WO2005094865A1 - Compound having angiogenic effect - Google Patents

Abstract

It is intended to search for a substance having a promoted angiogenic ability compared with a natural angiogenic peptide (SVVYGLR). Namely, a composition for angiogenesis which contains the sequence VVXGL, wherein X represents a modified aromatic amino acid, and comprises a peptide; a composition for forming a blood vessel network which contains the sequence VVXGL, wherein X represents a modified aromatic amino acid, and comprises a peptide; a method for angiogenesis which comprises administering the above-described composition to a site of a patient with a need for angiogenesis; and a method for blood vessel network formation which comprises administering the above-described composition to a site of a patient with a need for blood vessel network formation.

Description

 Specification

 Angiogenic compounds

 Technical field

 [0001] The present invention is in the field of medicine. More particularly, the present invention relates to a medicament for forming a novel angiogenic agent.

 Background fields

 [0002] There have been reports of various active peptides, and they have begun to be used as medicines in various fields.

 [0003] In fields involving blood vessels, the search for such active peptides has become active. In particular, various factors have been investigated for factors controlling angiogenesis, angiogenesis, etc., and both inhibitory and active effects are effective. Treatment of various diseases and disorders (eg, cancer, ischemic disease, etc.) It is used for prevention and prevention.

 [0004] At present, diseases caused by ischemia caused by occlusion of blood vessels such as myocardial infarction and cerebral infarction account for a large number of deaths in developed countries. In addition, some diseases, such as obstructive atherosclerosis, which require amputation of the lower limbs and cause a loss of quality of life (QOL), even if they do not cause death. For these ischemic diseases, there is great expectation for vascular network formation therapy by forming new blood vessels, but as described above, vascular formation alone is not always sufficient, It is desirable that blood vessels be formed so that nutrients can be sufficiently transmitted to the ends and material exchange can be performed smoothly.

 [0005] On the other hand, peptides that have great advantages in safety, metabolism, etc., including side effects, are relatively easy to design, and highly efficient synthetic methods and assay methods have been established. In addition, amino acid derivatives are convenient building units for combinatorial chemical libraries, and their structure can be optimized in a short time by solid-phase synthesis. Therefore, if a compound that promotes angiogenesis more strongly exists, it can be advantageously synthesized and administered alone or in combination.

[0006] Conventionally, experiments have been attempted in which a peptide sequence is modified (Patent Document 1). However, such modification at the peptide level alone has a limitation in enhancing the activity. Special In Patent Document 1, modifications other than peptides have been attempted, but compounds having angiogenic activity exceeding those of peptides have been found.

 [0007] Therefore, in the art, angiogenesis has been enhanced more than natural peptides, and the appearance of a compound has been awaited.

 Patent document l: WO 03Z030925

 Disclosure of the invention

 Problems to be solved by the invention

[0008] Therefore, an object of the present invention is to search for a substance having an enhanced angiogenic ability compared to a natural peptide (SVVYGLR (SEQ ID NO: 2)). Another object of the present invention is to provide a novel angiogenic agent containing such a substance as an active ingredient and which can be used clinically for human treatment and the like.

 The present inventors have conducted intensive studies and found that a peptide having a specific modification of an aromatic group in a specific amino acid sequence unexpectedly has enhanced angiogenic activity. Was completed.

 Accordingly, the present invention provides the following.

 (1) A composition for angiogenesis, comprising a peptide, comprising a sequence of VVXGL (SEQ ID NO: 1), wherein X is a variant of an aromatic amino acid.

 (2) The composition according to item 1, wherein X is a modified form of phenalanine or tyrosine.

 (3) The composition according to item 1, wherein the modification includes a peptide in which the para, ortho, or meta position of the side chain of the aromatic amino acid is substituted with an electron-withdrawing substituent.

 (4) The composition according to item 1, wherein X is a group in which an aromatic group of the aromatic amino acid is substituted with a hydrophobic group.

 (5) The composition according to item 1, wherein X is an aromatic group of the aromatic amino acid substituted with an alkyl group.

 (6) The composition according to item 1, wherein X is substituted with an alkyl group at the para-position of the aromatic group of the aromatic amino acid.

(7) The X is an aromatic amino acid wherein the aromatic group is substituted with a C1-C6 alkyl group, Item 1. The composition according to Item 1.

 (8) The composition according to item 1, wherein in X, the aromatic group of the aromatic amino acid is substituted with a C1-C3 alkyl group.

 (9) The composition according to item 1, wherein X is a group in which the aromatic group of the aromatic amino acid is substituted with a methyl group.

 (10) The composition according to item 1, wherein X is substituted with a methyl group at the para-position of the aromatic group of the aromatic amino acid.

 (11) The composition according to item 1, wherein in X, the aromatic group of the aromatic amino acid is a phenyl group, and the aromatic group is substituted with a phenyl group.

 (12) A composition for forming a vascular network, comprising a peptide comprising the sequence WXGL, wherein X is a variant of an aromatic amino acid.

 (13) The composition according to item 12, wherein the X is a modified form of phenalanine or tyrosine.

 (14) The composition according to item 12, wherein the modification includes a peptide in which the para, ortho, or meta position of the aromatic group of the aromatic amino acid is substituted with an electron-withdrawing substituent.

(15) The composition according to item 12, wherein X is a group in which the aromatic group of the aromatic amino acid is substituted with a hydrophobic group.

 (16) The composition according to item 12, wherein X is an aromatic amino acid in which the aromatic group is substituted with an alkyl group.

 (17) The composition according to item 12, wherein in X, the para-position of the aromatic group of the aromatic amino acid is substituted with an alkyl group.

 (18) The composition according to item 12, wherein in X, the aromatic group of the aromatic amino acid is substituted with a C1-C6 alkyl group.

 (19) The composition according to item 12, wherein in X, the aromatic group of the aromatic amino acid is substituted with a C1-C3 alkyl group.

 (20) The composition according to item 12, wherein X is a group in which the aromatic group of the aromatic amino acid is substituted with a methyl group.

(21) The X is substituted with a methyl group at the para-position of the aromatic group of the aromatic amino acid, Item 13. The composition according to Item 12.

 (22) The composition according to item 12, wherein in X, the aromatic group of the aromatic amino acid is a phenyl group, and the aromatic group is substituted with a methyl group at the para position.

 (23) A peptide comprising the sequence of WXGL (SEQ ID NO: 1), wherein X is a variant of an aromatic amino acid.

 (24) The peptide according to item 23, wherein X is a modified form of phenalanine or tyrosine.

 (25) The peptide according to item 23, wherein the modification includes a peptide in which the para, ortho, or meta position of the side chain of the aromatic amino acid is substituted with an electron-withdrawing substituent.

 (26) The peptide according to item 23, wherein X is one in which an aromatic group of the aromatic amino acid is substituted with a hydrophobic group.

 (27) The peptide according to item 23, wherein X is an aromatic amino acid in which the aromatic group is substituted with an alkyl group.

 (28) The peptide according to item 23, wherein in X, the para position of the aromatic group of the aromatic amino acid is substituted with an alkyl group.

 (29) The peptide according to item 23, wherein in X, the aromatic group of the aromatic amino acid is substituted with a C1-C6 alkyl group.

 (30) The peptide according to item 23, wherein in X, the aromatic group of the aromatic amino acid is substituted with a C1-C3 alkyl group.

 (31) The peptide according to item 23, wherein X is a group in which an aromatic group of the aromatic amino acid is substituted with a methyl group.

 (32) The peptide according to item 23, wherein X is a para-substituted aromatic group of the aromatic amino acid substituted with a methyl group.

 (33) The peptide according to item 23, wherein in X, the aromatic group of the aromatic amino acid is a phenyl group, and the amino position is substituted with a methyl group.

 (34) A method for forming a blood vessel, which comprises administering the composition according to any one of Items 1 to L1 to a site of a patient where angiogenesis is desired.

(35) Any of items 12 to 22 at the site of the patient where formation of a vascular network is desired A method for forming a vascular network, comprising administering the composition according to claim 1.

 (36) Use of a peptide comprising the sequence WXGL, wherein X is a modified aromatic amino acid or a modified variant thereof or a salt thereof, for the manufacture of a pharmaceutical composition for angiogenesis.

 (37) Use of a peptide called WXGL, wherein X is a modified aromatic amino acid or a modified variant thereof or a salt thereof, for use in the production of a pharmaceutical composition for forming a vascular network of a peptide. .

 [0011] Accordingly, it is understood that these and other advantages of the present invention will become apparent to those skilled in the art upon reading and understanding the following detailed description, with reference to the accompanying drawings.

 The invention's effect

 [0012] According to the present invention, a peptide having a strong angiogenic action was found, and a novel angiogenic agent containing the peptide as an active ingredient was provided for the first time. The angiogenesis agent of the present invention is useful for regenerating and repairing organs using biological substitutes such as artificial bones, and for ischemic diseases such as myocardial infarction, cerebral infarction, and obstructive aortic sclerosis, which are a large part of lifestyle-related diseases. It is also useful for treatment.

 Brief Description of Drawings

 FIG. 1 shows a schematic diagram of the action of adhering cells to form a lumen between them.

 FIG. 2 shows an example of the action of forming a lumen by comparing VEGF and peptide SWYGLR (SEQ ID NO: 2).

 [FIG. 3] FIG. 3 is a diagram showing the state of a tissue around a microcell observed with a microscope using a DAS assay 5 days later in Example 3.

 FIG. 4 shows the state of angiogenesis and formation of a vascular network when peptide SWYGLR (SEQ ID NO: 2) was used.

 FIG. 5 shows the state of angiogenesis and vascular network formation using peptide SWYGLR (SEQ ID NO: 2) and the modified peptide of the present invention.

FIG. 6 shows a balance sheet of the vascular network ability of the peptide of the present invention. Description of Sequence Listing

SEQ ID NO: 1 is an amino acid sequence WXGL showing the basic structure of the peptide of the present invention. Where X is a variant of an aromatic amino acid.

SEQ ID NO: 2 is a peptide (SVVYGLR) capable of angiogenesis in osteobontin.

SEQ ID NO: 3: SWFGLR

SEQ ID NO: 4: SWF (pMe) GLR (where F (pMe) is a phenyl group of phenylalanine substituted with a methyl group at the para-position)

SEQ ID NO: 5: SWF (pF) GLR (where F (pF) is obtained by substituting a fluoro group at the para-position of the phenyl group of phenylalanine)

SEQ ID NO: 6: SWF (pNO) GLR (where F (pNO) is a phenylalanine phenol

 twenty two

With-toro group substituted at the para position of the group)

SEQ ID NO: 7: SWF (D) GLR (where F (D) is the D-form of phenalanine) SEQ ID NO: 8: SWF (pEt) GLR (where F (pEt) is The phenyl group of realanine is substituted with an ethyl group at the para position.)

SEQ ID NO: 9: SWF (pPr) GLR (where F (pPr) is a phenyl group in which a propyl group is substituted with a propyl group at the para position)

SEQ ID NO: 10: SWF (pHex) GLR (where, F (pHex) is phenylylalanine in which a hexyl group is substituted at the para-position of the phenyl group)

SEQ ID NO: 11: SWYGL

SEQ ID NO: 12: SWFGL

SEQ ID NO: 13: WYGLR

SEQ ID NO: 14: WFGLR

SEQ ID NO: 15: WYGL

SEQ ID NO: 16: WFGL

SEQ ID NO: 17: \ ^? (^^) 00 ^ (where F (pMe) is a phenyl group of phenylalanine substituted with a methyl group at the para-position)

SEQ ID NO: 18: SWF (pMe) GL (where F (pMe) is a phenyl group of phenylalanine With a methyl group substituted at the para-position)

 SEQ ID NO: 19: VVF (pMe) GL (where F (pMe) is a phenylalanine in which a methyl group is substituted at the para-position of the phenyl group)

 SEQ ID NO: 20 is another exemplary sequence of the peptide of the present invention.

 BEST MODE FOR CARRYING OUT THE INVENTION

 Hereinafter, the present invention will be described. It should be understood that throughout this specification, the use of the singular includes the plural concept unless specifically stated otherwise. Therefore, it is to be understood that singular articles (eg, "a", "an", "the", etc. in English) also include the concept of the plural unless specifically stated otherwise. . It is to be understood that the terms used in the present specification are used in a meaning commonly used in the art unless otherwise specified. Thus, unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. . In case of conflict, the present specification, including definitions, will control.

 [0016] (term)

 The terms "protein", "polypeptide", "oligopeptide" and "peptide" as used herein are used interchangeably herein and refer to a polymer of amino acids of any length. The polymer may be linear or branched or cyclic. The amino acids may be naturally occurring or non-naturally occurring or modified amino acids. These terms may also include those assembled into a complex of multiple polypeptide chains. The term also embraces natural or artificially modified amino acid polymers. Such modifications include, for example, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification (eg, conjugation with a labeling component). This definition also includes, for example, polypeptides containing one or more analogs of an amino acid (eg, including unnatural amino acids, etc.), peptide-like conjugates (eg, peptoids) and those known in the art. Other modifications are included.

[0017] As used herein, the terms "polynucleotide", "oligonucleotide" and " "Nucleic acid" is used interchangeably herein and refers to a polymer of nucleotides of any length. The term also includes "derivative oligonucleotides" or "derivative polynucleotides." “Derivative oligonucleotide” or “derivative polynucleotide” refers to an oligonucleotide or polynucleotide having an unusual force or a bond between nucleotides containing a derivative of a nucleotide, and is used interchangeably. Specific examples of such an oligonucleotide include, for example, 2 ′ O-methyl-ribonucleotide, a derivative oligonucleotide in which a phosphodiester bond in an oligonucleotide is converted to a phosphorothioate bond, and a phosphorus in an oligonucleotide. Derivative oligonucleotide in which acid diester bond is converted to N3,1-P5, phosphoramidate bond, derivative oligonucleotide in which ribose and phosphodiester bond in oligonucleotide are converted to peptide nucleic acid bond, and peracil in oligonucleotide is C 5 Derivative oligonucleotide substituted with propylperacyl, derivative oligonucleotide in which peracyl in oligonucleotide is substituted with C-5 thiazole peracyl, cytosine in oligonucleotide substituted with C-5 propynylcytosine Derivative oligonucleotides, derivative oligonucleotides in which cytosine in the oligonucleotide is replaced by phenoxazine-modified cytosine, derivative oligonucleotides in which ribose in DNA is replaced by 2, -O-propylribose, and oligonucleotides Derivative oligonucleotides in which ribose in nucleotides is substituted with 2, -methoxyethoxy ribose are exemplified. Unless otherwise indicated! /, A particular nucleic acid sequence also has a conservatively modified version thereof (e.g., a degenerate codon substitution), similar to the explicitly indicated sequence. ) And complementary sequences are contemplated. Specifically, degenerate codon substitutions create a sequence in which the third position of one or more selected (or all) codons is replaced with a mixed base and a Z or deoxyinosine residue. (Batzer et al., Nucleic Acid Res. 19: 5081 (1991); Ohtsuka et al., J. Biol. Chem. 260: 2605- 2608 (1985); Rossolini et al., Mol Cell. Probes 8: 91- 98 (1994)

) o

As used herein, the term “complex molecule” refers to a molecule formed by linking a plurality of molecules such as polypeptides, polynucleotides, lipids, and sugars. Such complex molecules include, for example, Examples include, but are not limited to, glycolipids, glycopeptides, and the like. It is understood that the peptide of the present invention includes a complex in which an alkyl group, a PEG group, a sugar chain, and the like are bound.

 [0019] As used herein, an "isolated" biological agent (eg, a nucleic acid or protein) is defined as another biological agent in a cell of an organism in which the biological agent naturally exists. Factors (e.g., when it is a nucleic acid, a nucleic acid containing a factor other than the nucleic acid and a nucleic acid sequence other than the target nucleic acid; when it is a protein, it contains an factor other than the protein and an amino acid sequence other than the target protein) Protein or the like). "Isolated" nucleic acids and proteins include nucleic acids and proteins that have been purified by standard purification methods. Thus, isolated nucleic acids and proteins include chemically synthesized nucleic acids and proteins.

 [0020] As used herein, a "purified" biological agent (eg, a nucleic acid or a protein) is at least a part of a factor naturally associated with the biological agent. Means that has been removed. Thus, typically, the purity of the biological agent in the purified biological agent is higher (ie, more concentrated) than the state in which the biological agent is normally present.

 [0021] As used herein, "purified" and "isolated" preferably refer to at least 75% by weight, more preferably at least 85% by weight, even more preferably at least 95% by weight. %, And most preferably at least 98% by weight, of the same type of biological agent.

[0022] As used herein, "homology" of a gene (eg, a nucleic acid sequence, an amino acid sequence, etc.) refers to the degree of identity between two or more gene sequences. Thus, the higher the homology between two genes, the higher the identity or similarity between their sequences. Whether the two genes have homology can be determined by direct sequence comparison or, in the case of nucleic acids, by stringent conditions under stringent conditions. As used herein, the term “similarity” of a gene (eg, a nucleic acid sequence, an amino acid sequence, etc.) refers to two or more similarities when conservative substitutions are regarded as positive (identical) in the above homology. It refers to the degree of identity between gene sequences. Therefore, if there is a conservative substitution, Identity and similarity differ depending on the presence of When there is no conservative substitution, identity and similarity show the same numerical value.

 [0023] In the present specification, "amino acid" is used in a meaning commonly used in the art, and refers to an organic compound having a carboxyl group and an amino group. As used herein, amino acids may be natural or unnatural amino acids.

 [0024] As used herein, "natural amino acid" refers to the L-isomer of a natural amino acid. Natural amino acids include glycine, alanine, norin, leucine, isoleucine, serine, methionine, threonine, phenylalanine, tyrosine, tryptophan, cysteine, proline, histidine, aspartic acid, asparagine, glutamic acid, glutamine, γ-force Xyglutamic acid, arginine, ortin, and lysine. Unless otherwise indicated, all amino acids referred to in the present specification usually mean L-form, but the present invention is not limited thereto, and forms using D-form amino acids are also within the scope of the present invention. It is understood that.

 [0025] As used herein, the term "amino acid variant" refers to a molecule that is not a natural amino acid but is similar in physical properties and / or function to a natural amino acid. Examples of the modified amino acids include a hydrophobic group, phenylalanine having a benzyl side chain (para-, meta-, ortho-position, etc.) to which an alkyl group, a halo group, a nitro group, etc. are bonded, etyonine, canavanine , 2-methylglutamine and the like. It is understood that, in the present invention, amino acid variants may include unnatural amino acids and amino acid mimetics.

 [0026] As used herein, "unnatural amino acid" means an amino acid that is not normally found in nature in a protein. Examples of unnatural amino acids include norleucine, para-nitrophenylalanine, homophenylalanine, para-fluorophenylalanine, 3-amino-2-benzylpropionic acid, the D- or L-form of homoarginine, and D-phenylalanine. No.

 [0027] As used herein, the term "amino acid mimetic" refers to a compound that has a structure different from the general chemical structure of an amino acid, but that functions in a manner similar to a naturally occurring amino acid.

[0028] In the present specification, "nucleotide" may be a natural or non-natural one as long as it has an ability to encode an amino acid. [0029] As used herein, the term "fragment" refers to a polypeptide or polynucleotide having a sequence length of l to n-1 relative to a full-length polypeptide or polynucleotide (having a length of n). Refers to tide. It is understood that variants of the peptides of the invention also include such fragments. The length of the fragment can be appropriately changed depending on the purpose.For example, as a lower limit of the length, in the case of a polypeptide, 3, 4, 5, 6, 7, 8, 9, 10, 1, 5, 20, 25, 30, 40, 50 and more amino acids, and integer lengths not specifically listed herein (eg, 11) are also suitable as lower limits. possible. In the case of a polynucleotide, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 40, 50, 75, 100 and more nucleotides are mentioned, and specific listings are given here. Lengths expressed as unintended integers (eg, 11, etc.) may also be appropriate as lower limits. In the present specification, the lengths of polypeptides and polynucleotides can be represented by the numbers of amino acids or nucleic acids, respectively, as described above, but the above numbers are not absolute and have the same function. As far as it has, the above-mentioned number as an upper limit or adjustment is intended to include a few above and below (or, for example, 10% above and below) the number. In order to express such an intention, in this specification, "about" may be used before a number. However, it should be understood that the presence or absence of "about" herein does not affect the interpretation of that number.

 [0030] In the present specification, the "corresponding" amino acid or nucleic acid refers to an action similar to a predetermined amino acid in a polypeptide or polynucleotide serving as a reference for comparison with respect to a certain polypeptide molecule or polynucleotide molecule, respectively. Refers to an amino acid or nucleic acid predicted to have, or particularly to an enzyme molecule, an amino acid present at a similar position in an active site and making a similar contribution to catalytic activity. For example, an antisense molecule of a polynucleotide can be a similar portion of an ortholog corresponding to a particular portion of the antisense molecule. In the case of the peptide of the present invention, the ability of the specific sequence in human osteopontin to be used. Is understood.

[0031] As used herein, the term "corresponding" gene refers to, in a certain species, Refers to a gene that has or is expected to have the same action as the predetermined gene in the above, and when there are a plurality of genes having such an action, refers to those having the same evolutionary origin. Thus, the corresponding gene for a gene may be an ortholog or species homolog of that gene. Such corresponding genes can be identified using techniques well known in the art. Thus, for example, the corresponding gene in an animal can be obtained by using the sequence of the reference gene for the corresponding gene (for example, the mouse osteopontin gene) or the peptide of the present invention or its coding sequence itself as a query sequence (eg, It can be found by searching the sequence database of humans and rats.

 [0032] Amino acids may be referred to herein by either their commonly known three letter symbols or by the one letter symbols recommended by the IUPAC- IUB Biochemica 1 Nomenclature Commission. Nucleotides may also be referred to by the generally recognized one-letter code.

 [0033] The character codes are as follows.

 Amino acids

 3-letter symbol 1-letter symbol Meaning

 Ala A

 Cys C Sistine

 Asp D Asno laginic acid

 Glu E glutamic acid

 Phe F Hue-Lualanin

 Gly G glycine

 His H histidine

 He I isoleucine

 Lys K Lysine

 Leu L Leucine

 Met M Methionin

Asn N Pro P Proline

 Gin Q Glutamine

 Arg R Anoreginin

 Ser S Serine

 Thr T threonine

 Val V

 Trp w Tribute fan

 Tyr Y tyrosine

 Asx asparagine or aspartic acid

Glx gnoretamine or glutamic acid

Xaa Unknown or other amino acid.

Base

Symbol Meaning

a Adenine

g Guanin

 Cytosine

 Thymine

 Peracil

 Guanine or adenine purine Adenine or cytosine amino group

 Guanine or thymine Uracilketo group Guanine or cytosine

 Adenine or thymine Z ゥ racil

Guanine or cytosine or thymine ゥ lasinole adenine or guanine or thymine ゥ lasinole adenine or cytosine or thymine Z ゥ racil adenine or guanine or cytosine n Adenine or guanine or cytosine or thymine Z-racil, unknown or other base.

 [0034] In the present specification, the comparison of similarity, identity, and homology between amino acid sequences and base sequences is calculated using BLAST, a sequence analysis tool, with default parameters.

 [0035] The molecular biological techniques, biochemical techniques, and microbiological techniques used herein are well known and commonly used in the art, and are described, for example, in Sambrook J. et al. (, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor and its 3rd Ed. (2001); Ausubel, FM (1987) .Current Protocols in Molecular Biology, Greene Pub. AssociatESand Wiley—Inter science; Ausubel, FM (1989). ). short Protocols in Molecular Biology: A Compendium of Methods from Current Protocols in Molecular

Biology, Greene Pub. Associat ESand Wiley-Interscience; Innis, MA (1990) .PCR Protocols: A Guide to Methods and Applications, Academic Press; Ausubel, FM (1992) .Short Protocols in Molecular Biology: A Compendium of Methods from Current Protocols in Molecular Biology, Greene Pub. Associates; Ausubel, FM (1995) .Short

Protocols in Molecular Biology: A Compendium of Methods from Current Protocols in Molecular Biology, Greene Pub. Associates; Inn is, MA et al. (1995) .PCR Strategies, Academic Press; Ausubel, FM (1999). Short Protocols in Molecular Biology : A Compendium of

Methods from Current Protocols in Molecular Biology, Wiley, and annual updates; Sninsky, JJ et al. (1999) .PCR Applications: Protocols for Functional Genomics, Academic Press, Separate Investigator "Yodosha, 1997, etc., which are hereby incorporated by reference in their entirety (which may be all).

[0036] (Modification)

In the present invention, it is understood that variants also fall within the scope of the present invention. That's it In designing such modifications, the hydropathic index of amino acids can be considered. The importance of the hydrophobic amino acid index in conferring interactive biological functions on peptides is generally recognized in the art (Kyte. J and Doolittle, RFJ Mol. Biol. 157 (1): 105-). 132, 1982). The hydrophobic nature of amino acids contributes to the secondary structure of the resulting peptide, which in turn defines the interaction of the peptide with other molecules (eg, enzymes, substrates, receptors, DNA, antibodies, antigens, etc.). Each amino acid is assigned a hydrophobicity index based on its hydrophobicity and the nature of the charge. They are: isoleucine (+4.5); valine (+4.2); leucine (+3.8); hueralanine (+2.8); cysteine Z cystine (+2.5); methionine ( Alanine (+1.8); glycine (0.4); threonine (0.7); serine (0.8); tryptophan (0.9); tyrosine (1.1). 3); Proline (1-1.6); Histidine (1.3.2); Glutamic acid (13.5); Glutamine (13.5); Aspartic acid (13.5); Asparagine (1-3) 5); lysine (1-3.9); and arginine (1-4.5)).

 [0037] The ability to substitute one amino acid for another amino acid having a similar hydrophobicity index and still produce a peptide having a similar biological function (eg, a peptide equivalent in enzymatic activity) Are well known in the art. In such amino acid substitutions, the hydrophobicity index is preferably within ± 2, more preferably within ± 1, and even more preferably within ± 0.5. It is understood in the art that such substitution of amino acids based on hydrophobicity is efficient.

[0038] The hydrophilicity index can also be considered in designing polypeptides. As described in US Pat. No. 4,554,101, the following hydrophilicity indices have been assigned to amino acid residues: arginine (+3.0); lysine (+3.0); aspartic acid Glutamic acid (+ 3.0 ± 1); Serine (+0.3); Asparagine (+0.2); Glutamine (+0.2); Glycine (0); Threonine (+ 3.0 ± 1); Proline (0.5 ± 1); alanine (0.5); histidine (0.5); cysteine (1.1); methionine (1.3); valine ( I-1.5); Leucine (1-1.8); Isoloisin (1-1.8); Tyrosine (1-2.3); Fehlalanine (1-2.5); and Tryptophan (3.4). It is understood that an amino acid can be substituted for another that has a similar hydrophilicity index and still provide a bioisostere. In such amino acid substitutions, the hydrophilicity index is preferably within ± 2, more preferably within ± 1, and Even more preferably, it is within ± 0.5.

 [0039] In the present invention, "conservative substitution" refers to an amino acid substitution in which the hydrophilicity index or Z and hydrophobicity index of the original amino acid and the amino acid to be substituted are similar to those described above. And permutation. Examples of conservative substitution include, for example, those having a hydrophilicity index or a hydrophobicity index of 2 or less, preferably ± 1 or less, more preferably ± 0.5 or less. But not limited to them.

 [0040] In the present specification, conservative substitutions include, for example, hydrophobic amino acids (such as alanine, palin, leucine, and isoleucine), acidic amino acids (such as glutamic acid, aspartic acid, 4-carboxyglutamic acid, and aminocunic acid). It is understood that it can also be prepared by exchanging basic amino acids (arginine, histidine, lysine, etc.) with each other and aromatic amino acids (phenylalanine, tyrosine, tryptophan, etc.). Thus, examples of conservative substitutions are well known to those skilled in the art and include, for example, substitutions within each of the following groups: arginine and lysine; glutamic and aspartic acid; serine and threonine; glutamine and asparagine; Isoleucine, and the like, are not limited thereto.

[0041] In the present specification, the "variant" when referring to a polypeptide or polynucleotide is a substance in which a part of the substance such as the original polypeptide or polynucleotide is modified. Say. Such variants include substitutional variants, addition variants, deletion variants, truncated variants, allelic variants, and the like. Alleles refer to genetic variants that belong to the same locus and are distinct from each other. Therefore, “allelic variant” refers to a variant that has an allelic relationship to a certain gene. Such allelic variants usually have sequences identical or very similar to their corresponding alleles, and usually have rarely different biological May have activity. “Species homologue or homolog” means homology (preferably 60% or more homology, more preferably 80% or more) at the amino acid level or nucleotide level with a certain gene in a certain species. , 85% or more, 90% or more, 95% or more homology). Methods for obtaining such species homologs will be apparent from the description herein. "Ortholog" is an orthologue Also referred to as an orthologous gene! /,,, Are genes derived from speciation from a common ancestor that has two genes. For example, in the case of the hemoglobin gene family having a multigene structure, the human and mouse α-hemoglobin genes are orthologs.The human human hemoglobin gene and β-hemoglobin gene are paralogs (genes generated by gene duplication). . Orthologs are useful for estimating molecular phylogenetic trees. Orthologs of the present invention may also be useful in the present invention, as orthologs can usually perform the same function in another species as the original species.

As used herein, the term "conservative (modified) variant" applies to both amino acid sequences and nucleic acid sequences. With respect to a particular nucleic acid sequence, a conservatively modified variant refers to a nucleic acid that encodes the same or essentially the same amino acid sequence, and essentially the same if the nucleic acid does not encode an amino acid sequence. Sequence. Because of the degeneracy of the genetic code, many functionally identical nucleic acids encode any given protein. For example, the codons GCA, GCC, GCG, and GCU all encode the amino acid alanine. Thus, at every position where an alanine is specified by a codon, that codon can be changed to any of the corresponding codons described, without altering the encoded polypeptide. Such nucleic acid variation is a "silent modification (mutation)," which is one type of conservatively modified mutation. Every nucleic acid sequence herein which encodes a polypeptide also describes every possible silent variation of the nucleic acid. In the art, each codon in the nucleic acid (except AUG, which is usually the only codon for methionine, and TGG, which is usually the only codon for tryptophan) 1S Produces functionally identical molecules It will be understood that this can be modified. Accordingly, each silent variation of a nucleic acid which encodes a polypeptide is implicit in each described sequence. Preferably, such modifications may be made to avoid substitution of the amino acid cysteine, which greatly affects the conformation of the polypeptide. Examples of such base sequence modification methods include cleavage with a restriction enzyme or the like, ligation treatment with a DNA polymerase, Klenow fragment, DNA ligase, or the like, site-specific base substitution using a synthetic oligonucleotide, or the like. (Site-directed mutagenesis; Mark Zoller and Michael Smith, Methods in Enzymology, 100, 468-500 (1983)) Modifications can also be made by methods commonly used in the field of molecular biology.

 [0043] In the present specification, in order to produce a functionally equivalent peptide, in addition to amino acid substitution, amino acid addition, deletion, or modification can also be performed. Amino acid substitution refers to substitution of the original peptide with one or more, for example, 1 to 3 amino acids. The addition of an amino acid refers to the addition of one or more, for example, 1 to 3 amino acids to the original peptide chain. Amino acid deletion refers to the deletion of one or more, for example, 1 to 3 amino acids from the original peptide. Amino acid modifications include, but are not limited to, amidation, carboxylation, sulfation, halogenation, alkylation, glycosylation, phosphorylation, hydroxylation, acylation (eg, acetylation). The amino acid to be substituted or added may be a natural amino acid or a non-natural amino acid or an amino acid analog. Natural amino acids are preferred.

 [0044] As used herein, the term "substitution, addition or deletion" of a polypeptide or polynucleotide refers to an amino acid or its substitute, or a nucleotide, respectively, relative to an original polypeptide or polynucleotide. Or its substitute power means to be replaced, added or removed. Techniques for such substitution, addition or deletion are well known in the art, and examples of such techniques include site-directed mutagenesis techniques. The number of substitutions, additions, or deletions may be any number as long as the number is one or more. ) Can be much as long as For example, such a number can be one or several, and preferably can be within 20%, 10%, or 3 or less, 2 or less, 1 or less of the total length, etc. .

 [0045] (Organic chemistry)

 In the present specification, the term "hydrophobic group" is used as an index for determining hydrophobicity. The hydrophobicity index (Kyte and Doolittle, J. Mol. Biol., 157, 105-132 (1982)) Also, any substituent that increases the logP (1-octanol—the logarithm of the distribution coefficient of water) from the group before substitution. Examples of such a hydrophobic group include, but are not limited to, an alkyl group and an alkyl group.

Hereinafter, organic substituents that may be contained in the compound of the present invention will be described. [0047] As used herein, the term "alkyl" refers to a monovalent group generated by the loss of one hydrogen atom in an aliphatic hydrocarbon (alkane) such as methane, ethane, and propane. CH

 n 2n + l is represented by one (where n is a positive integer). Alkyl can be straight or branched. As used herein, the term "substituted alkyl" refers to an alkyl in which H of the alkyl is substituted by a substituent defined below. Examples of these are C1-C2 alkyl, C1-C3 alkyl, C1-C4 alkyl, C1-C5 alkyl, C1-C6 alkyl, C1-C7 alkyl, C1-C8 alkyl, C1-C9 alkyl, C1-C10 alkyl C1-C12 alkyl or C1-C12 alkyl, C1-C2-substituted alkyl, C1-C3-substituted alkyl, C1-C4-substituted alkyl, C1-C5-substituted alkyl, C1-C6-substituted Alkyl, C1-C7 substituted alkyl, C1-C8 substituted alkyl, C1-C9 substituted alkyl, C1-C10 substituted alkyl, C1-C11 substituted alkyl or C1-C12 substituted alkyl. Or alkyl. Here, for example, C 1 -C 10 alkyl means a linear or branched alkyl having 1 to 10 carbon atoms, such as methyl (CH—), ethyl (CH-1), n-propyl (CH CH CH—), iso

 3 2 5 3 2 2 Propyl ((CH) CH-), n-butyl (CH CH CH CH-), n-pentyl (CH C

 3 2 3 2 2 2 3

H CH CH CH 1), n-hexyl (CH CH CH CH CH CH 1), n-heptyl

2 2 2 2 3 2 2 2 2 2

 (CH CH CH CH CH CH CH-), n-octyl (CH CH CH CH CH CH CH C

3 2 2 2 2 2 2 3 2 2 2 2 2

H CH—), n—nonyl (CH CH CH CH CH CH CH CH CH—), n—decyl

2 2 3 2 2 2 2 2 2 2 2

 (CH CH CH CH CH CH CH CH CH CH I), C (CH) CH CH CH (C

3 2 2 2 2 2 2 2 2 2 3 2 2 2

H), —CH CH (CH) and the like. Also, for example, C1-C10 substituted

3 2 2 3 2

 Alkyl is C1-C10 alkyl in which one or more hydrogen atoms have been replaced by substituents.

 [0048] In the present specification, "optionally substituted alkyl" means that "alkyl" or "substituted alkyl" as defined above may be different from the above.

 [0049] As used herein, the term "alkylene" refers to a divalent group formed by the loss of two hydrogen atoms in an aliphatic hydrocarbon (alkane) such as methylene, ethylene, and propylene. — CH — (where n is a positive integer). Alkylene is straight chain or branched n 2n

Can be a chain. As used herein, the term "substituted alkylene" refers to a substituent defined below. Alkylene in which H of alkylene is substituted by a group. Specific examples of these include Cl-C2 alkylene, C1-C3 alkylene, C1-C4 alkylene, C1-C5 alkylene, C1-C6 alkylene, C1-C7 alkylene, C1-C8 alkylene, C1-C9 alkylene, C1-C10 Alkylene, 1 to 11 alkylene or 1 to 12 alkylene, C1 to C2 substituted alkylene, C1 to C3 substituted alkylene, C1 to C4 substituted alkylene, C1 to C5 substituted alkylene, C1-C6-substituted alkylene, C1-C7-substituted alkylene, C1-C8-substituted alkylene, C1-C9-substituted alkylene, C1-C10-substituted alkylene, C1-C11-substituted It can be alkylene or C1-C12 substituted alkylene. Here, for example, C1-C10 alkylene means a straight-chain or branched alkylene having 1 to LO carbon atoms, and methylene (1-CH-),

 2 Ethylene (one CH—), n propylene (one CH CH CH one), isopropylene (one CH

 2 4 2 2 2 3

) C-), n-butylene (one CH CH CH CH one), n-pentylene (one CH CH CH C

2 2 2 2 2 2 2 2

H CH-one), n-hexylene (one CH CH CH CH CH CH one), n-heptylene (one

2 2 2 2 2 2 2 2

 CH CH CH CH CH CH CH CH) n-octylene (CH CH CH CH CH CH C

2 2 2 2 2 2 2 2 2 2 2 2

H CH CH one), n-nonylene (one CH CH CH CH CH CH CH CH CH —), n

2 2 2 2 2 2 2 2 2 2 2 2 Decylene (one CH CH CH CH CH CH CH CH CH CH one), CH C (CH)

 2 2 2 2 2 2 2 2 2 2 2 3

— And the like. Also, for example, C1-C10 substituted alkylene refers to C1-C1

2

 O alkylene, of which one or more hydrogen atoms are substituted with a substituent. As used herein, “alkylene” may include one or more atoms selected from an oxygen atom and a sulfur atom.

[0050] In the present specification, the term "substituted or alkylene" may be a shift of "alkylene" or "substituted alkylene" defined above. means.

[0051] In the present specification, "cycloalkyl" refers to alkyl having a cyclic structure. “Substituted cycloalkyl” refers to cycloalkyl in which H of cycloalkyl is replaced by a substituent defined below. Specific examples include C3-C4 cycloalkyl, C3-C5 cycloalkyl, C3-C6 cycloalkyl, C3-C7 cycloalkyl, C3-C8 cycloalkyl, C3-C9 cycloalkyl, C3-C10 cycloalkyl, C3-C11 Cycloalkyl, C3-C12 cycloalkyl, C3-C4-substituted cycloalkyl, C3-C5-substituted Cycloalkyl, C3-C6-substituted cycloalkyl, C3-C7-substituted cycloalkyl, C3-C8-substituted cycloalkyl, C3-C9-substituted cycloalkyl, C3-C10-substituted cycloalkyl , C3-C11 substituted cycloalkyl or C3-C12 substituted cycloalkyl. For example, examples of cycloalkyl include cyclopropyl and cyclohexyl.

[0052] In the present specification, the "optionally substituted cycloalkyl" means that the "cycloalkyl" or the "substituted cycloalkyl" defined above may be shifted. You.

 [0053] In the present specification, "alkenyl" refers to a monovalent group generated by the loss of one hydrogen atom from an aliphatic hydrocarbon having one double bond in the molecule, and is generally represented by CH1.

 n 2n- l (where n is a positive integer greater than or equal to 2). The term "substituted alkenyl" refers to an alkenyl in which H of the alkenyl is substituted by a substituent defined below. Specific examples include C2-C3 anore keninole, C2-C4 anore keninore, C2-C5 anore keninore, C2-C6 alkalle, C2-C7 alkalle, C2-C8 alkalle, C2-C9 alkalle, C2-C10 scale, J2-J11 scale or J2-J12 scale, C2-C3-substituted scale, C2-C4-substituted scale, C2-C5 Substituted alkyl, C2-C6 substituted alkyl, C2-C7 substituted alkyl, C2-C8 substituted alkyl, C2-C9 substituted alkyl, C2-C10 It may be a substituted alkyl, a C2-C11 substituted alkyl, or a C2-C12 substituted alkyl. Here, for example, C2-C10 alkyl means a straight-chain or branched alkaryl containing 2 to L0 carbon atoms, such as butyl (CH = CH), aryl (CH = CHCH-), CH C

 2 2 2 3

H = CH is exemplified. In addition, for example, C2-C10 substituted alkyl is

C2 to C10 alkyl, in which one or more hydrogen atoms have been replaced by substituents.

[0054] In the present specification, the term "substituted or optionally substituted" refers to the above-defined

This means that the deviation of the "-" or "substituted calendar" may be different.

[0055] In the present specification, "alkenylene" refers to a divalent group formed by losing two hydrogen atoms from an aliphatic hydrocarbon having one double bond in the molecule, and generally a CH 1 so

n 2n-2 (Where n is a positive integer greater than or equal to 2). The term "substituted alkelenes" refers to alkelenes in which the H of the alkene is substituted with a substituent as defined below. Specific examples include C2-C25 alkylenes and C2-C25-substituted alkylenes, particularly C2-C3 alkylenes, C2-C4 alkylenes, C2-C5 alkylenes. , C2-C6 alkalene, C2-C7 alkalene, C2-C8 alkalene, C2-C9 alkalene, C2-C10 alkalene, 2-11 alkalene or 2-12 Alkalene, C2-C3-substituted alkelenes, C2-C4-substituted alkelenes, C2-C5-substituted alkelenes, C2-C6-substituted alkelenes, C2-C7 substituted Alkalene, C2-C8 substituted alkene, C2-C9 substituted alkene, C2-C10 substituted alkene, C2-C11 substituted alkene or C2-C12 substituted Preferred is alkenylene. Here, for example, C2-C10 alkyl means a straight-chain or branched alkylene containing 2 to 10 carbon atoms, -CH = CH-, one CH = CHCH-,-(CH) C = CH— and the like. Also examples

 twenty three

 For example, C2-C10 substituted alkylene refers to C2-C10 alkylene, in which one or more hydrogen atoms are substituted with a substituent. As used herein, “alkene” includes one or more atoms that are also selected from oxygen and sulfur nuclear! / ヽ.

 [0056] In the present specification, the "optionally substituted alkenylene" may be a deviation from the above defined "alkylene" or "substituted alkenylene". Means

[0057] In the present specification, the term "cycloalkyl" refers to an alkyl having a cyclic structure. The “substituted cycloalkyl” refers to a cycloalkyl in which H of the cycloalkyl is substituted by a substituent defined below. Specific examples include C3-C4 cycloalkyl, C3-C5 cycloalkyl, C3-C6 cycloalkyl, C3-C7 cycloalkyl, C3-C8 cycloalkyl, C3-C9 cycloalkyl, C3-C10 Cycloalkyl, C3-C11 cycloalkyl, C3-C12 cycloalkyl, C3-C4-substituted cycloalkyl, C3-C5-substituted cycloalkyl, C3-C6-substituted cycloalkyl, C3 -C7 substituted cycloalkyl, C3-C8 substituted cycloalkyl, C3-C9 substituted cycloalkyl, C3-C10 substituted cycloalkyl , A 3- to 11-substituted cycloalkyl or a 3- to 12-substituted cycloalkyl. For example, preferred cycloalkyls include 1-cyclopentyl, 2-cyclohexyl and the like.

[0058] In the present specification, the "cycloalkyl which may be substituted" may be a shift of the "cycloalkyl" or the "substituted cycloalkyl" as defined above. Means

 [0059] As used herein, the term "alkynyl" refers to a monovalent group such as acetylene formed by the loss of one hydrogen atom from an aliphatic hydrocarbon having one triple bond in the molecule. Generally represented by CH (where n is a positive integer greater than or equal to 2). "Substituted al n 2n_3

 “Kyl” refers to an alkyl in which H of the alkyl is substituted by a substituent defined below. Specific examples include C2-C3 alkyl, C2-C4 alkyl, C2-C5 alkyl, C2-C6 alkyl, C2-C7 alkyl, C2-C8 alkyl, C2- C9 alkyl, C2-C10 alkyl, C2-C11 alkyl, C2-C12 alkynyl, C2-C3-substituted alkyl, C2-C4-substituted alkyl, C2-C5-substituted Alkyl, C2-C6-substituted alkyl, C2-C7-substituted alkyl, C2-C8-substituted alkyl, C2-C9-substituted alkyl, C2-C10-substituted Or a C2-C11 substituted alkyl or a C2-C12 substituted alkyl. Here, for example, C2-C10 alkyl means, for example, a linear or branched alkyl containing 2 to 10 carbon atoms, such as ethur (CH≡C—), 1 propynyl ( CH C≡C). Also, for example, C2-C10 substituted alkyl

 Three

 Nyl refers to C2-C10 alkyl in which one or more hydrogen atoms are substituted with a substituent.

[0060] As used herein, the term "substituted or optionally, alkyl" is defined as "alkoxy" as defined above.

This means that the difference between “-alkyl” and “substituted alkyl” may be different.

[0061] In the present specification, "alkoxy" refers to a monovalent group generated by losing a hydrogen atom of a hydroxy group of an alcohol, and is generally represented by CHO (where n is 1 or more).

 n 2n + l

Integer). “Substituted alkoxy” refers to alkoxy in which H of the alkoxy is substituted by a substituent defined below. Specific examples include C1-C2 alkoxy, C1 ~ C3 alkoxy, C1 ~ C4 alkoxy, C1 ~ C5 alkoxy, C1 ~ C6 alkoxy, C1 ~ C7 alkoxy, C1 ~ C8 alkoxy, C1 ~ C9 alkoxy, C1 ~ C10 alkoxy, C1 ~ C11 alkoxy, C1 ~ C12 alkoxy, C1-C2-substituted alkoxy, C1-C3-substituted alkoxy, C1-C4-substituted alkoxy, C1-C5-substituted alkoxy, C1-C6-substituted alkoxy, C1-C7-substituted alkoxy, C1 It may be -C8-substituted alkoxy, C1-C9-substituted alkoxy, C1-C10-substituted alkoxy, C1-C11-substituted alkoxy or C1-C12-substituted alkoxy. Here, for example, C1-C10 alkoxy means a linear or branched alkoxy containing 1 to L0 carbon atoms, such as methoxy (CHO), ethoxy (CHO), n-propyl.

 3 2 5

 Mouth oxy (CH CH CH O) and the like are exemplified.

 3 2 2

 [0062] In the present specification, "optionally substituted alkoxy" means that the "alkoxy" or "substituted alkoxy" defined above may be shifted.

 [0063] As used herein, the term "heterocycle (group)" refers to a group having a cyclic structure including carbon and hetero atoms. Here, the hetero atom is selected from the group consisting of 0, S, and N forces, and may be the same or different, may contain one, or may contain two or more. Heterocyclic groups can be aromatic or non-aromatic, and can be monocyclic or polycyclic. Heterocyclic groups may be substituted.

 As used herein, the term “substituted or optionally a heterocycle (group)” refers to a “heterocyclic ring (group)” or a “substituted heterocycle (group)” as defined above. Base) ”means that it may be misaligned.

[0065] In the present specification, "alcohol" refers to an organic compound in which one or more hydrogen atoms of an aliphatic hydrocarbon are substituted with a hydroxyl group. In this specification, it is also referred to as ROH. Here, R is an alkyl group. Preferably, R may be C1-C6 alkyl. Examples of the alcohol include, but are not limited to, methanol, ethanol, 1-propanol, 2-prono-Vole and the like.

[0066] In the present specification, the term "carbocyclic group" refers to a group containing a cyclic structure containing only carbon, and includes the above-mentioned "cycloalkyl", "substituted cycloalkyl", "cycloalkyl" And "substituted cycloalkyl". Carbocyclic groups can be aromatic or non-aromatic And can be monocyclic or polycyclic. The term “substituted carbocyclic group” refers to a carbocyclic group in which H of the carbocyclic group is substituted by a substituent defined below. Specific examples include C3-C4 carbocyclic group, C3-C5 carbocyclic group, C3-C6 carbocyclic group, C3-C7 carbocyclic group, C3-C8 carbocyclic group, C3-C9 carbocyclic group, C3-C10 Carbocyclic group, C3-C11 carbocyclic group, C3-C12 carbocyclic group, C3-C4-substituted carbocyclic group, C3-C5-substituted carbocyclic group, C3-C6-substituted carbocyclic group, C3- C7-substituted carbocycle, C3-C8-substituted carbocycle, C3-C9-substituted carbocycle, C3-C10-substituted carbocycle, C3-C11-substituted carbocycle or C3 It may be a -C12 substituted carbocyclic group. The carbocyclic group can also be a C4-C7 carbocyclic group or a C4-C7 substituted carbocyclic group. Examples of the carbon ring group include one in which one phenyl group hydrogen atom is deleted. Here, the hydrogen deletion position may be any position that is chemically possible, whether on an aromatic ring or on a non-aromatic ring.

 [0067] As used herein, the term "substituted or optionally a carbocyclic group" refers to a deviation from the above-defined "carbocyclic group" or "substituted carbocyclic group". It also means.

[0068] In the present specification, the term "heterocyclic group" refers to a group having a cyclic structure including carbon and hetero atoms. Here, the heteroatom is selected from the group consisting of 0, S, and N forces, and may be the same or different, and may include one or two or more. Heterocyclic groups can be aromatic or non-aromatic, and can be monocyclic or polycyclic. “Substituted heterocyclic group” refers to a heterocyclic group in which H of the heterocyclic group is substituted by a substituent defined below. Specific examples include C3-C4 carbocyclic group, C3-C5 carbocyclic group, C3-C6 carbocyclic group, C3-C7 carbocyclic group, C3-C8 carbocyclic group, C3-C9 carbocyclic group, C3-C10 Carbocyclic group, C3-C11 carbocyclic group, C3-C12 carbocyclic group, C3-C4-substituted carbocyclic group, C3-C5-substituted carbocyclic group, C3-C6-substituted carbocyclic group, C3 -C7 substituted carbocyclic group, C3-C8 substituted carbocyclic group, C3-C9 substituted carbocyclic group, C3-C10 substituted carbocyclic group, J3-J11 substituted carbocycle It may be a group or a 3- to C12-substituted carbocyclic group in which one or more carbon atoms have been substituted with a heteroatom. The heterocyclic group may also be a C4-C7 carbocyclic group or a C4-C7 substituted carbocyclic group in which one or more carbon atoms have been substituted with one or more heteroatoms. As a heterocyclic group, Examples thereof include a phenyl group, a pyrrolyl group, a furyl group, an imidazolyl group, and a pyridyl group. The position of deletion of hydrogen may be any position that is chemically possible, and may be on an aromatic ring or on a non-aromatic ring.

 [0069] In the present specification, a carbocyclic group or a heterocyclic group may be substituted with a divalent substituent in addition to being substituted with a monovalent substituent as defined below. Such a divalent substitution can be an oxo substitution (= O) or a thioxo substitution (= S).

 [0070] In this specification, "halogen" refers to a monovalent group of elements such as fluorine (F), chlorine (C1), bromine (Br), and iodine (I) belonging to Group 7B of the periodic table. Say.

 [0071] In the present specification, "hydroxy" refers to a group represented by OH. The term “substituted hydroxy” refers to a compound in which H of hydroxy is substituted by a substituent defined below.

 [0072] In the present specification, "thiol" is a group in which an oxygen atom of a hydroxy group is substituted with a sulfur atom (mercapto group), and is represented by SH. The term “substituted thiol” refers to a group in which H of mercapto is substituted with a substituent defined below.

 [0073] In the present specification, "cyano" refers to a group represented by -CN. "Nitro" means -NO

 Refers to the group represented by 2. "Amino" means -NH

 Refers to the group represented by 2. The term "substituted amino" refers to an amino in which H of the amino is substituted with a substituent as defined below.

[0074] In the present specification, "carboxy" refers to a group represented by -COOH. "Substituted carboxy" refers to carboxy in which H is substituted with a substituent as defined below.

[0075] In the present specification, "thiocarboxy" refers to a group in which an oxygen atom of a carboxy group is substituted with a sulfur atom, and is represented by C (= S) OH, —C (= 0) SH or —CSSH. Can be represented. “Substituted thiocarboxy” refers to thiocarboxy in which H is substituted with a substituent as defined below.

 [0076] As used herein, the term "acyl" refers to a monovalent group formed by removing OH from a carboxylic acid. Representative examples of acetyl groups include acetyl (CH 2 CO 3) and benzoyl (CH 2 CO 3)

 3 6 5

 Is mentioned. “Substituted acyl” refers to the hydrogen of an acyl substituted with a substituent as defined below.

[0077] As used herein, the term "amide" refers to a group in which hydrogen of ammonia is substituted with an acid group (acyl group). And preferably represented by -CONH. "Substituted amide" refers to an amide substituted

 2

 Means

 [0078] In the present specification, the term "carbon" refers to one (C) which is a characteristic group of aldehyde and ketone.

 = o). "Substituted carbonyl" refers to a carbonyl group substituted with a substituent selected as described below.

 [0079] In the present specification, "thiocarbol" is a group in which an oxygen atom in carbonyl is substituted by a sulfur atom, and includes a characteristic group-(C = S)-. Thiocarbol includes thioketone and thioaldehyde. "Substituted thiocarbol" means thiocarbonyl substituted with a substituent selected as described below.

 [0080] In this specification, "sulfol" is a generic term for a substance containing SO 2 as a characteristic group.

 2

 Means “Substituted sulfol” means a sulfol substituted with a substituent selected below.

 [0081] In the present specification, "sulfiel" is a generic term for a substance containing SO- which is a characteristic group. "Substituted sulfiel" means sulfiel which has been replaced by a substituent selected below.

 [0082] In the present specification, "aryl" refers to a group formed by the removal of one hydrogen atom bonded to the ring of an aromatic hydrocarbon, and is included in the present specification as a carbocyclic group.

 [0083] In this specification, unless otherwise specified, the term "substitution" refers to replacement of one or more hydrogen atoms in an organic compound or a substituent with another atom or atomic group. It is also possible to remove one hydrogen atom and substitute with a monovalent substituent, and it is also possible to remove two hydrogen atoms and substitute with a divalent substituent.

 [0084] In this specification, unless otherwise specified, the term "substitution" refers to replacing one or more hydrogen atoms in an organic compound or a substituent with another atom or atomic group. It is also possible to remove one hydrogen atom and substitute with a monovalent substituent, and it is also possible to remove two hydrogen atoms and substitute with a divalent substituent.

[0085] Examples of the substituent in the present invention include alkyl, cycloalkyl, alkenyl, cycloalkyl, alkyl, alkoxy, carbocyclic group, heterocyclic group, halogen, hydroxy, thiol, nitro nitro, Carboxy with ami, rubamoyl, asil, asilamino, chio Examples include, but are not limited to, carboxy, amide, substituted carboyl, substituted thiocarbol, substituted sulfonyl or substituted sulfinyl, or any inorganic substituent (e.g., a silicon-containing substituent). Not limited. In the present invention, such substituents can be appropriately used in designing linkers and biomolecules.

 [0086] Preferably, when a plurality of substituents are present, each may independently be a hydrogen atom or an alkyl or any inorganic substituent (for example, a silicon-containing substituent). It cannot be a hydrogen atom. More preferably, independently, when there are a plurality of substituents, each may be independently selected from the group consisting of hydrogen and a C1-C6 alkyl group or any inorganic substituent (eg, a silicon-containing substituent). The substituents may all have a substituent other than hydrogen, but preferably have at least one hydrogen, more preferably 2 to n (where n is the number of substituents) hydrogen. Can have. It may be preferable that the number of hydrogen atoms in the substituent is large. This is because a large substituent or a polar substituent may impair the effect (binding property) of the present invention. Accordingly, the substituents other than hydrogen are preferably C1-C6 alkyl, C1-C5 alkyl, C1-C4 alkyl, C1-C3 alkyl, C1-C2 alkyl, methyl or any inorganic substituent (e.g., (Containing substituent). However, it may be preferable to have a large substituent since the effect of the present invention may be enhanced.

 [0087] In the present specification, Cl, C2, ..., Cn represent the number of carbon atoms. Accordingly, C1 is used to represent a substituent having one carbon atom.

 [0088] As used herein, the term "optical isomer" refers to one or a pair of non-superimposable compounds in which the structure of the crystal or molecule is a mirror image. It is a form of stereoisomer that differs only in optical rotation, despite other properties being the same. In the present invention, those having high optical isomer purity can be preferably used.

 [0089] As used herein, the term "protection reaction" refers to a reaction in which a protecting group such as Boc is added to a functional group for which protection is desired. By protecting the functional group with the protecting group, the reaction of the functional group having higher reactivity can be suppressed, and only the functional group having lower reactivity can be reacted. The protection reaction can be performed, for example, by a dehydration reaction.

[0090] As used herein, the term "deprotection reaction" refers to a reaction for eliminating a protecting group such as Boc. . Examples of the deprotection reaction include a reaction such as a reduction reaction using PdZC. The deprotection reaction can be performed, for example, by hydrolysis.

 [0091] In the present specification, "protecting group" includes, for example, a fluorenylmethoxycarbol (Fmoc) group, an acetyl group, a benzyl group, a benzoyl group, a t-butoxycarbol group, t-butyldimethyl group, silyl group, trimethylsilylethyl group, N-phthalimidyl group, trimethylsilylethyloxycarbonyl group, 2-tro-5,5-dimethoxybenzyl group, 2-nitro-4,5-dimethoxy Typical protective groups include a benzyloxycarbol group and a sulfamate group. Protecting groups can be used to protect moieties that are not involved in binding in biomolecules. The protecting group can be used, for example, to protect a reactive functional group such as an amino group and a carboxyl group. Various protecting groups can be used depending on the reaction conditions and purpose. The protecting group for the hydroxy group includes acetyl, benzyl, silyl and derivatives thereof, and the protecting group for the amino group includes acetyl, benzyloxycarbyl, t-butoxycarbol and the like. Inductors and the like can be used. As the protecting group for the aminooxy group and the N-alkylaminooxy group, a trimethylsilylethyloxycarboxy group, a 2-toro-4,5 dimethoxybenzyloxycarboxy group or a derivative thereof is preferable.

 [0092] In each method of the present invention, the target product is obtained by removing contaminants (unreacted weight loss, by-products, solvent, etc.) from the reaction solution by a method commonly used in the art (for example, extraction, distillation, After removal by washing, concentration, precipitation, filtration, drying, etc.), isolation by a combination of post-treatment methods commonly used in the art (eg, adsorption, elution, distillation, precipitation, precipitation, chromatography, etc.). obtain.

 [0093] (Angiogenic activity)

 As used herein, the term "angiogenesis" refers to the formation of new blood vessels and the activity of such formation.

 [0094] As used herein, the term "vascular network formation" refers to the formation of new blood vessels or existing vascular forces and the activity of forming such networks! , U.

[0095] As used herein, the vascular network forming ability is determined by the angiogenesis index and the length of the newly formed blood vessel, or the vascular network forming ability or the vascular network forming index. Indicated by Angiogenesis can be determined by observing whether the reticulated force has formed (e.g., the bifurcation of a blood vessel joining more blood vessels and increasing the number of its junctions). For simplicity, the vascular network formation index is calculated as follows in this specification.

 [0096] First, the number of new blood vessels is counted. The count is determined by the number of blood vessels per 0.79 cm. Blood

 2

 When the number of tubes is the following number, it is converted to the right score.

 0: 0

 1 to: LO: l

 11-20: 2

 21-30: 3

 31-40: 4

 41 or more: 5

 Next, the length of the new blood vessel is determined. The length is measured as follows. Observation was made with a stereomicroscope (Olympus, SZX12, Japan). The obtained image was read by Photoshop (registered trademark) (Adobe, Japan). Count in pixels.

 Less than 100: 1

 100 to less than 125: 2

 125 to less than 150: 3

 150 to less than 175: 4

 175 to less than 200: 5

 More than 200: 6.

 [0097] Since both of these can be regarded as having a good score! And those having a high vascular network forming ability, it can be considered that the product thereof is used to evaluate the vascular network forming ability. Monkey

[0098] Regarding the ability to form a network, the state of angiogenesis in a tissue can be observed with a stereoscopic microscope (Olympus, SZX12, Japan). The obtained image is read by Photoshop (registered trademark) (Adobe, Japan) and the network ability is scored as follows. The following scores are referred to herein as “vascular network forming ability” or “vascular network forming ability”. Also called "index" or simply "network index!"

[0099] Nwl: Network formation Although angiogenesis is observed as described above, each new blood vessel is a single fe.

 [0100] Nw2: In the middle stage of network formation, each new blood vessel is in a state in which a side branch on a ladder is engaged.

 [0101] Nw3: The network is in the final stage, and the ladder upper branch has more side branches.

[0102] Nw4: Network maturation stage, showing a wide range of neovascular plexus.

[0103] As demonstrated in the present invention, the ability to form a vascular network usually means such a vascular network formation index force of at least 2, preferably 2.5 or more, and more preferably 2.5 or more. It is understood that it is preferably 3 or more, more preferably 3.5 or more.

 [0104] The term "blood vessel" in the present specification is used in a sense commonly used in the art, and includes not only ordinary arteries and veins, but also capillaries.

 [0105] As used herein, the term "cell adhesion molecule" or "adhesion molecule" is used interchangeably and refers to the approach of two or more cells to each other (cell adhesion) or the relationship between a substrate and a cell. Refers to molecules that mediate adhesion between Generally, a molecule involved in cell-cell adhesion (cell-cell adhesion) (cell-cell adhesion molecule) and a molecule involved in cell-extracellular matrix adhesion (cell-substrate adhesion) (cell-substrate adhesion molecule) Divided into In the present invention, any molecule is useful and can be used effectively. Therefore, in the present specification, the cell adhesion molecule includes a protein on the substrate side during cell-substrate adhesion, but in the present specification, a protein on the cell side (for example, integrin) is also included, and other than the protein, Even a molecule, as long as it mediates cell adhesion, falls within the concept of a cell adhesion molecule or cell adhesion molecule herein.

 [0106] Regarding cell-cell adhesion, cadherin, many molecules belonging to the immunoglobulin superfamily (NCAM, Ll, ICAM, facyclin Π, ΙΠ, etc.), selectins, and the like are known, and cell membranes are formed by unique molecular reactions. It is also known to combine

[0107] On the other hand, the major cell adhesion molecule that works for cell-substrate adhesion is integrin, which recognizes and binds to various proteins contained in the extracellular matrix. These cell adhesion molecules Are all located on the cell membrane surface and can be regarded as a type of receptor (cell adhesion receptor). Accordingly, such receptors located on the cell membrane can also be used in the tissue pieces of the present invention. Such receptors include, but are not limited to, for example, α integrin, j8 intedarin, CD44, syndecan, and aggrecan.

[0108] In the present specification, extracellular matrix molecules (cell adhesion proteins such as fibronectin and laminin), which are binding partners such as integrins, are also included in the category of cell adhesion molecules. The function of each adhesion receptor in cell-cell adhesion and cell-substrate adhesion is not strict, and varies depending on the distribution of partner molecules (ligands). For example, some integrins are also involved in cell-cell adhesion, such as adhesion between blood cells. It is also known that when growth factors, cytokins, and the like are present as cell membrane proteins, they react with their receptors distributed on other cells and consequently adhere the cells. Such growth factors and cytokins can also be used as biomolecules contained in the tissue piece of the present invention.

 [0109] Since a wide variety of molecules are involved in cell adhesion as described above and their functions are different, those skilled in the art may appropriately determine the molecules to be included in the tissue piece of the present invention according to the purpose. You can choose. Techniques relating to cell adhesion are well known in addition to those described above, and are described, for example, in Extracellular Matrix Clinical Application-Medical Review.

[0110] Whether a molecule is a cell adhesion molecule can be determined by biochemical quantification (SDS-PAG method, labeled collagen method), immunological quantification (enzyme antibody method, fluorescent antibody method, immunohistological examination) PD The determination can be made by determining a positive result in an assay such as the R method or the hybridization method. Such cell adhesion molecules include collagen, integrin, fibronectin, laminin, vitronectin, fibrinogen, the immunoglobulin parfamily (e.g., CD2, CD4, CD8, ICM1, ICAM2, VCAM1), selectin, cadherin, and the like. But not limited thereto. Many of these cell adhesion molecules transmit auxiliary signals for cell activation by cell-cell interaction simultaneously with cell adhesion. Therefore, the adhesion factors used in the tissue piece of the present invention include Those that transmit such an auxiliary signal for cell activation into a cell are preferred. This is because, after being applied to an injured site in a certain tissue or organ as a tissue piece, cell activation can promote the proliferation of cells aggregated therein and / or cells in the tissue or organ. Whether such an auxiliary signal can be transmitted into cells is determined by biochemical quantification (SDS-PAGE, labeled collagen method), immunological quantification (enzyme-linked immunosorbent assay, immunofluorescent assay, immunohistochemical assay). Investigation) It can be determined by determining positive in the PDR method and the hybridization method.

 [0111] Examples of the cell adhesion molecule include cadherin, which is widely known as a cell adhesion molecule in tissue-fixing cell lines, and cadherin can be used in a preferred embodiment of the present invention. On the other hand, in cells of the non-fixed blood 'immune system, examples of cell adhesion molecules include immunoglobulin superfamily molecules (CD2, LFA-3, ICAM-1, CD2, CD4, CD8, ICM1, ICAM2, VCAM1). Integrin family molecules (LFA-1, Mac-1, gpllbllla, pl50, 95, VLA1, VLA2, VLA3, VLA4, VLA5, VLA6, etc.); selectin family molecules (L-selectin, E-selectin, P— Selectin) and the like, but are not limited thereto. Thus, such molecules may be particularly useful for treating tissues or organs of the blood-immune system.

 [0112] In order for non-fixed cells to work in a specific tissue, the cell adhesion molecule needs to adhere to that tissue. In this case, it is thought that the adhesion between cells is gradually strengthened by the primary adhesion by the constantly expressed selectin molecule and the like, followed by the secondary adhesion of the activated integrin molecule and the like. Therefore, as a cell adhesion molecule used in the present invention, such a factor that mediates primary adhesion, a factor that mediates secondary adhesion, or both may be used together.

[0113] As used herein, "cell adhesive protein" refers to a protein having a function of mediating cell adhesion as described above. Therefore, in the present specification, the cell adhesive protein includes a protein on the substrate side during cell-substrate adhesion, but herein also includes a protein on the cell side (eg, integrin). For example, when cultured cells are seeded under serum-free conditions on a substrate (glass or plastic) to which the protein on the substrate side has been adsorbed, the integrin, a receptor, recognizes the cell adhesion protein and the cells Glue to the quality. The active site of the cell adhesion protein has been elucidated at the amino acid level, and RGD, YIGSR and the like are known (these are also collectively referred to as the RGD sequence). Thus, in one preferred embodiment, the protein contained in the tissue piece of the present invention may advantageously include an RGD sequence such as RGD, YIGSR. Normally, cell adhesion proteins are present in extracellular matrices, cultured cell surfaces, and in plasma 'serum' various body fluids. Its functions in vivo include not only the adhesion of cells to the extracellular matrix, but also the movement, proliferation, morphology, and tissue construction of cells. Apart from cellular actions, some proteins exhibit a function of regulating blood coagulation and complement action. In the present invention, proteins having such functions may also be useful. Examples of such cell adhesion proteins include, but are not limited to, fibronectin, collagen, vitronectin, laminin, and the like.

 [0114] As used herein, the term "RGD molecule" refers to a protein molecule containing the amino acid sequence RGD (Arg-Gly-Asp) or a functionally identical sequence thereof. The RGD molecule is characterized by containing RGD, which is an amino acid sequence useful as an amino acid sequence of a cell adhesion active site of a cell adhesion protein, or another amino acid sequence which is functionally equivalent. The RGD sequence was discovered as a cell adhesion site of fibronectin, and was later found on a number of molecules exhibiting cell adhesion activities, such as type I collagen, laminin, vitronectin, fibrinogen, von Willebrand factor, and entactin. Since the chemically synthesized RGD peptide exhibits cell adhesion activity when immobilized on a solid phase, the biomolecule in the present invention may be a chemically synthesized RGD molecule. Such RGD molecules include, but are not limited to, for example, the GRGDSP peptide, in addition to the naturally occurring molecules described above. Since the RGD sequence is recognized by integrin (eg, the receptor for fibronectin), which is a cell-adhesive molecule (and also a receptor), a functionally equivalent molecule of RGD may use such an integrin. It can be identified by examining the interaction.

[0115] As used herein, "integrin" refers to a transmembrane glycoprotein that is a receptor involved in cell adhesion. Integrins are present on the cell surface and function when cells adhere to the extracellular matrix. It is known that it is involved in cell-cell adhesion in blood cell systems. Such integrins include, for example, fibronectin, vitronectin Receptor, such as protein and collagen, platelet lib / Ilia, macrophage Mac-1, lymphocyte LFA-1 and VLA-1-6, and Drosophila PSA, but are not limited thereto. Integrin usually has a heterodimer structure in which a α chain having a molecular weight of 130 to 210 kDa and a j8 chain having a molecular weight of 95 to 130 kDa are non-covalently and one-to-one associated with a j8 chain. Is it an α chain and f row? J, Q;, Q;, Q;, Q;, Q;, Q;, Q; ', Q;, α, α, ^αv , α α, and the like, but are not limited thereto. As the j8 chain, for example, j8, β, β, β,

 1234 β, β, β, and the like, but are not limited thereto.

 5 6 7

[0116] Examples of such a heterodimer include, in addition to GpIIbllla, VLA-1, VLA-2, VLA-3, VLA-4, VLA-5, VLA-6, CD51 / CD29, LFA- 1, Mac- 1, pl50, 90 , vitronectin receptor, j8 ⁴ subfamilies, j8 ⁵ subfamily, j8 ⁶ sub family, LPAM-1, HML- but are not limited to such 1. Usually, the extracellular domain of the α chain has a divalent cation binding site, the extracellular domain of the β chain has a cysteine-rich region, and the intracellular domain of the β chain has a tyrosine phosphorylation site in many cases. The recognition site in the binding ligand is often an RGD sequence. Thus, an integrin may be an RGD molecule.

 [0117] (Detailed description)

 The description of the preferred embodiments is set forth below, but it should be understood that these embodiments are exemplary of the present invention and that the scope of the present invention is not limited to such preferred embodiments.

 [0118] In one aspect, the present invention provides a peptide comprising the sequence VVXGL (SEQ ID NO: 1), wherein X is a variant of an aromatic amino acid.

[0119] In another aspect, the present invention provides a composition for angiogenesis, comprising a peptide comprising the sequence VVXGL, wherein X is a variant of an aromatic amino acid.

[0120] In another aspect, the present invention provides a composition for forming a vascular network, comprising a peptide comprising the sequence VVXGL, wherein X is a variant of an aromatic amino acid.

[0121] In the peptide of the present invention, the composition for angiogenesis and the composition for forming a vascular network of the present invention, X represents an amino acid having an aromatic ring in a side chain or a variant thereof. Advantageously, Thus, it was found for the first time in the present invention that the angiogenesis ability and the ability to form a vascular network were enhanced by modifying the side chain. Accordingly, the present invention provides an angiogenic agent and a composition for forming a vascular network (also referred to as a vascular network forming agent), comprising the above-mentioned peptide. This sequence was designed based on the pentapeptide WYGL contained in SEQ ID NO: 2, and it is known to those skilled in the art that any variant other than WYGL may be within the scope of the present invention. it is obvious.

 [0122] In a preferred embodiment, X is a modified form of phenylalanine or tyrosine. This angiogenic agent, when naturally occurring, has a tyrosine at X. Therefore, it is advantageous to be a variant of the natural amino acids phenalanine or tyrosine. In the present invention, it was demonstrated that the modification of tyrosine in the side chain fuel group has a strong angiogenic activity of a peptide having tyrosine in X and feniralanine which can also be seen as a variant thereof. Based on that. In the present invention, it has been demonstrated in the present invention that the vascular network-forming activity of the peptide having phenalanine, which can also be regarded as a cysteine in X and a variant thereof, was also strong. Therefore, it is understood that by making similar modifications to the peptide of the present invention, both the angiogenic activity and the vascular network forming activity can be enhanced.

 [0123] Here, the angiogenesis ability can be indicated by the number of angiogenesis. In this specification! As defined, the angiogenic potential can be assessed by scoring, wherein the peptide of the invention or a variant thereof has a score of at least 3, preferably a score of 3.5, More preferably, it has a score of 4, and even more preferably has a score of 4.5.

[0124] Here, the vascular network forming ability can be indicated by a vascular network forming ability index. The ability of the peptide of the invention to form a vascular network is at least 5, preferably at least 7, more preferably at least 8, more preferably at least 9, more preferably at least 10, More preferably at least 11, more preferably at least 12, more preferably at least 13, more preferably at least 14, more preferably at least 15. Such a value is already remarkable when the vascular network forming ability index is actually, for example, about 8 or more. It is clear from the fact that the appearance of a reticulated blood vessel is observed almost always.

 [0125] In one embodiment, it is understood that the peptide of the present invention may contain an exogenous sequence as long as it contains the above specific sequence and has a vascular potential and a Z vascular network forming ability.

 [0126] In a preferred embodiment, the peptide of the present invention may be a peptide in which the para, ortho, or meta position of the side chain of the aromatic amino acid is substituted with an electron-withdrawing substituent.

 [0127] In another preferred embodiment, X in the peptide of the present invention may be a substituent in which an aromatic group of an aromatic amino acid is substituted with a hydrophobic group. By having a hydrophobic group (for example, an alkyl group such as a methyl group) instead of the hydroxyl group of tyrosine, the angiogenesis ability and the vascular network formation ability are enhanced. Thus, the present invention may advantageously have any hydrophobic group substituted, especially in the para position, instead of the hydroxyl group of tyrosine. Here, the hydrophobic group preferably increases the hydrophobicity index more preferably by 0.5, more preferably by 1.0, and more preferably by 1.5 or 2. Advantageously, it should be raised by zero. It is understood that such a hydrophobic group can be, for example, an alkyl group, but is not limited thereto. One preferred such hydrophobic group can include, but is not limited to, for example, an alkyl group or a substituted alkyl group. Even more preferably, such hydrophobic groups are C1-C6 alkyl groups, even more preferably C1-C3 alkyl groups, and in one preferred embodiment may be methyl groups.

 [0128] Preferably, in the embodiment, the hydrophobic group (eg, an alkyl group) can be substituted with a nora group of an aromatic group.

 [0129] Preferably, in the embodiment, the aromatic side chain is preferably a phenyl group, but is not limited thereto. It is understood that another example is also preferable, such as a side chain of tributophan. Is done.

 [0130] The amino acid sequence of the peptide of the present invention further comprises an amino acid sequence X -X -X -X -X

 1 2 3 4 5

X -X (where X = serine (S), threonine (T) or a variant thereof or not present

6 7 1

V, or X = valine (V), alanine (A), glycine (G), leucine (L), isoleucine (I) or Are variants thereof, where X = valine (V), alanine (A), glycine (G), leucine (L),

 Three

 X is an amino acid having an aromatic ring in the side chain or a leucine (I) or a variant thereof,

 Four

 X = glycine (G) or a variant thereof, X = oral isin (L), arani

 5 6

 (A), glycine (G), norin (V), isoleucine (I) or a variant thereof, and X = arginine (R), lysine (K) or a variant thereof. However, the peptide may be a peptide containing the amino acid sequence represented by the above or a variant thereof.

[0131] In one preferred embodiment, X is serine or a variant thereof, and more preferably, X is serine.

[0132] In another preferred embodiment, X is palin or a variant thereof, more preferably

 2

 Or, X is Norin.

 2

 [0133] In another preferred embodiment, X is palin or a variant thereof, more preferably

 Three

 Or, X is Norin.

 Three

 [0134] In another preferred embodiment, it is understood that the above X should have a specific modification in the side chain similarly to the above X.

[0135] In another preferred embodiment, X is glycine or a variant thereof, more preferably

 Five

 In a preferred embodiment, X is glycine.

 Five

 [0136] In another preferred embodiment, X is leucine or a variant thereof, more preferably

 6

 In a preferred embodiment, X is leucine.

 6

 [0137] In another preferred embodiment, X is arginine or a variant thereof, and in a more preferred embodiment, X is arginine.

 [0138] Therefore, it is understood that the peptide of the present invention may have a sequence of the combination of the above X to X, preferably of the embodiment.

In general, in a peptide having a physiological activity, when one or more amino acids in the amino acid sequence are substituted or deleted, or one or more amino acids are inserted or added to the amino sequence. Even so, it is well known that the physiological activity may be maintained. Therefore, in the amino acid sequence shown in SEQ ID NO: 1, one to three amino acids are substituted, or one or two amino acids located at one or both ends are deleted, or the amino acid Other amino acids at one or both ends of the sequence Peptides having an amino acid sequence to which a sequence is added (hereinafter sometimes referred to as “peptide variants” for convenience) and having an angiogenic action and a Z or vascular network forming action are also included in the scope of the present invention. It is. The modified peptide of the present invention can be modified by modifying the side chain of the aromatic amino acid with a natural amino acid in another amino acid sequence (for example, an alkyl group or a nitro group on the side chain of the amino acid). , Introduction of halogen, etc.). Further, such an amino acid may be a D-type amino acid. A peptide having an amino acid sequence in which one amino acid at the N-terminus or C-terminus of the amino acid sequence of WXGL has been deleted has an angiogenesis effect and a Z- or vascular network-forming effect comparable to those of the peptide having the amino acid sequence represented by VVXGL. It is understood that they can be activated and fall within the scope of the present invention. In the past, WXGL Pico !, a peptide in which X was substituted for natural tyrosine by alanine lost the angiogenic effect, so it was thought that it would be better not to change this tyrosine residue. In addition, a stronger effect was found, especially when modified with a hydrophobic group. Therefore, such an effect is considered to be an effect achieved for the first time by the present invention.

 [0140] In another preferred embodiment, among the peptide variants of the present invention, in the amino acid sequence shown in VVXGL, one or two amino acids are substituted in the sequence other than X in the amino acid sequence shown in VVXGL. Or a peptide having an amino acid sequence in which one amino acid located at one or both ends is deleted, or another amino acid sequence is added at one or both ends of the amino acid sequence, Peptides having a neoplastic and Z or vascular network forming effect may be provided. In the “amino acid having an aromatic ring in the side chain”, the number of substituents on the aromatic ring, preferably on the phenol group, is 1 to 5, and preferably 1 to 3. In the peptide of the present invention, the aromatic ring is preferably a condensed ring such as a benzene ring or a naphthalene ring containing a benzene ring (may be a heterocyclic ring such as a tributophan side chain), and particularly preferably a fluor group. is there.

[0141] Accordingly, the upper limit of the size of the peptide used in the present invention is not limited at all. However, if it is too large, production becomes difficult, handling is inconvenient, and vascular new life per unit weight is required. Therefore, the total number of amino acids of the peptide is usually 4 to 350, preferably 4 to 50, more preferably 5 to 20, and more preferably 5 to 10 And more preferably 6 to 10. Amino shown in X—X—X—X—X—X—X

 1 2 3 4 5 6 7

 Preference is also given to peptides in which not more than 10 amino acids have been added to the acid sequence or to one or both ends of the amino acid sequence, respectively.

 [0142] Whether the peptide of the present invention has an angiogenic action and an action of forming a Z or vascular network was determined by specifically ascertaining whether or not a microcell filled with a peptide solution was placed on the back of a mouse as shown in the Examples below. It can be examined by observing the state of formation of capillaries in the tissue surrounding the implanted tissue.

 [0143] The peptide or variant of the present invention can be easily synthesized manually or by a conventional method using a commercially available peptide synthesizer. In addition, a large-sized peptide is produced by a conventional method using genetic engineering, then modifying the side chain or modifying the genetic engineering technique to use a modified tRNA (Taki M. et al., FEBS Lett. 507, 35-38, 2001; Hohsaka T. et al., J. Am. Chem. Soc, 121, 34-40, 1999; and Hohsa ka T. et al., J. Am. Chem. Soc. , 118, 9778—9779, 1996).

 [0144] The peptide used in the present invention is designed based on amino acids that constitute a natural protein, and is degraded to amino acids by the action of peptidase in vivo. High safety. In fact, no toxicity was observed in in vivo experiments using mice performed in the following examples. This indicates that the amount of use that exerts its medicinal effect is not so toxic, and that it has been used! /

 [0145] In a preferred embodiment, the angiogenic agent and the vascular network forming agent of the present invention may be targeted to capillaries. This is because the capillaries can have the normal physiological activity that they have naturally by being networked in a network. The present invention provides, for the first time, a conjugate having enhanced angiogenic activity and Z or vascular network forming ability, thereby achieving a powerful therapeutic effect, stabilizing effect, and prevention that could not be achieved by conventional angiogenic agents. The effect can be demonstrated.

[0146] Angiogenesis can also be promoted by binding the peptide to a carrier and implanting the carrier with the peptide bound into a living body. This can selectively act on the necessary parts because it is fixed to the carrier, and a new DDS (drug delivery system) M) as a potential. Local administration of the angiogenesis agent of the present invention to a biomaterial implanted portion by application, spraying or the like promotes angiogenesis and promotes postoperative healing. Here, examples of the carrier include, but are not particularly limited to, biomacromolecules such as fats and proteins used for bone substitutes, tooth substitutes, and artificial organs. By bonding the peptide to the resin, when the resin is implanted in a living body, angiogenesis in peripheral tissues in contact with the resin is promoted, and the affinity of the resin with the living body is further improved. In a more preferred embodiment, a protein (in the present specification, when used as a carrier, unless otherwise specified, the term "protein" includes a protein-containing complex such as a glycoprotein and a phosphoprotein). Can be used as a carrier.

 [0147] Here, the protein used as a carrier may be a biocompatible, non-uniform protein, and in particular, a cell-adhesive protein in order to improve bonding with a biological tissue. Preferably, there is. Preferred examples of the cell adhesion protein include, but are not limited to, collagen (gelatin), fibronectin, vitronectin, laminin, and the like, and partial hydrolyzate thereof. In addition, these proteins are preferably purified proteins from which allergens have been removed, and are also preferable in terms of preventing allergic reactions. For example, various collagens derived from animals are commercially available as collagen. However, these contain allergens with low purity and are poor in reproducibility of quality, so that it is not preferable to apply them to clinical use. Since gelatin obtained by partially hydrolyzing animal-derived collagen and removing allergens is commercially available for clinical use, it is preferable to use such purified collagen or a partially hydrolyzed product thereof.

 [0148] The amount of the peptide bound to the carrier is not particularly limited and can be appropriately selected. Usually, the weight ratio of the carrier to the peptide (carrier: peptide) is about 100: 1 to 1: 1. And preferably about 20: 1 to 5: 1.

[0149] The bond between the carrier and the peptide is preferably a covalent bond. Bonding can be easily carried out, for example, by bonding the amino group at the N-terminus of the peptide to any amino group in the carrier using a bonding cross-linking agent such as dartalaldehyde. An example of a suitable bonding method is described. In the case of binding to a resin such as an artificial organ, a monomer containing a group that can be used for binding to a peptide, such as an amino group, is contained in the resin. Can be previously copolymerized, and the amino group or the like can be bonded to the N-terminal amino group of the peptide. Another peptide having an arbitrary amino acid sequence at one or both ends of a peptide having the amino acid sequence shown in SEQ ID NO: 1 or a peptide variant having an angiogenic action, in which a part of the amino acids is substituted or deleted. It is also preferable to employ a compound in which this arbitrary peptide is bound to a carrier.

[0150] The carrier to which the peptide is bound can be embedded in a living body as it is, in addition to spraying the coated text. When a cell-adhesive protein is employed as the carrier, the peptide-bound carrier can be used alone or in combination with other medicinal ingredients as a suture, various orthopedic materials, a wound adhesion promoter, and the like. Further, a mixture of the carrier protein bound with the peptide and other materials such as carbonate apatite or the cell adhesive protein not bound with the peptide of the present invention can be used as a bone substitute or the like. In this case, although the amount of the peptide contained in the final biomaterial such as bone substitute is not particularly limited, it is usually about 0.1 to: LOmg per 100 g of biomaterial.

[0151] The carriers used herein are preferably pharmaceutically acceptable, such as antioxidants, preservatives, coloring agents, flavorings, and diluents, milk匕 includes, but is not limited to, narcotics, suspending agents, solvents, fillers, bulking agents, buffers, delivery vehicles, diluents, excipients and Z or pharmaceutical adjuvants. Typically, the medicament of the invention will be in the form of a composition comprising a peptide of the invention, or a variant or derivative thereof, together with one or more physiologically acceptable carriers, excipients or diluents. Is administered. For example, suitable vehicles may be water for injection, physiological solutions, or artificial cerebrospinal fluid, which may be supplemented with other materials common in compositions for parenteral delivery. is there.

[0152] As used herein, an acceptable carrier, excipient or stabilizer is non-toxic to the recipient, and preferably inert at the dosages and concentrations employed. E.g., phosphates, citrates or other organic acids; ascorbic acid, a-tocopherol; low molecular weight polypeptides; proteins (e.g., serum albumin, gelatin or immunoglobulins); hydrophilic polymers (e.g. Amino acids (eg, glycine, glutamine, asparagine, arginine or lysine); monosaccharides; Disaccharides and other carbohydrates (including glucose, mannose or dextrin); chelating agents (eg, EDTA); sugar alcohols (eg, mannitol or sorbitol); salt-forming counterions (eg, sodium); Non-ionic surface activators (eg, Tween, pluronic or polyethylene glycol (PEG)) and the like, but are not limited thereto.

 [0153] Further exemplary suitable carriers include neutral buffered saline or saline mixed with serum albumin. Preferably, the product is formulated as a lyophilizate using a suitable excipient (eg, sucrose). Other standard carriers, diluents and excipients may be included as desired. Other exemplary compositions include Tris buffer at pH 7.0-8.5 or acetate buffer at pH 4.0-5.5, which further comprises sorbitol or a suitable alternative thereof. May be included.

 [0154] In the present specification, the carrier is preferably a protein, more preferably a cell adhesion protein. Preferably, the cell adhesion protein used in the present invention may be collagen.

 [0155] The peptide of the present invention can be locally administered to a tissue in which angiogenesis is desired, alone or in the form of an injection solution dissolved in a physiological buffer. Local administration of the angiogenic agent of the present invention to a wound or the like caused by surgery or trauma by injection, application, spraying, etc. promotes angiogenesis and promotes wound healing. Here, the peptide concentration in the peptide solution used for injection or application or spraying is not particularly limited. Usually, the concentration is about 1 ng (nanogram) to (/ zg (microgram) ZmL. The dose can be appropriately selected depending on the size and depth of the wound or the like, but it is sufficient that the whole wound is covered with the peptide solution. It can be administered once to several times every day to several days until the wound heals. Further, the injection solution may contain various components usually contained in a remedy for wounds, such as other disinfectants and anti-inflammatory analgesics.

[0156] The medicament of the present invention can be administered orally or parenterally. Alternatively, the medicament of the present invention can be administered intravenously or subcutaneously. When administered systemically, the medicament used in the present invention may be in the form of a pyrogen-free, pharmaceutically acceptable aqueous solution. The preparation of such pharmaceutically acceptable compositions takes into account pH, isotonicity, stability, etc. By doing so, those skilled in the art can easily carry out. In the present specification, the administration method includes oral administration, parenteral administration (for example, intravenous administration, intramuscular administration, subcutaneous administration, intradermal administration, mucosal administration, rectal administration, vaginal administration, local administration to the affected area, Skin administration). Formulations for such administration may be provided in any formulation. Such preparations include, for example, solutions, injections, and sustained-release preparations.

[0157] The medicament of the present invention may contain a physiologically acceptable carrier, excipient or stabilizing agent (Japanese Pharmacopoeia, 14th edition, its supplement or its latest edition, Remington's Pharmaceutical Sciences, if necessary). , 18th Edition, AR Lrennaro, ed., Mack Publisnmg Company, 1990, etc.) and a glycan composition having the desired degree of purity to form a lyophilized cake or aqueous solution. Can be prepared and stored.

 [0158] The amount of the composition of the present invention to be administered depends on the purpose of use, target disease (type, severity, etc.), age, weight, sex, medical history, cell morphology or type of the patient, and the like. Thus, those skilled in the art can easily determine. The frequency of applying the treatment method of the present invention to the subject (or patient) also depends on the purpose of use, target disease (type, severity, etc.), patient age, weight, sex, medical history, and course of treatment. In consideration of the above, a person skilled in the art can easily determine. Frequently, for example, once every few months (eg, once a week – once a month). It is preferable to administer once a week to once a month while monitoring the progress

[0159] In another aspect, the present invention provides a method for generating a blood vessel, which comprises administering the composition for angiogenesis of the present invention to a site of a patient in which angiogenesis is desired. I do.

 [0160] In another aspect, the present invention forms a vascular network, which comprises administering the composition for forming a vascular network of the present invention to a site of a patient where formation of a vascular network is desired. To provide a way to:

[0161] It is understood that the compositions used in the above methods can take any of the forms detailed herein. It is understood that the administration method used herein can also use any form. [0162] In a preferred embodiment, the present invention provides a composition for forming an angiogenesis and Z or vascular network of the invention at a site in a patient where angiogenesis and Z or vascular network formation is desired. Provided for angiogenesis and forming a Z or vascular network.

 [0163] As used herein, "patient" refers to an organism to which the treatment of the present invention is applied, and is also referred to as "subject" or "subject". The patient can preferably be a human.

 [0164] The present invention provides methods of treatment, inhibition and prevention by administering to a patient an effective amount of a composition of the present invention. In a preferred aspect, the compositions of the present invention can be substantially purified (including, for example, conditions in which there is substantially no ability to limit its effects or substances that produce undesirable side effects).

 [0165] The animal targeted by the present invention may be any organism (eg, an animal (eg, a vertebrate, an invertebrate)) as long as it has an immune system or a similar system. Preferably, it is a vertebrate animal (e.g., a metal eel, alfalfa, chondrichthyes, teleosts, amphibians, reptiles, birds, mammals, etc.), and more preferably, a mammal (e.g., a monotreme, marsupial) , Rodents, dermis, skin wings, carnivores, carnivores, carnivores, longnoses, ungulates, artiodactyla, tubulars, squamata, squids, cetaceans, primates, rodents Teeth, egrets). Illustrative patients include, but are not limited to, animals such as, for example, horses, pigs, horses, birds, cats, dogs, and the like. More preferably, primates (eg, chimpanzees, diphones, humans) are targeted. Most preferably, humans are targeted.

[0166] Various delivery systems are known for prophylaxis and therapy, and the present invention contemplates any route of administration. Techniques that can be used to administer the compositions and the like of the present invention include, for example, aqueous solutions, ribosomes, microparticles, and microcapsules. Thus, the compositions of the present invention can be administered orally or parenterally. Such administration methods include oral administration, parenteral administration (e.g., intravenous, intramuscular, subcutaneous, intradermal, mucosal (intranasal, intravaginal, sub-airway, intrathecal, rectal) Mucosal and intestinal mucosa), topical administration to the affected area, dermal administration, etc.). When administered systemically, the compositions used in the present invention are preferably pyrogen-free. The preparation of such pharmaceutically acceptable compositions depends on the skill of the art in considering pH, isotonicity, stability and the like. Can be easily performed. Formulations for such administration may be provided in any formulation. Such preparation forms include, for example, liquid preparations, injections, and sustained release preparations. Methods of introduction include, but are not limited to, administration as an oral agent, inhalation (eg, lungs), syringe, catheter, injection using a tube, needleless injection, gene gun, and the like. In this case, it can be administered together with other biologically active agents.

 [0167] The amount of the medicament used in the prophylactic method of the present invention can be easily determined by those skilled in the art in consideration of the purpose of use, target disease (eg, type), age, weight, and medical history of the patient. I can do it. The frequency of applying the treatment method of the present invention to a patient (or patient) also depends on the purpose of use, target disease (type, severity, etc.), age, weight, medical history, and course of the patient, and the like. Those skilled in the art can easily determine. The frequency may include, for example, daily-once-monthly administration (eg, once a week, once a month), or once every year before the epidemic. It is advantageous to boost at least about a week at intervals of at least about one week, which is preferably administered once a week-monthly while monitoring the progress. More preferably, the interval between boosts can be at least about 3 weeks. The dose of the composition or the like of the present invention varies depending on the age, body weight, symptoms, administration method, and the like of the patient, and is not particularly limited.

[0168] As used herein, "administering" refers to giving the medicament or the like of the present invention or a pharmaceutical composition containing the same, alone or in combination with another therapeutic agent, to a host to be treated. means. The combination may be administered, for example, either as a mixture, simultaneously, separately but simultaneously or concurrently; or sequentially. This includes the indication that the combined agents are administered together as a therapeutic mixture, and the procedure in which the combined agents are administered separately but simultaneously (e.g., through separate mucous membranes to the same individual). Also included. "Combination" administration further includes separately administering one of the compounds or agents given first, followed by the second.

 [0169] The administration of the medicament of the present invention may be performed by any method, but it is advantageous to use needleless injection. This is a drug that exempts the patient from administration without undue burden.

[0170] Here, the needleless syringe in the present invention refers to a gas pressure or an elastic part without using a needle. It refers to a medical device that moves a piston by the force of a material to inject a drug solution onto the skin and administers a drug component subcutaneously, more preferably, into cells under the skin.

 [0171] Specifically, for example, ShimaJet ™ (manufactured by Shimadzu Corporation), Medi-Jector Vision, (Elite medical Inc. ^), Henget (Pejet) (manufactured by Penjet) Is commercially available.

 [0172] In another aspect, the present invention relates to a medicament for angiogenesis, comprising a peptide called VVXGL, wherein X is a modified aromatic amino acid, a peptide or a modified variant thereof, or a salt thereof. There is provided a use for the manufacture of a composition.

 [0173] In another aspect, the present invention comprises a pharmaceutical composition for forming a vascular network, comprising a sequence of WXGL, wherein X is a variant of an aromatic amino acid, a peptide or a variant thereof, or a salt thereof. Provide use for manufacturing.

 [0174] It is understood that the compositions used in the above uses may take any of the forms detailed herein. In addition, it is understood that any technique of cultivated land can be used as a technique for producing a pharmaceutical composition in the art.

 [0175] (Compound design)

Since the region having an angiogenic activity and Z or vascular network forming activity is included in the amino acid sequence shown in WXL, the peptide having the amino acid sequence shown in VVXGL or a partial amino acid thereof is substituted or its terminal. The modified peptide having an angiogenic effect and Z or a vascular network-forming activity in which a partial region has been deleted, even if one or both ends of the modified peptide have an angiogenic effect and Z or a vascular network forming activity, the peptide still has an angiogenic effect and Z or It can exert a vascular network forming activity. This indicates that the cell-adhesive action of cell-adhesive peptides such as fibronectin and laminin is exerted by RGD, a region consisting of only three amino acids, YIGSR, and a region consisting of only five amino acids. It can be inferred from the fact that they are things. Therefore, since the region having an angiogenic activity is included in the amino acid sequence shown in VVXGL, the peptide having the amino acid sequence shown in WXGL or a partial amino acid thereof has been substituted or the terminal region has been deleted. Even if a long amino acid sequence is added to one or both ends of a peptide variant having an angiogenesis effect, the above-mentioned peptide or peptide variant region may have an interaction. Is considered to exert an angiogenic effect as long as it is exposed. At present, the three-dimensional structure of an artificial peptide can be easily predicted from its primary sequence using commercially available computer software. The peptides whose regions are exposed can be easily designed.

 [0176] Computer programs for performing computer modeling can also be used in the process of selecting fragments or chemicals.

 [0177] Once the appropriate compounds or fragments (compound species) are selected, they can be assembled into a single compound or complex. Prior to construction, a visual inspection of the relevance of the fragments to one another can be performed on a three-dimensional image displayed on a computer screen in relation to the structural coordinates of the angiogenic peptides of the invention. This is followed by manual model building using software such as Quanta or Sybyl [Tripos Associates, St. Louis, MO].

 [0178] Useful programs that can be used in linking individual chemicals or fragments include:

 [0179] 1. CAVEAT (PA Bartlett et al., "CAVEAT: A Program to Facilitate the Structure-Derived Design of Biologically Active MoleculesJ (Molecular Recognition in Chemical and Biological Problems, Special Pu b., Royal Chem. Soc., 78 G, Lauri and PA Barlett, "CAVEAT: a Program to Facilitate the Design of Organic Moleculesj, J. Comput. Aided Mol. Des., 8, 51-66 (1994). CAVEAT is available from the University of California, Berkeley, CA.

 [0180] 2. A 3D database system such as ISIS (MDL Information Systems, San Leandro, CA). This field is reviewed by Y. C. Martin, "3D Database Searching in Drug Designj, J. Med. Chem., 35, 2145-2154 (1992).

[0181] 3. HOOK (MB Eisen et al., "HOOK: A Program for Finding Novel Molecular Architectures that Satisfy the Chemical and Steric Requirements of a Macromolecule Binding Site", Proteins: Struct., Funct., Genet., 19, 199-221 (1994)). HOOK is available from Molecular Simulations, San Diego, CA.

 [0182] Instead of proceeding to build one fragment or chemical at a time, such as an angiogenic activator or inhibitor, in a stepwise manner, as described above, it can be designed globally or de novo. There are many new ligand design methods, including:

 [0183] 1. LUDI (H. — J. Bohm, “The Computer Program LUDI: A New Met hod for the De Novo Design of Enzyme InhibitorsJ, J. Comp. Aid.

Molec. Design, 661-78 (1992)). LUDI is available from Molecular Simulations Incorporated, San Diego, CA.

[0184] 2. LEGEND (Y. Nishibata et al., Tetrahedron, 47, p. 8985 (1991)). LEGEN

D is available from Molecular Simulations Incorporated, San Diego, CA.

 [0185] 3. LeapFrog (available from Tripos Associates, St. Louis, MO).

 [0186] 4. SPROUT (V. Gillet et al., "SPROUT: A Program for Structure Generation", J. Comput. Aided Mol. Design, 7, 127-153 (1993)). SPROUT is available from the University of Leeds, UK.

 [0187] Other molecular modeling techniques may also be used in accordance with the present invention [eg, NC Cohen et al., "Molecular Modeling Software and Metnods for Medicinal Chemistryj, J. Med. Chem., 33, 883—894 ( 1990); see also MA Na via and MA Murcko, "The Use of Structural Information in Drug Design J, Current Opinions in Structural Biology, 2, 202-210 (1992); LM Balbes et al., `` A Perspective of Modern Method s in Computer-Aided Drug Design J, (Reviews in Computational Chemistry, vol. 5, KB Lipkowitz and DB Boyd, eds., VCH, New York, pp. 337-380 (1994)); WC Guida, "Software For Structure -Based Dmg DesignJ, Curr. Opin. Struct. Biology., 4, 777-781 (1994)].

[0188] Once the strength or design of the conjugate is determined by the above method, the material is optimized Competitive angiogenic potential can be tested and optimized by computational evaluation.

 [0189] The substance of the present invention can be further optimized by calculation, for example, so that there is no electrostatic repulsion interaction with surrounding water molecules. Such non-complementary electrostatic interactions include charge-charge repulsion interactions, dipole-dipole repulsion interactions, and charge-dipole repulsion interactions.

[0190] Certain computer software is available in the field of evaluating compound deformation energy and electrostatic interactions. Examples of programs designed for such use include: Gaussian 94, revision C (MJ Frisch, Gaussian, Inc., Pittsburgh, PA 1995); AMBER, version 4.1 (PA Kol lman, University of California at San Francisco, 1995); QUANTA / CHARMM (Molecular Simulations, Inc., San Diego, CA 1995); Insert Il / Discover, (Molecular Simulations, Inc., San Diego, CA 1995); (Molecular Simulations, Inc., San Diego, CA 1995); and AM SOL (Quantum Chemistry Program Exchange, Indiana University). These programs can be executed using, for example, a Silicon Graphics workstation (eg, Indigo ² with “IMP ACT” graphics). Other hardware systems and software packages are also known to those skilled in the art.

 [0191] Another approach possible with the present invention is the computational screening of small molecule databases for chemicals or compounds that can bind, in whole or in part, to LOX-1. In this screen, the quality of the fit of such a material to the binding site can be determined from either shape complementarity or the estimated interaction energy [EC Meng et al., J. Comp. Chem. , 16, 505-524 (1992)].

 [0192] (Combinatorial chemistry)

The compound library used in the present invention can be prepared or obtained by any means including, but not limited to, combinatorial chemistry technology, fermentation methods, plant and cell extraction procedures, and the like. be able to. Methods for creating combinatorial libraries are well known in the art. For example, ER Fel der, Chimia 1994, 48, 512-541; Gallop et al., Med. Chem. 1994, 37, 1233-1251; RA Houghten, Trends Genet. 1993, 9, 235-239; Houghten et al., Nature 1991, 354, 84-86; Lam et al., Nature 1991, 354, 82-84; Carell et al., Chem. Biol. 1995, 3, 171-183; Madden et al., Perspectives in Drug Discovery and Design 2, 269-282; Cwirla et al., Biochemistry. 1990, 87, 6 378-6382; Brenner et al., Proc. Natl. Acad. Sci. USA 1992, 89, 5381-5383; Gordon et al., Med. Chem. 1994, 37, 1385-1401; Lebl et al., Biopolymers.

1995, 37 177—198; and the references cited therein. These references are incorporated by reference herein in their entirety.

 [0193] The computer used may be any computer as long as the program can be executed, for example, Windows (registered trademark) -based, UNIX (registered trademark) -based, Mac (registered trademark) OS-based, LINUX-based, and the like. But not limited to them.

 The present invention has been described above with reference to the preferred embodiments for easy understanding. Hereinafter, the present invention will be described based on examples. However, the above description and the following examples are provided for illustrative purposes only, and are not provided for limiting the present invention. Therefore, it should be understood that the scope of the present invention is not limited to the embodiments or examples specifically described herein, but only by the appended claims. . Example

 [0195] Hereinafter, the present invention will be described more specifically based on examples.

 (Example 1 Synthesis of Peptide)

 High-efficiency solid-phase method by Fmoc-Danigaku (K. Nokihara, et al., Innovation and Perspectives in Solid—Phase Synthesis 1992, ed., R. Epton, Intercept Limited, Andover, UK, 445—448, 1992 , Design and Applications of a Novel simultaneous Multiple Solid-Phase Peptide Synthesizer; Based on the above, a representative example of a peptide having the amino acid sequence shown in SEQ ID NO: 1 was synthesized using an automatic peptide synthesizer.

[0197] The modification of the aromatic side chain of the aromatic amino acid was performed as follows, or Was used.

 [0198] F (pMe) GL: (product name) B— 3335: Fmoc— p— Me— Phe— OH

 F (pF) GL: (product name) B— 2835: Fmoc— p— fluoro— Phe— OH

 F (pNO): (brand name) B— 1395: Fmoc— p— nitro— Phe— OH

 2

 All sources: Bachem AG (Switzerland).

[0199] The peptide was synthesized using a solid phase synthesis method developed by Merrifield (Merrifield, RB (1963) Solid phase peptide synthesis. I. the synthesis of a tetrapeptide. J. Am. Chem. Soc. 85, 2149-2154), and the Fmoc method using a 9-fluorenylmethycarboxyl (Fmoc) group as a protecting group for the CK-amino group.

[0200] The synthesized peptides are as follows, and include, in principle, those having WXGL (SEQ ID NO: 1) and SWYGLR (SEQ ID NO: 2) which is a natural type as a control. SEQ ID NO: 2: SWYGLR

 SEQ ID NO: 3: SWFGLR

 SEQ ID NO: 4: SVVF (pMe) GLR (where F (pMe) is obtained by substituting a methyl group at the para-position of the phenyl group of phenylalanine)

 SEQ ID NO: 5: SWF (pF) GLR (where F (pF) is a phenyl group of phenylalanine with a fluoro group substituted at the para-position)

 SEQ ID NO: 6: SWF (pNO) GLR (where F (pNO) is phenyl of phenylalanine)

 twenty two

 With-toro group substituted at the para position of the group)

 SEQ ID NO: 7: SWF (D) GLR (where F (D) is the D-form of phenylalanine) SEQ ID NO: 8: SWF (pEt) GLR (where F (pEt) is fenilalanine With a phenyl group substituted with an ethyl group at the para-position)

 SEQ ID NO: 9: SWF (pPr) GLR (where F (pPr) is a phenyl group in which the propyl group is substituted at the para-position of the phenyl group of hua- / realanine)

 SEQ ID NO: 10: SWF (pHex) GLR (where F (pHex) is a phenyl group substituted with a hexyl group at the para-position of the phenyl group)

SEQ ID NO: 11: SWYGL SEQ ID NO: 12: SWFGL

SEQ ID NO: 13: WYGLR

SEQ ID NO: 14: WFGLR

SEQ ID NO: 15: WYGL

SEQ ID NO: 16: WFGL

SEQ ID NO: 17: VVF (pMe) GLR (where F (pMe) is a phenylalanine in which a methyl group is substituted at the para-position of the phenyl group)

SEQ ID NO: 18: SWF (pMe) GL (where F (pMe) is phenylalanine in which a methyl group is substituted at the para-position of the phenyl group)

SEQ ID NO: 19: ^^ 1 ^^) 0 (where? 1 ^ 6) is the substitution of a methyl group at the para-position of the phenyl group of phenylalanine

SEQ ID NO: 21: SWF (pMe) GLR (where, F (pMe) is a compound obtained by substituting a methyl group at the ortho-position of the phenyl group of phenylalanine)

SEQ ID NO: 22: SWF (pF) GLR (where, F (pF) is a ferrule having a fluoro group substituted at the ortho position of the ferule group)

SEQ ID NO: 23: SWF (pNO) GLR (where F (pNO) is

twenty two

In which the -toro group is substituted at the ortho position of the

SEQ ID NO: 24: SWF (pMe) GLR (where F (pMe) is a phenylalanine in which a methyl group is substituted at the meta position of the phenyl group)

SEQ ID NO: 25: SWF (pF) GLR (where F (pF) is a ferrule having a fluoro group substituted at the meta-position of the ferule group)

SEQ ID NO: 26: SWF (pNO) GLR (where F (pNO) is

 twenty two

In which the -toro group is substituted at the meta position of the

 Furthermore, in addition to SEQ ID NOS: 8 to 10, the following variants are prepared for feralanine in SVVFGLR at the ortho, meta or para position of the side chain.

 Ethyl group

 1 propyl group

2-propyl group (iso-propyl group) 1-butyl group

 2 Butynole group (iso Butynole group)

 2-Methylpropyl group (sec butyl group)

 1, 1-dimethylethyl group (tert-butyl group)

 1 Pentyl group

 2—pentyl group

 3—pentyl group

 1 Methylbutyl group (iso pentyl group)

 2-methylbutyl group

 3-methylbutyl group

 2, 2-dimethylpropyl group (neo pentyl group)

 Black mouth group

 Bromo group

 Edo group.

 [0202] The obtained peptide was assayed with a liquid chromatography-coupled mass spectrum (LCMS) system to confirm that the peptide was highly pure (single component, consistent with the theoretical mass value).

 [0203] According to such a procedure, peptides used in the following Examples were produced.

 [0204] (Example 2 Confirmation of angiogenic action of synthetic peptide)

 Rat vascular endothelial cells are three-dimensionally implanted in the presence of the peptide of the present invention synthesized in Example 1.

 [0205] This operation was specifically performed as follows. The cells used were transformed rat lung endothelial cells (TRLEC cells). TRLEC cells were seeded in the collagen I layer mixed with a peptide solution having a concentration of lOngZml, and cultured in a carbon dioxide incubator for 14 days. Controls were performed with no factor (1) and with VEGF (+), a protein known conventionally as an angiogenic factor.

[0206] After 14 days, the cultured cells were observed under a microscope. As a result, the control showed no luminal formation. The peptide of the present invention and VEGF formed a lumen, and cells surrounding the lumen were adhered to each other. Observation of the lumen with an electron microscope at 7000x magnification As a result, it was confirmed that a plurality of micropirai (fine cell projections) were formed on the inner wall of the lumen. Further, at a magnification of 15,000, the junction of the vascular endothelial cells surrounding the lumen was observed. As a result, a region where the endothelial cells were tightly bonded, that is, a tight junction was observed. These findings indicate that the endothelial cells acquired polarity and formed a lumen. Polarity is a property of cells having a part that shares functions such as head and tail.Even if endothelial cells are normally cultured, they lose polarity and do not form a lumen, so it was induced by this peptide. Is shown. The peptide of the present invention was significantly superior in tube formation length to VEGF. From these results, the peptide of the present invention has the effect of adhering cells to each other in a tissue composed of vascular endothelial cultured cells to form a lumen between them (in vivo, this lumen is Blood vessels). The schematic diagram is shown in FIG. FIG. 2 shows a comparison between VEGF and the peptide SVVYG LR (SEQ ID NO: 2).

 (Example 3 Confirmation of In Vivo Angiogenic Action by DAS Atssay)

A solution obtained by dissolving a peptide at a concentration of 10 gZ ml in DMEM (Dulbecco's modified Eagle medium) used as a cell culture solution was obtained. After filling the upper and lower cylinder diameter 0. ⁴⁵ mm Millipore filter (MILLIPORE, USA, DAS Assay) microcells the back of the mouse consists blocking, the pair of peptides or the like of the present invention in a microcell flop A tide solution and VEGF solution were injected. As a control, a phosphate buffer (PBS) containing no peptide alone was also injected (control). Five days later, the state of the tissue around the microcell was observed with a microscope. The schematic diagram is shown in FIG.

[0208] FIGS. 4 and 5 show the state of angiogenesis when peptide SWYGLR (SEQ ID NO: 2) was used. As shown in FIG. 4 and FIG. 5, it was clarified that the blood vessels were newly formed, and the newly formed blood vessels were renewed and began to be reticulated. Therefore, it was revealed that the peptide of the present invention is useful as an angiogenic agent and a Z or vascular network forming agent. When this was compared with VEGF and the control, peptide SVVYGLR (SEQ ID NO: 2) showed angiogenesis and reticulation, whereas VEGF showed angiogenesis but no reticulation. I got it. In the control, neither neovascularization nor reticulation was observed. Therefore, the peptide of the present invention has an angiogenic action and a vascular network. It has been shown that the ability to form stalk may be enhanced.

 [0209] The results obtained by measuring the angiogenesis length of the peptide synthesized in Example 1 are shown below.

 [0210] Control: 0.00 ± 0

 VEGF: 0.00 ± 0

 Peptide SWYGLR (SEQ ID NO: 2) (wild type): 3.7

 Peptide SWFGLR (SEQ ID NO: 3): 4.4

 Peptide SWF (pMe) GLR (SEQ ID NO: 4): 4.6

 Peptide S WF (pF) GLR (SEQ ID NO: 5): 4.1

 Peptide SWF (pNO) GLR (SEQ ID NO: 6): 4.1.

 2

 [0211] The results of counting the number of angiogenesis of the peptide synthesized in Example 1 are shown below. The average number of pixels per new blood vessel was observed with a stereoscopic microscope (Olympus, SZX12, Japan). The obtained image was read by Photoshop (registered trademark) (Adobe, Japan). Counted in pixels. The scores were as follows.

[0212] Score less than 1: 100

 Score 2: 100 or more and less than 125

 Score 3: 125 or more and less than 150

 Score 4: 150 or more and less than 175

 Score 5: 175 or more and less than 200

 Score 6: 200 or more

 Controller: 0.00 ± 0

 VEGF: 2.75 ± 1.03

 Peptide SWYGLR (SEQ ID NO: 2) (wild type): 4.1

 Peptide SWYGLR (SEQ ID NO: 2) (50 ° C): 3.0

 Peptide SWYGLR (SEQ ID NO: 2) (60 ° C): 3.5

 Peptide SWYGLR (SEQ ID NO: 2) (90 ° C): 2.1

 Peptide SWFGLR (SEQ ID NO: 3): 3.8

Peptide SWF (pMe) GLR (SEQ ID NO: 4): 4.5 Peptide SWF (pF) GLR (SEQ ID NO: 5): 4.1

 Peptide SWF (pNO) GLR (SEQ ID NO: 6): 4.1

 2

 On the other hand, in the case of substitution with fenilalanine, angiogenesis and angiogenesis length were increased.

 [0213] Next, the state of angiogenesis in the tissue was observed with a stereoscopic microscope (Olympus, SZX12, Japan). The obtained image was read by Photoshop (registered trademark) (Adobe, Japan) and the network ability was scored as follows.

 [0214] Nwl: Network formation Although neovascularization was observed in the above, each new blood vessel was a single fe.

 [0215] Nw2: In the middle stage of network formation, each new blood vessel is in a state in which a side branch on a ladder is engaged.

 [0216] Nw3: The network is in the final stage, and the upper branch of the ladder has more side branches.

[0217] Nw4: Network maturation stage, showing a wide range of neovascular plexus.

[0218] Network index measurement results are shown below. The network index of peptide SVVYGLR on day 5 was as low as 1.6, and it was 3.0 on the 10th day, when the network was incomplete, indicating that network formation was observed. Peptides SVVF (pF) GLR and SWF (pNO) GLR were also almost equivalent to SVVYGLR on days 5 and 10.

2

 It was observed to show an index. In contrast, SWF (pMe) GLR and SW FGLR showed higher network index capacity than SVVYGLR on both days 5 and 10.

 [0219] As the temperature increased, the angiogenic potential decreased. At 50 ° C and 60 ° C, the average was 3, 3.5, and both had a score of 3 or more. At 90 ° C, the average value was 2.1, which was much lower than the wild type (SEQ ID NO: 1) (average value 4.1).

[0220] On the other hand, when phenylalanine was substituted, angiogenesis and angiogenesis length were increased. In addition, when the para-position of the phenyl group of phenylalanine was substituted with a methyl group, the ability to form a vascular network (20.7) was further increased. Therefore, it is understood that substitution with a hydrophobic group is appropriate for enhancing the ability to form angiogenesis and vascular networks. [0221] Potential of the peptide of the present invention to be applied to various biomaterials It was found that when a peptide is bound to a biomaterial, its angiogenesis ability is maintained if the treatment temperature is up to 60 ° C. Was. Among SWYGLR (SEQ ID NO: 2), VVYGLR (SEQ ID NO: 13) or SVVYGL (SEQ ID NO: 12) was confirmed to have an angiogenic ability and a vascular network-forming ability. It is understood that variants of these tyrosine residues also have enhanced angiogenesis and vascular network formation capabilities. Therefore, in the present invention, it is shown that it is preferable that the middle 5 amino acids of the 7 amino acids are included, and further that the substituent of ferulalanine therein is replaced by a hydrophobic group.

 FIG. 6 shows a diagram summarizing these results. This is sometimes called a balance sheet in this specification.

 Example 4 In Vivo Angiogenesis Action

 As a carrier protein, FreAlagin AD type (manufactured by Miyagi-Danigaku Kogyo Co., Ltd., molecular weight 2000 to 20000), which is a gelatin obtained by partially hydrolyzing animal-derived collagen and removing allergens, was used. The FreAlagin AD type has been approved for clinical use.

 [0224] 100 mg of FreAlagin AD (carrier protein) was dissolved in 2.5 mL of MilliQ water.

 1.20-1.32 mg of the peptide of the present invention was dissolved in 1 mL of 0.1 M phosphate buffer (pH 7.0), and the solution was added to the carrier protein under ice-cooling. Daltaraldehyde (25% solution diluted 10-fold with 0.1 M phosphate buffer (pH 7.0), 0.15 mL is added dropwise to the resulting mixture (4 ° C), and the reaction mixture is cooled to 4 ° C. Thus, the N-terminal amino group of the peptide prepared in Example 1 and the amino group of the carrier protein were covalently bonded.

 [0225] Confirmation of complete binding was performed by thin layer chromatography (TLC) when the raw materials disappeared.

 [0226] The reaction mixture was desalted with a G10 column (Pharmacia) using 5% acetic acid as an eluent. The main peak was lyophilized to give a bound compound almost quantitatively.

[0227] Rat vascular endothelial cells were three-dimensionally cultured in the presence of the gelatin-binding peptide thus prepared. This operation was specifically performed as follows. Transformed rat lung endothelial cells (TRLEC cells) were used. Gelatin-binding peptide (conjugate) and collagen type I are mixed at a ratio of 1:10, and 10 ng (nanogram) / m TRLEG cells were seeded in the conjugate 'collagen mixed solution mixed layer at a concentration of 1 and cultured in a carbon dioxide incubator for 14 days. The control was no factor (1), and the protein VEGF (+) conventionally known as an angiogenic factor was used as a positive control. After 14 days, when observed under a microscope, the control was vigorous, forming no lumen at all. The peptide conjugate of the present invention and VEGF formed a lumen, and cells surrounding the lumen were adhered to each other. In particular, as shown in SEQ ID NO: 4, the adhesive activity of the side chain phenyl group substituted with a hydrophobic group was enhanced as compared with the natural type (SEQ ID NO: 2).

[0228] Further, upon observing the lumen portion with an electron microscope at a magnification of 7000 times, it was confirmed that a plurality of microvillages (fine cell projections) were formed on the inner wall of the lumen. Furthermore, when the junction of vascular endothelial cells surrounding the lumen was observed at a magnification of 15,000, a region where the endothelial cells were firmly connected, that is, a tight junction was observed. The tube formation length was significantly better with gelatin-binding peptide than with VEGF. In particular, as shown in SEQ ID NO: 4, although the phenyl group of the side chain was substituted with a hydrophobic group, the tube forming activity was enhanced compared to the native type (SEQ ID NO: 2). Based on these results, gelatin-binding peptide has the effect of adhering cells to each other and forming a lumen between them in a tissue composed of cultured vascular endothelial cells (in vivo, this lumen It is evident that the effect of the peptide modification seen in vitro will be retained in vivo.

 [0229] A gelatin-binding peptide having a concentration of 100ngZml was dissolved in collagen I at a ratio of 1:10 to obtain a peptide-binding gelatin-collagen mixed solution having a concentration of 100ngZml. After embedding a microcell consisting of a 0.45 mm diameter cylinder up and down with a Millipore filter (MILLIPORE, USA, DAS Assay kit) in the back of the mouse, the gelatin-binding peptide and VEGF solution were placed in the microcell. Was injected. As a control, a phosphate buffer (PBS) containing no peptide alone was also injected (control). Five days later, the state of the tissue around the microcell was observed with a microscope.

 [0230] The obtained vascular network forming ability is shown.

[0231] Gelatin binding peptide (using SEQ ID NO: 2): 19.0 Gelatin binding peptide (using SEQ ID NO: 3): 20.0

 VEGF: 5.5

 = ι Control: 0

 Thus, the present invention was demonstrated to have angiogenic ability even in vivo. Such an effect is a force that was not seen in VEGF, which has a remarkable angiogenesis ability, and it can be said that it provides a powerful effect that has not been obtained conventionally.

[0232] (Example 5: Combination with biosubstitute material)

 It is confirmed whether the peptide of the present invention has a strong angiogenic action and a Z or vascular network forming ability, and can be used for a biological substitute material such as an artificial bone, an implant in an artificial organ, and a repair of the organ. A bone substitute is made of hydroxyapatite, a representative of artificial bone, to which a gelatin-binding peptide is attached as shown in Example 4. By implanting this in place of bone, it became possible to form a vascular network around it.

(Example 6: Heart treatment)

 In the present example, a piece of tissue was prepared using polylactic glycolic acid (PLGA) as a support, the peptide SWYGLR (SEQ ID NO: 2) produced in Example 1 and its variant as a biomolecule, The angiogenic ability and the vascular network forming ability of the peptide of the present invention were demonstrated.

[0234] (Method 'Results')

 <Scaffold design>

 Vycryl voriractin 910 mesh (copolymer of glycolic acid to lactate 90:10, PLGA), a biodegradable synthetic polymer, has a nitted mesh on the lumen side and a woven mesh on the outside (Woven mesh) A total of three sheets (0.2 mm each, 0.6 mm in total) were stacked, and a collagen-crosslinked PLGA-collagen composite membrane was used as a scaffold. A group in which only collagen type I was cross-linked using collagen as a cross-linking agent, and a group in which collagen SW was further cross-linked to peptide SWYGLR (SEQ ID NO: 2) were prepared. The crosslinking method is as follows. A 20 mm diameter patch is sewn to the main trunk of the pulmonary artery.

[0235] <Cross-linking method> The support is impregnated with a collagen solution. Next, freeze-dry and crosslink with dartal aldehyde saturated steam at 37 ° C for about 4 hours. Finally, the mixture was shaken three times in a 0.1 M glycine aqueous solution for 15 minutes, washed three times with distilled water, and freeze-dried. Thereby, a collagen-containing support is produced. The peptide SVVYGLR is immersed in this solution and crosslinked similarly.

 [0236] <Mechanical strength>

The strength of the support was measured with a tensile tester. A strip material having a width of 5 mm and a length of 30 mm was loaded in the short axis direction at a speed of 10 mmZ, and the load at break and the elastic modulus were measured. Were compared with (TENS ILLON ORIENTEC) ₀ control as glutaraldehyde treatment © Ma pericardium. The tensile strength was 75 ± 5N for the PLGA-collagen composite membrane and 34 ± 11N for dataraldehyde-treated pericardium. Membranes to which the angiogenic peptide was bound also had comparable tensile strengths.

 [0237] <Efficiency of cell adhesion>

 In order to confirm cell engraftment on the support, vascular endothelial cells (VECs) and smooth muscle cells (VSMCs) labeled with fluorescent antibodies (PKH-26 (SIGMA)) were injected into collagen at the in-vitro mouth. Cell adhesion efficiency was compared between a PLGA-collagen composite membrane with only cross-linking treatment and a PLGA-collagen composite membrane with collagen type I and cross-linked type IV. When comparing the coloring area (%) of the fluorescent dye per visual field with a fluorescence microscope, it was found that the type of vascular endothelial cells (VECs) and smooth muscle cells (VSMCs), and those of the slipped cells were also collagen type I and type IV. Cell engraftment was observed in the crosslinked-treated support of the present invention, in which the coloring region of the fluorescent dye was significantly large.

 [0238] From the above results, it was revealed that the prepared support maintained or enhanced the cell adhesion efficiency.

[0239] <Factor VIII staining>

The number of blood vessels can be determined by performing immunohistological staining with a factor VIII-related antigen or the like and then counting. In this counting method, a sample is fixed with 10% buffered formalin, embedded in paraffin, several serial sections are prepared from each sample, and frozen. The frozen sections were then treated with a 2% paraformaldehyde solution in PBS for 5 minutes at room temperature. Fix and immerse in methanol containing 3% hydrogen peroxide for 15 minutes, then wash with PBS. Cover the sample with a serum albumin solution for approximately 10 minutes to block nonspecific reactions. Specimens are incubated overnight with an EPOS conjugated antibody against factor VIII-related antigen, which binds to HRP. After washing the samples with PBS, they are immersed in a diaminobenzidine solution (eg, 0.3 mg Zml diaminobenzidine in PBS) to obtain positive staining. The stained vascular endothelial cells are counted, for example, under an optical microscope at a magnification of 200 times, and the counting result is expressed, for example, as the number of blood vessels per square millimeter. After a particular treatment, the presence of factor VIII is confirmed by determining whether the number of blood vessels is statistically significantly increased in calories, thereby confirming the presence of, for example, endothelial cells and vascular new life. Gender can be determined.

 [0240] Elastica 'Fan' Gieson Stain>

 Elastica 'Van' Giesson staining was performed to stain the elastic fibers. The procedure is as follows. If necessary, deparaffinize (for example, with pure ethanol), wash with water, and immerse the sample in a resorcin fuchsin solution available from Mutoh Chemical for 40-60 minutes. The sample is then washed with 70% alcohol and soaked in omni hematoxylin for 15 minutes. After that, rinse with running water for 5 minutes, and soak in Fan (1) 'Gison's solution for 2 minutes. Wash, quickly dehydrate, clear, enclose and confirm staining.

 [0241] Hematoxylin and eosin (HE) staining>

 HE staining was performed to observe the fixation of the support in the cells. The procedure is as follows. If necessary, the sample was deparaffinized (for example, with pure ethanol), washed with water, and immersed in omni hematoxylin for 10 minutes. After that, the sample was washed with running water, and colored with ammonia water for 30 seconds. After that, it is washed with running water for 5 minutes, stained with a 10-fold diluted solution of eosin hydrochloride for 2 minutes, dehydrated, cleared, sealed, and the staining can be confirmed.

 [0242] <von Kossa staining>

The cells were stained by the von Kossa method to observe calcification. The procedure is as follows. If necessary, remove paraffin (for example, with pure ethanol), wash with water (distilled water), and immerse in a 25% silver nitrate solution (under indirect light) for 2 hours. Then, wash with distilled water and soak in 42% 2% sodium thiosulfate (hypo) for 5 minutes. Then, wash with running water for 5 minutes Then soak for 5 minutes in Kärnechteroth. After that, it is washed with running water for 5 minutes, dehydrated, transparent, sealed, and stained.

[0243] <Porting>

 The prepared support and the support in which autologous vascular endothelial cells (VECs) and smooth muscle cells (VSMCs) were seeded on the composite membrane were prepared, and partially blocked in the main trunk of the pulmonary artery of adult beagle dogs (8 to: LOkg) (partial clamp).

[0244] Cells were isolated from the lower limb superficial veins of adult beagle dogs and isolated and cultured for vascular endothelial cells and squamous muscle cells (VSMCs). Each was seeded at a density of 1.3 × 10 ⁶ cellZcm ² . Two weeks, two months, and six months after transplantation, extirpation and histological examination are performed.

 [0245] (In vivo: 2 weeks after transplantation)

 Both the prepared support and the support seeded with autologous cells showed no apparent thrombus formation. HE staining showed PLGA remaining, with intervening connective tissue. In the PLGA collagen composite membrane seeded with autologous vascular endothelial cells and smooth muscle cells, the seeded vascular endothelial cells labeled with the fluorescent antibody are only scattered on the luminal side, and most of the cells are from the PLGA-collagen composite membrane. It was suggested that they had dropped out.

 [0246] (In vivo: 2 months after transplantation)

 In both groups, the prepared support and the PLGA collagen composite membrane seeded with autologous cells, the luminal surface was macroscopically smooth, and PLGA was completely absorbed by HE staining, which is inferior to that of normal blood vessels. It was an organizational structure.

 [0247] Vascular endothelial cells were examined by factor VIII staining and smooth muscle cell ex SMA immunostaining. In both groups, a continuous monolayer of vascular endothelial cells was observed by factor VIII immunostaining, and oriented smooth muscle cells were observed in the lumen by α-SΜΑ immunostaining.

 [0248] Further, the elastic fibers of the blood vessels were examined by elastica-van Gieson staining. In both groups, the expression of elastic fibers in the vascular lining was observed.

 [0249] Observation of the angiogenesis ability and the ability to form a vascular network reveals that the formation of the vascular network is enhanced even in the pulmonary artery of the heart.

[0250] As described above, the present invention has been exemplified using the preferred embodiment of the present invention. The description should not be construed as being limited to this embodiment. It is understood that the scope of the present invention should be construed only by the appended claims. It is understood that those skilled in the art can implement equivalent ranges based on the description of the present invention and common general knowledge from the description of the specific preferred embodiments of the present invention. Patents, patent applications, and references cited herein should be incorporated by reference in their entirety, as if the content itself were specifically described herein. Is understood.

Industrial applicability

 The present invention is useful in any aspect where it is desired to form an angiogenic ability and a vascular network (medicine, medicine, dentistry, etc.). In particular, the present invention is also useful for treating lifestyle-related diseases (eg, myocardial infarction, cerebral infarction, ischemic diseases such as obstructive aortic sclerosis).

Claims

The scope of the claims  [I] A composition for angiogenesis, comprising a peptide, comprising a VVXGL (SEQ ID NO: 1) t, wherein X is a variant of an aromatic amino acid.  [2] The composition according to claim 1, wherein the X is a modified form of hueralanine or tyrosine.  [3] The composition according to claim 1, wherein the modification includes a peptide in which the para, ortho, or meta position of the side chain of the aromatic amino acid is substituted with an electron-withdrawing substituent.  [4] The composition according to claim 1, wherein in X, an aromatic group of the aromatic amino acid is substituted with a hydrophobic group.  [5] The composition according to claim 1, wherein in X, an aromatic group of the aromatic amino acid is substituted with an alkyl group.  [6] The composition according to claim 1, wherein in X, the para-position of the aromatic group of the aromatic amino acid is substituted with an alkyl group. [7] The composition according to claim 1, wherein in X, the aromatic group of the aromatic amino acid is substituted with a C1-C6 alkyl group. [8] The composition according to claim 1, wherein in X, the aromatic group of the aromatic amino acid is substituted with a C1-C3 alkyl group. [9] The composition according to claim 1, wherein in X, an aromatic group of the aromatic amino acid is substituted with a methyl group.  [10] The composition according to claim 1, wherein the X is substituted with a methyl group at a para position of an aromatic group of the aromatic amino acid.  [II] The composition according to claim 1, wherein in X, the aromatic group of the aromatic amino acid is a phenyl group, and the aromatic amino acid is substituted with a para-methyl group.  [12] A thread-forming composition for forming a vascular network, comprising a peptide, which is a variant of an aromatic amino acid, comprising VVXGL and a sequence. 13. The composition according to claim 12, wherein said X is a modified form of phenalanine or tyrosine. [14] The modification is such that the para-, ortho-, or meta-position of the aromatic group of the aromatic amino acid has electron absorption. 13. The composition according to claim 12, comprising a peptide substituted with an attractive substituent. 15. The composition according to claim 12, wherein in X, an aromatic group of the aromatic amino acid is substituted with a hydrophobic group.  16. The composition according to claim 12, wherein in X, an aromatic group of the aromatic amino acid is substituted with an alkyl group.  17. The composition according to claim 12, wherein in X, the para-position of the aromatic group of the aromatic amino acid is substituted with an alkyl group. [18] The composition according to claim 12, wherein in X, the aromatic group of the aromatic amino acid is substituted with a C1-C6 alkyl group. [19] The composition according to claim 12, wherein in X, the aromatic group of the aromatic amino acid is substituted with a C1-C3 alkyl group. [20] The composition according to claim 12, wherein in X, an aromatic group of the aromatic amino acid is substituted with a methyl group.  [21] The composition according to claim 12, wherein the X is substituted with a methyl group at a para position of an aromatic group of the aromatic amino acid.  22. The composition according to claim 12, wherein X is an aromatic group of the aromatic amino acid, wherein the aromatic group is a phenyl group, and the para-position is substituted with a methyl group. [23] A peptide comprising VVXGL (SEQ ID NO: 1) t, wherein X is a modified aromatic amino acid.  24. The peptide according to claim 23, wherein said X is a modified form of phenalanine or tyrosine.  25. The peptide according to claim 23, wherein the modification includes a peptide in which the para, ortho, or meta position of the side chain of the aromatic amino acid is substituted with an electron-withdrawing substituent.  26. The peptide according to claim 23, wherein in X, an aromatic group of the aromatic amino acid is substituted with a hydrophobic group.  [27] The peptide according to claim 23, wherein X is such that an aromatic group of the aromatic amino acid is substituted with an alkyl group. [28] The above-mentioned X, wherein the para-position of the aromatic group of the aromatic amino acid is substituted with an alkyl group. 24. The peptide according to claim 23. 29. The peptide according to claim 23, wherein in X, the aromatic group of the aromatic amino acid is substituted with a C1-C6 alkyl group. [30] The peptide according to claim 23, wherein X is one in which an aromatic group of the aromatic amino acid is substituted with a C1-C3 alkyl group. [31] The peptide according to claim 23, wherein X is such that an aromatic group of the aromatic amino acid is substituted with a methyl group.  32. The peptide according to claim 23, wherein said X is substituted with a methyl group at a para position of an aromatic group of said aromatic amino acid.  33. The peptide according to claim 23, wherein in X, the aromatic group of the aromatic amino acid is a phenyl group, and the aromatic amino group is substituted with a para-methyl group. [34] A method for generating a blood vessel, which comprises administering the composition according to any one of claims 1 to 11 to a site of a patient in which angiogenesis is desired. [35] A method for forming a vascular network, comprising administering the composition according to any one of claims 12 to 22 to a site of a patient where formation of a vascular network is desired. [36] Use of a peptide comprising a sequence of VVXGL, wherein X is a variant of an aromatic amino acid or a variant or a salt thereof, for the production of a pharmaceutical composition for angiogenesis.  [37] For the production of a pharmaceutical composition for forming a vascular network of a peptide, which comprises VVXGL and a sequence, wherein X is a variant of an aromatic amino acid or a variant or a salt thereof. Use of.