WO2005094865A1 - Composé ayant un effet angiogénique - Google Patents

Composé ayant un effet angiogénique Download PDF

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Publication number
WO2005094865A1
WO2005094865A1 PCT/JP2005/006124 JP2005006124W WO2005094865A1 WO 2005094865 A1 WO2005094865 A1 WO 2005094865A1 JP 2005006124 W JP2005006124 W JP 2005006124W WO 2005094865 A1 WO2005094865 A1 WO 2005094865A1
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Prior art keywords
group
substituted
amino acid
peptide
aromatic
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PCT/JP2005/006124
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English (en)
Japanese (ja)
Inventor
Yoshiki Sawa
Satoshi Taketani
Shigeru Miyagawa
Junzo Takahashi
Nariaki Matsuura
Yoshinosuke Hamada
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Cardio Incorporated
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Publication of WO2005094865A1 publication Critical patent/WO2005094865A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
    • A61K38/08—Peptides having 5 to 11 amino acids
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/06—Linear peptides containing only normal peptide links having 5 to 11 amino acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system

Definitions

  • the present invention is in the field of medicine. More particularly, the present invention relates to a medicament for forming a novel angiogenic agent.
  • amino acid derivatives are convenient building units for combinatorial chemical libraries, and their structure can be optimized in a short time by solid-phase synthesis. Therefore, if a compound that promotes angiogenesis more strongly exists, it can be advantageously synthesized and administered alone or in combination.
  • Patent Document 1 Conventionally, experiments have been attempted in which a peptide sequence is modified (Patent Document 1). However, such modification at the peptide level alone has a limitation in enhancing the activity. Special In Patent Document 1, modifications other than peptides have been attempted, but compounds having angiogenic activity exceeding those of peptides have been found.
  • angiogenesis has been enhanced more than natural peptides, and the appearance of a compound has been awaited.
  • Patent document l WO 03Z030925
  • the present inventors have conducted intensive studies and found that a peptide having a specific modification of an aromatic group in a specific amino acid sequence unexpectedly has enhanced angiogenic activity. Was completed.
  • the present invention provides the following.
  • a composition for angiogenesis comprising a peptide, comprising a sequence of VVXGL (SEQ ID NO: 1), wherein X is a variant of an aromatic amino acid.
  • composition according to item 1 wherein X is a modified form of phenalanine or tyrosine.
  • composition according to item 1 wherein the modification includes a peptide in which the para, ortho, or meta position of the side chain of the aromatic amino acid is substituted with an electron-withdrawing substituent.
  • composition according to item 1 wherein X is a group in which an aromatic group of the aromatic amino acid is substituted with a hydrophobic group.
  • composition according to item 1 wherein X is an aromatic group of the aromatic amino acid substituted with an alkyl group.
  • composition according to item 1 wherein X is substituted with an alkyl group at the para-position of the aromatic group of the aromatic amino acid.
  • the X is an aromatic amino acid wherein the aromatic group is substituted with a C1-C6 alkyl group, Item 1.
  • composition for forming a vascular network comprising a peptide comprising the sequence WXGL, wherein X is a variant of an aromatic amino acid.
  • composition according to item 12 wherein the modification includes a peptide in which the para, ortho, or meta position of the aromatic group of the aromatic amino acid is substituted with an electron-withdrawing substituent.
  • composition according to item 12 wherein X is a group in which the aromatic group of the aromatic amino acid is substituted with a hydrophobic group.
  • composition according to item 12 wherein X is an aromatic amino acid in which the aromatic group is substituted with an alkyl group.
  • composition according to item 12 wherein X is a group in which the aromatic group of the aromatic amino acid is substituted with a methyl group.
  • a method for forming a blood vessel which comprises administering the composition according to any one of Items 1 to L1 to a site of a patient where angiogenesis is desired.
  • a method for forming a vascular network comprising administering the composition according to claim 1.
  • WXGL a peptide called WXGL, wherein X is a modified aromatic amino acid or a modified variant thereof or a salt thereof, for use in the production of a pharmaceutical composition for forming a vascular network of a peptide.
  • angiogenesis agent of the present invention is useful for regenerating and repairing organs using biological substitutes such as artificial bones, and for ischemic diseases such as myocardial infarction, cerebral infarction, and obstructive aortic sclerosis, which are a large part of lifestyle-related diseases. It is also useful for treatment.
  • FIG. 1 shows a schematic diagram of the action of adhering cells to form a lumen between them.
  • FIG. 2 shows an example of the action of forming a lumen by comparing VEGF and peptide SWYGLR (SEQ ID NO: 2).
  • FIG. 3 is a diagram showing the state of a tissue around a microcell observed with a microscope using a DAS assay 5 days later in Example 3.
  • FIG. 4 shows the state of angiogenesis and formation of a vascular network when peptide SWYGLR (SEQ ID NO: 2) was used.
  • FIG. 5 shows the state of angiogenesis and vascular network formation using peptide SWYGLR (SEQ ID NO: 2) and the modified peptide of the present invention.
  • FIG. 6 shows a balance sheet of the vascular network ability of the peptide of the present invention. Description of Sequence Listing
  • SEQ ID NO: 1 is an amino acid sequence WXGL showing the basic structure of the peptide of the present invention. Where X is a variant of an aromatic amino acid.
  • SEQ ID NO: 2 is a peptide (SVVYGLR) capable of angiogenesis in osteobontin.
  • SEQ ID NO: 4 SWF (pMe) GLR (where F (pMe) is a phenyl group of phenylalanine substituted with a methyl group at the para-position)
  • SEQ ID NO: 5 SWF (pF) GLR (where F (pF) is obtained by substituting a fluoro group at the para-position of the phenyl group of phenylalanine)
  • SEQ ID NO: 6 SWF (pNO) GLR (where F (pNO) is a phenylalanine phenol
  • SEQ ID NO: 7 SWF (D) GLR (where F (D) is the D-form of phenalanine)
  • SEQ ID NO: 8 SWF (pEt) GLR (where F (pEt) is The phenyl group of realanine is substituted with an ethyl group at the para position.)
  • SEQ ID NO: 9 SWF (pPr) GLR (where F (pPr) is a phenyl group in which a propyl group is substituted with a propyl group at the para position)
  • SEQ ID NO: 10 SWF (pHex) GLR (where, F (pHex) is phenylylalanine in which a hexyl group is substituted at the para-position of the phenyl group)
  • SEQ ID NO: 18 SWF (pMe) GL (where F (pMe) is a phenyl group of phenylalanine With a methyl group substituted at the para-position)
  • VVF (pMe) GL (where F (pMe) is a phenylalanine in which a methyl group is substituted at the para-position of the phenyl group)
  • SEQ ID NO: 20 is another exemplary sequence of the peptide of the present invention.
  • protein protein
  • polypeptide oligopeptide
  • peptide refers to a polymer of amino acids of any length.
  • the polymer may be linear or branched or cyclic.
  • the amino acids may be naturally occurring or non-naturally occurring or modified amino acids. These terms may also include those assembled into a complex of multiple polypeptide chains.
  • the term also embraces natural or artificially modified amino acid polymers. Such modifications include, for example, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification (eg, conjugation with a labeling component).
  • This definition also includes, for example, polypeptides containing one or more analogs of an amino acid (eg, including unnatural amino acids, etc.), peptide-like conjugates (eg, peptoids) and those known in the art. Other modifications are included.
  • polypeptides containing one or more analogs of an amino acid eg, including unnatural amino acids, etc.
  • peptide-like conjugates eg, peptoids
  • polynucleotide As used herein, the terms “polynucleotide”, “oligonucleotide” and “ “Nucleic acid” is used interchangeably herein and refers to a polymer of nucleotides of any length. The term also includes “derivative oligonucleotides” or “derivative polynucleotides.” “Derivative oligonucleotide” or “derivative polynucleotide” refers to an oligonucleotide or polynucleotide having an unusual force or a bond between nucleotides containing a derivative of a nucleotide, and is used interchangeably.
  • oligonucleotide examples include, for example, 2 ′ O-methyl-ribonucleotide, a derivative oligonucleotide in which a phosphodiester bond in an oligonucleotide is converted to a phosphorothioate bond, and a phosphorus in an oligonucleotide.
  • Derivative oligonucleotide substituted with propylperacyl derivative oligonucleotide in which peracyl in oligonucleotide is substituted with C-5 thiazole peracyl, cytosine in oligonucleotide substituted with C-5 propynylcytosine
  • a particular nucleic acid sequence also has a conservatively modified version thereof (e.g., a degenerate codon substitution), similar to the explicitly indicated sequence. ) And complementary sequences are contemplated. Specifically, degenerate codon substitutions create a sequence in which the third position of one or more selected (or all) codons is replaced with a mixed base and a Z or deoxyinosine residue. (Batzer et al., Nucleic Acid Res. 19: 5081 (1991); Ohtsuka et al., J. Biol. Chem. 260: 2605- 2608 (1985); Rossolini et al., Mol Cell. Probes 8: 91- 98 (1994)
  • complex molecule refers to a molecule formed by linking a plurality of molecules such as polypeptides, polynucleotides, lipids, and sugars.
  • Such complex molecules include, for example, Examples include, but are not limited to, glycolipids, glycopeptides, and the like. It is understood that the peptide of the present invention includes a complex in which an alkyl group, a PEG group, a sugar chain, and the like are bound.
  • an "isolated" biological agent eg, a nucleic acid or protein
  • a nucleic acid or protein is defined as another biological agent in a cell of an organism in which the biological agent naturally exists.
  • Factors e.g., when it is a nucleic acid, a nucleic acid containing a factor other than the nucleic acid and a nucleic acid sequence other than the target nucleic acid; when it is a protein, it contains an factor other than the protein and an amino acid sequence other than the target protein
  • Protein or the like Protein or the like.
  • isolated nucleic acids and proteins include nucleic acids and proteins that have been purified by standard purification methods. Thus, isolated nucleic acids and proteins include chemically synthesized nucleic acids and proteins.
  • a "purified" biological agent eg, a nucleic acid or a protein
  • a purified biological agent is at least a part of a factor naturally associated with the biological agent. Means that has been removed.
  • the purity of the biological agent in the purified biological agent is higher (ie, more concentrated) than the state in which the biological agent is normally present.
  • purified and isolated preferably refer to at least 75% by weight, more preferably at least 85% by weight, even more preferably at least 95% by weight. %, And most preferably at least 98% by weight, of the same type of biological agent.
  • homology of a gene refers to the degree of identity between two or more gene sequences.
  • similarity refers to two or more similarities when conservative substitutions are regarded as positive (identical) in the above homology. It refers to the degree of identity between gene sequences. Therefore, if there is a conservative substitution, Identity and similarity differ depending on the presence of When there is no conservative substitution, identity and similarity show the same numerical value.
  • amino acid is used in a meaning commonly used in the art, and refers to an organic compound having a carboxyl group and an amino group.
  • amino acids may be natural or unnatural amino acids.
  • natural amino acid refers to the L-isomer of a natural amino acid. Natural amino acids include glycine, alanine, norin, leucine, isoleucine, serine, methionine, threonine, phenylalanine, tyrosine, tryptophan, cysteine, proline, histidine, aspartic acid, asparagine, glutamic acid, glutamine, ⁇ -force Xyglutamic acid, arginine, ortin, and lysine. Unless otherwise indicated, all amino acids referred to in the present specification usually mean L-form, but the present invention is not limited thereto, and forms using D-form amino acids are also within the scope of the present invention. It is understood that.
  • amino acid variant refers to a molecule that is not a natural amino acid but is similar in physical properties and / or function to a natural amino acid.
  • modified amino acids include a hydrophobic group, phenylalanine having a benzyl side chain (para-, meta-, ortho-position, etc.) to which an alkyl group, a halo group, a nitro group, etc. are bonded, etyonine, canavanine , 2-methylglutamine and the like. It is understood that, in the present invention, amino acid variants may include unnatural amino acids and amino acid mimetics.
  • unnatural amino acid means an amino acid that is not normally found in nature in a protein.
  • unnatural amino acids include norleucine, para-nitrophenylalanine, homophenylalanine, para-fluorophenylalanine, 3-amino-2-benzylpropionic acid, the D- or L-form of homoarginine, and D-phenylalanine. No.
  • amino acid mimetic refers to a compound that has a structure different from the general chemical structure of an amino acid, but that functions in a manner similar to a naturally occurring amino acid.
  • nucleotide may be a natural or non-natural one as long as it has an ability to encode an amino acid.
  • fragment refers to a polypeptide or polynucleotide having a sequence length of l to n-1 relative to a full-length polypeptide or polynucleotide (having a length of n). Refers to tide. It is understood that variants of the peptides of the invention also include such fragments.
  • the length of the fragment can be appropriately changed depending on the purpose.
  • a lower limit of the length in the case of a polypeptide, 3, 4, 5, 6, 7, 8, 9, 10, 1, 5, 20, 25, 30, 40, 50 and more amino acids, and integer lengths not specifically listed herein (eg, 11) are also suitable as lower limits. possible.
  • a polynucleotide 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 40, 50, 75, 100 and more nucleotides are mentioned, and specific listings are given here. Lengths expressed as unintended integers (eg, 11, etc.) may also be appropriate as lower limits.
  • the lengths of polypeptides and polynucleotides can be represented by the numbers of amino acids or nucleic acids, respectively, as described above, but the above numbers are not absolute and have the same function. As far as it has, the above-mentioned number as an upper limit or adjustment is intended to include a few above and below (or, for example, 10% above and below) the number. In order to express such an intention, in this specification, "about” may be used before a number. However, it should be understood that the presence or absence of "about” herein does not affect the interpretation of that number.
  • the "corresponding" amino acid or nucleic acid refers to an action similar to a predetermined amino acid in a polypeptide or polynucleotide serving as a reference for comparison with respect to a certain polypeptide molecule or polynucleotide molecule, respectively.
  • an antisense molecule of a polynucleotide can be a similar portion of an ortholog corresponding to a particular portion of the antisense molecule.
  • the ability of the specific sequence in human osteopontin to be used is understood.
  • the term "corresponding" gene refers to, in a certain species, Refers to a gene that has or is expected to have the same action as the predetermined gene in the above, and when there are a plurality of genes having such an action, refers to those having the same evolutionary origin.
  • the corresponding gene for a gene may be an ortholog or species homolog of that gene.
  • Such corresponding genes can be identified using techniques well known in the art.
  • the corresponding gene in an animal can be obtained by using the sequence of the reference gene for the corresponding gene (for example, the mouse osteopontin gene) or the peptide of the present invention or its coding sequence itself as a query sequence (eg, It can be found by searching the sequence database of humans and rats.
  • Amino acids may be referred to herein by either their commonly known three letter symbols or by the one letter symbols recommended by the IUPAC- IUB Biochemica 1 Nomenclature Commission. Nucleotides may also be referred to by the generally recognized one-letter code.
  • the hydrophobicity index is preferably within ⁇ 2, more preferably within ⁇ 1, and even more preferably within ⁇ 0.5. It is understood in the art that such substitution of amino acids based on hydrophobicity is efficient.
  • hydrophilicity index can also be considered in designing polypeptides. As described in US Pat. No. 4,554,101, the following hydrophilicity indices have been assigned to amino acid residues: arginine (+3.0); lysine (+3.0); aspartic acid Glutamic acid (+ 3.0 ⁇ 1); Serine (+0.3); Asparagine (+0.2); Glutamine (+0.2); Glycine (0); Threonine (+ 3.0 ⁇ 1); Proline (0.5 ⁇ 1); alanine (0.5); histidine (0.5); cysteine (1.1); methionine (1.3); valine ( I-1.5); Leucine (1-1.8); Isoloisin (1-1.8); Tyrosine (1-2.3); Fehlalanine (1-2.5); and Tryptophan (3.4).
  • an amino acid can be substituted for another that has a similar hydrophilicity index and still provide a bioisostere.
  • the hydrophilicity index is preferably within ⁇ 2, more preferably within ⁇ 1, and Even more preferably, it is within ⁇ 0.5.
  • conservative substitution refers to an amino acid substitution in which the hydrophilicity index or Z and hydrophobicity index of the original amino acid and the amino acid to be substituted are similar to those described above. And permutation.
  • conservative substitution include, for example, those having a hydrophilicity index or a hydrophobicity index of 2 or less, preferably ⁇ 1 or less, more preferably ⁇ 0.5 or less. But not limited to them.
  • conservative substitutions include, for example, hydrophobic amino acids (such as alanine, palin, leucine, and isoleucine), acidic amino acids (such as glutamic acid, aspartic acid, 4-carboxyglutamic acid, and aminocunic acid). It is understood that it can also be prepared by exchanging basic amino acids (arginine, histidine, lysine, etc.) with each other and aromatic amino acids (phenylalanine, tyrosine, tryptophan, etc.).
  • hydrophobic amino acids such as alanine, palin, leucine, and isoleucine
  • acidic amino acids such as glutamic acid, aspartic acid, 4-carboxyglutamic acid, and aminocunic acid. It is understood that it can also be prepared by exchanging basic amino acids (arginine, histidine, lysine, etc.) with each other and aromatic amino acids (phenylalanine, tyrosine, tryptophan, etc.).
  • conservative substitutions are well known to those skilled in the art and include, for example, substitutions within each of the following groups: arginine and lysine; glutamic and aspartic acid; serine and threonine; glutamine and asparagine; Isoleucine, and the like, are not limited thereto.
  • the "variant" when referring to a polypeptide or polynucleotide is a substance in which a part of the substance such as the original polypeptide or polynucleotide is modified.
  • Such variants include substitutional variants, addition variants, deletion variants, truncated variants, allelic variants, and the like.
  • Alleles refer to genetic variants that belong to the same locus and are distinct from each other. Therefore, “allelic variant” refers to a variant that has an allelic relationship to a certain gene. Such allelic variants usually have sequences identical or very similar to their corresponding alleles, and usually have rarely different biological May have activity.
  • “Species homologue or homolog” means homology (preferably 60% or more homology, more preferably 80% or more) at the amino acid level or nucleotide level with a certain gene in a certain species. , 85% or more, 90% or more, 95% or more homology). Methods for obtaining such species homologs will be apparent from the description herein.
  • "Ortholog” is an orthologue Also referred to as an orthologous gene! /,,, Are genes derived from speciation from a common ancestor that has two genes.
  • the human and mouse ⁇ -hemoglobin genes are orthologs.
  • the human human hemoglobin gene and ⁇ -hemoglobin gene are paralogs (genes generated by gene duplication).
  • Orthologs are useful for estimating molecular phylogenetic trees. Orthologs of the present invention may also be useful in the present invention, as orthologs can usually perform the same function in another species as the original species.
  • conservatively modified variant refers to a nucleic acid that encodes the same or essentially the same amino acid sequence, and essentially the same if the nucleic acid does not encode an amino acid sequence. Sequence. Because of the degeneracy of the genetic code, many functionally identical nucleic acids encode any given protein. For example, the codons GCA, GCC, GCG, and GCU all encode the amino acid alanine.
  • nucleic acid variation is a "silent modification (mutation)," which is one type of conservatively modified mutation.
  • Every nucleic acid sequence herein which encodes a polypeptide also describes every possible silent variation of the nucleic acid.
  • each codon in the nucleic acid except AUG, which is usually the only codon for methionine, and TGG, which is usually the only codon for tryptophan
  • AUG which is usually the only codon for methionine
  • TGG which is usually the only codon for tryptophan
  • each silent variation of a nucleic acid which encodes a polypeptide is implicit in each described sequence.
  • modifications may be made to avoid substitution of the amino acid cysteine, which greatly affects the conformation of the polypeptide.
  • base sequence modification methods include cleavage with a restriction enzyme or the like, ligation treatment with a DNA polymerase, Klenow fragment, DNA ligase, or the like, site-specific base substitution using a synthetic oligonucleotide, or the like. (Site-directed mutagenesis; Mark Zoller and Michael Smith, Methods in Enzymology, 100, 468-500 (1983)) Modifications can also be made by methods commonly used in the field of molecular biology.
  • amino acid addition, deletion, or modification in addition to amino acid substitution, amino acid addition, deletion, or modification can also be performed.
  • Amino acid substitution refers to substitution of the original peptide with one or more, for example, 1 to 3 amino acids.
  • the addition of an amino acid refers to the addition of one or more, for example, 1 to 3 amino acids to the original peptide chain.
  • Amino acid deletion refers to the deletion of one or more, for example, 1 to 3 amino acids from the original peptide.
  • Amino acid modifications include, but are not limited to, amidation, carboxylation, sulfation, halogenation, alkylation, glycosylation, phosphorylation, hydroxylation, acylation (eg, acetylation).
  • the amino acid to be substituted or added may be a natural amino acid or a non-natural amino acid or an amino acid analog. Natural amino acids are preferred.
  • substitution, addition or deletion of a polypeptide or polynucleotide refers to an amino acid or its substitute, or a nucleotide, respectively, relative to an original polypeptide or polynucleotide. Or its substitute power means to be replaced, added or removed. Techniques for such substitution, addition or deletion are well known in the art, and examples of such techniques include site-directed mutagenesis techniques.
  • the number of substitutions, additions, or deletions may be any number as long as the number is one or more. ) Can be much as long as For example, such a number can be one or several, and preferably can be within 20%, 10%, or 3 or less, 2 or less, 1 or less of the total length, etc. .
  • hydrophobic group is used as an index for determining hydrophobicity.
  • the hydrophobicity index (Kyte and Doolittle, J. Mol. Biol., 157, 105-132 (1982))
  • Examples of such a hydrophobic group include, but are not limited to, an alkyl group and an alkyl group.
  • alkyl refers to a monovalent group generated by the loss of one hydrogen atom in an aliphatic hydrocarbon (alkane) such as methane, ethane, and propane.
  • n 2n + l is represented by one (where n is a positive integer).
  • Alkyl can be straight or branched.
  • substituted alkyl refers to an alkyl in which H of the alkyl is substituted by a substituent defined below.
  • C1-C2 alkyl C1-C3 alkyl, C1-C4 alkyl, C1-C5 alkyl, C1-C6 alkyl, C1-C7 alkyl, C1-C8 alkyl, C1-C9 alkyl, C1-C10 alkyl C1-C12 alkyl or C1-C12 alkyl, C1-C2-substituted alkyl, C1-C3-substituted alkyl, C1-C4-substituted alkyl, C1-C5-substituted alkyl, C1-C6-substituted Alkyl, C1-C7 substituted alkyl, C1-C8 substituted alkyl, C1-C9 substituted alkyl, C1-C10 substituted alkyl, C1-C11 substituted alkyl or C1-C12 substituted alkyl.
  • C 1 -C 10 alkyl means a linear or branched alkyl having 1 to 10 carbon atoms, such as methyl (CH—), ethyl (CH-1), n-propyl (CH CH CH—), iso
  • Alkyl is C1-C10 alkyl in which one or more hydrogen atoms have been replaced by substituents.
  • optionally substituted alkyl means that “alkyl” or “substituted alkyl” as defined above may be different from the above.
  • alkylene refers to a divalent group formed by the loss of two hydrogen atoms in an aliphatic hydrocarbon (alkane) such as methylene, ethylene, and propylene. — CH — (where n is a positive integer). Alkylene is straight chain or branched n 2n
  • substituted alkylene refers to a substituent defined below.
  • Alkylene in which H of alkylene is substituted by a group include Cl-C2 alkylene, C1-C3 alkylene, C1-C4 alkylene, C1-C5 alkylene, C1-C6 alkylene, C1-C7 alkylene, C1-C8 alkylene, C1-C9 alkylene, C1-C10 Alkylene, 1 to 11 alkylene or 1 to 12 alkylene, C1 to C2 substituted alkylene, C1 to C3 substituted alkylene, C1 to C4 substituted alkylene, C1 to C5 substituted alkylene, C1-C6-substituted alkylene, C1-C7-substituted alkylene, C1-C8-substituted alkylene, C1-C9-substituted alkylene, C
  • C1-C10 substituted alkylene refers to C1-C1
  • alkylene of which one or more hydrogen atoms are substituted with a substituent.
  • alkylene may include one or more atoms selected from an oxygen atom and a sulfur atom.
  • substituted or alkylene may be a shift of "alkylene” or "substituted alkylene” defined above. means.
  • cycloalkyl refers to alkyl having a cyclic structure. “Substituted cycloalkyl” refers to cycloalkyl in which H of cycloalkyl is replaced by a substituent defined below.
  • C3-C4 cycloalkyl C3-C5 cycloalkyl, C3-C6 cycloalkyl, C3-C7 cycloalkyl, C3-C8 cycloalkyl, C3-C9 cycloalkyl, C3-C10 cycloalkyl, C3-C11 Cycloalkyl, C3-C12 cycloalkyl, C3-C4-substituted cycloalkyl, C3-C5-substituted Cycloalkyl, C3-C6-substituted cycloalkyl, C3-C7-substituted cycloalkyl, C3-C8-substituted cycloalkyl, C3-C9-substituted cycloalkyl, C3-C10-substituted cycloalkyl , C3-C11 substituted cycloalkyl or C3-C12
  • the "optionally substituted cycloalkyl” means that the "cycloalkyl” or the “substituted cycloalkyl” defined above may be shifted. You.
  • alkenyl refers to a monovalent group generated by the loss of one hydrogen atom from an aliphatic hydrocarbon having one double bond in the molecule, and is generally represented by CH1.
  • substituted alkenyl refers to an alkenyl in which H of the alkenyl is substituted by a substituent defined below.
  • C2-C3 anore keninole C2-C4 anore keninore, C2-C5 anore keninore, C2-C6 alkalle, C2-C7 alkalle, C2-C8 alkalle, C2-C9 alkalle, C2-C10 scale, J2-J11 scale or J2-J12 scale, C2-C3-substituted scale, C2-C4-substituted scale, C2-C5 Substituted alkyl, C2-C6 substituted alkyl, C2-C7 substituted alkyl, C2-C8 substituted alkyl, C2-C9 substituted alkyl, C2-C10 It may be a substituted alkyl, a C2-C11 substituted alkyl, or a C2-C12 substituted alkyl.
  • H CH is exemplified.
  • C2-C10 substituted alkyl is
  • alkenylene refers to a divalent group formed by losing two hydrogen atoms from an aliphatic hydrocarbon having one double bond in the molecule, and generally a CH 1 so
  • substituted alkelenes refers to alkelenes in which the H of the alkene is substituted with a substituent as defined below. Specific examples include C2-C25 alkylenes and C2-C25-substituted alkylenes, particularly C2-C3 alkylenes, C2-C4 alkylenes, C2-C5 alkylenes.
  • C2-C10 substituted alkylene refers to C2-C10 alkylene, in which one or more hydrogen atoms are substituted with a substituent.
  • alkene includes one or more atoms that are also selected from oxygen and sulfur nuclear! / ⁇ .
  • the "optionally substituted alkenylene” may be a deviation from the above defined “alkylene” or "substituted alkenylene”.
  • cycloalkyl refers to an alkyl having a cyclic structure.
  • substituted cycloalkyl refers to a cycloalkyl in which H of the cycloalkyl is substituted by a substituent defined below.
  • C3-C4 cycloalkyl C3-C5 cycloalkyl, C3-C6 cycloalkyl, C3-C7 cycloalkyl, C3-C8 cycloalkyl, C3-C9 cycloalkyl, C3-C10 Cycloalkyl, C3-C11 cycloalkyl, C3-C12 cycloalkyl, C3-C4-substituted cycloalkyl, C3-C5-substituted cycloalkyl, C3-C6-substituted cycloalkyl, C3 -C7 substituted cycloalkyl, C3-C8 substituted cycloalkyl, C3-C9 substituted cycloalkyl, C3-C10 substituted cycloalkyl , A 3- to 11-substituted cycloalkyl or a 3- to 12-substituted cycloalkyl
  • cycloalkyl which may be substituted may be a shift of the "cycloalkyl” or the "substituted cycloalkyl” as defined above.
  • alkynyl refers to a monovalent group such as acetylene formed by the loss of one hydrogen atom from an aliphatic hydrocarbon having one triple bond in the molecule. Generally represented by CH (where n is a positive integer greater than or equal to 2). "Substituted al n 2n_3
  • “Kyl” refers to an alkyl in which H of the alkyl is substituted by a substituent defined below. Specific examples include C2-C3 alkyl, C2-C4 alkyl, C2-C5 alkyl, C2-C6 alkyl, C2-C7 alkyl, C2-C8 alkyl, C2- C9 alkyl, C2-C10 alkyl, C2-C11 alkyl, C2-C12 alkynyl, C2-C3-substituted alkyl, C2-C4-substituted alkyl, C2-C5-substituted Alkyl, C2-C6-substituted alkyl, C2-C7-substituted alkyl, C2-C8-substituted alkyl, C2-C9-substituted alkyl, C2-C10-substituted Or a C2-C11 substituted alkyl or a C2-C12 substituted alkyl.
  • C2-C10 alkyl means, for example, a linear or branched alkyl containing 2 to 10 carbon atoms, such as ethur (CH ⁇ C—), 1 propynyl ( CH C ⁇ C). Also, for example, C2-C10 substituted alkyl
  • Nyl refers to C2-C10 alkyl in which one or more hydrogen atoms are substituted with a substituent.
  • alkyl is defined as “alkoxy” as defined above.
  • alkoxy refers to a monovalent group generated by losing a hydrogen atom of a hydroxy group of an alcohol, and is generally represented by CHO (where n is 1 or more).
  • Substituted alkoxy refers to alkoxy in which H of the alkoxy is substituted by a substituent defined below. Specific examples include C1-C2 alkoxy, C1 ⁇ C3 alkoxy, C1 ⁇ C4 alkoxy, C1 ⁇ C5 alkoxy, C1 ⁇ C6 alkoxy, C1 ⁇ C7 alkoxy, C1 ⁇ C8 alkoxy, C1 ⁇ C9 alkoxy, C1 ⁇ C10 alkoxy, C1 ⁇ C11 alkoxy, C1 ⁇ C12 alkoxy, C1-C2-substituted alkoxy, C1-C3-substituted alkoxy, C1-C4-substituted alkoxy, C1-C5-substituted alkoxy, C1-C6-substituted alkoxy, C1-C7-substituted alkoxy, C1 It may be -C8-substituted alkoxy, C
  • Mouth oxy CH CH CH O
  • Mouth oxy CH CH CH O
  • optionally substituted alkoxy means that the "alkoxy" or “substituted alkoxy” defined above may be shifted.
  • heterocycle (group) refers to a group having a cyclic structure including carbon and hetero atoms.
  • the hetero atom is selected from the group consisting of 0, S, and N forces, and may be the same or different, may contain one, or may contain two or more.
  • Heterocyclic groups can be aromatic or non-aromatic, and can be monocyclic or polycyclic. Heterocyclic groups may be substituted.
  • substituted or optionally a heterocycle refers to a “heterocyclic ring (group)” or a “substituted heterocycle (group)” as defined above.
  • Base means that it may be misaligned.
  • alcohol refers to an organic compound in which one or more hydrogen atoms of an aliphatic hydrocarbon are substituted with a hydroxyl group.
  • ROH is also referred to as ROH.
  • R is an alkyl group.
  • R may be C1-C6 alkyl.
  • examples of the alcohol include, but are not limited to, methanol, ethanol, 1-propanol, 2-prono-Vole and the like.
  • Carbocyclic group refers to a group containing a cyclic structure containing only carbon, and includes the above-mentioned “cycloalkyl”, “substituted cycloalkyl”, “cycloalkyl” And “substituted cycloalkyl”.
  • Carbocyclic groups can be aromatic or non-aromatic And can be monocyclic or polycyclic.
  • substituted carbocyclic group refers to a carbocyclic group in which H of the carbocyclic group is substituted by a substituent defined below.
  • C3-C4 carbocyclic group C3-C5 carbocyclic group, C3-C6 carbocyclic group, C3-C7 carbocyclic group, C3-C8 carbocyclic group, C3-C9 carbocyclic group, C3-C10 Carbocyclic group, C3-C11 carbocyclic group, C3-C12 carbocyclic group, C3-C4-substituted carbocyclic group, C3-C5-substituted carbocyclic group, C3-C6-substituted carbocyclic group, C3- C7-substituted carbocycle, C3-C8-substituted carbocycle, C3-C9-substituted carbocycle, C3-C10-substituted carbocycle, C3-C11-substituted carbocycle or C3 It may be a -C12 substituted carbocyclic group.
  • the carbocyclic group can also be a C4-C7 carbocyclic group or a C4-C7 substituted carbocyclic group.
  • Examples of the carbon ring group include one in which one phenyl group hydrogen atom is deleted.
  • the hydrogen deletion position may be any position that is chemically possible, whether on an aromatic ring or on a non-aromatic ring.
  • substituted or optionally a carbocyclic group refers to a deviation from the above-defined “carbocyclic group” or “substituted carbocyclic group”. It also means.
  • heterocyclic group refers to a group having a cyclic structure including carbon and hetero atoms.
  • the heteroatom is selected from the group consisting of 0, S, and N forces, and may be the same or different, and may include one or two or more.
  • Heterocyclic groups can be aromatic or non-aromatic, and can be monocyclic or polycyclic.
  • “Substituted heterocyclic group” refers to a heterocyclic group in which H of the heterocyclic group is substituted by a substituent defined below.
  • C3-C4 carbocyclic group C3-C5 carbocyclic group, C3-C6 carbocyclic group, C3-C7 carbocyclic group, C3-C8 carbocyclic group, C3-C9 carbocyclic group, C3-C10 Carbocyclic group, C3-C11 carbocyclic group, C3-C12 carbocyclic group, C3-C4-substituted carbocyclic group, C3-C5-substituted carbocyclic group, C3-C6-substituted carbocyclic group, C3 -C7 substituted carbocyclic group, C3-C8 substituted carbocyclic group, C3-C9 substituted carbocyclic group, C3-C10 substituted carbocyclic group, J3-J11 substituted carbocycle It may be a group or a 3- to C12-substituted carbocyclic group in which one or more carbon atoms have been substituted with
  • the heterocyclic group may also be a C4-C7 carbocyclic group or a C4-C7 substituted carbocyclic group in which one or more carbon atoms have been substituted with one or more heteroatoms.
  • a heterocyclic group examples thereof include a phenyl group, a pyrrolyl group, a furyl group, an imidazolyl group, and a pyridyl group.
  • the position of deletion of hydrogen may be any position that is chemically possible, and may be on an aromatic ring or on a non-aromatic ring.
  • a carbocyclic group or a heterocyclic group may be substituted with a divalent substituent in addition to being substituted with a monovalent substituent as defined below.
  • halogen refers to a monovalent group of elements such as fluorine (F), chlorine (C1), bromine (Br), and iodine (I) belonging to Group 7B of the periodic table.
  • hydroxy refers to a group represented by OH.
  • substituted hydroxy refers to a compound in which H of hydroxy is substituted by a substituent defined below.
  • thiol is a group in which an oxygen atom of a hydroxy group is substituted with a sulfur atom (mercapto group), and is represented by SH.
  • substituted thiol refers to a group in which H of mercapto is substituted with a substituent defined below.
  • cyano refers to a group represented by -CN.
  • Niro means -NO
  • substituted amino refers to an amino in which H of the amino is substituted with a substituent as defined below.
  • carboxy refers to a group represented by -COOH.
  • substituted carboxy refers to carboxy in which H is substituted with a substituent as defined below.
  • acyl refers to a monovalent group formed by removing OH from a carboxylic acid.
  • Representative examples of acetyl groups include acetyl (CH 2 CO 3) and benzoyl (CH 2 CO 3)
  • substituted acyl refers to the hydrogen of an acyl substituted with a substituent as defined below.
  • amide refers to a group in which hydrogen of ammonia is substituted with an acid group (acyl group). And preferably represented by -CONH. "Substituted amide” refers to an amide substituted
  • carbon refers to one (C) which is a characteristic group of aldehyde and ketone.
  • Substituted carbonyl refers to a carbonyl group substituted with a substituent selected as described below.
  • Thiocarbol includes thioketone and thioaldehyde.
  • Substituted thiocarbol means thiocarbonyl substituted with a substituent selected as described below.
  • sulfol is a generic term for a substance containing SO 2 as a characteristic group.
  • Substituted sulfol means a sulfol substituted with a substituent selected below.
  • sulfiel is a generic term for a substance containing SO- which is a characteristic group.
  • substituted sulfiel means sulfiel which has been replaced by a substituent selected below.
  • aryl refers to a group formed by the removal of one hydrogen atom bonded to the ring of an aromatic hydrocarbon, and is included in the present specification as a carbocyclic group.
  • substitution refers to replacement of one or more hydrogen atoms in an organic compound or a substituent with another atom or atomic group. It is also possible to remove one hydrogen atom and substitute with a monovalent substituent, and it is also possible to remove two hydrogen atoms and substitute with a divalent substituent.
  • substitution refers to replacing one or more hydrogen atoms in an organic compound or a substituent with another atom or atomic group. It is also possible to remove one hydrogen atom and substitute with a monovalent substituent, and it is also possible to remove two hydrogen atoms and substitute with a divalent substituent.
  • Examples of the substituent in the present invention include alkyl, cycloalkyl, alkenyl, cycloalkyl, alkyl, alkoxy, carbocyclic group, heterocyclic group, halogen, hydroxy, thiol, nitro nitro, Carboxy with ami, rubamoyl, asil, asilamino, chio Examples include, but are not limited to, carboxy, amide, substituted carboyl, substituted thiocarbol, substituted sulfonyl or substituted sulfinyl, or any inorganic substituent (e.g., a silicon-containing substituent). Not limited. In the present invention, such substituents can be appropriately used in designing linkers and biomolecules.
  • each when a plurality of substituents are present, each may independently be a hydrogen atom or an alkyl or any inorganic substituent (for example, a silicon-containing substituent). It cannot be a hydrogen atom. More preferably, independently, when there are a plurality of substituents, each may be independently selected from the group consisting of hydrogen and a C1-C6 alkyl group or any inorganic substituent (eg, a silicon-containing substituent).
  • the substituents may all have a substituent other than hydrogen, but preferably have at least one hydrogen, more preferably 2 to n (where n is the number of substituents) hydrogen. Can have. It may be preferable that the number of hydrogen atoms in the substituent is large.
  • the substituents other than hydrogen are preferably C1-C6 alkyl, C1-C5 alkyl, C1-C4 alkyl, C1-C3 alkyl, C1-C2 alkyl, methyl or any inorganic substituent (e.g., (Containing substituent).
  • any inorganic substituent e.g., (Containing substituent).
  • Cl, C2, ..., Cn represent the number of carbon atoms. Accordingly, C1 is used to represent a substituent having one carbon atom.
  • optical isomer refers to one or a pair of non-superimposable compounds in which the structure of the crystal or molecule is a mirror image. It is a form of stereoisomer that differs only in optical rotation, despite other properties being the same. In the present invention, those having high optical isomer purity can be preferably used.
  • the term "protection reaction” refers to a reaction in which a protecting group such as Boc is added to a functional group for which protection is desired. By protecting the functional group with the protecting group, the reaction of the functional group having higher reactivity can be suppressed, and only the functional group having lower reactivity can be reacted.
  • the protection reaction can be performed, for example, by a dehydration reaction.
  • the term "deprotection reaction” refers to a reaction for eliminating a protecting group such as Boc.
  • Examples of the deprotection reaction include a reaction such as a reduction reaction using PdZC.
  • the deprotection reaction can be performed, for example, by hydrolysis.
  • protecting group includes, for example, a fluorenylmethoxycarbol (Fmoc) group, an acetyl group, a benzyl group, a benzoyl group, a t-butoxycarbol group, t-butyldimethyl group, silyl group, trimethylsilylethyl group, N-phthalimidyl group, trimethylsilylethyloxycarbonyl group, 2-tro-5,5-dimethoxybenzyl group, 2-nitro-4,5-dimethoxy
  • Typical protective groups include a benzyloxycarbol group and a sulfamate group.
  • Protecting groups can be used to protect moieties that are not involved in binding in biomolecules.
  • the protecting group can be used, for example, to protect a reactive functional group such as an amino group and a carboxyl group.
  • Various protecting groups can be used depending on the reaction conditions and purpose.
  • the protecting group for the hydroxy group includes acetyl, benzyl, silyl and derivatives thereof, and the protecting group for the amino group includes acetyl, benzyloxycarbyl, t-butoxycarbol and the like. Inductors and the like can be used.
  • a trimethylsilylethyloxycarboxy group, a 2-toro-4,5 dimethoxybenzyloxycarboxy group or a derivative thereof is preferable.
  • the target product is obtained by removing contaminants (unreacted weight loss, by-products, solvent, etc.) from the reaction solution by a method commonly used in the art (for example, extraction, distillation, After removal by washing, concentration, precipitation, filtration, drying, etc.), isolation by a combination of post-treatment methods commonly used in the art (eg, adsorption, elution, distillation, precipitation, precipitation, chromatography, etc.). obtain.
  • a method commonly used in the art for example, extraction, distillation, After removal by washing, concentration, precipitation, filtration, drying, etc.
  • isolation by a combination of post-treatment methods commonly used in the art (eg, adsorption, elution, distillation, precipitation, precipitation, chromatography, etc.). obtain.
  • angiogenesis refers to the formation of new blood vessels and the activity of such formation.
  • vascular network formation refers to the formation of new blood vessels or existing vascular forces and the activity of forming such networks! , U.
  • the vascular network forming ability is determined by the angiogenesis index and the length of the newly formed blood vessel, or the vascular network forming ability or the vascular network forming index.
  • Indicated by Angiogenesis can be determined by observing whether the reticulated force has formed (e.g., the bifurcation of a blood vessel joining more blood vessels and increasing the number of its junctions).
  • the vascular network formation index is calculated as follows in this specification.
  • the length of the new blood vessel is determined.
  • the length is measured as follows. Observation was made with a stereomicroscope (Olympus, SZX12, Japan). The obtained image was read by Photoshop (registered trademark) (Adobe, Japan). Count in pixels.
  • Nwl Network formation Although angiogenesis is observed as described above, each new blood vessel is a single fe.
  • Nw2 In the middle stage of network formation, each new blood vessel is in a state in which a side branch on a ladder is engaged.
  • Nw3 The network is in the final stage, and the ladder upper branch has more side branches.
  • Nw4 Network maturation stage, showing a wide range of neovascular plexus.
  • the ability to form a vascular network usually means such a vascular network formation index force of at least 2, preferably 2.5 or more, and more preferably 2.5 or more. It is understood that it is preferably 3 or more, more preferably 3.5 or more.
  • blood vessel in the present specification is used in a sense commonly used in the art, and includes not only ordinary arteries and veins, but also capillaries.
  • cell adhesion molecule or "adhesion molecule” is used interchangeably and refers to the approach of two or more cells to each other (cell adhesion) or the relationship between a substrate and a cell. Refers to molecules that mediate adhesion between Generally, a molecule involved in cell-cell adhesion (cell-cell adhesion) (cell-cell adhesion molecule) and a molecule involved in cell-extracellular matrix adhesion (cell-substrate adhesion) (cell-substrate adhesion molecule) Divided into In the present invention, any molecule is useful and can be used effectively.
  • the cell adhesion molecule includes a protein on the substrate side during cell-substrate adhesion, but in the present specification, a protein on the cell side (for example, integrin) is also included, and other than the protein, Even a molecule, as long as it mediates cell adhesion, falls within the concept of a cell adhesion molecule or cell adhesion molecule herein.
  • cadherin many molecules belonging to the immunoglobulin superfamily (NCAM, Ll, ICAM, facyclin ⁇ , ⁇ , etc.), selectins, and the like are known, and cell membranes are formed by unique molecular reactions. It is also known to combine
  • the major cell adhesion molecule that works for cell-substrate adhesion is integrin, which recognizes and binds to various proteins contained in the extracellular matrix.
  • integrin the major cell adhesion molecule that works for cell-substrate adhesion
  • These cell adhesion molecules Are all located on the cell membrane surface and can be regarded as a type of receptor (cell adhesion receptor). Accordingly, such receptors located on the cell membrane can also be used in the tissue pieces of the present invention.
  • Such receptors include, but are not limited to, for example, ⁇ integrin, j8 intedarin, CD44, syndecan, and aggrecan.
  • extracellular matrix molecules such as fibronectin and laminin
  • integrins binding partners
  • the function of each adhesion receptor in cell-cell adhesion and cell-substrate adhesion is not strict, and varies depending on the distribution of partner molecules (ligands).
  • partner molecules ligands
  • some integrins are also involved in cell-cell adhesion, such as adhesion between blood cells.
  • growth factors, cytokins, and the like are present as cell membrane proteins, they react with their receptors distributed on other cells and consequently adhere the cells.
  • growth factors and cytokins can also be used as biomolecules contained in the tissue piece of the present invention.
  • Whether a molecule is a cell adhesion molecule can be determined by biochemical quantification (SDS-PAG method, labeled collagen method), immunological quantification (enzyme antibody method, fluorescent antibody method, immunohistological examination) PD The determination can be made by determining a positive result in an assay such as the R method or the hybridization method.
  • cell adhesion molecules include collagen, integrin, fibronectin, laminin, vitronectin, fibrinogen, the immunoglobulin parfamily (e.g., CD2, CD4, CD8, ICM1, ICAM2, VCAM1), selectin, cadherin, and the like. But not limited thereto.
  • the adhesion factors used in the tissue piece of the present invention include Those that transmit such an auxiliary signal for cell activation into a cell are preferred. This is because, after being applied to an injured site in a certain tissue or organ as a tissue piece, cell activation can promote the proliferation of cells aggregated therein and / or cells in the tissue or organ. Whether such an auxiliary signal can be transmitted into cells is determined by biochemical quantification (SDS-PAGE, labeled collagen method), immunological quantification (enzyme-linked immunosorbent assay, immunofluorescent assay, immunohistochemical assay). Investigation) It can be determined by determining positive in the PDR method and the hybridization method.
  • Examples of the cell adhesion molecule include cadherin, which is widely known as a cell adhesion molecule in tissue-fixing cell lines, and cadherin can be used in a preferred embodiment of the present invention.
  • examples of cell adhesion molecules include immunoglobulin superfamily molecules (CD2, LFA-3, ICAM-1, CD2, CD4, CD8, ICM1, ICAM2, VCAM1).
  • Integrin family molecules LFA-1, Mac-1, gpllbllla, pl50, 95, VLA1, VLA2, VLA3, VLA4, VLA5, VLA6, etc.
  • selectin family molecules L-selectin, E-selectin, P— Selectin
  • such molecules may be particularly useful for treating tissues or organs of the blood-immune system.
  • the cell adhesion molecule In order for non-fixed cells to work in a specific tissue, the cell adhesion molecule needs to adhere to that tissue. In this case, it is thought that the adhesion between cells is gradually strengthened by the primary adhesion by the constantly expressed selectin molecule and the like, followed by the secondary adhesion of the activated integrin molecule and the like. Therefore, as a cell adhesion molecule used in the present invention, such a factor that mediates primary adhesion, a factor that mediates secondary adhesion, or both may be used together.
  • cell adhesive protein refers to a protein having a function of mediating cell adhesion as described above. Therefore, in the present specification, the cell adhesive protein includes a protein on the substrate side during cell-substrate adhesion, but herein also includes a protein on the cell side (eg, integrin). For example, when cultured cells are seeded under serum-free conditions on a substrate (glass or plastic) to which the protein on the substrate side has been adsorbed, the integrin, a receptor, recognizes the cell adhesion protein and the cells Glue to the quality.
  • the protein contained in the tissue piece of the present invention may advantageously include an RGD sequence such as RGD, YIGSR.
  • RGD sequence such as RGD, YIGSR.
  • cell adhesion proteins are present in extracellular matrices, cultured cell surfaces, and in plasma 'serum' various body fluids. Its functions in vivo include not only the adhesion of cells to the extracellular matrix, but also the movement, proliferation, morphology, and tissue construction of cells. Apart from cellular actions, some proteins exhibit a function of regulating blood coagulation and complement action. In the present invention, proteins having such functions may also be useful. Examples of such cell adhesion proteins include, but are not limited to, fibronectin, collagen, vitronectin, laminin, and the like.
  • RGD molecule refers to a protein molecule containing the amino acid sequence RGD (Arg-Gly-Asp) or a functionally identical sequence thereof.
  • the RGD molecule is characterized by containing RGD, which is an amino acid sequence useful as an amino acid sequence of a cell adhesion active site of a cell adhesion protein, or another amino acid sequence which is functionally equivalent.
  • the RGD sequence was discovered as a cell adhesion site of fibronectin, and was later found on a number of molecules exhibiting cell adhesion activities, such as type I collagen, laminin, vitronectin, fibrinogen, von Willebrand factor, and entactin.
  • the biomolecule in the present invention may be a chemically synthesized RGD molecule.
  • RGD molecules include, but are not limited to, for example, the GRGDSP peptide, in addition to the naturally occurring molecules described above.
  • integrin eg, the receptor for fibronectin
  • a functionally equivalent molecule of RGD may use such an integrin. It can be identified by examining the interaction.
  • integrin refers to a transmembrane glycoprotein that is a receptor involved in cell adhesion. Integrins are present on the cell surface and function when cells adhere to the extracellular matrix. It is known that it is involved in cell-cell adhesion in blood cell systems. Such integrins include, for example, fibronectin, vitronectin Receptor, such as protein and collagen, platelet lib / Ilia, macrophage Mac-1, lymphocyte LFA-1 and VLA-1-6, and Drosophila PSA, but are not limited thereto.
  • Integrin usually has a heterodimer structure in which a ⁇ chain having a molecular weight of 130 to 210 kDa and a j8 chain having a molecular weight of 95 to 130 kDa are non-covalently and one-to-one associated with a j8 chain. Is it an ⁇ chain and f row? J, Q;, Q;, Q;, Q;, Q;, Q; ', Q;, ⁇ , ⁇ , ⁇ v , ⁇ ⁇ , and the like, but are not limited thereto.
  • the j8 chain for example, j8, ⁇ , ⁇ , ⁇ , ⁇ ,
  • Examples of such a heterodimer include, in addition to GpIIbllla, VLA-1, VLA-2, VLA-3, VLA-4, VLA-5, VLA-6, CD51 / CD29, LFA- 1, Mac- 1, pl50, 90 , vitronectin receptor, j8 4 subfamilies, j8 5 subfamily, j8 6 sub family, LPAM-1, HML- but are not limited to such 1.
  • the extracellular domain of the ⁇ chain has a divalent cation binding site
  • the extracellular domain of the ⁇ chain has a cysteine-rich region
  • the intracellular domain of the ⁇ chain has a tyrosine phosphorylation site in many cases.
  • the recognition site in the binding ligand is often an RGD sequence.
  • an integrin may be an RGD molecule.
  • the present invention provides a peptide comprising the sequence VVXGL (SEQ ID NO: 1), wherein X is a variant of an aromatic amino acid.
  • the present invention provides a composition for angiogenesis, comprising a peptide comprising the sequence VVXGL, wherein X is a variant of an aromatic amino acid.
  • the present invention provides a composition for forming a vascular network, comprising a peptide comprising the sequence VVXGL, wherein X is a variant of an aromatic amino acid.
  • the composition for angiogenesis and the composition for forming a vascular network of the present invention represents an amino acid having an aromatic ring in a side chain or a variant thereof.
  • X represents an amino acid having an aromatic ring in a side chain or a variant thereof.
  • the present invention provides an angiogenic agent and a composition for forming a vascular network (also referred to as a vascular network forming agent), comprising the above-mentioned peptide.
  • This sequence was designed based on the pentapeptide WYGL contained in SEQ ID NO: 2, and it is known to those skilled in the art that any variant other than WYGL may be within the scope of the present invention. it is obvious.
  • X is a modified form of phenylalanine or tyrosine.
  • This angiogenic agent when naturally occurring, has a tyrosine at X. Therefore, it is advantageous to be a variant of the natural amino acids phenalanine or tyrosine.
  • the modification of tyrosine in the side chain fuel group has a strong angiogenic activity of a peptide having tyrosine in X and feniralanine which can also be seen as a variant thereof. Based on that.
  • the vascular network-forming activity of the peptide having phenalanine which can also be regarded as a cysteine in X and a variant thereof, was also strong. Therefore, it is understood that by making similar modifications to the peptide of the present invention, both the angiogenic activity and the vascular network forming activity can be enhanced.
  • the angiogenesis ability can be indicated by the number of angiogenesis.
  • the angiogenic potential can be assessed by scoring, wherein the peptide of the invention or a variant thereof has a score of at least 3, preferably a score of 3.5, More preferably, it has a score of 4, and even more preferably has a score of 4.5.
  • the vascular network forming ability can be indicated by a vascular network forming ability index.
  • the ability of the peptide of the invention to form a vascular network is at least 5, preferably at least 7, more preferably at least 8, more preferably at least 9, more preferably at least 10, More preferably at least 11, more preferably at least 12, more preferably at least 13, more preferably at least 14, more preferably at least 15.
  • Such a value is already remarkable when the vascular network forming ability index is actually, for example, about 8 or more. It is clear from the fact that the appearance of a reticulated blood vessel is observed almost always.
  • the peptide of the present invention may contain an exogenous sequence as long as it contains the above specific sequence and has a vascular potential and a Z vascular network forming ability.
  • the peptide of the present invention may be a peptide in which the para, ortho, or meta position of the side chain of the aromatic amino acid is substituted with an electron-withdrawing substituent.
  • X in the peptide of the present invention may be a substituent in which an aromatic group of an aromatic amino acid is substituted with a hydrophobic group.
  • a hydrophobic group for example, an alkyl group such as a methyl group
  • the present invention may advantageously have any hydrophobic group substituted, especially in the para position, instead of the hydroxyl group of tyrosine.
  • the hydrophobic group preferably increases the hydrophobicity index more preferably by 0.5, more preferably by 1.0, and more preferably by 1.5 or 2.
  • hydrophobic group can be, for example, an alkyl group, but is not limited thereto.
  • One preferred such hydrophobic group can include, but is not limited to, for example, an alkyl group or a substituted alkyl group.
  • such hydrophobic groups are C1-C6 alkyl groups, even more preferably C1-C3 alkyl groups, and in one preferred embodiment may be methyl groups.
  • the hydrophobic group eg, an alkyl group
  • the hydrophobic group can be substituted with a nora group of an aromatic group.
  • the aromatic side chain is preferably a phenyl group, but is not limited thereto. It is understood that another example is also preferable, such as a side chain of tributophan. Is done.
  • amino acid sequence of the peptide of the present invention further comprises an amino acid sequence X -X -X -X -X -X
  • X is an amino acid having an aromatic ring in the side chain or a leucine (I) or a variant thereof,
  • the peptide may be a peptide containing the amino acid sequence represented by the above or a variant thereof.
  • X is serine or a variant thereof, and more preferably, X is serine.
  • X is palin or a variant thereof, more preferably
  • X is Norin.
  • X is palin or a variant thereof, more preferably
  • X is Norin.
  • X is glycine or a variant thereof, more preferably
  • X is glycine
  • X is leucine or a variant thereof, more preferably
  • X is leucine
  • X is arginine or a variant thereof, and in a more preferred embodiment, X is arginine.
  • the peptide of the present invention may have a sequence of the combination of the above X to X, preferably of the embodiment.
  • peptide having a physiological activity when one or more amino acids in the amino acid sequence are substituted or deleted, or one or more amino acids are inserted or added to the amino sequence. Even so, it is well known that the physiological activity may be maintained. Therefore, in the amino acid sequence shown in SEQ ID NO: 1, one to three amino acids are substituted, or one or two amino acids located at one or both ends are deleted, or the amino acid Other amino acids at one or both ends of the sequence Peptides having an amino acid sequence to which a sequence is added (hereinafter sometimes referred to as “peptide variants” for convenience) and having an angiogenic action and a Z or vascular network forming action are also included in the scope of the present invention. It is.
  • the modified peptide of the present invention can be modified by modifying the side chain of the aromatic amino acid with a natural amino acid in another amino acid sequence (for example, an alkyl group or a nitro group on the side chain of the amino acid). , Introduction of halogen, etc.). Further, such an amino acid may be a D-type amino acid.
  • a peptide having an amino acid sequence in which one amino acid at the N-terminus or C-terminus of the amino acid sequence of WXGL has been deleted has an angiogenesis effect and a Z- or vascular network-forming effect comparable to those of the peptide having the amino acid sequence represented by VVXGL. It is understood that they can be activated and fall within the scope of the present invention.
  • one or two amino acids are substituted in the sequence other than X in the amino acid sequence shown in VVXGL.
  • a peptide having an amino acid sequence in which one amino acid located at one or both ends is deleted, or another amino acid sequence is added at one or both ends of the amino acid sequence Peptides having a neoplastic and Z or vascular network forming effect may be provided.
  • the number of substituents on the aromatic ring, preferably on the phenol group is 1 to 5, and preferably 1 to 3.
  • the aromatic ring is preferably a condensed ring such as a benzene ring or a naphthalene ring containing a benzene ring (may be a heterocyclic ring such as a tributophan side chain), and particularly preferably a fluor group. is there.
  • the upper limit of the size of the peptide used in the present invention is not limited at all. However, if it is too large, production becomes difficult, handling is inconvenient, and vascular new life per unit weight is required. Therefore, the total number of amino acids of the peptide is usually 4 to 350, preferably 4 to 50, more preferably 5 to 20, and more preferably 5 to 10 And more preferably 6 to 10. Amino shown in X—X—X—X—X—X—X
  • Whether the peptide of the present invention has an angiogenic action and an action of forming a Z or vascular network was determined by specifically ascertaining whether or not a microcell filled with a peptide solution was placed on the back of a mouse as shown in the Examples below. It can be examined by observing the state of formation of capillaries in the tissue surrounding the implanted tissue.
  • the peptide or variant of the present invention can be easily synthesized manually or by a conventional method using a commercially available peptide synthesizer.
  • a large-sized peptide is produced by a conventional method using genetic engineering, then modifying the side chain or modifying the genetic engineering technique to use a modified tRNA (Taki M. et al., FEBS Lett. 507, 35-38, 2001; Hohsaka T. et al., J. Am. Chem. Soc, 121, 34-40, 1999; and Hohsa ka T. et al., J. Am. Chem. Soc. , 118, 9778—9779, 1996).
  • the peptide used in the present invention is designed based on amino acids that constitute a natural protein, and is degraded to amino acids by the action of peptidase in vivo. High safety. In fact, no toxicity was observed in in vivo experiments using mice performed in the following examples. This indicates that the amount of use that exerts its medicinal effect is not so toxic, and that it has been used! /
  • the angiogenic agent and the vascular network forming agent of the present invention may be targeted to capillaries. This is because the capillaries can have the normal physiological activity that they have naturally by being networked in a network.
  • the present invention provides, for the first time, a conjugate having enhanced angiogenic activity and Z or vascular network forming ability, thereby achieving a powerful therapeutic effect, stabilizing effect, and prevention that could not be achieved by conventional angiogenic agents. The effect can be demonstrated.
  • Angiogenesis can also be promoted by binding the peptide to a carrier and implanting the carrier with the peptide bound into a living body. This can selectively act on the necessary parts because it is fixed to the carrier, and a new DDS (drug delivery system) M) as a potential.
  • Local administration of the angiogenesis agent of the present invention to a biomaterial implanted portion by application, spraying or the like promotes angiogenesis and promotes postoperative healing.
  • the carrier include, but are not particularly limited to, biomacromolecules such as fats and proteins used for bone substitutes, tooth substitutes, and artificial organs.
  • a protein in the present specification, when used as a carrier, unless otherwise specified, the term "protein” includes a protein-containing complex such as a glycoprotein and a phosphoprotein). Can be used as a carrier.
  • the protein used as a carrier may be a biocompatible, non-uniform protein, and in particular, a cell-adhesive protein in order to improve bonding with a biological tissue.
  • a cell adhesion protein include, but are not limited to, collagen (gelatin), fibronectin, vitronectin, laminin, and the like, and partial hydrolyzate thereof.
  • these proteins are preferably purified proteins from which allergens have been removed, and are also preferable in terms of preventing allergic reactions.
  • various collagens derived from animals are commercially available as collagen. However, these contain allergens with low purity and are poor in reproducibility of quality, so that it is not preferable to apply them to clinical use.
  • gelatin obtained by partially hydrolyzing animal-derived collagen and removing allergens is commercially available for clinical use, it is preferable to use such purified collagen or a partially hydrolyzed product thereof.
  • the amount of the peptide bound to the carrier is not particularly limited and can be appropriately selected.
  • the weight ratio of the carrier to the peptide is about 100: 1 to 1: 1. And preferably about 20: 1 to 5: 1.
  • the bond between the carrier and the peptide is preferably a covalent bond. Bonding can be easily carried out, for example, by bonding the amino group at the N-terminus of the peptide to any amino group in the carrier using a bonding cross-linking agent such as dartalaldehyde. An example of a suitable bonding method is described. In the case of binding to a resin such as an artificial organ, a monomer containing a group that can be used for binding to a peptide, such as an amino group, is contained in the resin. Can be previously copolymerized, and the amino group or the like can be bonded to the N-terminal amino group of the peptide.
  • the carrier to which the peptide is bound can be embedded in a living body as it is, in addition to spraying the coated text.
  • the peptide-bound carrier can be used alone or in combination with other medicinal ingredients as a suture, various orthopedic materials, a wound adhesion promoter, and the like.
  • a mixture of the carrier protein bound with the peptide and other materials such as carbonate apatite or the cell adhesive protein not bound with the peptide of the present invention can be used as a bone substitute or the like.
  • the amount of the peptide contained in the final biomaterial such as bone substitute is not particularly limited, it is usually about 0.1 to: LOmg per 100 g of biomaterial.
  • the carriers used herein are preferably pharmaceutically acceptable, such as antioxidants, preservatives, coloring agents, flavorings, and diluents, milk ⁇ includes, but is not limited to, narcotics, suspending agents, solvents, fillers, bulking agents, buffers, delivery vehicles, diluents, excipients and Z or pharmaceutical adjuvants.
  • the medicament of the invention will be in the form of a composition comprising a peptide of the invention, or a variant or derivative thereof, together with one or more physiologically acceptable carriers, excipients or diluents. Is administered.
  • suitable vehicles may be water for injection, physiological solutions, or artificial cerebrospinal fluid, which may be supplemented with other materials common in compositions for parenteral delivery. is there.
  • an acceptable carrier, excipient or stabilizer is non-toxic to the recipient, and preferably inert at the dosages and concentrations employed.
  • phosphates, citrates or other organic acids e.g., phosphates, citrates or other organic acids; ascorbic acid, a-tocopherol; low molecular weight polypeptides; proteins (e.g., serum albumin, gelatin or immunoglobulins); hydrophilic polymers (e.g.
  • Amino acids eg, glycine, glutamine, asparagine, arginine or lysine
  • monosaccharides eg., glutamine, asparagine, arginine or lysine
  • Disaccharides and other carbohydrates including glucose, mannose or dextrin
  • chelating agents eg, EDTA
  • sugar alcohols eg, mannitol or sorbitol
  • salt-forming counterions eg, sodium
  • Non-ionic surface activators eg, Tween, pluronic or polyethylene glycol (PEG)
  • exemplary suitable carriers include neutral buffered saline or saline mixed with serum albumin.
  • the product is formulated as a lyophilizate using a suitable excipient (eg, sucrose).
  • suitable excipient eg, sucrose
  • Other standard carriers, diluents and excipients may be included as desired.
  • Other exemplary compositions include Tris buffer at pH 7.0-8.5 or acetate buffer at pH 4.0-5.5, which further comprises sorbitol or a suitable alternative thereof. May be included.
  • the carrier is preferably a protein, more preferably a cell adhesion protein.
  • the cell adhesion protein used in the present invention may be collagen.
  • the peptide of the present invention can be locally administered to a tissue in which angiogenesis is desired, alone or in the form of an injection solution dissolved in a physiological buffer.
  • Local administration of the angiogenic agent of the present invention to a wound or the like caused by surgery or trauma by injection, application, spraying, etc. promotes angiogenesis and promotes wound healing.
  • the peptide concentration in the peptide solution used for injection or application or spraying is not particularly limited. Usually, the concentration is about 1 ng (nanogram) to (/ zg (microgram) ZmL.
  • the dose can be appropriately selected depending on the size and depth of the wound or the like, but it is sufficient that the whole wound is covered with the peptide solution. It can be administered once to several times every day to several days until the wound heals.
  • the injection solution may contain various components usually contained in a remedy for wounds, such as other disinfectants and anti-inflammatory analgesics.
  • the medicament of the present invention can be administered orally or parenterally.
  • the medicament of the present invention can be administered intravenously or subcutaneously.
  • the medicament used in the present invention may be in the form of a pyrogen-free, pharmaceutically acceptable aqueous solution.
  • the preparation of such pharmaceutically acceptable compositions takes into account pH, isotonicity, stability, etc. By doing so, those skilled in the art can easily carry out.
  • the administration method includes oral administration, parenteral administration (for example, intravenous administration, intramuscular administration, subcutaneous administration, intradermal administration, mucosal administration, rectal administration, vaginal administration, local administration to the affected area, Skin administration).
  • Formulations for such administration may be provided in any formulation.
  • Such preparations include, for example, solutions, injections, and sustained-release preparations.
  • the medicament of the present invention may contain a physiologically acceptable carrier, excipient or stabilizing agent (Japanese Pharmacopoeia, 14th edition, its supplement or its latest edition, Remington's Pharmaceutical Sciences, if necessary). , 18th Edition, AR Lrennaro, ed., Mack Publisnmg Company, 1990, etc.) and a glycan composition having the desired degree of purity to form a lyophilized cake or aqueous solution. Can be prepared and stored.
  • the amount of the composition of the present invention to be administered depends on the purpose of use, target disease (type, severity, etc.), age, weight, sex, medical history, cell morphology or type of the patient, and the like. Thus, those skilled in the art can easily determine.
  • the frequency of applying the treatment method of the present invention to the subject (or patient) also depends on the purpose of use, target disease (type, severity, etc.), patient age, weight, sex, medical history, and course of treatment. In consideration of the above, a person skilled in the art can easily determine. Frequently, for example, once every few months (eg, once a week – once a month). It is preferable to administer once a week to once a month while monitoring the progress
  • the present invention provides a method for generating a blood vessel, which comprises administering the composition for angiogenesis of the present invention to a site of a patient in which angiogenesis is desired. I do.
  • the present invention forms a vascular network, which comprises administering the composition for forming a vascular network of the present invention to a site of a patient where formation of a vascular network is desired.
  • compositions used in the above methods can take any of the forms detailed herein. It is understood that the administration method used herein can also use any form.
  • the present invention provides a composition for forming an angiogenesis and Z or vascular network of the invention at a site in a patient where angiogenesis and Z or vascular network formation is desired. Provided for angiogenesis and forming a Z or vascular network.
  • patient refers to an organism to which the treatment of the present invention is applied, and is also referred to as “subject” or “subject”.
  • the patient can preferably be a human.
  • compositions of the present invention provide methods of treatment, inhibition and prevention by administering to a patient an effective amount of a composition of the present invention.
  • the compositions of the present invention can be substantially purified (including, for example, conditions in which there is substantially no ability to limit its effects or substances that produce undesirable side effects).
  • the animal targeted by the present invention may be any organism (eg, an animal (eg, a vertebrate, an invertebrate)) as long as it has an immune system or a similar system.
  • it is a vertebrate animal (e.g., a metal eel, alfalfa, chondrichthyes, teleosts, amphibians, reptiles, birds, mammals, etc.), and more preferably, a mammal (e.g., a monotreme, marsupial) , Rodents, dermis, skin wings, carnivores, carnivores, carnivores, longnoses, ungulates, artiodactyla, tubulars, squamata, squids, cetaceans, primates, rodents Teeth, egrets).
  • a vertebrate animal e.g., a metal eel, alfalfa, chondrichthyes,
  • Illustrative patients include, but are not limited to, animals such as, for example, horses, pigs, horses, birds, cats, dogs, and the like. More preferably, primates (eg, chimpanzees, diphones, humans) are targeted. Most preferably, humans are targeted.
  • animals such as, for example, horses, pigs, horses, birds, cats, dogs, and the like.
  • primates eg, chimpanzees, diphones, humans
  • humans are targeted.
  • compositions of the present invention can be administered orally or parenterally.
  • administration methods include oral administration, parenteral administration (e.g., intravenous, intramuscular, subcutaneous, intradermal, mucosal (intranasal, intravaginal, sub-airway, intrathecal, rectal) Mucosal and intestinal mucosa), topical administration to the affected area, dermal administration, etc.).
  • compositions used in the present invention are preferably pyrogen-free.
  • the preparation of such pharmaceutically acceptable compositions depends on the skill of the art in considering pH, isotonicity, stability and the like. Can be easily performed.
  • Formulations for such administration may be provided in any formulation.
  • Such preparation forms include, for example, liquid preparations, injections, and sustained release preparations.
  • Methods of introduction include, but are not limited to, administration as an oral agent, inhalation (eg, lungs), syringe, catheter, injection using a tube, needleless injection, gene gun, and the like. In this case, it can be administered together with other biologically active agents.
  • the amount of the medicament used in the prophylactic method of the present invention can be easily determined by those skilled in the art in consideration of the purpose of use, target disease (eg, type), age, weight, and medical history of the patient. I can do it.
  • the frequency of applying the treatment method of the present invention to a patient (or patient) also depends on the purpose of use, target disease (type, severity, etc.), age, weight, medical history, and course of the patient, and the like. Those skilled in the art can easily determine.
  • the frequency may include, for example, daily-once-monthly administration (eg, once a week, once a month), or once every year before the epidemic.
  • the interval between boosts can be at least about 3 weeks.
  • the dose of the composition or the like of the present invention varies depending on the age, body weight, symptoms, administration method, and the like of the patient, and is not particularly limited.
  • administering refers to giving the medicament or the like of the present invention or a pharmaceutical composition containing the same, alone or in combination with another therapeutic agent, to a host to be treated. means.
  • the combination may be administered, for example, either as a mixture, simultaneously, separately but simultaneously or concurrently; or sequentially. This includes the indication that the combined agents are administered together as a therapeutic mixture, and the procedure in which the combined agents are administered separately but simultaneously (e.g., through separate mucous membranes to the same individual). Also included.
  • “Combination” administration further includes separately administering one of the compounds or agents given first, followed by the second.
  • the administration of the medicament of the present invention may be performed by any method, but it is advantageous to use needleless injection. This is a drug that exempts the patient from administration without undue burden.
  • the needleless syringe in the present invention refers to a gas pressure or an elastic part without using a needle. It refers to a medical device that moves a piston by the force of a material to inject a drug solution onto the skin and administers a drug component subcutaneously, more preferably, into cells under the skin.
  • ShimaJet TM manufactured by Shimadzu Corporation
  • Medi-Jector Vision (Elite medical Inc. ⁇ )
  • Henget manufactured by Penjet
  • Penjet manufactured by Penjet
  • the present invention relates to a medicament for angiogenesis, comprising a peptide called VVXGL, wherein X is a modified aromatic amino acid, a peptide or a modified variant thereof, or a salt thereof.
  • a use for the manufacture of a composition comprising a peptide called VVXGL, wherein X is a modified aromatic amino acid, a peptide or a modified variant thereof, or a salt thereof.
  • the present invention comprises a pharmaceutical composition for forming a vascular network, comprising a sequence of WXGL, wherein X is a variant of an aromatic amino acid, a peptide or a variant thereof, or a salt thereof.
  • a pharmaceutical composition for forming a vascular network comprising a sequence of WXGL, wherein X is a variant of an aromatic amino acid, a peptide or a variant thereof, or a salt thereof.
  • compositions used in the above uses may take any of the forms detailed herein.
  • any technique of cultivated land can be used as a technique for producing a pharmaceutical composition in the art.
  • the region having an angiogenic activity and Z or vascular network forming activity is included in the amino acid sequence shown in WXL, the peptide having the amino acid sequence shown in VVXGL or a partial amino acid thereof is substituted or its terminal.
  • cell-adhesive action of cell-adhesive peptides such as fibronectin and laminin is exerted by RGD, a region consisting of only three amino acids, YIGSR, and a region consisting of only five amino acids. It can be inferred from the fact that they are things. Therefore, since the region having an angiogenic activity is included in the amino acid sequence shown in VVXGL, the peptide having the amino acid sequence shown in WXGL or a partial amino acid thereof has been substituted or the terminal region has been deleted.
  • the above-mentioned peptide or peptide variant region may have an interaction. Is considered to exert an angiogenic effect as long as it is exposed.
  • the three-dimensional structure of an artificial peptide can be easily predicted from its primary sequence using commercially available computer software. The peptides whose regions are exposed can be easily designed.
  • Computer programs for performing computer modeling can also be used in the process of selecting fragments or chemicals.
  • Useful programs that can be used in linking individual chemicals or fragments include:
  • CAVEAT PA Bartlett et al., "CAVEAT: A Program to Facilitate the Structure-Derived Design of Biologically Active MoleculesJ (Molecular Recognition in Chemical and Biological Problems, Special Pu b., Royal Chem. Soc., 78 G, Lauri and PA Barlett, "CAVEAT: a Program to Facilitate the Design of Organic Moleculesj, J. Comput. Aided Mol. Des., 8, 51-66 (1994). CAVEAT is available from the University of California, Berkeley, CA.
  • HOOK (MB Eisen et al., "HOOK: A Program for Finding Novel Molecular Architectures that Satisfy the Chemical and Steric Requirements of a Macromolecule Binding Site", Proteins: Struct., Funct., Genet., 19, 199-221 (1994)). HOOK is available from Molecular Simulations, San Diego, CA.
  • LUDI H. — J. Bohm, “The Computer Program LUDI: A New Met hod for the De Novo Design of Enzyme InhibitorsJ, J. Comp. Aid.
  • LUDI is available from Molecular Simulations Incorporated, San Diego, CA.
  • the material is optimized Competitive angiogenic potential can be tested and optimized by computational evaluation.
  • the substance of the present invention can be further optimized by calculation, for example, so that there is no electrostatic repulsion interaction with surrounding water molecules.
  • Such non-complementary electrostatic interactions include charge-charge repulsion interactions, dipole-dipole repulsion interactions, and charge-dipole repulsion interactions.
  • Another approach possible with the present invention is the computational screening of small molecule databases for chemicals or compounds that can bind, in whole or in part, to LOX-1.
  • the quality of the fit of such a material to the binding site can be determined from either shape complementarity or the estimated interaction energy [EC Meng et al., J. Comp. Chem. , 16, 505-524 (1992)].
  • the compound library used in the present invention can be prepared or obtained by any means including, but not limited to, combinatorial chemistry technology, fermentation methods, plant and cell extraction procedures, and the like. be able to. Methods for creating combinatorial libraries are well known in the art. For example, ER Fel der, Chimia 1994, 48, 512-541; Gallop et al., Med. Chem. 1994, 37, 1233-1251; RA Houghten, Trends Genet. 1993, 9, 235-239; Houghten et al., Nature 1991, 354, 84-86; Lam et al., Nature 1991, 354, 82-84; Carell et al., Chem. Biol.
  • the computer used may be any computer as long as the program can be executed, for example, Windows (registered trademark) -based, UNIX (registered trademark) -based, Mac (registered trademark) OS-based, LINUX-based, and the like. But not limited to them.
  • the peptide was synthesized using a solid phase synthesis method developed by Merrifield (Merrifield, RB (1963) Solid phase peptide synthesis. I. the synthesis of a tetrapeptide. J. Am. Chem. Soc. 85, 2149-2154), and the Fmoc method using a 9-fluorenylmethycarboxyl (Fmoc) group as a protecting group for the CK-amino group.
  • the synthesized peptides are as follows, and include, in principle, those having WXGL (SEQ ID NO: 1) and SWYGLR (SEQ ID NO: 2) which is a natural type as a control.
  • SEQ ID NO: 2 SWYGLR
  • SEQ ID NO: 4 SVVF (pMe) GLR (where F (pMe) is obtained by substituting a methyl group at the para-position of the phenyl group of phenylalanine)
  • SEQ ID NO: 5 SWF (pF) GLR (where F (pF) is a phenyl group of phenylalanine with a fluoro group substituted at the para-position)
  • SEQ ID NO: 6 SWF (pNO) GLR (where F (pNO) is phenyl of phenylalanine)
  • SEQ ID NO: 7 SWF (D) GLR (where F (D) is the D-form of phenylalanine)
  • SEQ ID NO: 8 SWF (pEt) GLR (where F (pEt) is fenilalanine With a phenyl group substituted with an ethyl group at the para-position)
  • SEQ ID NO: 9 SWF (pPr) GLR (where F (pPr) is a phenyl group in which the propyl group is substituted at the para-position of the phenyl group of hua- / realanine)
  • SEQ ID NO: 10 SWF (pHex) GLR (where F (pHex) is a phenyl group substituted with a hexyl group at the para-position of the phenyl group)
  • SEQ ID NO: 17 VVF (pMe) GLR (where F (pMe) is a phenylalanine in which a methyl group is substituted at the para-position of the phenyl group)
  • SEQ ID NO: 18 SWF (pMe) GL (where F (pMe) is phenylalanine in which a methyl group is substituted at the para-position of the phenyl group)
  • SEQ ID NO: 19 ⁇ 1 ⁇ ) 0 (where? 1 ⁇ 6) is the substitution of a methyl group at the para-position of the phenyl group of phenylalanine
  • SEQ ID NO: 21 SWF (pMe) GLR (where, F (pMe) is a compound obtained by substituting a methyl group at the ortho-position of the phenyl group of phenylalanine)
  • SEQ ID NO: 22 SWF (pF) GLR (where, F (pF) is a ferrule having a fluoro group substituted at the ortho position of the ferule group)
  • SEQ ID NO: 23 SWF (pNO) GLR (where F (pNO) is
  • SEQ ID NO: 24 SWF (pMe) GLR (where F (pMe) is a phenylalanine in which a methyl group is substituted at the meta position of the phenyl group)
  • SEQ ID NO: 25 SWF (pF) GLR (where F (pF) is a ferrule having a fluoro group substituted at the meta-position of the ferule group)
  • SEQ ID NO: 26 SWF (pNO) GLR (where F (pNO) is
  • Rat vascular endothelial cells are three-dimensionally implanted in the presence of the peptide of the present invention synthesized in Example 1.
  • TRLEC cells transformed rat lung endothelial cells
  • TRLEC cells were seeded in the collagen I layer mixed with a peptide solution having a concentration of lOngZml, and cultured in a carbon dioxide incubator for 14 days. Controls were performed with no factor (1) and with VEGF (+), a protein known conventionally as an angiogenic factor.
  • the cultured cells were observed under a microscope. As a result, the control showed no luminal formation.
  • the peptide of the present invention and VEGF formed a lumen, and cells surrounding the lumen were adhered to each other. Observation of the lumen with an electron microscope at 7000x magnification As a result, it was confirmed that a plurality of micropirai (fine cell projections) were formed on the inner wall of the lumen. Further, at a magnification of 15,000, the junction of the vascular endothelial cells surrounding the lumen was observed. As a result, a region where the endothelial cells were tightly bonded, that is, a tight junction was observed.
  • the endothelial cells acquired polarity and formed a lumen.
  • Polarity is a property of cells having a part that shares functions such as head and tail.Even if endothelial cells are normally cultured, they lose polarity and do not form a lumen, so it was induced by this peptide. Is shown.
  • the peptide of the present invention was significantly superior in tube formation length to VEGF. From these results, the peptide of the present invention has the effect of adhering cells to each other in a tissue composed of vascular endothelial cultured cells to form a lumen between them (in vivo, this lumen is Blood vessels).
  • FIG. FIG. 2 shows a comparison between VEGF and the peptide SVVYG LR (SEQ ID NO: 2).
  • FIGS. 4 and 5 show the state of angiogenesis when peptide SWYGLR (SEQ ID NO: 2) was used. As shown in FIG. 4 and FIG. 5, it was clarified that the blood vessels were newly formed, and the newly formed blood vessels were renewed and began to be reticulated. Therefore, it was revealed that the peptide of the present invention is useful as an angiogenic agent and a Z or vascular network forming agent. When this was compared with VEGF and the control, peptide SVVYGLR (SEQ ID NO: 2) showed angiogenesis and reticulation, whereas VEGF showed angiogenesis but no reticulation. I got it. In the control, neither neovascularization nor reticulation was observed. Therefore, the peptide of the present invention has an angiogenic action and a vascular network. It has been shown that the ability to form stalk may be enhanced.
  • VEGF 0.00 ⁇ 0
  • Peptide S WF (pF) GLR (SEQ ID NO: 5): 4.1
  • Peptide SWF (pNO) GLR SEQ ID NO: 6: 4.1.
  • Score 2 100 or more and less than 125
  • VEGF 2.75 ⁇ 1.03
  • Peptide SWF (pMe) GLR SEQ ID NO: 4
  • Peptide SWF (pF) GLR SEQ ID NO: 5
  • Nwl Network formation Although neovascularization was observed in the above, each new blood vessel was a single fe.
  • Nw2 In the middle stage of network formation, each new blood vessel is in a state in which a side branch on a ladder is engaged.
  • Nw3 The network is in the final stage, and the upper branch of the ladder has more side branches.
  • Nw4 Network maturation stage, showing a wide range of neovascular plexus.
  • SWF (pMe) GLR and SW FGLR showed higher network index capacity than SVVYGLR on both days 5 and 10.
  • SWYGLR SEQ ID NO: 2
  • VVYGLR SEQ ID NO: 13
  • SVVYGL SEQ ID NO: 12
  • FIG. 6 shows a diagram summarizing these results. This is sometimes called a balance sheet in this specification.
  • FreAlagin AD type As a carrier protein, FreAlagin AD type (manufactured by Miyagi-Danigaku Kogyo Co., Ltd., molecular weight 2000 to 20000), which is a gelatin obtained by partially hydrolyzing animal-derived collagen and removing allergens, was used.
  • the FreAlagin AD type has been approved for clinical use.
  • FreAlagin AD carrier protein
  • reaction mixture was desalted with a G10 column (Pharmacia) using 5% acetic acid as an eluent.
  • the main peak was lyophilized to give a bound compound almost quantitatively.
  • Rat vascular endothelial cells were three-dimensionally cultured in the presence of the gelatin-binding peptide thus prepared. This operation was specifically performed as follows. Transformed rat lung endothelial cells (TRLEC cells) were used. Gelatin-binding peptide (conjugate) and collagen type I are mixed at a ratio of 1:10, and 10 ng (nanogram) / m TRLEG cells were seeded in the conjugate 'collagen mixed solution mixed layer at a concentration of 1 and cultured in a carbon dioxide incubator for 14 days. The control was no factor (1), and the protein VEGF (+) conventionally known as an angiogenic factor was used as a positive control.
  • TRLEC cells Transformed rat lung endothelial cells
  • Gelatin-binding peptide (conjugate) and collagen type I are mixed at a ratio of 1:10, and 10 ng (nanogram) / m TRLEG cells were seeded in the conjugate 'collagen mixed solution mixed layer at a
  • the control was vigorous, forming no lumen at all.
  • the peptide conjugate of the present invention and VEGF formed a lumen, and cells surrounding the lumen were adhered to each other.
  • the adhesive activity of the side chain phenyl group substituted with a hydrophobic group was enhanced as compared with the natural type (SEQ ID NO: 2).
  • gelatin-binding peptide has the effect of adhering cells to each other and forming a lumen between them in a tissue composed of cultured vascular endothelial cells (in vivo, this lumen It is evident that the effect of the peptide modification seen in vitro will be retained in vivo.
  • a gelatin-binding peptide having a concentration of 100ngZml was dissolved in collagen I at a ratio of 1:10 to obtain a peptide-binding gelatin-collagen mixed solution having a concentration of 100ngZml.
  • a microcell consisting of a 0.45 mm diameter cylinder up and down with a Millipore filter (MILLIPORE, USA, DAS Assay kit) in the back of the mouse, the gelatin-binding peptide and VEGF solution were placed in the microcell.
  • MILLIPORE Millipore filter
  • a phosphate buffer (PBS) containing no peptide alone was also injected (control). Five days later, the state of the tissue around the microcell was observed with a microscope.
  • Gelatin binding peptide using SEQ ID NO: 2: 19.0
  • Gelatin binding peptide using SEQ ID NO: 3: 20.0
  • the present invention was demonstrated to have angiogenic ability even in vivo.
  • Such an effect is a force that was not seen in VEGF, which has a remarkable angiogenesis ability, and it can be said that it provides a powerful effect that has not been obtained conventionally.
  • the peptide of the present invention has a strong angiogenic action and a Z or vascular network forming ability, and can be used for a biological substitute material such as an artificial bone, an implant in an artificial organ, and a repair of the organ.
  • a bone substitute is made of hydroxyapatite, a representative of artificial bone, to which a gelatin-binding peptide is attached as shown in Example 4. By implanting this in place of bone, it became possible to form a vascular network around it.
  • a piece of tissue was prepared using polylactic glycolic acid (PLGA) as a support, the peptide SWYGLR (SEQ ID NO: 2) produced in Example 1 and its variant as a biomolecule, The angiogenic ability and the vascular network forming ability of the peptide of the present invention were demonstrated.
  • PLGA polylactic glycolic acid
  • Vycryl voriractin 910 mesh (copolymer of glycolic acid to lactate 90:10, PLGA), a biodegradable synthetic polymer, has a nitted mesh on the lumen side and a woven mesh on the outside (Woven mesh)
  • a total of three sheets (0.2 mm each, 0.6 mm in total) were stacked, and a collagen-crosslinked PLGA-collagen composite membrane was used as a scaffold.
  • a group in which only collagen type I was cross-linked using collagen as a cross-linking agent, and a group in which collagen SW was further cross-linked to peptide SWYGLR (SEQ ID NO: 2) were prepared.
  • the crosslinking method is as follows. A 20 mm diameter patch is sewn to the main trunk of the pulmonary artery.
  • ⁇ Cross-linking method The support is impregnated with a collagen solution. Next, freeze-dry and crosslink with dartal aldehyde saturated steam at 37 ° C for about 4 hours. Finally, the mixture was shaken three times in a 0.1 M glycine aqueous solution for 15 minutes, washed three times with distilled water, and freeze-dried. Thereby, a collagen-containing support is produced. The peptide SVVYGLR is immersed in this solution and crosslinked similarly.
  • the strength of the support was measured with a tensile tester.
  • a strip material having a width of 5 mm and a length of 30 mm was loaded in the short axis direction at a speed of 10 mmZ, and the load at break and the elastic modulus were measured.
  • the tensile strength was 75 ⁇ 5N for the PLGA-collagen composite membrane and 34 ⁇ 11N for dataraldehyde-treated pericardium.
  • Membranes to which the angiogenic peptide was bound also had comparable tensile strengths.
  • VECs vascular endothelial cells
  • VSMCs smooth muscle cells
  • PSH-26 fluorescent antibodies
  • Cell adhesion efficiency was compared between a PLGA-collagen composite membrane with only cross-linking treatment and a PLGA-collagen composite membrane with collagen type I and cross-linked type IV.
  • coloring area (%) of the fluorescent dye per visual field it was found that the type of vascular endothelial cells (VECs) and smooth muscle cells (VSMCs), and those of the slipped cells were also collagen type I and type IV.
  • Cell engraftment was observed in the crosslinked-treated support of the present invention, in which the coloring region of the fluorescent dye was significantly large.
  • the number of blood vessels can be determined by performing immunohistological staining with a factor VIII-related antigen or the like and then counting.
  • a sample is fixed with 10% buffered formalin, embedded in paraffin, several serial sections are prepared from each sample, and frozen. The frozen sections were then treated with a 2% paraformaldehyde solution in PBS for 5 minutes at room temperature. Fix and immerse in methanol containing 3% hydrogen peroxide for 15 minutes, then wash with PBS. Cover the sample with a serum albumin solution for approximately 10 minutes to block nonspecific reactions. Specimens are incubated overnight with an EPOS conjugated antibody against factor VIII-related antigen, which binds to HRP.
  • vascular endothelial cells After washing the samples with PBS, they are immersed in a diaminobenzidine solution (eg, 0.3 mg Zml diaminobenzidine in PBS) to obtain positive staining.
  • a diaminobenzidine solution eg, 0.3 mg Zml diaminobenzidine in PBS
  • the stained vascular endothelial cells are counted, for example, under an optical microscope at a magnification of 200 times, and the counting result is expressed, for example, as the number of blood vessels per square millimeter.
  • the presence of factor VIII is confirmed by determining whether the number of blood vessels is statistically significantly increased in calories, thereby confirming the presence of, for example, endothelial cells and vascular new life. Gender can be determined.
  • Elastica 'Van' Giesson staining was performed to stain the elastic fibers.
  • the procedure is as follows. If necessary, deparaffinize (for example, with pure ethanol), wash with water, and immerse the sample in a resorcin fuchsin solution available from Mutoh Chemical for 40-60 minutes. The sample is then washed with 70% alcohol and soaked in omni hematoxylin for 15 minutes. After that, rinse with running water for 5 minutes, and soak in Fan (1) 'Gison's solution for 2 minutes. Wash, quickly dehydrate, clear, enclose and confirm staining.
  • HE staining was performed to observe the fixation of the support in the cells.
  • the procedure is as follows. If necessary, the sample was deparaffinized (for example, with pure ethanol), washed with water, and immersed in omni hematoxylin for 10 minutes. After that, the sample was washed with running water, and colored with ammonia water for 30 seconds. After that, it is washed with running water for 5 minutes, stained with a 10-fold diluted solution of eosin hydrochloride for 2 minutes, dehydrated, cleared, sealed, and the staining can be confirmed.
  • the cells were stained by the von Kossa method to observe calcification.
  • the procedure is as follows. If necessary, remove paraffin (for example, with pure ethanol), wash with water (distilled water), and immerse in a 25% silver nitrate solution (under indirect light) for 2 hours. Then, wash with distilled water and soak in 42% 2% sodium thiosulfate (hypo) for 5 minutes. Then, wash with running water for 5 minutes Then soak for 5 minutes in Kärnmüroth. After that, it is washed with running water for 5 minutes, dehydrated, transparent, sealed, and stained.
  • VECs autologous vascular endothelial cells
  • VSMCs smooth muscle cells
  • VSMCs vascular endothelial cells and squamous muscle cells
  • Both the prepared support and the support seeded with autologous cells showed no apparent thrombus formation.
  • HE staining showed PLGA remaining, with intervening connective tissue.
  • the seeded vascular endothelial cells labeled with the fluorescent antibody are only scattered on the luminal side, and most of the cells are from the PLGA-collagen composite membrane. It was suggested that they had dropped out.
  • Vascular endothelial cells were examined by factor VIII staining and smooth muscle cell ex SMA immunostaining. In both groups, a continuous monolayer of vascular endothelial cells was observed by factor VIII immunostaining, and oriented smooth muscle cells were observed in the lumen by ⁇ -S ⁇ immunostaining.
  • the present invention is useful in any aspect where it is desired to form an angiogenic ability and a vascular network (medicine, medicine, dentistry, etc.).
  • the present invention is also useful for treating lifestyle-related diseases (eg, myocardial infarction, cerebral infarction, ischemic diseases such as obstructive aortic sclerosis).

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Abstract

Il est prévu de rechercher une substance ayant une capacité angiogénique favorisée, par comparaison avec un peptide angiogénique naturel (SVVYGLR). En fait, une composition pour l'angiogénèse qui contient la séquence VVXGL, où X représente un acide aminé aromatique modifié, et comprend un peptide ; une composition pour former un réseau de vaisseaux sanguins qui contient la séquence VVXGL, où X représente un acide aminé aromatique modifié, et comprend un peptide ; une méthode pour l'angiogénèse, qui implique d'administrer la composition susmentionnée sur le site d'un patient en attente d'angiogénèse ; une méthode pour la formation d'un réseau de vaisseaux sanguins qui implique d'administrer la composition susmentionnée sur le site d'un patient en attente de la formation d'un réseau de vaisseaux sanguins.
PCT/JP2005/006124 2004-03-31 2005-03-30 Composé ayant un effet angiogénique WO2005094865A1 (fr)

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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2008026634A1 (fr) * 2006-08-31 2008-03-06 Osaka University Stimulateur de proliferation de cellule mesenchymateuse et biomateriau du systeme squelettique
EA010734B1 (ru) * 2006-06-22 2008-10-30 Общество С Ограниченной Ответственностью "Сиа Пептайдс" Средство, нормализующее функции кровеносных сосудов, и способ его получения
WO2011119008A3 (fr) * 2010-03-26 2012-04-26 Industry-Academic Cooperation Foundation, Sookmyung Women's University Peptides pour favoriser l'angiogenèse et leur utilisation
WO2020116062A1 (fr) * 2018-12-03 2020-06-11 株式会社ハイペップ研究所 Nouveau composé et agent angiogénique le comprenant

Citations (1)

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Publication number Priority date Publication date Assignee Title
WO2003003925A1 (fr) * 2001-07-04 2003-01-16 Adam James Procede et appareil de suture

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Publication number Priority date Publication date Assignee Title
WO2003003925A1 (fr) * 2001-07-04 2003-01-16 Adam James Procede et appareil de suture

Non-Patent Citations (2)

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Title
ASOU Y. ET AL.: "Osteopontin facilitates angiogenesis, accumulation of osteoclastst and resorption in ectopic bone.", ENDOCRINOLOGY, vol. 142, no. 3, 2001, pages 1325 - 1332, XP002994514 *
HAMADA Y. ET AL.: "Angiogenic activity of osteopontin-derived peptide SVVYGLR", BIOCHEM BOPHYS RES.COMMUN., vol. 310, no. 1, 2003, pages 153 - 157, XP004457198 *

Cited By (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EA010734B1 (ru) * 2006-06-22 2008-10-30 Общество С Ограниченной Ответственностью "Сиа Пептайдс" Средство, нормализующее функции кровеносных сосудов, и способ его получения
WO2008026634A1 (fr) * 2006-08-31 2008-03-06 Osaka University Stimulateur de proliferation de cellule mesenchymateuse et biomateriau du systeme squelettique
US8222216B2 (en) 2006-08-31 2012-07-17 Osaka University Mesenchymal cell proliferation promoter and skeletal system biomaterial
WO2011119008A3 (fr) * 2010-03-26 2012-04-26 Industry-Academic Cooperation Foundation, Sookmyung Women's University Peptides pour favoriser l'angiogenèse et leur utilisation
US9249185B2 (en) 2010-03-26 2016-02-02 Industry-Academic Cooperation Foundation, Sookmyung Women's University Peptides for promoting angiogenesis and an use thereof
WO2020116062A1 (fr) * 2018-12-03 2020-06-11 株式会社ハイペップ研究所 Nouveau composé et agent angiogénique le comprenant
CN113366013A (zh) * 2018-12-03 2021-09-07 株式会社海培普研究所 新型化合物和包含其的血管生成剂
EP3907235A4 (fr) * 2018-12-03 2023-01-11 HiPep Laboratories Nouveau composé et agent angiogénique le comprenant
JP7475051B2 (ja) 2018-12-03 2024-04-26 株式会社ハイペップ研究所 新規化合物及びそれを含む血管新生剤

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