Peptide complex having vegfr -2 agonist activity

Abstract

[Problem] To provide a new compound having VEGFR -2 agonist activity. [Solution] Provided is peptide complex, or a pharmaceutically acceptable salt thereof, comprising a first peptide and having VEGFR -2 agonist activity, wherein 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 has an amino acid sequence represented by SEQ ID NO: 1 in which 1-3 amino acids are replaced, deleted, added or inserted.

Description

Peptide conjugates with VEGFR-2 agonist activity

[0001] The present invention relates to a novel peptide complex having VEGFR-2 agonist activity and a cell culture composition containing the peptide complex. The present invention also relates to a novel peptide complex having cell proliferation ability derived from VEGFR-2 agonist activity and a cell culture composition containing the peptide complex.

[0002] Angiogenesis is important in normal physiological processes, including embryonic development, follicular development, and wound healing, and vascular endothelial growth factor (VEGF) is a major mediator of angiogenesis. VEGFR, the VEGF receptor, is a type of receptor tyrosine kinase, and there are three types of proteins, VEGFR-1 to VEGFR-3. In particular, binding of VEGF to VEGFR-2 induces receptor dimerization and autophosphorylation of tyrosine residues, which triggers the activation of several signal transduction cascades, resulting in the induction of endothelial cell differentiation, proliferation, and migration, vascular permeability, and angiogenesis. There have been reported studies using VEGFR-2 agonists for wound treatment, treatment of liver dysfunction and liver disease, protection from liver damage, and treatment of kidney disease (Patent Documents 1, 2, 3, and 4), but the VEGFR-2 agonists used in these studies are natural VEGF or variants thereof, and no chemically synthesizable VEGFR-2 agonist peptides have been reported.

[0003] Patent Publication No. 2008-546707, Patent Publication No. 2010-159266, Patent No. 5111729, Patent Publication No. 2009-541207

[0004] Rom J Morphol Embryol 2018, 59, 455. Biomolecules 2020, 10, 1673. Biology Methods and Protocols, 2023, 8, bpac034. Scientific Reports 2020, 10, 17937. Scientific Reports 4: 6716 doi: 10.1038 / srep06716. Developmental Cell 2020, 54, 516. Cell Reports 2018, 23, 1620.

[0005] One aspect of the present invention provides novel compounds having VEGFR-2 agonist activity and also novel compounds having cell proliferation potency resulting from VEGFR-2 agonist activity.

[0006] The present invention includes, but is not limited to, the following aspects: [1] A peptide conjugate or a pharmaceutically acceptable salt thereof, which comprises a first peptide and has VEGFR-2 agonist activity, wherein the first peptide is １ -W-X ２ -X ３ -X ４ -X ５ -X ６ -X ７ -X ８ -Y-X ９ -X １０ -X １１ -C (SEQ ID NO: 1), or the amino acid sequence of SEQ ID NO: 1 in which 1 to 3 amino acids are substituted, deleted, added or inserted, １ is an amino acid having, in the side chain, a chain alkyl group which may be substituted with a polar group, or an aromatic ring which may be substituted, ２ is an amino acid having an alkyl group which may be substituted on the side chain, ３ is an amino acid having an optionally substituted chain alkyl group at the side chain, ４ is an amino acid having an alkyl group which may be substituted on the side chain,５ is any amino acid, and X ６ is an amino acid having a chain alkyl group in the side chain which may be substituted with a polar group, or having no side chain, ７ is an amino acid having an optionally substituted chain alkyl group at the side chain, ８ is an amino acid having an optionally substituted chain alkyl group at the side chain, ９ is an amino acid having an aromatic ring which may be substituted in the side chain, １０ is an amino acid having an alkyl group which may be substituted on the side chain, １１ [2] The peptide conjugate or pharmaceutically acceptable salt thereof according to [1], wherein X is any amino acid. ２ is an amino acid having a chain or cyclic alkyl group or a chain alkyl group optionally substituted with an aromatic ring in the side chain, ３ is an amino acid having a chain alkyl group in the side chain which may be substituted with a polar group, ４ is an amino acid having a chain or cyclic alkyl group or a chain alkyl group optionally substituted with a polar group in the side chain, ７ is an amino acid having a chain alkyl group which may be substituted with a polar group or a chain alkyl group which may be branched in the side chain, ８ is an amino acid having a chain alkyl group which may be substituted with a polar group or a chain alkyl group which may be branched in the side chain, １０ [3] The peptide complex or pharmaceutically acceptable salt thereof according to [2], wherein X is an amino acid having a chain or cyclic alkyl group in the side chain, or a chain alkyl group substituted with an aromatic ring or a heterocycle. １ is W, Hcit, 4Py2NH2, 3Py6NH2, F4aao, or W7N, and X ２ is V, Tbg, Chg, or Hty; X ３ is D, Q, or MetO2; X４ is V, Tbg, a1T, or Chg; X ５ is Q, Ahp, W, Hph, Cha, F4COO, or K; ６ is D, G, or N; X ７ is D or V, and X ８ is L, N, or Atb; ９ is F, Bph, Yph, F4G, or F4C, and X １０ is V, Hph, Hty, Chg, or H4Py; X １１ [4] The peptide conjugate or pharmaceutically acceptable salt thereof according to [1], wherein X is D, A, S, F, F4aao, Har, or Hyp. ２ is an amino acid having a linear or cyclic alkyl group in the side chain, ４ is an amino acid having a chain alkyl group which may be substituted with a polar group or a chain alkyl group which may be branched in the side chain, ６ is an amino acid having a chain alkyl group in the side chain which may be substituted with a polar group, ７ is an amino acid having a chain alkyl group in the side chain which may be substituted with a polar group, ８ is an amino acid having a chain alkyl group which may be substituted with a polar group or a chain alkyl group which may be branched in the side chain, １０ [5] The peptide complex or pharmaceutically acceptable salt thereof according to [4], wherein X is an amino acid having a chain or cyclic alkyl group in the side chain, or a chain alkyl group optionally substituted with an aromatic ring. １ is W or W7N, X ２ is V, Tbg, or Chg; X ３ is D and X ４ is V or aIT, and X ５ is Ahp, Cha or K, and X ６ is D or N, and X ７ is D and X８ is L, N or Atb, ９ is F or F4G, and X １０ is V, Hty or Chg, X １１is D or S. [6] The peptide complex or pharmaceutically acceptable salt thereof according to [1], wherein the first peptide has the amino acid sequence represented by W-W-V-D-V-Q-D-D-LY-F-V-D-C (SEQ ID NO: 2), or consists of the amino acid sequence represented by SEQ ID NO: 2 in which 1 to 3 amino acids have been substituted, deleted, added, or inserted. [7] The peptide complex or 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 pharmaceutically acceptable salt thereof according to [1], wherein the first peptide is a peptide consisting of the amino acid sequence represented by any of SEQ ID NOs: 3 to 51. [9] The peptide complex or pharmaceutically acceptable salt thereof according to [1], wherein the first peptide is a cyclic peptide.

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

[11] The peptide complex or pharmaceutically acceptable salt thereof according to [1], wherein the peptide complex comprises the first peptide, a second peptide, and a linker connecting the first peptide and the second peptide, and the second peptide may be the same as or different from the first peptide, and has the amino acid sequence set forth in SEQ ID NO: 1, or an amino acid sequence in which 1 to 3 amino acids have been substituted, deleted, added, or inserted in the amino acid sequence set forth in SEQ ID NO: 1.

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

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

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

[11] , wherein the first peptide and the second peptide have the same sequence.

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

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

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

[11] , wherein the linker is a PEG linker or a linker in which 1 to 6 amino acids are added 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 composition for cell culture, comprising the peptide complex according to [1] or a pharmaceutically acceptable salt thereof and a carrier, and used for cell culture.

[18] A composition, comprising the peptide complex according to [1] or a pharmaceutically acceptable salt thereof and a carrier, and used for medical, diagnostic or research purposes.

[0007] According to the present invention, novel compounds having VEGFR-2 agonistic activity can be provided, and further, the present invention can provide novel compounds having cell proliferation ability derived from VEGFR-2 agonistic activity.

[0008] Figure 1 shows the results of a Human Phospho-RTK Array assay using the peptide conjugate of the present invention and VEGF165a. Figure 1A shows an array map of the target factors, and Figure 1B shows the results of the assay.

[0009] The present invention includes, but is not limited to, the following embodiments. Unless otherwise specified herein, 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 merely illustrative and are not intended to be limiting. When referring to "in one embodiment" in this specification, it means that the embodiment is not limited, i.e., is not limited.

[0010] The first invention of this specification relates to a peptide conjugate or a pharmaceutically acceptable salt thereof. This peptide conjugate or a pharmaceutically acceptable salt thereof comprises a first peptide and has VEGFR-2 agonist activity. Furthermore, this peptide conjugate or a pharmaceutically acceptable salt thereof has cell proliferation ability derived from the VEGFR-2 agonist activity. The pharmaceutically acceptable salt refers to a pharmaceutically acceptable salt of the peptide conjugate. Pharmaceutically acceptable salts are as described below. Hereinafter, for simplicity, the peptide conjugate or a pharmaceutically acceptable salt thereof may be simply referred to as the peptide conjugate.

[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 ability derived from VEGFR-2 agonist activity. The peptide complex is a peptide, peptide-containing compound, or pharmaceutically acceptable salt thereof, comprising a first peptide and another peptide or compound. The peptide complex may comprise one or more (three or more, or four or more) first peptides. The peptide complex may further comprise one or more partial peptides different from the first peptide. In the peptide complex, the first peptide or partial peptide is preferably linked via a linker. The peptide complex may be a homomultimer containing only peptides having the same amino acid sequence. The peptide complex may be a heteromultimer containing peptides having different amino acid sequences. The peptide complex is preferably a homodimer containing two first peptides having the same amino acid sequence, the two first peptides being linked via a linker. In a homodimer, the two peptide moieties may have the exact same amino acid sequence or may have substantially the same amino acid sequence. The peptide complex has VEGFR-2 agonist activity, and the first peptide or partial peptide constituting the peptide complex may also have VEGFR-2 agonist activity. As shown in the examples, the first peptide exhibits VEGFR-2 agonist activity by forming a peptide complex structure via a linker.

[0012] In one embodiment, the peptide complex of the present invention may comprise a first peptide, a second peptide, and a linker connecting the first and second peptides. In this case, the second peptide may be identical to or different from the first peptide, and preferably has the amino acid sequence set forth in SEQ ID NO: 1 or an amino acid sequence in which 1 to 3 amino acids have been substituted, deleted, added, or inserted in the amino acid sequence set forth in SEQ ID NO: 1. Furthermore, the peptide complex is preferably one in which the homology between the first and second peptides is 90% or more and 100% or less, and more preferably one in which the first and second peptides are substantially identical and are a homodimer.

[0013] First Peptide In one embodiment, the first peptide comprises X １ -W-X ２ -X ３ -X ４ -X ５ -X ６ -X ７ -X ８ -Y-X ９ -X １０ -X １１ -C (SEQ ID NO: 1), or consisting of the amino acid sequence of SEQ ID NO: 1 in which 1 to 3 (1, 2, or 3) amino acids have been substituted, deleted, added, or inserted. The substituted amino acids include pharmaceutically acceptable salts of the amino acids. A preferred example of a peptide having an amino acid sequence in which amino acids have been substituted, deleted, added, or inserted in the amino acid sequence of SEQ ID NO: 1 is a peptide having an amino acid sequence in which conservative amino acid substitutions have been made.

[0014] Conservative Amino Acid Substitutions Conservative amino acid substitutions are preferred when one, two, or three amino acid residues are substituted, deleted, added, or inserted from a specific sequence. A "conservative amino acid substitution" refers to a substitution with a functionally equivalent or similar amino acid. Conservative amino acid substitutions in a peptide result in a static change to the amino acid sequence of the peptide. For example, one or more amino acids with similar polarity act functionally equivalently, resulting in a static change to the amino acid sequence of the peptide. In general, substitutions within a group can be considered conservative in terms of structure and function. However, as will be apparent to those skilled in the art, the role played by a particular amino acid residue can be determined by its relationship to the three-dimensional structure of the molecule containing that amino acid. For example, cysteine ​​residues can adopt an oxidized (disulfide) form, which is less polar than its reduced (thiol) form. The long aliphatic portion of the arginine side chain can constitute a structurally and functionally important feature. Additionally, side chains containing aromatic rings (tryptophan, tyrosine, phenylalanine) can contribute to ion-aromatic interactions or cation-pi interactions. In such cases, substitution of amino acids with those containing these side chains with amino acids belonging to acidic or nonpolar groups can be structurally and functionally conservative. Residues such as proline, glycine, and cysteine ​​(disulfide form) can have a direct effect on the conformation of the main chain and often cannot be substituted without structural distortion. Conservative amino acid substitutions include specific substitutions based on side chain similarity (L. Lehninger, Biochemistry, 2nd edition, 1975, pp. 73-75; L. Lehninger, Biochemistry, 2nd edition, pp. 73-75, Worth Publisher, New York (1975)) and typical substitutions, as shown below.

[0015] Furthermore, conservative amino acid substitution is preferably, for example, substitution with an amino acid that belongs to the same group as a certain amino acid, where natural amino acids are divided into groups based on the properties of their common side chains, as follows: Hydrophobic (also called non-polar) amino acids: Amino acids that exhibit hydrophobicity (non-polarity), and include alanine (also written as "Ala" or simply "A"), glycine (also written as "Gly" or simply "G"), valine (also written as "Val" or simply "V"), leucine (also written as "Leu" or simply "L"), isoleucine (also written as "Ile" or simply "I"), proline (also written as "Pro" or simply "P"), phenylalanine (also written as "Phe" or simply "F"), tryptophan (also written as "Trp" or simply "W"), tyrosine (also written as "Tyr" or simply "Y"), and methionine (also written as "Met" or simply "M"). Hydrophobic amino acids can be further divided into the following groups: Aliphatic amino acids: Amino acids having a fatty acid or hydrogen in the side chain, including Ala, Gly, Val, Ile, and Leu. Aliphatic / branched-chain amino acids: Amino acids having a branched fatty acid in the side chain, including Val, Ile, and Leu. Aromatic amino acids: Amino acids having an aromatic ring in the side chain, including Trp, Tyr, and Phe. Hydrophilic (also called polar) amino acids: Amino acids that exhibit hydrophilicity (polarity), including serine (also referred to as "Ser" or simply "S"), threonine (also referred to as "Thr" or simply "T"), cysteine ​​(also referred to as "Cys" or simply "C"), asparagine (also referred to as "Asn" or simply "N"), glutamine (also referred to as "Gln" or simply "Q"), aspartic acid (also referred to as "Asp" or simply "D"), glutamic acid (also referred to as "Glu" or simply "E"), lysine (also referred to as "lysine", "Lys" or simply "K"), arginine (also referred to as "Arg" or simply "R"), and histidine (also referred to as "His" or simply "H"). Hydrophilic amino acids can also be further divided into the following groups: Acidic amino acids: amino acids whose side chains are acidic, including Asp and Glu. Basic amino acids: amino acids whose side chains are basic, including Lys, Arg, and His.Neutral amino acids: Amino acids whose side chains are neutral, including Ser, Thr, Asn, Gln, and Cys. 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 their side chains, such as Cys and Met, can also be classified as "sulfur-containing amino acids."

[0016] As used herein, the term "amino acid" includes not only natural amino acids but also unnatural amino acids. Unnatural amino acids include, for example, N-alkylamino acids in which the above-described natural amino acids are N-alkylated, and amino acids in which the nitrogen atom forming the peptide bond is modified with a branched or unbranched lower (e.g., C1-C5, preferably C1-C3, more preferably C1) alkyl group. N-Alkylamino acids are preferably N-ethylamino acids, N-butylamino acids, or N-methylamino acids, and more preferably N-methylamino acids. Unnatural amino acids also include chemically modified amino acids such as D-amino acids (also referred to as D-amino acids), β-amino acids, γ-amino acids, amino acid mutants, and amino acid derivatives, as well as amino acids that do not serve as building blocks of proteins in vivo, such as norleucine and ornithine. Furthermore, naturally occurring amino acids include amino acids in which a functional group has been added to the side chain or which have been substituted with another functional group (e.g., amino acids having a substitution or addition to an arylene group, alkylene group, or other moiety in the side chain; amino acids in which the arylene group, alkylene group, or alkyl group in the side chain has an increased carbon number; amino acids having a substitution in an aromatic ring in the side chain; and heterocyclized or condensed cyclized amino acids). The addition or substitution of a functional group or other structure to the side chain of a naturally occurring amino acid can impart properties different from those of naturally occurring amino acids. For example, A4p is an alanine with a piperidyl group added to the side chain, and due to the addition of the piperidyl group, it exhibits basic polarity, unlike alanine, which belongs to the nonpolar amino acid group. In other words, unnatural amino acids with similar side chain properties can be included in the aforementioned groups, which are obtained by dividing naturally occurring amino acids based on the properties of their common side chains. For example, N-methylarginine (MeR), an N-methylated amino acid of arginine, which belongs to the basic amino acids, is an unnatural amino acid, but exhibits basicity and can therefore be classified as a basic amino acid. In this way, non-natural amino acids that exhibit side chain properties similar to those of a certain amino acid can also be included as targets for conservative amino acid substitution.Non-naturally occurring amino acids include, but are not limited to, N-methyl amino acids, 4Py, a1T, Cit, P4Sh, F4CON, F4COO, 3Py, HyP, SMe, A4paa, Atp, Hgl, KAc, Na11, W6N, W7N, PeG, etc. N-methyl amino acids can also be classified as N-alkyl amino acids, or can be classified according to the properties of the side chain of the original non-N-methylated amino acid.

[0017] Peptide Length: In one embodiment, the first peptide may contain additional amino acid residues in addition to, for example, the amino acid sequences set forth in SEQ ID NOS: 2 to 51, or the amino acid sequences corresponding to positions 1 to 14 of those amino acid sequences. The additional amino acid residues may be contained in a peptide that forms a cyclic structure, or further amino acid residues may be added to the cyclic peptide in the form of a linker. The number of amide bonds (number of amino acids / length) in the peptide or peptide moiety is not particularly limited, but the total number of amino acid residues (referring to the number of amino acid residues contained in the peptide that forms a cyclic structure; if further amino acid residues are added to the cyclic peptide in the form of a linker, these amino acids are not included) is preferably 20 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, and preferably 19 or less or 18 or less amino acid residues. More preferred peptide lengths are 13 to 16 amino acid residues, and most preferably 14 or 15 amino acids.

[0018] A preferred example of the first peptide is SEQ ID NO: 1 (X １ -W-X ２ -X ３ -X ４ -X ５ -X ６ -X ７ -X ８ -Y-X ９ -X １０ -X １１ -C) A peptide having an amino acid sequence represented by X １ is an amino acid having, in the side chain, a chain alkyl group which may be substituted with a polar group, or an aromatic ring which may be substituted, ２is an amino acid having an alkyl group which may be substituted on the side chain, ３ is an amino acid having an optionally substituted chain alkyl group at the side chain, ４ is an amino acid having an alkyl group which may be substituted on the side chain, ５ is any amino acid, and X ６ is an amino acid having a chain alkyl group in the side chain which may be substituted with a polar group, or having no side chain, ７ is an amino acid having an optionally substituted chain alkyl group at the side chain, ８ is an amino acid having an optionally substituted chain alkyl group at the side chain, ９ is an amino acid having an aromatic ring which may be substituted in the side chain, １０ is an amino acid having an alkyl group which may be substituted on the side chain, １１ is any amino acid.

[0019] A preferred example of the first peptide is a peptide having the amino acid sequence shown in SEQ ID NO: 1, ２ is an amino acid having a chain or cyclic alkyl group or a chain alkyl group optionally substituted with an aromatic ring in the side chain, ３ is an amino acid having a chain alkyl group in the side chain which may be substituted with a polar group, ４ is an amino acid having a chain or cyclic alkyl group or a chain alkyl group optionally substituted with a polar group in the side chain, ７ is an amino acid having a chain alkyl group which may be substituted with a polar group or a chain alkyl group which may be branched in the side chain, ８ is an amino acid having a chain alkyl group which may be substituted with a polar group or a chain alkyl group which may be branched in the side chain, １０ is an amino acid having a linear or cyclic alkyl group in the side chain, or a linear alkyl group substituted with an aromatic ring or heterocyclic ring.

[0020] A preferred example of the first peptide is a peptide having the amino acid sequence shown in SEQ ID NO: 1, １ is W, Hcit, 4Py2NH2, 3Py6NH2, F4aao, or W7N, and X ２ is V, Tbg, Chg, or Hty; X ３ is D, Q, or MetO2; X ４ is V, Tbg, a1T, or Chg; X ５ is Q, Ahp, W, Hph, Cha, F4COO, or K; ６ is D, G, or N; X ７ is D or V, and X ８ is L, N, or Atb; ９ is F, Bph, Yph, F4G, or F4C, and X １０ is V, Hph, Hty, Chg, or H4Py; １１ is D, A, S, F, F4aao, Har, or Hyp.

[0021] A preferred example of the first peptide is a peptide having the amino acid sequence shown in SEQ ID NO: 1, ２ is an amino acid having a linear or cyclic alkyl group in the side chain, ４ is an amino acid having a chain alkyl group which may be substituted with a polar group or a chain alkyl group which may be branched in the side chain, ６ is an amino acid having a chain alkyl group in the side chain which may be substituted with a polar group, ７ is an amino acid having a chain alkyl group in the side chain which may be substituted with a polar group, ８ is an amino acid having a chain alkyl group which may be substituted with a polar group or a chain alkyl group which may be branched in the side chain, １０ is an amino acid having a linear or cyclic alkyl group or a linear alkyl group optionally substituted with an aromatic ring in the side chain. Conjugates containing these peptides have been shown in the Examples to have VEGFR-2 agonist activity and cell proliferation activity.

[0022] A preferred example of the first peptide is a peptide having the amino acid sequence shown in SEQ ID NO: 1, １ is W or W7N, X ２ is V, Tbg, or Chg; X ３ is D and X ４ is V or aIT, and X ５ is Ahp, Cha or K, and X ６ is D or N, and X ７ is D and X ８ is L, N or Atb, ９ is F or F4G, and X １０ is V, Hty or Chg, X １１ is D or S.

[0023] As used herein, the term "optionally substituted" means unsubstituted or substituted with any number of optional substituents.

[0024] Amino Acid Having an Optionally Substituted Alkyl Group on the Side Chain "An amino acid having an Optionally Substituted Alkyl Group on the Side Chain" refers to an amino acid having an alkyl group on the side chain, such as, but not limited to, alanine, proline, isoleucine, leucine, valine, etc., where the alkyl group may be linear or cyclic, and the side chain of the amino acid having an alkyl group on the side chain may be further substituted with a substituent such as an alkyl group, an aromatic ring, a heterocycle, a hydroxyl group, or the like, or may be unsubstituted.

[0025] Amino Acid Having an Acyclic Alkyl Group Optionally Substituted on the Side Chain "Amino acid having an Acyclic Alkyl Group Optionally Substituted on the Side Chain" refers to an amino acid having an Acyclic Alkyl Group on the side chain, such as, but not limited to, alanine, isoleucine, leucine, valine, etc., in which the side chain of the amino acid having an Acyclic Alkyl Group on the side chain is further substituted with a substituent such as an alkyl group or a polar group, such as, but not limited to, a carboxy group, an amide group, a methanesulfonyl group, etc., or is unsubstituted.

[0026] Amino Acid Having a Chain Alkyl Group in the Side Chain Which May Be Substituted with a Polar Group "An amino acid having a chain alkyl group in the side chain which may be substituted with a polar group" refers to an amino acid having the chain alkyl group in the side chain, in which the side chain of the amino acid having the chain alkyl group is further substituted with a polar group such as, but not limited to, a urea group, an amino group, a carboxy group, an amide group, a hydroxyl group, a methanesulfonyl group, etc., or is not substituted with a polar group, with the former being preferred.

[0027] Amino Acid Having No Side Chain The term "amino acid having no side chain" refers to glycine or a glycine derivative.

[0028] Amino Acid Having a Linear or Cyclic Alkyl Group in the Side Chain An "amino acid having a linear or cyclic alkyl group in the side chain" is an amino acid having the alkyl group in the side chain, and the alkyl group may be linear or cyclic.

[0029] Amino Acid Having a Linear Alkyl Group in the Side Chain Which May Be Substituted with an Aromatic Ring "An amino acid having a linear alkyl group in the side chain which may be substituted with an aromatic ring" refers to an amino acid having the above-mentioned linear alkyl group in the side chain, in which the side chain of the amino acid having a linear alkyl group in the side chain is either further substituted with an aromatic ring or is not substituted with an aromatic ring, with the former being preferred.

[0030] Amino acids having an optionally branched chain alkyl group in the side chain "Amino acids having an optionally branched chain alkyl group in the side chain" are amino acids having the aforementioned chain alkyl group in the side chain, and the side chains of these amino acids may be further branched. The amino acids having an optionally branched chain alkyl group in the side chain are preferably amino acids having a branched chain alkyl group.

[0031] Amino Acids Having an Aromatic Ring Optionally Substituted on the Side Chain "An amino acid having an aromatic ring substituted on the side chain" refers to an amino acid having an aromatic ring on the side chain, in which the carbon atoms constituting the aromatic ring or the functional group bonded to the aromatic ring may or may not be substituted, with the former being preferred. The aromatic ring substituted on the side chain may have a fused ring structure such as a benzene ring, indole ring, or naphthalene ring, in which some of the carbon atoms may be substituted with other molecules such as nitrogen. The aromatic ring may also be a heterocycle. Furthermore, the amino acid having an aromatic ring substituted on the side chain may be, for example, an amino acid in which the hydroxyl group on 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 selected from alkyl groups, phenyl groups, cycloalkyl groups, hydroxyl groups, halogens, and the like. The amino acid having an aromatic ring substituted on the side chain includes, for example, tyrosine, tryptophan, histidine, phenylalanine, etc. Furthermore, the amino acid having an aromatic ring optionally substituted at the side chain may be a non-natural amino acid, such as 4Py2NH2, 3Py6NH2, F4aao, W7N, Bph, Yph, F4G, or F4C.

[0032] Amino Acid Having an Acyclic Alkyl Group Substituted with an Aromatic Ring or Heterocycle in the Side Chain "An amino acid having an Acyclic Alkyl Group Substituted with an Aromatic Ring or Heterocycle in the Side Chain" refers to an amino acid having the above-mentioned Acyclic Alkyl Group in the side chain, and the side chain of these amino acids is further substituted with an aromatic ring or heterocycle.

[0033] A preferred example of the first peptide is a peptide having the amino acid sequence shown by W-W-V-D-V-Q-D-D-LY-F-V-D-C (SEQ ID NO: 2), or an amino acid sequence in which 1 to 3 amino acids have been substituted, deleted, added, or inserted in the amino acid sequence shown by SEQ ID NO: 2. When an amino acid is 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 of SEQ ID NO: 2.

[0034] A preferred example of the first peptide is a peptide having an amino acid sequence in which a glycine residue is further added to the C-terminus. In addition, in the peptides of SEQ ID NOs: 14 to 35 and 37 to 51, the carboxylic acid at the C-terminus of the 15th glycine residue is preferably converted to an amide.

[0035] A preferred example of the first peptide is a peptide consisting of an amino acid sequence set forth in any one of SEQ ID NOS: 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 salt and potassium salt, alkaline earth metal salts such as calcium salt and magnesium salt, aluminum salt, and ammonium salt. Examples of organic base salts include diethylamine salt, diethanolamine salt, meglumine salt, and N,N'-dibenzylethylenediamine salt. Examples of acidic amino acid salts include aspartate and glutamate. Examples of basic amino acid salts include arginine salts, lysine salts, and ornithine salts. The first peptide and the peptide complex may be in the form of a pharmaceutically acceptable salt or solvate. An example of a solvate is a hydrate.

[0036] 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, the peptide contains a cysteine ​​residue, and the N-terminal amino acid residue and the cysteine ​​residue are bonded to each other.

[0037] Cyclic Peptide As used herein, a cyclic peptide refers to a peptide in which two amino acids are bonded together, forming a ring structure in whole or in part. The term also includes peptides in which amino acids form a cross-linked structure, peptides in which a ring structure is formed by lactam ring formation or macrocyclization, and peptides having a lasso peptide-like structure. That is, as used herein, a cyclic peptide may be any peptide in which a portion forms a ring structure, and may also have a linear portion.

[0038] As used herein, some amino acids may be modified for peptide cyclization. The term "peptide" as used herein also encompasses peptides containing amino acids with such partial modifications. An example of a modification for cyclization is adding a chloroacetyl group to the amino acid at the N-terminus, which is then bonded to a cysteine ​​residue in the peptide for cyclization. Peptides containing various (natural / unnatural) amino acids to which a chloroacetyl group has been added are also encompassed by the term "peptide" as used herein.

[0039] Peptides generally have poor metabolic stability in vivo and, due to their large size, have the drawback of being difficult to penetrate cell membranes. To address these issues, peptide cyclization has been used. It has been suggested that cyclization of peptides improves protease resistance, metabolic stability, and restricts conformational changes, thereby increasing rigidity and improving membrane permeability and affinity for target proteins.

[0040] Peptide cyclization can be carried out according to known methods. For example, but not limited to, by designing a peptide to contain two or more cysteine ​​residues, a cyclic structure can be formed by disulfide bonds after translation. Alternatively, cyclization can be achieved by synthesizing a peptide with a chloroacetyl group at the N-terminus and placing a cysteine ​​residue in the peptide using genetic code reprogramming technology (Y. Goto, et al. ACS Chem. Biol. 3 120-129 (2008)). This allows spontaneous nucleophilic attack of the mercapto group on the chloroacetyl group after translation, resulting in cyclization of the peptide via a thioether bond. Using genetic code reprogramming technology, other combinations of amino acids that bond to form a cyclic structure can also be placed in the peptide for cyclization. Alternatively, a peptide with a cycloamide at the N-terminus can be synthesized, and an L-2-aminoadipic acid residue can be placed in the peptide, followed by bonding between them to form cyclization. Thus, any known cyclization method can be used without particular limitations.

[0041] Linker In one embodiment, a linker may be added to the first peptide or the partial peptide. The linker may be a structure that connects multiple peptides to each other in the peptide complex. Examples of the linker include an amino acid linker (peptide linker), a chemical linker, a fatty acid linker, a nucleic acid linker, and a sugar chain linker. The linker may also be a complex of a chemical linker and a peptide linker. An example of a chemical linker is a PEG (Polyethyleneglycol) linker. The PEG linker may be a linker consisting of 1 to 36 ethylene glycol units. The linker may also be a fatty acid linker containing a divalent chemical moiety derived from a fatty acid. The amino acid (peptide) linker is a linker containing at least one amino acid. For example, a glycine-rich peptide such as a peptide having the sequence [Gly-Gly-Gly-Gly-Ser]n (where n is 1, 2, 3, 4, 5, or 6) as described in U.S. Pat. No. 7,271,149, or a serine-rich peptide linker as described in U.S. Pat. No. 5,525,491, can be used. Addition of a linker may alter the physical properties (e.g., solubility) of the peptide. Furthermore, the linker may be a combination of the above linkers. For example, a Gly-Lys amino acid linker may be bonded, with a PEG linker attached to the side chain terminus of the Lys. Alternatively, the linker may have a structure in which amino acids and PEG are bonded alternately, 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 are attached to the PEG linker moiety. This linker may have a structure in which an amino acid is added to one end of the PEG linker moiety, or may have a structure in which amino acids are added to both ends of the PEG linker moiety. A preferred example of the amino acid added to the PEG moiety is K (Lys), but is not limited to K and may be other amino acids. A preferred example of the linker is a linker having a structure shown in Table 3. The linker may be added to any position on the peptide.For example, the linker may be attached to a Cys residue located at the C-terminus of the peptide and bonded to the first amino acid to form a cyclic structure, or to an amino acid contained in the cyclic peptide. This is not a limitation. Preferably, the linker is bonded to a Cys residue located at the C-terminus or to the side chain of an amino acid contained in the cyclic peptide. More preferably, the linker is bonded to a Cys residue located at the C-terminus or to the side chain of the sixth amino acid contained in the cyclic peptide. For example, in the amino acid sequences represented by SEQ ID NOs: 2 to 51, the 15th Gly residue can also be considered a linker. For example, in a non-limiting example, in a cyclic peptide in which the first amino acid in the amino acid sequence represented by SEQ ID NOs: 2 to 51 is bonded to the 14th amino acid, Cys, two cyclic peptide structures in which a glycine is further added to the 14th Cys residue form a dimer structure via the added glycine residue and a structure shown in Table 2, the linker structure can be considered to be the structure shown in Table 2, or can be considered to be a structure in which a structure shown in Table 2 is bonded to glycine. The dimer may be one in which the sixth amino acid of the first peptide is linked to the sixth amino acid of the second peptide via a linker, or one in which the C-terminus of the first peptide is linked to the C-terminus of the second peptide via a linker, with the former being preferred.

[0042] VEGFR-2 VEGFR-2, also known as KDR (kinase insert domain receptor) or Flk-1 (fetal liver kinase 1), is a member of the tyrosine kinase receptor family. VEGFR-2 is highly expressed in vascular endothelial cells and lymphatic vessels, and has a higher affinity for its VEGF ligands, VEGF-A and VEGF-E, and a lower affinity for VEGF-C and VEGF-D (Non-Patent Document 1). Like other tyrosine kinase receptors, VEGFR-2 has an intracellular tyrosine kinase domain. When VEGF binds to the extracellular domain of VEGFR-2, autophosphorylation of tyrosine residues occurs, and by activating several signals and mechanisms, angiogenesis and non-pathological or pathological angiogenesis are induced. (Non-Patent Document 1) On the other hand, VEGF-A is an important ligand for angiogenesis and plays an important role in vasculogenesis and neovascularization. VEGF-A binds to VEGFR-1 and VEGFR-2. The binding affinity of VEGF-A to VEGFR-2 is lower than that of VEGF-A to VEGFR-1, but the tyrosine kinase activity of VEGFR-2 is higher than that of VEGFR-1. Therefore, taking everything into consideration, VEGFR-2 is considered to be the main signal transduction factor receptor for VEGF-A binding. (Non-Patent Documents 1 and 2)

[0043] VEGFR-2 Agonist Activity In one embodiment, the peptide conjugate of the present invention preferably has VEGFR-2 agonist activity. VEGFR-2 agonist activity refers to the ability to produce an effect similar to the effect of VEGF, a naturally occurring VEGFR-2 agonist, on VEGFR-2. Specifically, it refers to the ability to specifically activate VEGFR-2 dimerization and signal transduction pathways. "VEGFR-2 agonist activity" may be evaluated, for example, by a kinase assay system using VEGFR-2 as the target protein. For example, but not limited to, evaluation can be performed by AlphaLISA assay or NFAT assay, which detect phosphorylation of VEGFR-2 or ERK. Non-limiting examples of such assays include evaluation using AlphaLISA® SureFire® Ultra VEGFR-2 (p-Tyr1175) Assay Kit (PerkinElmer), AlphaLISA® SureFire® Ultra p-ERK1 / 2 (T202 / Y204) kit (PerkinElmer), VEGFR2 / NFAT Reporter-HEK293 Recombinant Cell line (BPS Bioscience), and VEGF Bioassay kit (Promega). In any evaluation system, if VEGFR-2 agonist activity can be detected using the peptide or peptide complex at an optimal concentration under optimal conditions according to standard procedures, the peptide or peptide complex is said to have VEGFR-2 agonist activity. The agonist activity of a peptide can be expressed, without limitation, by EC50.

[0044] This specification also describes nucleic acids encoding the first peptide or peptide complex. As used herein, "nucleic acid" may be natural or non-natural, and includes, but is not limited to, DNA, RNA, and chimeras thereof. The nucleic acid can be designed and produced by known methods based on the sequence of the first peptide or peptide complex.

[0045] Peptide Drug Conjugates (PDCs) In one aspect, the present invention relates to a conjugate (peptide drug conjugate) comprising the peptide or peptide conjugate of the present invention, a substance to be delivered to VEGFR-2, and a linker for linking the substance to the peptide or peptide conjugate. The peptide conjugate has VEGFR-2 agonist activity and is therefore believed to bind to VEGFR-2. Therefore, the peptide or peptide conjugate is capable of delivering a substance to VEGFR-2. The substance may be any substance desired by those skilled in the art as long as it is a substance desired to be delivered to VEGFR-2. Examples of such substances include, but are not limited to, the following: Compounds: may be low-molecular-weight compounds or medium-molecular-weight compounds, such as known low-molecular-weight drugs. Peptides: may be peptides that bind to targets in the body and exert some effect, such as cyclic peptides. RI: may be any compound labeled with a radioisotope, such as a low-molecular-weight compound, medium-molecular-weight compound, or antibody labeled with a radioisotope. For example, compounds for PET scans may be used. Proteins: Any proteins that exhibit useful functions in the body, such as antibodies and enzymes, are acceptable. Examples include enzymes used in enzyme replacement therapy. Nucleic acids: They may be DNA, RNA, or chimeras thereof, and are not particularly limited. Examples include nucleic acid pharmaceuticals. Molecules used in drug delivery systems (DDS): They may be known molecules used in DDS, such as liposomes and micelles. The DDS molecules may further contain compounds such as pharmaceuticals. Furthermore, the substance desired to be delivered to VEGFR-2 may be a complex of the substances listed above.

[0046] In one aspect, the present invention relates to a composition comprising the peptide conjugate and a carrier, such as water (e.g., sterile water, pure water, distilled water, etc.), physiological saline, glucose solution, alcohol (e.g., ethanol, etc.), polyalcohol (e.g., glycerol, propylene glycol, polyethylene glycol, etc.), sterile organic solvent, aqueous starch, or PBS, or a mixture of two or more of these.

[0047] Cell Culture Composition In one aspect, the present invention relates to a cell culture composition. The cell culture composition is a composition used for cell culture. Because the peptide complex has VEGFR-2 agonist activity, it can also be used as a medium reagent or additive for cell culture, preferably for mammalian cells, and more preferably for human cells. The cell culture medium reagent or additive may also be a medium reagent or additive for culturing cells for the production of cell-cultured meat. The peptide complex can also be used as a reagent or additive in a medium for producing liver organoids for the treatment of liver disease. The liver organoids are created by co-culturing hepatic endoderm cells, vascular endothelial cells, and mesenchymal cells induced from pluripotent stem cells, but are not limited thereto. The peptide complex can be used in a differentiation induction system for vascular endothelial cells (Non-Patent Documents 3 and 4), but is not limited thereto. VEGF, a naturally occurring VEGFR-2 agonist, induces differentiation of pluripotent stem cells into vascular endothelial cells and vascular wall cells, and contributes to the formation of higher-order structures and the establishment and maintenance of organoids when co-cultured with cardiomyocytes, intestinal epithelial cells, pancreatic islets, etc. (Non-Patent Documents 5, 6, 7). Because VEGF plays such a fundamental role in tissue creation, the peptide complex can be used as a reagent or additive for producing the various cells described above.

[0048] The medium is not particularly limited as long as it is a medium for culturing cells or tissues. The medium may be a serum medium, preferably a serum-free medium or a low-serum medium. In one embodiment, the culture medium additive may be in the form of a solution or a dried solid (e.g., solid, powder, etc.). When in the form of a solution, it may be used as a culture medium as is, or it may be diluted with a solvent and, if necessary, the above-mentioned additives may be added to the solution and used as a culture medium. Examples of solvents used for dilution include water, buffer solutions, physiological saline solutions, and media used for various cell and tissue cultures. These may be used alone or in combination of two or more. When the culture medium additive is in the form of a dry solid, it may be dissolved in a solvent such as water, buffer solutions, physiological saline solutions, or media used for various cell and tissue cultures, and, if necessary, the above-mentioned additives may be added to the solution and used as a culture medium. The content of the peptide conjugate of the present invention in a medium for culturing the cells or tissues, or in a medium for cells obtained therefrom, can be, for example, about 0.01 to about 10,000 nmol / L, preferably about 0.1 to about 1,000 nmol / L, more preferably about 0.5 to about 1,000 nmol / L, and even more preferably about 1 to about 100 nmol / L, as a final concentration relative to the total volume of the composition or the total volume of the medium.

[0049] Medical Composition In one aspect, the present invention relates to a medical composition. In one embodiment, the medical composition comprises the peptide conjugate of the present invention or a pharmaceutically acceptable salt or solvate thereof (hereinafter, for simplicity, these will also be referred to simply as "peptide"). The medical composition preferably contains an effective amount of the peptide conjugate as an active ingredient. The disease targeted by the medical composition refers to any disease caused by, exacerbated by, or otherwise related to increased or decreased expression or activity of VEGFR-2, or any disorder caused by, exacerbated by, or otherwise related to decreased VEGF signaling or any other intracellular signaling cascade activated via VEGFR-2. Examples of wounds include chronic wounds, acute wounds, and ordinary wounds. Chronic wounds refer to wounds that do not heal, and include, but are not limited to, arterial ulcers, diabetic ulcers, pressure ulcers, venous ulcers, etc. Acute wounds can develop into chronic wounds. Acute wounds include, but are not limited to, wounds caused by thermal injury, trauma, surgery, extensive skin cancer excision, deep fungal and bacterial infections, vasculitis, scleroderma, penicillin, and toxic epidermal necrolysis. Normal wounds refer to wounds that undergo normal wound healing and repair. Pathological conditions of liver dysfunction and disease are related to any structural and / or functional liver abnormality, including, but not limited to, liver failure, hepatitis, cirrhosis, toxic liver damage, drug-induced liver damage, hepatic encephalopathy, hepatic coma, and hepatic necrosis. Protection from liver damage refers to protection from structural or functional liver damage resulting directly or indirectly from internal or external factors (e.g., chemical agents, biological agents, etc.), or a combination thereof. Kidney diseases include, but are not limited to, inflammatory kidney disease, nephritis, glomerulosclerosis, glomerulonephritis, focal segmental glomerulosclerosis, etc. Also included are infectious diseases that cause kidney disease.

[0050] The administration route of the medical composition is not particularly limited and may be oral or parenteral. Examples of parenteral administration include injections such as intramuscular injection, intravenous injection, and subcutaneous injection, as well as transdermal and transmucosal administration (nasal, oral, ocular, pulmonary, vaginal, and rectal). The peptides in the medical composition can be modified in various ways to accommodate their susceptibility to metabolism and excretion. For example, polyethylene glycol (PEG) or sugar chains can be added to the polypeptide to increase its blood residence time and reduce its antigenicity. Furthermore, biodegradable polymers such as polylactic acid glycol (PLGA), porous hydroxyapatite, liposomes, surface-modified liposomes, emulsions prepared with unsaturated fatty acids, nanoparticles, nanospheres, and the like can be used as sustained-release bases, and the polypeptides can be encapsulated within them. For transdermal administration, a weak electric current can be applied to the skin surface to penetrate the stratum corneum (iontophoresis).

[0051] The medical composition may contain the active ingredient as is, or may be formulated by adding pharmaceutically acceptable carriers, excipients, additives, etc. Dosage forms include, for example, liquids (e.g., injections), dispersions, suspensions, tablets, pills, powders, suppositories, powders, fine granules, granules, capsules, syrups, lozenges, inhalants, ointments, eye drops, nasal drops, ear drops, and poultices. The formulation may be carried out by a conventional method using, for example, excipients, binders, disintegrants, lubricants, solubilizers, solubilizers, colorants, flavorings, stabilizers, emulsifiers, absorption enhancers, surfactants, pH adjusters, preservatives, antioxidants, etc. as appropriate.

[0052] Examples of ingredients used in the formulation include, but are not limited to, purified water, saline, phosphate buffer, dextrose, glycerol, ethanol and other pharmaceutically acceptable organic solvents, animal and vegetable oils, lactose, mannitol, glucose, sorbitol, crystalline cellulose, hydroxypropyl cellulose, starch, corn starch, silicic anhydride, magnesium aluminum silicate, collagen, polyvinyl alcohol, polyvinylpyrrolidone, carboxyvinyl polymer, sodium carboxymethylcellulose, sodium polyacrylate, sodium alginate, water-soluble dextran, sodium carboxymethyl starch, pectin, methylcellulose, ethylcellulose, xanthan gum, gum arabic, tragacanth, casein, agar, polyethylene glycol, diglycerin, glycerin, propylene glycol, petrolatum, paraffin, octyldodecyl myristate, isopropyl myristate, higher alcohols, stearyl alcohol, stearic acid, human serum albumin, trehalose, polysorbate and the like.

[0053] The absorption enhancer can be used to improve the absorption of poorly absorbed drugs. Examples of the absorption enhancer that can be used include surfactants such as polyoxyethylene lauryl ethers, sodium lauryl sulfate, and saponin; bile salts such as glycocholic acid, deoxycholic acid, and taurocholic acid; chelating agents such as EDTA and salicylic acids; fatty acids such as caproic acid, capric acid, lauric acid, oleic acid, linoleic acid, and mixed micelles; enamine derivatives, N-acyl collagen peptides, N-acyl amino acids, cyclodextrins, chitosans, and nitric oxide donors.

[0054] The pills or tablets may be coated with sugar, gastric, or enteric coating materials. The injections may contain distilled water for injection, physiological saline, propylene glycol, polyethylene glycol, vegetable oil, alcohols, etc. Furthermore, wetting agents, emulsifiers, dispersants, stabilizers, solubilizers, solubilizers, preservatives, etc. may be added.

[0055] In one embodiment, the medical composition of the present invention may be administered in combination with other medications or treatments useful for the disease.

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

[0057] Diagnostic Composition In one aspect, the present invention relates to a diagnostic composition. The peptide conjugate has VEGFR-2 agonist activity and is therefore believed to bind to VEGFR-2. Therefore, it can also be used as a diagnostic agent for detecting VEGFR-2. The diagnostic agent may be a detection agent for detecting the expression level of VEGFR-2, and when used as a detection agent, the peptide conjugate of the present invention may be detectably labeled. In this way, the peptide conjugate or a composition containing it can be used as a diagnostic agent for detecting VEGFR-2.

[0058] In one aspect, the present invention relates to a composition for research. The peptide conjugate has VEGFR-2 agonist activity and is therefore believed to bind to VEGFR-2. Therefore, it can be preferably used in research involving VEGFR-2.

[0059] In one aspect, the present invention provides a method for testing a peptide and / or a conjugate comprising a peptide and / or a substance further conjugated to a conjugate of a peptide and a linker, the method testing at least one of: a) solubility in a solvent; b) ability to bind to VEGFR-2; c) toxicity to cells and / or tissues; and d) toxicity to laboratory animals, wherein the peptide has an amino acid sequence obtained by deleting, substituting, inserting, and / or adding 1 to 3 amino acid residues in the amino acid sequence of the peptide of the present invention.

[0060] Regarding the test method, the test for the solubility of the peptide and / or complex in a solvent may be a solubility measurement. When measuring solubility, the solvent is not limited and may be freely selected depending on the purpose. Furthermore, a known method for measuring solubility can be appropriately selected depending on the type of solvent. The test for binding ability to VEGFR-2 may be a measurement of binding ability to VEGFR-2, and known methods such as, but not limited to, surface plasmon resonance (SPR) assay, Scatchard analysis, and / or competitive binding assays such as radioimmunoassay (RIA), enzyme immunoassay (EIA), and sandwich competition assays, can be preferably used. The test for toxicity to cells and / or tissues may be a known toxicity evaluation test using cells and / or tissues, for example, an in vitro method. The cells and / or tissues may be cells and / or tissues typically used in toxicity evaluation tests for pharmaceuticals, and are not limited thereto. The test method for toxicity in laboratory animals may be a known toxicity evaluation test using laboratory animals. The experimental animals are not particularly limited as long as they are commonly used, and examples thereof include mice, rats, guinea pigs, gerbils, hamsters, ferrets, rabbits, dogs, cats, pigs, goats, horses, cows, birds (e.g., chickens, quails, etc.), monkeys, and non-human primates (e.g., cynomolgus monkeys, marmosets, rhesus monkeys, etc.). The toxicity evaluation tests may be, but are not limited to, safety tests that are commonly conducted in non-clinical studies of pharmaceuticals, and examples thereof include general toxicity tests (single-dose toxicity tests / repeated-dose toxicity tests), genotoxicity tests (Ames tests / chromosomal aberration tests / in vitro micronucleus tests), carcinogenicity tests, reproductive and developmental toxicity tests (ICH-I, II, III), local irritation tests (eye irritation tests, skin irritation tests, etc.), other toxicity tests (skin sensitization tests, phototoxicity tests, antigenicity tests), chemical analysis / biological analysis (TK / PK), etc.

[0061] Abbreviations (general) Å as Angstrom (unit); BSA as bovine serum albumin; Boc as tert-butoxycarbonyl group; ClAc as chloroacetyl; ClAcOSu as (2,5-dioxopyrrolidin-1-yl) 2-chloroacetate; DCM as dichloromethane or methylene chloride; DIPCI as N,N'-diisopropylcarbodiimide; DIPEA or DIEA as N,N-diisopropylethylamine; DMSO as dimethylsulfoxide; DMF as N,N-dimethylformamide; DODT as 3,6-dioxa-1,8-octanedithiol; DMEM as Dulbecco's modified Eagle's medium; EC50 as 50% effective concentration; FBS as fetal bovine serum; Fmoc as 9-fluorenylmethyloxycarbonyl; N ２ , N ６ Fmoc-Lys(Fmoc)-OH as -bis(((9H-fluoren-9-yl)methoxy)carbonyl)-L-lysine; g as grams; HATU as O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate; HPLC as high performance liquid chromatography; LC-MS or LC / MS as liquid chromatography mass spectrometry; M as molar; MeCN as acetonitrile; mg as milligrams; min as minutes; mL as milliliters; mM as millimolar; mm as millimeters; Mpe group as O-3-methyl-pent-3-yl group; NHS as N-hydroxysuccinimide; nm as nanometers; μL as microliters; OSu as oxysuccinimide; Pbf as 2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl group; PEG as polyethylene glycol; rpm as revolutions per minute (units); Sub as dibenzosuberyl group; tBu as tert-butyl group; TEAA as triethylamine acetate; TFA as trifluoroacetic acid; TIS as triisopropylsilane; Trt or Tr as trityl group;

[0062] Abbreviations (unnatural amino acids) Ahp (S)-2-aminoheptanoic acid (CAS number: 44902-02-5) alt allo-threonine (CAS number: 28954-12-3) Atb (S)-2-amino-4,4-dimethylpentanoic acid (CAS number: 57224-50-7) Atp (2S)-2-amino-3-(oxan-4-yl)propanoic acid (CAS number: 1344910-91-3) Bph (S)-3-([1,1'-biphenyl]-4-yl)-2-aminopropanoic acid (CAS number: 155760-02-4) Cha (S)-2-amino-3-cyclohexylpropanoic acid (CAS number: 27527-05-5) Chg (S)-2-Amino-2-cyclohexylacetic acid (CAS number: 14328-51-9) Cit L-Citrulline (CAS number: 372-75-8) cPEG9c 4,7,10,13,16,19,22,25,28-Nonaoxahentriacontanedioic acid (CAS number: 1268488-70-5) cPEG13c 4,7,10,13,16,19,22,25,28,31,34,37,40-Tridecaoxatritetracontanedioic acid (CAS number: 2225903-66-0) cPEG17c 4, 7, 10, 13, 16, 19, 22, 25, 28, 31, 34, 37, 40, 43, 46, 49,52-Heptadecaoxapentacontanedioic acid (CAS number: 2226897-74-9) F4aao (S)-2-Amino-3-(4-(carboxymethoxy)phenyl)propanoic acid (CAS number: 24558-63-2) F4c (S)-2-Amino-3-(4-chlorophenyl)propanoic acid (CAS number: 14173-39-8) F4COO 4-Carboxyl-L-phenylalanine (CAS number: 126109-42-0) F4G (S)-2-Amino-3-(4-guanidinophenyl)propanoic acid (CAS number: 59574-11-7) H4Py (S)-2-Amino-4-(pyridin-4-yl)butanoic acid (CAS number: 1240588-62-8) Har N6-carbamimidoyl-L-lysine (CAS number: 156-86-5) Hcit N6-carbamoyl-L-lysine (CAS number: 1190-49-4) Hgl L-2-aminoadipic acid (CAS number: 1118-90-7) Hph (S)-2-Amino-4-phenylbutanoic acid (CAS number: 943-73-7) Hty Homo-L-tyrosine (CAS number: 221243-01-2) Hyp Hydroxyproline (CAS number: 51-35-4) MetO2 (S)-2-Amino-4-(methylsulfonyl)butanoic acid (CAS number: 7314-32-1) NHS-cPEG9c-NHS bis(2,5-dioxopyrrolidin-1-yl) 4,7,10,13,16,19,22,25,28-nonaoxahentriacontanedioate (CAS number: 1008402-79-6) NHS-cPEG13c-NHS bis(2,5-dioxopyrrolidin-1-yl) 4,7,10,13,16,19,22,25,28,31,34,37,40-tridecaoxatritetracontanedioate (Bis-PEG13-NHS ester CAS number: 2221949-00-2) NHS-cPEG17c-NHS bis(2,5-dioxopyrrolidin-1-yl) 4,7,10,13,16,19,22,25,28,31,34,37,40,43,46,49,52-Heptadecaoxapentacontanedioate (Bis-PEG17-NHS ester CAS number: 2221948-93-0) NHS-OCOPEG13OCO-NHS Bis(2,5-dioxopyrrolidin-1-yl)(3,6,9,12,15,18,21,24,27,30,33-undecaoxapentatriacontane-1,35-diyl)biscarbonate (TS-L12-TS NOF Corporation) NHS-OCOPEG17OCO-NHS Bis(2,5-dioxopyrrolidin-1-yl)(3,6,9,12,15,18,21,24,27,30,33,36,39,42,45-pentadecaoxaheptatetracontane-1,47-diyl)biscarbonate (TS-L16-TS NOF Corporation) OCOPEG13OCO 3,6,9,12,15,18,21,24,27,30,33-undecaoxapentatriacontane-1,35-diyl bis(hydrogen carbonate) (NOF Corporation) OCOPEG17OCO 3,6,9,12,15,18,21,24,27,30,33,36,39,42,45-pentadecaoxaheptatetracontane-1,47-diyl Bis(hydrogen carbonate) (NOF Corporation) PEG8c 1-amino-3,6,9,12,15,18,21,24-octaoxaheptacosane-27-oic acid (CAS number: 756526-04-2) PEG12c 1-amino-3,6,9,12,15,18,21,24,27,30,33,36-dodecaoxanonatriacontan-39-oic acid (CAS number: 1415408-69-3) 3Py6NH2 (S)-2-amino-3-(6-aminopyridin-3-yl)propanoic acid (CAS number: 1269968-61-7) 4Py2NH2 (S)-2-amino-3-(2-aminopyridin-4-yl)propanoic acid (CAS number: 1269969-46-1) Tbg (S)-2-amino-3,3-dimethylbutanoic acid (CAS number: 20859-02-3) W7N (S)-2-amino-3-(1H-pyrrolo[2,3-b]pyridin-3-yl)propanoic acid (CAS number: 49758-35-2) Yph (S)-2-amino-3-(4-phenoxyphenyl)propanoic acid (CAS number: 150351-64-7),

[0063] The present invention will be described in detail below with reference to examples, but the present invention is not limited to these examples. Those skilled in the art can easily modify and alter the present invention based on the description in this specification, and such modifications and alterations are within the technical scope of the present invention.

[0064] Chemical Synthesis All raw materials, building blocks, reagents, acids, bases, solid-phase resins, and solvents used in the chemical synthesis in the following examples were either commercially available or could be synthesized by those skilled in the art using organic chemistry techniques. Unless otherwise specified, commercially available amino acids containing protecting groups were used as is. Peptide chain elongation on solid-phase resins was carried out using the resins described in each example as starting materials under commonly used peptide coupling reaction conditions and Fmoc removal reaction conditions. The reactions were carried out using an automated peptide synthesizer, such as a Biotage Syro I, a Biotage Syro II, a CEM Liberty Blue, a CEM Liberty Blue HT12, or a CEM Liberty Prime, according to the manufacturer's instructions. The resin used was NovaPEG Rink Amide resin or Seiber Amide resin, and the amount used ranged from 5 mg to 2 g depending on the peptide. The reagent cocktail used for side chain deprotection and cleavage from the solid-phase resin was 4 mL to 50 mL depending on the peptide, and a solution with the following composition was used: A: TFA / H ２ O / TIS / DODT (92.5 / 2.5 / 2.5 / 2.5) B:TFA / H ２ O / TIS / DODT (90 / 2.5 / 2.5 / 5) Common amino acids used are listed below with side chain protecting groups in parentheses: Table 1. Common amino acids used in the examples

[0065]

[0066] ​The resulting crude peptides were purified using one of the following reversed-phase preparative purification systems A) / B) / C) / D) unless otherwise specified: A): Shimadzu prep-HPLC system (LC-20AP, SPD-M20A, CTO-20AC, and CBM-20A); B): Waters AutoPurification System; C): Waters AutoPurification System with SQD; D): Waters Preparative HPLC System; The column used was one of the following a) to n) unless otherwise specified: a) Jeanious One-Column 20 mm I.D. x150mmL 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] The structure of the chemically synthesized peptides was determined by ESI-MS(+) mass spectrometry, where the molecular weight was calculated based on the amino acids used in the target sequence and the building blocks used as needed. "ESI-MS(+)" refers to electrospray ionization mass spectrometry performed in positive ion mode. Detected masses were reported in "m / z" units. Compounds with molecular weights greater than approximately 1000 were frequently detected as multiply charged ions.

[0068] Basic analytical equipment and basic conditions For mass spectral analysis of the peptides synthesized in the following examples, the following basic analytical equipment and basic conditions were used, unless otherwise specified: Analysis was performed using either of the conditions for gradient B (%) x / y.

[0069] Equipment: Shimadzu LC / MS system (LC-20ADXR, CTO-20AC, SPD-M20A, SIL-20AXR, CBM-20A and LCMS-2020) Column: Kinetex EVO C18 2.6 μm 2.1x150mm, 100Å Column temperature: 60°C Mobile phase A: 0.025% TFA in H ２ O Mobile phase B: 0.025% TFA in MeCN Flow rate: 0.5 mL / min Wavelength: 225 nm PDA Gradient B (%): x: 20-60% / 7.15 min, 60-95% / 0.3 min, 95-95% / 1.55 ​​min; y: 40-80% / 7.15min, 80-95% / 0.3min, 95-95% / 1.55min.

[0070] Example 1 Synthesis of Peptide Conjugate (Dimer Structure No. 52) In this example, a peptide conjugate having the following structure (a conjugate of peptide SEQ ID NO: 47 and linker structure No. 6, dimer structure No. 52 in Table 4) was synthesized.

[0071]

[0072] The target peptide was synthesized using Sieber amide resin (Watanabe Chemical, 0.60 mmol / g) by the general method described above, starting with the removal of the Fmoc group. A CEM Liberty Blue HT12 solid-phase synthesizer was used, and the synthesis was performed according to the manufacturer's instructions. To introduce each residue, Fmoc-AA / DIPCI / Oxyma pure (4.2 equivalents / 8 equivalents / 4 equivalents) was used per equivalent of resin, and the reaction was carried out once for 3 minutes at 90°C in DMF. The third and ninth residues were reacted once for 10 minutes at 90°C. The 14th residue was reacted once for 15 minutes at 50°C. Fmoc removal was carried out by reacting with a 10% pyrrolidine solution in DMF at 90°C for 1 minute or at 50°C for 90 seconds. The introduction of the chloroacetyl group was carried out by adding a DMF solution of ClAcOSu (5 equivalents) to the solid-phase resin and shaking for 60 minutes at room temperature. The side chain deprotection and cleavage from the solid-phase resin were carried out by first washing the resin obtained after the chloroacetyl group introduction step with DMF, followed by methylene chloride, and then diethyl ether, and then drying under reduced pressure. The reaction vessel containing the solid-phase resin was then charged with reagent cocktail A (TFA / H ２ A mixture of 92.5:2.5:2.5:2.5 by volume of DISO / TIS / DODT was added and the mixture was shaken at room temperature for 60 minutes. The reaction solution was filtered through a frit. When the filtrate was added to a chilled excess of a mixed solvent of diethyl ether / hexane (1 / 1), a cloudy white precipitate was formed. This mixture was centrifuged and the solution was decanted. The resulting solid was washed again with chilled diethyl ether and then dried under reduced pressure. The resulting solid was used in the subsequent cyclization reaction. The peptide cyclization reaction was carried out in DMSO / H2SO4 at a final peptide concentration of 4.2 mM based on the molar number of the solid phase resin. ２ After dissolving the residue in HCl (9 / 1), triethylamine (20 equivalents) was added, and the mixture was shaken at room temperature for 1 hour. After that, acetic acid was added. The resulting reaction solution was concentrated under reduced pressure using a Genevac HT-12. The resulting crude product was purified using the following conditions (column: Waters XSelect C18 5 μm 50 × 250 mm; mobile phase: A = 20 mM TEAA in H ２ O, B = 20mM TEAA in MeCN, C = 0.2M TEAA in H２ 0, D = MeCN; Temperature: 50°C; Gradient (% A conc): 0.1% over 5.0 min, then 0.1-100% over 0.1 min, (100% - % B) from 5.1 min onwards; (% B conc): 0% over 5.1 min, then 0-4.2% over 1.9 min, then 4.2-20.5% over 3 min, then 20.5-25.6% over 15.5 min, then 25.6-60% over 1.5 min, then 60% over 4 min; (% C conc): 99.9% over 5.0 min, then 99.9-0% over 0.1 min, 0% from 5.1 min onwards; (% D conc): 0%. Flow rate: 18 mL / min over 5.1 min, then (18 mL / min - 118 mL / min) over 1.9 min, then 118 mL / min. After lyophilization, 0.1% TFA in H ２ O-MeCN (1:1, 100 mL) was added and the mixture was lyophilized again. The resulting peptide A was used for the synthesis of peptide conjugates. To synthesize the peptide conjugates, peptide A was dissolved in DMSO to a final concentration of 25 mM, and then NHS-OCOPEG17OCO-NHS (0.4 equivalents) and DIEA (10 equivalents) were added. The mixture was shaken at room temperature for 1 hour, and then acetic acid was added. The resulting crude product was purified using the following conditions (column: Waters XBridge C18, 19 x 150 mm; mobile phase: A = 0.1% TFA in H ２ O, B = 0.1% TFA in MeCN; temperature: 50°C; gradient (% B conc): 15-40% over 3 minutes, then 40-45% over 8 minutes, then 45-60% over 1 minute; flow rate: 17 mL / min. The purity of the target product was calculated from the area ratio of the LC / MS (UV wavelength 225 nm) chromatogram under the following analytical conditions and was found to be 94.86%. Analytical conditions: retention time = 5.7 min; column: Kinetex EVO C18 2.6 μm 2.1 x 150 mm, 100 Å; mobile phase: A = 0.025% TFA in H ２0, B = 0.025% TFA in MeCN; temperature: 60°C; gradient (% B conc): 20-60% over 7.15 min, then 60-95% over 0.30 min, then 95-95% over 1.55 min; flow rate: 0.5 mL / min; ESI-MS (+) observed m / z = 1197 (M+4H)4+.

[0073] Example 2 Synthesis of Peptide Conjugate (Dimer Structure No. 1) In this example, a peptide conjugate having the following structure (a conjugate of peptide SEQ ID NO: 3 and linker structure No. 1, dimer structure No. 1 in Table 4) was synthesized.

[0074]

[0075] The target peptide was synthesized using Sieber amide resin (Watanabe Chemical, 0.65 mmol / g) by the general method described above, starting with the removal of the Fmoc group. Following the introduction of Fmoc-Lys(Fmoc)-OH, the linker moiety PEG and each Fmoc amino acid were introduced. A Biotage Syro I solid-phase synthesizer was used, and the synthesis was performed according to the manufacturer's instructions. Each residue was introduced using Fmoc-AA / HATU / DIEA (9.45 equivalents / 9 equivalents / 18.8 equivalents) per equivalent of resin, with two 20-minute reactions in DMF at 75°C. The second residue was introduced three times at 75°C for 20 minutes. The fourth residue was introduced twice at room temperature for 60 minutes. The 14th residue was introduced 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. Fmoc removal was carried out by reacting with a 20% DMF solution at room temperature for 5 minutes, removing the solution, and then reacting again with a 20% DMF solution at room temperature for 15 minutes. The introduction of a chloroacetyl group was carried out by removing the Fmoc group from the α-amino group of the solid-phase resin carrying the Fmoc-protected peptide obtained in the previous step using the method described above, followed by adding chloroacetic acid (0.5 M DMF solution, 10 equivalents), HCTU (0.49 M DMF solution, 9.8 equivalents), and DIEA (0.5 M DMF solution, 10 equivalents) to the solid-phase resin and shaking at room temperature for 30 minutes. The deprotection of the side chain and the cleavage from the solid-phase resin were carried out by adding a reagent cocktail A (TFA / H ２ A mixture of 92.5:2.5:2.5:2.5 by volume of DISO / TIS / DODT was added and shaken well, followed by shaking at room temperature for 60 minutes. The reaction solution was filtered through a frit. The filtrate was added to a chilled excess of a mixed solvent of diisopropyl ether / hexane (1 / 1) to produce a cloudy white precipitate. This mixture was centrifuged and the solution was decanted. The resulting solid was washed again with a chilled mixed solvent of diisopropyl ether / hexane (1 / 1) and then dried under reduced pressure for 60 minutes. The resulting solid was used in the subsequent cyclization reaction. The peptide cyclization reaction was carried out in DMSO / MeCN / H2SO4 at a final peptide concentration of 1 mM based on the molar number of the solid phase resin.２ After dissolving the residue in 18 / 1 / 1 ethanol, triethylamine (20 equivalents) was added and the mixture was stirred at room temperature for 3 hours. The resulting reaction solution was concentrated under reduced pressure using a Genevac EZ-2 Elite. The resulting crude product was purified using the following conditions: (Column: Waters XBridge® C18, 50 x 150 mm; Mobile phase: A = 0.1% TFA in H ２ O, B = 0.1% TFA in MeCN; temperature: 40°C; gradient (% B conc): 9-34% over 3 minutes, then 34-39% over 8 minutes, then 39-60% over 1 minute; flow rate: 120 mL / min. The purity of the target product was calculated from the area ratio of the LC / MS (UV wavelength 225 nm) chromatogram under the following analytical conditions and was found to be 86.83%. Analytical conditions: retention time = 5.3 minutes; column: Kinetex EVO C18, 2.6 μm, 2.1 × 150 mm, 100 Å; mobile phase: A = 0.025% TFA in H ２ 0, B = 0.025% TFA in MeCN; temperature: 60°C; gradient (% B conc): 20-60% over 7.15 min, then 60-95% over 0.30 min, then 95-95% over 1.55 min; flow rate: 0.5 mL / min; ESI-MS (+) observed m / z = 1023 (M+5H)5+.

[0076] Example 3 Synthesis of Peptide Conjugate (Dimer Structure No. 43) In this example, a peptide conjugate having the following structure (a conjugate of peptide SEQ ID NO: 42 and linker structure No. 4, dimer structure No. 43 in Table 4) was synthesized.

[0077]

[0078] The target peptide was synthesized using Sieber amide resin (Watanabe Chemical, 0.57 mmol / g) by the general method described above, starting with the removal of the Fmoc group. A Biotage Syro I solid-phase synthesizer was used, and the synthesis was performed according to the manufacturer's instructions. To introduce each residue, Fmoc-AA / HATU / DIPEA (4.2 equivalents / 4 equivalents / 8.4 equivalents) was used per equivalent of resin, and the reaction was carried out twice at 75°C for 20 minutes in DMF. 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. Fmoc removal was carried out by reacting the solid-phase resin with a 20% piperidine solution in DMF at room temperature for 5 minutes, removing the solution, and then reacting again with a 20% piperidine solution in DMF at room temperature for 15 minutes. The introduction of a chloroacetyl group was carried out by removing the Fmoc group from the α-amino group of the solid-phase resin carrying the Fmoc-protected peptide obtained in the previous step using the method described above, followed by stirring DIPCI (10 equivalents), HOSu (10 equivalents), and chloroacetic acid (10 equivalents) in DCM, adding an equal amount of DMF to the DCM, preparing a DCM / DMF solution of ClAcOSu, and adding the resulting solution to the solid-phase resin, followed by shaking for 60 minutes at room temperature. Deprotection of the side chain and cleavage from the solid-phase resin were carried out by adding reagent cocktail A (TFA / H ２ A mixture of 92.5:2.5:2.5:2.5 by volume of DISO / TIS / DODT was added and the mixture was shaken at room temperature for 60 minutes. The reaction solution was filtered through a frit. When the filtrate was added to an excess of chilled diisopropyl ether, a cloudy white precipitate was formed. This mixture was centrifuged and the solution was decanted. The resulting solid was washed again with chilled diethyl ether and then dried under reduced pressure. The resulting solid was used in the subsequent cyclization reaction. The peptide cyclization reaction was carried out in DMSO / H2SO4 at a final peptide concentration of 5 mM based on the molar number of the solid phase resin. ２After dissolving the product in DMSO (9 / 1), triethylamine (10 equivalents) was added and the mixture was stirred at room temperature for 15 hours. The resulting reaction solution was concentrated under reduced pressure using a Genevac EZ-2 Elite to obtain crude cyclic peptide B. The peptide conjugate was synthesized by dissolving the above-obtained peptide B in DMSO to a concentration of 25 mM, adding 10 equivalents of diisopropylethylamine, followed by 0.4 equivalents of NHS-cPEG17c-NHS, shaking at room temperature for 30 minutes, and then adding acetic acid. The resulting crude product was purified using the following conditions: (Column: Kinetex EVO C18 5 μm 21.2 × 150 mm; Mobile phase: A = 0.1% TFA in H ２ O, B = 0.1% TFA in MeCN; temperature: 60°C; gradient (% B conc): 5-30% over 3 minutes, then 30-35% over 8 minutes, then 35-60% over 1 minute; flow rate: 20 mL / min. The purity of the target product was calculated from the area ratio of the LC / MS (UV wavelength 225 nm) chromatogram under the following analytical conditions and was found to be 87.55%. Analytical conditions: retention time = 4.5 minutes; column: Kinetex EVO C18, 2.6 μm, 2.1 × 150 mm, 100 Å; mobile phase: A = 0.025% TFA in H ２ 0, B = 0.025% TFA in MeCN; temperature: 60°C; gradient (% B conc): 20-60% over 7.15 min, then 60-95% over 0.30 min, then 95-95% over 1.55 min; flow rate: 0.5 mL / min; ESI-MS (+) observed m / z = 1194 (M+4H)4+.

[0079] Example 4: Synthesis of Various Peptides In this example, various peptide conjugates were chemically synthesized in the same manner as in Examples 1-3. Table 2 shows the sequences of the synthesized cyclic peptides, Table 3 shows the linker structures, and Table 4 shows peptide conjugates in which the cyclic peptides are dimerized via the linker. The conjugation number in Table 4 indicates the position of the amino acid to which the linker is attached in the amino acid sequence indicated by each peptide SEQ ID NO. It is preferable that the C-terminal carboxylic acid of the 15th Gly in peptide SEQ ID NOs: 14-35 and 37-51 in Tables 2 and 4 be converted to an amide. The synthesized peptide conjugates were analyzed using the basic analytical equipment and under any of the analytical conditions described in the basic conditions above, and their structures were confirmed by ESI-MS(+) in mass spectrometry. Table 4 shows the ESI-MS(+) observed values, retention times, charge numbers, and the concentration gradient (%) of mobile phase B used in the analysis.

[0080] Table 2. Sequences of synthesized cyclic peptides

[0081] Table 3. Linker structures

[0082] Table 4. Peptide conjugates

[0083] Example 5 Evaluation of VEGFR-2 Agonist Activity by pVEGFR AlphaLISA Assay To evaluate the VEGFR-2 activation ability of the VEGFR-2 agonist peptide of the present invention, phosphorylation of VEGFR-2 was examined. ＴＭThe cells were cultured in Endothelial Cell Growth Medium-2 Bullet Kit (LONZA; CC-3162). After detachment using Tryple (ThermoFisher Scientific), they were seeded onto a 96-well PDL-coated plate for adherent cells (Corning: 354461) at 24,000 cells per well 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 (included in the above LONZA; CC-3162), and 0.1% Gentamicin (Nacalai), and the cells were cultured for 18 hours. Recombinant Human VEGF165a Protein (R&D systems) or peptide was then added, and the cells were cultured for 37 hours in a CO2 incubator. ｏC. After 10 minutes of stimulation, cells were lysed with the lysis buffer included in the AlphaLISA SureFire Ultra VEGFR-2 (p-Tyr1175) Assay Kit (PerkinElmer). The assay was performed according to the kit's protocol, and a SpectraMax Paradigm multimode microplate reader (Molecular Devices) was used for signal detection. The resulting signals were analyzed using GraphPad Prism, and the % activity was calculated by setting the maximum signal induced by VEGF165a as 100% and the unstimulated signal as 0%. The VEGF165a concentration was evaluated at five 1 / 4-fold dilutions (0.08, 0.31, 1.25, 5, and 20 nM) starting from 20 nM, and the maximum activity (100%) was determined. The peptide concentrations added were 3.2, 16, and 80 nM for dimer structure number 1, 10 and 100 nM for dimer structure numbers 2 to 41, and 1.25, 5, and 20 nM for dimer structure numbers 42 to 57. Regarding % activity, samples with 50% or more activity when 3.2 nM of peptide was added were designated 1-A, those with 50% or more activity when 16 nM of peptide was added were designated 1-B, those with 50% or more activity when 80 nM of peptide was added were designated 1-C, and those with 1% or more but less than 50% activity when 80 nM of peptide was added were designated 1-D. Those that showed 50% or more activity when 10 nM of peptide was added were designated 2-A, those that showed 50% or more activity when 100 nM of peptide was added were designated 2-B, and those that showed 1% to less than 50% activity when 100 nM of peptide was added were designated 2-C. Those that showed 50% or more activity when 1.25 nM of peptide was added were designated 3-A, those that showed 50% or more activity when 5 nM of peptide was added were designated 3-B, those that showed 50% or more activity when 20 nM of peptide was added were designated 3-C, and those that showed 1% to less than 50% activity when 20 nM of peptide was added were designated 3-D.

[0084] The results are shown in Table 5. None of the peptides fell into the above categories 1-A to 1-C and 3-A. As shown in Table 5, the synthesized peptide conjugates were shown to have VEGFR-2 agonist activity that induces phosphorylation of VEGFR-2.

[0085] Table 5. VEGFR-2 agonist activity evaluation

[0086] Example 6 Evaluation of VEGFR-2 Agonist Activity by pERK AlphaLISA Assay To evaluate the intracellular signal activation ability of the VEGFR-2 agonist peptides of the present invention, ERK phosphorylation was examined. Using the lysate used in pVEGFR AlphaLISA, the assay was performed according to the protocol of the AlphaLISA SureFire Ultra p-Erk1 / 2 (Thr202 / Tyr204) Assay Kit (PerkinElmer), and signals were detected using a SpectraMax Paradigm multimode microplate reader (Molecular Devices). The resulting signals were analyzed using GraphPad Prism, and the percent activity was calculated by setting the maximum signal induced by VEGF165a as 100% and the unstimulated signal as 0%. VEGF165a was added at five concentrations (0.08, 0.31, 1.25, 5, and 20 nM) diluted 1 / 4 from 20 nM to determine the maximum (100%). The peptide concentrations added were 3.2, 16, and 80 nM for dimer structure number 1, 10 and 100 nM for dimer structure numbers 2 to 41, and 1.25, 5, and 20 nM for dimer structure numbers 42 to 57. Regarding % activity, peptides with 50% or more activity when added at 3.2 nM were designated 1-A, peptides with 50% or more activity when added at 16 nM were designated 1-B, peptides with 50% or more activity when added at 80 nM were designated 1-C, and peptides with 1% to less than 50% activity when added at 80 nM were designated 1-D. Peptides with 50% or more activity when added at 10 nM were designated 2-A, peptides with 50% or more activity when added at 100 nM were designated 2-B, and peptides with 1% to less than 50% activity when added at 100 nM were designated 2-C. Peptides with 50% or more activity when added at 1.25 nM were designated 3-A, peptides with 50% or more activity when added at 5 nM were designated 3-B, peptides with 50% or more activity when added at 20 nM were designated 3-C, and peptides with 1% to less than 50% activity when added at 20 nM were designated 3-D.

[0087] The results are shown in Table 5. No compounds were found that fell into the above categories 1-A to 1-C, 3-A, or 3-D. As shown in Table 5, the synthesized peptide complexes were shown to have the ability to induce not only VEGFR-2 phosphorylation but also ERK phosphorylation, an intracellular signal.

[0088] Example 7 Evaluation of HUVEC proliferation promotion To evaluate the biological activity of the VEGFR-2 agonist peptide of the present invention, HUVEC proliferation promotion evaluation was carried out. HUVECs cultured in the same manner as above were detached using Tryple (ThermoFisher Scientific), and then plated in a Nunc plate for adherent cells and a Nunc plate for luminescence detection at 10,000 cells per well. ＴＭ MicroWell ＴＭ The cells were seeded onto a 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 (included in the above LONZA; CC-3162), and 0.1% Gentamicin (Nacalai) for starvation, and the cells were cultured for 18 hours. Recombinant Human VEGF165a Protein (R&D systems) or peptide was then added, and the cells were incubated for 37 hours in a CO2 incubator. ｏC, cultured for 2 days. The procedure was performed according to the CellTiter-Glo (Promega) kit protocol, and a SpectraMax Paradigm multimode microplate reader (Molecular Devices) was used for signal detection. The resulting signals were analyzed using GraphPad Prism, and the % activity was calculated by setting the maximum signal induced by VEGF165a as 100% and unstimulated as 0%. VEGF165a was added at six concentrations (0.006, 0.03, 0.16, 0.8, 4, and 20 nM) at 1 / 5 dilutions starting from 20 nM, and the maximum value (100%) was determined. The peptide concentrations added were 1, 10, and 100 nM for dimer structure numbers 2, 5, 13, 14, 25, 27, 29, 31, and 33, and four concentrations (0.31, 1.25, 5, and 20 nM) diluted 1 / 4-fold from 20 nM for dimer structure numbers 42 to 53. Regarding % activity, peptides with 50% or more activity when added at 1 nM were designated 4-A, peptides with 50% or more activity when added at 10 nM were designated 4-B, peptides with 50% or more activity when added at 100 nM were designated 4-C, and peptides with 1% or more but less than 50% activity when added at 100 nM were designated 4-D. The peptides that showed 50% or more activity when 0.31 nM of peptide was added were designated 5-A, those that showed 50% or more activity when 1.25 nM of peptide was added were designated 5-B, those that showed 50% or more activity when 5 nM of peptide was added were designated 5-C, those that showed 50% or more activity when 20 nM of peptide was added were designated 5-D, and those that showed 1% or more but less than 50% activity when 20 nM of peptide was added were designated 5-E.

[0089] The results are shown in Table 6. None of the peptides fell into the above categories 4-A, 4-C, or 5-E. As shown in Table 6, the synthesized peptide complexes were shown to have the ability to promote HUVEC proliferation.

[0090] Table 6. Evaluation of HUVEC proliferation promotion

[0091] Example 8 Evaluation of VEGFR-specific activation ability by RTK array assay 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, including VEGFR-2, was examined. HUVECs cultured in the same manner as above were detached using Tryple (ThermoFisher Scientific), seeded into a 6-well plate for adherent cells at 720,000 cells per well, 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 (included in the above LONZA; CC-3162), and 0.1% Gentamicin (Nacalai), and the cells were cultured for 18 hours. Then, Recombinant Human VEGF165a Protein (R&D Systems) or peptide was added, and the cells were incubated for 37 h in a CO2 incubator. ｏ C. After 10 minutes of stimulation, cells were lysed with the lysis buffer included in the Human Phospho-RTK Array Kit (R&D). The assay was performed according to the kit's protocol, and C-Digit (Li-COR) was used for signal detection. The resulting signals were analyzed using the Transparency Overlay Template image provided with the kit. The VEGF165a concentration was 5 nM, and the peptide concentration was 20 nM.

[0092] The results are shown in Figure 1B. As shown in Figure 1B, the synthesized peptide complexes (dimer structure numbers 48 and 52 in Table 4) exhibited VEGFR-2 specific activation ability similar to that of recombinant VEGF protein.

[0093] The invention can be used in the pharmaceutical and biotechnology industries.

Claims

1. A peptide complex or a pharma- ceutical acceptable salt thereof comprising a first peptide and having VEGFR-2 agonist activity, wherein the first peptide is １ -W-X ２ -X ３ -X ４ -X ５ -X ６ -X ７ -X ８ -Y-X ９ -X １０ -X １１ -C (SEQ ID NO: 1), or the amino acid sequence of SEQ ID NO: 1 in which 1 to 3 amino acids have been substituted, deleted, added or inserted, １ is an amino acid having, in the side chain, a chain alkyl group which may be substituted with a polar group, or an aromatic ring which may be substituted, ２ is an amino acid having an optionally substituted alkyl group on the side chain, ３ is an amino acid having an optionally substituted chain alkyl group at the side chain, ４ is an amino acid having an optionally substituted alkyl group on the side chain, ５ is any amino acid, ６ is an amino acid having a chain alkyl group which may be substituted with a polar group in the side chain or having no side chain, ７ is an amino acid having an optionally substituted chain alkyl group at the side chain, ８ is an amino acid having an optionally substituted chain alkyl group at the side chain, ９ is an amino acid having an aromatic ring which may be substituted in the side chain, １０ is an amino acid having an optionally substituted alkyl group on the side chain, １１ is any amino acid, or a pharma- ceutically acceptable salt thereof.

2. The peptide conjugate according to claim 1 or a pharma- ceutically acceptable salt thereof, ２ is an amino acid having a linear or cyclic alkyl group or a linear alkyl group which may be substituted with an aromatic ring in the side chain, ３ is an amino acid having a chain alkyl group in the side chain which may be substituted with a polar group, ４ is an amino acid having a linear or cyclic alkyl group or a linear alkyl group which may be substituted with a polar group in the side chain; ７ is an amino acid having a chain alkyl group which may be substituted with a polar group or a chain alkyl group which may be branched at the side chain, ８ is an amino acid having a chain alkyl group which may be substituted with a polar group or a chain alkyl group which may be branched at the side chain, １０ is an amino acid having a linear or cyclic alkyl group in the side chain, or a linear alkyl group substituted with an aromatic ring or a heterocyclic ring, or a peptide conjugate or a pharma- ceutically acceptable salt thereof.

3. The peptide conjugate according to claim 2 or a pharma- ceutically acceptable salt thereof, １ is W, Hcit, 4Py2NH2, 3Py6NH2, F4aao, or W7N; ２ is V, Tbg, Chg, or Hty; X ３ is D, Q, or MetO2; X ４ is V, Tbg, aIT, or Chg; ５ is Q, Ahp, W, Hph, Cha, F4COO, or K; ６ is D, G, or N; ７ is D or V; X ８ is L, N, or Atb; ９ is F, Bph, Yph, F4G, or F4C; １０ is V, Hph, Hty, Chg, or H4Py; １１ is D, A, S, F, F4aao, Har, or Hyp; or a peptide conjugate or a pharma- ceutically acceptable salt thereof.

4. The peptide conjugate according to claim 1 or a pharma- ceutically acceptable salt thereof, ２ is an amino acid having a linear or cyclic alkyl group in the side chain; ４ is an amino acid having a chain alkyl group which may be substituted with a polar group or a chain alkyl group which may be branched at the side chain, ６ is an amino acid having a chain alkyl group in the side chain which may be substituted with a polar group, ７ is an amino acid having a chain alkyl group in the side chain which may be substituted with a polar group, ８ is an amino acid having a chain alkyl group which may be substituted with a polar group or a chain alkyl group which may be branched at the side chain, １０ is an amino acid having a linear or cyclic alkyl group, or a linear alkyl group which may be substituted with an aromatic ring, in the side chain; or a peptide conjugate or a pharma- ceutically acceptable salt thereof.

5. The peptide conjugate according to claim 4 or a pharma- ceutically acceptable salt thereof, １ is W or W7N; X ２ is V, Tbg, or Chg; ３ is D, and X ４ is V or alT; X ５ is Ahp, Cha or K; ６ is D or N; X ７ is D, and X ８ is L, N or Atb; ９ is F or F4G, X １０ is V, Hty or Chg; X １１ is D or S; or a pharma- ceutically acceptable salt thereof.

6. The peptide conjugate or pharma- ceutically acceptable salt thereof according to claim 1, wherein the first peptide has an amino acid sequence represented by W-W-V-D-V-Q-D-D-L-Y-F-V-D-C (SEQ ID NO: 2), or is an amino acid sequence represented by SEQ ID NO: 2 in which 1 to 3 amino acids have been substituted, deleted, added or inserted.

7. A peptide conjugate or a pharma- ceutically acceptable salt thereof according to claim 1, wherein the first peptide has an amino acid sequence with glycine added to the C-terminus.

8. The peptide complex or pharma- ceutical acceptable salt thereof according to claim 1, wherein the first peptide is a peptide consisting of an amino acid sequence shown in any one of SEQ ID NOs: 3 to 51.

9. The peptide conjugate or pharma- ceutically acceptable salt thereof according to claim 1, wherein the first peptide is a cyclic peptide.

10. A peptide complex or a pharma- ceutically acceptable salt thereof according to claim 1, wherein the first peptide is a cyclic peptide in which the N-terminal amino acid residue is a chloroacetylated amino acid residue, the peptide has a cysteine ​​residue, and the N-terminal amino acid residue and the cysteine ​​residue are bound to each other.

11. A peptide complex or a pharma- ceutically acceptable salt thereof according to claim 1, wherein the peptide complex comprises the first peptide, a second peptide, and a linker connecting the first peptide and the second peptide, and the second peptide may be the same as or different from the first peptide, and has an amino acid sequence as shown in SEQ ID NO:1, or comprises an amino acid sequence in which 1 to 3 amino acids have been substituted, deleted, added, or inserted in the amino acid sequence as shown in SEQ ID NO:

1.

12. The peptide complex or pharma- ceutically acceptable salt thereof according to claim 11, wherein the homology between the first peptide and the second peptide is 90% or more and 100% or less.

13. The peptide conjugate or a pharma- ceutically acceptable salt thereof according to claim 11, wherein the first peptide and the second peptide are peptides having the same sequence.

14. The peptide conjugate or pharma- ceutically acceptable salt thereof according to claim 11, wherein the sixth amino acid of the first peptide and the sixth amino acid of the second peptide, or the C-terminus of the first peptide and the C-terminus of the second peptide, are linked via the linker.

15. The peptide conjugate or a pharma- ceutically acceptable salt thereof according to claim 11, wherein the linker is a PEG linker or a linker in which 1 to 6 amino acids are added to a PEG linker.

16. A composition comprising the peptide conjugate of claim 1 or a pharma- ceutically acceptable salt thereof and a carrier.

17. A composition for cell culture, which comprises the peptide complex or a pharma- ceutically acceptable salt thereof according to claim 1 and a carrier and is used for cell culture.

18. A composition for medical, diagnostic or research use comprising the peptide conjugate of claim 1 or a pharma- ceutically acceptable salt thereof and a carrier.

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