WO2005099742A1 - Vascular network forming agent - Google Patents

Abstract

An object of the present invention is to search for a substance having an ability to form a vascular network. The present invention provides a novel vascular network forming agent comprising such a substance as an effective ingredient, which can be used in clinical applications, such as therapy for humans, and the like. A composition is provided for forming a network of blood vessels, comprising a peptide having an amino acid sequence X1-X2-X3-X4-X5-X6-X7 (SEQ ID NO.: 14), where X1 is serine (S), threonine (T), or a variant thereof, or is absent, X2 is valine (V), alanine (A), glycine (G), leucine (L), isoleucine (I), or a variant thereof, X3 is valine(V), alanine (A), glycine (G), leucine (L), isoleucine (I), or a variant thereof, X4 is an amino acid having an aromatic ring as a side chain or a variant thereof, X5 is glycine (G) or a variant thereof, X6 is leucine (L), alanine (A), glycine (G), valine (V), isoleucine (I), or a variant thereof, X7 is arginine (R), lysine (K), or a variant thereof, or is absent; or a variant or salt thereof.

Description

DESCRIPTION

VASCULAR NETWORK FORMING AGENT TECHNICAL FIELD

The present invention relates to a vascular network forming agent having an activity of forming a network of blood vessels. The vascular network forming agent of the present invention is useful for regeneration and repair of various tissue failures as well as treatment of ischemic diseases .

BACKGROUND ART

Various researches have been made to find agents involved in vascular network formation, vascular formation, or the like. Both inhibitory action and activating action have been utilized for the treatment and prophylaxis of various diseases and disorders (e.g., cancer, ischemic disease, etc.). However, despite angiogenesis, sufficient circulation cannot be obtained unless a peripheral network is formed. Ischemic diseases caused by occlusion off blood vessels, such as myocardial infarction, brain infaarction, or the like, are currently major causes of death in de-veloped countries. Ischemia may not cause death but may -lead to diseases which impair quality of life (QOL ) , such as occlusive aortosclerosis which forces the patient to have his/her lower limb amputated. Vascular network forming therapy by newly forming blood vessels is greatly expected to trea^~t these ischemic diseases . Simple vascular formation is not necessarily sufficient. It is desirable that bloodvessels actually form a network so that sufficient nutrients reach peripheral sites and materials are smoothly exchanged. Peptides have great advantages in terms of safety against side effects, metabolism, and the like. Peptides are relatively easy to design. Highly-efficient synthesis techniques and testing methods have been established for peptides. In addition, amino acid derivatives are convenient building units for construction of combinatorial chemical libraries and can be used to optimize the structure of peptides in a short time using a solid-phase synthesis method- Therefore, if a relatively low molecular weight peptide having a network forming activity exists, it can be advantageously synthesized and administered singly or in combination with other materials. However, no low molecular weight peptide or peptidometic compound having a network forming activity has been previously known. DISCLOSURE OF THE INVENTION

Therefore, an object of the present invention is to search for a substance having an ability to form a vascular network. Another object of the present invention is to provide a novel vascular network forming agent comprising such a substance as an effective ingredient , which can be used in. clinical applications , such as therapy for humans , and the like. After diligent research, the present inventors unexpectedly found a peptide having a particular amino acid sequence, which has a vascular network forming activity. The present invention provides the following.

1. A composition for forming a network of blood vessels, comprising: a peptide having an amino acid sequence

Xι-X₂-X₃-X₄-X₅-X₆-X₇ (SEQ ID NO.: 14), where Xi is serine (S), threonine (T), or a variant thereof, or is absent, X₂ is valine (V) , alanine (A) , glycine (G), leucine (L), isoleucine (I), or a variant thereof, X₃ is valine(V), alanine (A), glycine (G), leucine (L), isoleucine (I), or a variant thereof, X₄ is an amino acid having an aromatic ring as a side chain or a variant thereof, X₅ is glycine (G) or a variant thereof, X₆ is leucine (L), alanine (A), glycine (G) , valine (V), isoleucine (I), or a variant thereof, X is arginine (R) , lysine (K) , or a variant thereof, or is absent, or a variant or salt thereof.

2. A composition according to item 1, wherein the peptide or avariant thereof has a vascular network formation activity of 3.0 or more according to the vascular network formation index.

3. A composition according to item 1, wherein Xx is serine or a variant thereof.

4. A composition according to item 1, wherein X₂ is valine or a variant thereof .

5. A composition according to item 1, wherein X₃ is valine or a variant thereof .

6. A composition according to item 1, wherein X is phenylalanine, tyrosine, or a variant thereof.

7. A composition according to item 1, wherein X is phenylalanine or a variant thereof.

8. A composition according to item 1, wherein X₄ is tyrosine or a variant thereof .

9. A composition according to item 1, wherein X₅ is glycine or a variant thereof.

10. A composition according to item 1, wherein Xe is leucine or a variant thereof .

11. A composition according to item 1 , wherein X₇ is arginine or a variant thereof .

12. A composition according to item 1, wherein the peptide or a variant thereof is an amino acid sequence set forth in SEQ ID NO. : 1, or an amino acid sequence set forth in SEQ ID NO.: 1 having 1 to 3 amino acid substitutions, or 1 or 2 amino acid deletions at one or both ends thereof, or another amino acid sequence added to or at least one amino acid addition at one or both ends thereof, and has a vascular network forming activity.

13. A composition according to item 1, wherein the peptide or avariant thereof: is apeptide having an amino acid sequence set forth in SEQ ID NO. : 1, or an amino acid sequence set forth in SEQ ID NO. : 1 having 1 or 2 amino acid substitutions wherein the fourth tyrosine residue is a tyrosine residue or an amino acid having an aromatic ring as a side chain, or 1 amino acid deletion at one or both ends thereof, or another amino acid sequence added to or at least one amino acid addition at one or both ends thereof, and has a vascular network forming activity.

14. A composition according to item 1, wherein the amino acid having an aromatic ring as a side chain is phenylalanine or chemically modified phenylalanine having 1 or more substituents on a benzene ring thereof.

15. A composition according to item 1, wherein the peptide or a variant thereof is a peptide having an amino acid sequence set forth in SEQ ID NO.: 9, or an amino acid sequence set forth in SEQ ID NO. : 9 having 1 amino acid deletion at one or both ends thereof, or another amino acid sequence added to or at least one amino acid addition at one or both ends thereof after 1 amino aciddeletion at one orbothends thereof .

16. A composition according to item 1, wherein the peptide or a variant thereof is a peptide having an amino acid sequence set forth in any one of SEQ ID NOs . : 1 to 7, or an amino acid sequence set forth in any one of SEQ ID NOs . : 1 to 7 having another amino acid sequence added to or at least one amino acid addition at one or both ends thereof.

17. A composition according to item 1, wherein the peptide or a variant thereof is a peptide having an amino acid sequence set forth in any one of SEQ ID NOs.: 9 to 11, or an amino acid sequence set forth in any one of SEQ ID NOs.: 9 to 11 having another amino acid sequence added to or at least one amino acid addition at one or both ends thereof.

18. A composition according to item 1, wherein the peptide or a variant thereof is a peptide having an amino acid sequence set forth in SEQ ID NO. : 9, or an amino acid sequence set forth in SEQ ID NO. : 9 having another amino acid sequence added to or at least one amino acid addition at one or both ends thereof .

19. A composition according to item 1, wherein the peptide or a variant thereof comprises an amino acid sequence represented by SWX₄GL or WX₄GLR where X₄ is an amino acid having an aromatic ring as a side chain or a variant thereof.

20. A composition according to item 1, wherein the total number of amino acid residues in the peptide or a variant thereof is 4 to 350.

21. A composition according to item 1, wherein the total number of amino acid residues in the peptide or a variant thereof is 4 to 50.

22. A composition according to item 1, wherein the total number of amino acid residues in the peptide or a variant thereof is 5 to 20.

23. A composition according to item 1, wherein the peptide or a variant thereof is a pepticϋe having an amino acid sequence set forth in any one of SEQ ID NOs.: 1 to 7, or an amino acid sequence set forth in any one of SEQ ID NOs . : 1 to 7 having 10 or less amino acid additions at one or both ends thereof.

24. A composition according to item 1, wherein the peptide comprises an amino acid sequence set forth in any one of SEQ ID NOs . : 1 to 7. 25. A composition according to item 1, wherein the peptide or a variant thereof is a peptide having an amino acid sequence set forth in any one of SEQ ID NO.: 9 to 11, or an amino acid sequence set forth in any one of SEQ ID NO. : 9 to 11 having 10 or less amino acid additions at one or both ends thereof.

26. A composition according to item 1, wherein the peptide or a variant thereof comprises an amino acid sequence set forth in any one of SEQ ID NOs.: 9 to 11.

27. A composition according to item 1, wherein the blood vessel is a capillary blood vessel.

28. A composition according to item 1, wherein the peptide or a variant thereof is bound to a carrier.

29. A composition according to item 28, wherein the carrier is a protein.

30. A composition according to item 29, wherein the protein is a cell adhesion protein.

31. A composition according to item 30, wherein the cell adhesion protein is collagen or a partial hydrolysis product thereof .

32. A method for forming a network of blood vessels, comprising the step of: administering a composition according to any one of items 1 to 31 to a site in a patient in need of formation of the network of blood vessels. 33. Use of: a peptide having an amino acid sequence X1-X2-X3-X4-X5-X6-X7 (SEQ ID NO. : 14), where Xi is serine (S) , threonine (T) , or a variant thereof, or is absent, X₂ is valine (V), alanine (A), glycine (G) , leucine (L), isoleucine (I), or a variant thereof, X₃ ^' is valine(V), alanine (A), glycine (G), leucine (L), isoleucine (I), or a variant thereof, X₄ is an amino acid having an aromatic ring as a side chain or a variant thereof, X₅ is glycine (G) or a variant thereof, Xs is leucine (L), alanine (A), glycine (G) , valine (V) , isoleucine (I), or a variant thereof, X₇ is arginine (R), lysine (K) , or a variant thereof, or is absent; or a variant or salt thereof, for production of a medicament for forming a network of blood vessels. According to the present invention, a peptide having a potent vascular network forming activity was found, and a novel vascular network forming agent comprising the peptide as an effective ingredient was first provided. The vascular network forming agent of the present invention is useful for the regeneration/repair of organs using biological substituting materials, such as artificial bones and the like, and the treatment of ischemic diseases, such as myocardial infarction, brain infarction, occlusive aortosclerosis, and the like, which are predominantly implicated in lifestyle related diseases.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 shows the results of examination of a peptide synthesized in Example 1 using a coupled liquid chromatography mass spectrometry (LCMS) system. The result is consistent with a theoretical molecular weight of the peptide SWGLR. Figure 2 is a schematic diagram showing the formation of a lumen from adherent cells . Figure 3 compares the lumen forming activity of VEGF and peptide SWYGLR (SEQ ID NO.: 1).

Figure 4 schematically shows a protocol for a DAS assay.

Figure 5 shows angiogenesis induced by peptide SWYGLR (SEQ ID NO. : 1).

Figure 6 shows tissue around amicro cell 5 days after implantation using a DAS assay, which was observed under a microscope.

Figure 7 shows a DAS assay for alanine scanning. Figure 8 shows the presence or absence of heat resistance when temperature is increased.

Figure 9 shows comparison of the vascular network forming ability of the peptides of the present invention.

Figures 10A and 10B show exemplary modified aromatic amino acids. Ala scan assays or deletion assays revealed that Y plays an important role in angiogenesis . Attention was focused onto the benzene ring of Y. Y is substituted with, other amino acids (Figure 10A) . Tyr (Y) is obtained by adding -OH to the benzene ring of Phe (F) (Figure 10B) . -F, -CH₃, or -N0₂ is bound to the benzene ring of Phe (F) . Figure 11 shows an exemplary result of counting the number; of new blood vessels using a DAS assay.

Figure 12 shows another exemplary result of counting the number of new blood vessels using a DAS assay.

BRIEF DESCRIPTION OF THE SEQUENCE LISTING

SEQ ID NO. : 1 sets forth the sequence of a peptide (SWYGLR) having a vascular network forming ability in osteopontin .

SEQ ID NO. : 2 sets forth a peptide AWYGLR which is a modification of SEQ ID NO. : 1.

SEQ ID NO. : 3 sets forth a peptide SAVYGLR which is a modification of SEQ ID NO. : 1.

SEQ ID NO. : 4 sets forth a peptide SVAYGLR which is a modification of SEQ ID NO. : 1.

SEQ ID NO. : 5 sets forth a peptide SWYALR which is a modification of SEQ ID NO. : 1. SEQ ID NO. : 6 sets forth a peptide SWYGAR which is a modification of SEQ ID NO. : 1.

SEQ ID NO. : 7 sets forth a peptide SWYGLA which is a modification of SEQ ID NO. : 1.

SEQ ID NO . : 8 sets forth a peptide S AGLR which is a modification of SEQ ID NO . : 1 . SEQ ID NO- : 9 sets forth a peptide SWFGLR which is a modification of: SEQ ID NO. : 1.

SEQ ID NO- : 10 sets forth a peptide S YGL which is a modification off SEQ ID NO. : 1.

SEQ ID NO- : 11 sets forth a peptide YGLR which is a modification of SEQ ID NO. : 1. SEQ ID NO . : 12 sets forth a peptide SWYGLRC which is a modification of SEQ ID NO.: 1.

SEQ ID NO . : 13 sets forth a peptide GRGDS YGLR which is a modification of SEQ ID NO. : 1.

SEQ ID NO . : 14 sets forth a schematic sequence of the peptide of the present invention.

SEQ ID NO.: 15 sets forth an amino acid sequence SWX₄GL which is a preferable sequence of the peptide of the present invention .

SEQ ID NO . : 16 sets forth an amino acid sequence WX4GLR which is a preferable sequence of the peptide of the present invention.

SEQ ID NO . : 17 sets forth a peptide SWYG which is an exemplary modification of SEQ ID NO. : 1. SEQ ID NO . : 18 sets forth a peptide SWWGLR which is an exemplary modification of SEQ ID NO.: 1.

SEQ ID NO. : 19 sets forth a peptide SVVF(pF)GLR which is an exemplary modification of SEQ ID NO.: 1 (F(pF) represents a fluorine group at the para position on the side chain of phenylalanine ) . SEQIDNO.: 20 sets forth apeptide SWF (pMe)GLRwhich is an exemplary modification of SEQ ID NO. : 1 (F(pMe) represents a methyl group at the para position on the side chain of phenylalanine ) . SEQ ID NO.: 21 sets forth a peptide SWF (pN0₂ )GLR which is an exemplary modification of SEQ ID NO. : 1 (F(pN0₂) represents a nitro group at the para position on the side chain of phenylalanine ) . SEQ ID NO. : 22 sets forth a peptide WYGL which is an exemplary modification of SEQ ID NO.: 1.

SEQIDNO.: 23 sets forth a peptide WF(pMe)GL which is an exemplary modiffication of SEQ ID NO. : 1.

SEQ ID NO. : 24 sets forth a peptide WdFGL which is an exemplary modification of SEQ ID NO.: 1 (dF represents D-phenylalanine) . SEQ ID NO. : 25 sets forth a peptide WF(pF)GL which is an exemplary modiffication of SEQ ID NO. : 1.

SEQIDNO.: 26 sets forth a peptide WF(pN0₂)GL which is an exemplary modiffication of SEQ ID NO.: 1.

BEST MODE FOR CARRYING OUT THE INVENTION

The present invention will be described below. It should be understood throughout the present specification that articles for singular- forms include the concept of their plurality unless otherwise mentioned. Therefore, articles or adjectives for singular forms (e.g., "a", "an", "the", andthe like in English) include the concept of theirplurality unless otherwise specified. Also, it should be also understood that terms as used herein have definitions ordinarily used in the art unless otherwise mentioned. Therefore, all technical and scientific terms used herein have the same meanings as commonlyunderstoodby those skilled in the relevant art. Otlierwise, the present application (including definitions) takes precedence.

(Terms ) The terms "protein" , "polypeptide", "oligopeptide" and "peptide" as used herein have the same meaning and refer to an amino acid polymer having any length. This polymer may be a straight, branched or cyclic chain. An amino acid maybe a naturally-occurring or nonnaturally-occurring amino acid, or a variant amino acid. The term may include those assembled into a composite of a plurality of polypeptide chains. The term also includes a naturally-occurring or artificially modified amino acid polymer. Such modification includes, for; example, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification (e.g., conjugation with a labeling moiety) - This definition encompasses a polypeptide containing at least one amino acid analog (e.g. , nonnaturally-occurring amino acid, etc.), a peptide-like compound (e.g., peptoid) _M and other variants known in the art , for example .

The terms "polynucleotide", "oligonucleotide", and "nucleic acid" as used herein have the same meaning and refer to a nucleotide polymer having any length. This term also includes an "oligonucleotide derivative" or a "polynucleotide derivative". An "oligonucleotide derivative" or a "polynucleotide derivative" includes a nucleotide derivative, or refers to an oligonucleotide or a polynucleotide having different linkages between nucleotides fromtypical linkages , which are interchangeably used. Examples of such an oligon cleotide specifically include 2 ' -O-methyl-ribonucleotide, an oligonucleotide derivative in which a phosphodiester bond in an oligonucleotide is converted to a iphosphorothioate bond, an oligonucleotide derivative in which a phosphodiester bond in an oligonucleotide is converted to a N3^r-P5' phosphoroamidate bond, an oligonucleotide derivative in which aribose andaphosphodiesterbond in an oligonucleotide are converted to a peptide-nuσleic acid bond, an oligonucleotide derivative in which uracil in an oligonucleotide is substituted with. C-5 propynyl uracil, an oligonucleotide derivative in which uracil in an oligonucleotide is substituted with. C-5 thiazole uracil, an oligonucleotide derivative in which cytosine in an oligonucleotide is substituted with C-5 propynyl cytosine, an oligonucleotide derivative in which cytosine in an oligonucleotide is substituted with phenoxazine-modi ied cytosine, an oligonucleotide derivative in which ribose in DNA is substituted with 2'-0-propyl ribose, and an oligonucleotide derivative in which ribose in an oligonucleotide is substitutedwith 2 ' -methoxyethoxy ribose. Unless otherwise indicated, a parrticular nucleic acid sequence also implicitly encompasses conservatively-modified variants thereof (e.g. degenerate codon substitutions ) and complementary sequences as well as the sequence explicitly indicated. Specifically, degenerate codon substitutions may be produced by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed-base and/or deoxyinosine residues (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) ) .

As used herein, the term "composite molecule" refers to a molecule in which a plurality of molecules, such as polypeptides, polynucleotides, -Lipids, sugars, small molecules, or the like, are linked together. Examples of a composite molecule include, but: are not limited to, glycolipids, glycopeptides , and the like. The peptide of the present invention includes composite products obtained by linking such sugar chains .

As used herein, the term "isolated" biological agent (e.g., nucleic acid, protein, or the like) refers to a biological agent that is substantial ly separated or purified from other biological agentεs in cells of a naturally-occurring organism (e.g. , in the case of nucleic acids, agents other than nucleic acids and a nucleic acid having nucleic acid sequences other than an intended nucleic acid; and in the case of proteins, agents other than proteins and proteins having an amino acid sequence other than an intended protein) . The "isolated" nucleic acids and proteins include nucleic acids and proteins puri ied by a standard purification method. The isolated nucleic acids and proteins also include chemically synthesized nucleic acids and proteins .

As used herein, the term "purified" biological agent (e.g., nucleic acids, proteins, and the like) refers to one from which at least a part of naturally accompanying agents is removed. Therefore, ordinarily, the purity of a purified biological agent is higher than that of the biological agent in a normal state (i.e., concentrated).

As used herein, the terms "purified" and "isolated" mean that the same type of biological agent is present preferably 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.

As used herein, the term "homology" in relation to a gene (e.g., a nucleic acid sequence, an amino acid sequence, etc.) refers to the proportion of identity- between two or more gene sequences . Therefore , the great er the homology between two given genes , the greater tlie identity or similarity between their sequences . Whether: or not two genes have homology is determined by comparing their sequences directly or by a hybridization method under stringent conditions. As used herein, the term "similarity" in relation to a gene (e.g. , a nucleic acid sequence, an amino acid sequence, or the like) refers to the proportion of identity between two or more sequences when conservative substitution is regarded as positive (identical) in the above-described homology. Therefore, homology and similarity differ from each other in tlie presence of conservative substitutions. If no conservative substitutions are present, homology and similarity have the same value.

As used herein, the term "amino acid" is used in its broadest sense in the art and refers to an organic compound having a carboxy group and an amino group. Amino acids used herein may be either naturally-occurring or nonnaturally-oσcuring . The term "naturally-occurring amino acid'^r refers to an L-isomer of a naturally-occurring amino acid. The naturally-occurring amino acids are glycine, alanine, valine, leucine, isoleucine, serine, methionine, threonine, phenylalanine, tyrosine, tryptophan, cysteine, proline, histidine, aspartiσ acid, asparagine, glutamic acid, glutamine, γ-σairboxyglutamic acid, arginine, ornithine, and lysine. Unless otherwise indicated, all amino acids as used herein are L-isomers. An embodiment using a D-isomer of an amino acid falls within the scope of the present invention.

As used herein, the term "amino acid variant" refers to a molecule which is not a naturally-occurring amino acid and has a physical property and/or a function ssimilar to that of a naturally-occurring amino acid. Examples of amino acid variants include amino acids with a hydrophobic group (e.g., an alkyl group, etc. ) introduced into the side chain thereof; phenylalanine with an alkyl group, a halogen group, a nitro group or the like linked to a benzyl side chain thereof (para position, meta position, ortho position, etc.); ethionine; canavanine; 2-methylglutamine; and the like. In the present invention, it will be understood that amino acid variants encompass nonnaturally-occurring amino acids and amino acid mimics . The term "nonnaturally-occurring amino acid" refers to an amino acid which is ordinarily not found in nature. Examples of nonnaturally-oσcurring amino acids include norleuσine, paxra-nitrophenylalanine, homophenylalanine, para-fluorophenylalanine, 3-amino-2-benzyl propionic acid, D- or L-homoarginine, and D-phenylalanine.

As used herein, the term "amino acid mimic" refers to a compound which has a structure different from that of the general chemical structure of amino acids - but which functions in a manner similar to that of naturally—occurring amino acids . As used herein, the term "nucleotide" may be either naturally-occurring or nonnaturally-occurring as long as it has an ability to encode amino acids .

As used herein, the term "fragment" with irespect to a polypeptide or polynucleotide refer to a polyjpeptide or polynucleotide having a sequence length ranging from 1 to n-1 with respect to the full length of the reference polypeptide or polynucleotide (of length n) . The length of the fragment can be appropriately changed depending on the purpose . For example , in the case of polypeptides , the lower limit of the length of the fragment includes 3 , 4 , 5 , 6 , 7, 8, 9, 10, 15, 20, 25, 30, 40, 50 or more nucleotides. Lengths represented by integers which are not herein specified (e.g., 11 and the like) may be appropriate as a lower limit. For example, in the case of polynucleotides, the lower limit of the length of the fragment includes 5 , 6, 7, 8, 9, 10, 15, 20, 25, 30, 40, 50, 75, 100 ormore nαcleotides . Lengths represented by integers which are not herein specified (e.g., 11 and the like) may be appropriate as a lower limit. As used herein, the length of polypeptides or polynucleotides can be represented by the numbear of amino acids or nucleic acids, respectively. However, the above-described numbers are not absolute. The above-described numbers as the upper or lower limit are intended to include some greater or smaller numbers (e.g_ , ±10%) , as long as the same function is maintained. For this purpose, "about" may be herein put ahead of the numbers. However, it should be understood that the interpretation of numbers is not affected by the presence or absence of "about" in the present specification.

As used herein, the term "corresponding" amino acid or nucleic acid refers to an amino acid or nucleotide in a given polypeptide or polynucleotide molecule, which has, or is anticipated to have, a function similar to that of a predetermined amino acid or nucleotide in a polypepticϋe or polynucleotide as a reference for comparison.. Particularly, in the case of enzyme molecules, the term referrs to an amino acid which is present at a similar position in an active site and similarly contributes to catalytic activity. For example, in the case of antisense molecul s for a certain polynucleotide, the term refers to a similar portion in an ortholog corresponding to a particular portion of the antisense molecule. In the case of the peptide of the present invention, a particular sequence of human osteopontin is used. However, it will be understood that a particular sequence of osteopontin of other species animals may have "corresponding amino acids" corresponding to a portion of the peptide of the present invention.

As usedherein, the term "corresponding" gene (e.g. , a polypeptide or polynucleotide molecule) refers to a genie in a given species, which has, or is anticipated to have, a function similar to that of a predetermined gene in a species as a reference for comparison. When there are a plurality of genes having such a function, the term refers to a gerte having the same evolutionary origin. Therefore, a gene corresponding to a given gene may be an ortholog of the given gene. Such a corresponding gene can be identified by techniques well known in the art. Therefore, for example, a corresponding gene in a given animal canbe foundby searching a sequence database of the animal (e.g., human, rat) using the sequence of a reference gene (e.g., mouse osteopontin gene, etc.), or the peptide of the present invention or a code sequence thereof as a query sequence.

Amino acids may be referred to herein by either theix: commonly known three letter symbols or by the one-lettear symbols recommended by the IUPAC-IUB Biochemical

Nomenclature Commission. Nucleotides, likewise, may be referred to by their commonly accepted single-letter codes -

The symbols are described below.

Amino acids :

Three-letter One-letter Meaning symbol symbol

Ala A alanine

Cys C cysteine

Asp D aspartic acid

Glu E glutamic acid

Phe F phenylalanine

Gly G glycine

His H histidine

He I isoleucine

Lys K lysine

Leu L leucine

Met M methionine

Asn N asparagine Pro P proline Gin Q glutamine Arg R arginine Ser S serine Thr T threonine Val V valine Trp W tryptophan Tyr Y tyrosine Asx asparagine or asparagic acid

Glx glutamine or glutamic acid Xaa unknown or other amino acids .

Bases:

Symbol Meaning a adenine g guanine c cytosine t thymine u uracil r guanine or adenine purine y thymine/uracil or cytosine pyrimidine m adenine or cytosine amino group k guanine or thymine/uracil keto group s guanine or cytosine w adenine or thymine/uracil b guanine or cytosine or thymine/uracil d adenine or guanine or thymine/uracil h adenine or cytosine or thymine/uracil v adenine or guanine or cytosine n adenine or guanine or cytosine or thymine/uracil, unknown, or other bases, The similarity, identity and homology of amino acid sequences and base sequences are herein compared using BLAST (sequence analyzing tool) with the default parameters.

Molecular biological techniques , biochemical techniques, and microorganism techniques as used herein are well known in the art and commonly used, and are described in, for example, SambrookJ. etal. ( 1989 ), Molecular Cloning: ALaboratoryManual, ColdSpringHarbor and its 3rd Ed. (2001); Ausubel, F.M. (1987), Current Protocols inMolecularBiology, Greene Pub. Associates andWiley-interscienσer Ausubel, F.M. (1989), Short Protocols in Molecular Biology: A Compendium of Methods from Current Protocols in Molecular Biology, Greene Pub. Associates and Wiley-interscience; Innis, M.A. (1990) , PCR Protocols: A Guide to Methods and Applications, Academic Press; Ausubel, F.M. (1992), Short Protocols in Molecular Biology: A Compendium of Methods from Current Protocols in Molecular Biology, Greene Pub . Associates; Ausubel, F.M. (1995), Short Protocols in Molecular Biology: A Compendium of Methods from Current Protocols in Molecular Biology, Greene Pub. Associates; Innis, M.A. et al. (1995), PCR Strategies, Academic Press ; Ausubel, F.M. (1999), Short Protocols in Molecular Biology: A Compendium of Methods from Current Protocols in Molecular Biology, Wiley, and annual updates; Sninsky, J.J. et al. (1999), PCR Applications: Protocols for Functional Genomics, Academic Press; Special issue, Jikken Igaku [Experimental Medicine] ™ Idenshi Donyu & Hatsugenkaiseki Jikkenho [Experimental Method for Gene introduction & Expression Analysis ]" , Yodo-sha, 1997; and the like. Relevant portions (or possibly the entirety) of each of these publications are herein incorporated by reference . (Modification) It will be understood that the present invention encompasses modifications . Whenmodifications are designed, the hydrophobicity indices of amino acids may be taken into consideration. The hydrophobic amino acid indices play an important role in providing a protein with an interactive biological function, which is generally recognized in the art (Kyte, J. and Doolittle, R.F. , J. Mol. Biol. 157(1) :105-132, 1982) . The hydrophobic property of an amino acid contributes to the secondary structure of a protein and then regulates interactions between the protein and other molecules (e.g., enzymes, substrates, receptors, DNA, antibodies, antigens, etc.). Each amino acid is given a hydrophobicity index based on the hydrophobicity and charge properties thereof as follows: isoleucine (+4.5); valine (+4.2); leucine (+3.8); phenylalanine (+2.8); cysteine/cystine (+2.5); methionine (+1.9); alanine (+1.8) ; glycine (-0.4); threonine (-0.7); serine (-0.8); tryptophan (-0.9); tyrosine (-1.3); proline (-1.6); histidine (-3.2); glutamic acid (-3.5); glutamine (-3.5); asparticacid (-3.5); asparagine (-3.5); lysine (-3.9); and arginine (-4.5).

It is well known that if a given amino acid is substituted with another amino acid having a similar hydrophobicity index, the resultant protein may still have a biological function similar to that of the original protein (e.g. , a protein having an equivalent enzymatic activity) . For such an amino acid substitution, 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 an amino acid substitution based on hydrophobicity is efficient . A hydrophilicity index is also useful for modification of an amino acid sequence of the present invention. As described in US Patent No. 4,554,101, amino acidresidues are given the followinghydrophilicityindices arginine (+3.0); lysine (+3.0); aspartic acid (+3.0±1) glutamic acid (+3.0±1); serine (+0.3); asparagine (+0.2) glutamine (+0.2); glycine (0); threonine (-0.4); proline ( -0.5±1) ; alanine (-0.5); histidine (-0.5); cysteine (-1.0); methionine (-1.3); valine (-1.5); leucine (-1.8) ; isoleucine (-1.8); tyrosine (-2.3); phenylalanine (-2.5); and tryptophan (-3.4) . It is understood that an amino acid may be substituted with another amino acid which has a similar hydrophilicity index and can still provide a biological equivalent. For such an amino acid substitution, the hydrophilicityindex is preferablywithin ±2 , more preferably ± 1 , and even more preferably ±0.5.

The term "conservative substitution" as used herein refers to amino acid substitution in which a substituted amino acid and a substituting amino acid have similar hydrophilicity indices or/and hydrophobicity indices . For example, the conservative substitution is carriedout between amino acids having a hydrophilicity or hydrophobicity index of within ±2, preferablywithin ±1, andmore preferablywithin ±0.5.

It will be understood that conservative substitution may also be herein performed between, for example, hydrophobic amino acids (alanine, valine, leucine, isoleucine, etc. ) , between acidic amino acids (glutamic acid, aspartic acid, 4-carboxyglutamic acid, amino citric acid, etc. ) , betweenbasic amino acids (arginine, histidine, lysine. etc.), or between aromatic amino acids (phenylalanine, tyrosine, tryptophan, etc.). Examples of the conservative substitution include, but are not limited to, substitutions within each of the following residue pairs : arginine and lysine; glutamic acid and aspartic acid; serine and threonine; glutamine and asparagine; and valine, leucine, and isoleucine, which are well known to those skilled, in the art . As used herein, the term "hydrophobic group" refers to a substituent which increases a hydrophobicity index ( see Kyte, J. and Doolittle, R.F., J. Mol. Biol. 157( 1) : 105- 132 , 1982) or logP (the logarithm of the distribution coefficient of 1-octanol/water) higher than before substitut on. Examples of hydrophobic groups include, but are not limited to, an alkyl group, an alkenyl group, and the like. As iised herein, the term "hydrophobic amino acid" refers to an amino acid having a hydrophobic index of "-2" or more (see Kyte, J . and Doolittle, R.F. , J. Mol. Biol.157(1) :105-132, 1982) , excluding polar and neutral amino acids (e.g., naturally-occurring amino acids Ser, Thr, Tyr, and C^ys ) .

As used herein, the term "variant" refers to a substance, such as a polypeptide, polynucleotide, or the like, which differs partially from the original substance. Examples of such a variant include a substitution variant , an addition variant , a deletion variant , a truncatedvariant , an allelic variant , and the like . Examples of such a variant include, but are not limited to, a nucleotide or polypeptide having one or several substitutions, additions and/or deletions or a nucleotide or polypeptide having at least one substitution, addition and/or deletion. The term "allele" as used herein refers to a genetic variant located at a locus identical to a corresponding gene, where the two genes are distinguishedfrom each other. Therefore, the term "allelic variant" as used herein refers to a variant which has an allelic relationship with a given gene. Such an allelic variant ordinarily has a sequence the same as or highly similar to that of the corresponding allele, and ordinarily has almost the same biological activity, though it rarely has different biological activity. The term "species homolog" or "homolog" as used herein refers to one that has an amino acid or nucleotide homology with a given gene in a given species (preferably at least 60% homology, more preferably at least 80%, at least 85%, at least 90%, and at least 95% homology) . A method for obtaining such a species homolog is clearly understood from the description of the present specification. The term "orthologs" (also called orthologous genes) refers to genes in different species derived from a common ancestry (due to speciation) . For example, in the case of the hemoglobin gene family having multigene structure, human and mouse -hemoglobin genes are orthologs, while the human α-hemoglobin gene and the human β-hemoglobin gene are paralogs (genes arising from gene duplication) . Orthologs are useful for estimation of molecular phylogenetiσ trees. Usually, orthologs in different species may have a function similar to that of the original species. Therefore, orthologs of the present invention may be useful in the present invention.

As used herein, the term "conservative (or conservatively modified) variant" applies to both amino acid and nucleic acid sequences. With respect to particular nucleic acid sequences, conservatively modified variants refer to those nucleic acids which encode identical or essentially identical amino acid sequences. Because of the degeneracy of the genetic code, a large number of 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, the codon can be altered to any of the corresponding codons described without altering the encoded polypeptide. Such nucleic acid variations are "silent variations" which represent one species of conservatively modified variation . Every nucleic acid sequence herein which encodes a polypeptide also describes every possible silent variation of the nucleic acid. Those skilled in the art will recognize that each codon in a nucleic acid (except AUG, which is ordinarily the only codon for methionine, and TGG, which is ordinarily the only codon for tryptophan ) can be modified to yield a functionally identical molecule. Accordingly, each silent variation of a nucleic acidwhich encodes apolypeptide is implicit in each described sequence. Preferably, such modification may be performed while avoiding substitution of cysteine which is an amino acid capable of largely affecting the higher-order structure of a polypeptide . Examples of a method for such modification of a base sequence include cleavage using a restriction enzyme or the like; ligation or the like by treatment using DNA polymerase, Klenow fragments, DNA ligase, or the like; and a site specific base substitution method using synthesized oligonucleotides (specific-site directed mutagenesis; Mark Zoller and Michael Smith, Methods in Enzymology, 100, 468-500(1983)). Modification can be performed using methods ordinarily used in the field of molecular biology.

In order to prepare functionally equivalent polypeptides, amino acid additions, deletions, or modifications can be performed in addition to amino acid substitutions. Amino acid substitution ( s) refers to the replacement of at least one amino acid of an original peptide with different amino acids, such as the replacement of 1 to 3 amino acids with different amino acids . Amino acid addition(s) refers to the addition of at least one amino acid to an original peptide chain, such as the addition of 1 to 3 amino acids to an original peptide chain. Amino acid deletion(s) refers to the deletion of at least one amino acid, such as the deletion of 1 to 3 amino acids. Amino acid modification includes, but is not limited to, amidation, carboxylation, sulfation, halogenation, truncation, lipidation, alkylation, glycosylation, phosphorylation, hydroxylation, acylation (e.g., acetylation) , and the like. Amino acids to be substituted or added may be naturally-occurring or nonnaturally-occurring amino acids, or amino acid analogs. Naturally-occurring amino acids are preferable .

As used herein, the term "substitution, addition or deletion" for a polypeptide or a polynucleotide refers to the substitution, addition or deletion of an amino acid or its substitute, or a nucleotide or its substitute with respect to the original polypeptide or polynucleotide. This is achieved by techniques well known in the art, including a site-specific mutagenesis technique and the like. A polypeptide or a polynucleotide may have any number (>0) of substitutions, additions, or deletions. The number can be as large as a variant having such a number of substitutions , additions or deletions maintains an intended function (e.g. , vascular network formation activity, etc.). For example, such a number may be one or several, and preferably within

20% or 10% of the full length, or no more than 100, no more than 3 , no more than , no more than 1 , or the like . (Vascular network formation activity) As used herein, "angiogenesis" refers to the formation of new blood vessels and the activity of such formation.

As usedherein, the term "vascular network formation" refers to the process of forming a network of new bloo vessels or existing bloodvessels and the activity of such formation.

As used herein, the ability of vascular network formation is indicated by a vascular network formation index. Vascular network formation can be determined based on whether or not a network is formed, (e.g., branched blood vessels are coupled with other blood vessels, and the number of junction points is increased) . Briefly, vascular network formation index is herein calculated as follows .

Initially, the number of new blood vessels is counted per 0.79) ccmm²².. TThhee nnuummbbeerrss ooff bblloooodd vessels are converted to right-handed scores as follows.

0 : 0

1 to 10 : 1

11 to 20 : 2

21 to 30 : 3

31 to 40 : 4

41 or more : 5 Next, the length of new blood vessels is determined as follows . Thebloodvessels are imagedundera stereoscopic microscope (SZX12, Olympus , Japan) . The resultant image is analyzed using Photoshop (registered trademark, Adobe, Japan) . The number of pixels is counted and converted to the right-handed scores below.

100 or less: 1 100 to 125 or less: 2 125 to 150 or less: 3 150 to 175 or less: 4 175 to 200 or less: 5 200 or more: 6.

When both the number and length of new blood vessels have high scores , the vascular network forming activity is considered to be high. The product of the two scores is herein referred to as a vascular networ-k formation index. As demonstrated by the present invention, it will be understood that the presence of the vascular network forming activity means a vascular network formation index of at least 5, preferably 8 or more, more preferably 10 or more, and even more preferably 15 or more.

Network forming activitycan be examinedby observing the angiogenesis state of tissue using a stereoscopic microscope (SZX12, Olympus, Japan). The resultant image is analyzed using Photoshop (registered trademark, Adobe, Japan) . Network forming activity is represented by scores as follows . The score below is also referred to as a "vascular network forming activity" or "blood vessel network forming index", or simply "network index" - Nwl: the early stage of network formation.

Angiogenesis is observed but new blood vessels are not connected to one another. Nw2: the intermediate stage of network formation. New blood vessels are connected to one another like a ladder

Nw3 : the late stage of network formation . New blood vessels are multiply branched.

Nw4 : the mature stage of network formation. The plexus of new blood vessels is wide spread. As demonstrated in the present invention, the presence of vascularnetworkformingactivitytypicallymeans a vascular network forming index of at least 2, preferably at least 2.5, more preferably at least 3, and even more preferably 3.5 or more.

The term "bloodvessel" is used in its broadest sense in the art and includes capillary blood vessels as well as typical arteries, veins, and the like. As used herein, the terms "cell adhesion molecule" and "adhesion molecule" are used interchangeably, referring to a molecule capable of mediating the joining of two or more cells (cell adhesion) or adhesion between a substrate and a cell. In general, cell adhesion molecules are divided into two groups: molecules involved in cell-cell adhesion (intercellular adhesion) (cell-cell adhesion molecules ) and molecules involved in cell-extracellular matrix adhesion (cell-substrate adhesion) (cell-substrate adhesion molecules). For the present invention, any cell adhesion molecules are useful and can be effectively used. Therefore, cell adhesion molecules herein include a protein of a substrate and a protein of a cell (e.g. , integrin, etc . ) in cell-substrate adhesion. A molecule other than proteins falls within the concept of cell adhesion molecule as long as it can mediate cell adhesion.

For cell-cell adhesion, cadherin, a number of molecules belonging- in an immunoglobulin superfamil-y (NCAM,

LI, ICAM, fasciclin II, III, etc.), selectin, and the like are known, each of which is known to join cell membranes via a specific molecular reaction. On the other hand, a major cell adhesion molecule functioning for cell-substrate adhesion is integrin, which recognizes and binds to various proteins contained in extracellular matrices . These cell adhesion molecules are all located on cell membranes and can be regarded as a type of receptor (cell adhesion receptor) . Therefore, receptors present on cell membranes can also be used in an implant of the present invention . Examples of such areceptor include , but are not limited to, α-integrin, β-integrin., CD44, syndecan , aggrecan , and the like .

Note that extracellular matrix molecules (cellular adhesive protein, such as fibronectin, laminin, andthelike), which are bound by integrin or the like, herein fall within the category of cell adhesion molecules. A function shared by each adhesion receptor in cell-cell adhesion and cell-substrate adhesion is not strictly defined and varies depending on the dis tribution of binding molecules ( ligand) . For example, a certain integrin is involved in cell-cell adhesion, such as rxemocyte-hemoσyte adhesion or the like. It is known that when a growth factor, cytokine or the like is present as a cell membrane protein, a reaction with its receptor present on other cells eventually causes cell adhesion. Such a growth factor or cytokine can be used as a biological molecule contained in an implant of the present invention .

Thus, various molecules are involvedin cell adhesion and have different functions . Those skilled in the art can appropriately select a molecule to be contained in an implant of the present invention depending on the purpose . Techniques for cell adhesion are well known as described above and as described in, for example, "Saibogaimatorikkusu -Rinsho heno Oyo - [Extracellular matrix -Clinical Applications-], Medical Review.

It can be det erminedwhether or not a certain molecule is a cell adhesion molecule, by an assay, such as biochemical quantification (an SDS-PAGE method, a labeled-collagen method, etc.), imπvunological quantification (an enzyme antibody method, a fluorescent antibody method, an immunohistological study, etc.), a PDR method, a hybridization method, or the like, in which a positive reaction is detected. Examples of such a cell adhesion molecule include, but are not limited to, collagen, integrin , fibroneσtin, laminin, vitronectin, fibrinogen, an immunoglobulin superfamilymember (e.g. , CD2, CD4, CD8 , ICM1 , ICAM2, VCAM1), selectin, cadherin, and the like. Most of these cell adhesion molecules transmit into a cell an auxiliary signal for cell activation due to intercellular interaction as well as cell adhesion. Therefore, an adhesion factor for use in an implant of the present invention preferably transmits an auxiliary signal for cell activation into a cell. This is because cell activation can promote growth of cells originally present or aggregating in a tissue or organ at an injured site after application of an implant thereto. It can be determined whether or not such an auxiliary signal can be transmitted into a cell, by an assay, such as biochemical quantification (an SDS-PAG method, a labeled-collagen method, etc.), immunological quantification (an enzyme antibody method, a fluorescent antibody method, an immunohistological study, etc.), a I?DR method, a hybridization method, or the like, in which a positive reaction is detected.

An example of a cell adhesion molecule is cadherin which is present in many cells capable of being fixed to tissue. Cadherin can be used in a preferred embodiment of the present invention. Examples of a cell adhesion molecule in cells of blood and the immune system which are not fixed to tissue, include, but are not limited to, immunoglobulin superfamily molecules (CD 2, LFA-3, ICAM-1, CD2, CD4, CD8,

ICM1, ICAM2, VCAM1, etc. ) ; integrin family molecules (LFA-1,

Mac-1, gpllbllla, l50, p95, VLA1, VLA2 , VLA3 , VLA4, VLA5,

VLA6, etc.); selectin family molecules (L-seleσtin,

E-selectin, P-seleσtin, etc.), and the like. Therefore, such a molecule may be useful for treatment of a tissue or organ of blood and the immune system.

Nonfixedcells need to be adhered to a specific tissue in order to act on the tissue. In this case, it is believed that cell-cell adhesion is gradually enhanced via a first adhesionbya selectin molecule or the likewhich is constantly expressed and a second adhesion by a subsequently activated integrin molecule. Therefore, in the present invention, a cell adhesion molecule for mediating the first adhesion and another cell adhesion molecule for mediating the second adhesion may be used together.

As used herein, the term "cellular adhesive protein" refers to a protein capable of mediating cell adhesion as described above. Therefore, as used herein, the term "cellular adhesive protein" includes a protein (e.g., integrin, etc. ) of a cell as well as a protein of a substrate in cell-substrate adhesion. For example, when cultured cells are seeded on a substrate (glass or plastic) adsorbing a protein under serum-free conditions, a receptor integrin recognizes the cellular adhesive protein and adheres to the substrate. An active site off a cellular adhesive protein has been determined at the amino acid level . As such an active site, RGD, YIGSR or the like are known (these are collectively called "RGD sequences"). Therefore, in one preferred embodiment, a protein contained in an implant of the present invention may advantageously have an RGD sequence, such as RGD, YIGSR, or the like. Typically, a cellular adhesive protein is present in an extracellular matrix, the surface of a cultured cell, and body fluid (plasma, serum, etc.). It is known that the In vivo function of cellular adhesive proteins include migration, growth, morphological regulation, tissue construction and the like of cells as well as adhesion of cells to an extracellular matrix. In addition to action on cells , some proteins are capable of regulating blood coagulation and complement action. Such proteins may be useful in the present invention. Examples of such a cellular adhesive protein include, but are not limited to, fibronectin, collagen, vitronectin, laminin, and the like.

As used herein, the term "RGD molecule" refers to a protein molecule comprising an amino acid sequence RGD

(Arg-Gly-Asp) or a sequence having the same function as that of the sequence RGD. RGD molecules are characterized by comprising an amino acid sequence RGD which is useful as an amino acid sequence of a cell adhesion active site of a cellular adhesive protein or another amino acid sequence having an equivalent function. The RGD sequence was found as a cell adhesion site of fibrronectin, and subsequently, a number of molecules having cellular adhesive activity were found, including collagen type I, laminin, vitronectin, fibrinogen, the von illebrandL factor, entactin, and the like. If a chemically synthesized RGD peptide is attached to a solidphase, the peptide exhibits cell adhesion activity. A biological molecule of the present invention may be a chemically synthesized RGD molecule. Examples of such an RGD molecule include, but are not limited to, a GRGDSP peptide in addition to the above-described naturally-occurring molecules . The RGD sequence is recognizedby integrin (e.g. , a receptor for fibronectin) which is a cell adhesion molecule (and also a receptor). Therefore, a molecule having a function equivalent to RGD can be identified by examining interaction with integrin. As used herein, the term "integrin" refers to a transmembrane glycoprotein which is a receptor involved in cell adhesion. Integrins are located on cell surfaces and function when a cell adheres to an extracellular matrix. It is known that integrins are involved in cell-cell adhesion in the hemocyte system. Examples of such integrins include, but are not limited to, receptors for fibronectin, vitronectin, collagen, or the like; Ilb/IIla in platelets; Mac-1 in macrophages; LFA-1, VLA-1 to 6 in lymphocytes; PSA in fruit flies; and the like. Typically, integrins have a hetero dimer structure in which an α chain having a molecular weight of 130 kDa to 210 kDa and a β chain having a molecular weight of 95 kDa to 130 kDa are associated via a non-covalent bond. Examples of the α chain include, but are not limited to, α¹, α², α³, α⁴, α⁵, α⁶, ct^L, α^M, α^x, α^IIb, α^v, α^E, and the like. Examples of the β chain include, but are not limited to, βi, β₂, β₃, β₄, β₅, βs, β?, and the like. Examples of such a hetero dimer include , but are not limited to, Gp lib Ilia, VLA-1, VLA-2, VLA-3, VLA-4, VLA-5, VLA-6, CD51/CD29, LFA-1, Mac-1, pl50, p90, a vitronectin receptor, β⁴ subfamily, β⁵ subfamily, β⁶ subfamily, LPAM-1, HML-1, and the like. Typically, it is often that the extracellular domain of the α chain has a divalent cation binding site, and the extracellular domain of the β chain has a σysteine-rich domain and the intrracellular domain of the β chain has a tyrosine phosphorylation site. A recognition site of a binding ligand is of ten the RGD sequence . Therefore, integrin may be an RGD molecule.

(Detailed Description of the Invention) Hereinafter, the present invent ion will be described by way of examples . Examples described, below are provided only for illustrative purposes. Accordingly, the scope of the present invention is not limited except as by the appended claims .

In one aspect, the present invention provides a peptide comprising: a peptide having an amino acid sequence X1-X2-X3-X4-X5-X6-X7 (SEQ ID NO. : 14), where Xi is serine (S) , threonine (T ) , or a variant thereof , or is absent , X₂ is valine (V), alanine (A), glycine (G), leucine (L), isoleucine (I), or a variant thereof, X₃ is valine(V), alanine (A), glycine (G), leucine (L), isoleucine (I), or a variant thereof, X₄ is an amino acid having an aromatic ring as a side chain or a variant thereof, X₅ is glycine (G) or a variant thereof, X₆ is leucine (L), alanine (A), glycine (G), valine (V), isoleucine (I) , or a variant thereof, X₇ is arginine (R), lysine (K), or a variant thereof, or is absent ; or a variant or salt thereof. The present invention is the first to find that such a peptide has an ability to form a vascular network. Therefore, the present invention provides a composition for vascular network formation, comprising the peptide or a variant thereof (herein also referred to as avascular network forming agent) . The above-described sequence was designed basedon SWYGLR set forth in SEQ ID NO. : 1. Itwillbe clearly understood by those skilled in the art that the above-described sequence may have the vascular network forming activity.

The vascular network forming activity can be represented by a vascular network formation index. The vascular network forming activity possessed try the peptide of the present invention may be at least 2 , preferably at least 2.5, more preferablyat least 3 , andevenmorepreferably 3.5 or more. Actually, when the vascular network forming activity index: is about 2.5 or more (preferably 3.0 or more) , the formation of a significant network of blood vessels is substantially always observed.

In one embodiment, it will be understood that the peptide of the present invention may comprise a foreign sequence as long as it comprises the above-described specific sequence and the vascular network forming activity.

In one preferred embodiment , Xi is serine or avariant thereof, and more preferably, Xi is serine.

In another preferred embodiment, X₂ is valine or a variant thereof, and more preferably, X₂ is valine. In another preferred embodiment , X₃ is valine or a variant thereof, and more preferably, X₃ is -valine.

In another preferred embodiment , X₄ is phenylalanine, tyrosine, or a variant thereof, andmore preferably, tyrosine or a variant thereof, and more preferably phenylalanine or a variant (the side chain of phenylalanine is substituted with a hydrophobic group (methyl group) .

In another preferred embodiment , X₄ is phenylalanine or a variant thereof . In a more preferred embodiment , X₄ is phenylalanine. In another preferred embodiment, X₄ is tyrosine or a variant thereof. In a more preferred embocliment, X is tyrosine.

In another preferred embodiment, X₅ is glycine or a variant thereof. In a more preferred emboόliment, X₅ is glycine .

In another preferred embodiment, X₆ is leucine or a variant thereof. In a more preferred embocliment , X₆ is leucine.

In another preferred embodiment, X₇ is arginine or a variant thereof. In a more preferred embocliment , X₇ is arginine .

Therefore, it will be understood that the peptide of the present invention may have a sequence comprising the above-described preferred embodiments of Xi to X₇. In a more preferred embodiment, the peptide off the present invention or a variant thereof is an amino acid sequence set forth in SEQ ID NO . : 1 , or an amino acid sequence set forth in SEQ ID NO. : 1 having 1 to 3 amino acid substitutions, 1 or 2 amino acid deletions at one or both ends thereof , or another amino acid sequence added t o or at least one amino acid addition at one or both ends thereof. Preferably, the peptide or a variant thereof advantageously has a vascular network forming activity.

In a more preferred embodiment, the peptide off the present invention or a variant thereof is a peptide having an amino acid sequence set forth in SEQ ID NO. : 1, α*r an amino acid sequence set forth in SEQ ID NO. : 1 having 1 or 2 amino acid substitutions wherein the fourth tyrosine residue is a tyrosine residue or an amino acid having an aromatic ring as a side chain, 1 amino acid deletion at one or both ends thereof, or another amino acid sequence added to or at least one amino acid addition at one or both ends thereof. Preferably, the peptide or a variant thereoff has a vascular network forming activity.

In a more preferred embodiment, in the pepticLe of the present invention or a variant thereof, the amino acid having an aromatic ring as a side chain is phenylalanine or chemically modified phenylalanine having 1 or more substituents on the benzene ring thereof. In a more preferred embodiment, the peptide off the present invention or a variant thereof is a peptide having an amino acid sequence set forth in SEQ ID NO.: 9, or an amino acid sequence set forth in SEQ ID NO. : 9 having 1 amino acid deletion at one or both ends thereof, or another amino acid sequence added to orr at least one amino acid addition at one or both ends thereof after 1 amino acid deletion at one or both ends thereof. Preferably, the peptide or a variant thereof has a vascular network forming activity .

In a more preferxred embodiment, the peptide of the present invention or a variant thereof is a peptide havin g an amino acid sequence set forth in any one of SEQ ID NOs . : 1 to 7, or an amino acid sequence set forth in any one of SEQ ID NOs.: 1 to 7 having another amino acid sequence added to or at least one amino acid addition at one or both ends thereof. Preferably, the peptide or a variant thereof has a vascular network forming activity.

In a more preferred embodiment, the peptide of th-e present invention or a variant thereof is a peptide havin g an amino acid sequence set forth in any one of SEQ ID NOs. : 9 to 11, or an amino acid sequence set forth in any one of SEQ ID NOs . : 9 to 11 having another amino acid sequence added to or at least one amino acid addition at one or both end-s thereof. Preferably, the peptide or a variant thereof h s a vascular network forming activity. In a more preferrred embodiment, the peptide of th-e present invention or a variant thereof is a peptide having an amino acid sequence set forth in SEQ ID NO.: 9, or an amino acid sequence set forth in SEQ ID NO. : 9 having another amino acid sequence addedto orat least one amino acidadditio n at one or both ends thereof. Preferably, the peptide or a variant thereof has a vascular network forming activity.

In another embodiment, the peptide of the present invention may comprise an amino acid sequence represented by SWX₄GL (SEQ ID NO. : 15) or WX₄GLR (SEQ ID NO. : 16) where X₄ is defined as above. More preferably, the peptide of the present invention may comprise an amino acid sequence represented by SWYGL (SEQ ID NO.: 10) or an amino acid sequence represented by WYGLR (SEQ ID NO. : 11), and more preferably an amino acid sequence represented by SWFGLR (SEQ ID NO.: 10) or an amino acid sequence represented by SWYGLR (SEQ ID NO. : 1).

As specifically described in the examples below, the present inventors found that a peptide having an amino acid sequence set forth in SEQ ID NO. : 1 has a vascular network forming activity. Therefore, a preferable example of the vascular network forming agent of the present invention comprises the peptide having the amino acid sequence set forth in SEQ ID NO. : 1 as an effective ingredient.

It is generally well known that a physiologically active peptide retains its physiological activity even when the amino acid sequence thereof has one or more amino acid substitutions, deletions, insertions or additions.

Therefore, the present invention encompasses a peptide which has an amino acid sequence set forth in SEQ ID NO. : 1 having 1 to 3 amino acid substitutions , or 1 or 2 amino acid deletions at one or both ends thereof, or another amino acid sequence added to or at least one amino acid addition at one or both ends thereof (hereinafter also referred to as a "peptide variant" for the sake of simplicity) andhas avascular network forming activity. Amino acids contained in the peptide variant of the present invention are not limited to amino acids constituting naturally-occurring proteins, and include amino acids obtained by chemically modifying naturally-occurring amino acids (e.g., introduction of a nitro group, a halogen atom, or the like into a side chain of an amino acid, etc. ) . The amino acid may be D type. As specifically confirmed in the examples below, ixi a preferred embodiment of the present invention, a peptide having an amino acid sequence (SEQ ID NO. : 1) having 1 amino acid deletion at the N- or C-terminal thereof has substantially the samevascularnetwork formingeffect as that of the peptide having the amino acid sequence set forth in SEΪQ ID NO. : 1. As specifically described in the examples below, a peptide (SEQ ID NO.: 8) having an amino acid sequence set forth in SEQ ID NO. : 1 in which tyrosine is substituted with alanine at position 4 lacks a vascular network forming activity. Therefore, a tyrosine residue at position 4 is considered to be important. It is preferable not to substitute it with one whose side chain has a significantly altered chemical structure . Insteadof naturally-occurring L-t^ e tyrosine, D-type tyrosine or tyrosine with a halogen atom or nitro group introduced into the phenol ring of a side chain thereof may be typically designed using medicinal chemistry as a substitute for tyrosine. Such tyrosine is known to often have a more potent effect or an effect which is unaltered. It is also believed that other amino acids (e.g., phenylalanine, etc.) having an aromatic ring in the side chain thereof have substantially the same effect. As specifically described in the examples below, it was confirmed that a peptide modified by substituting phenylalanine for tyrosine at position 4 has a better effect . Therefore, an exemplary preferable peptide variant is a peptide, which has an amino acid sequence set forth in SEQ ID NO. : 1 having 1 or 2 amino acid substitution , or 1 amino acid deletion at one or both ends thereof, or another amino acid sequence added to or at least one amino acid addition at one or both ends thereof, wherein an amino acid residue at position 4 is a tyrosine residue or an amino acid having an aromatic ring in the side chain thereof, or preferably phenylalanine, and has a vascular network forming activity. Note that the "amino acid having the arromatic ring in the side chain thereof" is not necessarily limited to amino acids constituting naturally-occurring proteins, and includes tyrosine or phenylalanine derivatives in which at least one substituent selected from the group consisting of a nitro group, a halogen atom, an alkyl group h.aving 1 to 5 carbon atoms , and an acyl group having 1 to 5 carbon atoms is bound to an aromatic ring of tyrosine or phenylalanine. If any, the number of substituents on an aromatic ring or preferably abenzene ring thereof is preferably 1 to 5., andmorepreferably 1 to 3. In the peptide of the present invention, the above-described aromatic ring may be preferably a benzene ring or a fused ring, such as a naphthalene ring comprising benzene rings (or may be a heterocycle, such as a tryptophan side chain). Particularly, a benzene ring is preferable.

As specifically described in the examples below, the present inventors found that a peptide having an amino acid sequence set forth in SEQ ID NO. : 9, in which tyrosine at position 4 is substitutedwith phenylalanine in SEQ ID NO. : 1, has a better vascular network forming ef ect than the peptide having the amino acid sequence set forth in SEQ ID NO. : 1. Note that phenylalanine is an amino acicl having an aromatic ring (benzene ring) in the side chain thereof. Therefore, in a preferred embodiment of the present invention, avascular network forming agent is provided, which comprises a peptide having an amino acid sequence set forth in SEQ ID NO. : 9 or an amino acid sequence set forth in SEQ ID NO. : 9 having 1 amino acid deletion at one or both ends thereof, or another amino acid sequence addedto or at least one amino acidaddition at one or both ends thereof after 1 amino acid deletion at one or both ends thereof . Therefore, the upper limit of the size of a peptide used in the present invention is not limited, though an excessively large peptide is difficult to produce and inconvenient to handle, and is also considered to have a reduced vascular network formation activity per unit weight . Therefore, the total number of amino acids in the peptide is typically 4 to 350, preferably 4 to 50 , more preferably

5 to 20, evenmore preferably 5 to 10, and still more preferably

6 to 10. In addition, a peptide having an amino acid sequence set forth in SEQ ID NO. : 14 (e.g. , any one of SEQ ID NO. : 1 to 7 and 9 to 11) or an amino acid sequence set forth in SEQ ID NO. : 14 having 10 or less amino acid additions at one or both ends thereof. For example, it has been confirmed that peptides having amino acid sequences set forth in SEQ ID NOs.: 12 and 13, in which another amino acid sequence is added at one end of the amino acid sequence set forth in SEQ ID NO. : 1, each have an excellent vascular network formation activity. Note that it was speci ically confirmed in the examples below that the peptide having the amino acid sequence set forth in SEQ ID NO. : 14 (representatively, SEQ ID NOs. : 1 to 7 and 9 to 11) has a vascular network forming activity. Therefore, the above-described peptides are preferable.

Note that it canbe deterrainedwhether ornot apeptide has a vascular network forming activity, by embedding a micro cell filled with peptide solution into the dorsum of a mouse and subsequently observing the process of formation of capillary blood vessels in tissue around the embedded site. as specifically described in the examples below.

The peptide of the present invention or a variant thereof can be easily synthesized with a commonly used method manually or using a commercially available peptide synthesizer. Large size peptides can also be produced in a genetically engineered manner with a commonly used method.

The peptide of the present invention comprises amino acids constituting naturally-occurring proteins, and therefore, is subsequently degraded into amino acids In vivo by the action of peptidase . Thus , the peptide of the present invention is highly safe. In fact, no toxicity was found due to the peptide of the present invention in In vivo experiments conducted using mice in the examples below. It was observed that no toxicity was observed when the peptide of the present invention was used in an effective amount .

In a preferred embodiment, a target of the vascular network forming agent of the present invention may be a capillary blood vessel. This is because capillary blood vessels acquire normal naturally-occurring physiological activity by forming a network. To date there have been several known compounds having a vascular network forming activity. However, it is also known that thevascularnetwork forming activity does not necessarily lead to tissue regeneration. To date the reason has not been clarified.

As compounds having a vascular network forming activity do not always have a network formation activity, it is considered that conventional compounds having avascularnetworkforming activity do not fully construct a network of capillary blood vessels . The present invention is the first to provide a compound having a network formation activity, thereby obtaining therapeutic effects, stabilizing effects, and prophylactic effects which could not be achieved by conventional parameters of vascular network formation. In addition, when the peptide is coupled with a carrier and the peptide with the carrier is embedded into an organism, vascular network formation can be promoted. The carrier allows the peptide to be selectively applied to a site in need of therapy, thereby potentially providing a novel DDS (drug delivery system) . By locally administering the vascular network forming agent of the present invention into a site, into which a biological sample is implanted, with a method, such as application, spraying, or the like, vascular network formation is promoted so that postoperative recovery is accelerated. Examples of carriers include, but are not limited to, resins as used in bone substitute, tooth substitute, artificial organs, or the like, and biological polymers, such as proteins and the like. By coupling the above -described peptide with a resin, when the resin is embedded into an organism, vascular network formation is promoted in surrounding tissue contacting the resin, so that the af inity of the resin to the organism is further improved. In a more preferred embodiment, a protein can be used as a carrier (as used herein, unless otherwise specified, the term "protein" encompasses protein-containing complexes, such as glycoproteins , phosphoproteins, and the like).

Proteins used as carriers may be any biocompatible proteins, particularly preferably cell adhesion proteins for obtaining satisfactory junction withbiological tissues . Examples of preferable cell adhesion proteins include, but are not limited to, collagen (gelatin), fibronectin, vitronectin, laminin, and partial hydrolysis products thereof, and the like. Note that these proteins may be preferably purified to remove allergens from a viewpoint of preventing allergic reactions. For example, various animal-derived collagens are commercially available, but their purities are low, i.e., they contain allergens. The reproducibility of results using these collagens is low. Therefore, these collagens are not suitable for clinical applications . Gelatins obtained by partial hydrolysis of animal-derivedcollagen to remove allergens are commercially available for clinical applications. Such purified collagens or partial hydrolysis products are preferably employed.

The amount of a peptide coupled with a carrier may be selected as appropriate without limitation. Such an amount is typically about 100:1 to 1:1 in a weight ratio of the carrier to the peptide (carrier:peptide) , and preferably about 20:1 to 5:1. A carrier is preferably coupled with a peptide via a covalent bond. Coupling may be easily performed by, for example, coupling an amino group at the N-terminus of the peptide with any amino group of the carrier using a coupling crosslinking agent, such as glutaraldehyde or the like. An exemplary coupling method is described in detail in the examples below. When a peptide is coupled with a resin of an artificial organ or the like, a monomer comprising a group (e.g. , an amino group, etc. ) , which can be used for coupling with the peptide, is copolymerized with the resin so that the amino group or the like can be coupled with an amino group at the N-terminus of the peptide. A carrier may be preferably coupled with a peptide comprising a peptide having any amino acid sequence linked at one or both ends of a peptide an amino acid sequence set forth in SEQ ID NO. : 1 or a peptide variant thereof having some amino acid substitution(s) or deletion(s) . Carriers coupled with peptides can be embedded in organisms by application or spraying, or directly. When a cell adhesionprotein is usedas a carrier, the carrier coupled with the peptide can be used as suture, various orthopedic surgery materials, agents for promoting adhesion of wounds, singly or in combination with other effective ingredients. In addition, a carrier protein coupled with a peptide, which is mixed with carbonated apatite or other materials (e.g., cell adhesion proteins which are not coupled with the peptide of the present invention) , can be used as a bone substitute and the like. In this case, the amount of a peptide contained in a final biological material, such as bone substitute or the like, is typically, without limitation, 0.1 to 10 mg per 100 g of the biological material. A carrierusedhe ein is preferablypharmaceutically acceptable. Examples of such a pharmaceutically acceptable carrier include, but are not limited to, antioxidants, preservatives, colorants, flavoring agents, diluents, emulsifiers, suspending agents, solvents, fillers, bulking agents, buffers, delivery vehicles, agricultural or pharmaceutical adjuvants , and the like. In the case of the medicament of the present invention comprising the peptide of the present invention, or a variant or analog thereof, representatively, a support and a biological molecule are administered with at least one physiologically acceptable carrier, excipient or diluent in the form of a composition. Examples of an appropriate vehicle may include an injection solvent, a physiological solution, or artificial cerebrospinal fluid. Other materials which are commonly used in a composition for implantation can be added to the above-described materials. Acceptable carriers , excipients or stabilizers used herein preferably are nontoxic to recipients and are preferably inert at the dosages and concentrations employed, and preferably include phosphate, citrate, or other organic acids; ascorbic acid, α-tocopherol; low molecular weight polypeptides; proteins (e.g. , serum albumin, gelatin, or immunoglobulins); hydrophilic polymers (e.g., polyvinylpyrrolidone) ; amino acids (e.g., glycine, glutamine, asparagine, arginine or lysine) ; monosaccharides, disaccharides , and other carbohydrates (glucose, mannose, ordextrins); chelating agents (e.g., EDTA); sugar alcohols (e.g. , mannitolor sorbitol) ; salt-forming counterions (e.g., sodium) ; and/or nonionic surfactants (e.g. , Tween, pluronics or polyethylene glycol (PEG) ) . Examples of appropriate additional carriers include neutral buffered saline or saline mixed with serum albumin. Preferably, the product is formulated as a lyophilizate using appropriate excipients (e.g., sucrose). Other standard carriers, diluents, and excipients may be included as desired. Other exemplary compositions comprise Tris buffer of about pH 7.0-8.5, or acetate buffer of about pH 4.0-5.5, which may further include sorbitol or a suitable substitute therefor. A carrier used herein is preferably a protein, and more preferably a cell adhesion protein. Preferably, a cell adhesion protein used herein may be collagen. The peptide of the present invention can be locally administered into tissue in need of vascular network formation singly or in the form of, for example, an injection solution obtained by dissolving the peptide in a physiological buffer. By locally administering the vascular network forming agent of the present invention into the vicinity of wounds or the like, which are caused by operation or injury, by injection, application , spraying, or the like, vascular network formation is promoted to accelerate the recovery of the wound. The concentration of the peptide solutionusedin injection, application, spraying, or the like is typically, without limitation, about 1 to 10 μg/mL. The dose of the peptide solution can be selected as appropriate depending on the size or depth off the wound. Preferably, such a dose of the peptide solution may cover the entire wound. The peptide solution can be administered once to several times per day to several days until the wound has healed. The injection solution may contain various ingredients which are typically contained in therapeutic agents for wounds, such as disinfectants, anti-inflammatory agents, analgesics, and the like.

The medicament of the present invention may be administered orally or parenterally. Alternatively, the medicament of the present invention may be administered intravenously or subcutaneously. When systemiσally administered, the medicament for use in the present invention may be in the form of a pyrogen-free, pharmaceutically acceptable aqueous solution. The preparation of such pharmaceutically acceptable compositions, with, due regard to pH, isotonicity, stability and the like, is within the skill of the art . Administration methods may be berein oral, parenteral administration (e.g./ intravenous, intramuscular, subcutaneous, intradermal, to mucosa, intrarectal, vaginal, topical to an affected site, to the skin, etc . ) . A prescription for such administration may be provided in any formulation form. Such a formulation form includes liquid formulations, injections, sustained preparations, and the like.

The medicament of the present invention may be prepared for storage by mixing a sugar chain composition having the desired degree of purity with optional physiologically acceptable carriers, excipients, or stabilizers ( Japanese Pharmacopeia ver. 14, or a supplement thereto or the latest version; Remington's Pharmaceutical Sciences, 18th Edition, A. R. Gennaro, ed. , Mack Publishing Company, 1990; and the like), in the form of lyophilized cake or aqueous solutions .

The amount of thecomposition of thepresent invention used in the treatment method of the present invention can be easily determinedby those skilled in the art with reference to the purpose of use, a target disease (type, severity, and the like), the patient ' s age, weight , sex, andcase history, the form or type of the cell, and the like. The frequency of the treatment method of the present invention applied to a subject (or patient) is also determined by those skilled in the art with respect to the purpose of use, target disease (type, severity, and the like), the patient's age, weight, sex, and case history, the progression of the therapy, and the like- Examples of the frequency include once per day to several months (e.g., once per week to once per month). Preferably, administration is performed once per week to month with reference to the progression. In another aspect, the present invention provides a method for forming a network of blood vessels, comprising the step of administering the composition of the present invention for vascular network formation into a patient having a site in need of formation of a vascular network. It will be -understood that the peptide may be in any of the forms described herein above. It will be understood that administration methods can be performed in any form. As used herein, the term "patient" refers to an organism to which treatment of the present invention is applied and is also referred to as "specimen" or "subject". A subject may be preferably a human. The present invention provides amethodfor treatment, inhibition, and prophylaxis, comprising administrating an effective amount of the composition of the present invention into patients. In a preferred embodiment, the composition of the present invention may be substantially purified (e.g. , substantially free from substances which limit the effect of the composition or elicit adverse side effects).

Animals targeted by the present invention include any organism as long as it has an immune system or a similar system (e.g., animals (e.g., vertebrates, invertebrate)). Preferably, the animal is a vertebrate (e.g. , Myxiniformes , Petronyzoniformes, Chondrichthyes , Osteichthyes , amphibian, reptilian, avian, mammalian, etc.), more preferably mammalian (e.g., monotremata, marsupialia, edentate, dermoptera, chiroptera, carnivore, insectivore, proboscidea, perissodaσtyla, artiodactyla, tubulidentata, pholidota, sirenia, cetacean, primates, rodentia, lagomorpha, etc.). Illustrative examples of a patient include, but are not limited to, animals, such as cattle, pigs, horses, chickens, cats, dogs, and the like. More preferably, primates (e.g., chimpanzee, Japanese monkey, human, etc.) are used. Most preferably, a human is used.

Various delivery systems are known for prophylasis and therapy. In the present invention, any administration routes are intended- Examples of techniques for administering the composition and the like of the present invention include aqueous solutions, liposomes, microparticles, microcapsules , and the like. Therefore, the composition of the present invention may be administered orally or parenterally. Administration methods may be herein oral, parenteral administration (e.g. , intravenously, intramuscularly, subcutaneously, intradermally, intramucosally (nasally, varginally, bronchially, orally, rectally, intestinally , etc. ) , topical to an affected site, to the skin, etc. When systemically administered, a composition used in the present invention may be preferably free from pyrogens - The preparation of such pharmaceutically acceptable compositions, with due regard to pH, isotonicity, stability and the like, is within the skill of the art. A prescription for such administration may be provided in any formulation. Such a formulation includes liquid formulations, injections, sustained preparations, and the like. Examples of introduction methods include, but are not limited to, administration as oral drugs, ventilation or aspiration (e.g., lung), and injection using a syringe, a catheter, a tube, a needleless syringe, a gene gun, or the like. In these cases, the composition of the present invention may be administered in combinationwith other biologicallyactive pharmaceutical agents. The amount of a medicament used in the prophylaxis method of the present invention can be easily determined by those skilled in the art with reference to the purpose of use, a target disease (type, severity, and the like), the patient's age, weight, sex, and case history, the form or type of the cell, and the like. The frequency of the treatment method of thepresent invention applied to a subject (or patient) is also determined by those skilled in the art with respect to the purpose of use, target disease (type, severity, and the like), the patient's age, weight, sex, and case history, the progression of the therapy, and the like. Examples of the frequency include once per day to several months (e.g., once per week to once per month), or once before an epidemic outbreak every year. Preferably, administration is performed once per week to month with reference to the progression. Booster immunization may be advantageously given at an interval of at least about one week. More preferably, the interval of booster immunization may be at least about 3 weeks. The dose of the composition of the present invention varies depending on the patient ' s age, weight, condition or its administration method, or the like, and is not particularly limited. As used herein, the term "administer" means that the medicament or the like of the present invention, or a pharmaceutical composition containing it , is incorporated into hosts in need of treatment either alone or in combination with other therapeutic agents. Combinations may be administered either conσomitantly (e.g. , as an admixture) , separately but simultaneously or concurrently; or sequentially. This includes presentations in which the combined agents are administered together as a therapeutic mixture, and also procedures in which the combined agents are administered separately but simultaneously (e.g., as through separate mucosas into the same individual) . Administration "in combination" further includes the separate administration of one of the compounds or agents given first, followed by a second compound or agent.

The medicament of the present invention may be administered by any technique, preferably using a needless syringe- This is because administration can be performed without placing undue stress onto patients.

As used herein, the term "needleless syringe" refers to a medical instrument which is used without a needle to spray a pharmaceutical liquid to the skin by the movement of a piston by means of gas pressure or the elasticity of an elastic material so as to administer a pharmaceutical ingredient subcutaneously, and more preferably into subcutaneous cells .

Specifically, for example, ShimaJET (manufactured by Shimadzu) , Medi-Jector Vision™ (manu actured by Elite medical) , PenJet™ (manufactured by PenJet), and the like are commercially available.

In another aspect, the present invention provides use of a peptide having an amino acid sequence Xι-X₂-X3- 4-X₅-X6-X₇ (SEQ ID NO.: 14), where X_x is serine (S), threonine ( T ) , or a variant thereof , or is absent , X₂ is valine (V), alanine (A), glycine (G) , leucine (L), isoleucine (I), or a variant thereof, X₃ is valine(V) , alanine (A), glycine (G), leucine (L), isoleucine (I), or a variant thereof, X₄ is an amino acid having an aromatic ring as a side chain or a variant thereof , X₅ is glycine ( G ) or a variant thereof , Xβ is leucine (L), alanine (A), glycine (G) , valine (V) , isoleucine (I), or a variant thereof, X₇ is arginine (R) , lysine (K) , or a variant thereof, or is absent; or a variant or salt thereof, for production of a medicament for forming a network of blood vessels. It will be understood that the above-described peptide may be in any of the forms described herein above with respect to pepticles . (Design of compounds) A region having a vascular network forming activity is a part of the amino acid sequence set forth in SEQ ID NO. : 1. Therefore, a peptide having the amino acid sequence set forth in SEQ ID NO. : 1 or an amino acid sequence set forth in SEQ ID NO.: 1 having amino acid substitution s) , amino acid deletion(s) at the end(s ) thereof, or another amino acid sequence addedto or at least one amino acidaddition at one or both ends thereof , may have vascular network forming ac ivity. This is also inferred from, the fact that the cell adhesion action of cell adhesive peptides, such as fibronectin, laminin, and the like, is attributed to a region con sisting of only three amino acids , R.GD , a region consisting of only 5 amino acids, YIGSR, or trie like. Therefore, a peptide having the amino acid sequence set forth in SEQ ID NO. : 1 or an amino acid sequence set forth in SEQ ID NO. : 1 having amino acid substitution(s ) , amino acid deletion(s) at the end(s) thereof, or another amino acid sequence added to or at least one amino acid addition at one or both ends thereof, is considered to have vascular network forming activity as long as the above-described peptide or peptide variant region is exposed for interaction. Note that at present, the three-dimensional structure of artificial peptides can be easily predicted from the primary sequences using commerciallyavailable computer software . Therefore, even large-size peptides can be easily designed to have the above-described peptide or peptide variant region exposed. A computer program for computer modeling may be used in the process of selecting fragments or chemical materials .

Once appropriate chemical materials or fragments (compound species) are selected, they can be assembled into a single compound or a complex. Prior to construction, the relationship between the fragments may be conducted with viral inspection on the 3D images displayed on a computer screen concerning the structural coordinates of the vascular network formation peptide of the present invention. Following this, software, such as Quanta or Sybyl (Tripos Associates, St. Louis, MO), is used to manually construct models .

Examples of useful programs, which may be used for joining chemical materials or fragments , include:

1. CAVEAT (P.A. Bartlett et al., "CAVEAT: A Program to Facilitate the Structure-Derived Design of Biologically Active Molecules" (Molecular Recognition in Chemical and Biological Problems, Special Pub., Royal Chem. Soc, 78, pp. 182-196 (1989)); G, Lauri and P.A. Bartlett, "CAVEAT: a Program to Facilitate the Design of Organic Molecules", J. Comput. Aided Mol. Des . , 8, pp. 51-66 (1994)). CAVEAT is available from University of California, Berkeley, CA.

2. A 3D database system, suchasISΣS (MDL Information Systems, San Leandro, CA) . This field is reviewed in, for example, Y. C . Martin , "3D Database Searching in Drug Design" , J. Med. Chem., 35, pp. 2145-2154 (1992).

3. HOOK (M.B. 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, pp. 199-221 (1994) ) . HOOK is available from Molecular Simulations, San Diego, CA. Instead of construction of an activator or inhibitor by assembling fragments or chemical materials one by one, i.e., in a stepwise manner, the activator or inhibitor may be designed globally or de novo. A number of methods for designing novel ligands are available, for example:

1. LUDI (H.-J. Bohm, "The Computer Program LUDI: A New Method for the De IMovo Design of Enzyme Inhibitors", J. Comp. Aid. Molec. Design, 6, pp. 61-78 (1992)). LUDI is available fromMolecular- Simulations Incorporated, Sean Diego , CA.

2. LEGEND (Y. Nishibata et al.. Tetrahedron, 47, p. 8985 (1991)). LEGEND is available from Molecular Simulations Incorporated, San Diego, CA.

3. LeapFrog (available from Tripos Associa es, St. Louis, MO).

4. SPROUT (V. Gillet et al . , "SPROUT: A Prog- am for Structure Generation", J. Comput. Aided Mol. Design, 7, pp. 127-153 (1993)). SPROUT is available from University of Leeds, UK. Other molecular modeling techniques may be used in the present invention (e.g., N.C. Cohen et al., "Molecular Modeling Software and Methods for Medicinal Chemistry", J. Med. Chem. ,33, pp. 883-894 (1990);M.A. NaviaandM.A . Murcko, "The Use of Structural Information in Drug Design" , Current Opinions in Structural Biology, 2, pp. 202-210 (1992); L.M. Balbes et al., "A Perspective of Modern Methiods in Computer-Aided Drug Design", (Reviews in Computational Chemistry, vol. 5, K.B. Lipkowitz andD.B. Boyd, eds., VCH, New York, pp. 337-380 (1994)); W.C. Guida, "Software For Structure-Based Drug Design" , Curr. Opin. Struct. Biology., 4, pp. 777-781 (1994)).

Once a compound is designed or selected by the above-described method, the compound is evaluated by calculation to determine whether or not the compound has an optimum vascular network forming activity, resulting in optimization. The material of the present invention may be calculated and optimized so as not to have an electrostatic repulsive interaction with surrounding water molecules. Such a non-complementary electrostatic interaction includes charge-charge repulsive interaction, dipolev-dipole repulsive interaction, and charge-dipole repulsive interaction.

Specific computer software is available in the field of evaluating chemical deformation energy and electrostatic interaction. Examples of programs for such applications include: Gaussian 94, revision C (M.J. Frisch, Gaussian, Inc. , Pittsburgh, PA 1995) ; AMBER, version 4.1 (P.A. ICollman, University of California at San Francisco, 1995); QUANTA/CHARMM (Molecular Simulations , Inc., San Diego, CA 1995); Insight II/Discover (Molecular Simulations, Inc., San Diego, CA 1995); DelPhi (Molecular Simulations, Inc., San Diego, CA 1995); and AMSOL (Quantum Chemistry Program Exchange, Indiana University). These programs may be executed using, for example, a workstation ( Indigo2 with "IMPACT" graphics, Silicon Graphics). Other hardware systems and software packages ar-e known to those skilled in the art .

Another approach realized by the present invention is computational screening using a low molecular weight molecule database for chemical materials or compounds which may bind to the whole or a part of ZLOX-1. In this screening, the fitting level of the material to a binding site may be determined based on either morphological complementarity or estimated interaction energy E.C. Meng et al., J. Comp. Chem., 16, pp. 505-524 (1992)). (Combinatorial chemistry) Compound libraries used in the present invention can be produced by techniques, such as, without limitation, combinatorial chemistry techniques, fermentation techniques, plant and cell extraction techniques, or can be available from any supply source. Techniques for producing combinatorial libraries are well known in the art.

See, for example, E.R. Felder, Chimia, 1994, 48, 512-541;

Gallop et al. , J. Med. Chem., 1994, 37, 1233-1251; R.A.

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 etal.. Perspectives in DrugDiscovery and Design, 2, 269-282;

Cwirla et al.. Biochemistry, 1990, 87, 6378-6382; Brenner et al., Proc. Natl. Acad. Sci. USA, 1992, 89, 5381-5383; Gordon et al., J. Med. Chem., 1994, 37, 1385-1401; Lebl et al., Biopolymers, 1995, 37, 177-198; and references cited therein. These documents are herein incorporated by reference in their entirety.

Computers used herein may be any computers which can execute programs, including, without limitation, Windows

(registered trademark) -based computers, UNIX-based computers, Mac (registered trademark) OS-based computers,

LINUX-based computers, and the like.

The preferred embodiments have ^"been heretofore described for a better understanding of the pr-esent invention . Hereinafter, the present invention will be described by way of examples. The above descriptions and the examples below are provided only for illustrative purposes. The present invention is not limited thereto. Therefore, it should be understood that the scope of the present invention is not limitedto the embodiments orexamples specificallydescribed herein except as by the appended claims .

Examples Hereinafter, the present invention will be specifically described by way of examples .

(Example 1: Synthesis of peptide) A peptide having an amino acid secjuence set forth in SEQ ID NO. : 1 was synthesized using an automatic peptide synthesizer with a high-efficiency solid- phase technique based on Fmoc chemistry (K. Nokihara, et al., "Innovation and Perspectives in Solid-Phase Synthesis", 1992, ed. , R. Epton, Intercept Limited, Andover, UK, 445-4=48, 1992, Design andApplications of aNovel Simultaneous Multiple Solid-Phase Peptide Synthesizer; Kiyoshi Nokihara, Journal of Synthetic Organic Chemistry, Japan, 52, 34"7-358, 1994, "Kokoritsu Peputido Gosei: Tahinshumoku Dojijidogosei to Peputido Laiburari [Organic Peptide Synthesis: Multiple-item Simultaneous Automatic Synthesis cand Peptide Libraries]). The resultant peptide was examinecd using a coupled liquid chromatographymass spectrometry (L.CMS) system and confirmed to be highly pure (single ingredient, consistent with theoretical value) . An example of the result is shown in Figure 1. Figure 1 shows an example of the result of analysis of the peptide SWYGLR (SEQ ID NO.: 1).

With the above-described procedure, peptides used in the examples below were produced.

SEQ ID NO. : 2 sets forth a peptide AWYGLR which is a modification of SEQ ID NO. : 1. SEQ ID NO. : 3 sets forth a peptide SAVYGLR which is a modification of SEQ ID NO. : 1.

SEQ ID NO. : 4 sets forth a peptide SVAYGLR which is a modification of SEQ ID NO. : 1.

SEQ ID NO. : 5 sets forth a peptide SWYALR which is a modification of SEQ ID NO. : 1.

SEQ ID NO. : 6 sets forth a peptide SWYGAR which is a modification of SEQ ID NO. : 1.

SEQ ID NO. : 7 sets forth a peptide SWYGLA which is a modification of SEQ ID NO. : 1. SEQ ID NO. : 8 sets forth a peptide SWAGLR which is a modification of SEQ ID NO. : 1.

SEQ ID NO. : 9 sets forth a peptide SWFGLR which is a modification of SEQ ID NO. : 1.

SEQ ID NO. : 10 sets forth a pepticϋe SWYGL which is a modification of SEQ ID NO. : 1.

SEQ ID NO. : 11 sets forth a pepticϋe WYGLR which is a modification of SEQ ID NO. : 1.

SEQ ID NO. : 12 sets forth a peptide SWYGLRC which is a modification of SEQ ID NO.: 1.

SEQ ID NO. : 13 sets forth a peptide GRGDSWYGLR which is a modification of SEQ ID NO. : 1. (Example 2: Confirmation of angiogenesis action of synthesized peptide) Rat endothelial cells were three-dimensionally cultured in the presence of the peptide of the present invention synthesized in Example 1. To achieve this, the following procedure was specifically conducted. Transformed rat lung endothelial cells ( TRLEC cells) were used. TRLEC cells were disseminated in 10 μg/ml peptide solution blended with a collagen I layer in a carbon acid gas incubator for 14 days. A control (-: lacking a vascular network forming agent andVEGF ( + : aprotein Jnown as avascular network forming agent) were used.

After 14 days , the cultured cells were observed under a microscope to find that the control did not form a lumen. In the case of the peptide of the present invention and VEGF, lumens were formed and cells were adhered around the lumens . The lumen was observed under an electron microscope (x7000) to find that a plurality of microvilli (micro cell out growth) were formed on the inner wall of the lumen. Furtber, the interface between endothelial cells surrounding the lumen was observed under the electron microscope (xl500O) to find that the endothelial cells bound together tightly, i.e., tight junction. These findings indicated that the endothelial cells acquired the polarity and formed the lumen . Note that the polarity is defined as a property of a cell that the cell comes to have distinct portions, sixch as a headandatail. Endothelial cells typicallyhave nopolarity in culture and therefore do not form a lumen. Th-us, the above-described result indicated that the peptide induced lumen formation. The length of the lumen induced by the peptide of the present invention was superior to that induced by VEGF. According to these results, the peptide of the present invention was confirmed to have an action of adhering together vascular endothelial cells constituting tissue to form a lumen ( the lumen becomes a blood vessel in organisms ) . A schematic diagram showing this lumen formation is shown in Figure 2. Figure 3 shows a comparison between VEGF and the peptide SWYGLR (SEQ ID NO.: 1) in terms of lumen formation.

(Example 3 : Confirmation of In vivovascular network forming activity by DAS assay) The peptide of the present invention was dissolved in Dulbecσo ' s Modified Eagle ' s medium (DMEM) for cell culture at a concentration of 10 μg/ml. A micro cell comprising a 0.45 mm diameter cylinder with opposite ends being- sealed with a Millipore filter (trade name) was embedded in the dorsum of a mouse. The above-described solution containing the peptide of the present invention or a VEGF solution was injected into the micro cell. As a control, phosphate buffer solution without the peptide was injected to a mouse. After 5 days, tissue around the micro cell was observed under a microscope. A protocol for the DAS assay is schematicjally shown in Figure 4. The results of the DAS assay are shown in Figure 6.

Figure 5 shows angiogenesis when the peptide SWYGLR (SEQIDNO.: 1) was used. As clearly shown in Figure 5 , blood vessels were newly formed, and the newly formed blood essels were forminganetwork. Therefore, thepeptide of thepresent invention was demonstrated to be useful as a vascular network forming agent. In the case of the peptide SWYGLR (SEQ ID NO. : 1) , the formation of a network was seen. In contrast, no such network formation was observed with VEGF anal the control. Therefore, it was found that the present invention has a vascular network forming activitywhich is notpossessed by conventional angiogenesis agents .

Peptides synthesized as above, which were subjected to alanine scanning, and peptides shortened or elongated at the N- or C-terminus were examined for their network formation indices . Their sequences are described in the table below.

Peptide AWYGLR(SEQ ID NO. : 2) Peptide SAVYGLR (SEQ ID NO.: 3) Peptide SVAYGLR ( SEQ ID NO . : 8 ) Peptide SWAGLR (SEQ ID NO. : 4) Peptide SWYALR (SEQ ID NO. : 5) Peptide S YGAR (SEQ ID NO. : 6) Peptide SWYGLA (SEQ ID NO. : 7) Peptide SWYGL (SEQ ID NO. : 10) Peptide S YG (SEQ ID NO. : 17) Peptide WYGLR (SEQ ID NO.: 11) Peptide SWYGLRC (SEQ ID NO.: 12) Peptide GRGDSWYGLR (SEQ ID NO. : 13)

For alanine scanning, a photograph of an actual DAS assay is shown in Figure 7.

In addition, it was determined whether or not these peptides had heat resistance when temperature was increased (Figure 8) .

The vessel formation numbers calculated are described below.

control: O.OO±O VEGF: 2.75±1.03 peptide SWYGLR (SEQ ID NO.: l)(wild type): 3.75±0.75 peptide SWYGLR (SEQ ID NO.: 1) (50°C): 3.0 peptide SWYGLR (SEQ ID NO. : 1) (60°C): 3.5 peptide SWYGLR (SEQ ID NO. : 1) (90°C): 2.1 peptide SWYGLRC (SEQ ID NO. : 12): 3.9

As the temperature was increased, the angiogenesis ability was decreased. At 50°C and 60°C, the averages were 3 and 3.5, respectively, and the scores were 3 or more. At 90°C, the average was 2.1, which was significantly lower than that of the wild type (SEQ ID NO. : 1) (average: 4.1). With heat treatmen , the angiogenesis ability of the peptide SWYGLR was decreased. However, vascularization ability remained stable up to 60°C. SWYGLRC had an average of 3.9, which was only slightly different from that of the wild type ( SEQ ID NO . : 1 ) ( 4.1 ) . The difference between SWYGLRC ( SEQ ID NO.: 12) and SWYGLR (SEQ ID NO. : 1) was conducted to be attributed to the purity of the former which was lower than that of the latter wild type peptide. There is a possibility that SWYGLRC (SEQ ID NO. : 12) can be linked to other materials via the C residue. The peptide of the present invention can be applied to various biological materials . It was found that when the peptide was linked to a biological material, the angiogenesis ability was maintained up to a treatment temperature of 60°C. It was confirmed that WYGLR (SEQ ID NO. : 10) or SWYGL (SEQ ID NO.: 11) had angiogenesis ability.

Next, the length of new blood vessels is determined as follows. The number of pixels per new blood vessel is counted under a stereoscopic microscope (SZX12, Olympus, Japan) . The resultant image is read using Photoshop (registered trademark, Adobe, Japan). Pixels are counted. The number of pixels is converted to right-handed scores below.

score 1: 100 or less score 2: 100 or more and 125 or less score 3: 125 or more and 150 or less score 4: 150 or more and 175 or less score 5: 175 or more and 200 or less score 6: 200 or more

The results of measurement of the length of vessels formed are described as follows . control : O.OO±O

VEGF: 2.8+0 peptide SWYGLR (SEQ ID NO. : 1) (wild type) 4.1 peptide SWYGLR (SEQ ID NO. : 1) (50°C): 3.0 peptide SWYGLR (SEQ ID NO.: 1) (60°C): 3.5 peptide SWYGLR (SEQ ID NO. : 1) (90°C): 2.1 peptide SWFGLR (SEQ ID NO. : 9) 3.8 peptide SWF(pF)GLR (SEQ ID NO.: 19, pF represents parafluorine) : 4.1 peptide SWF (pMe ) GLR ( SEQ ID NO . : 20 , pMe represents paramethyl) : 4.5 peptide SWF (pN0₂ ) GLR ( SEQ ID NO . : 21 , pN0₂ represents paranitro ) : 4 . 1 peptide VVYGL (SEQ ID NO. : 22): 3.6 peptide WF(pMe)GL (SEQ ID NO. : 23): 3.5

The above-described results are summarized in Figure 9. This diagrammay be herein referred to as a balance sheet. In addition, vascular network forming acitivitv is observedforWYGLR (SEQIDNO. : 10 ) or SWYGL (SEQ IDNO. : 11). peptide WdFGL (dF: D-phenylalanine) (SEQ ID NO.: 24), peptide WF(pF)GL (SEQIDNO.: 25 ), peptide WF(pN0₂)GL (SEQ ID NO. : 26) were also demonstrated to have vascular network forming activity. Note that peptides having a D-isomer seem to have a slightly redueced level of vascular network forming activity.

Next, the length of new blood vessels is determined as follows . Thebloodvessels are imagedunder a stereoscopic microscope (SZX12 , Olympus, Japan) . The resultant imag-e is analyzed using Photoshop (registered trademark, Adobe,

Japan) . The number of pixels is counted and converted to the right-handed scores below.

Nwl : the early stage of network formation. Angiogenesis is observed but new blood vessels are not connected to one another.

Nw2 : the intermediate stage of network formation. New blood vessels are connected to one another like a ladder Nw3 : the late stage of network formation. New blood vessels are multiply branched.

Nw4 : the mature stage of network formation. The plexus of new blood vessels is wide spread.

The results of measurement of vascular- network forming index are described below. The vascular network forming index of the peptide SWYGLR was 3.0 and network formation was observed. In addition, SWFGLR hacL a higher vascular network forming index than that of SWYGLR. In contrast, such activity has not been generally observed for the conventional angiogenesis peptide VEGF. Therefore, it is understood that angiogenesis activity is not correlated with vascular network forming activity. Note that the vascular network forming index of a control was 1.0. Therefore, the present invention was the first to find peptides having vascular network forming activity.

Thus , it was revealed that at least the N-terminus and the C-terminus were not necessary for thevascular network forming activity. In the case of the peptide (SEQ ID NO. : 8) in which tyrosine at position 4 was substituted with alanine in SEQ ID NO.: 1, vascular network formation was not substantially observed. Therefore, it was revealed that when the N- or C-terminus of SEQ ID NO.: 1 was used as a linkage site to carriers, the angiogenesis action was not impaired-

(Example 4: Effect of chemically modi ication) NText, as shown in Figures 10A and 10B, it was investigated whether or not modification of an aromatic amino acid increases the vascular network formation activity.

E n the sequence of SEQ ID NO. : 1 , naturally-occurring tyrosinewas substitutedwith tryptophan (W) orphenylalanine (F) to produce a sequence as set forth in SEQ ID NO. : 15 or SEQ ID MO.: 9 , respectively. This procedure was performed using a peptide synthesizer as described in Example 1. Further, the side chain of the benzene ring of phenylalanine was modified. For example, a fluorine atom, a methyl group, or a nitro group was introduced into the para position of the benzene ring. Such a modified peptide was examined for its vascular network forming activity (Figures 11 and 12) . The results are shown below.

SWYGLR (SEQ ID NO.: 1): 3.0

SWFGLR (SEQ ID NO.: 9): 3.0 or more Substituted with fluorine (peptide SWF(pG)GLR) (SEQ ID

NO.: 19 )r 3.0

Substituted with methyl group (peptide SWF(pMe)GLR) (SEQ

ID NO. : 20) : 3.02

Substituted with nitro group (peptide SWF(pN0₂) GLR) (SEQ ID NO. : 21) : 3.0

I was observed that peptides SWF(pMe)GLR, SWF(pF)SLR, and SWF(pN0₂)GLR also had an vascular network forming index of 3.0 or mor , which is substantially the same as that of SWYGLR.

Accordingly, it was revealed that the vascular network forming activity was maintained or increased in any of the substituted sequences .

(Example 5: In vivo vascular network forming activity) FreAlagin AD type gelatin (manufactured by Miyagi

Kagaku Kogyo K.K. , molecular weight: 2000 to 20000), which is obtained by partial hydrolysis of animal-derived collagen to remove an allergenic portion, was used. The FreAlagin AD type gelatin is suitable for clinical applications .

FreAlagin AD or carrier protein (100 mg) was dissolved in 2.5 mL of MilliQ water. The peptide of the present invention (1.20 to 1.32 mg) was dissolved in 1 mL of 0.1 M phosphate buffer solution (pH 7.0) . The resultant mixture was added to the carrier protein, while cooling on ice. Glutaraldehide solution (25%) was diluted by a factor of 10 with 0.1 M phosphate buffer solution (pH 7.0). The solution (0.15 mL) was dropped into the above-described mixture (4°C). The reaction mixture was stirred at 4°C for 3 to 4 hours. Thereby, the amino group at the N-terminus of thepeptide of thepresent inventionwas boundviaacovalent bond to an amino group of the carrier protein.

Confirmation that the peptide was conjugated to the carrier protein was performed by thin layer chromatography

(TLC) based on absence of starting material. The reaction mixture was desalted using a G10 column (Pharmacia) where

5% acetic acid was used as an eluant . The main peak was lyophilized to quantitatively obtain the bound substance.

Rat endothelial cells were three-dimensionally cultured in the presence of the gelatin-conjugated vascular network formation peptide. This procedure was specifically performedas describedbelow. The cellusedwas atransformed rat lung endothelial cell (TRLEC) . The gelatin-conjugated vascular network formation peptide (conjugate) and collagen type I were mixed in a ratio of 1:10 to a concentration of 10 μg/ml (conjugate + collagen). The TRLEC cells were dispersed in the mixed layer of the mixed solution, and incubated in carbon acid gas for 14 days . A control ( - : lackingavascularnetwork formingagent andVEGF ( + : aprotein known as a vascular network forming agent) were used. After 14 days, the cultured cells were observed under a microscope to find that the controls did not form a lumen. In the case of the peptide conjugate of the present invention and VEGF, lumens were formed and cells were adhered around the lumens . The lumen was observed under an electron microscope (x7000) to find that a plurality of microvilli (micro cell outgrowth) were formed on the inner wall of the lumen. Further, the interface between endothelial cells surrounding the lumen was observed under the electron microscope (xl5000) to find that the endothelial cells bound together tightly, i.e., tight junction. The length of the lumen induced by the gelatin-conjugated vascular network forming agent was superiorto that inducedbyVEGF . Accordingto theseresults , the gelatin-conjugated vascular network forming agent was confirmed to have an action of adhering together vascular endothelial cells constituting tissue to form a lumen (the lumen becomes a blood vessel in organisms ) .

Gelatin-conjugated vascular network formation peptide solution (100 μg/ml) was blended with collagen I in a ratio of 1:10 to obtain a mixture having a concentration of 10 μg/ml(gelatin-conjugated peptide and collagen). A micro cell comprising a 0-45 mm diameter cylinder with opposite ends being sealed with a Mikawapore filter (trade name) was embedded in the dorsum of a mouse. The above-described solution containing the gelatin-conjugated vascular network formation peptide solution or a VEGF solution was injected into the micro cell. As a control, phosphate buffer solution without the peptide was injected into a mouse. After 5 days , tissue around the micro cell was observed under a microscope.

The resultant vascular network forming activity is shown below.

Gelatin-conjugated vascular network formation peptide (SEQ ID NO. : 1): 3.0 VEGF: 2 or less Control: 1

Thus, it was demonstrated that the present invention has the vascular network forming activity In vivo . This effect was not observed for VEGF, despite this protein having angiogenesis ability. The present invention can be said to provide the significant effect which cannot be achieved by conventional techniques . (Example 6: Use in combination of biological substitute materials) It is determined whether or not the vascular network forming agent of the present invention has a potent vascular network forming activity and can he used for embedding of biological substitute materials (e.g., artificial bones, etc . ) , artificial organs , and the like , and repair of organs . Hydroxyapatite (representative artificial bone) is used to prepare a bone substitute, and the gelatin-conjugated vascular network formation pepticLe is adhered thereto as described in Example 5. When this artificial bone is implanted, a vascular network can b>e formed around the bone. (Example 7: Treatment of Heart) In Example 7, a poly(lactic acid-co-glycoliσ acid) (ϊ>LGA) was used as a support, and the peptide SWYGLR (SEQ ID NO. : 1) produced in Example 1 and a variant thereof were used as biological molecules to prepare an implant. As a result , the ability to form a network of the present invention was demonstrated in the heart .

(Methods and Results) <Design of Scaffold> A sheet of knitted mesh was attached to two sheets of woven mesh (0.2 mm thick for each, a total of 0.6 mm thick). When a resultant patch is implanted into an organism, the knitted mesh faces the luminal side thereof while the woven mesh faces the outside thereof. These three sheets of mesh were made of a Vycryl polyglactin 910 mesh (PLGA (a copolymer having a glycolic acid-to-laσtic acid ratio of 90 : 10) ) , which is a biodegradable synthetic macromolecule. The resultant structure was subjected to collagen σrosslinking treatment to obtain a PLGA-collagen composite film which was used as a scaffold. Two groups of scaffolds were prepared: A) only type I collagen was used as a crosslinking agent in the σrosslinking treatment ; andB) type I collagen and the peptide SWYGLR (SEQ ID NO.: 1) were used. A. crosslinking method will be described below. A 20 mm diameter patch was stitched to the pulmonary artery trunk. <Crosslinking Method> The above-described support was impregnatedwith the collagen solution, followed by lyophilization. A crosslinking treatment was conducted or about 4 hours using 37°C glutaraldehyde saturated vapor . Finally, the support was shaken in 0.1-M aqueous glycine solution for 15 min 3 times, followed by washing with distilled water 3 times, and then lyophilization. With this procedure, a collagen-containing support was prepared. The peptide SWYGLR was added to the solution, and a crosslinking treatment was similarly performed.

<Mechanical Strength> The strength of the support was measured using a tension tester. A weight was loaded on a strip material having a width of 5 mm and a length of 30 mm in a minor axis direction at a rate of 10 mm/min so as to measure the strain at break and the modulus of elasticity thereof (TENSILLON ORIENTEC) . As a control, a glutaraldehyde-treated horse pericardium was used for comparison.. The PLGA-collagen composite film had a tension strength of 75±5 N, while the glutaraldehyde-treated horse pericardium had a tension strength of 34+11 N. Thus, the angiogenesis peptide-bound film had a tension strength comparable to that of these films . <Efficiency of Cell Adhesion> The cell acceptance ability of the support was determined as follows. The cell adhesion efficiency of vascular endothelial cells (VECs) andvascular smooth muscle cells (VSMCs) labeled with a fluorescent antibody (PKH-26 (SIGMA)) was compared In vitro between a PLGA-collagen composite film subjected to crosslinking treatment with only type I collagen and a PLGA-collagen composite film subjected to crosslinking treatment with type I and type IV collagens . The cell adhesion efficiency was determined! by the color development area (%) of a fluorescent pigment per visual field of a fluorescence microscope. For both vascular endothelial cells (VECs) and vascular smooth muscle cells (VSMCs) , the PLGA-collagen composite film subjected to type I and type IV collagen crosslinking treatment exhibited a significantly larger color development area of the fluorescent pigment, and cell acceptance was confirmed. The above-described result demonstrated that the support maintained or improved the cell adhesion efficiency.

<Factor VIII Staining> The number of blood vessels can be determined by immunohistochemically staining blood vessels with a Factor Vlll-relevant antigen or the like and counting the stained bloodvessels. Specifically, specimens are ixed with 10% buffered formalin, followed by paraffin embedding. Several continuous slices are prepared from each specimen, followed by freezing. Next, the frozen slices are fixed with 2% paraformaldehyde in PBS for 5 min at room temperature and immersed in methanol containing 3% hydrogen peroxide for 15 min, followed by washing with PBS . This sample is covered with bovine serum albumin solution for about 10 min to block non-specific reactions. The specimen is coupled with HRP, followed by incubation overnight with an EPOS-conjugated antibody for the Factor VIII-relevant antigen. After the sample is washed with PBS, the sample is immersed in diaminobenzidine solution (e.g., 0-3 mg/ml diaminobenzidine in PBS) to obtain positive staining. Stained vascular endothelial cells are counted under, for example, an optical microscope (x 200 magnification) . For example, the result of counting is represented by th-e number of blood vessels per square millimeters. After a specific treatment, it is determined whether or not the number of blood vessels statistically significantly increased, so as to confirmthepresence of FactorVIII . Thereby, forexample, the presence and angiogenesis activity of -vascular endothelial cells can be determined.

<Elastica van Gieson Staining> Elastic fiber was stained by elastica van Gieson staining. The procedure is described as follows. A sample is optionally deparaffinized (e.g., with pure ethanol), followed by washing with water. The sample is immersed in resorcin fuchsin solution (available from Muto Chemical, etc.) for 40 to 60 min. Thereafter, the sample is washed with 70% alcohol and is immersed in Omni's hematoxylin for 15 min. Thereafter, the sample is washed with running water for 5 min and is immersed in van Gieson solution for 2 min. The sample is washed, immediately followed by dehydration, clearing, and mounting.

<Hematoxylin ^• Eosin (HE) Staining> The fixation of cells on a support was observed by HE staining. The procedure is described as follows. A sample is optionallydeparaffinized (e.g. , withpure ethanol) , followed by washing with water. The sample is immersed in Omni's hematoxylin for 10 min. Thereafter, the sample is washed with running -water, followed by color development with ammonia water for 30 sec. Thereafter, the sample is washed with running water for 5 min and is stained with eosin hydrochloride solution (10-fold dilution) for 2 min, followed by dehydration, clearing, and mounting. <von Kossa Staining> Cells were stained by von Kossa method in order to observe the calcification in the cell. The procedure is described as follows . A sample is optionally deparaffinized (e.g., with pure ethanol), followed by washing with water (distilled water). The sample is immersed in 25% silver nitrate solution (under indirect light) for 2 hours. Thereafter, the sample is washed with distilled wat r and is immersed in 42% sodium thiosulfate (hypo) for 5 min. Thereafter, the sample is washed with running water for- 5 min and is immersed in Kolnechterot for 5 min. Thereafter, the sample is washed with running water for 5 min, folio-wed by dehydration, clearing, and mounting.

<Implantation> A thus-obtained support and a support obtained by seeding, onto such a composite film, self vascular endothelial cells (VECs) and self vascular smooth muscle cells (VSMCs), were prepared. These films were implanted into the pulmonary artery trunk of adult beagle dogs ( 8 to 10 kg) under partial clamping.

The cells were prepared as follows . A vein was extracted from the anterior surface of a lower limb of an adult beagle dogof the same type . Vascular endothelial cells (VECs) andvascular smoothmuscle cells (VSMCs) were isolated from the vein, followed by culture . The vascular endothelial cells and the vascular smooth muscle cells were seeded onto the PLGA-collagen composite film at a density of 1.3 x 10⁶ cells/cm², respectively. After implantation, the film was removed and histologically examined after two weeks , two months , and 6 months . <In vivo: Two Weeks after Implantation> Forboth the support andthe self cell-seeded support , no clear thrombus formation was observed using the naked eye. In the case of HE staining, PLGA residues were observed and connective tissue was present therebetween. In the PLGA-collagen composite film having- the seeded self vascular endothelial cells and vascular smooth muscle cells, only seeded fluorescent antibody-labeled vascular endothelial cells were scattered on the lumina side. Therefore, it is suggested that most of the cells were detached from the PLGA-collagen composite film.

<In vivo: Two Months after Implantation> Both the support and the self cell-seeded support had a smooth luminal surface observable with the naked eye. HE staining indicated complete absorption of PLGA anda tissue structure comparable to normal blood vessels.

Thevascular endothelial cells were studiedbyFactor VIII staining and the vascular smooth muscle cells were studied by -SMA (smooth muscle aσtin) immunostaining. In both of the supports. Factor VIII immunostaining indicated a monolayer of continuous vascular endothelial cells and the α-SMA immunostaining indicated the smooth muscle cells aligned on the luminal side.

Moreover, the vascular el stic fiber was studied by elastica van Gieson staining. In both of the supports, elastic fiber was observed in the luminal layer of a blood vessel .

The vascular network forming activity was examined to reveal that vascular network formation was enhanced in the pulmonary artery of the heart .

Although certain preferred embodiments have been described herein, it is not intended that such embodiments be construed as limitations on the scope of the invention except as set forth in the appended claims. Various other modifications and equivalents will be apparent to and can be readily made by those skilled in the art, after reading the description herein, without departing from the scope and spirit of this invention. All patents, published patent applications and publications cited herein are incorporated by reference as if set forth fully herein.

INDUSTRIAL APPLICABILITY The present invention is -useful in any situation

(medical, therapeutic, and odontological situations, etc.) in which vascular network formation is desired. Particularly, the present invention is useful in therapy of lifestyle related diseases, such as ischemic diseases (e.g., myocardial infarction, brain infarction, occlusive aortosclerosis, etc.), and the like.

Claims

1. A composition for forming a network of blood vessels, comprising: a peptide having an amino acid sequence

Xi-X2-X₃-X-X₅-X6-X7 (SEQ ID NO.: 14), where Xi is serine ( S ) , threonine ( ) , or a variant thereof, or is absent, X₂ is valine (V) , alanine (A) , glycine (G), leucine (L), isoleucine (I), or a variant thereof, X₃ is valine(V), alanine (A), glycine CG), leucine (L), isoleucine (I), or a variant thereof, X.₄ is an amino acid having an aromatic ring as a side chain or a variant thereof, X₅ is glycine (G) or a variant thereof, Xβ is leucine (L), alanine (A), glycine (G) , valine (V), isoleucine (I), or a variant thereof, X₇ is arginine (R) , lysine (K) , or a variant thereof, or is absent, or a variant or salt thereof.

2. A composition according to claim 1, wherein the peptide or avariant thereof has avascular network formation activity of 3.0 or more according to the vascular network formation index.

3. A composition according to claim 1, wherein Xi is serine or a variant thereof.

4. A composition according to claim 1, wherein X₂ is valine or a variant thereof .

5. A composition according to claim 1, wherein X₃ is valine or a variant thereof .

6. A composition according to claim 1, wherein X is phenylalanine, tyrosine, or a variant thereof.

7. A composition according to claim 1 , wherein X₄ is phenylalanine or a variant thereof.

8. A composition according to claim 1 , wherein X is tyrosine or a variant thereof.

9. A composition according to claim 1, wherein X₅ is glycine or a variant thereof.

10. A composition according to claim 1 , wherein X₆ is leucine or a variant thereof .

11. Acomposition according to claim 1, whereinX₇ is arginine or a variant thereof .

12. A composition according to claim 1, wherein the peptide or a variant thereof is an amino acid sequence set forth in SEQ ID NO.: 1, or an amino acid sequence set forth in SEQ ID NO. : 1 having 1 to 3 amino acid substitutions, or 1 or 2 amino acid deletions at one or both ends thereof, or another amino acid sequence added to or at least one- amino acid addition at one or both ends thereof, and has a vascular network forming activity.

13. A composition according to claim 1, wherein the peptide or a variant thereof is a peptide having an amino acid sequence set forth in SEQ ID NO. : 1, or an amino acid sequence set forth in SEQ ID NO. : 1 having 1 or 2 amino acid substitutions wherein the fourth tyrosine residue is a tyrosine residue or an amino acid having an aromatic ring as a side chain, or 1 amino acid deletion at one or both ends thereof, or another amino acid sequence added to or at least one amino acid addition at one or both ends thereof, and has a vascular network forming activity.

14. A composition according to claim 1, wherein the amino acid having an aromatic ring as a side chain is phenylalanine or chemically modified phenylalanine having 1 or more substituents on a benzene ring thereof.

15. A composition according to claim 1, wherein the peptide or a variant thereof is a peptide having an amino acid sequence set forth in SEQ ID NO.: 9, or an amino acid sequence set forth in SEQ ID NO. : 9 having 1 amino acid deletion at one or both ends thereof, or another amino aoid sequence added to or at least one amino acid addition at one or both ends thereof after 1 amino aciddeletion at one orboth ends thereof .

16. A composition according to claim 1, wherein the peptide or a variant thereof is a peptide having an amino acid sequence set forth in any one of SEQ ID NOs.: 1 to 7, or an amino acid sequence set forth in any one of SEIQ ID NOs . : 1 to 7 having another amino acid sequence added to or at least one amino acid addition at one or both ends thereof .

17. A composition according to claim 1, wherein the peptide or a variant thereof is a peptide having an amino acid sequence set forth in any one of SEQ ID NOs.: 9 to 11, or an amino acid sequence set forth in any one of SEQ ID NOs. : 9 to 11 having another amino acid sequence added to or at least one amino acid addition at one or both ends thereof.

18. A composition according to claim 1, wherein the peptide or a variant thereof is a peptide having an amino acid sequence set forth in SEQ ID NO. : 9, or an amino acid sequence set forth in SEQ ID NO. : 9 having another amino acid sequence added to or at least one amino acid addition at one or both ends thereof .

19. A composition according to claim 1, wherein the peptide or a variant thereof comprises an amino acid sequence represented by SWXGL or WX₄GLR where X₄ is an amino acid having an aromatic ring as a side chain or a variant thereof .

20. A composition according to claim 1, wherein the total number of amino acid residues in the peptide or a variant thereof is 4 to 350.

21. A composition according to claim 1, wherein the total number of amino acid residues in the peptide or a variant thereof is 4 to 50.

22. A composition according to claim 1, wherein the total number of amino acid residues in the peptide or a variant thereof is 5 to 20.

23. A composition according to claim 1, wherein the peptide or a variant thereof is a peptide having an amino acid sequence set forth in any one of SEQ ID NOs.: 1 to 7, or an amino acid sequence set forth in any one of SEQ ID NOs . : 1 to 7 having 10 or less amino acid additions at one or both ends thereof .

24. A composition according to claim 1, wherein the peptide comprises an amino acid sequence set forth in any one of SEQ ID NOs. : 1 to 7.

25. A composition according to claim 1, wherein the peptide or a variant thereof is a peptide having an amino acid sequence set forth in any one of SEQ ID NO.: 9 to 11, or an amino acid sequence set forth in any one of SEQ ID NO. : 9 to 11 having 10 or less amino acid additions at one or both ends thereof .

26. A composition according to claim 1, wherein the peptide or a variant thereof comprises an amino acid sequence set forth in any one of SEQ ID NOs.: 9 to 11.

27. A composition according to claim 1, wherein the blood vessel is a capillary blood vessel.

28. A composition according to claim 1, wherein the peptide or a variant thereof is bound to a carrier.

29. A composition according to claim 2 S , wherein the carrier is a protein.

30. A composition according to claim 29 , wherein the protein is a cell adhesion protein.

31. A composition according to claim 30, wherein the cell adhesion protein is collagen or a partial hydrolysis product thereof .

32. A method for forming a network of blood vessels, comprising the step of: administering a composition according to any one of claims 1 to 31 to a site in a patient in need of formation of the network of blood vessels.

33. Use of: a peptide having an amino acid sequence X1-X2-X3-X4-X5-X6-X7 (SEQ ID NO. : 14 ) , where Xi is serine (S) , threonine (T) , or a variant thereof or is absent , X₂ is valine (V), alanine (A), glycine (G) , leuoine (L), isoleucine (I), or a variant thereof, X₃ is valineCV), alanine (A), glycine (G), leucine (L), isoleucine (I), or a variant thereof, X is an amino acid having an aromatic ring as a side chain or a variant thereof, X₅ is glycine (G) or a variant thereof, X₆ is leucine (L), alanine (A), cjlycine (G), valine (V), isoleucine (I), or a variant thereof, X₇ is arginine (R), lysine (K) , or a variant thereof, or is absent; or a variant or salt thereof, for production of a medicament for forming a network of blood vessels.