limited, but the total number of amino acid residues (referring to the number of amino acid residues contained in a cyclic peptide, excluding these amino acids when further chain-like addition occurs from the cyclic peptide) is preferably 20 residues or less. Preferred peptide lengths are 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, or 11 or more amino acid residues; more preferably, 19 or less or 18 or less amino acid residues. Further preferred peptide lengths are 13 or more and 16 or less amino acid residues; most preferably, 14 or 15 amino acid residues.

[0018] A preferred example of the first peptide is one having sequence number 1 (X). 1 -WX 2 -X 3 -X 4 -X 5 -X 6 -X 7 -X 8 -YX 9 -X 10 -X 11 The peptide with the amino acid sequence shown in -C) X 1 For amino acids with chain alkyl groups that can be substituted by polar groups on their side chains or with aromatic rings that can be substituted, X 2 For amino acids with alkyl groups on their side chains that can be substituted, X 3 For amino acids with substituted chain alkyl groups in their side chains, X 4 For amino acids with alkyl groups on their side chains that can be substituted, X 5 For any amino acid, X 6 For amino acids with chain alkyl groups that can be substituted by polar groups on the side chain, or amino acids without side chains, X 7 For amino acids with substituted chain alkyl groups in their side chains, X 8 For amino acids with substituted chain alkyl groups in their side chains, X 9For amino acids with an aromatic ring in the side chain that can be substituted, X 10 For amino acids with alkyl groups on their side chains that can be substituted, X 11 It can be any amino acid.

[0019] Furthermore, a preferred example of the first peptide is a peptide having the amino acid sequence shown in sequence number 1. X 2 Amino acids having chain-like or cyclic alkyl groups in their side chains, or having chain-like alkyl groups that can be substituted with aromatic rings, X 3 For amino acids with chain-like alkyl groups on the side chain that can be replaced by polar groups, X 4 Amino acids having chain-like or cyclic alkyl groups in their side chains, or having chain-like alkyl groups that can be substituted by polar groups. X 7 For amino acids with chain alkyl groups on the side chain that can be substituted by polar groups or with chain alkyl groups that can be branched, X 8 For amino acids with chain alkyl groups on the side chain that can be substituted by polar groups or with chain alkyl groups that can be branched, X 10 Amino acids having chain-like or cyclic alkyl groups in their side chains, or having chain-like alkyl groups that can be substituted with aromatic or heterocyclic rings.

[0020] Furthermore, a preferred example of the first peptide is a peptide having the amino acid sequence shown in sequence number 1. X 1 The possible values ​​are W, Hcit, 4Py2NH2, 3Py6NH2, F4aao, or W7N. X 2 For V, Tbg, Chg, or Hty, X 3 For D, Q, or MetO2, X 4 For V, Tbg, alT, or Chg, X 5 For Q, Ahp, W, Hph, Cha, F4COO, or K, X 6 For D, G, or N, X 7 For D or V, X 8 For L, N, or Atb, X 9 For F, Bph, Yph, F4G, or F4C, X 10For V, Hph, Hty, Chg, or H4Py, X 11 The options are D, A, S, F, F4aao, Har, or Hyp.

[0021] Furthermore, a preferred example of the first peptide is a peptide having the amino acid sequence shown in sequence number 1. X 2 For amino acids with chain-like or cyclic alkyl groups in their side chains, X 4 For amino acids with chain alkyl groups on the side chain that can be substituted by polar groups or with chain alkyl groups that can be branched, X 6 For amino acids with chain-like alkyl groups on the side chain that can be replaced by polar groups, X 7 For amino acids with chain-like alkyl groups on the side chain that can be replaced by polar groups, X 8 For amino acids with chain alkyl groups on the side chain that can be substituted by polar groups or with chain alkyl groups that can be branched, X 10 These are amino acids having chain-like or cyclic alkyl groups in their side chains, or chain-like alkyl groups that can be substituted with aromatic rings. Examples show that complexes containing these peptides possess VEGFR-2 agonist activity and cell proliferation capacity.

[0022] Furthermore, a preferred example of the first peptide is a peptide having the amino acid sequence shown in sequence number 1. X 1 For W or W7N, X 2 For V, Tbg, or Chg, X 3 D, X 4 For V or alT, X 5 For Ahp, Cha, or K, X 6 For D or N, X 7 D, X 8 For L, N, or Atb, X 9 For F or F4G, X 10 For V, Hty, or Chg, X 11 It can be D or S.

[0023] In this specification, "substitutable" means either unsubstituted or substituted by any number of substituents.

[0024] Amino acids with substituted alkyl groups in their side chains The term "amino acid having a substituted alkyl group in its side chain" refers to an amino acid having an alkyl group in its side chain, such as, without limitation, alanine, proline, isoleucine, leucine, valine, etc., and the alkyl group can be chain-like or cyclic. The side chain of the amino acid having an alkyl group in its side chain is further substituted by substituents such as alkyl, aromatic ring, heterocyclic, hydroxyl, etc., or is an unsubstituted amino acid.

[0025] Amino acids with substituted chain alkyl groups in their side chains The term "amino acid having a substituted chain alkyl group in its side chain" refers to an amino acid having a chain alkyl group in its side chain, such as alanine, isoleucine, leucine, valine, etc., without limitation, and is an amino acid in which the side chain of the amino acid having a chain alkyl group in its side chain is further substituted by substituents such as alkyl groups, polar groups, etc., such as carboxyl groups, amide groups, methanesulfonyl groups, etc., or is unsubstituted.

[0026] Amino acids with chain-like alkyl groups in their side chains that can be replaced by polar groups. The term "amino acid having a chain-like alkyl group in its side chain that can be replaced by a polar group" means an amino acid having the chain-like alkyl group in its side chain, and is an amino acid whose side chain has been further replaced by a polar group such as, but not limited to, urea, amino, carboxyl, amide, hydroxyl, methanesulfonyl, etc., or is not replaced by a polar group, but the former is preferred.

[0027] Amino acids without side chains The term "amino acid without side chains" refers to glycine or glycine derivatives.

[0028] Amino acids with chain-like or cyclic alkyl groups in their side chains "Amino acids having chain-like or cyclic alkyl groups in their side chains" refers to amino acids having said alkyl groups in their side chains, where the alkyl groups can be chain-like or cyclic.

[0029] Amino acids with chain-like alkyl groups on their side chains that can be substituted with aromatic rings The term "amino acid having a chain-like alkyl group in its side chain that can be substituted with an aromatic ring" means an amino acid having such a chain-like alkyl group in its side chain, and that the side chain of the amino acid having such a chain-like alkyl group in its side chain can be further substituted with an aromatic ring or not substituted with an aromatic ring, but preferably the former.

[0030] Amino acids with branchable chain alkyl groups in their side chains "Amino acids having branchable chain alkyl groups in their side chains" refers to amino acids having said chain alkyl groups in their side chains, and these amino acids' side chains can be further branched. Preferably, the amino acids having branchable chain alkyl groups in their side chains are amino acids having already branched chain alkyl groups.

[0031] Amino acids with substituted aromatic rings in their side chains The term "amino acid with a substituted aromatic ring in its side chain" refers to an amino acid with an aromatic ring in its side chain, wherein the carbon atoms constituting the aromatic ring or the functional groups already bonded to the aromatic ring are either substituted or unsubstituted, preferably the former. The substituted aromatic ring in the side chain may have a fused ring structure, such as a benzene ring, indole ring, or naphthalene ring, and a portion of its carbon atoms (C, etc.) may be substituted with nitrogen atoms (N, etc.). The aromatic ring may be a heterocyclic ring. Furthermore, the amino acid with a substituted aromatic ring in its side chain may, for example, be an amino acid where the hydroxyl group in the side chain of tyrosine is substituted with another functional group. The type and position of the substituted functional group are not particularly limited, and can be arbitrarily selected from alkyl, phenyl, cycloalkyl, hydroxyl, halogen, etc. Examples of amino acids with a substituted aromatic ring in their side chain include tyrosine, tryptophan, histidine, and phenylalanine. In addition, the amino acid having a substituted aromatic ring in the side chain can be a non-natural amino acid, such as 4Py2NH2, 3Py6NH2, F4aao, W7N, Bph, Yph, F4G, F4C, etc.

[0032] Amino acids with chain-like alkyl groups on their side chains that have been substituted with aromatic or heterocyclic rings. The term "amino acids having a chain-like alkyl group substituted with an aromatic ring or heterocycle in the side chain" means amino acids having the chain-like alkyl group in the side chain, and amino acids whose side chains are further substituted with an aromatic ring or heterocycle.

[0033] Furthermore, a preferred example of the first peptide is a peptide having the amino acid sequence shown in WWVDVQDDLYFVDC (Sequence Number 2), or a peptide composed of amino acid sequences consisting of 1 to 3 amino acids substituted, deleted, added, or inserted in the amino acid sequence shown in Sequence Number 2. When amino acids are substituted or deleted, the position of the amino acid is preferably any one of the 1st, 3rd, 4th, 5th, 6th, 7th, 8th, 9th, 11th, 12th, and 13th amino acids in the amino acid sequence shown in Sequence Number 2.

[0034] A preferred example of the first peptide is a peptide having an amino acid sequence in which glycine has been further added to the C-terminus. Furthermore, in peptides numbered 14-35 and 37-51, the carboxylic acid at the C-terminus of the 15th glycine is preferably converted to an amide.

[0035] A preferred example of the first peptide is a peptide composed of any one of the amino acid sequences shown in sequence numbers 2 to 51, or a pharmaceutically acceptable salt thereof. Examples of pharmaceutically acceptable salts include inorganic acid salts, organic acid salts, inorganic base salts, organic base salts, and acidic or basic amino acid salts. Examples of inorganic acid salts include hydrochloride, hydrobromide, sulfate, nitrate, and phosphate. Examples of organic acid salts include acetate, succinate, fumarate, maleate, tartrate, citrate, lactate, stearate, benzoate, methanesulfonate, and p-toluenesulfonate. Examples of inorganic base salts include alkali metal salts such as sodium and potassium salts; alkaline earth metal salts such as calcium and magnesium salts; aluminum salts and ammonium salts. Examples of organic base salts include diethylamine salts, diethanolamine salts, meglumine salts, and N,N'-dibenzylethylenediamine salts. Examples of acidic amino acid salts include aspartate and glutamate. Examples of basic amino acid salts include arginine, lysine, and ornithine. The first peptide and the peptide complex can be pharmaceutically acceptable salts or solvates. An example of a solvate is a hydrate.

[0036] A preferred example of the first peptide is a cyclic peptide. A preferred example of the first peptide is a cyclic peptide in which the N-terminal amino acid residue is a chloroacetylated amino acid residue, a cysteine ​​residue is present within the peptide, and the N-terminal amino acid residue is bound to the cysteine ​​residue.

[0037] Cyclic peptides In this specification, a cyclic peptide refers to a peptide in which two amino acids are linked together, forming a ring structure, either wholly or partially. It should be noted that this specification also includes peptides in which amino acids form a cross-linked structure, peptides that form a cyclic structure through the formation of a lactam ring or a macrocyclization reaction, and peptides with a lasso peptide-like structure. That is, in this specification, a cyclic peptide may have a linear portion, provided that only a portion of it forms a cyclic structure.

[0038] In this specification, there are cases where a portion of the amino acids in a peptide is altered due to cyclization. The peptides in this specification also include peptides containing such altered amino acids. As an example of alteration due to cyclization, there is the following: the addition of a chloroacetyl group to the N-terminal amino acid, which then cyclizes with a cysteine ​​residue in the peptide. The peptides in this specification also include peptides containing various (natural / non-natural) amino acids with added chloroacetyl groups.

[0039] Generally, peptides exhibit poor metabolic stability in vivo and, due to their large size, struggle to cross cell membranes. To address this issue, methods for cyclizing peptides have been developed. Cycling peptides enhances protease resistance and metabolic stability without restricting conformational changes, suggesting increased rigidity and improved membrane permeability and affinity for target proteins.

[0040] Cyclation of peptides can be performed using well-known methods. While not limited to these, for example, by designing the peptide to contain two or more cysteine ​​residues, a cyclic structure can be formed post-translationally via disulfide bonds. Alternatively, following the method of Goto et al. (Y. Goto, et al. ACS Chem. Biol. 3 120-129 (2008)), peptides with an N-terminal chloroacetyl group can be synthesized using genetic code reprogramming techniques, and cysteine ​​residues can be incorporated into the peptide, thereby enabling cyclization. In this way, the thiol group automatically performs a nucleophilic attack on the chloroacetyl group post-translational, and the peptide cyclizes via a thioether bond. Cyclation can also be achieved by incorporating combinations of other amino acids that bind to form a cyclic structure into the peptide using genetic code reprogramming techniques. Alternatively, peptides with an N-terminal cyclic amide can be synthesized, and L-2-aminoadipic acid residues can be incorporated into the peptide, allowing cyclization through binding between them. Thus, any well-known cyclization method can be used without special restrictions.

[0041] connector In one embodiment, the first peptide or the fractional peptide may be joined with a linker. The linker may also be a structure that links multiple peptides together in the peptide complex. Examples of linkers include amino acid linkers (peptide linkers), chemical linkers, fatty acid linkers, nucleic acid linkers, and glycan linkers; additionally, complexes of chemical linkers and peptide linkers may be used. An example of a chemical linker is a PEG (Polyethylene glycol) linker. A PEG linker may be a linker composed of 1 to 36 ethylene glycol units. Alternatively, the linker may be a fatty acid linker containing a divalent chemical moiety derived from a fatty acid. An amino acid (peptide) linker is a linker containing at least one amino acid. For example, glycine-rich peptides such as those with the sequence [Gly-Gly-Gly-Gly-Ser]n (where n is 1, 2, 3, 4, 5, or 6) as described in U.S. Patent No. 7,271,149, or serine-rich peptide linkers as described in U.S. Patent No. 5,525,491, can be used. Non-limitingly, there are cases where the physical properties (e.g., solubility) of the peptide change due to the addition of the linker. Furthermore, the linker can be a combination of the linkers described above. For example, as an amino acid linker, it can be a form formed by combining Gly-Lys and further combining a PEG linker at the side chain end of the Lys. Additionally, the linker can be a structure where amino acids and PEG are alternately linked, such as PEG-amino acid-PEG. Here, PEG refers to a PEG linker. Another example of a linker is a linker in which 1 to 6 amino acids have been added to the PEG linker portion. The linker can be a linker having a structure formed by adding an amino acid to one end of the PEG linker portion, or a linker having a structure formed by adding amino acids to both ends of the PEG linker portion. While K (Lys) is a preferred example of the amino acid added to the PEG portion, it is not limited to K and can be other amino acids. A preferred example of the linker is a linker having the structure shown in Table 3. The linker can be added at any position on the peptide. For example, at the C-terminus of the peptide, it can bind to the first amino acid and form a cyclic structure (Cys), or it can bind to any amino acid contained in the cyclic peptide. Although not limited, it is preferred that the linker binds to the C-terminal Cys or to the side chain of an amino acid contained in the cyclic peptide. More preferably, the linker binds to the C-terminal Cys or to the side chain of the sixth amino acid contained in the cyclic peptide. It should be noted that, for example, in the amino acid sequences represented by sequence numbers 2 to 51, the 15th Gly can also be considered as a linker. Non-limitingly, for example, in the case where the cyclic peptide structure formed by the first amino acid in the amino acid sequences represented by sequence numbers 2 to 51 being combined with the 14th amino acid (Cys), i.e., the 14th Cys being further added to glycine, consists of two structures, and a dimer structure is formed from the added glycine using the structure shown in Table 2, the linker structure can also be the structure shown in Table 2. Alternatively, it can be considered as a structure formed by glycine combined with the structure shown in Table 2. The dimer can be a dimer formed by the 6th amino acid of the first peptide and the 6th amino acid of the second peptide linked by a linker, or a dimer formed by the C-terminus of the first peptide and the C-terminus of the second peptide linked by a linker. The former is preferred.

[0042] VEGFR-2 VEGFR-2 is a member of the tyrosine kinase receptor family, also known as KDR (kinase insert domain receptor) or Flk-1 (fetal liver kinase 1). VEGFR-2 is highly expressed in vascular endothelial cells and lymphatic vessels. Among its ligands, VEGF, it has a higher affinity for VEGF-A and VEGF-E, and a lower affinity for VEGF-C and VEGF-D (Non-Patent Literature 1). Like other tyrosine kinase receptors, VEGFR-2 has an intracellular tyrosine kinase domain. If VEGF binds to the extracellular domain of VEGFR-2, it causes autophosphorylation of tyrosine residues, which induces vasculogenesis, non-pathological or pathological angiogenesis through the activation of some signals and mechanisms (Non-Patent Literature 1). On the other hand, VEGF-A is an important ligand in angiogenesis, playing a crucial role in both angiogenesis and angiogenesis. VEGF-A binds to both VEGFR-1 and VEGFR-2. While the binding affinity of VEGF-A to VEGFR-2 is lower than that to VEGFR-1, the tyrosine kinase activity of VEGFR-2 is higher than that of VEGFR-1. Therefore, considering all factors, VEGFR-2 is considered the primary signal transduction receptor for VEGF-A binding (Non-Patent Literature 1, 2).

[0043] VEGFR-2 agonist activity In one embodiment, the peptide complex of the present invention preferably has VEGFR-2 agonist activity. The term "VEGFR-2 agonist activity" refers to the ability of naturally occurring VEGFR-2 agonists, namely VEGF, to produce effects similar to those caused by VEGFR-2. More specifically, it refers to the ability of VEGFR-2 to specifically activate VEGFR-2 dimerization and signal transduction pathways. "VEGFR-2 agonist activity" can be evaluated, for example, using a kinase assay system targeting VEGFR-2. For instance, non-limitingly, it can be evaluated using AlphaLISA assays, NFAT assays, etc., that detect phosphorylation of VEGFR-2 or ERK. Non-limitingly, examples of evaluation using, for instance, the AlphaLISA SureFire Ultra VEGFR-2 (p-Tyr1175) assay kit (PerkinElmer), the AlphaLISA SureFire Ultra p-ERK1 / 2 (T202 / Y204) assay kit (PerkinElmer), the VEGFR2 / NFAT Reporter-HEK293 recombinant cell line (BPS Bioscience), and the VEGF bioassay kit (Promega). When the peptide or peptide complex exhibits VEGFR-2 agonist activity, it is determined by following standard procedures and using optimal concentrations of the peptide or peptide complex under optimal conditions in any evaluation system. Non-limitingly, the agonist activity of a peptide can be represented using EC50.

[0044] Nucleic acid This specification also describes a nucleic acid encoding the first peptide and the peptide complex. In this specification, "nucleic acid" can be natural or non-natural and includes, but is not limited to, DNA, RNA, and their chimeras. The nucleic acid can be designed and manufactured based on the sequence of the first peptide and the peptide complex using known methods.

[0045] Peptide-drug complex (PDC) In one embodiment, the present invention relates to a peptide or peptide complex comprising the present invention, a desired substance to be delivered to VEGFR-2, and a complex (peptide-drug complex) for binding the peptide or peptide complex to the substance. The peptide complex is considered to bind to VEGFR-2 due to its VEGFR-2 agonist activity. Therefore, the peptide or peptide complex can deliver the substance to VEGFR-2. The substance described can be any substance desired to be delivered to VEGFR-2, provided it is intended to be such that it is not limited to that intended for delivery to VEGFR-2. Examples of such substances are not limited, but the following substances may be listed: Compounds: can be low molecular weight compounds or medium molecular weight compounds, and examples include well-known low molecular weight pharmaceuticals. Peptide: A peptide that can bind to a target in the body and exert a certain effect; for example, it can be a cyclic peptide. RI: Low-molecular-weight and medium-molecular-weight compounds, antibodies, etc., labeled with radioactive isotopes; any compound labeled with a radioactive isotope is acceptable. Examples include compounds used in PET scans. Proteins: This includes any protein that performs a useful function in the body, such as antibodies or enzymes. Examples include enzymes used in enzyme replacement therapy. Nucleic acids: can be DNA, RNA, or their chimeras, without particular limitation. Examples include nucleic acid pharmaceuticals. The molecules used in a drug delivery system (DDS) can be liposomes, micelles, or other known molecules used in DDS. The DDS molecule may also contain pharmaceutical compounds. In addition, the substance that is desired to be delivered to VEGFR-2 can be a complex of the substances listed above.

[0046] Composition In one embodiment, the present invention relates to a composition comprising the peptide complex and a carrier. Examples of the carrier include: water such as sterile water, pure water, and distilled water; physiological saline; glucose solution; alcohols such as ethanol; polyols such as glycerol, propylene glycol, and polyethylene glycol; sterile organic solvents; or aqueous starch; and any one or a mixture of two or more of PBS.

[0047] Compositions for cell culture In one respect, the present invention relates to compositions for cell culture. The cell culture composition is a composition for cell culture. Because the peptide complex has VEGFR-2 agonist activity, it can also be used as a reagent or additive for culture media for cell culture, preferably for mammalian cells, and more preferably for culture media for human cells. The culture media reagent or additive for cell culture can be a reagent or additive for culture media used to culture cells for the production of cultured meat. Additionally, the peptide complex can also be used as a reagent or additive in a culture medium for preparing liver organoids for the treatment of liver diseases. These liver organoids are not limited to being created by co-culturing hepatic endodermal cells, vascular endothelial cells, and mesenchymal cells induced from pluripotent stem cells. While the peptide complex can be used in systems induced to differentiate into vascular endothelial cells (Non-Patent Literature 3, 4), it is not limited thereto. Naturally occurring VEGFR-2 agonists, such as VEGF, can be induced to differentiate from pluripotent stem cells into vascular endothelial cells and vascular wall cells, and co-cultured with cardiomyocytes, intestinal epithelial cells, pancreatic islets, etc., thereby promoting the formation of higher-order structures and the establishment / maintenance of organoids (Non-Patent Literature 5, 6, 7). Thus, since VEGF is a molecule that plays a fundamental role in tissue creation, the peptide complex can be used as a reagent or additive for the production of various cells as described above.

[0048] The culture medium is not particularly limited as long as it is used for culturing cells or tissues. The culture medium can be a serum culture medium, preferably a serum-free culture medium or a low-serum culture medium. In one embodiment, the culture medium additive can be in the form of a solution or a dried solid (e.g., solid, powder, etc.). In the case of a solution, it can be used directly as a culture medium, or the substance obtained by diluting it with a solvent and adding the aforementioned additive as needed can be used as a culture medium. Examples of solvents used for dilution include water, buffer solutions, physiological saline, and various culture media used for cell or tissue culture; these can be used alone or in combination of two or more. When the culture medium additive is in the form of a dry solid, for example, a substance obtained by dissolving in solvents such as water, buffer solution, physiological saline, and various cell or tissue culture media, and adding the above-mentioned additives as needed, can be used as a culture medium. The content of the peptide complex of the present invention in the culture medium for culturing cells or tissues or in the culture medium for cells derived therefrom, as a final concentration, for example, relative to the total amount of the composition or the total amount of the culture medium, can be from about 0.01 to about 10,000 nmol / L, preferably from about 0.1 to about 1,000 nmol / L, more preferably from about 0.5 to about 1,000 nmol / L, and even more preferably from about 1 to about 100 nmol / L.

[0049] Medical Composition In one embodiment, the present invention relates to a medical composition. This medical composition, as one embodiment, comprises the peptide complex of the present invention, a pharmaceutically acceptable salt thereof, or a solvate thereof (for brevity, these will be referred to simply as peptides below). Preferably, the medical composition comprises an effective amount of the peptide complex as an active ingredient. The disease to which the medical composition is the object is a disease caused by an increase or decrease in the expression or activity of VEGFR-2, a disease that is aggravated as a result, or any other related disease; or a disorder caused by a decrease in VEGF signaling or any other intracellular signaling cascade activated by VEGFR-2, a disorder that is aggravated as a result, or any other related disorder. Examples of trauma include chronic trauma, acute trauma, and general trauma. Chronic trauma refers to unhealed trauma, including, but not limited to, arterial ulcers, diabetic ulcers, bedsores, and venous ulcers. Acute trauma can develop into chronic trauma. Acute trauma includes, but is not limited to, trauma caused by, for example, heat injury, external injury, surgery, extensive excision of skin cancer, deep fungal and bacterial infections, vasculitis, scleroderma, pemphigus, and toxic epidermal necrolysis. General trauma refers to trauma that has healed and repaired through conventional trauma treatment. Liver dysfunction, pathological states of liver disease, and all structural and / or functional abnormalities of the liver are associated with, but are not limited to, liver failure, hepatitis, cirrhosis, toxic liver injury, drug-induced liver injury, hepatic encephalopathy, hepatic coma, and liver necrosis. Prevention of liver injury refers to the prevention of structural or functional liver damage directly or indirectly caused by internal or external factors (e.g., chemical agents, biological agents, etc.) or combinations thereof. Kidney diseases include, but are not limited to, inflammatory kidney disease, nephritis, glomerulosclerosis, glomerulonephritis, and focal segmental glomerulosclerosis. Additionally, they also include infections that cause kidney disease.

[0050] The method of administration of the medical composition is not particularly limited; it can be administered orally or non-orally. Examples of non-oral administration include intramuscular injection, intravenous injection, subcutaneous injection, transdermal administration, and transmucosal administration (via nose, mouth, eye, lung, vagina, or rectum). The peptides in the medical composition can be modified in various ways to facilitate metabolism and excretion. For example, the addition of polyethylene glycol (PEG) or sugar chains to the peptide can prolong its retention time in the blood and reduce its antigenicity. Additionally, biodegradable polymers such as polylactic-co-glycolic acid copolymer (PLGA), porous hydroxyapatite, liposomes, surface-modified liposomes, emulsions prepared from unsaturated fatty acids, nanoparticles, and nanospheres can be used as sustained-release agents to encapsulate the peptides. In the case of transdermal administration, a weak current can be passed through the skin surface to allow it to penetrate the stratum corneum (iontophoresis).

[0051] The medical composition can be used directly with the active ingredient, or it can be formulated by adding pharmaceutically acceptable carriers, excipients, additives, etc. Examples of dosage forms include, for instance, liquids (e.g., injections), dispersants, suspensions, tablets, pills, powders, suppositories, powders, granules, capsules, syrups, lozenges, inhalers, ointments, eye drops, nasal drops, ear drops, and pastes. The formulation may be carried out by using appropriate excipients, binders, disintegrants, lubricants, solvents, dissolution aids, colorants, flavoring and odor-correcting agents, stabilizers, emulsifiers, absorption promoters, surfactants, pH adjusters, preservatives, antioxidants, etc., and by conventional methods.

[0052] Examples of ingredients used in the formulation include, but are not limited to, pharmaceutically acceptable organic solvents such as purified water, saline solution, phosphate buffer, D-glucose, glycerol, and ethanol; animal and vegetable oils; lactose; mannitol; glucose; sorbitol; crystalline cellulose; hydroxypropyl cellulose; starch; corn starch; silica; magnesium aluminum silicate; collagen; polyvinyl alcohol; polyvinylpyrrolidone; carboxyvinyl polymers; sodium carboxymethyl cellulose; sodium polyacrylate; sodium alginate; water-soluble dextran; sodium carboxymethyl starch; pectin; methylcellulose; ethylcellulose; xanthan gum; gum arabic; tragali gum; casein; agar; polyethylene glycol; diglycerides; glycerol; propylene glycol; petrolatum; paraffin; octyl dodecyl myristate; isopropyl myristate; higher alcohols; stearyl alcohol; stearic acid; human serum albumin; trehalose; and polysorbate.

[0053] The absorption enhancer can be used to improve the absorption of poorly absorbed drugs. As the absorption enhancer, the following surfactants can be used: polyoxyethylene lauryl ethers, sodium lauryl sulfate, saponins, etc.; bile salts such as glycocholic acid, deoxycholic acid, taurocholic acid, etc.; chelating agents such as EDTA, salicylic acid, etc.; fatty acids such as hexanoic acid, decanoic acid, lauric acid, oleic acid, linolenic acid, mixed micelles, etc.; enamine derivatives, N-acyl collagen peptides, N-acyl amino acids, cyclodextrins, chitosans, nitric oxide donors, etc.

[0054] The pills or tablets may also be coated with sugar, gastric-soluble, or enteric-soluble substances. The injectable formulation may contain distilled water for injection, physiological saline, propylene glycol, polyethylene glycol, vegetable oil, alcohols, etc. Furthermore, wetting agents, emulsifiers, dispersants, stabilizers, solubilizers, dissolving aids, preservatives, etc., may be added.

[0055] In one respect, the medical composition of the present invention can be administered in combination with other medicines or treatments useful for the said disease.

[0056] The dosage of the medical composition of the present invention administered to mammals (e.g., humans, mice, rats, guinea pigs, rabbits, dogs, horses, monkeys, pigs, sheep, etc.), especially humans, varies depending on the symptoms, the patient's age, sex, weight, tolerance differences, method of administration, dosing interval, type of active ingredient, and type of formulation, and is not particularly limited. However, for example, 30 μg to 1000 mg, 100 μg to 500 mg, or 100 μg to 100 mg can be administered once or divided into several doses. In the case of injection administration, 1 μg / kg to 3000 μg / kg or 3 μg / kg to 1000 μg / kg can be administered once or divided into several doses, depending on the patient's weight.

[0057] Diagnostic Compositions In one respect, the present invention relates to diagnostic compositions. The peptide complex, having VEGFR-2 agonist activity, is believed to bind to VEGFR-2. Therefore, it can also be used as a diagnostic agent for detecting VEGFR-2. As a diagnostic agent, it can be used as a detector for detecting the expression level of VEGFR-2; when used as a detector, the peptide complex of the present invention can also be detectably labeled. Thus, the peptide complex or compositions comprising them can be used as diagnostic agents for detecting VEGFR-2.

[0058] Research Composition In one respect, the present invention relates to a composition for research use. The peptide complex, having VEGFR-2 agonist activity, is believed to bind to VEGFR-2. Therefore, it is preferably used for research related to VEGFR-2.

[0059] As one approach, the present invention provides a method for testing at least one of a peptide and / or complex, said complex comprising a peptide and a substance further bound to a linker: a) Solubility in solvent b) Binding ability with VEGFR-2 c) Toxicity to cells and / or tissues, d) Toxicity in laboratory animals, The peptide is a peptide having an amino acid sequence in which 1 to 3 amino acid residues have been deleted, substituted, inserted and / or added in the amino acid sequence of the peptide of the present invention.

[0060] For the aforementioned test method, the solubility of peptides and / or complexes in a solvent can be measured as a solubility test. When measuring solubility, the solvent is not limited and can be freely selected according to the purpose. Furthermore, regarding the solubility measurement method, a known method can be appropriately selected depending on the type of solvent. The binding ability test for VEGFR-2 can be used to measure the binding ability with VEGFR-2. Although there are no limitations, known methods such as surface plasmon resonance (SPR) analysis, Scatchard analysis and / or radioimmunoassay (RIA), enzyme immunoassay (EIA), and sandwich competition assay are preferred. The toxicity test for cells and / or tissues can be a well-known toxicity evaluation test using cells and / or tissues, for example, a method performed in vitro. The cells and tissues can be those used in toxicity evaluation tests for general pharmaceutical products, without limitation. The toxicity testing methods for laboratory animals can be any well-known toxicity evaluation tests using laboratory animals. There are no particular limitations on the laboratory animals used, as long as they are commonly used animals. Examples include mice, rats, guinea pigs, gerbils, hamsters, ferrets, rabbits, dogs, cats, pigs, goats, horses, cattle, birds (e.g., chickens, quails, etc.), monkeys, and primates excluding humans (e.g., cynomolgus monkeys, marmosets, rhesus monkeys, etc.). While the above-mentioned toxicity evaluation tests are not limited, they can be any safety tests commonly performed in non-clinical trials of pharmaceuticals. Examples include general toxicity tests (single-dose toxicity tests / repeated-dose toxicity tests), genotoxicity tests (Ames test / chromosomal abnormality test / in vitro micronucleus test), carcinogenicity tests, reproductive and developmental toxicity tests (ICH-I, II, III), local irritation tests (eye irritation test, skin irritation test, etc.), other toxicity tests (skin sensitization test, phototoxicity test, antigenicity test), chemical analysis / biological analysis (TK / PK), etc.

[0061] Abbreviation (general) Angstrom (unit: Å) Bovine serum albumin is BSA; The tert-butyloxycarbonyl group is Boc; The chloroacetyl group is ClAc; (2,5-Dioxopyrrolidone-1-yl)2-chloroacetic acid ester is ClAcOSu; Dichloromethane or methylene chloride is called DCM; N,N'-diisopropylcarbodiimide is DIPCI; N,N-diisopropylethylamine is DIPEA or DIEA; Dimethyl sulfoxide is DMSO; N,N-dimethylformamide is DMF; 3,6-Dioxa-1,8-octanedithiol is DODT; Dulbecco's modified eagle medium was DMEM. 50% effective concentration is EC50; Fetal bovine serum is FBS; 9-fluorenylmethyloxycarbonyl group is Fmoc; N 2 N 6 -Bis(((9H-fluorene-9-yl)methoxy)carbonyl)-L-lysine is Fmoc-Lys(Fmoc)-OH; Grams (unit: g) are units of measurement. O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethylurea hexafluorophosphate is HATU; High-performance liquid chromatography (HPLC) is the method used. The liquid chromatography-mass spectrometry instrument is either LC-MS or LC / MS; The mole (unit) is M; Acetonitrile is MeCN; Milligram (unit) is mg; Minutes (unit) are in min: Milliliters (units) are expressed in mL; Millimoles (units) are expressed in mM; Millimeters (unit) are measured in mm; O-3-methyl-pentan-3-yl is Mpe group N-hydroxysuccinimide is NHS; Nanometers (unit: nm) are measured in nanometers. Microliters (units) are expressed in μL; Oxysuccinimide is OSu; 2,2,4,6,7-Pentamethyldihydrobenzofuran-5-sulfonyl group is Pbf; Polyethylene glycol is PEG; Rotation speed per minute (unit: rpm); Dibenzocycloheptyl is Sub tert-butyl is tBu; Triethylamine acetate is TEAA; Trifluoroacetic acid is TFA; Triisopropylsilane is TIS; Triphenylmethyl is either Trt or Tr;

[0062] Abbreviation (non-natural amino acids) Ahp (S)-2-aminoheptanoic acid (CAS No.: 44902-02-5) alT allothreonine (CAS No.: 28954-12-3) Atb (S)-2-amino-4,4-dimethylvaleric acid (CAS No.: 57224-50-7) Atp(2S)-2-amino-3-(tetrahydropyran-4-yl)propionic acid (CAS No.: 1344910-91-3) Bph (S)-3-([1,1'-biphenyl]-4-yl)-2-aminopropionic acid (CAS No.: 155760-02-4) Cha (S)-2-amino-3-cyclohexylpropionic acid (CAS No.: 27527-05-5) Chg (S)-2-amino-2-cyclohexylacetic acid (CAS No.: 14328-51-9) Cit L-citrulline (CAS No.: 372-75-8) cPEG9c 4,7,10,13,16,19,22,25,28-Nonoxatrionedioleic acid (CAS No.: 1268488-70-5) cPEG13c 4,7,10,13,16,19,22,25,28,31,34,37,40-Trinoxatritetradecanedioleic acid (CAS No.: 2225903-66-0) cPEG17c 4,7,10,13,16,19,22,25,28,31,34,37,40,43,46,49,52-Heptacyclopentadecanedioleic acid (CAS No.: 2226897-74-9) F4aao (S)-2-amino-3-(4-(carboxymethoxy)phenyl)propionic acid (CAS No.: 24558-63-2) F4c (S)-2-amino-3-(4-chlorophenyl)propionic acid (CAS No.: 14173-39-8) F4COO 4-Carboxy-L-phenylalanine (CAS No.: 126109-42-0) F4G (S)-2-amino-3-(4-guanidinophenyl)propionic acid (CAS No.: 59574-11-7) H4Py (S)-2-amino-4-(pyridin-4-yl)butyric acid (CAS No.: 1240588-62-8) Har N6-carbamimidoyl-L-lysine (CAS No.: 156-86-5) Hcit N6-carbamoyl-L-lysine (CAS No.: 1190-49-4) Hgl L-2-aminohexanoic acid (CAS No.: 1118-90-7) Hph (S)-2-amino-4-phenylbutyric acid (CAS No.: 943-73-7) Hty (homo-L-tyrosine) (CAS No.: 221243-01-2) Hyp Hydroxyproline (CAS No.: 51-35-4) MetO2 (S)-2-amino-4-(methanesulfonyl)butyric acid (CAS No.: 7314-32-1) NHS-cPEG9c-NHS Bis(2,5-dioxopyrrolidone-1-yl)4,7,10,13,16,19,22,25,28-nonoxatrione dicester (CAS No.: 1008402-79-6) NHS-cPEG13c-NHS bis(2,5-dioxopyrrolidone-1-yl)4,7,10,13,16,19,22,25,28,31,34,37,40-tetraoxatetradecanoic acid ester (Bis-PEG13-NHS ester CAS No.: 2221949-00-2) NHS-cPEG17c-NHS bis(2,5-dioxopyrrolidone-1-yl)4,7,10,13,16,19,22,25,28,31,34,37,40,43,46,49,52-heptadecoxapentadecane ester (Bis-PEG17-NHS ester CAS No.: 2221948-93-0) NHS-OCOPEG13OCO-NHS Bis(2,5-dioxopyrrolidine-1-yl)(3,6,9,12,15,18,21,24,27,30,33-undecoxapentadecane-1,35-diyl) dicarbonate (TS-L12-TS Nippon Oil Co., Ltd.) NHS-OCOPEG17OCO-NHS Bis(2,5-dioxopyrrolidine-1-yl)(3,6,9,12,15,18,21,24,27,30,33,36,39,42,45-pentadecaoxaheptadecane-1,47-diyl) dicarbonate (TS-L16-TS Nippon Oil Co., Ltd.) OCOPEG13OCO 3,6,9,12,15,18,21,24,27,30,33-Undecanoxapentadecane-1,35-Dimethylbis(bicarbonate) (Nippon Oil Co., Ltd.) OCOPEG17OCO 3,6,9,12,15,18,21,24,27,30,33,36,39,42,45-Decadecoxaheptadecane-1,47-Dimethylbis(bicarbonate) (Nippon Oil Co., Ltd.) PEG8c 1-Amino-3,6,9,12,15,18,21,24-octaoxaheptadecane-27-oleic acid (CAS No.: 756526-04-2) PEG12c 1-Amino-3,6,9,12,15,18,21,24,27,30,33,36-dodecoxane-39-oleic acid (CAS No.: 1415408-69-3) 3Py6NH2 (S)-2-amino-3-(6-aminopyridin-3-yl)propionic acid (CAS No.: 1269968-61-7) 4Py2NH2 (S)-2-amino-3-(2-aminopyridin-4-yl)propionic acid (CAS No.: 1269969-46-1) Tbg (S)-2-amino-3,3-dimethylbutyric acid (CAS No.: 20859-02-3) W7N (S)-2-amino-3-(1H-pyrrolo[2,3-b]pyridin-3-yl)propionic acid (CAS No.: 49758-35-2) Yph (S)-2-amino-3-(4-phenoxyphenyl)propionic acid (CAS No.: 150351-64-7) Example

[0063] The present invention has been described in detail below based on embodiments, but the invention is not limited to these embodiments. Those skilled in the art can readily make modifications and alterations to the invention based on the description herein, and these modifications are included within the scope of the invention.

[0064] Chemical synthesis In the following examples, all raw materials, structural units, reagents, acids, bases, solid resins, and solvents used in the chemical synthesis are commercially available or can be synthesized by those skilled in the art using organic chemical methods. It should be noted that amino acids containing protecting groups are commercially available unless otherwise specified. The resins described in each example were used as starting materials, and peptide chain elongation in the solid-phase resin was performed using conventional peptide coupling reaction conditions and Fmoc removal reaction conditions. The reactions were conducted using automated peptide synthesizers, namely Biotage Syro I, Biotage Syro II, CEM Liberty Blue, CEM Liberty BlueHT12, or CEM Liberty Prime, according to the manufacturer's manual. The resin used is either NovaPEG Rink amide resin or Seiber amide resin, and the amount used ranges from 5 mg to 2 g depending on the peptide. The reaction mixture (cocktail) used for deprotection of the side chains and cleavage from the solid resin is 4 mL to 50 mL depending on the peptide, and uses a solution with the following composition. A: TFA / H2O / TIS / DODT (92.5 / 2.5 / 2.5 / 2.5) B: TFA / H2O / TIS / DODT (90 / 2.5 / 2.5 / 5) The common amino acids used are listed below, with side-chain protecting groups shown in parentheses. Table 1. Common amino acids used in the examples

[0065] [Table 1-1] [Table 1-2]

[0066] Unless otherwise specified, any of the following reverse-phase preparation purification apparatuses (A), B), C), and D) may be used as the purification method for the obtained crude purified peptide. A): Shimadzu preparative high-performance liquid chromatography (prep-HPLC) systems (LC-20AP, SPD-M20A, CTO-20AC, and CBM-20A); B): Waters AutoPurification System; C): Waters AutoPurification System with SQD; D): Waters Preparative HPLC System; Unless otherwise specified, any one of the following columns (a) to (n) shall be used. a) Jeanious One-Column 20mm I.D. x 150mm L b) Kinetex EVO C18 5μm 21.2x150mm c) Waters XBridge BEH Prep OBD Amide 5μm 19x150mm d) Waters XBridge C18 19x150mm e) Waters XBridge C18 5μm 19x150mm f) Waters XBridge C18 5μm 30x150mm g) Waters XBridge C18 5μm 50x150mm h) Waters XSelect C18 19x150mm i) Waters XSelect C18 30x150mm j) Waters XSelect C18 50x150mm k) Waters XSelect C18 5μm 19x150mm l) Waters XSelect C18 5μm 30x150mm m) Waters XSelect CSH Prep C18 5μm OBD 30x150mm n) Waters XSelect CSH Prep C18 5μm OBD 50x250mm

[0067] For the structural confirmation of chemically synthesized peptides, the molecular weight was confirmed by ESI-MS(+) mass spectrometry, taking into account the amino acids used in the target sequence and the structural units used as required. It should be noted that "ESI-MS(+)" indicates electrospray ionization mass spectrometry performed in positive ion mode. The detected mass is expressed in m / z units. It should be noted that compounds with molecular weights greater than approximately 1000 were detected frequently as multivalent ions.

[0068] Basic analytical apparatus and basic conditions In the mass spectrometry analysis of the synthesized peptides in the following examples, unless otherwise specified, the following basic analytical apparatus and conditions were used. Gradient B (%) was analyzed using any of the x / y conditions.

[0069] Equipment: Shimadzu LC / MS system (LC-20ADXR, CTO-20AC, SPD-M20A, SIL-20AXR, CBM-20A and LCMS-2020) Column: Kinetex EVO C18 2.6μm 2.1x150mm, 100Å Column temperature: 60℃ Mobile phase A: 0.025% TFA aqueous solution Mobile phase B: 0.025% TFA acetonitrile solution Flow rate: 0.5 mL / min Wavelength: 225nm PDA Gradient B (%): x: 20-60% / 7.15min, 60-95% / 0.3min, 95-95% / 1.55min; y: 40-80% / 7.15min, 80-95% / 0.3min, 95-95% / 1.55min.

[0070] Example 1 Synthesis of peptide complex (dimer structure number 52) In this embodiment, a peptide complex with the following structure was synthesized (the complex of peptide sequence number 47 and linker structure number 6, and the dimer structure number 52 in Table 4).

[0071] [Chemical Formula 1]

[0072] The target peptide was synthesized using Sieber amide resin (Watanabe Chemicals, 0.60 mmol / g) following the general method described above, starting with the removal of the Fmoc group. A Liberty Blue HT12 solid-phase synthesizer from CEM was used, and the synthesis was performed according to the manufacturer's manual. For each residue introduction, Fmoc-AA / DIPCI / Oxyma pure (4.2 / 8 / 4 equivalents) was used relative to 1 resin equivalent, and the reaction was carried out once every 3 minutes in DMF at 90°C. However, the 3rd and 9th residues were reacted once every 10 minutes at 90°C. The 14th residue was reacted once every 15 minutes at 50°C. Fmoc removal is achieved by reacting it with a 10% pyrrolidine DMF solution at 90°C for 1 minute or at 50°C for 90 seconds. The introduction of chloroacetyl groups was carried out by adding a DMF solution of ClAcOSu (5 equivalents) to the solid-phase resin and shaking at room temperature for 60 minutes. Regarding the deprotection of the side chains and their cleavage from the solid resin, firstly, the chloroacetyl group was introduced into the resin obtained after the previous step and washed with DMF, then with methylene dichloro, followed by washing with diethyl ether. The resin was then dried under reduced pressure. In the reaction vessel containing the solid resin, reactant mixture A (a mixture of TFA / H2O / TIS / DODT in a volume ratio of 92.5:2.5:2.5:2.5) was added, and the mixture was shaken at room temperature for 60 minutes. The reaction solution was recovered by filtration through a sieve plate. The filtrate was added to an excess of cooled diethyl ether / hexane (1 / 1) mixed solvent, producing a white turbid precipitate. This mixture was centrifuged, and the solution was decanted. The resulting solid was washed again with cooled diethyl ether and dried under reduced pressure. The resulting solid was used in the subsequent cyclization reaction. For the peptide cyclization reaction, the peptide was dissolved in DMSO / H2O (9 / 1) at a final concentration of 4.2 mM based on the molar amount of the solid-phase resin. Triethylamine (20 equivalents) was added, and the mixture was shaken at room temperature for 1 hour, followed by the addition of acetic acid. The resulting reaction solution was concentrated under reduced pressure using a Genevac HT-12 column. The crude product was purified using the following conditions (column: Waters XSelect C18 5µm 50x250mm). Mobile phases: A = 20 mM TEAA aqueous solution, B = 20 mM TEAA acetonitrile solution, C = 0.2 M TEAA aqueous solution, D = MeCN; Temperature: 50℃; Gradient (%A conc): 5.0 minutes, 0.1%; then 0.1 minutes, 0.1%-100%; after 5.1 minutes, (100%-%B); (%B conc): 5.1 minutes, 0%; then 1.9 minutes, 0%-4.2%; then 3 minutes, 4.2%-20.5%; then 15.5 minutes, 20.5%-25.6%; then 1.5 minutes, 25.6%-60%; then 4 minutes, 60%; (%C conc): 5.0 minutes, 99.9%; then 0.1 minutes, 99.9%-0%; after 5.1 minutes, 0%; (%D conc): 0%. Flow rate: 18 mL / min for 5.1 minutes, then 118 mL / min for 1.9 minutes (18 mL / min - 118 mL / min), then 118 mL / min. After freeze-drying, 0.1% TFA water-acetonitrile solution (1:1, 100 mL) was added, and the mixture was freeze-dryed again. The resulting peptide A was used for the synthesis of peptide complexes. Regarding the synthesis of the peptide complex, peptide A was dissolved in DMSO to achieve a final concentration of 25 mM. Then, NHS-OCOPEG17OCO-NHS (0.4 equivalents) and DIEA (10 equivalents) were added. After shaking at room temperature for 1 hour, acetic acid was added. The crude product was purified using the following conditions (column: Waters XBridge C18, 19x150mm; mobile phase: A = 0.1% TFA aqueous solution, B = 0.1% TFA acetonitrile solution; temperature: 50℃; gradient (%B conc): 15-40% for 3 minutes, then 40-45% for 8 minutes, then 45-60% for 1 minute; flow rate: 17 mL / min). The purity of the target analyte was calculated from the area ratio of the LC / MS (UV wavelength 225 nm) chromatogram under the following analytical conditions and was 94.86%. Analytical conditions: Residence time = 5.7 min; Column: Kinetex EVO C18 2.6 μm 2.1 x 150 mm, 100 Å; Mobile phase: A = 0.025% TFA aqueous solution, B = 0.025% TFA acetonitrile solution; Temperature: 60 °C; Gradient (%B conc): 7.15 min 20–60%, then 0.30 min 60–95%, then 1.55 min 95–95%; Flow rate: 0.5 mL / min; ESI-MS (+) observation m / z = 1197 (M+4H)4+.

[0073] Example 2 Synthesis of peptide complex (dimer structure number 1) In this embodiment, a peptide complex with the following structure was synthesized (a complex of peptide sequence number 3 and linker structure number 1, and dimer structure number 1 in Table 4).

[0074] [Chemical Formula 2]

[0075] Using Sieber amide resin (Watanabe Chemicals, 0.65 mmol / g), the target peptide was synthesized by starting with the removal of the Fmoc group, introducing Fmoc-Lys(Fmoc)-OH, followed by the introduction of the linker moiety PEG and each Fmoc amino acid, according to the general method described above. The synthesis was performed using a Biotage Syro I solid-phase synthesizer according to the manufacturer's manual. For each residue introduction, Fmoc-AA / HATU / DIEA (9.45 equivalences / 9 equivalences / 18.8 equivalences) were used relative to 1 resin equivalent, and the reaction was carried out twice in DMF at 75°C for 20 minutes. However, the second residue was reacted three times at 75°C for 20 minutes. The fourth residue was reacted twice at room temperature for 60 minutes. The fourteenth residue was reacted twice at room temperature for 30 minutes. The introduction of the 15th residue, the linker moiety Fmoc-PEG12c, and Fmoc-Lys(Fmoc)-OH was carried out twice at 75°C for 20 minutes. Regarding Fmoc removal, after reacting with a 20% piperidine DMF solution for 5 minutes at room temperature, the solution was removed, and then reacted again with a 20% piperidine DMF solution for 15 minutes at room temperature. Regarding the introduction of chloroacetyl groups, for a solid-phase resin containing the Fmoc-protected peptide obtained in the previous step, after removing the Fmoc group of the α-amino group using the method described above, chloroacetic acid (0.5M DMF solution, 10 equivalents), HCTU (0.49M DMF solution, 9.8 equivalents) and DIEA (0.5M DMF solution, 10 equivalents) are added to the solid-phase resin and shaken at room temperature for 30 minutes, thereby proceeding. Regarding the deprotection of side chains and cleavage from the solid resin, a reaction mixture-A (a mixture of TFA / H2O / TIS / DODT in a volume ratio of 92.5:2.5:2.5:2.5) was added to a reaction vessel containing the solid resin. After careful shaking, the mixture was incubated at room temperature for 60 minutes. The reaction solution was recovered by filtration through a sieve plate. This filtrate was added to an excess of cooled diisopropyl ether / hexane (1 / 1) mixed solvent, resulting in a white turbid precipitate. This mixture was centrifuged, and the solution was decanted. The resulting solid was washed again with cooled diisopropyl ether / hexane (1 / 1) mixed solvent and dried under reduced pressure for 60 minutes. The resulting solid was used for subsequent cyclization reactions. For the peptide cyclization reaction, the peptide was dissolved in DMSO / MeCN / H2O (18 / 1 / 1) at a final concentration of 1 mM based on the molar amount of solid resin, followed by the addition of triethylamine (20 equivalents) and stirring at room temperature for 3 hours. The resulting reaction solution was then concentrated under reduced pressure using Genevac EZ-2 Elite. Purify the crude product under the following conditions: (Column: Waters XBridge (registered trademark) C18, 50x150mm; Mobile phase: A = 0.1% TFA aqueous solution, B = 0.1% TFA acetonitrile solution; Temperature: 40℃; Gradient (%Bconc): 3 min 9-34%, then 8 min 34-39%, then 1 min 39-60%; Flow rate: 120 mL / min). The purity of the target analyte was calculated from the area ratio of the LC / MS (UV wavelength 225 nm) chromatogram under the following analytical conditions and was 86.83%. Analytical conditions: Residence time = 5.3 min; Column: Kinetex EVO C18, 2.6 μm, 2.1 x 150 mm, 100 Å; Mobile phase: A = 0.025% TFA aqueous solution, B = 0.025% TFA acetonitrile solution; Temperature: 60 °C; Gradient (%B conc): 7.15 min 20–60%, then 0.30 min 60–95%, then 1.55 min 95–95%; Flow rate: 0.5 mL / min; ESI-MS (+) observation m / z = 1023(M+5H)⁵⁺.

[0076] Example 3 Synthesis of peptide complex (dimer structure number 43) In this embodiment, a peptide complex with the following structure was synthesized (the complex of peptide sequence number 42 and linker structure number 4, and the dimer structure number 43 in Table 4).

[0077] [Chemical Formula 3]

[0078] The target peptide was synthesized using Sieber amide resin (Watanabe Chemicals, 0.57 mmol / g) via the general method described above, starting with the removal of the Fmoc group. A Biotage SyroI solid-phase synthesizer was used, and the synthesis was performed according to the manufacturer's manual. For each residue introduction, Fmoc-AA / HATU / DIPEA (4.2 equivalences / 4 equivalences / 8.4 equivalences) were used relative to 1 resin equivalent, and the reaction was carried out twice in DMF at 75°C for 20 minutes. However, the 13th residue was reacted twice at 75°C for 30 minutes. The 14th residue was reacted once at room temperature for 30 minutes. The 15th residue was reacted once at 75°C for 20 minutes. Regarding Fmoc removal, after reacting with a 20% piperidine DMF solution for 5 minutes at room temperature, the solution was removed, and then reacted again with a 20% piperidine DMF solution for 15 minutes at room temperature. Regarding the introduction of chloroacetyl groups, for a solid-phase resin that has retained the Fmoc-protected peptide obtained in the previous step, after removing the Fmoc group of the α-amino group using the method described above, DIPCI (10 equivalents), HOSu (10 equivalents), and chloroacetic acid (10 equivalents) are stirred in DCM, and DMF of the same amount as DCM is added. The DCM / DMF solution of ClAcOSu is adjusted, and the resulting substance is added to the solid-phase resin. The mixture is then shaken at room temperature for 60 minutes. Regarding the deprotection of the side chains and their cleavage from the solid resin, a reaction mixture-A (a mixture of TFA / H2O / TIS / DODT in a volume ratio of 92.5:2.5:2.5:2.5) was added to a reaction vessel containing the solid resin, and the mixture was shaken at room temperature for 60 minutes. The reaction solution was recovered by filtration through a sieve plate. The filtrate was added to excess, cooled diisopropyl ether, resulting in a white, turbid precipitate. This mixture was centrifuged, and the solution was decanted. The resulting solid was washed again with cooled diethyl ether and dried under reduced pressure. The resulting solid was used for subsequent cyclization reactions. For the peptide cyclization reaction, the peptide was dissolved in DMSO / H2O (9 / 1) at a final concentration of 5 mM based on the molar amount of solid-phase resin, and then triethylamine (10 equivalents) was added. The mixture was stirred at room temperature for 15 hours. The resulting reaction solution was concentrated under reduced pressure using Genevac EZ-2 Elite to obtain unpurified cyclic peptide B. Regarding the synthesis of the peptide complex, the peptide B obtained above was dissolved in DMSO at a concentration of 25 mM, 10 equivalents of diisopropylethylamine were added, followed by 0.4 equivalents of NHS-cPEG17c-NHS. After shaking at room temperature for 30 minutes, acetic acid was added. Purify the crude product under the following conditions: (Column: Kinetex EVO C18 5μm 21.2x150mm; Mobile phase: A = 0.1% TFA aqueous solution, B = 0.1% TFA acetonitrile solution; Temperature: 60℃; Gradient (%B conc): 3 min 5-30%, then 8 min 30-35%, then 1 min 35-60%; Flow rate: 20 mL / min). The purity of the target analyte was calculated from the area ratio of the LC / MS (UV wavelength 225 nm) chromatogram under the following analytical conditions and was 87.55%. Analytical conditions: Residence time = 4.5 min; Column: Kinetex EVO C18, 2.6 μm, 2.1 x 150 mm, 100 Å; Mobile phase: A = 0.025% TFA aqueous solution, B = 0.025% TFA acetonitrile solution; Temperature: 60 °C; Gradient (%B conc): 7.15 min 20–60%, then 0.30 min 60–95%, then 1.55 min 95–95%; Flow rate: 0.5 mL / min; ESI-MS (+) observation m / z = 1194(M+4H)4+.

[0079] Example 4 Synthesis of various peptides In this embodiment, similar to Examples 1-3, various peptide complexes were chemically synthesized. The sequences of the synthesized cyclic peptides are shown in Table 2, the structures of the linkers are shown in Table 3, and the peptide complexes dimerized from the cyclic peptides via linkers are shown in Table 4. The binding numbers in Table 4 indicate the positions of the amino acids added by the linkers in the amino acid sequences shown by the peptide sequence numbers. It should be noted that in peptide sequences 14-35 and 37-51 in Tables 2 and 4, the C-terminal carboxylic acid of the 15th Gly is preferably replaced with an amide. The synthesized peptide complex was analyzed using any of the analytical conditions described above for the basic analytical apparatus and conditions, and its structure was confirmed by ESI-MS(+) mass spectrometry. The obtained ESI-MS(+) observations, along with the retention time, valence, and concentration gradient (%) of mobile phase B used in the analysis, are shown in Table 4.

[0080] Table 2. Sequences of the synthesized cyclic peptides [Table 2-1] [Table 2-2]

[0081] Table 3. Structure of connectors [Table 3]

[0082] Table 4. Peptide complexes [Table 4-1] [Table 4-2]

[0083] Example 5: Evaluation of VEGFR-2 agonist activity by pVEGFR AlphaLISA analysis To evaluate the VEGFR-2 activation ability of the VEGFR-2 agonist peptide of the present invention, VEGFR-2 phosphorylation was verified. In EGM... TM HUVECs were cultured in an Endothelial Cell Growth Medium-2 Bullet Kit (LONZA; CC-3162). After cell dissection using a Tryple (ThermoFisher Scientific), cells were seeded at a rate of 24,000 cells per well into a 96-well PDL-coated plate (Corning; 354461) for adherent cells and cultured for 2 days. For starvation, the medium was replaced with EBM-2 medium (LONZA) containing 0.5% FBS (ThermoFisher Scientific), 0.1% heparin (as per the LONZA; CC-3162 instructions above), and 0.1% gentamicin (Nacalai), and cultured for 18 hours. Recombinant human VEGF165a protein (R&D systems) or peptide was then added. After stimulation at 37°C for 10 minutes in a CO2 incubator, cells were lysed with the lysis buffer provided with the AlphaLISA SureFire Ultra VEGFR-2 (p-Tyr1175) assay kit (PerkinElmer). The procedure was followed according to the kit instructions, and signal detection was performed using a SpectraMax Paradigm multi-mode microplate reader (Molecular Devices). The obtained signals were analyzed using a GraphPad Prism, with the maximum value of the VEGF165a-induced signal set to 100% and no stimulation set to 0%, to calculate the percentage activity. The concentration of VEGF165a was evaluated at 5 points (0.08, 0.31, 1.25, 5, 20 nM) using a 1 / 4-fold dilution starting from 20 nM, and the maximum value (100%) was determined. Regarding the peptide concentration, experiments were conducted at 3.2, 16, and 80 nM for dimer structure number 1; at 10 and 100 nM for dimer structures numbers 2–41; and at 1.25, 5, and 20 nM for dimer structures numbers 42–57. Regarding the percentage of activity, peptides with more than 50% activity when 3.2 nM of peptide is added are designated as 1-A, peptides with more than 50% activity when 16 nM of peptide is added are designated as 1-B, peptides with more than 50% activity when 80 nM of peptide is added are designated as 1-C, and peptides with more than 1% but less than 50% activity when 80 nM of peptide is added are designated as 1-D. Peptides with more than 50% activity when 10 nM of peptide is added are designated as 2-A, peptides with more than 50% activity when 100 nM of peptide is added are designated as 2-B, and peptides with more than 1% but less than 50% activity when 100 nM of peptide is added are designated as 2-C. Peptides that exhibit more than 50% activity when 1.25 nM of peptide is added are designated as 3-A; peptides that exhibit more than 50% activity when 5 nM of peptide is added are designated as 3-B; peptides that exhibit more than 50% activity when 20 nM of peptide is added are designated as 3-C; and peptides that exhibit more than 1% but less than 50% activity when 20 nM of peptide is added are designated as 3-D.

[0084] The results are shown in Table 5. It should be noted that no peptides conforming to the above classification (1-A to 1-C, 3-A) were found. As shown in Table 5, the synthesized peptide complexes exhibit VEGFR-2 agonist activity that induces VEGFR-2 phosphorylation.

[0085] Table 5. Evaluation of VEGFR-2 agonist activity [Table 5-1] [Table 5-2]

[0086] Example 6 Evaluation of VEGFR-2 agonist activity by pERK AlphaLISA analysis To evaluate the intracellular signal activation ability of the VEGFR-2 agonist peptide of this invention, ERK phosphorylation was verified. Lysate from pVEGFR was used in the AlphaLISA assay, and the procedure was performed according to the PerkinElmer AlphaLISA SureFire Ultrap-Erk1 / 2 (Thr202 / Tyr204) assay kit. Signal detection was performed using a SpectraMax Paradigm multi-mode microplate reader (Molecular Devices). The obtained signals were analyzed using GraphPadPrism, with the maximum value of the VEGF165a-induced signal set to 100% and no stimulation set to 0%, and the % activity was calculated. The concentration of VEGF165a was evaluated at 5 points (0.08, 0.31, 1.25, 5, 20 nM) using a 1 / 4-fold dilution starting from 20 nM, and the maximum value (100%) was determined. Regarding the peptide concentration, experiments were conducted at 3.2, 16, and 80 nM for dimer structure number 1; at 10 and 100 nM for dimer structures numbers 2–41; and at 1.25, 5, and 20 nM for dimer structures numbers 42–57. Regarding the percentage of activity, peptides with more than 50% activity when 3.2 nM of peptide is added are designated as 1-A, peptides with more than 50% activity when 16 nM of peptide is added are designated as 1-B, peptides with more than 50% activity when 80 nM of peptide is added are designated as 1-C, and peptides with more than 1% but less than 50% activity when 80 nM of peptide is added are designated as 1-D. Peptides with more than 50% activity when 10 nM of peptide is added are designated as 2-A, peptides with more than 50% activity when 100 nM of peptide is added are designated as 2-B, and peptides with more than 1% but less than 50% activity when 100 nM of peptide is added are designated as 2-C. Peptides that exhibit more than 50% activity when 1.25 nM of peptide is added are designated as 3-A; peptides that exhibit more than 50% activity when 5 nM of peptide is added are designated as 3-B; peptides that exhibit more than 50% activity when 20 nM of peptide is added are designated as 3-C; and peptides that exhibit more than 1% but less than 50% activity when 20 nM of peptide is added are designated as 3-D.

[0087] The results are shown in Table 5. It should be noted that peptides conforming to the above classification of 1-A to 1-C, 3-A, and 3-D did not exist. As shown in Table 5, the synthesized peptide complexes exhibit not only VEGFR-2 phosphorylation induction ability but also the ability to inducing phosphorylation of the intracellular signal ERK.

[0088] Example 7 Evaluation of HUVEC proliferation promotion To evaluate the bioactivity of the VEGFR-2 agonist peptide of the present invention, a proliferation-promoting assay of HUVECs was performed. HUVECs cultured in the same manner as described above were peeled using a Tryple (ThermoFisher Scientific) cell line and seeded at a density of 10,000 cells per well into adherent cell line and Nunc luminescence assay. TM MicroWell TM 96-well Nunclon Delta-Treated Flat-Bottom Microplate (cat. # 136101). After 24 hours, the medium was replaced with EBM-2 medium (LONZA) containing 0.5% FBS (ThermoFisher Scientific), 0.1% heparin (LONZA; CC-3162), and 0.1% gentamicin (Nacalai) for 18 hours to starve the cells. Recombinant human VEGF165a protein (R&D systems) or peptide was then added, and the cells were incubated at 37°C for 2 days in a CO2 incubator. The procedure was performed according to the CellTiter-Glo (Promega) kit instructions, and signal detection was performed using a SpectraMax Paradigm multi-mode microplate reader (Molecular Devices). The resulting signals were analyzed using a GraphPad Prism, with the maximum value of the VEGF165a-induced signal set to 100% and no stimulation set to 0%, to calculate the % activity. The concentration of VEGF165a was evaluated at 6 points (0.006, 0.03, 0.16, 0.8, 4, 20 nM) with a 1 / 5-fold dilution starting from 20 nM, and the maximum value (100%) was determined. Regarding the peptide concentration, experiments were conducted at 1, 10, and 100 nM for dimer structures numbered 2, 5, 13, 14, 25, 27, 29, 31, and 33, and at 4 points (0.31, 1.25, 5, 20 nM) with a 1 / 4-fold dilution starting from 20 nM for dimer structures numbered 42–53. Regarding the percentage of activity, peptides with more than 50% activity when 1 nM of peptide is added are designated as 4-A, peptides with more than 50% activity when 10 nM of peptide is added are designated as 4-B, peptides with more than 50% activity when 100 nM of peptide is added are designated as 4-C, and peptides with more than 1% but less than 50% activity when 100 nM of peptide is added are designated as 4-D. Peptides exhibiting more than 50% activity upon the addition of 0.31 nM peptides are designated as 5-A; peptides exhibiting more than 50% activity upon the addition of 1.25 nM peptides are designated as 5-B; peptides exhibiting more than 50% activity upon the addition of 5 nM peptides are designated as 5-C; peptides exhibiting more than 50% activity upon the addition of 20 nM peptides are designated as 5-D; and peptides exhibiting more than 1% but less than 50% activity upon the addition of 20 nM peptides are designated as 5-E.

[0089] The results are shown in Table 6. It should be noted that peptides conforming to the above classification of 4-A, 4-C, and 5-E were not found. As shown in Table 6, the synthesized peptide complexes exhibited HUVEC proliferation-promoting capabilities.

[0090] Table 6. Evaluation of HUVEC proliferation promotion [Table 6]

[0091] Example 8 Evaluation of VEGFR-specific activation ability by RTK array analysis To evaluate the VEGFR-specific activation ability of the VEGFR-2 agonist peptide of the present invention, phosphorylation of 49 types of tyrosine kinase receptors containing VEGFR-2 was verified. HUVECs cultured in the same manner as described above were peeled using Tryple (ThermoFisher Scientific) and seeded at 720,000 cells per well into 6-well adherent cell plates, and cultured for 2 days. For starvation, the medium was replaced with EBM-2 (LONZA) containing 0.5% FBS (ThermoFisher Scientific), 0.1% heparin (LONZA; CC-3162 as described above), and 0.1% gentamicin (Nacalai), and cultured for 18 hours. Then, recombinant human VEGF165a protein (R&D systems) or peptides were added, and the cells were stimulated at 37°C for 10 minutes in a CO2 incubator. Cells were then lysed with the lysis buffer provided with the Human Phospho-RTK Array kit (R&D). The procedure was followed according to the kit instructions, and signal detection was performed using C-Digit (Li-COR). The obtained signals were analyzed using images from the Transparency Overlay Template provided with the kit. The concentration of VEGF165a was evaluated at 5 nM, and the concentration of peptides was evaluated at 20 nM.

[0092] The results are shown in Figure 1B .like Figure 1BAs shown, the synthesized peptide complexes (dimer structures numbered 48 and 52 in Table 4) exhibited VEGFR-2 specific activation ability similar to the VEGF of the recombinant protein. Industrial availability

[0093] This invention has been applied in the pharmaceutical and biotechnology industries.

Claims

1. A peptide complex or a pharmaceutically acceptable salt thereof, comprising a first peptide and having VEGFR-2 agonist activity, wherein, The first peptide has X as sequence number 1. 1 -WX 2 -X 3 -X 4 -X 5 -X 6 -X 7 -X 8 -YX 9 -X 10 -X 11 The amino acid sequence indicated by -C, or a sequence consisting of 1 to 3 amino acids substituted, deleted, added, or inserted in the amino acid sequence indicated by sequence number 1, may be used. X 1 For amino acids having chain-like alkyl groups that are substituted or unsubstituted with polar groups on their side chains, or having aromatic rings that are substituted or unsubstituted, X 2 For amino acids with substituted or unsubstituted alkyl groups in their side chains, X 3 For amino acids with substituted or unsubstituted chain alkyl groups in their side chains, X 4 For amino acids with substituted or unsubstituted alkyl groups in their side chains, X 5 For any amino acid, X 6 For amino acids with substituted or unsubstituted polar alkyl groups on the side chain, or amino acids without side chains, X 7 For amino acids with substituted or unsubstituted chain alkyl groups in their side chains, X 8 For amino acids with substituted or unsubstituted chain alkyl groups in their side chains, X 9 For amino acids with substituted or unsubstituted aromatic rings in their side chains, X 10 For amino acids with substituted or unsubstituted alkyl groups in their side chains, X 11 It can be any amino acid.

2. The peptide complex according to claim 1, or a pharmaceutically acceptable salt thereof, wherein, X 2 Amino acids having chain-like or cyclic alkyl groups in their side chains, or having chain-like alkyl groups that are substituted with or unsubstituted with aromatic rings, X 3 For amino acids with chain-like alkyl groups on their side chains that are either substituted with polar groups or not, X 4 Amino acids having chain-like or cyclic alkyl groups in their side chains, or having chain-like alkyl groups that are substituted or unsubstituted with polar groups. X 7 Amino acids having chain alkyl groups on their side chains that are substituted or unsubstituted with polar groups, or chain alkyl groups that are branched or unbranched. X 8 Amino acids having chain alkyl groups on their side chains that are substituted or unsubstituted with polar groups, or chain alkyl groups that are branched or unbranched. X 10 Amino acids having chain-like or cyclic alkyl groups in their side chains, or having chain-like alkyl groups that are substituted with or not substituted with aromatic or heterocyclic rings.

3. The peptide complex according to claim 2, or a pharmaceutically acceptable salt thereof, wherein, X 1 The possible values ​​are W, Hcit, 4Py2NH2, 3Py6NH2, F4aao, or W7N. X 2 For V, Tbg, Chg, or Hty, X 3 For D, Q, or MetO2, X 4 For V, Tbg, alT, or Chg, X 5 For Q, Ahp, W, Hph, Cha, F4COO, or K, X 6 For D, G, or N, X 7 For D or V, X 8 For L, N, or Atb, X 9 For F, Bph, Yph, F4G, or F4C, X 10 For V, Hph, Hty, Chg, or H4Py, X 11 The options are D, A, S, F, F4aao, Har, or Hyp.

4. The peptide complex of claim 1 or a pharmaceutically acceptable salt thereof, wherein, X 2 For amino acids with chain-like or cyclic alkyl groups in their side chains, X 4 Amino acids having chain alkyl groups on their side chains that are substituted or unsubstituted with polar groups, or chain alkyl groups that are branched or unbranched. X 6 For amino acids with chain-like alkyl groups on their side chains that are either substituted with polar groups or not, X 7 For amino acids with chain-like alkyl groups on their side chains that are either substituted with polar groups or not, X 8 Amino acids having chain alkyl groups on their side chains that are substituted or unsubstituted with polar groups, or chain alkyl groups that are branched or unbranched. X 10 Amino acids having chain-like or cyclic alkyl groups in their side chains, or having chain-like alkyl groups that are substituted with or not substituted with aromatic rings.

5. The peptide complex of claim 4 or a pharmaceutically acceptable salt thereof, wherein, X 1 For W or W7N, X 2 For V, Tbg, or Chg, X 3 D, X 4 For V or alT, X 5 For Ahp, Cha, or K, X 6 For D or N, X 7 D, X 8 For L, N, or Atb, X 9 For F or F4G, X 10 For V, Hty, or Chg, X 11 It can be D or S.

6. The peptide complex of claim 1 or a pharmaceutically acceptable salt thereof, wherein, The first peptide has the amino acid sequence shown as sequence number WWVDVQDDLYFVDC, or is composed of an amino acid sequence in which 1 to 3 amino acids are substituted, deleted, added or inserted in the amino acid sequence shown as sequence number 2.

7. The peptide complex of claim 1 or a pharmaceutically acceptable salt thereof, wherein, The first peptide has an amino acid sequence in which glycine has been added to the C-terminus.

8. The peptide complex of claim 1 or a pharmaceutically acceptable salt thereof, wherein, The first peptide is a peptide composed of any one of the amino acid sequences shown in sequence numbers 3 to 51.

9. The peptide complex of claim 1 or a pharmaceutically acceptable salt thereof, wherein, The first peptide is a cyclic peptide.

10. The peptide complex of claim 1 or a pharmaceutically acceptable salt thereof, wherein, The first peptide is an N-terminal amino acid residue that is chloroacetylated, and has an intrapeptide cysteine ​​residue and a cyclic peptide in which the N-terminal amino acid residue is bound to the cysteine ​​residue.

11. The peptide complex of claim 1 or a pharmaceutically acceptable salt thereof, wherein, The peptide complex consists of a first peptide, a second peptide, and a linker connecting the first peptide and the second peptide. The second peptide may be the same as or different from the first peptide, having the amino acid sequence shown in Sequence Number 1, or consisting of an amino acid sequence in which 1 to 3 amino acids in the amino acid sequence shown in Sequence Number 1 are substituted, deleted, added or inserted.

12. The peptide complex of claim 11 or a pharmaceutically acceptable salt thereof, wherein, The homology between the first peptide and the second peptide is more than 90% and less than 100%.

13. The peptide complex of claim 11 or a pharmaceutically acceptable salt thereof, wherein, The first peptide and the second peptide are peptides with the same sequence.

14. The peptide complex of claim 11 or a pharmaceutically acceptable salt thereof, wherein, The sixth amino acid of the first peptide is linked to the sixth amino acid of the second peptide, or the C-terminus of the first peptide is linked to the C-terminus of the second peptide via the linker.

15. The peptide complex of claim 11 or a pharmaceutically acceptable salt thereof, wherein, The linker is a PEG linker or a linker that has been added with 1 to 6 amino acids to a PEG linker.

16. A composition comprising the peptide complex of claim 1 or a pharmaceutically acceptable salt thereof and a carrier.

17. A cell culture composition comprising the peptide complex of claim 1 or a pharmaceutically acceptable salt thereof and a carrier, and for use in cell culture.

18. A composition comprising the peptide complex of claim 1 or a pharmaceutically acceptable salt thereof and a carrier, for medical, diagnostic or research use.

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