WO2005095443A1 - Drug delivery system using peptide modification - Google Patents

Drug delivery system using peptide modification Download PDF

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Publication number
WO2005095443A1
WO2005095443A1 PCT/JP2005/006127 JP2005006127W WO2005095443A1 WO 2005095443 A1 WO2005095443 A1 WO 2005095443A1 JP 2005006127 W JP2005006127 W JP 2005006127W WO 2005095443 A1 WO2005095443 A1 WO 2005095443A1
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Prior art keywords
peptide
amino acid
bioactive
composition according
amino acids
Prior art date
Application number
PCT/JP2005/006127
Other languages
French (fr)
Japanese (ja)
Inventor
Yoshiki Sawa
Satoshi Taketani
Shigeru Miyagawa
Junzo Takahashi
Nariaki Matsuura
Original Assignee
Cardio Incorporated
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Publication of WO2005095443A1 publication Critical patent/WO2005095443A1/en

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    • 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
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/16—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing nitrogen, e.g. nitro-, nitroso-, azo-compounds, nitriles, cyanates
    • A61K47/18—Amines; Amides; Ureas; Quaternary ammonium compounds; Amino acids; Oligopeptides having up to five amino acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/04—Centrally acting analgesics, e.g. opioids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/06—Antihyperlipidemics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/575—Hormones
    • C07K14/62—Insulins
    • 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
    • C07K7/08—Linear peptides containing only normal peptide links having 12 to 20 amino acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the present invention is in the field of medicine. More particularly, the present invention relates to a drug delivery system.
  • Patent Document 1 discloses a method for protecting proteazeca using a GA repeat. The purpose of this application is to protect a protein from a ubiquitin-mediated protease degradation system, and a partner to which GA repeat is added is a protein long enough to be recognized by ubiquitin. Therefore, it does not provide a mechanism for protecting small-molecular-weight peptides and the like in vivo, and does not provide such guidelines, discloses or suggests them.
  • Patent Document 1 W098Z22577
  • the present invention has found that an unexpected sustained release effect has been achieved by adding an amino acid such as glycine to a bioactive peptide, and completed a sustained release system using the addition of such an amino acid. I let it. In particular, it is noteworthy that a composition containing a plurality of such modified peptides had a remarkable sustained release effect.
  • the present invention provides the following.
  • a peptide comprising a bioactive peptide and at least one amino acid not derived from the bioactive peptide.
  • the peptide according to item 1 wherein the bioactive peptide has a molecular weight of 10 kDa or less.
  • the physiologically active peptide is a peptide having a length of 300 amino acids or less or a variant thereof.
  • the above-mentioned physiologically active peptide has an effect selected from the group consisting of angiogenesis, angiogenesis inhibition, bone regeneration, anti-inflammatory action (integrin inhibition), diabetes treatment, analgesic action, and anti-dementia.
  • the peptide according to any one of the above.
  • the above physiologically active peptides include peptide SVVYGLR, peptide SIKVAV, peptide IKVAV (laminin-derived peptide), peptide YIGSR (laminin-derived peptide), peptide ALKRQGRTLYGFGG (osteogenic growth peptide; OGP), peptide GDGRHDL ( Integrin inhibitor), peptide FVNQHLCGSHL VEAL YLVCHERGFFYTPKT GIVEQCCTSICSLYQLENYCN (insulin), peptide YGGFM (Met-enkephalin), YGGFL (Leu-enkephalin), peptide MLARMLNTTI SACFLS LLAF SSACYFQNCP RGGCFGRPS S ICCADELGCF VGTAEALRCQ EENYLPSPCQ SGQKPCGSGG R CAAVGICCS DESCVAEPEC HDGFFRLTRA REPSNATQLD GPAR ALLLRL VQLAGTRESV DSAK
  • a pharmaceutical composition comprising the peptide according to item 1.
  • composition comprising at least two kinds of peptides containing the amino acid sequence of a physiologically active peptide, wherein at least one kind of the peptide contains at least one amino acid not derived from the physiologically active peptide.
  • composition according to item 22 wherein the peptide includes at least two kinds of amino acids, and the amino acid portion includes at least two kinds of amino acids having different numbers of amino acids.
  • composition according to item 22 wherein the peptide includes at least two kinds of amino acids, and the amino acid portion includes at least two kinds of amino acids having different positions including amino acids.
  • composition according to item 22 wherein the bioactive peptide has a molecular weight of 30 kDa or less.
  • composition according to item 22, wherein the bioactive peptide has a molecular weight of 20 kDa or less.
  • composition according to item 22, wherein the bioactive peptide has a molecular weight of 10 OkDa or less.
  • the bioactive peptide is a peptide of 300 amino acids or less or a variant thereof.
  • composition according to item 22, wherein the physiologically active peptide is a peptide having a length of 200 amino acids or less or a variant thereof.
  • composition according to item 22 wherein the physiologically active peptide is a peptide having a length of 100 amino acids or less or a variant thereof.
  • composition according to item 22 wherein the amino acid includes at least one glycine.
  • composition according to item 22 wherein the amino acid is bound to at least one selected from the group consisting of C-terminal and N-terminal forces of the bioactive peptide.
  • composition according to item 22 wherein the amino acid is bound to both the C-terminus and the N-terminus of the bioactive peptide.
  • composition according to item 22 wherein the amino acid includes a plurality of glycines.
  • composition according to item 22 wherein the amino acid contains 3 to 10 glycines.
  • composition according to item 22 wherein the amino acid contains 3 to 6 glycines.
  • composition according to item 22 wherein the amino acid comprises a sequence different from a natural sequence of the bioactive peptide.
  • composition according to item 22, wherein the amino acid comprises a sequence different from a natural sequence of the leader sequence of the bioactive peptide.
  • composition according to item 22 wherein the composition comprises a physiologically active peptide having at least one kind of natural sequence.
  • the physiologically active peptide has an effect selected from the group consisting of angiogenesis, angiogenesis inhibition, bone regeneration, anti-inflammatory action (integrin inhibition), diabetes treatment, analgesic action, and anti-dementia.
  • the physiologically active peptides include peptide SVVYGLR, peptide SIKVAV, peptide IKVAV (laminin-derived peptide), peptide YIGSR (laminin-derived peptide), peptide ALKRQGRTLYGFGG (osteogenic growth peptide; OGP), peptide GDGRHDL ( Integrin inhibitor), peptide FVNQHLCGSHL VEAL YLVCHERGFFYTPKT GIVEQCCTSICSLYQLENYCN (insulin), peptide YGGFM (Met-enkephalin), peptide YGGFL (Leu-enkephalin), peptide MLARMLNTTI S ACFLSLLAF SSACYFQNCP RGGKRAISDM ELRQCLPCGP GGKG RCFGPS ICCADELGCF VGTAEALRCQ EENYLPSPCQ SGQKPCG SGG RCAAVGICCS DESCVAEPEC HDGFFRLTRA REPSNATQ
  • composition according to item 22, wherein the physiologically active peptide has a half-life in vivo of 2 days or less when present as the peptide itself.
  • composition according to item 22 wherein two or more kinds of the bioactive peptides are present.
  • composition according to item 22 further comprising an additional sustained-release component.
  • ⁇ ) a step of administering a peptide comprising a physiologically active peptide and at least one amino acid not derived from the physiologically active peptide to a subject intended for sustained release;
  • a method comprising:
  • a step of administering a peptide-containing composition comprising a bioactive peptide and at least one amino acid not derived from the bioactive peptide to a subject intended for sustained release, wherein the composition comprises: A plurality of the amino acids in the peptide.
  • the present invention provides a sustained-release agent using a physiologically active peptide.
  • This sustained release agent The sustained release effect was achieved while maintaining the activity of the active peptide and having biocompatibility. It is understood that this technique can be applied to any bioactive peptide.
  • the present invention can be designed as an amino acid sequence to be added so as to include (only) a sequence that does not have a biological side reaction (does not function). The design of the release agent becomes possible. This is also effective if it is possible to design freely which could not be realized with the conventional system using biological peptides.
  • FIG. 1 shows a schematic diagram of the action of adhering cells to form a lumen between them.
  • FIG. 2 is a diagram showing a state of a tissue around a microcell observed with a microscope by a DAS assay 5 days later in Example 3.
  • FIG. 3 shows the state of angiogenesis (day 5) when peptide SWYGLR (SEQ ID NO: 1) alone was used.
  • FIG. 4 shows the state of angiogenesis in the case of 6G alone (day 5).
  • FIG. 5 shows the state of angiogenesis in the case of using WG alone (day 5).
  • Figure 6 shows the state of angiogenesis in the case of the combination of the peptides SWYGLR and WG (
  • FIG. 7 shows the state of angiogenesis (day 5) in the case of a combination of G6 and peptide SWYGLR.
  • FIG. 8 shows the state of angiogenesis (day 5) in the case of a combination of G6 and WG.
  • FIG. 9 shows the state of angiogenesis of the three combinations (day 5).
  • FIG. 10 shows the state of angiogenesis of the combination of G6 and W10 on day 10.
  • FIG. 11 shows the state of angiogenesis in the case of the three combinations on day 10.
  • FIG. 12 shows the detailed evaluation of the number of angiogenesis in Example 3.
  • SEQ ID NO: 1 Angiogenesis peptide SVVYGLR derived from osteopontin
  • SEQ ID NO: 2 Osteobontin-derived angiogenic peptide variant GGGGGG—SW YGLR (also referred to herein as G6!)
  • SEQ ID NO: 3 Osteobontin-derived angiogenic peptide variant SVVYGLR—GG GGGG
  • SEQ ID NO: 4 Osteobontin-derived angiogenic peptide variant GGG—SVVYGL R-GGG (in this specification, both WG and WG! /)
  • SEQ ID NO: 5 Modified angiogenesis peptide derived from osteobontin GGG—SVVYGL
  • SEQ ID NO: 6 modified angiogenesis peptide derived from osteobontin SVVYGLR—GGG
  • SEQ ID NO: 7 Laminin-derived angiogenesis inhibitory peptide YIGSR
  • SEQ ID NO: 8 variant of laminin-derived angiogenesis inhibitory peptide GGGGGG—YIGSR
  • SEQ ID NO: 9 variant of laminin-derived angiogenesis inhibitory peptide YIGSR—GGGGGG
  • SEQ ID NO: 10 variant of laminin-derived angiogenesis inhibitory peptide GGG—YIGSR—GG G
  • SEQ ID NO: 11 A variant of laminin-derived angiogenesis inhibitory peptide GGG-YIGSR SEQ ID NO: 12: A variant of laminin-derived angiogenesis inhibitory peptide YIGSR-GGG SEQ ID NO: 13: Osteovontin-derived variant of angiogenesis peptide SVVFGLR SEQ ID NO: 14: Osteopontin Modified angiogenic peptide derived from AAAAAA— SV VYGLR
  • SEQ ID NO: 15 Osteobontin-derived angiogenic peptide variant SVVYGLR—A AAAAA
  • SEQ ID NO: 16 Angiogenesis peptide variant derived from osteopontin AAA—SVVYG LR-AAA
  • SEQ ID NO: 17 Osteopontin-derived angiogenic peptide variant AAA—SVVYG LR
  • SEQ ID NO: 18 Modified angiogenesis peptide derived from osteobontin SVVYGLR—A AA
  • SEQ ID NO: 19 Laminin-derived angiogenic peptide SIKVAV
  • SEQ ID NO: 20 Laminin-derived angiogenic peptide 'IKVAV
  • SEQ ID NO: 21 Vasopressin MLARMLNTTI SACFLSLLAF SSACYFQNC P RGGKRAISDM ELRQCLPCGP GGKGRCFGPS ICCADELGCF V GTAEALRCQ EENYLPSPCQ SGQKPCGSGG RCAAVGICCS DESC VAEPEC HDGFFRLTRA REPSNATQLD GPARALLLRL VQLPRV ES
  • SEQ ID NO: 22 Osteogenic growth peptide ALKRQGRTLYGFGG
  • SEQ ID NO: 23 Integrin inhibitor GDGRHDL
  • SEQ ID NO: 24 Insulin peptide FVNQHLCGSHL VEAL YLVCHERGFFYT PKTGIVEQCCTSICSLYQLENYCN
  • SEQ ID NO: 25 Met—Enkephalin YGGFM
  • SEQ ID NO: 26 Leu—Enkephalin YGGFL
  • SEQ ID NO: 27 A variant of laminin-derived angiogenesis peptide GGGGGG—SIKVAV SEQ ID NO: 28: A variant of laminin-derived angiogenesis peptide SIKVAV—GGGGGG SEQ ID NO: 29: A variant of laminin-derived angiogenesis peptide GGG—SIKVAV-GGG SEQ ID NO: 30 : Variant of laminin-derived angiogenic peptide GGG—SIKVAV SEQ ID NO: 31: Variant of laminin-derived angiogenic peptide SIKVAV—GGG SEQ ID NO: 32: Variant of laminin-derived angiogenic peptide GGGGGG—IKVAV SEQ ID NO: 33: Laminin-derived angiogenesis Modified peptide IKVAV-GGGGGG SEQ ID NO: 34: Modified laminin-derived angiogenic peptide GGG- IKVAV-GGG SEQ ID NO: 35: Modified laminin-
  • SEQ ID NO: 38 Variant of osteogenic growth peptide ALKRQGRTLYGFGG—GGGGGG
  • SEQ ID NO: 39 Variant of osteogenic growth peptide GGG—ALKRQGRTLYGFGG—GGG
  • SEQ ID NO: 40 Variant of osteogenic growth peptide GGG—ALKRQGRTLYGFGG
  • SEQ ID NO: 41 Modified osteogenic growth peptide ALKRQGRTLYGFGG—GGG
  • SEQ ID NO: 42 Modified angiogenesis peptide derived from osteopontin GLGLGL—SW YGLR
  • SEQ ID NO: 43 Modified angiogenesis peptide derived from osteobontin SVVYGLR—GL GLGL
  • SEQ ID NO: 44 Modified angiogenesis peptide derived from osteopontin GLG—SVVYGL R-GLG
  • SEQ ID NO: 45 Osteopontin-derived modified angiogenic peptide GLG—SVVYGL
  • SEQ ID NO: 46 Modified angiogenesis peptide derived from osteopontin SVVYGLR—GLG
  • SEQ ID NO: 47 A variant of laminin-derived angiogenic peptide GLGLGL—SIKVAV SEQ ID NO: 48: A variant of laminin-derived angiogenic peptide SIKVAV—GLGLGL SEQ ID NO: 49: A variant of laminin-derived angiogenic peptide GLG—SIKVAV-GLG SEQ ID NO: 50 : A variant of laminin-derived angiogenic peptide GLG—SIKVAV SEQ ID NO: 51: A variant of laminin-derived angiogenic peptide SIKVAV—GLG SEQ ID NO: 52: A variant of laminin-derived angiogenic peptide GLGLGL—IKVAV SEQ ID NO: 53: Laminin-derived angiogenesis Variant of peptide IKVAV—GLGLGL SEQ ID NO: 54: Variant of laminin-derived angiogenic peptide GLG—IKVAV—GLG SEQ ID NO: 55: Vari
  • SEQ ID NO: 56 Variant of laminin-derived angiogenic peptide IKVAV—GLG
  • the gene vaccine of the present invention can be implemented using a known technique in this field.
  • the production of such vaccines can be performed by applying molecular biological, biochemical and genetic engineering techniques as described elsewhere herein.
  • DNA synthesis technology for producing artificially synthesized genes and nucleic acid synthesis are described in, for example, Gait, MJ (1985). Oligonucleotide Synthesis: A Practical. Oligonucleotide Synthesis: A Practical Approach, IRL Press; Eckstein, F. (1991) .Oliigonucleotides and Analogues: A PracticalApproach, IRL Press; Adams, RL et al. (1992). The Biochemistry of the Nucleic Acids, Chapman &Hall; Shabarova, Z. et al. (1994) .AdvancedOrganic Chemistry of Nucleic Acids, Weinheim; Blackburn, GM et al. (1996) .Nucleic Acids in Chemistry and Biology, Oxford University Press; Hermanson, GT (1996).
  • the terms "protein,” “polypeptide,” “oligopeptide,” and “peptide” are used interchangeably herein and refer to a polymer of amino acids of any length. .
  • the polymer may be linear or branched or cyclic.
  • the amino acids may be naturally occurring or non-naturally occurring or modified amino acids.
  • the term is preferably, but not limited to, usually linear and in the form of naturally occurring amino acids alone, since it is preferably in a form translated by a nucleic acid molecule.
  • the term may also include those assembled into a complex of multiple polypeptide chains.
  • the term also embraces naturally 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.), peptidomimetic compounds (eg, peptoids), and other compounds known in the art. Changes are included.
  • protein refers to a polymer of amino acids having a relatively large molecular weight or a variant thereof, and when referred to as “peptide”, has a relatively small molecular weight. It is understood that it may refer to a polymer of amino acids or a variant thereof. Should be. Examples of such a molecular weight include, but are not limited to, about 30 kDa, preferably about 20 kDa, more preferably about 10 kDa.
  • nucleic acid molecule As used herein, the terms “nucleic acid molecule”, “polynucleotide”, “oligonucleotide” and “nucleic acid” are used interchangeably herein and refer to polymers of nucleotides of any length (e.g., cDNA, mRNA, genomic DNA, etc.). The term also includes “oligonucleotide derivatives” or “polynucleotide derivatives”. "Oligonucleotide derivative” or “polynucleotide derivative” 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, 2,1 O-methyl-ribonucleotide, an oligonucleotide derivative in which a phosphoric ester bond in the oligonucleotide is converted to a phosphorothioate bond, and an oligonucleotide in the oligonucleotide.
  • a particular nucleic acid sequence may also be a conservatively modified version (e.g., a degenerate codon substitution) and a conservatively modified version thereof, similar to the explicitly indicated sequence. It is contemplated to include the complementary sequence. 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.
  • a nucleic acid molecule encoding a gene sequence also includes "splice variants (variants, variants).”
  • a particular protein encoded by a nucleic acid includes any protein encoded by a splice variant of the nucleic acid.
  • splice variants are the products of alternative splicing of a gene. After transcription, the initial nucleic acid transcript may be spliced such that different (alternate) nucleic acid splice products encode different polypeptides. The mechanism of production of splice variants varies, but involves alternative splicing of exons.
  • polypeptide derived from the same nucleic acid by read-through transcription is also included in this definition. Any product of a splicing reaction, including recombinant forms of the splice product, is included in this definition. Therefore, the gene of the present invention may also include splice variants thereof. Such variants are useful in the prevention and treatment of the present invention. It is understood that the peptides of the present invention can usually be delivered in vivo by this polynucleotide form.
  • the term "gene” refers to a factor that defines a genetic trait. Those that define the primary structure of a protein are called structural genes, and those that control its expression are called regulatory genes (eg, promoters). As used herein, a gene includes a structural gene and a regulatory gene unless otherwise specified. Thus, a gene for a protein usually includes both transcriptional or translational regulatory sequences, such as the structural gene for the protein and a promoter for the protein. As used herein, “gene” generally refers to "polynucleotide”, “oligonucleotide”, “nucleic acid” and “nucleic acid molecule”.
  • gene product also refers to “polynucleotides”, “oligonucleotides”, “nucleic acids” and “nucleic acid molecules” expressed by genes and Z or “proteins”, “polypeptides”, “ “Oligopeptides” and “peptides”. Those skilled in the art can understand what the gene product is, depending on the situation.
  • homology of a gene refers to the degree of identity between two or more gene sequences.
  • similarity refers to the identity of two or more gene sequences to each other when conservative substitutions are regarded as positive in the above homology. The degree of. Thus, if there are conservative substitutions, identity and similarity will be different depending on the existence of the conservative substitution. 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 refers to 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 is different from the general chemical structure of an amino acid A compound that has a structure but 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.
  • nucleotide derivative or “nucleotide analog” refers to a nucleotide different from a naturally occurring nucleotide but having the same function as the original nucleotide.
  • nucleotide derivatives and nucleotide analogs are well-known in the art. Examples of such nucleotide derivatives and analogs include phosphorothioates, phosphoramidates, methylphosphonates, chiral methylphosphonates, 2-0-methylribonucleotides, peptides containing nucleic acids (PNA). It is not limited to these. In the present invention, any analog may be used as long as the gene product is expressed.
  • a genetic vaccine containing natural nucleotides is used. This is because the natural form is more likely to be translated into a peptide.
  • corresponding amino acids and nucleic acids refer to polypeptides and nucleic acids that serve as comparison standards in a given polypeptide and nucleic acid molecule, respectively. Refers to amino acids and nucleic acids that have, or are expected to have, the same action as a given amino acid and nucleic acid in a nucleic acid molecule. Refers to amino acids that make similar contributions and the nucleic acids that encode them. For example, if it is a nucleic acid sequence, it may be a portion that functions similarly to the nucleic acid sequence or the specific portion encoded by the nucleic acid sequence.
  • corresponding gene refers to a given gene in a species that serves as a reference for comparison in a certain species. It refers to a gene that has or is expected to have the same action as the offspring. When there are a plurality of genes having such an action, it refers to those having the same evolutionary origin. Thus, the corresponding gene for a gene may be an ortholog of that gene. Therefore, a gene corresponding to a gene such as a human cancer antigen can be found in other animals (mouse, rat, pig, mouse, etc.). Such corresponding genes can be identified using techniques well known in the art.
  • the corresponding gene in an animal can be determined by searching the sequence database of that animal (eg, mouse, rat) using the sequence of the reference gene for the corresponding gene (eg, a gene for a cancer antigen) as a query sequence. Or by screening the library in wet experiments.
  • search refers to the use of a certain nucleobase sequence electronically, biologically, or by another method to search for another nucleobase sequence having a specific function and Z or property.
  • Electronic searches include BLAST (Altschul et al, J. Mol. Biol. 215: 403-410 (1990)), FASTA (Pearson & Lipman, Proc. Natl. Acad. Sci., USA 85: 2444-2448 ( 1988, Smith and Waterman method (Smithand Waterman, J. Mol. Biol. 147: 195-197 (1981), and Needleman and Wunsch method (Needleman and Wunsch, J. Mol. Biol.
  • Examples of the biological search include stringent hybridization, a macroarray in which genomic DNA is attached to nylon membrane, or a microarray in which the DNA is attached to a glass plate. (Microarray assay), PCR, in situ hybridization, etc.
  • biosearch include stringent hybridization, a macroarray in which genomic DNA is attached to nylon membrane, or a microarray in which the DNA is attached to a glass plate. (Microarray assay), PCR, in situ hybridization, etc.
  • cancer antigens and the like are referred to by such electronic search and biological search. It is intended that the identified corresponding gene should also be included
  • fragment refers to a polypeptide or polynucleotide having a sequence length from l to n-1 relative to a full-length polypeptide or polynucleotide (length is n).
  • length is n
  • the length of the fragment can be appropriately changed depending on the purpose.For example, the lower limit of the length is 3, 4, 5, 6, 7, 8, 9, 10 for a polypeptide. , 15, 20, 25, 30, 40, 50 and more amino acids, and lengths represented by integers not specifically listed herein (for example, 11) are also suitable as lower limits.
  • polypeptide and polynucleotide can be represented by the number of amino acids or nucleic acids, respectively, as described above, but the above-mentioned numbers have the same function but are not absolute. As far as possible, the above-mentioned number as an upper limit or a lower limit is intended to include a few above and below (or, for example, 10% above and below) the number.
  • polynucleotide that hybridizes under stringent conditions refers to well-known conditions commonly used in the art. Using a polynucleotide selected from the polynucleotides of the present invention as a probe, colony 'hybridization method, plaque' hybridization method, Southern blot hybridization method or the like is used. Thus, such a polynucleotide can be obtained. Specifically, the polynucleotide to be hybridized under stringent conditions can be purified at 65 ° C in the presence of 0.7 to 1.OM NaCl using a filter on which DNA derived from colonies or plaques is immobilized.
  • a 0.1- to 2-fold concentration of SSC (saline-sodium citrate) solution (the composition of the 1-fold concentration SSC solution is 150 mM sodium salt sodium citrate, 15 mM sodium citrate).
  • SSC saline-sodium citrate
  • Hybridization is described in experimental books such as MolecularCloning 2nd ed., Current Protocols in Molecular Biology, supplement 1— «38, DNACloning l: Core Techniques, A Practical Approach, Second Edition, Oxford University Press (1995). It can be done according to the method.
  • sequences containing only the A sequence or only the T sequence are preferably excluded from the sequences that hybridize under stringent conditions.
  • Hybridizable polynucleotide refers to a polynucleotide that can hybridize to another polynucleotide under the above hybridization conditions.
  • Specific examples of the hybridizable polynucleotide include polynucleotides having at least 60% or more homology with the nucleotide sequence of DNA encoding the polypeptide having the amino acid sequence specifically shown in the present invention, preferably Polynucleotides having a homology of 80% or more, more preferably polynucleotides having a homology of 95% or more. Wear. It is understood that such homologous cell surface markers and the like can be used in the present invention.
  • Amino acids can 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.
  • Trp w Tribute fan Tyr Y tyrosine
  • the hydropathic index of amino acids can be considered.
  • the importance of the hydrophobic amino acid index in conferring interactive biological functions on peptides is generally recognized in the art (Kyte. J and Doolittle, RFJ Mol. Biol. 157 (1): 105-). 132, 1982).
  • the hydrophobic nature of amino acids contributes to the secondary structure of the resulting peptide, which in turn defines the interaction of the peptide with other molecules (eg, enzymes, substrates, receptors, DNA, antibodies, antigens, etc.).
  • Each amino acid is assigned a hydrophobicity index based on its hydrophobicity and the nature of the charge.
  • 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 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 (alanine, palin, leu). Syn, isoleucine, etc.), acidic amino acids (glutamic acid, aspartic acid, 4-carboxyglutamic acid, aminocunic acid, etc.), basic amino acids (arginine, histidine, lysine, etc.), aromatic amino acids (feralanine, tyrosine, It is understood that it can also be produced by exchanging with another person (such as tryptophan).
  • 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.
  • variants when referring to a polypeptide or polynucleotide refers to a substance in which a substance such as the original polypeptide or polynucleotide is partially 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.
  • the term "ortholog” refers to a gene derived from speciation from a common ancestor with two genes,! /,,, And both orthologous 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.
  • the term "conservative (modified) variants" applies to both amino acid sequences and nucleic acid sequences.
  • a conservatively modified variant refers to a nucleic acid that encodes the same or essentially the same amino acid sequence, and essentially the same if the nucleic acid does not encode an amino acid sequence. Sequence. Because of the degeneracy of the genetic code, many functionally identical nucleic acids encode any given protein. For example, the codons GCA, GCC, GCG, and GCU all encode the amino acid alanine. Thus, at every position where an alanine is specified by a codon, that codon can be changed to any of the corresponding codons described, without altering the encoded polypeptide.
  • 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
  • 1S Produces functionally identical molecules It will be understood that this can be modified.
  • each silent variation of a nucleic acid which encodes a polypeptide is implicit in each described sequence.
  • such modifications may be made to avoid substitution of the amino acid cysteine, which greatly affects the conformation of the polypeptide.
  • Examples of such base sequence modification methods include cleavage with a restriction enzyme or the like, ligation treatment with a DNA polymerase, Klenow fragment, DNA ligase, or the like, site-specific base substitution using a synthetic oligonucleotide, or the like. (Site-directed mutagenesis; Mark Zoller and Michael Smith, Methods in Enzymology, 100, 468-500 (1983)) You can do it too.
  • amino acid addition, deletion, or modification can also be performed to produce a functionally equivalent peptide.
  • 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 amidation, carboxylation, sulfation, Examples include, but are not limited to, logenation, 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 means amino acid or its substitute, or nucleotide or its substitute for the original polypeptide or polynucleotide, respectively. Physical ability 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 is
  • One or several, and preferably can be within 20%, 10%, or less than 3, less than 2, less than 1, etc. of the total length.
  • the term “cell biologically active substance” or “physiologically active substance” refers to a substance that acts on cells or tissues. Examples of such an action include, but are not limited to, control and change of the cell or the fibrous tissue.
  • Physiologically active substances include cytodynamics and growth factors.
  • the physiologically active substance may be a naturally occurring substance or a synthetic substance.
  • the physiologically active substance is produced by cells or has the same action as that of the bioactive substance, but may have a modified action.
  • the bioactive agent can be in a protein form or a nucleic acid form or other form, but at the time it actually acts, cytoforce usually refers to the protein form.
  • cytoforce in is defined in the same broadest sense as used in the art, and is produced by cellular forces acting on the same or different cells. Refers to a physiologically active substance. Cytokines are generally proteins or polypeptides that regulate immune response, regulate the endocrine system, regulate the nervous system, have antitumor and antiviral effects, regulate cell proliferation, regulate cell differentiation. Etc. As used herein, cytoforce in can be in protein or nucleic acid form or other forms, but at the time it actually works, cytoforce in usually refers to protein form.
  • growth factor or “cell growth factor” is used interchangeably herein and refers to a substance that promotes or controls cell growth. Growth factors are also called growth factors or growth factors. Growth factors can be added to the medium in cell or tissue culture to replace the action of serum macromolecules. Many growth factors have been found to function not only as cell growth but also as regulators of stagnation state.
  • the cytokins typically include interleukins, chemokines, hematopoietic factors such as colony stimulating factors, tumor necrosis factors, and interferons.
  • Typical growth factors include platelet-derived growth factor (PDGF), epidermal growth factor (EGF), fibroblast growth factor (FGF), hepatocyte growth factor (HGF), and vascular endothelial growth factor (VEGF )).
  • PDGF platelet-derived growth factor
  • EGF epidermal growth factor
  • FGF fibroblast growth factor
  • HGF hepatocyte growth factor
  • VEGF vascular endothelial growth factor
  • Biologically active substances such as cytodynamics and growth factors generally have other names and functions (eg, cell adhesion activity or cell-substrate adhesion activity, etc.) due to the phenomenon of redundancy.
  • Any of the cytokins or growth factors known in the above can be used in the present invention as long as they have the activity of the bioactive substance used in the present invention.
  • the cytodynamic force or growth factor has a preferable activity herein (for example, an activity to attract a host cell), it is preferable in the preferred embodiment of the tissue piece or the medicament of the present invention. Can be used.
  • bioactive peptides that can be used herein include antitumor agents, antibiotics, antipyretics, analgesics, anti-inflammatory agents, antitussive expectorants, sedatives, muscle relaxants, antiepileptics, antiulcer agents, antidepressants.
  • physiologically active peptide examples include luteinizing hormone-releasing hormone (LH-RH) , Insulin, somatostatin, growth hormone, growth hormone releasing hormone (GH-RH), prolatatin, erythropoietin, adrenocortical hormone, melanocyte stimulating hormone, thyroid hormone releasing hormone (TRH), thyroid stimulating hormone, luteinizing hormone, Follicle-stimulating hormone, vasopressin, oxytocin, calcitonin, gastrin, secretin, noncleozymine, cholecystokinin, angiotensin, human placental ratatogen, human chorionic gonadotropin, enkephalin, endorphin, kiyotorphin, tuftsin, thymocystin Thymic factor, blood thymic factor, tumor necrosis factor, colony-inducing factor, motilin, dinorphin, bombesin, neurotensin
  • Peptides having physiological activities include LH-RH antagonists (US Pat. Nos. 4,086,219, 4,124,577, 4,253,997, and 4,253,997). 4, 317, 815), but is not limited thereto.
  • examples of peptides having further physiological activity include insulin, somatostatin, somatostatin derivatives (US Pat. Nos. 4,087,390, 4,093,574, 4,100,117, No. 4,253,998), growth hormone, prolatatin, adrenocorticotropic hormone (ACTH), melanocyte stimulating hormone (MSH), thyroid hormone releasing hormone and its salts and derivatives (Japanese Patent Application Laid-Open No. 50-121273).
  • endorphin eg., ⁇ -type, j8-type, 0-type, etc.
  • interfe Lons eg, ⁇ -type, j8-type, 0-type, etc.
  • interleukins eg, I, II, ⁇ , etc.
  • tuftsin Thymopoietin thymosin, thymostymulin
  • THF thymic humoral factor
  • FTS blood thymic factor
  • its derivatives see US Pat. No. 4,229,438, and other thymic factors [Ayumi of Medicine, 125 Vol. 10, No.
  • Tumor necrosis factor (TNF), colony-inducing factor (CSF), motilin, dynorphin, bombesin, new mouth tensin, cellulin, bradycun, perokinase, asparaginase , Kallikrein, substance ⁇ ⁇ , nerve growth factor, cell growth factor, neurotrophic factor, blood coagulation factor VIII, IX, lysozyme chloride, polymyxin ⁇ , colistin, dalamicidin, bacitracin and erythropoietin ( ⁇ ), endothelin antagonist Peptides having an action (European Patent Publication Nos. 436189, 457195, and 496452; Nos. 94692 and 3-130299), but are not limited thereto.
  • the physiological activity of the peptide of the present invention can be determined by measuring whether or not the peptide retains the intrinsic biological activity. Therefore, it is understood that the peptide of the present invention can be determined for its sustained-release ability by any known assay for measuring the physiological activity (such as angiogenesis ability) of the physiologically active peptide to be contained therein. .
  • Such a physiological activity may be, for example, an angiogenesis ability, a vascular network formation ability.
  • 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 a network! , U.
  • angiogenesis is a force that can be determined by observing whether a reticulated force has formed (for example, the connection of a branched vessel to another blood vessel and the increase in the number of its junctions).
  • vascular network formation index is calculated in the present specification as follows.
  • the count is determined by the number of blood vessels per 0.79 cm.
  • 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.
  • the state of angiogenesis in a tissue can be observed with a stereoscopic microscope (Olympus, SZX12, Japan).
  • the obtained image is read by Photoshop (registered trademark) (Adobe, Japan) and the network ability is scored as follows.
  • the following scores are also referred to as “vascular network forming ability” or “vascular network forming index” or simply “network index”.
  • Nwl Network formation Although neovascularization is recognized 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 the side branch on the 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 is usually such a vascular network formation index force of at least 2, preferably 2.5 or more, It is understood that it is preferably 3 or more, more preferably 3.5 or more.
  • blood vessel is used in a meaning commonly used in the art, and includes a capillary, in addition to a normal artery, a vein, and the like.
  • deletion, substitution, or addition (including fusion) of the amino acid of the peptide of the present invention can be performed by site-directed mutagenesis or chemical synthesis, which is a well-known technique. Such deletion or substitution or addition of one or several amino acids is performed by Molecular Cloning, A Laboratory Manual, Second Edition, Cold spring Harbor Laboratory Press (1989), Current Protocols in Molecular Biology, Supplement 1-38, John Wiley & Sons (1987-1997), Nucleic Acids Research, 10, 64 87 (1982), Proc. Natl. Acad. Sci., USA, 79, 6409 (1982), Gene, 34, 315 (1985), Nucleic Acids Research. Natl. Acad. Sci USA, 82, 488 (1985), Proc. Natl. Acad. Sci., USA, 81, 5662 (198 4), Science, 224, 1431 (1984). ), PCT WO85 / 00817 (1985), Nature, 316, 601 (1985) and the like.
  • the compound used in the present invention, or a salt thereof or a hydrate thereof can be easily produced by a method known per se. Specific examples of the method include, for example, the following production method or a method analogous thereto.
  • salts with inorganic bases include alkali metal salts such as sodium salt and potassium salt, alkaline earth metal salts such as calcium salt, magnesium salt and barium salt, and aluminum salt and ammonium salt.
  • Examples of the salt with an organic base include trimethylamine, triethylamine, pyridine, picoline, ethanoylamine, jetanoylamine, triethanolamine, dicyclohexylamine, N, N, and dibenzylethylenediamine.
  • Examples of salts with inorganic acids include salts with hydrochloric acid, hydrofluoric acid, hydrobromic acid, nitric acid, sulfuric acid, phosphoric acid, perchloric acid, hydroiodic acid, and the like.
  • Salts with organic acids include formic acid, acetic acid, trifluoroacetic acid, fumaric acid, oxalic acid, tartaric acid, maleic acid, citric acid, succinic acid, malic acid, mandelic acid, ascorbic acid, lactic acid, dalconic acid, and methanesulfonic acid. , P toluene sulfonic acid, benzene sulfonic acid and the like.
  • Salts with basic amino acids include salts with arginine, lysine, ortin and the like.
  • Salts with acidic amino acids include salts with aspartic acid, glutamic acid and the like.
  • the factor of the present invention when used as a compound, it can be used in the form of a hydrate.
  • a pharmacologically acceptable hydrate is preferred, and a hydrate is also included. Specific examples include monohydrate, dihydrate, hexahydrate and the like.
  • Examples of this method include a method using an activated cellulose membrane (SPOTs manufactured by Sigma Genosys).
  • the method using the activated cellulose membrane is performed as follows. If the ⁇ -amino group is protected with 9-fluoromethoxycarbol (Fmoc) and the carboxyl group is also an active ester (Opfp), an amino acid derivative solution protected with (Odhbt) is placed at the marked position on the membrane. Spot about ⁇ 1 (equivalent to 100-200 g of Fmoc amino acid). A spacer arm is attached to this designated position, and since there is a free amino group at its tip, it is blocked. Colors blue with mufenol blue (BPB).
  • SPOTs activated cellulose membrane
  • amino acid derivatives are dissolved in 1-methyl-2-pyrrolidinone (NMF) before use.
  • NMF 1-methyl-2-pyrrolidinone
  • the amino group on the membrane reacts with the active ester to form an amide bond.
  • the unreacted amino group is treated with acetic anhydride ZDMF to acetylate the compound, thereby losing the reactivity (this is referred to as cabbing).
  • the amino group is protected by piperidine ZDMF which is a secondary amine, the Fmoc group is removed, and the amino group is released to carry out the next extension reaction.
  • the reactive side chains are t-butyl alcohol-based protecting groups (Pmc (2,2,5,7,8 pentamethylchroman-16-sulfol), OtBu (0-t-butoxy), trityl ( (Trt), tert-butoxycarbol (tBoc), tert-butoxy (tBu), etc.) to obtain the desired compound by removing the protecting group with trifluoroacetic acid (TFA) (deprotection) Can be.
  • Pmc t-butyl alcohol-based protecting groups
  • 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.
  • an analytical method using powder X-ray diffraction can be used to determine the physical properties of the obtained compound.
  • the compounds used in the present invention can be prepared or obtained by any means including, but not limited to, combinatorial chemistry techniques, fermentation methods, plant and cell extraction procedures, and the like. it can. Methods for making combinatorial libraries are well known in the art. For example, ER Felder, 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.
  • screening refers to selecting a target such as a target organism or substance having a specific property from a population including a large number by a specific operation Z evaluation method.
  • an agent eg, an antibody
  • polypeptide or nucleic acid molecule of the present invention can be used.
  • a library generated using an in silico (computer-based) system or a system using an existing substance such as in vitro or in vivo may be used.
  • the compounds obtained by the screening having the desired activity are also included in the scope of the present invention.
  • a drug by computer modeling is provided based on the disclosure of the present invention.
  • the present invention provides a candidate conjugate that modulates the ability to bind to the protein of the invention or the polypeptide of the invention, or a biologically active portion thereof, or to modulate these activities.
  • the present invention provides an assay for screening substances or test compounds.
  • Test compounds of the present invention can be obtained using any of a number of approaches in one of the combinatorial library methods known in the art, including: biological libraries; Accessible solid-phase or solution-phase libraries; one synthetic library requiring deconvolution; one single-bead-one compound library; and synthetic libraries using affinity chromatography selection.
  • biological libraries are limited to peptide libraries, while the other four approaches are applicable to small molecule libraries of peptides, non-peptide oligomers or compounds (Lam (1997) Anticancer Drug Des. 12 : 145).
  • a library of compounds is prepared in solution (eg, in Houghten (1992) BioTechniques).
  • preventing refers to reducing the ability of an organism to contract or develop an abnormal condition.
  • treating refers to having a therapeutic effect and the ability to reduce or at least partially alleviate an abnormal condition in an organism.
  • therapeutic effect refers to the ability to cause an abnormal condition or the ameliorating effect provided by an inhibitory factor or an active factor that contributes thereto.
  • the effect of treatment is to alleviate one or more of the symptoms of the abnormal condition.
  • a therapeutic effect can refer to one or more of the following: (a) cell proliferation, growth, and an increase in Z or S; (C) inhibition of degeneration; (d) alleviating one or more of the symptoms associated with the abnormal condition; and (e) affected cells.
  • Conjugates that exhibit efficacy against abnormal conditions can be identified as described herein.
  • the "subject” refers to any system (for example, an organism) to which the present invention is applied, and when an animal such as a human is the subject, it is also referred to as a "patient". .
  • the patient or subject can preferably be a human.
  • the subject may be used in therapy Often used herein to describe any system.
  • pathogenesis refers to a factor associated with a disease, disorder, or condition of a subject (in the present specification, also collectively referred to as “lesion” or as a disease in plants). Examples include, but are not limited to, causative pathogens (pathogenic factors), pathogens, diseased cells, pathogenic viruses, and the like.
  • the "disease” targeted by the present invention may be any disease.
  • diseases include lifestyle-related diseases (eg, periodontal disease, hypertension, hyperlipidemia, diabetes, heart disease, arteriosclerotic disease, etc.), cancer, infectious diseases, allergies, cerebral infarction, dementia. , Obesity, infertility, neuropsychiatric disorders, cataracts, progeria, ultraviolet radiation hypersensitivity, and the like.
  • the present invention for example, in the case of periodontal disease, which is a lifestyle-related disease of the nation, when alveolar bone resorption accompanied by poor gingival blood circulation is present, a peptide having a local angiogenesis ability or a peptide having a bone regeneration ability can be fixed. By acting for a period (1 to 2 months of alveolar bone regeneration), it is possible to improve poor gingival blood circulation and reconstruct the alveolar bone.
  • the "disorder" targeted by the present invention may be a disorder that deviates from its normal functional ability in an organism.
  • abnormal condition refers to a function of an organism in a cell or tissue that also deviates from its normal functioning ability.
  • the abnormal condition may be associated with cell proliferation, cell differentiation, cell signaling, or cell survival.
  • Abnormal conditions also include abnormal vascular formation, abnormal apoptosis, abnormalities in neurotransmission, obesity, complications of diabetes such as retinal degeneration, and irregularities in glucose uptake and metabolism, and fatty acid Irregularities in uptake and metabolism may be mentioned.
  • diagnosis, prophylactic, treatment or prognostically effective amount refers to an amount that is considered to be medically effective in diagnosis, prevention, treatment (or therapy) or prognosis, respectively. Say. Such an amount can be determined by one of ordinary skill in the art using techniques well known in the art and considering various parameters.
  • such a composition may further include a pharmaceutically acceptable carrier and the like.
  • Pharmaceutically acceptable carriers included in the medicament of the present invention include any substance known in the art.
  • the carrier used herein is preferably pharmaceutically acceptable.
  • Such carriers include antioxidants, preservatives, colorings, 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.
  • 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 recipients, 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, disaccharides and other carbohydrates including glucose, mannose, or dextrin
  • chelating agents eg, EDTA
  • sugar alcohols eg, , Mannitol or sorbitol
  • salt-forming counterion eg, sodium
  • Z or non-ionic surfactants 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.
  • polypeptide, polynucleotide and the like of the present invention are combined with a pharmaceutically acceptable carrier, and solid preparations such as tablets, capsules, granules, powders, powders and suppositories, or syrups and injections are prepared. It can be administered orally or parenterally as liquid preparations such as preparations, suspensions, solutions and sprays.
  • Pharmaceutically acceptable carriers include, as described above, excipients, lubricants, binders, disintegrants, disintegration inhibitors, absorption enhancers, adsorbents, humectants, solubilizing agents, Examples include stabilizers, solvents in liquid preparations, solubilizing agents, suspending agents, isotonic agents, buffers, soothing agents and the like. If necessary, additives for preparations such as preservatives, antioxidants, coloring agents, sweeteners and the like can be used.
  • the composition of the present invention can also contain substances other than the polynucleotide, polypeptide, etc. of the present invention. Parenteral routes of administration include, but are not limited to, intravenous injection, intramuscular injection, nasal, rectal, vaginal and transdermal.
  • excipient in the solid preparation examples include glucose, ratatose, sucrose, D-mantol, crystalline cellulose, starch, calcium carbonate, light caffeic anhydride, sodium chloride, kaolin and urea.
  • Examples of the lubricant in the solid preparation include, but are not limited to, magnesium stearate, calcium stearate, powdered boric acid, colloidal citric acid, talc, and polyethylene glycol.
  • binder in the solid preparation examples include water, ethanol, propanol, sucrose, D-mann-tol, crystalline cellulose, dextrin, methylcellulose, hydroxypropinoresenolerose, hydroxypropinolemethinoresenolerose, and canoleboximetinolole.
  • disintegrant in the solid preparation examples include starch, carboxymethylcellulose, carboxymethylcellulose calcium, agar powder, laminaran powder, croscarmellose sodium, sodium carboxymethyl starch, sodium alginate, sodium hydrogen carbonate, calcium carbonate, Polyoxyethylene sorbitan fatty acid esters, sodium lauryl sulfate, starch, monoglyceride stearate, ratatose and cellulose glycol Examples include, but are not limited to, calcium luate.
  • Preferable examples of the disintegration inhibitor in the solid preparation include, but are not limited to, hydrogenated oil, sucrose, stearin, cocoa butter, and hardened oil.
  • absorption enhancer in the solid preparation examples include, but are not limited to, quaternary ammonium bases and sodium lauryl sulfate.
  • Examples of the adsorbent in the solid preparation include, but are not limited to, starch, ratatose, kaolin, bentonite, and colloidal citric acid.
  • humectant in the solid preparation examples include, but are not limited to, glycerin and starch.
  • solubilizing agent in the solid preparation examples include, but are not limited to, arginine, glutamic acid, and aspartic acid.
  • Examples of the stabilizer in the solid preparation include, but are not limited to, human serum albumin, ratatose, and the like.
  • a film of a gastric or enteric substance sucrose, gelatin, hydroxypropylcellulose, hydroxypropylmethylcellulose phthalate, etc.
  • Tablets include tablets coated with usual coatings as necessary, such as sugar-coated tablets, gelatin-coated tablets, enteric-coated tablets, film-coated tablets or double tablets, and multilayer tablets.
  • Capsules include hard capsules and soft capsules.
  • Preferable examples of the solvent in the liquid preparation include water for injection, alcohol, propylene glycol, macrogol, sesame oil, corn oil and the like.
  • solubilizer in the liquid preparation include polyethylene glycol, propylene glycol, D-mantol, benzyl benzoate, ethanol, trisaminomethane, cholesterol, triethanolamine, sodium carbonate and Examples include, but are not limited to, sodium citrate.
  • suspending agent in the liquid preparation include stearyl triethanolamine, Surfactants such as sodium peryl sulfate, laurylaminopropionic acid, lecithin, benzalco-chloride, benzedonium chloride, glyceryl monostearate, and the like, for example, polyvinyl alcohol, polybutylpyrrolidone, sodium carboxymethylcellulose, methylcellulose, and hydroxy.
  • surfactants such as sodium peryl sulfate, laurylaminopropionic acid, lecithin, benzalco-chloride, benzedonium chloride, glyceryl monostearate, and the like, for example, polyvinyl alcohol, polybutylpyrrolidone, sodium carboxymethylcellulose, methylcellulose, and hydroxy.
  • hydrophilic polymers such as methinoresenololose, hydroxyethinoresenololose, hydroxyethinorescellulose, and hydroxypropylcellulose.
  • Preferable examples of the tonicity agent in the liquid preparation include, but are not limited to, sodium salt, glycerin, D-mantol and the like.
  • buffer in the liquid preparation include, but are not limited to, buffers such as phosphate, acetate, carbonate, and citrate.
  • Preferable examples of the soothing agent in the liquid preparation include, but are not limited to, benzyl alcohol, benzalco-dum chloride, and proforce hydrochloride.
  • Preferable examples of the preservative in the liquid preparation include, but are not limited to, paraoxybenzoic acid esters, chlorobutanol, benzyl alcohol, 2-phenylethyl alcohol, dehydroacetic acid, sorbic acid and the like. .
  • antioxidant in the liquid preparation include, but are not limited to, sulfite, ascorbic acid, a-tocopherol, and cysteine.
  • the liquid preparation and the suspension are preferably sterilized and isotonic with blood and blood.
  • these are sterilized by filtration using a nocteria retention filter or the like, blending of a bactericide or irradiation. Further, after these treatments, solidify by freeze-drying or the like.
  • sterile water or a sterile injectable diluent (lidocaine hydrochloride aqueous solution, physiological saline, glucose aqueous solution, ethanol or a mixed solution thereof) is used. Add some calories.
  • the pharmaceutical composition may contain a coloring agent, a preservative, a flavor, a flavoring agent, a sweetener, and the like, and other agents.
  • the pharmaceutical 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 Publisnm g Company, 1990, etc.) and a sugar chain composition having a desired degree of purity, can be prepared and stored in the form of a lyophilized cake or aqueous solution.
  • a physiologically acceptable carrier excipient or stabilizing agent
  • 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
  • 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 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 can be readily performed by those skilled in the art by considering pH, isotonicity, stability, and the like.
  • 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 peptide of the present invention can be locally administered alone or in the form of an injection solution dissolved in a physiological buffer to a tissue where delivery is desired.
  • Injectable solutions also contain other disinfectants and anti-inflammatory analgesics. Usually, it contains various components included in the remedy for wounds.
  • various delivery systems are known and can be used to administer the compounds of the invention (eg, ribosomes, microparticles, microcapsules, etc.).
  • Methods of introduction include, but are not limited to, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, and oral routes.
  • the compound or composition can be administered by any convenient route (eg, by infusion or bolus injection, by absorption through epithelial or mucosal linings (eg, oral, rectal and intestinal mucosa, etc.)) and other May be administered together with a biologically active agent. Administration can be systemic or local.
  • intraventricular injection can be, for example, an intraventricularly attached reservoir such as an Ommaya reservoir. (Which may be facilitated by a catheter) to introduce it into the central nervous system.
  • Pulmonary administration may also be used, for example, with the use of an inhaler or nebulizer, and formulation with an aerosolizing agent.
  • the compositions used in the present invention are preferably pyrogen free.
  • the preparation of such pharmaceutically acceptable compositions can be readily performed by those skilled in the art by considering pH, isotonicity, stability, and the like. Formulations for such administration may be provided in any formulation.
  • Such preparation forms include, for example, solutions, injections, and sustained-release preparations.
  • introduction method include, but are not limited to, administration as an oral preparation, inhalation (for example, lung), injection using a syringe, catheter, or tube, needleless injection, and a gene gun. In this case, it can be administered together with other biologically active agents.
  • polypeptides, polynucleotides or compositions of the invention may be desirable to administer locally to the area in need of treatment; For example, by local injection during surgery, topical application (eg, in combination with a post-operative wound dressing), by injection, by catheter, by suppository, or by an implant (this implant may be a sialastic membrane).
  • this implant may be a sialastic membrane.
  • membranes or fibers which are porous, non-porous, or glue-like materials.
  • a material to which the protein is not absorbed is used. Attention must be paid to! / ,.
  • the compound or composition can be delivered encapsulated in vesicles, particularly ribosomes (Langer, Science 249: 1527-1533 (1990); Treat et al., Liposomes mtne). fherapy of Infectious Disease and Ancer, Lo pez—Berestein and Fidler (eds.), Liss, New York, pp. 353-365 (1989); Lo pez—Berestein, pp. 317-327; see broadly the same book. That).
  • vesicles particularly ribosomes
  • the compound or composition can be delivered in a controlled release system.
  • a pump may be used (Langer, supra; Seft on, CRC Crit. Ref. Biomed. Eng. 14: 201 (1987); Buchwald et al., Surgery 88: 507 (1980); Saudek et al. , N. Engl. J. Med. 321: 574 (1989)).
  • polymeric materials can be used (Medical Applications of Controlled Release, Langer and Wise (eds.), CRC Pres., Boca Raton, Florida (1974); Controlled Drug Bioavailability, Drug Product Design and Performance, Smolen.
  • a controlled-release system can be placed near a therapeutic target and thus requires only a portion of the systemic dose (eg, Goodson, Medical Applications of Controlled Release). , (Supra), Vol. 2, pp. 115-138 (1984)).
  • the animal targeted by the present invention may be any organism (eg, animal (eg, vertebrate, invertebrate)) as long as it has a peptide metabolic system.
  • it is a vertebrate (e.g., a metal eel, alfalfa, chondrichthyes, teleosts, amphibians, reptiles, birds, mammals, etc.), and more preferably, a mammal (e.g., monotremes, marsupials, Poor Teeth, skin wings, winged hands, carnivores, insectivores, longnoses, hoofed hoofs, artiodactyla, tubulars, squamata, oxen, whales, primates, rodents, (Egret, etc.).
  • a vertebrate e.g., a metal eel, alfalfa, chondrichthyes, teleosts, amphibians, reptiles, birds, mammals, etc.
  • Exemplary 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, macaques, 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, macaques, humans
  • humans are targeted.
  • the amount of the drug 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, the target disease (eg, type), the patient's age, weight, and medical history. 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 to a host to be treated alone or in combination with another therapeutic agent. 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 in 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.
  • the "instruction” refers to a method of administering or diagnosing the medicament or the like of the present invention for a physician, a patient or the like who administers or diagnoses (possibly a patient). It is described.
  • This instruction describes a word indicating a procedure for administering the diagnostic agent, the medicine and the like of the present invention.
  • This instruction is prepared in accordance with the format prescribed by the competent authority of the country where the present invention is implemented (for example, the Ministry of Health, Labor and Welfare in Japan and the Food and Drug Administration (FDA) in the United States) and is issued by the competent authority. The approval is clearly stated. Instructions are so-called package inserts, which are usually provided on paper media, but are not limited to electronic media (eg, homepages (websites) provided on the Internet, emails, etc.). , SMS, voicemail, instant messaging).
  • Judgment of termination of treatment according to the methods of the present invention may be supported by standard clinical laboratory results or by the disappearance of clinical symptoms characteristic of the disease by using commercially available equipment or equipment. Treatment can be resumed upon recurrence of the disease.
  • the present invention also provides a pharmaceutical pack or kit comprising one or more containers filled with one or more components of the pharmaceutical composition of the present invention.
  • a notice in the form of a governmental body regulating the manufacture, use or sale of pharmaceuticals or biological products may optionally accompany such containers, and this notice may include the manufacture, use or sale of the product for use in humans. Represents approval by government agencies.
  • the present invention provides a peptide comprising a bioactive peptide and at least one amino acid not derived from the bioactive peptide.
  • the bioactive peptide contained in this peptide is preferably not recognized by ubiquitin. Peptides that are not recognized by ubiquitin will undergo degradation by various peptidases in the body.
  • a peptide containing an amino acid that is not derived from a bioactive peptide is unexpected.
  • Have a sustained release effect. Therefore, the present invention is useful for providing a sustained-release agent of a physiologically active peptide affected by peptidase (such as not affected by ubiquitin).
  • the present invention is used to achieve a sustained release effect of a bioactive peptide having a size of less than 35 kDa.
  • the size of the bioactive peptide used in the present invention is usually 30 kDa or less, preferably a molecular weight of 20 kDa or less, and more preferably 10 kDa or less. It is understood that it is possible.
  • the amino acid length of the bioactive peptide to be included in the present invention is usually 300 amino acids or less, preferably 200 amino acids or less, more preferably 100 amino acids or less, and further more preferably , 50 amino acids or less, and it is understood that the length may be any length as long as it retains the physiological activity.
  • the present invention preferably includes a continuous structure of at least two or more hydrophobic amino acids.
  • hydrophobic amino acid refers to a substance having a hydrophobicity index (described in Kyte and Doolittle, J. Mol. Biol., 157, 105-132 (1982)! Of these, those excluding polar and neutral amino acids (natural amino acids such as Ser, Thr, Tyr and Cys).
  • Advantages of adding a sequence containing a continuous sequence of hydrophobic amino acids are as follows. That is, since a sequence of continuous hydrophobic amino acids is not normally present in a living body, adding a sequence containing such a sequence of hydrophobic amino acids to the terminus causes the entire peptide to be degraded by peptidase. Receiving and delaying their disappearance Is one of the advantages.
  • the amino acids to be included in the peptide of the present invention advantageously include at least one glycine.
  • Glycine has little effect on bioactivity and has little effect on the three-dimensional structure.
  • Glycine is also a force that has been revealed by the present invention to be degraded by peptidases present in the living body, so that the activity of the actual bioactive peptide is exerted.
  • Glycine also has the advantage of not reducing the water solubility.
  • the amino acids to be included in the peptide of the present invention are bound to at least one selected from the group consisting of C-terminal and N-terminal forces of a bioactive peptide.
  • the amino acid to be contained in the peptide of the present invention is bound to both the C-terminus and the N-terminus of the bioactive peptide.
  • the amino acid to be contained in the peptide of the present invention is bound to both the C-terminus and the N-terminus of the bioactive peptide.
  • the amino acids to be included in the peptide of the present invention preferably include a plurality of glycines. It has been confirmed that the inclusion of a plurality of glycines enhances the sustained release effect as shown in the examples below. More preferably, the amino acid to be included in the peptide of the present invention contains 3 to 10, or 3 to 6, glycines.
  • the amino acids to be included in the peptide of the present invention include a sequence different from the natural sequence of the bioactive peptide.
  • Such peptides are non-naturally occurring and exhibit a more sustained release effect than naturally occurring peptides.
  • leader sequence is a naturally occurring form 1S Peptides of the invention have a more sustained release effect than forms with such leader sequence It is understood that.
  • the bioactive peptide to be included in the peptide of the present invention has an effect selected from the group consisting of angiogenesis, angiogenesis inhibition, bone regeneration, anti-inflammatory action (integrin inhibition), diabetes treatment, analgesic action, and anti-dementia. Is used. Specific examples of such a peptide include, for example, a laminin-derived peptide (having angiogenesis;
  • Specific amino acid sequences of such a physiologically active peptide include peptide SWYGLR, peptide SIKVAV (SEQ ID NO: 19), peptide, IKVAV (laminin-derived peptide) (SEQ ID NO: 20), peptide YIGSR (laminin-derived peptide) ( SEQ ID NO: 7), peptide ALKRQ GRTLYGFGG (osteoproliferative peptide; OGP) (SEQ ID NO: 22), peptide GDGRHD L (integrin inhibitor) (SEQ ID NO: 23), peptide FVNQHLCGSHLVEALYLVC
  • Peptide YGGFM Metal—Enkehuarin
  • YGGFL Leu—Enkehuarin
  • ICCADELGC F VGTAEALRCQ EENYLPSPCQ SGQRPGS DQAGS Vasopressin
  • the bioactive peptide to be included in the peptide of the present invention does not have an undesired activity.
  • the peptide is preferably as short as possible as long as the desired activity is maintained. Having a long array also increases the likelihood of another effect.
  • the half-life in vivo is generally within 2 months, preferably within 1 month, more preferably within 1 month. Preferably, it is within 15 days, within 7 days, more preferably within 2 days.
  • the present invention makes it possible to use such a peptide, which usually has a short half-life and cannot be used as a medicament, as a medicament by utilizing the sustained release effect.
  • the present invention provides a pharmaceutical composition comprising the peptide of the present invention. It is understood that such pharmaceutical compositions may include any other ingredients, carriers, and the like.
  • the pharmaceutical composition of the present invention may further contain an additional sustained release component.
  • sustained release component include, but are not limited to, ribosomes and the like.
  • the present invention relates to a composition
  • a composition comprising at least two peptides comprising the amino acid sequence of a bioactive peptide, wherein at least one of the peptides comprises at least one amino acid not derived from the bioactive peptide.
  • a composition comprising: This composition contains at least one kind of the peptide of the present invention, and it has been found that the peptidase resistance, metabolism, etc. are different for each kind, and that the combination administration has a sustained release effect. confirmed. As far as the inventors of the present invention have achieved the sustained release effect based on such an idea, the present invention has never been so far. Therefore, it can be said that the present invention has a remarkable effect.
  • the amino acid portion of the peptide to be included in the composition of the present invention has at least two types of amino acids having different numbers of amino acids, preferably at least three types, and more preferably at least three types. It is understood that there are 4, 5, 6, 7, 8, 9, 10, and more.
  • the amino acid portion of the peptide to be included in the composition of the present invention includes at least two types of amino acids differing in the position at which the amino acid is included, and preferably includes at least three types of amino acids, It is understood that more preferably at least 4, 5, 6, 7, 8, 9, 9, 10 or more.
  • the peptide to be included in the composition of the present invention is present in at least two, more preferably at least three compositions.
  • at least one of the peptides may be a bioactive peptide having a natural sequence. It has been confirmed in the present invention that the inclusion of three types achieves a sustained release effect that is more remarkable (for example, three to five times or more) than the effect achieved in the two types. Thus, it may be significantly advantageous for the present invention to include more than two, more than two, or more peptides.
  • composition of the present invention can take any of the forms described herein above.
  • a peptide derived from two or more types of bioactive peptides may be contained in the composition.
  • Such compositions are a desirable form when trying to achieve more than one bioactive effect.
  • the compositions of the present invention can be used to achieve a synergistic effect of two or more bioactive peptides.
  • composition of the present invention may further contain other components such as a sustained release component.
  • the present invention provides a method for sustained release of a bioactive peptide, comprising: A) a peptide comprising a bioactive peptide and at least one amino acid not derived from the bioactive peptide; Administering to a subject for which a sustained release is contemplated.
  • a sustained release is contemplated.
  • the peptides used herein can use any form as described herein above. It is also understood that the administration and the subject can also take any of the forms described herein.
  • the present invention relates to a method for sustained release of a bioactive peptide, comprising: A) a peptide comprising a bioactive peptide and at least one amino acid not derived from the bioactive peptide. Administering a composition to a subject for which sustained release is intended, wherein the composition comprises a plurality of the amino acids in the peptide. It is understood that the compositions used herein can use any form as described herein above. Also for administration and subject It is understood that any form described in this specification can be adopted.
  • the present invention relates to a method for producing a sustained-release formulation for producing a bioactive effect, comprising a bioactive peptide and at least one amino acid not derived from the bioactive peptide.
  • a sustained-release formulation for producing a bioactive effect comprising a bioactive peptide and at least one amino acid not derived from the bioactive peptide.
  • amino acid sequence of the synthesized peptide is shown below.
  • SEQ ID NO: 1 SVVYGLR
  • SEQ ID NO: 2 GGGGGG SVVYGLR (both in this specification and G6!)
  • SEQ ID NO: 3 SVVYGLR-GGGGGG
  • SEQ ID NO: 4 GGG-SVVYGLR-GGG (both in this specification and WG! / SEQ ID NO: 5) GGG-SVVYGLR
  • Rat vascular endothelial cells were three-dimensionally opened in the presence of the peptide of the present invention synthesized in Example 1.
  • synthesized peptides those containing SEQ ID NOS: 1, 2 and 4 were used singly or in combination of any two of them and combinations containing all three kinds.
  • 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 10 gZml, and cultured in a carbon dioxide incubator for 14 days. Controls were performed without factor (1) and with the protein VEGF (+), previously known as an angiogenic factor.
  • the cultured cells were observed under a microscope, and 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. Further, when the lumen was observed with an electron microscope at a magnification of 7000 times, 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.
  • Example 3 Confirmation of In Vivo Physiological Activity (Angiogenic Action) by DAS Atssay A solution was prepared by dissolving a peptide at a concentration of lOngZ ml in DMEM (Dulbecco's modified Eagle medium) used as a cell culture medium. Diameter 0. 45 mm Millipore filter the upper and lower cylinder (Millipore, USA) busy at, in the constructed microcell mice (BALBZc AnNCrj, 6 weeks old, female; CLEA Japan) after filling the back of microcells A peptide solution such as the peptide of the present invention and a VEGF solution were injected therein. As a control, phosphate buffer (PBS) alone containing no peptide was also injected (control). Five days later, the state of the tissue around the microcell was observed with a microscope. The schematic diagram is shown in FIG.
  • DMEM Dynabecco's modified Eagle medium
  • FIG. 3 shows the state of angiogenesis when peptide SWYGLR (SEQ ID NO: 1) alone was used.
  • Fig. 4 shows the state of angiogenesis in the case of 6G alone (day 5).
  • Figure 5 shows the state of angiogenesis (day 5) in the case of WG alone.
  • FIG. 6 shows the state of angiogenesis (day 5) in the case of the combination of peptide SWYGLR and WG.
  • Figure 7 shows the state of angiogenesis (day 5) in the case of the combination of G6 and the peptide SWYGLR.
  • Figure 8 shows the state of angiogenesis in the combination of G6 and WG (day 5).
  • Figure 9 shows the angiogenesis of the three combinations (day 5).
  • FIG. 10 shows the angiogenesis of the combination of G6 and W10 on day 10.
  • FIG. 11 shows the state of angiogenesis for the three combinations on day 10.
  • Fig. 3- As is clear from the photograph shown in L1, even when the sustained release structure is used, the three effects of SWYGL R, namely, the angiogenesis and vascular network forming ability are exhibited. Was revealed. This is also evident from the remarkable appearance of the newly formed blood vessels, and the newly formed blood vessels are beginning to renew and begin to reticulate. Therefore, it became clear that the peptide of the present invention can be effective in retaining a plurality of activities in a peptide having a plurality of activities.
  • Score 2 100 or more and less than 125
  • Figure 12 shows the results of the evaluation of the vascular network evaluation.
  • the state of angiogenesis in a tissue was observed with a stereoscopic microscope (Olympus, SZX12, Japan), and the obtained image was analyzed using Photoshop (registered trademark) (Adob e, Japan), the reading network ability was scored as follows.
  • Nwl Network formation Angiogenesis is observed as described above, but 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 the side branch on the ladder is engaged.
  • Nw3 The network is in its final stage, and the ladder upper branch has further extended the side branch.
  • Nw4 Network maturation stage, showing a wide range of neovascular plexus.
  • Example 4 Another bioactive peptide (laminin-derived angiogenesis inhibitory peptide))
  • a bioactive peptide having an angiogenesis inhibitory action SEQ ID NO: 7
  • a modified product thereof using amino acid-adhesive caspase was prepared. It was produced according to Example 1.
  • the fabricated ones are as follows.
  • amino acid sequence of the synthesized peptide is shown below.
  • SEQ ID NO: 7 Native YIGSR
  • SEQ ID NO: 8 GGGGGG— YIGSR
  • SEQ ID NO: 9 YIGSR-GGGGGG
  • SEQ ID NO: 10 GGG— YIGSR— GGG
  • SEQ ID NO: 12 YIGSR—GGG
  • Example 1 The same bioactive peptide as in Example 1 was used.
  • a variant in which tyrosine was changed to phenalanine was also tested.
  • SEQ ID NO: 1 SVVYGLR
  • SEQ ID NO: 14 AAAAAAA SWYGLR
  • SEQ ID NO: 15 SVVYGLR AAAAAA
  • SEQ ID NO: 16 AAA SWYGLR AAA
  • SEQ ID NO: 17 AAA SWYGLR
  • SEQ ID NO: 18 SVVYGLR -AAAo
  • the peptide of the present invention can be used for biosubstitute materials such as artificial bones, for implanting artificial organs in the body, and for repairing organs.
  • biosubstitute materials such as artificial bones, for implanting artificial organs in the body, and for repairing organs.
  • FreAlagin AD type manufactured by Miyagi Chemical Industry Co., Ltd., molecular weight 2,000 to 20,000
  • the FreAlagin AD type has been approved for clinical use.
  • the bone substitute is attached to the gelatin-binding peptide as follows. Briefly, 100 mg of FreAlagin AD (carrier protein) was dissolved in 2.5 mL of MilliQ water. 1.20-1.32 mg of the peptide of the present invention was dissolved in 1 mL of 0.1 M phosphate buffer ( ⁇ 7.0), and added to the carrier protein under ice-cooling. Daltaraldehyde (25% solution Was diluted 10-fold with 0.1 M phosphate buffer (pH 7.0), 0.15 mL was added dropwise to the resulting mixture (4 ° C), and the reaction mixture was added at 3 ° C at 3 ° C. Stirred for 4 hours. As a result, the amino group at the N-terminus of the peptide having achieved the sustained release effect and the amino group of the carrier protein are covalently bonded. By implanting this in place of bone, it becomes possible to form a vascular network around it.
  • a physiologically active peptide having an angiogenic action (SEQ ID NO: 19) and another physiologically active peptide (SEQ ID NO: 20), and their modified forms with amino acid-added syrup were prepared according to Example 1. did.
  • the produced ones are as follows.
  • the amino acid sequence of the synthesized peptide is shown below.
  • SEQ ID NO: 19 native SIKVAV
  • SEQ ID NO: 27 GGGGGG SIKVAV
  • SEQ ID NO: 28 SIKVAV -GGGGGG
  • SEQ ID NO: 29 GGG SIKVAV GGG
  • SEQ ID NO: 30 GGG SIKVAV
  • SEQ ID NO: 20 Natural type IKVAV
  • SEQ ID NO: 32 GGGGGG -IKVAV
  • SEQ ID NO: 34 GGG-IKVAV -GGG
  • SEQ ID NO: 36 IKVAV -GGG
  • a physiologically active peptide having a bone regeneration action (SEQ ID NO: 22) and a variant obtained by adding an amino acid thereto were produced according to Example 1.
  • the produced ones are as follows.
  • the amino acid sequence of the synthesized peptide is shown below.
  • SEQ ID NO: 22 native ALKRQGRTLYGFGG
  • SEQ ID NO: 37 GGGGGG-ALKRQGRTLYGFGG
  • SEQ ID NO: 38 ALKRQGRTLYGFGG GGGGGG
  • SEQ ID NO: 39 GGG-ALKRQGRTLYGFGG GGG
  • SEQ ID NO: 40 GGG-ALKRQGRTLYGFGG
  • SEQ ID NO: 41 ALKRQGRTLYGFGG-GGG
  • Example 1 The sustained release effect was confirmed using an amino acid different from glycine.
  • the following synthetic peptides were synthesized according to Example 1. The same bioactive peptide was used as in Example 1.
  • SEQ ID NO: 1 SVVYGLR
  • SEQ ID NO: 42 GLGLGL-SWYGLR
  • SEQ ID NO: 43 SVVYGLR-GLGLGL
  • SEQ ID NO: 44 GLG SWYGLR -GLG
  • SEQ ID NO: 45 GLG-SWYGLR
  • SEQ ID NO: 46 SVVYGLR -GLG
  • SEQ ID NO: 48 SIKVAV-GLGLGL
  • SEQ ID NO: 49 GLG-SIKVAV -GLG
  • SEQ ID NO: 50 GLG SIKVAV
  • SEQ ID NO: 51 SIKVAV-GLG
  • SEQ ID NO: 52 GLGLGL -IKVAV
  • SEQ ID NO: 53 IKVAV-GLGLGL
  • SEQ ID NO: 54 GLG-IKVAV-GLG
  • SEQ ID NO: 56 IKVAV-GLG
  • the osteogenic peptide can achieve a remarkably sustained release effect by the inclusion of other kinds of amino acid-added syrup and plural kinds of peptides according to the present invention.
  • the present invention is useful in any (veterinary, pharmaceutical, medical, dentistry, etc.) utilizing a bioactive peptide.
  • the present invention is also useful for treating lifestyle-related diseases (eg, myocardial infarction, cerebral infarction, ischemic diseases such as obstructive aortic sclerosis). It is understood that the present invention achieves a remarkable effect in the field of medicine because it achieves a long-term effect with a single administration.

Abstract

It is aimed at developing a method and a system for achieving drug delivery control by using a physiologically active peptide. It has been found out that an unexpected sustained release effect is achieved by adding an amino acid such as glycine to a physiologically active peptide and thus a sustained release system with the use of the amino acid addition has been established. It has been further found out that a composition containing a plural number of modified peptides of the above type has an extremely remarkable sustained release effect. Thus, it is intended to provide a peptide comprising a physiologically active peptide together with at least one amino acid not originating in this physiologically active peptide; and a composition containing at least two types of physiologically active peptide amino acid sequences wherein at least one of them contains at least one amino acid not originating in this physiologically active peptide.

Description

明 細 書  Specification
ペプチド改変を利用したドラッグデリバリーシステム  Drug delivery system using peptide modification
技術分野  Technical field
[0001] 本発明は、医薬の分野にある。より詳細には、本発明は、ドラッグデリバリーシステム に関する。  [0001] The present invention is in the field of medicine. More particularly, the present invention relates to a drug delivery system.
背景分野  Background fields
[0002] 種々の活性ペプチドの報告があり、種々の領域において医薬として利用され始め ている。  [0002] There have been reports of various active peptides, and they have begun to be used as medicines in various fields.
[0003] 医薬品を有効に治療すべき部位に送達するためのシステム、いわゆる、ドラッグデリ ノリーシステムもまた、開発されてきた。  [0003] Systems for effectively delivering pharmaceuticals to the site to be treated, so-called drug delivery systems, have also been developed.
[0004] ゼラチンを使用したシステムが使用されている力 徐放に限界があるという問題があ り、最近はゼラチンを使用したシステムに対する限界が囁かれて!/ヽる。 [0004] Gelatin-based systems are used There is a problem in that there is a limit to sustained release, and recently the limitations on gelatin-based systems have been whispered!
[0005] ゼラチンなどの他成分を使用したシステム（例えば、特表 2003-519194など）は、活 性ペプチドの他に別個に成分を添加することを特徴としており、その成分自体の特性 およびその挙動も制御する必要があることなどから、望ましくな 、結果を生むこともあ る。特にゼラチンを用いた系では、安全性に問題があることが指摘されている。例え ば以下の 3点などが問題視され得る。まず、（1)副反応の問題:分解したゼラチン成 分が生物学的に機能 (例えば、インテグリン結合など)を有し得ることが多ぐそれが 引き起こす副反応が問題となり得る。（2)代謝性の問題:生体内での代謝がどのよう に行われるか未解明であり、残存ゼラチンが長期的に生体にどのような影響を与えう る力未解明な点が問題点として挙げられる。さらに、（3)生体適合性の問題:使用す るゼラチンのタイプ毎に、その生体適合性を検討する必要があることが別の問題点と して指摘される。 [0005] Systems using other components such as gelatin (for example, JP-T-2003-519194) are characterized by adding components separately in addition to the active peptide. May need to be controlled, and may produce undesirable results. Particularly, it has been pointed out that a system using gelatin has a problem in safety. For example, the following three points can be regarded as problems. First, (1) the problem of side reactions: Degraded gelatin components often have biological functions (eg, integrin binding, etc.), and the side reactions caused by them can be problematic. (2) Metabolic problems: It is unclear how metabolism in the living body is performed, and the unclear power of how residual gelatin can affect the living body in the long term is a problem. No. In addition, (3) Biocompatibility issues: Another issue is that it is necessary to study the biocompatibility of each type of gelatin used.
[0006] ペプチド自体に化学修飾を施す方法も提唱されて!、るが、ペプチド自体の改変は 、生理活性への影響が大きなことから、あまり好ましい例ではない。  [0006] A method of chemically modifying the peptide itself has also been proposed! However, the modification of the peptide itself is not a very preferable example because of its great influence on the physiological activity.
[0007] また、リボソームなどの別の製剤成分を利用する方法もある力 ー且リボソームが溶 解すると、含まれるすべての成分が溶解することから、制御の精度に欠ける。 [0008] また、特許文献 1は、 GAリピートを用いたプロテア一ゼカも保護する方法を開示す る。この出願は、ュビキチンを介するプロテアーゼ分解系からタンパク質を保護するこ とを目的としており、 GAリピートを付加する相手は、ュビキチンが認識するほどの長さ のタンパク質が意図されている。従って、分子量の小さなペプチドなどが生体内で保 護されるメカニズムは提供せず、そのような指針も開示も示唆もされていない。 [0007] In addition, there is also a method in which another pharmaceutical ingredient such as ribosome is used. When the ribosome is dissolved, all the components contained therein are dissolved, so that the control accuracy is lacking. [0008] Further, Patent Document 1 discloses a method for protecting proteazeca using a GA repeat. The purpose of this application is to protect a protein from a ubiquitin-mediated protease degradation system, and a partner to which GA repeat is added is a protein long enough to be recognized by ubiquitin. Therefore, it does not provide a mechanism for protecting small-molecular-weight peptides and the like in vivo, and does not provide such guidelines, discloses or suggests them.
[0009] そのような中、生理的安定性を保ちつつ、比較的小さな分子量のペプチドなどの生 理活性ペプチドを用いて、徐放剤を調製するための技術の提供が待たれて 、る。 特許文献 1： W098Z22577号  [0009] Under such circumstances, there is a need to provide a technique for preparing a sustained-release agent using a physiologically active peptide such as a peptide having a relatively small molecular weight while maintaining physiological stability. Patent Document 1: W098Z22577
発明の開示  Disclosure of the invention
発明が解決しょうとする課題  Problems to be solved by the invention
[0010] このように、当該分野において、生理活性ペプチドを用いて、薬物送達の制御を達 成するための方法およびシステムの開発が待たれており、本発明では、その課題を 解決することを目的とする。 [0010] As described above, in this field, development of a method and system for achieving control of drug delivery using a bioactive peptide has been awaited, and the present invention aims to solve the problem. Aim.
課題を解決するための手段  Means for solving the problem
[0011] 本発明は、グリシンのようなアミノ酸を生理活性ペプチドに加えることによって、予想 外に徐放効果が達成されたことを見出し、このようなアミノ酸の付加を用いた徐放シス テムを完成させた。特に、このような改変型ペプチドを複数種類含む組成物にもきわ めて顕著な徐放効果があったことは、特筆に価する。 [0011] The present invention has found that an unexpected sustained release effect has been achieved by adding an amino acid such as glycine to a bioactive peptide, and completed a sustained release system using the addition of such an amino acid. I let it. In particular, it is noteworthy that a composition containing a plurality of such modified peptides had a remarkable sustained release effect.
[0012] 従って、本発明は、以下を提供する。  Accordingly, the present invention provides the following.
(1)生理活性ペプチドと、上記生理活性ペプチドに由来しない少なくとも 1つのアミノ 酸とを含む、ペプチド。  (1) A peptide comprising a bioactive peptide and at least one amino acid not derived from the bioactive peptide.
(2)上記生理活性ペプチドは、ュビキチンによって認識されない、項目 1に記載のぺ プチド。  (2) The peptide according to item 1, wherein the bioactive peptide is not recognized by ubiquitin.
(3)上記生理活性ペプチドは、分子量 30kDa以下である、項目 1に記載のペプチド  (3) The peptide according to item 1, wherein the physiologically active peptide has a molecular weight of 30 kDa or less.
(4)上記生理活性ペプチドは、分子量 20kDa以下である、項目 1に記載のペプチド (4) The peptide according to item 1, wherein the physiologically active peptide has a molecular weight of 20 kDa or less.
(5)上記生理活性ペプチドは、分子量 lOkDa以下である、項目 1に記載のペプチド (6)上記生理活性ペプチドは、 300アミノ酸長以下のペプチドまたはその改変体であ る、項目 1に記載のペプチド。 (5) The peptide according to item 1, wherein the bioactive peptide has a molecular weight of 10 kDa or less. (6) The peptide according to item 1, wherein the physiologically active peptide is a peptide having a length of 300 amino acids or less or a variant thereof.
(7)上記生理活性ペプチドは、 200アミノ酸長以下のペプチドまたはその改変体であ る、項目 1に記載のペプチド。  (7) The peptide according to item 1, wherein the physiologically active peptide is a peptide having a length of 200 amino acids or less or a variant thereof.
(8)上記生理活性ペプチドは、 100アミノ酸長以下のペプチドまたはその改変体であ る、項目 1に記載のペプチド。  (8) The peptide according to item 1, wherein the physiologically active peptide is a peptide having a length of 100 amino acids or less or a variant thereof.
(9)上記アミノ酸は、少なくとも 1つのグリシンを含む、項目 1に記載のペプチド。 (9) The peptide according to item 1, wherein the amino acid contains at least one glycine.
(10)上記アミノ酸は、上記生理活性ペプチドの C末端および N末端力もなる群より選 択される少なくとも 1つに結合される、項目 1に記載のペプチド。 (10) The peptide according to item 1, wherein the amino acid is bound to at least one selected from the group consisting of C-terminal and N-terminal forces of the bioactive peptide.
(11)上記アミノ酸は、上記生理活性ペプチドの C末端および N末端の両方に結合さ れる、項目 1に記載のペプチド。  (11) The peptide according to item 1, wherein the amino acid is bound to both the C-terminus and the N-terminus of the bioactive peptide.
(12)上記アミノ酸は、複数のグリシンを含む、項目 1に記載のペプチド。  (12) The peptide according to item 1, wherein the amino acid includes a plurality of glycines.
(13)上記アミノ酸は、グリシンを 3〜： L0個含む、項目 1に記載のペプチド。  (13) The peptide according to item 1, wherein the amino acid contains 3 to L0 glycines.
(14)上記アミノ酸は、上記生理活性ペプチドの天然の配列とは異なる配列を含む、 項目 1に記載のペプチド。  (14) The peptide according to item 1, wherein the amino acid includes a sequence different from a natural sequence of the bioactive peptide.
(15)上記アミノ酸は、上記生理活性ペプチドのリーダー配列の天然の配列とは異な る配列を含む、項目 1に記載のペプチド。  (15) The peptide according to item 1, wherein the amino acid includes a sequence different from a natural sequence of a leader sequence of the bioactive peptide.
(16)上記生理活性ペプチドは、血管新生、血管新生阻害、骨再生、抗炎症作用 (ィ ンテグリン阻害)、糖尿病治療、鎮痛作用、抗痴呆からなる群より選択される効果を有 する、項目 1に記載のペプチド。  (16) The above-mentioned physiologically active peptide has an effect selected from the group consisting of angiogenesis, angiogenesis inhibition, bone regeneration, anti-inflammatory action (integrin inhibition), diabetes treatment, analgesic action, and anti-dementia. The peptide according to any one of the above.
(17)上記生理活性ペプチドは、ペプチド SVVYGLR、ペプチド SIKVAV、ぺプチ ド IKVAV (ラミニン由来ペプチド）、ペプチド YIGSR (ラミニン由来ペプチド）、ぺプチ ド ALKRQGRTLYGFGG (骨原性増殖ペプチド； OGP)、ペプチド GDGRHDL (ィ ンテグリン阻害剤）、ペプチド FVNQHLCGSHL VEAL YLVCHERGFFYTPKT GIVEQCCTSICSLYQLENYCN (インスリン）、ペプチド YGGFM (Met—エンケ フアリン）、 YGGFL (Leu—エンケフアリン）、ペプチド MLARMLNTTI SACFLS LLAF SSACYFQNCP RGGKRAISDM ELRQCLPCGP GGKGRCFGP S ICCADELGCF VGTAEALRCQ EENYLPSPCQ SGQKPCGSGG R CAAVGICCS DESCVAEPEC HDGFFRLTRA REPSNATQLD GPAR ALLLRL VQLAGTRESV DSAKPRVY (バソプレシン）またはその改変体であ る、項目 1に記載のペプチド。 (17) The above physiologically active peptides include peptide SVVYGLR, peptide SIKVAV, peptide IKVAV (laminin-derived peptide), peptide YIGSR (laminin-derived peptide), peptide ALKRQGRTLYGFGG (osteogenic growth peptide; OGP), peptide GDGRHDL ( Integrin inhibitor), peptide FVNQHLCGSHL VEAL YLVCHERGFFYTPKT GIVEQCCTSICSLYQLENYCN (insulin), peptide YGGFM (Met-enkephalin), YGGFL (Leu-enkephalin), peptide MLARMLNTTI SACFLS LLAF SSACYFQNCP RGGCFGRPS S ICCADELGCF VGTAEALRCQ EENYLPSPCQ SGQKPCGSGG R CAAVGICCS DESCVAEPEC HDGFFRLTRA REPSNATQLD GPAR ALLLRL VQLAGTRESV DSAKPRVY (vasopressin) or a variant thereof, the peptide according to item 1.
(18)上記生理活性ペプチドは、所望されない活性を有しない、項目 1に記載のぺプ チド。  (18) The peptide according to item 1, wherein the bioactive peptide does not have an undesired activity.
(19)上記生理活性ペプチドは、ペプチド自体で存在する場合、生体内半減期が、 2 日以下である、項目 1に記載のペプチド。  (19) The peptide according to item 1, wherein the physiologically active peptide has a half-life in vivo of 2 days or less when the peptide itself is present.
(20)項目 1に記載のペプチドを含む、薬学的組成物。  (20) A pharmaceutical composition comprising the peptide according to item 1.
(21)さらなる徐放成分をさらに含む、項目 20に記載の薬学的組成物。  (21) The pharmaceutical composition according to item 20, further comprising an additional sustained release component.
(22)生理活性ペプチドのアミノ酸配列を含む少なくとも 2種のペプチド含む組成物 であって、上記ペプチドの少なくとも 1種は、上記生理活性ペプチドに由来しない少 なくとも 1つのアミノ酸を含む、組成物。  (22) A composition comprising at least two kinds of peptides containing the amino acid sequence of a physiologically active peptide, wherein at least one kind of the peptide contains at least one amino acid not derived from the physiologically active peptide.
(23)上記ペプチドは、少なくとも 2種力 上記アミノ酸を含み、上記アミノ酸の部分は 、アミノ酸数が異なる少なくとも 2種が存在する、項目 22に記載の組成物。  (23) The composition according to item 22, wherein the peptide includes at least two kinds of amino acids, and the amino acid portion includes at least two kinds of amino acids having different numbers of amino acids.
(24)上記ペプチドは、少なくとも 2種力 上記アミノ酸を含み、上記アミノ酸の部分は 、アミノ酸が含まれる位置が異なる少なくとも 2種が存在する、項目 22に記載の組成 物。  (24) The composition according to item 22, wherein the peptide includes at least two kinds of amino acids, and the amino acid portion includes at least two kinds of amino acids having different positions including amino acids.
(25)上記ペプチドは、少なくとも 3種が存在する、項目 22に記載の組成物。  (25) The composition according to item 22, wherein at least three kinds of the peptides are present.
(26)上記生理活性ペプチドは、ュビキチンによって認識されない、項目 22に記載の 組成物。  (26) The composition according to item 22, wherein the bioactive peptide is not recognized by ubiquitin.
(27)上記生理活性ペプチドは、分子量 30kDa以下である、項目 22に記載の組成 物。  (27) The composition according to item 22, wherein the bioactive peptide has a molecular weight of 30 kDa or less.
(28)上記生理活性ペプチドは、分子量 20kDa以下である、項目 22に記載の組成 物。  (28) The composition according to item 22, wherein the bioactive peptide has a molecular weight of 20 kDa or less.
(29)上記生理活性ペプチドは、分子量 lOkDa以下である、項目 22に記載の組成 物。  (29) The composition according to item 22, wherein the bioactive peptide has a molecular weight of 10 OkDa or less.
(30)上記生理活性ペプチドは、 300アミノ酸長以下のペプチドまたはその改変体で ある、項目 22に記載の組成物。 (30) The bioactive peptide is a peptide of 300 amino acids or less or a variant thereof. 23. The composition according to item 22, wherein
(31)上記生理活性ペプチドは、 200アミノ酸長以下のペプチドまたはその改変体で ある、項目 22に記載の組成物。  (31) The composition according to item 22, wherein the physiologically active peptide is a peptide having a length of 200 amino acids or less or a variant thereof.
(32)上記生理活性ペプチドは、 100アミノ酸長以下のペプチドまたはその改変体で ある、項目 22に記載の組成物。  (32) The composition according to item 22, wherein the physiologically active peptide is a peptide having a length of 100 amino acids or less or a variant thereof.
(33)上記アミノ酸は、少なくとも 1つのグリシンを含む、項目 22に記載の組成物。 (33) The composition according to item 22, wherein the amino acid includes at least one glycine.
(34)上記アミノ酸は、上記生理活性ペプチドの C末端および N末端力もなる群より選 択される少なくとも 1つに結合される、項目 22に記載の組成物。 (34) The composition according to item 22, wherein the amino acid is bound to at least one selected from the group consisting of C-terminal and N-terminal forces of the bioactive peptide.
(35)上記アミノ酸は、上記生理活性ペプチドの C末端および N末端の両方に結合さ れる、項目 22に記載の組成物。  (35) The composition according to item 22, wherein the amino acid is bound to both the C-terminus and the N-terminus of the bioactive peptide.
(36)上記アミノ酸は、複数のグリシンを含む、項目 22に記載の組成物。  (36) The composition according to item 22, wherein the amino acid includes a plurality of glycines.
(37)上記アミノ酸は、グリシンを 3〜10個含む、項目 22に記載の組成物。  (37) The composition according to item 22, wherein the amino acid contains 3 to 10 glycines.
(38)上記アミノ酸は、グリシンを 3〜6個含む、項目 22に記載の組成物。  (38) The composition according to item 22, wherein the amino acid contains 3 to 6 glycines.
(39)上記アミノ酸は、上記生理活性ペプチドの天然の配列とは異なる配列を含む、 項目 22に記載の組成物。  (39) The composition according to item 22, wherein the amino acid comprises a sequence different from a natural sequence of the bioactive peptide.
(40)上記アミノ酸は、上記生理活性ペプチドのリーダー配列の天然の配列とは異な る配列を含む、項目 22に記載の組成物。  (40) The composition according to item 22, wherein the amino acid comprises a sequence different from a natural sequence of the leader sequence of the bioactive peptide.
(41)上記組成物は、少なくとも 1種の天然の配列を有する生理活性ペプチドを含む 、項目 22に記載の組成物。  (41) The composition according to item 22, wherein the composition comprises a physiologically active peptide having at least one kind of natural sequence.
(42)上記生理活性ペプチドは、血管新生、血管新生阻害、骨再生、抗炎症作用 (ィ ンテグリン阻害)、糖尿病治療、鎮痛作用、抗痴呆からなる群より選択される効果を有 する、項目 22に記載の組成物。  (42) The physiologically active peptide has an effect selected from the group consisting of angiogenesis, angiogenesis inhibition, bone regeneration, anti-inflammatory action (integrin inhibition), diabetes treatment, analgesic action, and anti-dementia. A composition according to claim 1.
(43)上記生理活性ペプチドは、ペプチド SVVYGLR、ペプチド SIKVAV、ぺプチ ド IKVAV (ラミニン由来ペプチド）、ペプチド YIGSR (ラミニン由来ペプチド）、ぺプチ ド ALKRQGRTLYGFGG (骨原性増殖ペプチド； OGP)、ペプチド GDGRHDL (ィ ンテグリン阻害剤）、ペプチド FVNQHLCGSHL VEAL YLVCHERGFFYTPKT GIVEQCCTSICSLYQLENYCN (インスリン）、ペプチド YGGFM (Met—エンケ フアリン）、ペプチド YGGFL (Leu—エンケフアリン）、ペプチド MLARMLNTTI S ACFLSLLAF SSACYFQNCP RGGKRAISDM ELRQCLPCGP GGKG RCFGPS ICCADELGCF VGTAEALRCQ EENYLPSPCQ SGQKPCG SGG RCAAVGICCS DESCVAEPEC HDGFFRLTRA REPSNATQLD GPARALLLRL VQLAGTRESV DSAKPRVY (バソプレシン）またはその改 変体である、項目 22に記載の組成物。 (43) The physiologically active peptides include peptide SVVYGLR, peptide SIKVAV, peptide IKVAV (laminin-derived peptide), peptide YIGSR (laminin-derived peptide), peptide ALKRQGRTLYGFGG (osteogenic growth peptide; OGP), peptide GDGRHDL ( Integrin inhibitor), peptide FVNQHLCGSHL VEAL YLVCHERGFFYTPKT GIVEQCCTSICSLYQLENYCN (insulin), peptide YGGFM (Met-enkephalin), peptide YGGFL (Leu-enkephalin), peptide MLARMLNTTI S ACFLSLLAF SSACYFQNCP RGGKRAISDM ELRQCLPCGP GGKG RCFGPS ICCADELGCF VGTAEALRCQ EENYLPSPCQ SGQKPCG SGG RCAAVGICCS DESCVAEPEC HDGFFRLTRA REPSNATQLD GPARALLLRL VQLAGTRESV DSAKPRVY in the composition of the item or the modified vasopressin in the item 22
(44)上記生理活性ペプチドは、所望されない活性を有しない、項目 22に記載の糸且 成物。  (44) The thread composition according to item 22, wherein the bioactive peptide does not have an undesired activity.
(45)上記生理活性ペプチドは、ペプチド自体で存在する場合、生体内半減期が、 2 日以下である、項目 22に記載の組成物。  (45) The composition according to item 22, wherein the physiologically active peptide has a half-life in vivo of 2 days or less when present as the peptide itself.
(46)上記生理活性ペプチドは、 2種類以上が存在する、項目 22に記載の組成物。 (46) The composition according to item 22, wherein two or more kinds of the bioactive peptides are present.
(47)さらなる徐放成分をさらに含む、項目 22に記載の組成物。 (47) The composition according to item 22, further comprising an additional sustained-release component.
(48)生理活性ペプチドを、徐放させる方法であって、  (48) a method for sustained release of a physiologically active peptide,
Α)生理活性ペプチドと、上記生理活性ペプチドに由来しない少なくとも 1つのアミノ 酸とを含む、ペプチドを、徐放が企図される被検体に投与する工程、  Α) a step of administering a peptide comprising a physiologically active peptide and at least one amino acid not derived from the physiologically active peptide to a subject intended for sustained release;
を包含する、方法。  A method comprising:
(49)生理活性ペプチドを、徐放させる方法であって、  (49) A method for sustainedly releasing a physiologically active peptide,
Α)生理活性ペプチドと、上記生理活性ペプチドに由来しない少なくとも 1つのアミノ 酸とを含む、ペプチドを含む組成物を、徐放が企図される被検体に投与する工程で あって、上記組成物は、上記ペプチド中の上記アミノ酸が複数種類存在する、工程、 を包含する、方法。  Ii) a step of administering a peptide-containing composition comprising a bioactive peptide and at least one amino acid not derived from the bioactive peptide to a subject intended for sustained release, wherein the composition comprises: A plurality of the amino acids in the peptide.
(50)生理活性ペプチドと、上記生理活性ペプチドに由来しない少なくとも 1つのアミ ノ酸とを含む、ペプチドの、生理活性作用を発揮させる徐放処方物の製造のための、 使用。  (50) Use of a peptide containing a bioactive peptide and at least one amino acid not derived from the bioactive peptide for the production of a sustained-release formulation exhibiting a bioactive action.
[0013] 以下に、本発明の好ましい実施形態を示すが、当業者は本発明の説明および当該 分野における周知慣用技術力もその実施形態などを適宜実施することができ、本発 明が奏する作用および効果を容易に理解することが認識されるべきである。  Hereinafter, preferred embodiments of the present invention will be described. However, those skilled in the art can appropriately implement the embodiments and the like using the description of the present invention and well-known common technical skills in the art, and the effects and advantages of the present invention can be achieved. It should be appreciated that the effect is easily understood.
発明の効果  The invention's effect
[0014] 本発明は、生理活性ペプチドを用いた徐放剤が提供される。この徐放剤は、生理 活性ペプチドの活性が保持され、生体適合性を有しつつ、徐放効果が達成された。 この技術は、任意の生理活性ペプチドの応用することができることが理解される。特 に、本発明では、生物学的に副反応のない (機能しない)ような配列（のみ)を含むよ うにも付加すべきアミノ酸配列として設計することができることから、安全性をより高め た徐放剤の設計が可能になる。これは、従来の生体ペプチドを用いた系では実現で きなかった自在な設計ができると!、う効果も奏する。 [0014] The present invention provides a sustained-release agent using a physiologically active peptide. This sustained release agent The sustained release effect was achieved while maintaining the activity of the active peptide and having biocompatibility. It is understood that this technique can be applied to any bioactive peptide. In particular, the present invention can be designed as an amino acid sequence to be added so as to include (only) a sequence that does not have a biological side reaction (does not function). The design of the release agent becomes possible. This is also effective if it is possible to design freely which could not be realized with the conventional system using biological peptides.
図面の簡単な説明  Brief Description of Drawings
[0015] [図 1]図 1は、細胞同士を接着させてそれらの間に管腔を形成する作用の模式図を示 す。  FIG. 1 shows a schematic diagram of the action of adhering cells to form a lumen between them.
[図 2]図 2は、実施例 3において、 5日後に、 DASアツセィによってマイクロセル周辺の 組織の様子を顕微鏡で観察した図を示す。  [FIG. 2] FIG. 2 is a diagram showing a state of a tissue around a microcell observed with a microscope by a DAS assay 5 days later in Example 3.
[図 3]図 3は、ペプチド SWYGLR (配列番号 1)単独を用いた場合の血管新生の様 子（5日目）を示す。  FIG. 3 shows the state of angiogenesis (day 5) when peptide SWYGLR (SEQ ID NO: 1) alone was used.
[図 4]図 4は、 6G単独の場合の血管新生の様子（5日目）を示す。  FIG. 4 shows the state of angiogenesis in the case of 6G alone (day 5).
[図 5]図 5は、 WG単独の場合の血管新生の様子（5日目）を示す。  [Fig. 5] Fig. 5 shows the state of angiogenesis in the case of using WG alone (day 5).
[図 6]図 6は、ペプチド SWYGLRと WGとの組み合わせの場合の血管新生の様子（ [Figure 6] Figure 6 shows the state of angiogenesis in the case of the combination of the peptides SWYGLR and WG (
5日目）を示す。 Day 5).
[図 7]図 7は、 G6とペプチド SWYGLRとの組み合わせの場合の血管新生の様子（5 日目）を示す。  FIG. 7 shows the state of angiogenesis (day 5) in the case of a combination of G6 and peptide SWYGLR.
[図 8]図 8は、 G6と WGとの組み合わせの場合の血管新生の様子（5日目）を示す。  FIG. 8 shows the state of angiogenesis (day 5) in the case of a combination of G6 and WG.
[図 9]図 9は、 3つの組み合わせの血管新生の様子（5日目）を示す。  [FIG. 9] FIG. 9 shows the state of angiogenesis of the three combinations (day 5).
[図 10]図 10は、 10日目の G6と W10との組み合わせの血管新生の様子を示す。  FIG. 10 shows the state of angiogenesis of the combination of G6 and W10 on day 10.
[図 11]図 11は、 10日目の 3つの組み合わせの場合の血管新生の様子を示す。  FIG. 11 shows the state of angiogenesis in the case of the three combinations on day 10.
[図 12]実施例 3の血管新生数の詳細な評価を示す。  FIG. 12 shows the detailed evaluation of the number of angiogenesis in Example 3.
配列表の説明  Description of Sequence Listing
[0016] 配列番号 1：ォステオポンチン由来の血管新生ペプチド SVVYGLR  [0016] SEQ ID NO: 1: Angiogenesis peptide SVVYGLR derived from osteopontin
配列番号 2：ォステオボンチン由来の血管新生ペプチド改変体 GGGGGG— SW YGLR (本明細書にぉ ヽて G6とも!/、う） 配列番号 3：ォステオボンチン由来の血管新生ペプチド改変体 SVVYGLR— GG GGGG SEQ ID NO: 2: Osteobontin-derived angiogenic peptide variant GGGGGG—SW YGLR (also referred to herein as G6!) SEQ ID NO: 3: Osteobontin-derived angiogenic peptide variant SVVYGLR—GG GGGG
配列番号 4：ォステオボンチン由来の血管新生ペプチド改変体 GGG— SVVYGL R-GGG (本明細書にぉ 、て WGとも!/、う）  SEQ ID NO: 4: Osteobontin-derived angiogenic peptide variant GGG—SVVYGL R-GGG (in this specification, both WG and WG! /)
配列番号 5：ォステオボンチン由来の血管新生ペプチド改変体 GGG— SVVYGL SEQ ID NO: 5: Modified angiogenesis peptide derived from osteobontin GGG—SVVYGL
R R
配列番号 6：ォステオボンチン由来の血管新生ペプチド改変体 SVVYGLR— GG G  SEQ ID NO: 6: modified angiogenesis peptide derived from osteobontin SVVYGLR—GGG
配列番号 7：ラミニン由来血管新生阻害ペプチド YIGSR  SEQ ID NO: 7: Laminin-derived angiogenesis inhibitory peptide YIGSR
配列番号 8 :ラミニン由来血管新生阻害ペプチドの改変体 GGGGGG— YIGSR 配列番号 9 :ラミニン由来血管新生阻害ペプチドの改変体 YIGSR— GGGGGG 配列番号 10 :ラミニン由来血管新生阻害ペプチドの改変体 GGG—YIGSR—GG G  SEQ ID NO: 8: variant of laminin-derived angiogenesis inhibitory peptide GGGGGG—YIGSR SEQ ID NO: 9: variant of laminin-derived angiogenesis inhibitory peptide YIGSR—GGGGGG SEQ ID NO: 10: variant of laminin-derived angiogenesis inhibitory peptide GGG—YIGSR—GG G
配列番号 11 :ラミニン由来血管新生阻害ペプチドの改変体 GGG—YIGSR 配列番号 12：ラミニン由来血管新生阻害ペプチドの改変体 YIGSR— GGG 配列番号 13：ォステオボンチン由来の血管新生ペプチド改変体 SVVFGLR 配列番号 14：ォステオポンチン由来の血管新生ペプチド改変体 AAAAAA— SV VYGLR  SEQ ID NO: 11: A variant of laminin-derived angiogenesis inhibitory peptide GGG-YIGSR SEQ ID NO: 12: A variant of laminin-derived angiogenesis inhibitory peptide YIGSR-GGG SEQ ID NO: 13: Osteovontin-derived variant of angiogenesis peptide SVVFGLR SEQ ID NO: 14: Osteopontin Modified angiogenic peptide derived from AAAAAA— SV VYGLR
配列番号 15：ォステオボンチン由来の血管新生ペプチド改変体 SVVYGLR— A AAAAA  SEQ ID NO: 15: Osteobontin-derived angiogenic peptide variant SVVYGLR—A AAAAA
配列番号 16：ォステオポンチン由来の血管新生ペプチド改変体 AAA— SVVYG LR-AAA  SEQ ID NO: 16: Angiogenesis peptide variant derived from osteopontin AAA—SVVYG LR-AAA
配列番号 17：ォステオポンチン由来の血管新生ペプチド改変体 AAA— SVVYG LR  SEQ ID NO: 17: Osteopontin-derived angiogenic peptide variant AAA—SVVYG LR
配列番号 18 :ォステオボンチン由来の血管新生ペプチド改変体 SVVYGLR— A AA  SEQ ID NO: 18: Modified angiogenesis peptide derived from osteobontin SVVYGLR—A AA
配列番号 19 :ラミニン由来血管新生ペプチド SIKVAV  SEQ ID NO: 19: Laminin-derived angiogenic peptide SIKVAV
配列番号 20：ラミニン由来血管新生ペプチド 'IKVAV 配列番号 21 :バソプレシン MLARMLNTTI SACFLSLLAF SSACYFQNC P RGGKRAISDM ELRQCLPCGP GGKGRCFGPS ICCADELGCF V GTAEALRCQ EENYLPSPCQ SGQKPCGSGG RCAAVGICCS DESC VAEPEC HDGFFRLTRA REPSNATQLD GPARALLLRL VQLAGTR ESV DSAKPRVY SEQ ID NO: 20: Laminin-derived angiogenic peptide 'IKVAV SEQ ID NO: 21: Vasopressin MLARMLNTTI SACFLSLLAF SSACYFQNC P RGGKRAISDM ELRQCLPCGP GGKGRCFGPS ICCADELGCF V GTAEALRCQ EENYLPSPCQ SGQKPCGSGG RCAAVGICCS DESC VAEPEC HDGFFRLTRA REPSNATQLD GPARALLLRL VQLPRV ES
配列番号 22：骨原性増殖ペプチド ALKRQGRTLYGFGG  SEQ ID NO: 22: Osteogenic growth peptide ALKRQGRTLYGFGG
配列番号 23：インテグリン阻害剤 GDGRHDL  SEQ ID NO: 23: Integrin inhibitor GDGRHDL
配列番号 24：インスリンペプチド FVNQHLCGSHL VEAL YLVCHERGFFYT PKTGIVEQCCTSICSLYQLENYCN  SEQ ID NO: 24: Insulin peptide FVNQHLCGSHL VEAL YLVCHERGFFYT PKTGIVEQCCTSICSLYQLENYCN
配列番号 25： Met—エンケファリン YGGFM  SEQ ID NO: 25: Met—Enkephalin YGGFM
配列番号 26 :Leu—エンケフアリン YGGFL  SEQ ID NO: 26: Leu—Enkephalin YGGFL
配列番号 27：ラミニン由来血管新生ペプチドの改変体 GGGGGG— SIKVAV 配列番号 28：ラミニン由来血管新生ペプチドの改変体 SIKVAV— GGGGGG 配列番号 29：ラミニン由来血管新生ペプチドの改変体 GGG— SIKVAV -GGG 配列番号 30：ラミニン由来血管新生ペプチドの改変体 GGG— SIKVAV 配列番号 31：ラミニン由来血管新生ペプチドの改変体 SIKVAV— GGG 配列番号 32：ラミニン由来血管新生ペプチドの改変体 GGGGGG— IKVAV 配列番号 33 :ラミニン由来血管新生ペプチドの改変体 IKVAV— GGGGGG 配列番号 34 :ラミニン由来血管新生ペプチドの改変体 GGG— IKVAV— GGG 配列番号 35 :ラミニン由来血管新生ペプチドの改変体 GGG— IKVAV 配列番号 36 :ラミニン由来血管新生ペプチドの改変体 IKVAV— GGG 配列番号 37：骨原性増殖ペプチドの改変体 GGGGGG— ALKRQGRTLYGFG G  SEQ ID NO: 27: A variant of laminin-derived angiogenesis peptide GGGGGG—SIKVAV SEQ ID NO: 28: A variant of laminin-derived angiogenesis peptide SIKVAV—GGGGGG SEQ ID NO: 29: A variant of laminin-derived angiogenesis peptide GGG—SIKVAV-GGG SEQ ID NO: 30 : Variant of laminin-derived angiogenic peptide GGG—SIKVAV SEQ ID NO: 31: Variant of laminin-derived angiogenic peptide SIKVAV—GGG SEQ ID NO: 32: Variant of laminin-derived angiogenic peptide GGGGGG—IKVAV SEQ ID NO: 33: Laminin-derived angiogenesis Modified peptide IKVAV-GGGGGG SEQ ID NO: 34: Modified laminin-derived angiogenic peptide GGG- IKVAV-GGG SEQ ID NO: 35: Modified laminin-derived angiogenic peptide GGG-IKVAV SEQ ID NO: 36: Modified laminin-derived angiogenic peptide IKVAV—GGG SEQ ID NO: 37: Variant of osteogenic growth peptide GGGGGG—ALKRQGRTLYGFGG
配列番号 38：骨原性増殖ペプチドの改変体 ALKRQGRTLYGFGG— GGGGG G  SEQ ID NO: 38: Variant of osteogenic growth peptide ALKRQGRTLYGFGG—GGGGGG
配列番号 39：骨原性増殖ペプチドの改変体 GGG— ALKRQGRTLYGFGG— G GG  SEQ ID NO: 39: Variant of osteogenic growth peptide GGG—ALKRQGRTLYGFGG—GGG
配列番号 40：骨原性増殖ペプチドの改変体 GGG— ALKRQGRTLYGFGG 配列番号 41：骨原性増殖ペプチドの改変体 ALKRQGRTLYGFGG— GGG 配列番号 42：ォステオポンチン由来の血管新生ペプチド改変体 GLGLGL— SW YGLR SEQ ID NO: 40: Variant of osteogenic growth peptide GGG—ALKRQGRTLYGFGG SEQ ID NO: 41: Modified osteogenic growth peptide ALKRQGRTLYGFGG—GGG SEQ ID NO: 42: Modified angiogenesis peptide derived from osteopontin GLGLGL—SW YGLR
配列番号 43：ォステオボンチン由来の血管新生ペプチド改変体 SVVYGLR— GL GLGL  SEQ ID NO: 43: Modified angiogenesis peptide derived from osteobontin SVVYGLR—GL GLGL
配列番号 44：ォステオポンチン由来の血管新生ペプチド改変体 GLG— SVVYGL R-GLG  SEQ ID NO: 44: Modified angiogenesis peptide derived from osteopontin GLG—SVVYGL R-GLG
配列番号 45：ォステオポンチン由来の血管新生ペプチド改変体 GLG— SVVYGL SEQ ID NO: 45: Osteopontin-derived modified angiogenic peptide GLG—SVVYGL
R R
配列番号 46：ォステオポンチン由来の血管新生ペプチド改変体 SVVYGLR— GL G  SEQ ID NO: 46: Modified angiogenesis peptide derived from osteopontin SVVYGLR—GLG
配列番号 47：ラミニン由来血管新生ペプチドの改変体 GLGLGL— SIKVAV 配列番号 48 :ラミニン由来血管新生ペプチドの改変体 SIKVAV— GLGLGL 配列番号 49：ラミニン由来血管新生ペプチドの改変体 GLG— SIKVAV- GLG 配列番号 50：ラミニン由来血管新生ペプチドの改変体 GLG— SIKVAV 配列番号 51：ラミニン由来血管新生ペプチドの改変体 SIKVAV— GLG 配列番号 52：ラミニン由来血管新生ペプチドの改変体 GLGLGL— IKVAV 配列番号 53 :ラミニン由来血管新生ペプチドの改変体 IKVAV— GLGLGL 配列番号 54：ラミニン由来血管新生ペプチドの改変体 GLG— IKVAV— GLG 配列番号 55 :ラミニン由来血管新生ペプチドの改変体 GLG— IKVAV  SEQ ID NO: 47: A variant of laminin-derived angiogenic peptide GLGLGL—SIKVAV SEQ ID NO: 48: A variant of laminin-derived angiogenic peptide SIKVAV—GLGLGL SEQ ID NO: 49: A variant of laminin-derived angiogenic peptide GLG—SIKVAV-GLG SEQ ID NO: 50 : A variant of laminin-derived angiogenic peptide GLG—SIKVAV SEQ ID NO: 51: A variant of laminin-derived angiogenic peptide SIKVAV—GLG SEQ ID NO: 52: A variant of laminin-derived angiogenic peptide GLGLGL—IKVAV SEQ ID NO: 53: Laminin-derived angiogenesis Variant of peptide IKVAV—GLGLGL SEQ ID NO: 54: Variant of laminin-derived angiogenic peptide GLG—IKVAV—GLG SEQ ID NO: 55: Variant of laminin-derived angiogenic peptide GLG—IKVAV
配列番号 56 :ラミニン由来血管新生ペプチドの改変体 IKVAV— GLG  SEQ ID NO: 56: Variant of laminin-derived angiogenic peptide IKVAV—GLG
発明を実施するための最良の形態 BEST MODE FOR CARRYING OUT THE INVENTION
以下、本発明を説明する。本明細書の全体にわたり、単数形の表現は、特に言及 しない限り、その複数形の概念をも含むことが理解されるべきである。従って、単数形 の冠詞 (例えば、英語の場合は「a」、「an」、「the」など）は、特に言及しない限り、そ の複数形の概念をも含むことが理解されるべきである。また、本明細書において使用 される用語は、特に言及しない限り、当上記分野で通常用いられる意味で用いられる ことが理解されるべきである。したがって、他に定義されない限り、本明細書中で使用 される全ての専門用語および科学技術用語は、本発明の属する分野の当業者によ つて一般的に理解されるのと同じ意味を有する。矛盾する場合、本明細書 (定義を含 めて）が優先する。 Hereinafter, the present invention will be described. It should be understood that throughout this specification, the use of the singular includes the plural concept unless specifically stated otherwise. Therefore, it is to be understood that singular articles (eg, "a", "an", "the", etc. in English) also include the concept of the plural unless specifically stated otherwise. . It should also be understood that the terms used in the present specification are used in the meanings usually used in the above-mentioned field, unless otherwise specified. Therefore, as used herein, unless otherwise defined. All technical and scientific terms used have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In case of conflict, the present specification, including definitions, will control.
[0018] (一般技術）  [0018] (General technology)
本明細書において用いられる分子生物学的手法、遺伝子工学的手法、生化学的 手法、微生物学的手法は、当該分野において周知であり慣用されるものであり、例え 【 、 Sambrook J. et al. (1989). Molecular Cloning: A Laboratory Manual,し old Spring Harborおよびその 3rd Ed. (2001); Ausubel, F. M. (1987). Current Protocolsin Molecular Biology, Greene Pub. Associates and Wiley— Interscience; Ausubel, F. M. (1989). Short Protocols in Molecular Biology: A Compendium of Methodsfrom Current Protocols in Molecular Biology, Greene Pub. Associates  The molecular biological techniques, genetic engineering techniques, biochemical techniques, and microbiological techniques used in the present specification are well known and commonly used in the art, and are described, for example, in Sambrook J. et al. (1989) .Molecular Cloning: A Laboratory Manual, old Spring Harbor and its 3rd Ed. (2001); Ausubel, FM (1987) .Current Protocols in Molecular Biology, Greene Pub.Associates and Wiley—Interscience; Ausubel, FM (1989). ) .Short Protocols in Molecular Biology: A Compendium of Methods from Current Protocols in Molecular Biology, Greene Pub. Associates
andWiley-Interscience; Innis, M. A. (1990). PCR Protocols: A Guide to Methods andApplications, Academic Press; Ausubel, F. M. (1992). Short Protocols inMolecular Biology: A Compendium of Methods from Current Protocols in  andWiley-Interscience; Innis, M.A. (1990) .PCR Protocols: A Guide to Methods and Applications, Academic Press; Ausubel, F.M. (1992) .Short Protocols in Molecular Biology: A Compendium of Methods from Current Protocols in
MolecularBiology, Greene Pub. Associates; Ausubel, F. M. (1995). Short Protocols inMolecular Biology: A Compendium of Methods from Current Protocols in  MolecularBiology, Greene Pub. Associates; Ausubel, F.M. (1995) .Short Protocols in Molecular Biology: A Compendium of Methods from Current Protocols in
MolecularBiology, Greene Pub. Associates; Innis, M. A. et al. (1995). PCR  MolecularBiology, Greene Pub. Associates; Innis, M.A. et al. (1995) .PCR
Strategies, Academic Press; Ausubel, F. M. (1999). bhort Protocols in Molecular Biology: ACompendium of Methods from Current Protocols in Molecular Biology, Wiley, andannual updates; Sninsky, J. J. et al. (1999). PCR Applications: Protocols forFunctional Genomics, Academic Press,別冊実験医学「遺伝子導入 &発現解析 実験法」羊土社、 1997などに記載されており、これらは本明細書において関連する 部分 (全部であり得る）が参考として援用される。本発明の遺伝子ワクチンは、当該分 野において公知の技術を用いて実施することができる。そのようなワクチンの作製は 、本明細書において他の場所において詳述される分子生物学的手法、生化学的手 法および遺伝子工学的手法を応用することによって実施することができる。  Strategies, Academic Press; Ausubel, FM (1999) .bhort Protocols in Molecular Biology: ACompendium of Methods from Current Protocols in Molecular Biology, Wiley, and annual updates; Academic Press, separate volume Experimental Medicine “Experimental Method for Gene Transfer & Expression Analysis,” Yodosha, 1997, etc., and the relevant portions (which may be all) are incorporated herein by reference. The gene vaccine of the present invention can be implemented using a known technique in this field. The production of such vaccines can be performed by applying molecular biological, biochemical and genetic engineering techniques as described elsewhere herein.
[0019] 人工的に合成した遺伝子を作製するための DNA合成技術および核酸ィ匕学にっ ヽ ては、例えば、 Gait, M. J. (1985). Oligonucleotide Synthesis: A Practical Approach, IRL Press; Gait, M. J. (1990). Oligonucleotide Synthesis: A Practical Approach, IRL Press; Eckstein, F. (1991). Oligonucleotides and Analogues: A PracticalApproach, IRL Press; Adams, R. L. et al. (1992). The Biochemistry of theNucleic Acids, Chapman & Hall; Shabarova, Z. et al. (1994). AdvancedOrganic Chemistry of Nucleic Acids, Weinheim; Blackburn, G. M. et al. (1996). Nucleic Acids in Chemistry and Biology, Oxford University Press; Hermanson, G.T. (1996). [0019] DNA synthesis technology for producing artificially synthesized genes and nucleic acid synthesis are described in, for example, Gait, MJ (1985). Oligonucleotide Synthesis: A Practical. Oligonucleotide Synthesis: A Practical Approach, IRL Press; Eckstein, F. (1991) .Oliigonucleotides and Analogues: A PracticalApproach, IRL Press; Adams, RL et al. (1992). The Biochemistry of the Nucleic Acids, Chapman &Hall; Shabarova, Z. et al. (1994) .AdvancedOrganic Chemistry of Nucleic Acids, Weinheim; Blackburn, GM et al. (1996) .Nucleic Acids in Chemistry and Biology, Oxford University Press; Hermanson, GT (1996).
Bioconjugate Techniques, Academic Pressなどに記載されており、これらは本明細書 において関連する部分が参考として援用される。  Bioconjugate Techniques, Academic Press, etc., and the relevant portions are incorporated herein by reference.
[0020] 以下に本発明において使用される定義、基本的技術などを説明する。  Hereinafter, definitions, basic techniques, and the like used in the present invention will be described.
[0021] (用語の定義）  [0021] (Definition of terms)
以下に本明細書において特に使用される用語の定義を列挙する。  The definitions of terms used particularly in the present specification are listed below.
[0022] 本明細書において使用される用語「タンパク質」、「ポリペプチド」、「オリゴペプチド」 および「ペプチド」は、本明細書において同じ意味で使用され、任意の長さのアミノ酸 のポリマーをいう。このポリマーは、直鎖であっても分岐していてもよぐ環状であって もよい。アミノ酸は、天然のものであっても非天然のものであってもよぐ改変されたァ ミノ酸であってもよい。本明細書において用いられる場合、この用語は、好ましくは、 核酸分子によって翻訳された形態であることから、通常、直鎖であり、天然のアミノ酸 のみ力も構成されるがそれに限定されない。この用語はまた、複数のポリペプチド鎖 の複合体へとアセンブルされたものを包含し得る。この用語はまた、天然または人工 的に改変されたアミノ酸ポリマーも包含する。そのような改変としては、例えば、ジスル フイド結合形成、グリコシル化、脂質化、ァセチル化、リン酸化または任意の他の操作 もしくは改変（例えば、標識成分との結合体化)。この定義にはまた、例えば、アミノ酸 の 1または 2以上のアナログを含むポリペプチド (例えば、非天然のアミノ酸などを含 む）、ペプチド様化合物（例えば、ぺプトイド）および当該分野において公知の他の改 変が包含される。  [0022] As used herein, the terms "protein," "polypeptide," "oligopeptide," and "peptide" are used interchangeably herein and refer to a polymer of amino acids of any length. . The polymer may be linear or branched or cyclic. The amino acids may be naturally occurring or non-naturally occurring or modified amino acids. As used herein, the term is preferably, but not limited to, usually linear and in the form of naturally occurring amino acids alone, since it is preferably in a form translated by a nucleic acid molecule. The term may also include those assembled into a complex of multiple polypeptide chains. The term also embraces naturally 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.), peptidomimetic compounds (eg, peptoids), and other compounds known in the art. Changes are included.
[0023] 本明細書では、特に言及するときは、「タンパク質」は、比較的大きな分子量を有す るアミノ酸のポリマーまたはその改変体を指し、「ペプチド」というときは、比較的小さな 分子量を有するアミノ酸のポリマーまたはその改変体を指すことがあることが理解され るべきである。そのような分子量としては、たとえば、約 30kDa、好ましくは約 20kDa 、より好ましくは約 lOkDaなどを挙げることができるがそれらに限定されない。 [0023] In the present specification, when specifically referred to, "protein" refers to a polymer of amino acids having a relatively large molecular weight or a variant thereof, and when referred to as "peptide", has a relatively small molecular weight. It is understood that it may refer to a polymer of amino acids or a variant thereof. Should be. Examples of such a molecular weight include, but are not limited to, about 30 kDa, preferably about 20 kDa, more preferably about 10 kDa.
本明細書において使用される用語「核酸分子」、「ポリヌクレオチド」、「オリゴヌタレ ォチド」および「核酸」は、本明細書において同じ意味で使用され、任意の長さのヌク レオチドのポリマー（例えば、 cDNA、 mRNA、ゲノム DNAなど）をいう。この用語は また、「オリゴヌクレオチド誘導体」または「ポリヌクレオチド誘導体」を含む。「オリゴヌ クレオチド誘導体」または「ポリヌクレオチド誘導体」とは、ヌクレオチドの誘導体を含 む力 またはヌクレオチド間の結合が通常とは異なるオリゴヌクレオチドまたはポリヌク レオチドをいい、互換的に使用される。そのようなオリゴヌクレオチドとして具体的には 、例えば、 2，一 O—メチルーリボヌクレオチド、オリゴヌクレオチド中のリン酸ジエステ ル結合がホスホロチォエート結合に変換されたオリゴヌクレオチド誘導体、オリゴヌク レオチド中のリン酸ジエステル結合が N3，一 P5，ホスホロアミデート結合に変換され たオリゴヌクレオチド誘導体、オリゴヌクレオチド中のリボースとリン酸ジエステル結合 とがペプチド核酸結合に変換されたオリゴヌクレオチド誘導体、オリゴヌクレオチド中 のゥラシルが C 5プロピニルゥラシルで置換されたオリゴヌクレオチド誘導体、オリゴ ヌクレオチド中のゥラシルが C— 5チアゾールゥラシルで置換されたオリゴヌクレオチド 誘導体、オリゴヌクレオチド中のシトシンが C 5プロピ-ルシトシンで置換されたオリ ゴヌクレオチド誘導体、オリゴヌクレオチド中のシトシンがフエノキサジン修飾シトシン（ phenoxazine- modified cytosine)で置換されたオリゴヌクレオチド誘導体、 DNA中のリ ボースが 2， -0 -プロピルリボースで置換されたオリゴヌクレオチド誘導体およびオリ ゴヌクレオチド中のリボースが 2，ーメトキシエトキシリボースで置換されたオリゴヌタレ ォチド誘導体などが例示される。他にそうではないと示されなければ、特定の核酸配 列はまた、明示的に示された配列と同様に、その保存的に改変された改変体 (例え ば、縮重コドン置換体)および相補配列を包含することが企図される。具体的には、 縮重コドン置換体は、 1またはそれ以上の選択された (または、すべての）コドンの 3番 目の位置が混合塩基および Zまたはデォキシイノシン残基で置換された配列を作成 することにより達成され得る（Batzeret al, Nucleic Acid Res.19:5081(1991); Ohtsuka et al., J. Biol. Chem.260:2605- 2608 (1985); Rossolini et al., Mol. Cell. Probes 8: 91-98 (1994)) oある遺伝子配列をコードする核酸分子はまた、「スプライス変異体 (バ リアント、改変体)」を包含する。同様に、核酸によりコードされた特定のタンパク質は 、その核酸のスプライス改変体によりコードされる任意のタンパク質を包含する。その 名が示唆するように「スプライス変異体」は、遺伝子のオルタナティブスプライシングの 産物である。転写後、最初の核酸転写物は、異なる (別の)核酸スプライス産物が異 なるポリペプチドをコードするようにスプライスされ得る。スプライス変異体の産生機構 は変化するが、ェキソンのオルタナティブスプライシングを含む。読み過し転写により 同じ核酸に由来する別のポリペプチドもまた、この定義に包含される。スプライシング 反応の任意の産物 (組換え形態のスプライス産物を含む）がこの定義に含まれる。し たがって、本発明の遺伝子には、そのスプライス変異体もまた包含され得る。このよう な変異体は、本発明の予防および治療において有用である。本発明のペプチドは、 通常、このポリヌクレオチド形態によって生体内に送達され得ることが理解される。 As used herein, the terms "nucleic acid molecule", "polynucleotide", "oligonucleotide" and "nucleic acid" are used interchangeably herein and refer to polymers of nucleotides of any length (e.g., cDNA, mRNA, genomic DNA, etc.). The term also includes “oligonucleotide derivatives” or “polynucleotide derivatives”. "Oligonucleotide derivative" or "polynucleotide derivative" refers to an oligonucleotide or polynucleotide having an unusual force or a bond between nucleotides containing a derivative of a nucleotide, and is used interchangeably. Specific examples of such an oligonucleotide include, for example, 2,1 O-methyl-ribonucleotide, an oligonucleotide derivative in which a phosphoric ester bond in the oligonucleotide is converted to a phosphorothioate bond, and an oligonucleotide in the oligonucleotide. Oligonucleotide derivatives in which phosphodiester bonds have been converted to N3,1P5, phosphoramidate bonds, oligonucleotide derivatives in which ribose and phosphodiester bonds have been converted to peptide nucleic acid bonds, and oligonucleotide derivatives in which oligonucleotide has a phosphodiester bond. Oligonucleotide derivative with peracyl substituted with C5 propynyl peracyl, oligonucleotide derivative with peracyl substituted with C-5 thiazole peracyl, cytosine in oligonucleotide substituted with C5 propyl-cytosine Oligonucleotide derivatives in which cytosine in the oligonucleotide has been replaced with phenoxazine-modified cytosine, oligonucleotide derivatives in which the ribose in DNA has been replaced by 2, -0-propylribose, and Oligonucleotide derivatives in which ribose in the oligonucleotide is substituted with 2, -methoxyethoxyribose are exemplified. Unless otherwise indicated, a particular nucleic acid sequence may also be a conservatively modified version (e.g., a degenerate codon substitution) and a conservatively modified version thereof, similar to the explicitly indicated sequence. It is contemplated to include the complementary sequence. Specifically, degenerate codon substitutions create a sequence in which the third position of one or more selected (or all) codons is replaced with a mixed base and a Z or deoxyinosine residue. (Batzer et al, Nucleic Acid Res. 19: 5081 (1991); Ohtsuka et al., J. Biol. Chem. 260: 2605-2608 (1985); Rossolini et al., Mol. Cell. Probes 8: 91-98 (1994)) o A nucleic acid molecule encoding a gene sequence also includes "splice variants (variants, variants)." Similarly, a particular protein encoded by a nucleic acid includes any protein encoded by a splice variant of the nucleic acid. As the name suggests, "splice variants" are the products of alternative splicing of a gene. After transcription, the initial nucleic acid transcript may be spliced such that different (alternate) nucleic acid splice products encode different polypeptides. The mechanism of production of splice variants varies, but involves alternative splicing of exons. Another polypeptide derived from the same nucleic acid by read-through transcription is also included in this definition. Any product of a splicing reaction, including recombinant forms of the splice product, is included in this definition. Therefore, the gene of the present invention may also include splice variants thereof. Such variants are useful in the prevention and treatment of the present invention. It is understood that the peptides of the present invention can usually be delivered in vivo by this polynucleotide form.
[0025] 本明細書において「遺伝子」とは、遺伝形質を規定する因子をいう。タンパク質の一 次構造を規定するものを構造遺伝子と!/ ヽ、その発現を左右するものを調節遺伝子 (たとえば、プロモーター）という。本明細書では、遺伝子は、特に言及しない限り、構 造遺伝子および調節遺伝子を包含する。したがって、あるタンパク質について遺伝 子というときは、通常、そのタンパク質の構造遺伝子およびそのタンパク質などのプロ モーターなどの転写または翻訳の調節配列の両方を包含する。本明細書では、「遺 伝子」は、通常「ポリヌクレオチド」、「オリゴヌクレオチド」、「核酸」および「核酸分子」 をさす。本明細書においてはまた、「遺伝子産物」は、遺伝子によって発現された「ポ リヌクレオチド」、「オリゴヌクレオチド」、「核酸」および「核酸分子」ならびに Zまたは「 タンパク質」、「ポリペプチド」、「オリゴペプチド」および「ペプチド」を包含する。当業 者であれば、遺伝子産物が何たるかはその状況に応じて理解することができる。  [0025] As used herein, the term "gene" refers to a factor that defines a genetic trait. Those that define the primary structure of a protein are called structural genes, and those that control its expression are called regulatory genes (eg, promoters). As used herein, a gene includes a structural gene and a regulatory gene unless otherwise specified. Thus, a gene for a protein usually includes both transcriptional or translational regulatory sequences, such as the structural gene for the protein and a promoter for the protein. As used herein, "gene" generally refers to "polynucleotide", "oligonucleotide", "nucleic acid" and "nucleic acid molecule". As used herein, "gene product" also refers to "polynucleotides", "oligonucleotides", "nucleic acids" and "nucleic acid molecules" expressed by genes and Z or "proteins", "polypeptides", " "Oligopeptides" and "peptides". Those skilled in the art can understand what the gene product is, depending on the situation.
[0026] 本明細書において遺伝子 (例えば、核酸配列、アミノ酸配列など）の「相同性」とは、 2以上の遺伝子配列の、互いに対する同一性の程度をいう。従って、ある 2つの遺伝 子の相同性が高いほど、それらの配列の同一性または類似性は高い。 2種類の遺伝 子が相同性を有するか否かは、配列の直接の比較、または核酸の場合ストリンジェン トな条件下でのノ、イブリダィゼーシヨン法によって調べられ得る。本明細書にぉ 、て、 遺伝子 (例えば、核酸配列、アミノ酸配列など）の「類似性」とは、上記相同性におい て、保存的置換をポジティブ（同一）とみなした場合の、 2以上の遺伝子配列の、互い に対する同一性の程度をいう。従って、保存的置換がある場合は、その保存的置換 の存在に応じて同一性と類似性とは異なる。また、保存的置換がない場合は、同一 性と類似性とは同じ数値を示す。 As used herein, “homology” of a gene (eg, a nucleic acid sequence, an amino acid sequence, etc.) refers to the degree of identity between two or more gene sequences. Thus, the higher the homology between two genes, the higher the identity or similarity between their sequences. Whether the two genes have homology can be determined by direct sequence comparison or, in the case of nucleic acids, by stringent conditions under stringent conditions. In this specification, “Similarity” of a gene (eg, nucleic acid sequence, amino acid sequence, etc.) refers to the identity of two or more gene sequences to each other when conservative substitutions are regarded as positive in the above homology. The degree of. Thus, if there are conservative substitutions, identity and similarity will be different depending on the existence of the conservative substitution. When there is no conservative substitution, identity and similarity show the same numerical value.
[0027] 本明細書において「アミノ酸」とは、当該分野において通常用いられる意味で用いら れ、カルボキシル基とアミノ基とを有する有機化合物をいう。本明細書においてァミノ 酸は、天然アミノ酸であっても非天然アミノ酸であっても良い。  [0027] In the present specification, the term "amino acid" is used in a meaning commonly used in the art, and refers to an organic compound having a carboxyl group and an amino group. As used herein, amino acids may be natural or unnatural amino acids.
[0028] 本明細書において使用される「天然のアミノ酸」とは、天然のアミノ酸の L—異性体 を意味する。天然のアミノ酸は、グリシン、ァラニン、ノ リン、ロイシン、イソロイシン、セ リン、メチォニン、トレオニン、フエ二ルァラニン、チロシン、トリプトファン、システィン、 プロリン、ヒスチジン、ァスパラギン酸、ァスパラギン、グルタミン酸、グルタミン、 γ—力 ルボキシグルタミン酸、アルギニン、オル-チン、およびリジンである。特に示されな い限り、本明細書でいう全てのアミノ酸は、通常 L体を意図するが、本発明はそれに 限定されず、 D体のアミノ酸を用いた形態もまた本発明の範囲内にあることが理解さ れる。  [0028] As used herein, "natural amino acid" refers to the L-isomer of a natural amino acid. Natural amino acids include glycine, alanine, norin, leucine, isoleucine, serine, methionine, threonine, phenylalanine, tyrosine, tryptophan, cysteine, proline, histidine, aspartic acid, asparagine, glutamic acid, glutamine, γ-force Xyglutamic acid, arginine, ortin, and lysine. Unless otherwise indicated, all amino acids referred to in the present specification usually mean L-form, but the present invention is not limited thereto, and forms using D-form amino acids are also within the scope of the present invention. It is understood that.
[0029] 本明細書にぉ 、て「アミノ酸改変体」とは、天然のアミノ酸ではな 、が、天然のァミノ 酸の物性および Ζまたは機能に類似する分子をいう。アミノ酸改変体としては、例え ば、疎水性基、フエ-ルァラニンのベンジル側鎖 (パラ位、メタ位、オルト位など）にァ ルキル基、ハロ基、ニトロ基などが結合したもの、ェチォニン、カナバニン、 2—メチル グルタミンなどが挙げられる。本発明では、アミノ酸改変体は、非天然アミノ酸および アミノ酸模倣物を包含することがあることが理解される。  [0029] As used herein, the term "amino acid variant" refers to a molecule that is not a natural amino acid but is similar in physical properties and / or function to a natural amino acid. Examples of the modified amino acids include a hydrophobic group, phenylalanine having a benzyl side chain (para-, meta-, ortho-position, etc.) to which an alkyl group, a halo group, a nitro group, etc. are bonded, etyonine, canavanine , 2-methylglutamine and the like. It is understood that, in the present invention, amino acid variants may include unnatural amino acids and amino acid mimetics.
[0030] 本明細書において「非天然アミノ酸」とは、タンパク質中で通常は天然に見出されな いアミノ酸を意味する。非天然アミノ酸の例として、ノルロイシン、パラ—ニトロフエニル ァラニン、ホモフエ-ルァラニン、パラ一フルオロフェ-ルァラニン、 3—アミノー 2—ベ ンジルプロピオン酸、ホモアルギニンの D体または L体および D—フエ-ルァラニンが 挙げられる。  [0030] As used herein, the term "unnatural amino acid" refers to an amino acid that is not normally found in nature in a protein. Examples of unnatural amino acids include norleucine, para-nitrophenylalanine, homophenylalanine, para-fluorophenylalanine, 3-amino-2-benzylpropionic acid, the D- or L-form of homoarginine, and D-phenylalanine. No.
[0031] 本明細書において「アミノ酸模倣物」とは、アミノ酸の一般的な化学構造とは異なる 構造を有するが、天然に存在するアミノ酸と同様な様式で機能する化合物をいう。 [0031] As used herein, "amino acid mimetic" is different from the general chemical structure of an amino acid A compound that has a structure but functions in a manner similar to a naturally occurring amino acid.
[0032] 本明細書において「ヌクレオチド」は、アミノ酸をコードする能力を有する限り、天然 のものでも非天然のものでもよ 、。  [0032] In the present specification, "nucleotide" may be a natural or non-natural one as long as it has an ability to encode an amino acid.
[0033] 本明細書にぉ 、て「ヌクレオチド誘導体」または「ヌクレオチドアナログ」とは、天然 に存在するヌクレオチドとは異なるがもとのヌクレオチドと同様の機能を有するものを いう。そのようなヌクレオチド誘導体およびヌクレオチドアナログは、当該分野におい て周知である。そのようなヌクレオチド誘導体およびヌクレオチドアナログの例としては 、ホスホロチォエート、ホスホルアミデート、メチルホスホネート、キラルメチルホスホネ ート、 2— 0—メチルリボヌクレオチド、ペプチド 核酸（PNA)が含まれる力 これら に限定されない。本発明では、遺伝子産物が発現される限り、どのようなアナログを 使用してもよい。好ましくは、天然型のヌクレオチドを含む遺伝子ワクチンが使用され る。天然型のものは、ペプチドに翻訳される可能性が高いからである。  [0033] As used herein, the term "nucleotide derivative" or "nucleotide analog" refers to a nucleotide different from a naturally occurring nucleotide but having the same function as the original nucleotide. Such nucleotide derivatives and nucleotide analogs are well-known in the art. Examples of such nucleotide derivatives and analogs include phosphorothioates, phosphoramidates, methylphosphonates, chiral methylphosphonates, 2-0-methylribonucleotides, peptides containing nucleic acids (PNA). It is not limited to these. In the present invention, any analog may be used as long as the gene product is expressed. Preferably, a genetic vaccine containing natural nucleotides is used. This is because the natural form is more likely to be translated into a peptide.
[0034] 本明細書にぉ 、て「対応する」アミノ酸および核酸 (例えば、本発明の SWYGLR における Yなどのアミノ酸）とは、それぞれあるポリペプチドおよび核酸分子において 、比較の基準となるポリペプチドおよび核酸分子における所定のアミノ酸および核酸 と同様の作用を有するか、または有することが予測されるアミノ酸および核酸をいい、 例えば、酵素においては、その酵素の活性中心と同様の位置に存在し触媒活性に 同様の寄与をするアミノ酸およびそれをコードする核酸をいう。例えば、核酸配列で あれば、その核酸配列またはそれがコードする特定の部分と同様の機能を発揮する 部分であり得る。  [0034] As used herein, "corresponding" amino acids and nucleic acids (for example, amino acids such as Y in SWYGLR of the present invention) refer to polypeptides and nucleic acids that serve as comparison standards in a given polypeptide and nucleic acid molecule, respectively. Refers to amino acids and nucleic acids that have, or are expected to have, the same action as a given amino acid and nucleic acid in a nucleic acid molecule. Refers to amino acids that make similar contributions and the nucleic acids that encode them. For example, if it is a nucleic acid sequence, it may be a portion that functions similarly to the nucleic acid sequence or the specific portion encoded by the nucleic acid sequence.
[0035] 本明細書にぉ 、て「対応する」遺伝子 (例えば、細胞表面マーカーなどのポリぺプ チド、核酸分子など）とは、ある種において、比較の基準となる種における所定の遺 伝子と同様の作用を有するか、または有することが予測される遺伝子をいい、そのよ うな作用を有する遺伝子が複数存在する場合、進化学的に同じ起源を有するものを いう。従って、ある遺伝子の対応する遺伝子は、その遺伝子のオルソログであり得る。 したがって、ヒトの癌抗原などの遺伝子に対応する遺伝子は、他の動物（マウス、ラッ ト、ブタ、ゥシなど）においても見出すことができる。そのような対応する遺伝子は、当 該分野において周知の技術を用いて同定することができる。したがって、例えば、あ る動物における対応する遺伝子は、対応する遺伝子の基準となる遺伝子 (例えば、 癌抗原などの遺伝子）の配列をクエリ配列として用いてその動物（例えばマウス、ラッ ト）の配列データベースを検索することによって、またはウエットの実験でライブラリー をスクリーニングすることによって見出すことができる。 As used herein, the term “corresponding” gene (eg, a polypeptide such as a cell surface marker, a nucleic acid molecule, etc.) refers to a given gene in a species that serves as a reference for comparison in a certain species. It refers to a gene that has or is expected to have the same action as the offspring. When there are a plurality of genes having such an action, it refers to those having the same evolutionary origin. Thus, the corresponding gene for a gene may be an ortholog of that gene. Therefore, a gene corresponding to a gene such as a human cancer antigen can be found in other animals (mouse, rat, pig, mouse, etc.). Such corresponding genes can be identified using techniques well known in the art. So, for example, The corresponding gene in an animal can be determined by searching the sequence database of that animal (eg, mouse, rat) using the sequence of the reference gene for the corresponding gene (eg, a gene for a cancer antigen) as a query sequence. Or by screening the library in wet experiments.
[0036] 本明細書において「検索」とは、電子的にまたは生物学的あるいは他の方法により 、ある核酸塩基配列を利用して、特定の機能および Zまたは性質を有する他の核酸 塩基配列を見出すことをいう。電子的な検索としては、 BLAST(Altschul et al, J. Mol. Biol. 215: 403-410 (1990)), FASTA (Pearson& Lipman, Proc. Natl. Acad. Sci., USA 85: 2444-2448(1988》、 Smith and Waterman法 (Smithand Waterman, J. Mol. Biol. 147: 195-197 (1981》、および Needleman and Wunsch法 (Needlemanand Wunsch, J. Mol. Biol. 48: 443-453 (1970))などが挙げられるがそれらに限定されない 。生物学的な検索としては、ストリンジェントハイブリダィゼーシヨン、ゲノム DNAをナ イロンメンプレン等に貼り付けたマクロアレイまたはガラス板に貼り付けたマイクロアレ ィ（マイクロアレイアツセィ）、 PCRおよび in situノヽイブリダィゼーシヨンなどが挙げ られるがそれらに限定されない。本明細書において、癌抗原などには、このような電 子的検索、生物学的検索によって同定された対応遺伝子も含まれるべきであること が意図される。  [0036] As used herein, the term "search" refers to the use of a certain nucleobase sequence electronically, biologically, or by another method to search for another nucleobase sequence having a specific function and Z or property. To find. Electronic searches include BLAST (Altschul et al, J. Mol. Biol. 215: 403-410 (1990)), FASTA (Pearson & Lipman, Proc. Natl. Acad. Sci., USA 85: 2444-2448 ( 1988, Smith and Waterman method (Smithand Waterman, J. Mol. Biol. 147: 195-197 (1981), and Needleman and Wunsch method (Needleman and Wunsch, J. Mol. Biol. 48: 443-453 (1970)) Examples of the biological search include stringent hybridization, a macroarray in which genomic DNA is attached to nylon membrane, or a microarray in which the DNA is attached to a glass plate. (Microarray assay), PCR, in situ hybridization, etc. In the present specification, cancer antigens and the like are referred to by such electronic search and biological search. It is intended that the identified corresponding gene should also be included
[0037] 本明細書にぉ 、て「フラグメント」とは、全長のポリペプチドまたはポリヌクレオチド（ 長さが n)に対して、 l〜n— 1までの配列長さを有するポリペプチドまたはポリヌクレオ チドをいう。フラグメントの長さは、その目的に応じて、適宜変更することができ、例え ば、その長さの下限としては、ポリペプチドの場合、 3、 4、 5、 6、 7、 8、 9、 10、 15, 2 0、 25、 30、 40、 50およびそれ以上のアミノ酸が挙げられ、ここの具体的に列挙して いない整数で表される長さ (例えば、 11など)もまた、下限として適切であり得る。また 、ポリヌクレオチドの場合、 5、 6、 7、 8、 9、 10、 15, 20、 25、 30、 40、 50、 75、 100 およびそれ以上のヌクレオチドが挙げられ、ここの具体的に列挙して!/、な!/、整数で表 される長さ（例えば、 11など)もまた、下限として適切であり得る。本明細書において、 ポリペプチドおよびポリヌクレオチドの長さは、上述のようにそれぞれアミノ酸または核 酸の個数で表すことができるが、上述の個数は絶対的なものではなぐ同じ機能を有 する限り、上限または下限としての上述の個数は、その個数の上下数個（または例え ば上下 10%)のものも含むことが意図される。そのような意図を表現するために、本 明細書では、個数の前に「約」を付けて表現することがある。しかし、本明細書では、「 約」のあるなしはその数値の解釈に影響を与えな ヽことが理解されるべきである。本 発明において用いられる、細胞表面マーカー、癌抗原などについても、その機能 (例 えば、抗体惹起能)を有する限り、このようなフラグメントもまた使用され得ることが理 解される。 [0037] As used herein, the term "fragment" refers to a polypeptide or polynucleotide having a sequence length from l to n-1 relative to a full-length polypeptide or polynucleotide (length is n). Say. The length of the fragment can be appropriately changed depending on the purpose.For example, the lower limit of the length is 3, 4, 5, 6, 7, 8, 9, 10 for a polypeptide. , 15, 20, 25, 30, 40, 50 and more amino acids, and lengths represented by integers not specifically listed herein (for example, 11) are also suitable as lower limits. Can be In the case of polynucleotides, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 40, 50, 75, 100 and more nucleotides are mentioned, and specific listings here are provided. The length represented by! /, Na! /, And an integer (eg, 11 or the like) may also be appropriate as the lower limit. In the present specification, the lengths of the polypeptide and polynucleotide can be represented by the number of amino acids or nucleic acids, respectively, as described above, but the above-mentioned numbers have the same function but are not absolute. As far as possible, the above-mentioned number as an upper limit or a lower limit 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. It should be understood, however, that the presence or absence of “about” does not affect the interpretation of that number. It is understood that such fragments may also be used for the cell surface markers, cancer antigens and the like used in the present invention, as long as they have the function (for example, the ability to induce antibodies).
本明細書にぉ 、て「ストリンジェントな条件でノヽイブリダィズするポリヌクレオチド」と は、当該分野で慣用される周知の条件をいう。本発明のポリヌクレオチド中から選択 されたポリヌクレオチドをプローブとして、コロニ一'ハイブリダィゼーシヨン法、プラー ク 'ハイブリダィゼーシヨン法あるいはサザンブロットハイブリダィゼーシヨン法等を用 いることにより、そのようなポリヌクレオチドを得ることができる。具体的には、ストリンジ ェントな条件でノ、イブリダィズするポリヌクレオチドは、コロニーあるいはプラーク由来 の DNAを固定化したフィルターを用いて、 0. 7〜1. OMの NaCl存在下、 65°Cでノヽ イブリダィゼーシヨンを行った後、 0. 1〜2倍濃度の SSC (saline- sodium citrate)溶 液（1倍濃度の SSC溶液の組成は、 150mM 塩ィ匕ナトリウム、 15mM クェン酸ナト リウムである）を用い、 65°C条件下でフィルターを洗浄することにより同定できるポリヌ クレオチドを意味する。ハイブリダィゼーシヨンは、 MolecularCloning 2nd ed.， Current Protocols in Molecular Biology, supplement 1— «38, DNACloning l:Core Techniques, A Practical Approach, Second Edition, OxfordUniversity Press(1995)等の実験書に 記載されている方法に準じて行うことができる。ここで、ストリンジェントな条件下でノヽ イブリダィズする配列からは、好ましくは、 A配列のみまたは T配列のみを含む配列が 除外される。「ハイブリダィズ可能なポリヌクレオチド」とは、上記ハイブリダィズ条件下 で別のポリヌクレオチドにハイブリダィズすることができるポリヌクレオチドを 、う。ハイ ブリダィズ可能なポリヌクレオチドとして具体的には、本発明で具体的に示されるアミ ノ酸配列を有するポリペプチドをコードする DNAの塩基配列と少なくとも 60%以上の 相同性を有するポリヌクレオチド、好ましくは 80%以上の相同性を有するポリヌクレオ チド、さらに好ましくは 95%以上の相同性を有するポリヌクレオチドを挙げることがで きる。本発明では、そのような相同性のある細胞表面マーカーなどが使用され得るこ とが理解される。 As used herein, the term “polynucleotide that hybridizes under stringent conditions” refers to well-known conditions commonly used in the art. Using a polynucleotide selected from the polynucleotides of the present invention as a probe, colony 'hybridization method, plaque' hybridization method, Southern blot hybridization method or the like is used. Thus, such a polynucleotide can be obtained. Specifically, the polynucleotide to be hybridized under stringent conditions can be purified at 65 ° C in the presence of 0.7 to 1.OM NaCl using a filter on which DNA derived from colonies or plaques is immobilized. After performing the hybridization, a 0.1- to 2-fold concentration of SSC (saline-sodium citrate) solution (the composition of the 1-fold concentration SSC solution is 150 mM sodium salt sodium citrate, 15 mM sodium citrate). ) Means a polynucleotide that can be identified by washing the filter at 65 ° C. Hybridization is described in experimental books such as MolecularCloning 2nd ed., Current Protocols in Molecular Biology, supplement 1— «38, DNACloning l: Core Techniques, A Practical Approach, Second Edition, Oxford University Press (1995). It can be done according to the method. Here, sequences containing only the A sequence or only the T sequence are preferably excluded from the sequences that hybridize under stringent conditions. "Hybridizable polynucleotide" refers to a polynucleotide that can hybridize to another polynucleotide under the above hybridization conditions. Specific examples of the hybridizable polynucleotide include polynucleotides having at least 60% or more homology with the nucleotide sequence of DNA encoding the polypeptide having the amino acid sequence specifically shown in the present invention, preferably Polynucleotides having a homology of 80% or more, more preferably polynucleotides having a homology of 95% or more. Wear. It is understood that such homologous cell surface markers and the like can be used in the present invention.
[0039] アミノ酸は、その一般に公知の 3文字記号力、または IUPAC— IUB Biochemica 1 Nomenclature Commissionにより推奨される 1文字記号のいずれかにより、本 明細書中で言及され得る。ヌクレオチドも同様に、一般に認知された 1文字コードによ り言及され得る。  [0039] Amino acids can 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.
[0040] その文字コードは以下のとおりである。  [0040] The character codes are as follows.
アミノ酸  Amino acids
3文字記号 1文字記号 意味  3-letter symbol 1-letter symbol Meaning
Ala A ァラニン  Ala A
Cys C システィン  Cys C Sistine
Asp D ァスノ ラギン酸  Asp D Asno laginic acid
Glu E グルタミン酸  Glu E glutamic acid
Phe F フエ-ルァラニン  Phe F Hue-Lualanin
Gly G グリシン  Gly G glycine
His H ヒスチジン  His H histidine
He I イソロイシン  He I isoleucine
Lys K リジン  Lys K Lysine
Leu L ロイシン  Leu L Leucine
Met M メチォニン  Met M Methionin
Asn N ァスノ ラギン  Asn N
Pro P プロリン  Pro P Proline
Gin Q グルタミン  Gin Q Glutamine
Arg R ァノレギニン  Arg R Anoreginin
Ser S セリン  Ser S Serine
Thr T トレオニン  Thr T threonine
Val V パリン  Val V Palin
Trp w トリブトファン Tyr Y チロシン Trp w Tribute fan Tyr Y tyrosine
Asx ァスパラギン又はァスパラギン酸  Asx asparagine or aspartic acid
Glx グルタミン又はグルタミン酸  Glx glutamine or glutamic acid
Xaa 不明又は他のアミノ酸。  Xaa Unknown or other amino acid.
塩基  Base
記号 意味  Symbol Meaning
a アデニン  a Adenine
g グァニン  g Guanin
c シトシン  c cytosine
t チミン  t thymine
U ゥラシル  U Peracil
r グァニン又はアデニンプリン m アデニン又はシトシンアミノ基  r Guanine or adenine purine m Adenine or cytosine amino group
k グァニン又はチミン Zゥラシノレケト基  k Guanine or thymine Z ゥ lasinoleketo group
s グァニン又はシトシン  s Guanine or cytosine
w アデニン又はチミン Zゥラシル  w Adenine or thymine Z ゥ racil
b グァニン又はシトシン又はチミン Zゥラシノレ  b Guanine or cytosine or thymine Z
d アデニン又はグァニン又はチミン Zゥラシル  d Adenine or guanine or thymine Z ゥ racil
h アデニン又はシトシン又はチミン Zゥラシル  h Adenine or cytosine or thymine Z ゥ racil
V アデニン又はグァニン又はシトシン  V Adenine or guanine or cytosine
n アデニン又はグァニン又はシトシン又はチミン Zゥラシル、不明、または他の塩 基。  n Adenine or guanine or cytosine or thymine Z-racil, unknown or other base.
[0041] 本明細書では、アミノ酸配列および塩基配列の類似性、同一性および相同性の比 較は、配列分析用ツールである BLASTを用いてデフォルトパラメータを用いて算出 される。  [0041] In the present specification, the comparison of similarity, identity and homology between amino acid sequences and nucleotide sequences is calculated using BLAST, a sequence analysis tool, with default parameters.
[0042] 本明細書において用いられる分子生物学的手法、生化学的手法、微生物学的手 法は、当該分野において周知であり慣用されるものであり、例えば、 Sambrook J. e t al. (1989) . Molecular Cloning： A Laboratory Manual, Cold Spring Harborおよびその 3rd Ed. (2001)； Ausubel, F. M. (1987) . Current Pro tocols in Molecular Biology, Greene Pub. AssociatESand Wiley— Inte rscience ; Ausubel, F. M. (1989) . Short Protocols in Molecular Biolog y : A Compendium of Methods from Current Protocols in Molecul ar Biology, Greene Pub. Associat ESand Wiley— Interscience ;Innis, M. A. (1990) . PCR Protocols： A Guide to Methods and Applicatio ns, Academic Press ; Ausubel, F. M. (1992) . Short Protocols in Mole cular Biology： A Compendium of Methods from Current Protocols in Molecular Biology, Greene Pub. Associates ; Ausubel, F. M. (1995 ) . Short Protocols in Molecular Biology： A Compendium of Metho ds from Current Protocols in Molecular Biology, Greene Pub. Asso ciates ;Innis, M. A. et al. (1995) . PCR Strategies, Academic Press ;Au subel, F. M. (1999) . Short Protocols in Molecular Biology: A Comp endium of Methods from Current Protocols in Molecular Biology, Wiley, and annual updates ; Sninsky, J. J. et al. (1999) . PCR Applicati ons : Protocols for Functional Genomics, Academic Press、另 ll冊実験医 学「遺伝子導入 &発現解析実験法」羊土社、 1997などに記載されており、これらは 本明細書において関連する部分 (全部であり得る）が参考として援用される。 [0042] The molecular biological techniques, biochemical techniques, and microbiological techniques used herein are well known and commonly used in the art, and are described, for example, in Sambrook J. e. t al. (1989). Molecular Cloning: A Laboratory Manual, Cold Spring Harbor and its 3rd Ed. (2001); Ausubel, FM (1987) .Current Protocols in Molecular Biology, Greene Pub. AssociatESand Wiley—Interscience; Ausubel , FM (1989) .Short Protocols in Molecular Biology: A Compendium of Methods from Current Protocols in Molecular Biology, Greene Pub. Associat ESand Wiley— Interscience; Innis, MA (1990) .PCR Protocols: A Guide to Methods and Applicatio ns, Academic Press; Ausubel, FM (1992) .Short Protocols in Molecular Biology: A Compendium of Methods from Current Protocols in Molecular Biology, Greene Pub.Associates; Ausubel, FM (1995) .Short Protocols in Molecular Biology: A Compendium of Methods from Current Protocols in Molecular Biology, Greene Pub.Asso ciates; Innis, MA et al. (1995) .PCR Strategies, Academic Press; Au subel, FM (1999) .Short Protocols in Molecular Biology: A Comp endium of Methods from Current Protocols in Molecular Biology, Wiley , and annual updates; Sninsky, JJ et al. (1999) .PCR Applications: Protocols for Functional Genomics, Academic Press, II. And the relevant portions (which may be all) are incorporated herein by reference.
(改変）  (Modification)
本発明においては、改変体もまた本発明の範囲内に入ることが理解される。そのよ うな改変を設計する際に、アミノ酸の疎水性指数が考慮され得る。ペプチドにおける 相互作用的な生物学的機能を与える際の疎水性アミノ酸指数の重要性は、一般に 当該分野で認められている（Kyte. Jおよび Doolittle, R. F. J. Mol. Biol. 157 (1 ) : 105- 132, 1982)。アミノ酸の疎水的性質は、生成したペプチドの二次構造に 寄与し、次いでそのペプチドと他の分子 (例えば、酵素、基質、レセプター、 DNA、 抗体、抗原など）との相互作用を規定する。各アミノ酸は、それらの疎水性および電 荷の性質に基づく疎水性指数を割り当てられる。それらは:イソロイシン（+4. 5)；バ リン（+4. 2)；ロイシン（ + 3. 8)；フエ-ルァラニン（ + 2. 8)；システィン Zシスチン（ + 2. 5)；メチォニン（ + 1. 9)；ァラニン（ + 1. 8)；グリシン（一0. 4)；スレオニン（一0 . 7) ;セリン（一0. 8)；トリプトファン（一0. 9) ;チロシン（一1. 3) ;プロリン（一1. 6) ;ヒ スチジン（一3. 2)；グルタミン酸（一 3. 5) ;グルタミン（一 3. 5)；ァスパラギン酸（一3 . 5)；ァスパラギン（一3. 5) ;リジン（一3. 9)；およびアルギニン（一4. 5) )である。 In the present invention, it is understood that variants also fall within the scope of the present invention. In designing such modifications, the hydropathic index of amino acids can be considered. The importance of the hydrophobic amino acid index in conferring interactive biological functions on peptides is generally recognized in the art (Kyte. J and Doolittle, RFJ Mol. Biol. 157 (1): 105-). 132, 1982). The hydrophobic nature of amino acids contributes to the secondary structure of the resulting peptide, which in turn defines the interaction of the peptide with other molecules (eg, enzymes, substrates, receptors, DNA, antibodies, antigens, etc.). Each amino acid is assigned a hydrophobicity index based on its hydrophobicity and the nature of the charge. They are: isoleucine (+4.5); Phosphorus (+4.2); Leucine (+3.8); Hueralanine (+2.8); Cystine Z cystine (+2.5); Methionine (+1.9); Alanine (+1.8) Glycine (10.4); Threonine (10.7); Serine (10.8); Tryptophan (10.9); Tyrosine (11.3); Proline (11.6); Histidine (1.3.2); Glutamic acid (13.5); Glutamine (13.5); Aspartic acid (13.5); Asparagine (13.5); Lysine (13.9); And arginine (1-4.5)).
[0044] あるアミノ酸を、同様の疎水性指数を有する他のアミノ酸により置換して、そして依 然として同様の生物学的機能を有するペプチド (例えば、酵素活性において等価な ペプチド)を生じさせ得ることが当該分野で周知である。このようなアミノ酸置換にお いて、疎水性指数が ± 2以内であることが好ましぐ ± 1以内であることがより好ましく 、および ±0. 5以内であることがさらにより好ましい。疎水性に基づくこのようなァミノ 酸の置換は効率的であることが当該分野において理解される。  [0044] The ability to substitute an amino acid for another amino acid having a similar hydrophobicity index and still yield a peptide having a similar biological function (eg, a peptide equivalent in enzymatic activity) Are well known in the art. In such amino acid substitutions, the hydrophobicity index is preferably within ± 2, more preferably within ± 1, and even more preferably within ± 0.5. It is understood in the art that such substitution of amino acids based on hydrophobicity is efficient.
[0045] 親水性指数もまた、ポリペプチドの設計にお!、て考慮され得る。米国特許第 4, 55 4, 101号に記載されるように、以下の親水性指数がアミノ酸残基に割り当てられてい る：アルギニン（ + 3. 0)；リジン（ + 3. 0)；ァスパラギン酸（ + 3. 0± 1)；グルタミン酸（ + 3. 0± 1)；セリン（ + 0. 3)；ァスパラギン（ + 0. 2) ;グルタミン（ + 0. 2) ;グリシン（0 )；スレオニン（一0. 4)；プロリン（一0. 5± 1)；ァラニン（一0. 5)；ヒスチジン（一0. 5) ；システィン（一1. 0)；メチォニン（一1. 3)；バリン（一 1. 5)；ロイシン（一1. 8)；ィソロ イシン（一 1. 8) ;チロシン（一2. 3)；フエ-ルァラニン（一2. 5)；およびトリプトファン（ 3. 4)。アミノ酸が同様の親水性指数を有しかつ依然として生物学的等価体を与え 得る別のものに置換され得ることが理解される。このようなアミノ酸置換において、親 水性指数が ± 2以内であることが好ましぐ ± 1以内であることがより好ましぐおよび ±0. 5以内であることがさらにより好ましい。  [0045] The hydrophilicity index can also be considered in designing polypeptides. As described in US Pat. No. 4,554,101, the following hydrophilicity indices have been assigned to amino acid residues: arginine (+3.0); lysine (+3.0); aspartic acid Glutamic acid (+ 3.0 ± 1); Serine (+0.3); Asparagine (+0.2); Glutamine (+0.2); Glycine (0); Threonine (+ 3.0 ± 1); Proline (0.5 ± 1); alanine (0.5); histidine (0.5); cysteine (1.1); methionine (1.3); valine ( I-1.5); Leucine (1-1.8); Isoloisin (1-1.8); Tyrosine (1-2.3); Fehlalanine (1-2.5); and Tryptophan (3.4). It is understood that an amino acid can be substituted for another that has a similar hydrophilicity index and still provide a bioisostere. In such amino acid substitutions, the hydrophilicity index is preferably within ± 2, more preferably within ± 1, and even more preferably within ± 0.5.
[0046] 本発明にお 、て、「保存的置換」とは、アミノ酸置換にぉ 、て、元のアミノ酸と置換さ れるアミノ酸との親水性指数または Zおよび疎水性指数が上記のように類似して 、る 置換をいう。保存的置換の例としては、例えば、親水性指数または疎水性指数が、士 2以内のもの同士、好ましくは ± 1以内のもの同士、より好ましくは ±0. 5以内のもの 同士のものが挙げられるがそれらに限定されない。  [0046] In the present invention, "conservative substitution" refers to an amino acid substitution in which the hydrophilicity index or Z and hydrophobicity index of the original amino acid and the amino acid to be substituted are similar to those described above. And permutation. Examples of conservative substitution include, for example, those having a hydrophilicity index or a hydrophobicity index of 2 or less, preferably ± 1 or less, more preferably ± 0.5 or less. But not limited to them.
[0047] 本明細書において保存的置換は、例えば、疎水性アミノ酸 (ァラニン、パリン、ロイ シン、イソロイシンなど）同士、酸性アミノ酸（グルタミン酸、ァスパラギン酸、 4—カルボ キシグルタミン酸。アミノクェン酸など）同士、塩基性アミノ酸 (アルギニン、ヒスチジン 、リジンなど）同士、芳香族アミノ酸 (フエ-ルァラニン、チロシン、トリプトファンなど）同 士で交換することによつても作製することができることが理解される。従って、保存的 置換の例は、当業者に周知であり、例えば、次の各グループ内での置換:アルギニン およびリジン；グルタミン酸およびァスパラギン酸；セリンおよびスレオニン；グルタミン およびァスパラギン；ならびにパリン、ロイシン、およびイソロイシン、などが挙げられる 力 Sこれらに限定されない。 [0047] As used herein, conservative substitutions include, for example, hydrophobic amino acids (alanine, palin, leu). Syn, isoleucine, etc.), acidic amino acids (glutamic acid, aspartic acid, 4-carboxyglutamic acid, aminocunic acid, etc.), basic amino acids (arginine, histidine, lysine, etc.), aromatic amino acids (feralanine, tyrosine, It is understood that it can also be produced by exchanging with another person (such as tryptophan). Thus, examples of conservative substitutions are well known to those skilled in the art and include, for example, substitutions within each of the following groups: arginine and lysine; glutamic and aspartic acid; serine and threonine; glutamine and asparagine; Isoleucine, and the like, are not limited thereto.
本明細書において、「改変体」とは、ポリペプチドまたはポリヌクレオチドに関して言 及するとき、もとのポリペプチドまたはポリヌクレオチドなどの物質に対して、一部が変 更されているものをいう。そのような改変体としては、置換改変体、付加改変体、欠失 改変体、短縮 (truncated)改変体、対立遺伝子変異体などが挙げられる。対立遺伝 子 (allele)とは、同一遺伝子座に属し、互いに区別される遺伝的改変体のことをいう 。従って、「対立遺伝子変異体」とは、ある遺伝子に対して、対立遺伝子の関係にある 改変体をいう。そのような対立遺伝子変異体は、通常その対応する対立遺伝子と同 一または非常に類似性の高い配列を有し、通常はほぼ同一の生物学的活性を有す る力 まれに異なる生物学的活性を有することもある。「種相同体またはホモログ (ho molog)」とは、ある種の中で、ある遺伝子とアミノ酸レベルまたはヌクレオチドレベル で、相同性 (好ましくは、 60%以上の相同性、より好ましくは、 80%以上、 85%以上、 90%以上、 95%以上の相同性)を有するものをいう。そのような種相同体を取得する 方法は、本明細書の記載から明らかである。「オルソログ（ortholog)」とは、オルソロ ガス遺伝子（orthologous gene)とも!/、 、、二つの遺伝子がある共通祖先からの種 分化に由来する遺伝子をいう。例えば、多重遺伝子構造をもつヘモグロビン遺伝子 ファミリーを例にとると、ヒトおよびマウスの αヘモグロビン遺伝子はオルソログである 力 ヒトのひヘモグロビン遺伝子および βヘモグロビン遺伝子はパラログ (遺伝子重 複で生じた遺伝子)である。オルソログは、分子系統樹の推定に有用である。オルソ ログは、通常別の種においてもとの種と同様の機能を果たしていることがあり得ること から、本発明のオルソログもまた、本発明において有用であり得る。 [0049] 本明細書にぉ 、て「保存的（に改変された）改変体」は、アミノ酸配列および核酸配 列の両方に適用される。特定の核酸配列に関して、保存的に改変された改変体とは 、同一のまたは本質的に同一のアミノ酸配列をコードする核酸をいい、核酸がァミノ 酸配列をコードしない場合には、本質的に同一な配列をいう。遺伝コードの縮重のた め、多数の機能的に同一の核酸が任意の所定のタンパク質をコードする。例えば、コ ドン GCA、 GCC、 GCG、および GCUはすべて、アミノ酸ァラニンをコードする。した がって、ァラニンがコドンにより特定される全ての位置で、そのコドンは、コードされた ポリペプチドを変更することなぐ記載された対応するコドンの任意のものに変更され 得る。このような核酸の変動は、保存的に改変された変異の 1つの種である「サイレン ト改変（変異）」である。ポリペプチドをコードする本明細書中のすべての核酸配列は また、その核酸の可能なすべてのサイレント変異を記載する。当該分野において、核 酸中の各コドン（通常メチォニンのための唯一のコドンである AUG、および通常トリプ トフアンのための唯一のコドンである TGGを除く） 1S 機能的に同一な分子を産生す るため〖こ改変され得ることが理解される。したがって、ポリペプチドをコードする核酸 の各サイレント変異は、記載された各配列において暗黙に含まれる。好ましくは、そ のような改変は、ポリペプチドの高次構造に多大な影響を与えるアミノ酸であるシステ インの置換を回避するようになされ得る。このような塩基配列の改変法としては、制限 酵素などによる切断、 DNAポリメラーゼ、 Klenowフラグメント、 DNAリガーゼなどに よる処理等による連結等の処理、合成オリゴヌクレオチドなどを用いた部位特異的塩 基置換法（特定部位指向突然変異法； Mark Zoller and Michael Smith, Me thods in Enzymology, 100, 468— 500 (1983) )力挙げ、られる力 この他にも 通常分子生物学の分野で用いられる方法によって改変を行うこともできる。 As used herein, the term "variant" when referring to a polypeptide or polynucleotide refers to a substance in which a substance such as the original polypeptide or polynucleotide is partially 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. The term "ortholog" refers to a gene derived from speciation from a common ancestor with two genes,! /,,, And both orthologous genes. For example, in the case of the hemoglobin gene family having a multigene structure, the human and mouse α-hemoglobin genes are orthologs.The human human hemoglobin gene and β-hemoglobin gene are paralogs (genes generated by gene duplication). . Orthologs are useful for estimating molecular phylogenetic trees. Orthologs of the present invention may also be useful in the present invention, as orthologs can usually perform the same function in another species as the original species. [0049] As used herein, the term "conservative (modified) variants" applies to both amino acid sequences and nucleic acid sequences. With respect to a particular nucleic acid sequence, a conservatively modified variant refers to a nucleic acid that encodes the same or essentially the same amino acid sequence, and essentially the same if the nucleic acid does not encode an amino acid sequence. Sequence. Because of the degeneracy of the genetic code, many functionally identical nucleic acids encode any given protein. For example, the codons GCA, GCC, GCG, and GCU all encode the amino acid alanine. Thus, at every position where an alanine is specified by a codon, that codon can be changed to any of the corresponding codons described, without altering the encoded polypeptide. Such nucleic acid variation is a "silent modification (mutation)," which is one type of conservatively modified mutation. Every nucleic acid sequence herein which encodes a polypeptide also describes every possible silent variation of the nucleic acid. In the art, each codon in the nucleic acid (except AUG, which is usually the only codon for methionine, and TGG, which is usually the only codon for tryptophan) 1S Produces functionally identical molecules It will be understood that this can be modified. Accordingly, each silent variation of a nucleic acid which encodes a polypeptide is implicit in each described sequence. Preferably, such modifications may be made to avoid substitution of the amino acid cysteine, which greatly affects the conformation of the polypeptide. Examples of such base sequence modification methods include cleavage with a restriction enzyme or the like, ligation treatment with a DNA polymerase, Klenow fragment, DNA ligase, or the like, site-specific base substitution using a synthetic oligonucleotide, or the like. (Site-directed mutagenesis; Mark Zoller and Michael Smith, Methods in Enzymology, 100, 468-500 (1983)) You can do it too.
[0050] 本明細書中において、機能的に等価なペプチドを作製するために、アミノ酸の置換 のほかに、アミノ酸の付加、欠失、または修飾もまた行うことができる。アミノ酸の置換 とは、もとのペプチドを 1つ以上、例えば、 1〜3個のアミノ酸で置換することをいう。ァ ミノ酸の付加とは、もとのペプチド鎖に 1つ以上、例えば、 1〜3個のアミノ酸を付加す ることをいう。アミノ酸の欠失とは、もとのペプチドから 1つ以上、例えば、 1〜3個のァ ミノ酸を欠失させることをいう。アミノ酸修飾は、アミド化、カルボキシル化、硫酸化、ハ ロゲン化、アルキル化、グリコシル化、リン酸化、水酸化、ァシル化（例えば、ァセチル ィ匕)などを含むが、これらに限定されない。置換、または付加されるアミノ酸は、天然 のアミノ酸であってもよぐ非天然のアミノ酸、またはアミノ酸アナログでもよい。天然の アミノ酸が好ましい。 [0050] In the present specification, in addition to amino acid substitution, amino acid addition, deletion, or modification can also be performed to produce a functionally equivalent peptide. 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 amidation, carboxylation, sulfation, Examples include, but are not limited to, logenation, 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.
[0051] 本明細書において、ポリペプチドまたはポリヌクレオチドの「置換、付加または欠失」 とは、もとのポリペプチドまたはポリヌクレオチドに対して、それぞれアミノ酸もしくはそ の代替物、またはヌクレオチドもしくはその代替物力 置き換わること、付け加わること または取り除かれることをいう。このような置換、付加または欠失の技術は、当該分野 において周知であり、そのような技術の例としては、部位特異的変異誘発技術などが 挙げられる。置換、付加または欠失は、 1つ以上であれば任意の数でよぐそのような 数は、その置換、付加または欠失を有する改変体において目的とする機能 (例えば、 血管新生活性など）が保持される限り、多くすることができる。例えば、そのような数は [0051] As used herein, "substitution, addition or deletion" of a polypeptide or polynucleotide means amino acid or its substitute, or nucleotide or its substitute for the original polypeptide or polynucleotide, respectively. Physical ability 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 is
、 1または数個であり得、そして好ましくは、全体の長さの 20%以内、 10%以内、また は 3個以下、 2個以下、 1個以下などであり得る。 , One or several, and preferably can be within 20%, 10%, or less than 3, less than 2, less than 1, etc. of the total length.
[0052]  [0052]
(生理活性ペプチド） (Bioactive peptide)
本明細書にぉ ヽて「細胞生理活性物質」または「生理活性物質」 (physiologically active substance)とは、細胞または組織に作用する物質をいう。そのような作用 としては、例えば、その細胞または糸且織の制御、変化などが挙げられるがそれに限定 されない。生理活性物質には、サイト力インおよび増殖因子が含まれる。生理活性物 質は、天然に存在するものであっても、合成されたものでもよい。好ましくは、生理活 性物質は、細胞が産生するものまたはそれと同様の作用を有するものであるが改変 された作用を持つものであってもよい。本明細書では、生理活性物質はタンパク質形 態または核酸形態あるいは他の形態であり得るが、実際に作用する時点にぉ 、ては 、サイト力インは通常はタンパク質形態を意味する。  As used herein, the term “cell biologically active substance” or “physiologically active substance” refers to a substance that acts on cells or tissues. Examples of such an action include, but are not limited to, control and change of the cell or the fibrous tissue. Physiologically active substances include cytodynamics and growth factors. The physiologically active substance may be a naturally occurring substance or a synthetic substance. Preferably, the physiologically active substance is produced by cells or has the same action as that of the bioactive substance, but may have a modified action. As used herein, the bioactive agent can be in a protein form or a nucleic acid form or other form, but at the time it actually acts, cytoforce usually refers to the protein form.
[0053] 本明細書において使用される「サイト力イン」は、当該分野において用いられる最も 広義の意味と同様に定義され、細胞力 産生され同じまたは異なる細胞に作用する 生理活性物質をいう。サイト力インは、一般にタンパク質またはポリペプチドであり、免 疫応答の制禦作用、内分泌系の調節、神経系の調節、抗腫瘍作用、抗ウィルス作用 、細胞増殖の調節作用、細胞分化の調節作用などを有する。本明細書では、サイト 力インはタンパク質形態または核酸形態あるいは他の形態であり得るが、実際に作用 する時点においては、サイト力インは通常はタンパク質形態を意味する。 [0053] As used herein, "cytoforce in" is defined in the same broadest sense as used in the art, and is produced by cellular forces acting on the same or different cells. Refers to a physiologically active substance. Cytokines are generally proteins or polypeptides that regulate immune response, regulate the endocrine system, regulate the nervous system, have antitumor and antiviral effects, regulate cell proliferation, regulate cell differentiation. Etc. As used herein, cytoforce in can be in protein or nucleic acid form or other forms, but at the time it actually works, cytoforce in usually refers to protein form.
[0054] 本明細書において用いられる「増殖因子」または「細胞増殖因子」とは、本明細書で は互換的に用いられ、細胞の増殖を促進または制御する物質をいう。増殖因子は、 成長因子または発育因子ともいわれる。増殖因子は、細胞培養または組織培養にお いて、培地に添加されて血清高分子物質の作用を代替し得る。多くの増殖因子は、 細胞の増殖以外に、分ィ匕状態の制御因子としても機能することが判明している。  [0054] As used herein, "growth factor" or "cell growth factor" is used interchangeably herein and refers to a substance that promotes or controls cell growth. Growth factors are also called growth factors or growth factors. Growth factors can be added to the medium in cell or tissue culture to replace the action of serum macromolecules. Many growth factors have been found to function not only as cell growth but also as regulators of stagnation state.
[0055] サイト力インには、代表的には、インターロイキン類、ケモカイン類、コロニー刺激因 子のような造血因子、腫瘍壊死因子、インターフェロン類が含まれる。増殖因子として は、代表的には、血小板由来増殖因子 (PDGF)、上皮増殖因子 (EGF)、線維芽細 胞増殖因子 (FGF)、肝実質細胞増殖因子 (HGF)、血管内皮増殖因子 (VEGF)の ような増殖活性を有するものが挙げられる。  [0055] The cytokins typically include interleukins, chemokines, hematopoietic factors such as colony stimulating factors, tumor necrosis factors, and interferons. Typical growth factors include platelet-derived growth factor (PDGF), epidermal growth factor (EGF), fibroblast growth factor (FGF), hepatocyte growth factor (HGF), and vascular endothelial growth factor (VEGF )).
[0056] サイト力インおよび増殖因子などの生理活性物質は一般に、機能重複現象 (redun dancy)があることから、他の名称および機能 (例えば、細胞接着活性または細胞— 基質間の接着活性など)で知られるサイト力インまたは増殖因子であっても、本発明 に使用される生理活性物質の活性を有する限り、本発明において使用され得る。ま た、サイト力インまたは増殖因子は、本明細書における好ましい活性 (例えば、宿主の 細胞を呼び寄せる活性)を有してさえ 、れば、本発明の組織片または医薬の好まし い実施形態において使用することができる。  [0056] Biologically active substances such as cytodynamics and growth factors generally have other names and functions (eg, cell adhesion activity or cell-substrate adhesion activity, etc.) due to the phenomenon of redundancy. Any of the cytokins or growth factors known in the above can be used in the present invention as long as they have the activity of the bioactive substance used in the present invention. In addition, as long as the cytodynamic force or growth factor has a preferable activity herein (for example, an activity to attract a host cell), it is preferable in the preferred embodiment of the tissue piece or the medicament of the present invention. Can be used.
[0057] 本明細書において用いられ得る生理活性ペプチドは、抗腫瘍剤、抗生物質、解熱 ,鎮痛，消炎剤、鎮咳去痰剤、鎮静剤、筋弛緩剤、抗てんかん剤、抗潰瘍剤、抗うつ 剤、抗アレルギー剤、強心剤、不整脈治療剤、血管拡張剤、降圧利尿剤、糖尿病治 療剤、抗凝血剤、止血剤、抗結核剤、ホルモン剤、麻薬拮抗剤、骨吸収抑制剤、血 管新生抑制剤などまたはこれらの組み合わせの作用を有し得る。  [0057] The bioactive peptides that can be used herein include antitumor agents, antibiotics, antipyretics, analgesics, anti-inflammatory agents, antitussive expectorants, sedatives, muscle relaxants, antiepileptics, antiulcer agents, antidepressants. Agents, antiallergic agents, inotropic agents, antiarrhythmic agents, vasodilators, antihypertensive diuretics, antidiabetic agents, anticoagulants, hemostatic agents, antituberculous agents, hormonal agents, narcotics antagonists, bone resorption inhibitors, blood vessels It may have the action of a newborn inhibitor or the like or a combination thereof.
[0058] 生理活性ペプチドとしては、例えば、黄体形成ホルモン放出ホルモン (LH—RH) 、インスリン、ソマトスタチン、成長ホルモン、成長ホルモン放出ホルモン（GH—RH) 、プロラタチン、エリスロポイエチン、副腎皮質ホルモン、メラノサイト刺激ホルモン、甲 状腺ホルモン放出ホルモン (TRH)、甲状腺刺激ホルモン、黄体形成ホルモン、卵胞 刺激ホルモン、バソプレシン、ォキシトシン、カルシトニン、ガストリン、セクレチン、ノ ンクレオザィミン、コレシストキニン、アンジォテンシン、ヒト胎盤ラタトーゲン、ヒト絨毛 性ゴナドトロピン、エンケフアリン、エンドルフィン、キヨウトルフィン、タフトシン、サイモ ボイエチン、サイモシン、サイモチムリン、胸腺液性因子、血中胸腺因子、腫瘍壊死 因子、コロニー誘導因子、モチリン、ディノルフィン、ボンべシン、ニューロテンシン、 セルレイン、ブラジキュン、心房性ナトリウム排泄増加因子、神経成長因子、細胞増 殖因子、神経栄養因子、エンドセリン拮抗作用を有するペプチド類など、その誘導体 、これらのフラグメントまたはフラグメントの誘導体などを挙げることができるがそれらに 限定されない。 [0058] Examples of the physiologically active peptide include luteinizing hormone-releasing hormone (LH-RH) , Insulin, somatostatin, growth hormone, growth hormone releasing hormone (GH-RH), prolatatin, erythropoietin, adrenocortical hormone, melanocyte stimulating hormone, thyroid hormone releasing hormone (TRH), thyroid stimulating hormone, luteinizing hormone, Follicle-stimulating hormone, vasopressin, oxytocin, calcitonin, gastrin, secretin, noncleozymine, cholecystokinin, angiotensin, human placental ratatogen, human chorionic gonadotropin, enkephalin, endorphin, kiyotorphin, tuftsin, thymocystin Thymic factor, blood thymic factor, tumor necrosis factor, colony-inducing factor, motilin, dinorphin, bombesin, neurotensin, caerulein, bradycune , Atrial natriuretic factor, nerve growth factor, cell growth factor, neurotrophic factor, peptides having endothelin antagonism, derivatives thereof, and fragments or derivatives of these fragments. Not done.
[0059] また、生理活性を有するペプチドとしては、 LH— RH拮抗物質 (米国特許第 4, 08 6, 219号，同第 4, 124, 577号，同第 4, 253, 997号，同第 4, 317, 815号参照） を挙げることができるがそれらに限定されないことが理解される。  [0059] Peptides having physiological activities include LH-RH antagonists (US Pat. Nos. 4,086,219, 4,124,577, 4,253,997, and 4,253,997). 4, 317, 815), but is not limited thereto.
[0060] また、さらに生理活性を有するペプチドとしては、たとえばインスリン，ソマトスタチン ,ソマトスタチン誘導体 (米国特許第 4, 087, 390号，同第 4, 093, 574号，同第 4, 100, 117号，同第 4, 253, 998号参照），成長ホルモン，プロラタチン，副腎皮質刺 激ホルモン (ACTH) ,メラノサイト刺激ホルモン（MSH) ,甲状腺ホルモン放出ホル モンならびにその塩およびその誘導体 (特開昭 50— 121273号，特開昭 52— 1164 65号公報参照），甲状腺刺激ホルモン (TSH) ,黄体形成ホルモン (LH) ,卵胞刺激 ホルモン（FSH) ,バソプレシン，バソプレシン誘導体 {デスモプレシン〔日本内分泌 学会雑誌，第 54卷第 5号第 676〜691頁（1978)〕参照 },ォキシトシン，カルシトニ ン，副甲状腺ホルモン，グルカゴン，ガストリン，セクレチン，ノ ンクレオザィミン，コレ シストキニン，アンジォテンシン，ヒト胎盤ラタトーゲン，ヒト絨毛性ゴナドトロピン (HC G) ,エンケフアリン，エンケフアリン誘導体〔米国特許第 4, 277, 394号，ヨーロッパ 特許出願公開第 31567号公報参照〕，エンドルフィン，キヨウトルフィン，インターフエ ロン類 (例、 α型， j8型， 0型等），インターロイキン類 (例、 I, II, ΙΠ等），タフトシ ン ,サイモポイエチン，サイモシン，サイモスチムリン，胸腺液性因子 (THF) ,血中胸腺 因子 (FTS)およびその誘導体 (米国特許第 4, 229, 438号参照），およびその他の 胸腺因子〔医学のあゆみ，第 125卷，第 10号， 835— 843頁（1983年)〕，腫瘍壊死 因子（TNF) ,コロニー誘発因子（CSF) ,モチリン，ダイノルフィン，ボムべシン，ニュ 一口テンシン，セルレイン，ブラジキュン，ゥロキナーゼ，ァスパラギナーゼ，カリクレイ ン，サブスタンス Ρ,神経成長因子，細胞増殖因子，神経栄養因子，血液凝固因子 の第 VIII因子，第 IX因子，塩化リゾチーム，ポリミキシン Β,コリスチン，ダラミシジン， バシトラシンおよびエリスロポエチン (ΕΡΟ) ,エンドセリン拮抗作用を有するペプチド 類 (ヨーロッパ特許公開第 436189号，同第 457195号，同第 496452号，特開平 3 94692号，同 3— 130299号公報参照）などを挙げることができるがそれらに限定 されない。 Further, examples of peptides having further physiological activity include insulin, somatostatin, somatostatin derivatives (US Pat. Nos. 4,087,390, 4,093,574, 4,100,117, No. 4,253,998), growth hormone, prolatatin, adrenocorticotropic hormone (ACTH), melanocyte stimulating hormone (MSH), thyroid hormone releasing hormone and its salts and derivatives (Japanese Patent Application Laid-Open No. 50-121273). Thyroid stimulating hormone (TSH), luteinizing hormone (LH), follicle stimulating hormone (FSH), vasopressin, vasopressin derivatives {desmopressin [Journal of the Japan Endocrine Society, Vol. 54, vol. No. 5, pp. 676-691 (1978)], oxitosine, calcitonin, parathyroid hormone, glucagon, gastrin, secretin, noncleozymine, cholecystokinin Angiotensin, human placental ratatogen, human chorionic gonadotropin (HCG), enkephalin, enkephalin derivative [see U.S. Pat. No. 4,277,394, European Patent Application Publication No. 31567], endorphin, kyotorphin, interfe Lons (eg, α-type, j8-type, 0-type, etc.), interleukins (eg, I, II, ΙΠ, etc.), tuftsin Thymopoietin, thymosin, thymostymulin, thymic humoral factor (THF), blood thymic factor (FTS) and its derivatives (see US Pat. No. 4,229,438), and other thymic factors [Ayumi of Medicine, 125 Vol. 10, No. 835-843 (1983)], Tumor necrosis factor (TNF), colony-inducing factor (CSF), motilin, dynorphin, bombesin, new mouth tensin, cellulin, bradycun, perokinase, asparaginase , Kallikrein, substance 神 経, nerve growth factor, cell growth factor, neurotrophic factor, blood coagulation factor VIII, IX, lysozyme chloride, polymyxin Β, colistin, dalamicidin, bacitracin and erythropoietin (ΕΡΟ), endothelin antagonist Peptides having an action (European Patent Publication Nos. 436189, 457195, and 496452; Nos. 94692 and 3-130299), but are not limited thereto.
[0061] 本明細書において、本発明のペプチドが有する生理活性は、そのペプチドが本来 有する生理活性を保持しているかどうかを測定することによって判定することができる 。従って、本発明のペプチドは、それに含まれるべき生理活性ペプチドが通常有する 生理活性 (血管新生能など)を測定する任意の公知のアツセィによって、徐放能を判 定することができることが理解される。  [0061] In the present specification, the physiological activity of the peptide of the present invention can be determined by measuring whether or not the peptide retains the intrinsic biological activity. Therefore, it is understood that the peptide of the present invention can be determined for its sustained-release ability by any known assay for measuring the physiological activity (such as angiogenesis ability) of the physiologically active peptide to be contained therein. .
[0062] そのような生理活性は、例えば、血管新生能、血管ネットワーク形成能であり得る。 [0062] Such a physiological activity may be, for example, an angiogenesis ability, a vascular network formation ability.
[0063] 本明細書にぉ 、て「血管新生」とは、血管が新たに形成されることおよびそのように 形成する活性をいう。 [0063] As used herein, "angiogenesis" refers to the formation of new blood vessels and the activity of such formation.
[0064] 本明細書にぉ 、て「血管ネットワーク形成」とは、新生された血管または既存の血管 力 網状になることおよびそのように形成する活性を！、う。  [0064] As used herein, "vascular network formation" refers to the formation of new blood vessels or existing vascular forces and the activity of forming such a network! , U.
[0065] 本明細書において、血管ネットワーク形成能力は、血管新生インデックスで示され る。血管新生は、網状が形成された力どうか (例えば、分岐した血管がさらに別の血 管と結合すること、およびその連接点の数の増カロ）を観察することによって判定するこ とができる力 簡便には、血管ネットワーク形成インデックスは、本明細書において以 下のようにして算出される。  [0065] In the present specification, the ability to form a vascular network is indicated by an angiogenesis index. Angiogenesis is a force that can be determined by observing whether a reticulated force has formed (for example, the connection of a branched vessel to another blood vessel and the increase in the number of its junctions). For simplicity, the vascular network formation index is calculated in the present specification as follows.
[0066] まず、新生血管数を、計数する。計数は、 0. 79cmあたりの血管本数で決める。血  First, the number of new blood vessels is counted. The count is determined by the number of blood vessels per 0.79 cm. Blood
2  2
管数が、以下の本数のとき、右のスコアであると換算する。 0 : 0 When the number of tubes is the following number, it is converted to the right score. 0: 0
1〜： L0 : 1  1 to: L0: 1
11〜20 : 2  11-20: 2
21〜30 : 3  21-30: 3
31〜40 : 4  31-40: 4
41以上： 5  41 or more: 5
次に、新生した血管の長さを決定する。長さは、以下のようにして測定する。実体顕 微鏡 (ォリンパス、 SZX12、 Japan)にて観察した。得られた画像を Photoshop (登録 商標）（Adobe、 Japan)にて読み取り。ピクセルで計数する。  Next, the length of the new blood vessel is determined. The length is measured as follows. Observation was made with a stereomicroscope (Olympus, SZX12, Japan). The obtained image was read by Photoshop (registered trademark) (Adobe, Japan). Count in pixels.
100未満： 1  Less than 100: 1
100〜125未満： 2  100 to less than 125: 2
125〜150未満： 3  125 to less than 150: 3
150〜175未満： 4  150 to less than 175: 4
175〜200未満： 5  175 to less than 200: 5
200以上： 6。  More than 200: 6.
[0067] これらのうち双方ともスコアがよ!、ものが血管ネットワーク形成能が高 、こととみなす ことができることから、本明細書では、それらを積を血管ネットワーク形成能として評価 することができる。  [0067] Of these, both have a good score, and those having high scores can be regarded as having a high ability to form a vascular network. Therefore, in the present specification, their product can be evaluated as the ability to form a vascular network.
[0068] ネットワーク形成能にっ 、ては、組織での血管新生の状態を実体顕微鏡 (ォリンパ ス、 SZX12、Japan)にて観察することができる。得られた画像を Photoshop (登録商 標）（Adobe、 Japan)にて読み取りネットワーク能を以下のようにスコア化する。以下 のスコアを本明細書にぉ 、て「血管ネットワーク形成能」または「血管ネットワーク形成 インデックス」あるいは単に「ネットワークインデックス」とも!/、う。  [0068] Regarding the ability to form a network, the state of angiogenesis in a tissue can be observed with a stereoscopic microscope (Olympus, SZX12, Japan). The obtained image is read by Photoshop (registered trademark) (Adobe, Japan) and the network ability is scored as follows. In the present specification, the following scores are also referred to as “vascular network forming ability” or “vascular network forming index” or simply “network index”.
[0069] Nwl：ネットワーク形成前記で血管新生は認められるが、各新生血管は単独の状 fe。  Nwl: Network formation Although neovascularization is recognized as described above, each new blood vessel is a single fe.
[0070] Nw2 :ネットワーク形成中期であり、各新生血管同士は、はしご上の側枝が係った 状態。  [0070] Nw2: In the middle stage of network formation, each new blood vessel is in a state in which the side branch on the ladder is engaged.
[0071] Nw3 :ネットワーク完成期であり、はしご上側枝がさらに側枝を出した状態。 [0072] Nw4 :ネットワーク成熟期であり、広範囲に新生血管叢を示す状態。 Nw3: The network is in the final stage, and the ladder upper branch has more side branches. [0072] Nw4: Network maturation stage, showing a wide range of neovascular plexus.
[0073] 本発明にお 、て実証されるように、血管ネットワーク形成能があるとは、通常、その ような血管ネットワーク形成インデックス力 少なくとも 2であり、好ましくは、 2. 5以上 であり、より好ましくは、 3以上であり、さらに好ましくは 3. 5以上であることを意味する ことが理解される。 [0073] As demonstrated in the present invention, the ability to form a vascular network is usually such a vascular network formation index force of at least 2, preferably 2.5 or more, It is understood that it is preferably 3 or more, more preferably 3.5 or more.
[0074] 本明細書における「血管」は、当該分野において通常使用される意味で用いられ、 通常の動脈、静脈などのほか、毛細管を含む。  [0074] In the present specification, "blood vessel" is used in a meaning commonly used in the art, and includes a capillary, in addition to a normal artery, a vein, and the like.
[0075]  [0075]
(変異型ポリペプチドの作製方法） (Method for producing mutant polypeptide)
本発明のペプチドのアミノ酸の欠失、置換もしくは付加 (融合を含む）は、周知技術 である部位特異的変異誘発法または化学合成により実施することができる。かかる 1 もしくは数個のアミノ酸が欠失、置換もしくは付加は、 Molecular Cloning, A Lab oratory Manual, Second Edition, Cold spring Harbor Laboratory Pr ess (1989)、 Current Protocols in Molecular Biology, Supplement 1〜 38, JohnWiley & Sons (1987— 1997)、 Nucleic Acids Research, 10, 64 87 (1982)、 Proc. Natl. Acad. Sci. , USA, 79, 6409 (1982)、 Gene, 34, 31 5 (1985)、 Nucleic Acids Research, 13, 4431 (1985)、 Proc. Natl. Acad. Sci USA, 82, 488 (1985) , Proc. Natl. Acad. Sci. , USA, 81, 5662 (198 4)、 Science, 224, 1431 (1984)、 PCT WO85/00817 (1985) , Nature, 31 6, 601 (1985)等に記載の方法に準じて調製することができる。  The deletion, substitution, or addition (including fusion) of the amino acid of the peptide of the present invention can be performed by site-directed mutagenesis or chemical synthesis, which is a well-known technique. Such deletion or substitution or addition of one or several amino acids is performed by Molecular Cloning, A Laboratory Manual, Second Edition, Cold spring Harbor Laboratory Press (1989), Current Protocols in Molecular Biology, Supplement 1-38, John Wiley & Sons (1987-1997), Nucleic Acids Research, 10, 64 87 (1982), Proc. Natl. Acad. Sci., USA, 79, 6409 (1982), Gene, 34, 315 (1985), Nucleic Acids Research. Natl. Acad. Sci USA, 82, 488 (1985), Proc. Natl. Acad. Sci., USA, 81, 5662 (198 4), Science, 224, 1431 (1984). ), PCT WO85 / 00817 (1985), Nature, 316, 601 (1985) and the like.
[0076] (合成化学）  (Synthetic chemistry)
本発明で用いられる化合物、もしくはその塩またはそれらの水和物は、それ自体公 知の方法により容易に製造することができる。該方法の具体例として、例えば以下の 製造方法またはそれに準じる方法が挙げられる。  The compound used in the present invention, or a salt thereof or a hydrate thereof can be easily produced by a method known per se. Specific examples of the method include, for example, the following production method or a method analogous thereto.
[0077] 従って、本明細書におけるペプチド、化学物質、低分子などの因子は、合成化学 技術を用いて合成することができる。そのような合成化学技術は、当該分野において 周知の技術を用いることができる。そのような周知技術としては、例えば、 Fiesers' Reagents for Organic Synthesis (Fieser's Reagents forOrganic Synthesis) Tse- Lok Ho, John Wiley & Sons Inc (2002)などを参照することができる。 [0077] Therefore, factors such as peptides, chemical substances, and small molecules in the present specification can be synthesized using synthetic chemistry techniques. As such synthetic chemistry techniques, techniques well known in the art can be used. Such well-known techniques include, for example, Fiesers' Reagents for Organic Synthesis (Fieser's Reagents for Organic Synthesis) Tse-Lok Ho, John Wiley & Sons Inc (2002) and the like can be referred to.
[0078] 本発明の因子が化合物として利用される場合、塩形態で用いることができる。「塩」 としては、製薬上許容される塩が好ましぐ例えば無機塩基との塩、有機塩基との塩、 無機酸との塩、有機酸との塩、塩基性または酸性アミノ塩とのなどが挙げられる。無 機塩基との塩としては、ナトリウム塩、カリウム塩などのアルカリ金属塩、カルシウム塩 、マグネシウム塩、バリウム塩などのアルカリ土類金属塩、ならびにアルミニウム塩、ァ ンモ -ゥム塩などが挙げられる。有機塩基との塩としては、トリメチルァミン、トリェチル ァミン、ピリジン、ピコリン、ェタノ一ルァミン、ジェタノ一ルァミン、トリエタノールァミン 、ジシクロへキシルァミン、 N, N，一ジベンジルエチレンジァミンなどとの塩が挙げら れる。無機酸との塩としては、塩酸、フッ化水素酸、臭化水素酸、硝酸、硫酸、リン酸 、過塩素酸、ヨウ化水素酸などとの塩が挙げられる。有機酸との塩としては、ギ酸、酢 酸、トリフルォロ酢酸、フマル酸、シユウ酸、酒石酸、マレイン酸、クェン酸、コハク酸、 リンゴ酸、マンデル酸、ァスコルビン酸、乳酸、ダルコン酸、メタンスルホン酸、 p トル エンスルホン酸、ベンゼンスルホン酸などとの塩が挙げられる。塩基性アミノ酸との塩 としては、アルギニン、リジン、オル-チンなどとの塩が挙げられ、酸性アミノ酸との塩 としては、ァスパラギン酸、グルタミン酸などとの塩が挙げられる。  When the factor of the present invention is used as a compound, it can be used in the form of a salt. As the "salt", a pharmaceutically acceptable salt is preferred, for example, a salt with an inorganic base, a salt with an organic base, a salt with an inorganic acid, a salt with an organic acid, a basic or acidic amino salt, and the like. Is mentioned. Salts with inorganic bases include alkali metal salts such as sodium salt and potassium salt, alkaline earth metal salts such as calcium salt, magnesium salt and barium salt, and aluminum salt and ammonium salt. . Examples of the salt with an organic base include trimethylamine, triethylamine, pyridine, picoline, ethanoylamine, jetanoylamine, triethanolamine, dicyclohexylamine, N, N, and dibenzylethylenediamine. Salts. Examples of salts with inorganic acids include salts with hydrochloric acid, hydrofluoric acid, hydrobromic acid, nitric acid, sulfuric acid, phosphoric acid, perchloric acid, hydroiodic acid, and the like. Salts with organic acids include formic acid, acetic acid, trifluoroacetic acid, fumaric acid, oxalic acid, tartaric acid, maleic acid, citric acid, succinic acid, malic acid, mandelic acid, ascorbic acid, lactic acid, dalconic acid, and methanesulfonic acid. , P toluene sulfonic acid, benzene sulfonic acid and the like. Salts with basic amino acids include salts with arginine, lysine, ortin and the like. Salts with acidic amino acids include salts with aspartic acid, glutamic acid and the like.
[0079] 本発明の因子が化合物として利用される場合、水和物形態で用いることができる。「 水和物」としては、薬理学的に許容される水和物が好ましぐまた、含水塩も含まれ。 具体的には、一水和物、二水和物、六水和物等が挙げられる。  When the factor of the present invention is used as a compound, it can be used in the form of a hydrate. As the “hydrate”, a pharmacologically acceptable hydrate is preferred, and a hydrate is also included. Specific examples include monohydrate, dihydrate, hexahydrate and the like.
[0080] ペプチドの化学合成には、以下のような 9-フルォレ -ルメトキシカルボ-ル（Fmoc) 法によるペプチド合成を用いることができる。  [0080] For the chemical synthesis of peptides, peptide synthesis by the following 9-fluoromethoxycarbol (Fmoc) method can be used.
[0081] この方法としては、活性化セルロース膜を用いる方法（SigmaGenosys社の SPOT s)が例示される。活性ィ匕セルロース膜を用いる方法は、以下のようにして行う。 αアミ ノ基を 9 フルォレ -ルメトキシカルボ-ル（Fmoc)で、同じくカルボキシル基を活性 エステル (Opfp)ある 、は（Odhbt)で保護したアミノ酸誘導体溶液を、膜のマークさ れた位置に 1 μ 1程度 (Fmocアミノ酸 100〜 200 gに相当）をスポットする。この指定 の位置にはスぺーサーアームが結合し、その先端に遊離のァミノ基があるためにブロ ムフエノールブルー（BPB)で青く呈色する。これらのアミノ酸誘導体は 1—メチル—2 —ピロリジノン (NMF)に溶解して用いる。膜上のアミノ基と活性エステルが反応し、 アミド結合が形成される。過剰のアミノ酸誘導体をジメチルホルムアミドで洗 、流した 後、未反応アミノ基を無水酢酸 ZDMFで処理することによりァセチル化し、反応性を 失わせる（これをキヤッビングという）。次いで第 2級ァミンであるピぺリジン ZDMFに よってアミノ基を保護して 、た Fmoc基を外し、アミノ基を遊離させて次の伸長反応を 行う。なお、反応性を有する側鎖は t ブチルアルコール系の保護基 (Pmc (2, 2, 5 , 7, 8 ペンタメチルクロマン一 6—スルホ-ル）， OtBu (0— t—ブトキシ）、トリチル (Trt) , tert ブトキシカルボ-ル（tBoc) , tert ブトキシ（tBu)など）で保護してお したトリフルォロ酢酸 (TFA)で保護基を外す (脱保護化)ことによって、 目的の化合 物を得ることができる。 [0081] Examples of this method include a method using an activated cellulose membrane (SPOTs manufactured by Sigma Genosys). The method using the activated cellulose membrane is performed as follows. If the α-amino group is protected with 9-fluoromethoxycarbol (Fmoc) and the carboxyl group is also an active ester (Opfp), an amino acid derivative solution protected with (Odhbt) is placed at the marked position on the membrane. Spot about μ1 (equivalent to 100-200 g of Fmoc amino acid). A spacer arm is attached to this designated position, and since there is a free amino group at its tip, it is blocked. Colors blue with mufenol blue (BPB). These amino acid derivatives are dissolved in 1-methyl-2-pyrrolidinone (NMF) before use. The amino group on the membrane reacts with the active ester to form an amide bond. After the excess amino acid derivative is washed with dimethylformamide, the unreacted amino group is treated with acetic anhydride ZDMF to acetylate the compound, thereby losing the reactivity (this is referred to as cabbing). Next, the amino group is protected by piperidine ZDMF which is a secondary amine, the Fmoc group is removed, and the amino group is released to carry out the next extension reaction. The reactive side chains are t-butyl alcohol-based protecting groups (Pmc (2,2,5,7,8 pentamethylchroman-16-sulfol), OtBu (0-t-butoxy), trityl ( (Trt), tert-butoxycarbol (tBoc), tert-butoxy (tBu), etc.) to obtain the desired compound by removing the protecting group with trifluoroacetic acid (TFA) (deprotection) Can be.
[0082] 本発明の各方法において、 目的とする生成物は、反応液から夾雑物 (未反応減量 、副生成物、溶媒など)を、当該分野で慣用される方法 (例えば、抽出、蒸留、洗浄、 濃縮、沈澱、濾過、乾燥など）によって除去した後に、当該分野で慣用される後処理 方法 (例えば、吸着、溶離、蒸留、沈澱、析出、クロマトグラフィーなど)を組み合わせ て処理して単離し得る。  [0082] In each method of the present invention, the target product is obtained by removing contaminants (unreacted weight loss, by-products, solvent, etc.) from the reaction solution by a method commonly used in the art (for example, extraction, distillation, After removal by washing, concentration, precipitation, filtration, drying, etc.), isolation by a combination of post-treatment methods commonly used in the art (eg, adsorption, elution, distillation, precipitation, precipitation, chromatography, etc.). obtain.
[0083] 本明細書にぉ 、て、得られたィ匕合物の物性を決定するために、粉末 X線回折を用 いた解析法が用いられ得る。  [0083] In the present specification, an analytical method using powder X-ray diffraction can be used to determine the physical properties of the obtained compound.
[0084] (コンビナトリアルケミストリ）  [0084] (Combinatorial Chemistry)
本発明で使用する化合物は、例えば、コンビナトリアルケミストリー技術、醱酵方法 、植物および細胞抽出手順などが挙げられるがこれらに限定されない、いずれかの 手段により、作製することができる力または入手することができる。コンビナトリアルライ ブラリを作成する方法は、当該技術分野で周知である。例えば、 E. R. Felder, Chi mia 1994, 48, 512— 541 ;Gallopら、 Med. Chem. 1994, 37, 1233— 125 1 ;R. A. Houghten, Trends Genet. 1993, 9, 235— 239 ;Houghtenら、 Nat ure 1991, 354, 84— 86 ; Lamら、 Nature 1991, 354, 82— 84 ;Carellら、 Ch em. Biol. 1995, 3, 171— 183 ;Maddenら、 Perspectives in Drug Discove ry and Design2, 269— 282 ; Cwirlaら、 Biochemistry 1990, 87, 6378-6 382 ;Brennerら、 Proc. Natl. Acad. Sci. USA 1992, 89, 5381— 5383 ;Gor donら、 Med. Chem. 1994, 37, 1385— 1401 ;Leblら、 Biopolymers 1995 , 37 177— 198 ;およびそれらで引用された参考文献を参照のこと。これらの参考 文献は、その全体を、本明細書中で参考として援用する。 The compounds used in the present invention can be prepared or obtained by any means including, but not limited to, combinatorial chemistry techniques, fermentation methods, plant and cell extraction procedures, and the like. it can. Methods for making combinatorial libraries are well known in the art. For example, ER Felder, Chimia 1994, 48, 512-541; Gallop et al., Med. Chem. 1994, 37, 1233-1251; RA Houghten, Trends Genet. 1993, 9, 235-239; Houghten et al., Nature. 1991, 354, 84-86; Lam et al., Nature 1991, 354, 82-84; Carell et al., Chem. Biol. 1995, 3, 171-183; Madden et al., Perspectives in Drug Discove. ry and Design2, 269-282; Cwirla et al., Biochemistry 1990, 87, 6378-6 382; Brenner et al., Proc. Natl. Acad. Sci. USA 1992, 89, 5381-5383; Gor don et al., Med. Chem. 1994. , 37, 1385-1401; Lebl et al., Biopolymers 1995, 37 177-198; and the references cited therein. These references are incorporated by reference herein in their entirety.
[0085] (スクリーニング）  [0085] (Screening)
本明細書において「スクリーニング」とは、 目的とするある特定の性質をもつ生物ま たは物質などの標的を、特定の操作 Z評価方法で多数を含む集団の中から選抜す ることをいう。スクリーニングのために、本発明の因子 (例えば、抗体）、ポリペプチドま たは核酸分子を使用することができる。スクリーニングは、インビトロ、インビボなど実 在物質を用いた系を使用してもよぐインシリコ（コンピュータを用いた系）の系を用い て生成されたライブラリーを用いてもよい。本発明では、所望の活性を有するスクリー ユングによって得られた化合物もまた、本発明の範囲内に包含されることが理解され る。また本発明では、本発明の開示をもとに、コンピュータモデリングによる薬物が提 供されることち企図される。  As used herein, the term “screening” refers to selecting a target such as a target organism or substance having a specific property from a population including a large number by a specific operation Z evaluation method. For screening, an agent (eg, an antibody), polypeptide or nucleic acid molecule of the present invention can be used. For screening, a library generated using an in silico (computer-based) system or a system using an existing substance such as in vitro or in vivo may be used. In the present invention, it is understood that the compounds obtained by the screening having the desired activity are also included in the scope of the present invention. In the present invention, it is contemplated that a drug by computer modeling is provided based on the disclosure of the present invention.
[0086] 1実施形態において、本発明は、本発明のタンパク質または本発明のポリペプチド 、あるいはその生物学的に活性な部分に結合する力、またはこれらの活性を調節す る、候補ィ匕合物もしくは試験化合物をスクリーニングするためのアツセィを提供する。 本発明の試験化合物は、当該分野において公知のコンビナトリアルライブラリ一法に おける多数のアプローチの任意のものを使用して得られ得、これには、以下が挙げら れる：生物学的ライブラリー；空間的にアクセス可能な平行固相もしくは溶液相ライブ ラリー;逆重畳を要する合成ライブラリ一法；「1ビーズ 1ィヒ合物」ライブラリ一法;およ びァフィユティークロマトグラフィー選択を使用する合成ライブラリ一法。生物学的ライ ブラリーアプローチはペプチドライブラリーに限定されるが、他の 4つのアプローチは 、ペプチド、非ペプチドオリゴマーもしくは化合物の低分子ライブラリーに適用可能で ある（Lam (1997) Anticancer Drug Des. 12 : 145)。  [0086] In one embodiment, the present invention provides a candidate conjugate that modulates the ability to bind to the protein of the invention or the polypeptide of the invention, or a biologically active portion thereof, or to modulate these activities. The present invention provides an assay for screening substances or test compounds. Test compounds of the present invention can be obtained using any of a number of approaches in one of the combinatorial library methods known in the art, including: biological libraries; Accessible solid-phase or solution-phase libraries; one synthetic library requiring deconvolution; one single-bead-one compound library; and synthetic libraries using affinity chromatography selection. One law. Biological library approaches are limited to peptide libraries, while the other four approaches are applicable to small molecule libraries of peptides, non-peptide oligomers or compounds (Lam (1997) Anticancer Drug Des. 12 : 145).
[0087] 分子ライブラリーの合成のための方法の例は、当該分野において、例えば以下に 見出され得る： DeWittら（1993) Proc. Natl. Acad. Sci. USA 90 : 6909 ;Erbら (1994) Proc. Natl. Acad. Sci. USA 91:11422; Zuckermann¾ ( 1994) J. M ed. Chem 37: 2678 ;Cho¾ (1993) Science 261:1303; Carrell¾ ( 1994) An gew Chem. Int. Ed. Engl.33:2059;Carell¾(1994) Angew Chem. Int. E d. Engl.33 :2061;および Gallopら（1994) Med. Chem 37:1233。 [0087] Examples of methods for the synthesis of molecular libraries can be found in the art, for example: DeWitt et al. (1993) Proc. Natl. Acad. Sci. USA 90: 6909; Erb et al. Natl. Acad. Sci. USA 91: 11422; Zuckermann¾ (1994) J. Med.Chem 37: 2678; Cho¾ (1993) Science 261: 1303; Carrell¾ (1994) Angew Chem. Int. Ed. Engl. 33: 2059; Carell¾ (1994) Angew Chem. Int. Ed. Engl. 33: 2061; and Gallop et al. (1994) Med. Chem 37: 1233.
[0088] 化合物のライブラリ一は、溶液中で（例えば、 Houghten(1992)BioTechniques  [0088] A library of compounds is prepared in solution (eg, in Houghten (1992) BioTechniques).
13:412〜421)、あるいはビーズ上（Lam(1991)Nature 354:82〜84)、チッ プ上（Fodor( 1993) Nature 364: 555〜556)、細菌（Ladner 米国特許第 5, 2 23, 409号）、胞子（Ladner、上記）、プラスミド（Cullら（1992) Proc. Natl. Acad. Sci. USA 89:1865〜1869)またはファージ上（3。01 ぉょび3111辻11(1990)3(^ nce 249 :386〜390; Devlin (1990) Science 249:404〜406;Cwirlaら（199 0) Proc. Natl. Acad. Sci. U. S. A.87:6378〜6382;Felici(1991)J Mol B iol 222 : 301〜310 ; Ladner上記）にお!/、て示され得る。 13: 412-421) or on beads (Lam (1991) Nature 354: 82-84), on chips (Fodor (1993) Nature 364: 555-556), and on bacteria (Ladner U.S. Pat. No. 409), spores (Ladner, supra), plasmids (Cull et al. (1992) Proc. Natl. Acad. Sci. USA 89: 185-1869) or on phage (3.01 び 3111 Tsuji 11 (1990) 3 ( ^ nce 249: 386-390; Devlin (1990) Science 249: 404-406; Cwirla et al. (1990) Proc. Natl. Acad. Sci. USA 87: 6378-6382; Felici (1991) J Mol Biol 222: 301-310; Ladner supra)!
[0089] (医薬、処置、予防）  [0089] (medicine, treatment, prevention)
本明細書にぉ 、て「予防 (する）」は、生物が病気にかかる（contract)力または異 常な状態を発生する可能性を減少させることを 、う。  As used herein, "preventing" refers to reducing the ability of an organism to contract or develop an abnormal condition.
[0090] 本明細書において「処置 (する）」は、治療効果を有すること、および生物における 異常な状態を少なくとも部分的に軽減する力または抑止することをいう。  [0090] As used herein, "treating" refers to having a therapeutic effect and the ability to reduce or at least partially alleviate an abnormal condition in an organism.
[0091] 本明細書において「治療効果」は、異常な状態を引き起こす力またはこれに寄与す る阻害因子または活性ィ匕因子がもたらす改善効果をいう。治療効果は、異常な状態 の症状の 1つ以上をある程度緩和する。異常な状態の処置に関して、治療効果とは 、以下の 1つ以上をいい得る：（a)細胞の増殖（proliferation)、増殖（growth)、お よび Zまたは分ィ匕における増加；（b)細胞死の阻害 (すなわち、遅らせることまたは停 止させること）；（c)変性の阻害；（d)異常な状態に関連する症状の 1つ以上をある程 度緩和する；および (e)罹患した細胞集団の機能を強化すること。異常な状態に対す る効力を示すィ匕合物は、本明細書中に記載されるように同定され得る。  [0091] As used herein, "therapeutic effect" refers to the ability to cause an abnormal condition or the ameliorating effect provided by an inhibitory factor or an active factor that contributes thereto. The effect of treatment is to alleviate one or more of the symptoms of the abnormal condition. For the treatment of an abnormal condition, a therapeutic effect can refer to one or more of the following: (a) cell proliferation, growth, and an increase in Z or S; (C) inhibition of degeneration; (d) alleviating one or more of the symptoms associated with the abnormal condition; and (e) affected cells. To strengthen the function of the group. Conjugates that exhibit efficacy against abnormal conditions can be identified as described herein.
[0092] 本明細書において「被検体」（subject)とは、本発明が適用される任意の系（例え ば、生物）をいい、ヒトなどの動物が対象である場合は「患者」ともいわれる。患者また は被検体は好ましくは、ヒトであり得る。被検体は、治療において用いられ得ることが 多いが、本明細書では、任意の系を記述するために用いられる。 [0092] In the present specification, the "subject" refers to any system (for example, an organism) to which the present invention is applied, and when an animal such as a human is the subject, it is also referred to as a "patient". . The patient or subject can preferably be a human. The subject may be used in therapy Often used herein to describe any system.
[0093] 本明細書において「病因」とは、被検体の疾患、障害または状態 (本明細書におい て、総称して「病変」ともいい、植物では病害ともいう）に関連する因子をいい、例えば 、原因となる病原物質 (病原因子）、病原体、病変細胞、病原ウィルスなどが挙げられ るがそれらに限定されない。  [0093] As used herein, the term "pathogenesis" refers to a factor associated with a disease, disorder, or condition of a subject (in the present specification, also collectively referred to as "lesion" or as a disease in plants). Examples include, but are not limited to, causative pathogens (pathogenic factors), pathogens, diseased cells, pathogenic viruses, and the like.
[0094] 本発明が対象とする「疾患」は、任意の疾患であり得る。そのような疾患としては、生 活習慣病 (例えば、歯周病、高血圧、高脂血症、糖尿病、心臓病、動脈硬化性疾患 、など）、癌、感染症、アレルギー、脳梗塞、痴呆症、肥満、不妊症、精神神経疾患、 白内障、早老症、紫外線放射線過敏症などが挙げられるがそれらに限定されない。 本発明は、例えば、国民の生活習慣病である歯周病において、歯肉の血行不良を 伴う歯槽骨の吸収が存在する場合、局所に血管新生能のあるペプチドまたは骨再生 能のあるペプチドを一定期間（歯槽骨再生の 1〜2ヶ月）作用させることによって歯肉 の血行不良の改善および歯槽骨の再建を図ることができる。  [0094] The "disease" targeted by the present invention may be any disease. Such diseases include lifestyle-related diseases (eg, periodontal disease, hypertension, hyperlipidemia, diabetes, heart disease, arteriosclerotic disease, etc.), cancer, infectious diseases, allergies, cerebral infarction, dementia. , Obesity, infertility, neuropsychiatric disorders, cataracts, progeria, ultraviolet radiation hypersensitivity, and the like. The present invention, for example, in the case of periodontal disease, which is a lifestyle-related disease of the nation, when alveolar bone resorption accompanied by poor gingival blood circulation is present, a peptide having a local angiogenesis ability or a peptide having a bone regeneration ability can be fixed. By acting for a period (1 to 2 months of alveolar bone regeneration), it is possible to improve poor gingival blood circulation and reconstruct the alveolar bone.
[0095] 本発明が対象とする「障害」は、生物におけるその正常な機能力 逸脱する意の障 害であり得る。  [0095] The "disorder" targeted by the present invention may be a disorder that deviates from its normal functional ability in an organism.
[0096] 本明細書において「異常な状態」は、生物におけるその正常な機能力も逸脱する、 生物の細胞または組織における機能をいう。異常な状態は、細胞増殖、細胞分化、 細胞シグナル伝達、または細胞生存に関連し得る。異常な状態としてはまた、血管形 成の異常、アポトーシスの異常、神経伝達における異常、肥満、網膜変性のような糖 尿病合併症、ならびにグルコースの取り込みおよび代謝における不規則性、ならび に脂肪酸の取り込みおよび代謝における不規則性が挙げられ得る。  [0096] As used herein, "abnormal condition" refers to a function of an organism in a cell or tissue that also deviates from its normal functioning ability. The abnormal condition may be associated with cell proliferation, cell differentiation, cell signaling, or cell survival. Abnormal conditions also include abnormal vascular formation, abnormal apoptosis, abnormalities in neurotransmission, obesity, complications of diabetes such as retinal degeneration, and irregularities in glucose uptake and metabolism, and fatty acid Irregularities in uptake and metabolism may be mentioned.
[0097] 本明細書において「診断、予防、処置または予後上有効な量」とは、それぞれ、診 断、予防、処置 (または治療)または予後において、医療上有効であると認められる 程度の量をいう。このような量は、当該分野において周知の技法を用いて当業者が 種々のパラメータを参酌しながら決定することができる。  [0097] As used herein, the term "diagnostic, prophylactic, treatment or prognostically effective amount" refers to an amount that is considered to be medically effective in diagnosis, prevention, treatment (or therapy) or prognosis, respectively. Say. Such an amount can be determined by one of ordinary skill in the art using techniques well known in the art and considering various parameters.
[0098] 本発明のペプチドが医薬として使用される場合、そのような組成物は、薬学的に受 容可能なキャリアなどをさらに含み得る。本発明の医薬に含まれる薬学的に受容可 能なキャリアとしては、当該分野において公知の任意の物質が挙げられる。 [0099] 本明細書において使用されるキャリアは、薬学的に受容可能であることが好ましぐ そのようなキャリアとしては、抗酸化剤、保存剤、着色料、風味料、および希釈剤、乳 ィ匕剤、懸濁化剤、溶媒、フィラー、増量剤、緩衝剤、送達ビヒクル、希釈剤、賦形剤お よび Zまたは薬学的アジュバントが挙げられるがそれらに限定されない。代表的には 、本発明の医薬は、本発明のペプチド、またはその改変体もしくは誘導体を、 1っ以 上の生理的に受容可能なキャリア、賦形剤または希釈剤とともに含む組成物の形態 で投与される。例えば、適切なビヒクルは、注射用水、生理的溶液、または人工脳脊 髄液であり得、これらには、非経口送達のための組成物に一般的な他の物質を補充 することが可能である。 [0098] When the peptide of the present invention is used as a medicament, such a composition may further include a pharmaceutically acceptable carrier and the like. Pharmaceutically acceptable carriers included in the medicament of the present invention include any substance known in the art. [0099] The carrier used herein is preferably pharmaceutically acceptable. Such carriers include antioxidants, preservatives, colorings, flavorings, and diluents, milk匕 includes, but is not limited to, narcotics, suspending agents, solvents, fillers, bulking agents, buffers, delivery vehicles, diluents, excipients and Z or pharmaceutical adjuvants. Typically, the medicament of the invention will be in the form of a composition comprising a peptide of the invention, or a variant or derivative thereof, together with one or more physiologically acceptable carriers, excipients or diluents. Is administered. For example, suitable vehicles may be water for injection, physiological solutions, or artificial cerebrospinal fluid, which may be supplemented with other materials common in compositions for parenteral delivery. is there.
[0100] 本明細書で使用される受容可能なキャリア、賦形剤または安定化剤は、レシピエン トに対して非毒性であり、そして好ましくは、使用される投薬量および濃度において不 活性であり、例えば、リン酸塩、クェン酸塩、または他の有機酸;ァスコルビン酸、 a —トコフエロール;低分子量ポリペプチド；タンパク質 (例えば、血清アルブミン、ゼラ チンまたは免疫グロブリン）；親水性ポリマー（例えば、ポリビュルピロリドン）；アミノ酸（ 例えば、グリシン、グルタミン、ァスパラギン、アルギニンまたはリジン）；モノサッカリド、 ジサッカリドおよび他の炭水化物（グルコース、マンノース、またはデキストリンを含む） ；キレート剤（例えば、 EDTA)；糖アルコール（例えば、マン-トールまたはソルビトー ル）；塩形成対イオン (例えば、ナトリウム）；ならびに Zあるいは非イオン性表面活性 化剤（例えば、 Tween、プル口ニック（pluronic)またはポリエチレングリコール（PEG ) )などが挙げられるがそれらに限定されな 、。  [0100] As used herein, an acceptable carrier, excipient, or stabilizer is non-toxic to recipients, and preferably inert at the dosages and concentrations employed. E.g., phosphates, citrates or other organic acids; ascorbic acid, a-tocopherol; low molecular weight polypeptides; proteins (e.g., serum albumin, gelatin or immunoglobulins); hydrophilic polymers (e.g. Amino acids (eg, glycine, glutamine, asparagine, arginine or lysine); monosaccharides, disaccharides and other carbohydrates (including glucose, mannose, or dextrin); chelating agents (eg, EDTA); sugar alcohols (eg, , Mannitol or sorbitol); salt-forming counterion (Eg, sodium); and Z or non-ionic surfactants (eg, Tween, pluronic or polyethylene glycol (PEG)), and the like.
[0101] さらなる例示の適切なキャリアとしては、中性緩衝化生理食塩水、または血清アル ブミンと混合された生理食塩水が挙げられる。好ましくは、その生成物は、適切な賦 形剤（例えば、スクロース）を用いて凍結乾燥剤として処方される。他の標準的なキヤ リア、希釈剤および賦形剤は所望に応じて含まれ得る。他の例示的な組成物は、 pH 7. 0— 8. 5の Tris緩衝剤または pH4. 0— 5. 5の酢酸緩衝剤を含み、これらは、さら に、ソルビトールまたはその適切な代替物を含み得る。  [0101] Further exemplary suitable carriers include neutral buffered saline or saline mixed with serum albumin. Preferably, the product is formulated as a lyophilizate using a suitable excipient (eg, sucrose). Other standard carriers, diluents and excipients may be included as desired. Other exemplary compositions include Tris buffer at pH 7.0-8.5 or acetate buffer at pH 4.0-5.5, which further comprises sorbitol or a suitable alternative thereof. May be included.
[0102] 以下に本発明の医薬組成物の一般的な調製法を示す。なお、動物薬組成物、医 薬部外品、水産薬組成物、食品組成物およびィヒ粧品組成物等についても公知の調 製法により製造することができる。 [0102] The general preparation method of the pharmaceutical composition of the present invention is shown below. It should be noted that veterinary drug compositions, quasi-drugs, marine drug compositions, food compositions, ligne cosmetic compositions, and the like are also known in the art. It can be manufactured by a manufacturing method.
[0103] 本発明のポリペプチド、ポリヌクレオチドなどは、薬学的に受容可能なキャリアと配 合し、錠剤、カプセル剤、顆粒剤、散剤、粉剤、座剤等の固形製剤、またはシロップ 剤、注射剤、懸濁剤、溶液剤、スプレー剤等の液状製剤として経口または非経口的 に投与することができる。薬学的に受容可能なキャリアとしては、上述のように、固形 製剤における賦形剤、滑沢剤、結合剤、崩壊剤、崩壊阻害剤、吸収促進剤、吸着剤 、保湿剤、溶解補助剤、安定化剤、液状製剤における溶剤、溶解補助剤、懸濁化剤 、等張化剤、緩衝剤、無痛化剤等が挙げられる。また、必要に応じ、防腐剤、抗酸ィ匕 剤、着色剤、甘味剤等の製剤添加物を用いることができる。また、本発明の組成物に は本発明のポリヌクレオチド、ポリペプチドなど以外の物質を配合することも可能であ る。非経口の投与経路としては、静脈内注射、筋肉内注射、経鼻、直腸、膣および経 皮等が挙げられるがそれらに限定されない。  [0103] The polypeptide, polynucleotide and the like of the present invention are combined with a pharmaceutically acceptable carrier, and solid preparations such as tablets, capsules, granules, powders, powders and suppositories, or syrups and injections are prepared. It can be administered orally or parenterally as liquid preparations such as preparations, suspensions, solutions and sprays. Pharmaceutically acceptable carriers include, as described above, excipients, lubricants, binders, disintegrants, disintegration inhibitors, absorption enhancers, adsorbents, humectants, solubilizing agents, Examples include stabilizers, solvents in liquid preparations, solubilizing agents, suspending agents, isotonic agents, buffers, soothing agents and the like. If necessary, additives for preparations such as preservatives, antioxidants, coloring agents, sweeteners and the like can be used. The composition of the present invention can also contain substances other than the polynucleotide, polypeptide, etc. of the present invention. Parenteral routes of administration include, but are not limited to, intravenous injection, intramuscular injection, nasal, rectal, vaginal and transdermal.
[0104] 固形製剤における賦形剤としては、例えば、グルコース、ラタトース、スクロース、 D マン-トール、結晶セルロース、デンプン、炭酸カルシウム、軽質無水ケィ酸、塩化 ナトリウム、カオリンおよび尿素等が挙げられる。  [0104] Examples of the excipient in the solid preparation include glucose, ratatose, sucrose, D-mantol, crystalline cellulose, starch, calcium carbonate, light caffeic anhydride, sodium chloride, kaolin and urea.
[0105] 固形製剤における滑沢剤としては、例えば、ステアリン酸マグネシウム、ステアリン 酸カルシウム、ホウ酸末、コロイド状ケィ酸、タルクおよびポリエチレングリコール等が 挙げられるがそれらに限定されない。  [0105] Examples of the lubricant in the solid preparation include, but are not limited to, magnesium stearate, calcium stearate, powdered boric acid, colloidal citric acid, talc, and polyethylene glycol.
[0106] 固形製剤における結合剤としては、例えば、水、エタノール、プロパノール、白糖、 D マン-トール、結晶セルロース、デキストリン、メチルセルロース、ヒドロキシプロピ ノレセノレロース、ヒドロキシプロピノレメチノレセノレロース、カノレボキシメチノレセノレロース、デ ンプン溶液、ゼラチン溶液、ポリビニルピロリドン、リン酸カルシウム、リン酸カリウム、 およびシェラック等が挙げられる。  [0106] Examples of the binder in the solid preparation include water, ethanol, propanol, sucrose, D-mann-tol, crystalline cellulose, dextrin, methylcellulose, hydroxypropinoresenolerose, hydroxypropinolemethinoresenolerose, and canoleboximetinolole. Senorelose, starch solution, gelatin solution, polyvinylpyrrolidone, calcium phosphate, potassium phosphate, shellac and the like.
[0107] 固形製剤における崩壊剤としては、例えば、デンプン、カルボキシメチルセルロース 、カルボキシメチルセルロースカルシウム、カンテン末、ラミナラン末、クロスカルメロ一 スナトリウム、カルボキシメチルスターチナトリウム、アルギン酸ナトリウム、炭酸水素ナ トリウム、炭酸カルシウム、ポリオキシエチレンソルビタン脂肪酸エステル類、ラウリル 硫酸ナトリウム、デンプン、ステアリン酸モノグリセリド、ラタトースおよび繊維素グリコー ル酸カルシウム等が挙げられるがそれらに限定されない。 [0107] Examples of the disintegrant in the solid preparation include starch, carboxymethylcellulose, carboxymethylcellulose calcium, agar powder, laminaran powder, croscarmellose sodium, sodium carboxymethyl starch, sodium alginate, sodium hydrogen carbonate, calcium carbonate, Polyoxyethylene sorbitan fatty acid esters, sodium lauryl sulfate, starch, monoglyceride stearate, ratatose and cellulose glycol Examples include, but are not limited to, calcium luate.
[0108] 固形製剤における崩壊阻害剤の好適な例としては、水素添加油、白糖、ステアリン 、カカオ脂および硬化油等が挙げられるがそれらに限定されない。  [0108] Preferable examples of the disintegration inhibitor in the solid preparation include, but are not limited to, hydrogenated oil, sucrose, stearin, cocoa butter, and hardened oil.
[0109] 固形製剤における吸収促進剤としては、例えば、第四級アンモ-ゥム塩基類および ラウリル硫酸ナトリウム等が挙げられるがそれらに限定されない。  [0109] Examples of the absorption enhancer in the solid preparation include, but are not limited to, quaternary ammonium bases and sodium lauryl sulfate.
[0110] 固形製剤における吸着剤としては、例えば、デンプン、ラタトース、カオリン、ベント ナイトおよびコロイド状ケィ酸等が挙げられるがそれらに限定されない。  [0110] Examples of the adsorbent in the solid preparation include, but are not limited to, starch, ratatose, kaolin, bentonite, and colloidal citric acid.
[0111] 固形製剤における保湿剤としては、例えば、グリセリン、デンプン等が挙げられるが それらに限定されない。  [0111] Examples of the humectant in the solid preparation include, but are not limited to, glycerin and starch.
[0112] 固形製剤における溶解補助剤としては、例えば、アルギニン、グルタミン酸、ァスパ ラギン酸等が挙げられるがそれらに限定されない。  [0112] Examples of the solubilizing agent in the solid preparation include, but are not limited to, arginine, glutamic acid, and aspartic acid.
[0113] 固形製剤における安定化剤としては、例えば、ヒト血清アルブミン、ラタトース等が挙 げられるがそれらに限定されない。  [0113] Examples of the stabilizer in the solid preparation include, but are not limited to, human serum albumin, ratatose, and the like.
[0114] 固形製剤として錠剤、丸剤等を調製する際には、必要により胃溶性または腸溶性 物質（白糖、ゼラチン、ヒドロキシプロピルセルロース、ヒドロキシプロピルメチルセル口 ースフタレート等）のフィルムで被覆していてもよい。錠剤には、必要に応じ通常の剤 皮を施した錠剤、例えば、糖衣錠、ゼラチン被包錠、腸溶被錠、フィルムコーティング 錠あるいは二重錠、多層錠が含まれる。カプセル剤にはハードカプセルおよびソフト カプセルが含まれる。座剤の形態に成形する際には、上記に列挙した添加物以外に 、例えば、高級アルコール、高級アルコールのエステル類、半合成グリセライド等を添 加することができるがそれらに限定されない。  [0114] When preparing tablets, pills, and the like as solid preparations, they are coated with a film of a gastric or enteric substance (sucrose, gelatin, hydroxypropylcellulose, hydroxypropylmethylcellulose phthalate, etc.) as necessary. Is also good. Tablets include tablets coated with usual coatings as necessary, such as sugar-coated tablets, gelatin-coated tablets, enteric-coated tablets, film-coated tablets or double tablets, and multilayer tablets. Capsules include hard capsules and soft capsules. When molding into a suppository form, for example, higher alcohols, higher alcohol esters, semi-synthetic glycerides, etc. can be added in addition to the additives listed above, but the invention is not limited thereto.
[0115] 液状製剤における溶剤の好適な例としては、注射用水、アルコール、プロピレンダリ コール、マクロゴール、ゴマ油およびトウモロコシ油等が挙げられる。  [0115] Preferable examples of the solvent in the liquid preparation include water for injection, alcohol, propylene glycol, macrogol, sesame oil, corn oil and the like.
[0116] 液状製剤における溶解補助剤の好適な例としては、ポリエチレングリコール、プロピ レングリコール、 D—マン-トール、安息香酸ベンジル、エタノール、トリスァミノメタン 、コレステロール、トリエタノールァミン、炭酸ナトリウムおよびクェン酸ナトリウム等が 挙げられるがそれらに限定されない。  [0116] Preferable examples of the solubilizer in the liquid preparation include polyethylene glycol, propylene glycol, D-mantol, benzyl benzoate, ethanol, trisaminomethane, cholesterol, triethanolamine, sodium carbonate and Examples include, but are not limited to, sodium citrate.
[0117] 液状製剤における懸濁化剤の好適な例としては、ステアリルトリエタノールァミン、ラ ゥリル硫酸ナトリウム、ラウリルアミノプロピオン酸、レシチン、塩化ベンザルコ-ゥム、 塩ィ匕べンゼトニゥム、モノステアリン酸グリセリン等の界面活性剤、例えば、ポリビュル アルコール、ポリビュルピロリドン、カルボキシメチルセルロースナトリウム、メチルセル ロース、ヒドロキシメチノレセノレロース、ヒドロキシェチノレセノレロース、ヒドロキシェチノレセ ルロース、ヒドロキシプロピルセルロース等の親水性高分子等が挙げられるがそれら に限定されない。 [0117] Preferable examples of the suspending agent in the liquid preparation include stearyl triethanolamine, Surfactants such as sodium peryl sulfate, laurylaminopropionic acid, lecithin, benzalco-chloride, benzedonium chloride, glyceryl monostearate, and the like, for example, polyvinyl alcohol, polybutylpyrrolidone, sodium carboxymethylcellulose, methylcellulose, and hydroxy. Examples include, but are not limited to, hydrophilic polymers such as methinoresenololose, hydroxyethinoresenololose, hydroxyethinorescellulose, and hydroxypropylcellulose.
[0118] 液状製剤における等張化剤の好適な例としては、塩ィ匕ナトリウム、グリセリン、 D- マン-トール等が挙げられるがそれらに限定されない。  [0118] Preferable examples of the tonicity agent in the liquid preparation include, but are not limited to, sodium salt, glycerin, D-mantol and the like.
[0119] 液状製剤における緩衝剤の好適な例としては、リン酸塩、酢酸塩、炭酸塩およびク ェン酸塩等の緩衝液等が挙げられるがそれらに限定されない。  [0119] Preferable examples of the buffer in the liquid preparation include, but are not limited to, buffers such as phosphate, acetate, carbonate, and citrate.
[0120] 液状製剤における無痛化剤の好適な例としては、ベンジルアルコール、塩化ベン ザルコ -ゥムおよび塩酸プロ力イン等が挙げられるがそれらに限定されない。  [0120] Preferable examples of the soothing agent in the liquid preparation include, but are not limited to, benzyl alcohol, benzalco-dum chloride, and proforce hydrochloride.
[0121] 液状製剤における防腐剤の好適な例としては、パラォキシ安息香酸エステル類、ク ロロブタノール、ベンジルアルコール、 2—フエ-ルエチルアルコール、デヒドロ酢酸、 ソルビン酸等が挙げられるがそれらに限定されない。  [0121] Preferable examples of the preservative in the liquid preparation include, but are not limited to, paraoxybenzoic acid esters, chlorobutanol, benzyl alcohol, 2-phenylethyl alcohol, dehydroacetic acid, sorbic acid and the like. .
[0122] 液状製剤における抗酸化剤の好適な例としては、亜硫酸塩、ァスコルビン酸、 a - トコフエロールおよびシスティン等が挙げられるがそれらに限定されない。  [0122] Preferable examples of the antioxidant in the liquid preparation include, but are not limited to, sulfite, ascorbic acid, a-tocopherol, and cysteine.
[0123] 注射剤として調製する際には、液剤および懸濁剤は殺菌され、かっ血液と等張で あることが好ましい。通常、これらは、ノ クテリア保留フィルタ一等を用いるろ過、殺菌 剤の配合または照射によって無菌化する。さらにこれらの処理後、凍結乾燥等の方 法により固形物とし、使用直前に無菌水または無菌の注射用希釈剤 (塩酸リドカイン 水溶液、生理食塩水、ブドウ糖水溶液、エタノールまたはこれらの混合溶液等）を添 カロしてちょい。  When prepared as an injection, the liquid preparation and the suspension are preferably sterilized and isotonic with blood and blood. Usually, these are sterilized by filtration using a nocteria retention filter or the like, blending of a bactericide or irradiation. Further, after these treatments, solidify by freeze-drying or the like. Immediately before use, sterile water or a sterile injectable diluent (lidocaine hydrochloride aqueous solution, physiological saline, glucose aqueous solution, ethanol or a mixed solution thereof) is used. Add some calories.
[0124] さらに、必要ならば、医薬組成物は、着色料、保存剤、香料、矯味矯臭剤、甘味料 等、ならびに他の薬剤を含んでいてもよい。  [0124] Further, if necessary, the pharmaceutical composition may contain a coloring agent, a preservative, a flavor, a flavoring agent, a sweetener, and the like, and other agents.
[0125] 本発明の医薬は、必要に応じて生理学的に受容可能なキャリア、賦型剤または安 定化剤 (日本薬局方第 14版、その追補またはその最新版、 Remington' s Pharm aceutical Sciences, 18th Edition, A. R. Lrennaro, ed. , Mack Publisnm g Company, 1990などを参照）と、所望の程度の純度を有する糖鎖組成物とを混 合することによって、凍結乾燥されたケーキまたは水溶液の形態で調製され保存され 得る。 [0125] The pharmaceutical 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 Publisnm g Company, 1990, etc.) and a sugar chain composition having a desired degree of purity, can be prepared and stored in the form of a lyophilized cake or aqueous solution.
[0126] 本発明の組成物の投与すべき量は、使用目的、対象疾患 (種類、重篤度など）、患 者の年齢、体重、性別、既往歴、細胞の形態または種類などを考慮して、当業者が 容易に決定することができる。本発明の処置方法を被検体 (または患者）に対して施 す頻度もまた、使用目的、対象疾患 (種類、重篤度など)、患者の年齢、体重、性別、 既往歴、および治療経過などを考慮して、当業者が容易に決定することができる。頻 度としては、例えば、毎日 数ケ月に 1回（例えば、 1週間に 1回ー1ヶ月に 1回）の投 与が挙げられる。 1週間ー1ヶ月に 1回の投与を、経過を見ながら施すことが好ましい  [0126] 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
[0127] 本明細書において「患者」とは、本発明の処置が適用される生物をいい、「被検体」 または「被験体」ともいわれる。患者は好ましくは、ヒトであり得る。 [0127] As used herein, "patient" refers to an organism to which the treatment of the present invention is applied, and is also referred to as "subject" or "subject". The patient can preferably be a human.
[0128] 本発明は、患者への有効量の本発明の組成物の投与による処置、阻害および予 防の方法を提供する。好ましい局面において、本発明の組成物は実質的に精製され たものであり得る（例えば、その効果を制限する力または望ましくない副作用を生じる 物質が実質的に存在しな 、状態が挙げられる)。  [0128] The present invention provides methods of treatment, inhibition and prevention by administering to a patient an effective amount of a composition of the present invention. In a preferred aspect, the compositions of the present invention can be substantially purified (including, for example, conditions in which there is substantially no ability to limit its effects or substances that produce undesirable side effects).
[0129] 本発明の医薬は、経口的または非経口的に投与され得る。あるいは、本発明の医 薬は、静脈内または皮下で投与され得る。全身投与されるとき、本発明において使用 される医薬は、発熱物質を含まない、薬学的に受容可能な水溶液の形態であり得る 。そのような薬学的に受容可能な組成物の調製は、 pH、等張性、安定性などを考慮 することにより、当業者は、容易に行うことができる。本明細書において、投与方法は 、経口投与、非経口投与 (例えば、静脈内投与、筋肉内投与、皮下投与、皮内投与 、粘膜投与、直腸内投与、膣内投与、患部への局所投与、皮膚投与など)であり得る 。そのような投与のための処方物は、任意の製剤形態で提供され得る。そのような製 剤形態としては、例えば、液剤、注射剤、徐放剤が挙げられる。本発明のペプチドは 、単独で、または生理緩衝液中に溶解した注射液等の形態で、送達が望まれる組織 に局所投与することができる。また、注射液には、他の消毒剤や消炎鎮痛剤など、通 常、傷の治療薬に含まれる種々の成分を含んで 、てもよ 、。 [0129] The medicament of the present invention can be administered orally or parenterally. Alternatively, the medicament of the present invention can be administered intravenously or subcutaneously. When administered systemically, the medicament used in the present invention may be in the form of a pyrogen-free, pharmaceutically acceptable aqueous solution. The preparation of such pharmaceutically acceptable compositions can be readily performed by those skilled in the art by considering pH, isotonicity, stability, and the like. In the present specification, the administration method includes oral administration, parenteral administration (for example, intravenous administration, intramuscular administration, subcutaneous administration, intradermal administration, mucosal administration, rectal administration, vaginal administration, local administration to the affected area, Skin administration). Formulations for such administration may be provided in any formulation. Such preparations include, for example, solutions, injections, and sustained-release preparations. The peptide of the present invention can be locally administered alone or in the form of an injection solution dissolved in a physiological buffer to a tissue where delivery is desired. Injectable solutions also contain other disinfectants and anti-inflammatory analgesics. Usually, it contains various components included in the remedy for wounds.
[0130] 上述のような医薬の投与のために、様々な送達系が公知であり、そして本発明の化 合物を投与するために用いられ得る（例えば、リボソーム、微粒子、マイクロカプセル など)。導入方法としては、皮内、筋内、腹腔内、静脈内、皮下、鼻腔内、硬膜外、お よび経口経路が挙げられるがそれらに限定されない。化合物または組成物は、任意 の好都合な経路により（例えば、注入またはボーラス注射により、上皮または粘膜内 層（例えば、口腔粘膜、直腸粘膜および腸粘膜など)を通しての吸収により）投与され 得、そして他の生物学的に活性な薬剤と一緒に投与され得る。投与は、全身的また は局所的であり得る。さらに、本発明の薬学的化合物または組成物を、任意の適切 な経路 (脳室内注射および髄腔内注射を包含し;脳室内注射は、例えば、 Ommaya リザーバのようなリザーバに取り付けられた脳室内カテーテルにより容易にされ得る） により中枢神経系に導入することが望まれ得る。例えば、吸入器または噴霧器の使 用、およびエアロゾル化剤を用いた処方により、肺投与もまた使用され得る。全身投 与されるとき、本発明において使用される組成物は、発熱物質を含まないことが好ま しい。そのような薬学的に受容可能な組成物の調製は、 pH、等張性、安定性などを 考慮することにより、当業者は、容易に行うことができる。そのような投与のための処 方物は、任意の製剤形態で提供され得る。そのような製剤形態としては、例えば、液 剤、注射剤、徐放剤が挙げられる。導入方法としては、経口剤としての投与、吸入（ 例えば、肺）、シリンジ、カテーテル、チューブを用いた注入、針無し注射、遺伝子銃 などが挙げられるがそれらに限定されない。この場合、他の生物学的に活性な薬剤と 一緒に投与され得る。  [0130] For the administration of medicaments as described above, various delivery systems are known and can be used to administer the compounds of the invention (eg, ribosomes, microparticles, microcapsules, etc.). Methods of introduction include, but are not limited to, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, and oral routes. The compound or composition can be administered by any convenient route (eg, by infusion or bolus injection, by absorption through epithelial or mucosal linings (eg, oral, rectal and intestinal mucosa, etc.)) and other May be administered together with a biologically active agent. Administration can be systemic or local. In addition, the pharmaceutical compounds or compositions of the present invention can be administered by any suitable route, including intraventricular and intrathecal injections; intraventricular injection can be, for example, an intraventricularly attached reservoir such as an Ommaya reservoir. (Which may be facilitated by a catheter) to introduce it into the central nervous system. Pulmonary administration may also be used, for example, with the use of an inhaler or nebulizer, and formulation with an aerosolizing agent. When administered systemically, the compositions used in the present invention are preferably pyrogen free. The preparation of such pharmaceutically acceptable compositions can be readily performed by those skilled in the art by considering pH, isotonicity, stability, and the like. Formulations for such administration may be provided in any formulation. Such preparation forms include, for example, solutions, injections, and sustained-release preparations. Examples of the introduction method include, but are not limited to, administration as an oral preparation, inhalation (for example, lung), injection using a syringe, catheter, or tube, needleless injection, and a gene gun. In this case, it can be administered together with other biologically active agents.
[0131] 特定の実施形態において、本発明のポリペプチド、ポリヌクレオチドまたは組成物を 、処置の必要な領域に局所的に投与することが望まれ得る;これは、制限する目的で はないが、例えば、手術中の局部注入、局所適用（例えば、手術後の創傷包帯との 組み合わせて）により、注射により、カテーテルにより、坐剤により、またはインプラント (このインプラントは、シァラスティック（sialastic)膜のような膜または繊維を含む、多 孔性、非多孔性、または膠様材料である）により達成され得る。好ましくは、抗体を含 む本発明のタンパク質を投与する際、タンパク質が吸収されない材料を使用するた めに注意が払われなければならな!/、。 [0131] In certain embodiments, it may be desirable to administer the polypeptides, polynucleotides or compositions of the invention locally to the area in need of treatment; For example, by local injection during surgery, topical application (eg, in combination with a post-operative wound dressing), by injection, by catheter, by suppository, or by an implant (this implant may be a sialastic membrane). Such as membranes or fibers, which are porous, non-porous, or glue-like materials). Preferably, when administering the protein of the present invention including the antibody, a material to which the protein is not absorbed is used. Attention must be paid to! / ,.
[0132] 別の実施形態において、化合物または組成物は、小胞、特に、リボソーム中に封入 された状態で送達され得る（Langer, Science 249 : 1527- 1533 (1990)； Treat ら, Liposomes m tne fherapy of Infectious Disease and し ancer, Lo pez— Beresteinおよび Fidler (編）， Liss, New York, 353〜365頁（1989) ;Lo pez— Berestein,同書 317〜327頁を参照のこと；広く同書を参照のこと）。  [0132] In another embodiment, the compound or composition can be delivered encapsulated in vesicles, particularly ribosomes (Langer, Science 249: 1527-1533 (1990); Treat et al., Liposomes mtne). fherapy of Infectious Disease and Ancer, Lo pez—Berestein and Fidler (eds.), Liss, New York, pp. 353-365 (1989); Lo pez—Berestein, pp. 317-327; see broadly the same book. That).
[0133] さらに別の実施形態において、化合物または組成物は、制御された徐放系中で送 達され得る。 1つの実施形態において、ポンプが用いられ得る（Langer (前出）； Seft on, CRC Crit. Ref. Biomed. Eng. 14 : 201 (1987)； Buchwaldら， Surgery 88 : 507 (1980) ; Saudekら， N. Engl. J. Med. 321 : 574 (1989)を参照のこと）。 別の実施形態において、高分子材料が用いられ得る（Medical Applications of Controlled Release, Langerおよび Wise (編) , CRC Pres. , Boca Raton, Florida ( 1974)； Controlled Drug Bioavailability, Drug Product Design and Performance, Smolenおよび Ball (編）， Wiley, New York (1984)； Ra ngerおよび Peppas, J.、 Macromol. Sci. Rev. Macromol. Chem. 23 : 61 (19 83)を参照のこと； Levyら， Science 228 : 190 (1985)； Duringら， Ann. Neurol . 25 : 351 (1989)； Howard^, J. Neurosurg. 71 : 105 (1989)もまた参照のこと）  [0133] In yet another embodiment, the compound or composition can be delivered in a controlled release system. In one embodiment, a pump may be used (Langer, supra; Seft on, CRC Crit. Ref. Biomed. Eng. 14: 201 (1987); Buchwald et al., Surgery 88: 507 (1980); Saudek et al. , N. Engl. J. Med. 321: 574 (1989)). In another embodiment, polymeric materials can be used (Medical Applications of Controlled Release, Langer and Wise (eds.), CRC Pres., Boca Raton, Florida (1974); Controlled Drug Bioavailability, Drug Product Design and Performance, Smolen. And Ball (Ed.), Wiley, New York (1984); Langer and Peppas, J., Macromol. Sci. Rev. Macromol. Chem. 23:61 (1983); Levy et al., Science 228: 190 (1985); During et al., Ann. Neurol. 25: 351 (1989); See also Howard ^, J. Neurosurg. 71: 105 (1989)).
[0134] さらに別の実施形態において、制御された徐放系は、治療標的の近くに置かれ得、 従って、全身用量の一部のみを必要とする（例えば、 Goodson, Medical Applica tions of Controlled Release, (前出），第 2卷， 115〜 138頁（1984)を参照の こと)。 [0134] In yet another embodiment, a controlled-release system can be placed near a therapeutic target and thus requires only a portion of the systemic dose (eg, Goodson, Medical Applications of Controlled Release). , (Supra), Vol. 2, pp. 115-138 (1984)).
[0135] 他の制御された徐放系は、 Langerにより総説において議論される（Science 249  [0135] Other controlled release systems are discussed in a review by Langer (Science 249
: 1527—1533 (1990) )。  : 1527-1533 (1990)).
[0136] 本発明が対象とする動物は、ペプチドの代謝系を有するものであれば、どの生物（ 例えば、動物 (たとえば、脊椎動物、無脊椎動物)）でもよい。好ましくは、脊椎動物（ たとえば、メタラウナギ類、ャッメゥナギ類、軟骨魚類、硬骨魚類、両生類、爬虫類、 鳥類、哺乳動物など)であり、より好ましくは、哺乳動物 (例えば、単孔類、有袋類、貧 歯類、皮翼類、翼手類、食肉類、食虫類、長鼻類、奇蹄類、偶蹄類、管歯類、有鱗 類、海牛類、クジラ目、霊長類、齧歯類、ゥサギ目など)であり得る。例示的な患者とし ては、例えば、ゥシ、ブタ、ゥマ、 -ヮトリ、ネコ、ィヌなどの動物が挙げられるがそれら に限定されない。さらに好ましくは、霊長類 (たとえば、チンパンジー、二ホンザル、ヒト )が対象とされる。最も好ましくはヒトが対象とされる。 [0136] The animal targeted by the present invention may be any organism (eg, animal (eg, vertebrate, invertebrate)) as long as it has a peptide metabolic system. Preferably, it is a vertebrate (e.g., a metal eel, alfalfa, chondrichthyes, teleosts, amphibians, reptiles, birds, mammals, etc.), and more preferably, a mammal (e.g., monotremes, marsupials, Poor Teeth, skin wings, winged hands, carnivores, insectivores, longnoses, hoofed hoofs, artiodactyla, tubulars, squamata, oxen, whales, primates, rodents, (Egret, etc.). Exemplary 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, macaques, humans) are targeted. Most preferably, humans are targeted.
[0137] 本発明の予防方法において使用される医薬の量は、使用目的、対象疾患 (種類な ど）、患者の年齢、体重、既往歴などを考慮して、当業者が容易に決定することがで きる。本発明の処置方法を患者 (または患者）に対して施す頻度もまた、使用目的、 対象疾患 (種類、重篤度など)、患者の年齢、体重、既往歴、および経過などを考慮 して、当業者が容易に決定することができる。頻度としては、例えば、毎日—数ケ月に 1回（例えば、 1週間に 1回 1ヶ月に 1回）の投与、あるいは毎年流行前に 1回の頻 度などが挙げられる。 1週間ー1ヶ月に 1回の投与を、経過を見ながら施すことが好ま しぐ少なくとも約 1週間の間隔をあけて追加免疫をすることが有利である。より好まし くは、追加免疫の間隔は少なくとも約 3週間であり得る。本発明の組成物などの投与 量は、患者の年齢、体重、症状または投与方法などにより異なり、特に限定されない  [0137] The amount of the drug 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, the target disease (eg, type), the patient's age, weight, and medical history. I can do it. The frequency of applying the treatment method of the present invention to a patient (or patient) also depends on the purpose of use, target disease (type, severity, etc.), age, weight, medical history, and course of the patient, and the like. Those skilled in the art can easily determine. The frequency may include, for example, daily-once-monthly administration (eg, once a week, once a month), or once every year before the epidemic. It is advantageous to boost at least about a week at intervals of at least about one week, which is preferably administered once a week-monthly while monitoring the progress. More preferably, the interval between boosts can be at least about 3 weeks. The dose of the composition or the like of the present invention varies depending on the age, body weight, symptoms, administration method, and the like of the patient, and is not particularly limited.
[0138] 本明細書中、「投与する」とは、本発明の医薬などまたはそれを含む医薬組成物を 、単独で、または他の治療剤と組み合わせて処置が意図される宿主に与えることを意 味する。組み合わせは、例えば、混合物として同時に、別々であるが同時にもしくは 並行して;または逐次的にかのいずれかで投与され得る。これは、組み合わされた薬 剤が、治療混合物としてともに投与される提示を含み、そして組み合わせた薬剤が、 別々であるが同時に (例えば、同じ個体へ別々の粘膜を通じての場合)投与される手 順もまた含む。「組み合わせ」投与は、第 1に与えられ、続いて第 2に与えられる化合 物または薬剤のうちの 1つを別々に投与することをさらに含む。 [0138] In the present specification, "administering" refers to giving the medicament or the like of the present invention or a pharmaceutical composition containing the same to a host to be treated alone or in combination with another therapeutic agent. 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.
[0139] 本発明における医薬の投与は、どのような手法を用いて行ってもよいが、好ましくは 、針無し注射を用いることが有利である。患者に過度の負担を与えることなく投与を 行免るカゝらである。  [0139] The administration of the medicament in 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.
[0140] ここで本発明における針無注射器とは、注射針を用いずに、ガス圧または弾性部 材の弹力によりピストンを移動させて薬液を皮膚に噴射し、薬剤成分を皮下、より好 ましくは皮下の細胞内に投与する医療機器を意味する。 [0140] Here, the needleless syringe in the present invention refers to a gas pressure or an elastic part without using a needle. It refers to a medical device that moves a piston by the force of a material to inject a drug solution onto the skin and administers a drug component subcutaneously, more preferably, into cells under the skin.
[0141] 具体的には例えば、シマジェット™ (島津製作所製)、メディ 'ジェクタ一ビジョン [0141] Specifically, for example, ShimaJet ™ (manufactured by Shimadzu Corporation), Medi'Jecta-I Vision
(Medi-JectorVision)™, (Elite medical社製)、ペンジェット (Penjet)™ (Penjet社製)など が市販されている。 (Medi-JectorVision) ™, (Elite medical), and Penjet ™ (Penjet) are commercially available.
[0142] 本明細書において「指示書」は、本発明の医薬などを投与する方法または診断する 方法などを医師、患者など投与を行う人、診断する人 (患者本人であり得る）に対して 記載したものである。この指示書は、本発明の診断薬、医薬などを投与する手順を指 示する文言が記載されている。この指示書は、本発明が実施される国の監督官庁（ 例えば、 日本であれば厚生労働省、米国であれば食品医薬品局 (FDA)など）が規 定した様式に従って作成され、その監督官庁により承認を受けた旨が明記される。指 示書は、いわゆる添付文書 (package insert)であり、通常は紙媒体で提供される 力 それに限定されず、例えば、電子媒体 (例えば、インターネットで提供されるホー ムページ（ウェブサイト）、電子メール、 SMS、ボイスメール、インスタントメッセージ）の ような形態でも提供され得る。  [0142] In the present specification, the "instruction" refers to a method of administering or diagnosing the medicament or the like of the present invention for a physician, a patient or the like who administers or diagnoses (possibly a patient). It is described. This instruction describes a word indicating a procedure for administering the diagnostic agent, the medicine and the like of the present invention. This instruction is prepared in accordance with the format prescribed by the competent authority of the country where the present invention is implemented (for example, the Ministry of Health, Labor and Welfare in Japan and the Food and Drug Administration (FDA) in the United States) and is issued by the competent authority. The approval is clearly stated. Instructions are so-called package inserts, which are usually provided on paper media, but are not limited to electronic media (eg, homepages (websites) provided on the Internet, emails, etc.). , SMS, voicemail, instant messaging).
[0143] 本発明の方法による治療の終了の判断は、商業的に利用できるアツセィもしくは機 器使用による標準的な臨床検査室の結果または疾患に特徴的な臨床症状の消滅に よって支持され得る。治療は、疾患の再発により再開することができる。  [0143] Judgment of termination of treatment according to the methods of the present invention may be supported by standard clinical laboratory results or by the disappearance of clinical symptoms characteristic of the disease by using commercially available equipment or equipment. Treatment can be resumed upon recurrence of the disease.
[0144] 本発明はまた、本発明の医薬組成物の 1つ以上の成分を満たした 1つ以上の容器 を備える薬学的パックまたはキットを提供する。医薬品または生物学的製品の製造、 使用または販売を規制する政府機関が定めた形式の通知が、このような容器に任意 に付属し得、この通知は、ヒトへの投与に対する製造、使用または販売に関する政府 機関による承認を表す。  [0144] The present invention also provides a pharmaceutical pack or kit comprising one or more containers filled with one or more components of the pharmaceutical composition of the present invention. A notice in the form of a governmental body regulating the manufacture, use or sale of pharmaceuticals or biological products may optionally accompany such containers, and this notice may include the manufacture, use or sale of the product for use in humans. Represents approval by government agencies.
[0145] (好ましい実施形態の説明）  (Description of Preferred Embodiment)
以下に本発明の好ましい実施形態を説明する。以下に提供される実施形態は、本 発明のよりよい理解のために提供されるものであり、本発明の範囲は以下の記載に 限定されるべきでないことが理解される。従って、当業者は、本明細書中の記載を参 酌して、本発明の範囲内で適宜改変を行うことができることは明らかである。 [0146] 1つの局面において、本発明は、生理活性ペプチドと、該生理活性ペプチドに由来 しない少なくとも 1つのアミノ酸とを含む、ペプチドを提供する。このペプチドに含まれ る生理活性ペプチドは、ュビキチンによって認識されないものが好ましい。ュビキチ ンによって認識されないようなペプチドは、体内で、種々のぺプチダーゼによる分解 を受けることになる。しかし、従来は、このようなぺプチダーゼ耐性を有するような製剤 の開発は遅れていた。本発明者らは、上述のように、生理活性ペプチドに由来しない アミノ酸を含む (好ましくは、ペプチド結合により、上記生理活性ペプチドの C端また は N端に結合している)ペプチドが、予想外に徐放効果を示すことを見出した。従つ て、本発明は、ぺプチダーゼの影響を受ける（ュビキチンの影響を受けないような）生 理活性ペプチドの徐放剤を提供するために有用である。 Hereinafter, preferred embodiments of the present invention will be described. The embodiments provided below are provided for a better understanding of the present invention, and it is understood that the scope of the present invention should not be limited to the following description. Therefore, it is apparent that those skilled in the art can appropriately make modifications within the scope of the present invention in view of the description in the present specification. [0146] In one aspect, the present invention provides a peptide comprising a bioactive peptide and at least one amino acid not derived from the bioactive peptide. The bioactive peptide contained in this peptide is preferably not recognized by ubiquitin. Peptides that are not recognized by ubiquitin will undergo degradation by various peptidases in the body. However, conventionally, development of such a drug having peptidase resistance has been delayed. The present inventors have found that, as described above, a peptide containing an amino acid that is not derived from a bioactive peptide (preferably, bound to the C-terminal or N-terminal of the bioactive peptide by a peptide bond) is unexpected. Have a sustained release effect. Therefore, the present invention is useful for providing a sustained-release agent of a physiologically active peptide affected by peptidase (such as not affected by ubiquitin).
[0147] 好ま 、実施形態では、本発明は、 35kDa未満のサイズの生理活性ペプチドの徐 放効果を達成するために使用される。本発明にお ヽて用いられる生理活性ペプチド のサイズは、通常 30kDa以下であり、好ましくは、分子量 20kDa以下であり、さらに 好ましくは lOkDa以下であり、生理活性を保有する限り、どれだけ小さなものであつ てもよいことが理解される。。アミノ酸長で計数する場合、本発明において含まれるベ き生理活性ペプチドのアミノ酸長は、通常 300アミノ酸以下であり、好ましくは、 200 アミノ酸以下であり、より好ましくは 100アミノ酸以下であり、さらに好ましくは、 50ァミノ 酸以下であり、生理活性を保有する限り、どれだけ短いものであってもよいことが理解 される。  [0147] Preferably, in an embodiment, the present invention is used to achieve a sustained release effect of a bioactive peptide having a size of less than 35 kDa. The size of the bioactive peptide used in the present invention is usually 30 kDa or less, preferably a molecular weight of 20 kDa or less, and more preferably 10 kDa or less. It is understood that it is possible. . When counting by amino acid length, the amino acid length of the bioactive peptide to be included in the present invention is usually 300 amino acids or less, preferably 200 amino acids or less, more preferably 100 amino acids or less, and further more preferably , 50 amino acids or less, and it is understood that the length may be any length as long as it retains the physiological activity.
[0148] 好ましい実施形態では、本発明は、少なくとも 2つ以上の疎水性アミノ酸が連続した 構造を含むことが好ましい。ここで「疎水性アミノ酸」とは、疎水性インデックス (Kyte and Doolittle, J. Mol. Biol. , 157, 105— 132 (1982)に記載されて! /、る）力 S「 2」以上のもののうち、極性かつ中性のアミノ酸（天然のアミノ酸であれば、 Ser、 Th r、 Tyrおよび Cys)をのぞくものをいう。  [0148] In a preferred embodiment, the present invention preferably includes a continuous structure of at least two or more hydrophobic amino acids. As used herein, the term "hydrophobic amino acid" refers to a substance having a hydrophobicity index (described in Kyte and Doolittle, J. Mol. Biol., 157, 105-132 (1982)! Of these, those excluding polar and neutral amino acids (natural amino acids such as Ser, Thr, Tyr and Cys).
[0149] 疎水性アミノ酸が連続した配列を含む配列を付加することの利点は、以下のような 点にある。すなわち、疎水性アミノ酸が連続した配列は、生体内に通常は存在しない ので、このような疎水性アミノ酸が連続した配列を含む配列を末端に付加することに よって、ペプチド全体がぺプチダーゼにより分解を受け、消失してしまうことを遅らせ ることができるという点が利点の一つである。 [0149] Advantages of adding a sequence containing a continuous sequence of hydrophobic amino acids are as follows. That is, since a sequence of continuous hydrophobic amino acids is not normally present in a living body, adding a sequence containing such a sequence of hydrophobic amino acids to the terminus causes the entire peptide to be degraded by peptidase. Receiving and delaying their disappearance Is one of the advantages.
[0150] 疎水性インデックスが高いイソロイシン (インデックス =4. 5)が少なくとも 2つ以上連 続した構造を人工的に作製すると、水溶液中には溶解し得ず、製剤化が比較的困難 であると考えられる。従って、疎水性アミノ酸が少なくとも 2つ以上連続する構造を人 ェ的に作製する場合、比較的疎水性インデックスが低いグリシン (インデックス = -0 . 4)などをカ卩えることが好ましいが、これに限定されない。  [0150] When an isoleucine having a high hydrophobicity index (index = 4.5) is artificially produced, a structure in which at least two or more areoleucins are connected cannot be dissolved in an aqueous solution and is relatively difficult to formulate. Conceivable. Therefore, when artificially producing a structure in which at least two or more hydrophobic amino acids are continuous, it is preferable to remove glycine (index = -0.4) having a relatively low hydrophobicity index. Not limited.
[0151] 好ましい実施形態において、本発明のペプチドに含まれるべきアミノ酸は、少なくと も 1つのグリシンを含むことが有利である。グリシンは、生理活性に影響を与えることが 少なぐ立体構造に影響を与えることが少ないからである。グリシンはまた、生体内に 存在するぺプチダーゼによって分解を受け、実際の生理活性ペプチドの活性が発揮 されるようになることが本発明によって明らかになった力 である。また、グリシンは、 水溶性を低減させな 、などの利点も併せ持って 、る。  [0151] In a preferred embodiment, the amino acids to be included in the peptide of the present invention advantageously include at least one glycine. Glycine has little effect on bioactivity and has little effect on the three-dimensional structure. Glycine is also a force that has been revealed by the present invention to be degraded by peptidases present in the living body, so that the activity of the actual bioactive peptide is exerted. Glycine also has the advantage of not reducing the water solubility.
[0152] 好ま 、実施形態では、本発明のペプチドに含まれるべきアミノ酸は、生理活性べ プチドの C末端および N末端力もなる群より選択される少なくとも 1つに結合される。  [0152] Preferably, in an embodiment, the amino acids to be included in the peptide of the present invention are bound to at least one selected from the group consisting of C-terminal and N-terminal forces of a bioactive peptide.
[0153] より好ましくは、本発明のペプチドに含まれるべきアミノ酸は、前記生理活性べプチ ドの C末端および N末端の両方に結合される。体内のぺプチダーゼは複数種類存在 し、その分解活性は、総合してみると、 C末端および N末端の両方から影響があること が通常である力 である。  [0153] More preferably, the amino acid to be contained in the peptide of the present invention is bound to both the C-terminus and the N-terminus of the bioactive peptide. There are several types of peptidases in the body, and their degradation activity is a force that, when taken together, usually has an effect from both the C-terminal and the N-terminal.
[0154] 本発明のペプチドに含まれるべきアミノ酸は、複数のグリシンを含むことが好ましい 。複数のグリシンを含むことによって、下記実施例において示されるように、徐放効果 が上がることが確認されている。より好ましくは、本発明のペプチドに含まれるべきアミ ノ酸は、グリシンを 3〜10個、あるいは 3〜6個含む。  [0154] The amino acids to be included in the peptide of the present invention preferably include a plurality of glycines. It has been confirmed that the inclusion of a plurality of glycines enhances the sustained release effect as shown in the examples below. More preferably, the amino acid to be included in the peptide of the present invention contains 3 to 10, or 3 to 6, glycines.
[0155] 好ましい実施形態において、本発明のペプチドに含まれるべきアミノ酸は、生理活 性ペプチドの天然の配列とは異なる配列を含む。このようなペプチドは、天然に存在 しないものであり、天然に存在するペプチドよりも徐放効果を示す。  [0155] In a preferred embodiment, the amino acids to be included in the peptide of the present invention include a sequence different from the natural sequence of the bioactive peptide. Such peptides are non-naturally occurring and exhibit a more sustained release effect than naturally occurring peptides.
[0156] より好ましくは、本発明のペプチドに含まれるべき生理活性ペプチドのリーダー配列 の天然の配列とは異なる配列を含む。リーダー配列は、天然に存在する形態である 1S 本発明のペプチドは、そのようなリーダー配列を有する形態よりも徐放効果がある ことが理解される。 [0156] More preferably, it contains a sequence different from the natural sequence of the leader sequence of the bioactive peptide to be included in the peptide of the present invention. Leader sequence is a naturally occurring form 1S Peptides of the invention have a more sustained release effect than forms with such leader sequence It is understood that.
[0157] 本発明のペプチドに含まれるべき生理活性ペプチドは、血管新生、血管新生阻害 、骨再生、抗炎症作用 (インテグリン阻害)、糖尿病治療、鎮痛作用、抗痴呆からなる 群より選択される効果を有するものが使用される。このようなペプチドの具体例として は、例えば、ラミニン由来ペプチド (血管新生を有する；  [0157] The bioactive peptide to be included in the peptide of the present invention has an effect selected from the group consisting of angiogenesis, angiogenesis inhibition, bone regeneration, anti-inflammatory action (integrin inhibition), diabetes treatment, analgesic action, and anti-dementia. Is used. Specific examples of such a peptide include, for example, a laminin-derived peptide (having angiogenesis;
J.Cell.Physiol.,1992;153:614- 625 ;Adv.Exp.Med.Biol.2000:;476:139- 154を参照）、ラ ミニン由来ペプチド（血管新生阻害活性を有する； Br.J.Cancer, 199673:589-595 ; Clin.Exp.Metastasis, 1998: 16:645-654などを参照）；（骨原性増殖ペプチド； OGP ; EMBOJ.1992;ll:1867-1873)；インテグリン阻害作用ペプチド（特開平 6-321988号）； インスリン;エンケフアリン;バソプレシンなどを挙げることができるがそれらに限定され ない。  J. Cell. Physiol., 1992; 153: 614-625; see Adv. Exp. Med. Biol. 2000 :; 476: 139-154), laminin-derived peptides (having angiogenesis inhibitory activity; Br. J Cancer, 199673: 589-595; Clin. Exp. Metastasis, 1998: 16: 645-654, etc.); (Osteogenic proliferating peptide; OGP; EMBOJ. 1992; ll: 1867-1873); Integrin inhibitory action Peptides (JP-A-6-321988); insulin; enkephalin; vasopressin, and the like, but are not limited thereto.
[0158] そのような生理活性ペプチドの具体的アミノ酸配列としては、ペプチド SWYGLR 、ペプチド SIKVAV (配列番号 19)、ペプチド、IKVAV (ラミニン由来ペプチド）（配列 番号 20)、ペプチド YIGSR (ラミニン由来ペプチド）（配列番号 7)、ペプチド ALKRQ GRTLYGFGG (骨原性増殖ペプチド； OGP) (配列番号 22)、ペプチド GDGRHD L (インテグリン阻害剤）（配列番号 23)、ペプチド FVNQHLCGSHLVEALYLVC  [0158] Specific amino acid sequences of such a physiologically active peptide include peptide SWYGLR, peptide SIKVAV (SEQ ID NO: 19), peptide, IKVAV (laminin-derived peptide) (SEQ ID NO: 20), peptide YIGSR (laminin-derived peptide) ( SEQ ID NO: 7), peptide ALKRQ GRTLYGFGG (osteoproliferative peptide; OGP) (SEQ ID NO: 22), peptide GDGRHD L (integrin inhibitor) (SEQ ID NO: 23), peptide FVNQHLCGSHLVEALYLVC
、ペプチド YGGFM (Met—エンケフアリン）（配列番号 25)、 YGGFL (Leu—エンケ フアリン）（配列番号 26)、ペプチド MLARMLNTTI SACFLSLLAF SSACYF QNCP RGGKRAISDM ELRQCLPCGP GGKGRCFGPS ICCADELGC F VGTAEALRCQ EENYLPSPCQ SGQKPCGSGG RCAAVGICCS D ESCVAEPEC HDGFFRLTRA REPSNATQLD GPARALLLRL VQLA GTRESV DSAKPRVY (バソプレシン）（配列番号 21)またはその改変体などを挙 げることができるがそれらに限定されない。 , Peptide YGGFM (Met—Enkehuarin) (SEQ ID NO: 25), YGGFL (Leu—Enkehuarin) (SEQ ID NO: 26), Peptide MLARMLNTTI SACFLSLLAF SSACYF QNCP RGGKRAISDM ELRQCLPCGP GGKGRCFGPS ICCADELGC F VGTAEALRCQ EENYLPSPCQ SGQRPGS DQAGS Vasopressin) (SEQ ID NO: 21) or a variant thereof, but is not limited thereto.
[0159] 好ま 、実施形態では、本発明のペプチドに含まれるべき生理活性ペプチドは、所 望されない活性を有しない。このような作用を実現するには、ペプチドは、所望の活 性を保持する限りできるだけ短い方が好ましい。長い配列を有することによって、別の 効果が出てくる可能性が高まる力もである。 [0160] 1つの実施形態では、本発明のペプチドに含まれるべき生理活性ペプチドは、ぺプ チド自体で存在する場合、生体内半減期が、通常 2ヶ月以内、好ましくは、 1ヶ月以内 、より好ましくは、 15日以内、 7日以内、さらに好ましくは 2日以内であるものが対象と され得る。本発明は、このような通常であれば、半減期が短く医薬として使用できない ようなペプチドでさえ、徐放効果を利用して医薬として使用することを可能にする。 [0159] Preferably, in an embodiment, the bioactive peptide to be included in the peptide of the present invention does not have an undesired activity. In order to achieve such an effect, the peptide is preferably as short as possible as long as the desired activity is maintained. Having a long array also increases the likelihood of another effect. [0160] In one embodiment, when the physiologically active peptide to be included in the peptide of the present invention is present in the form of the peptide itself, the half-life in vivo is generally within 2 months, preferably within 1 month, more preferably within 1 month. Preferably, it is within 15 days, within 7 days, more preferably within 2 days. The present invention makes it possible to use such a peptide, which usually has a short half-life and cannot be used as a medicament, as a medicament by utilizing the sustained release effect.
[0161] 従って、本発明は、本発明のペプチドを含む薬学的組成物を提供することが理解さ れる。このような薬学的組成物には、任意の他の成分、キャリアなどを含んでもよいこ とが理解される。  [0161] Therefore, it is understood that the present invention provides a pharmaceutical composition comprising the peptide of the present invention. It is understood that such pharmaceutical compositions may include any other ingredients, carriers, and the like.
[0162] 本発明の薬学的組成物は、さらなる徐放成分をさらに含んでいてもよい。そのような 徐放成分としては、例えば、リボソームなどを挙げることができるがそれらに限定され ない  [0162] The pharmaceutical composition of the present invention may further contain an additional sustained release component. Examples of such a sustained release component include, but are not limited to, ribosomes and the like.
別の局面において、本発明は、生理活性ペプチドのアミノ酸配列を含む少なくとも 2 種のペプチド含む組成物であって、該ペプチドの少なくとも 1種は、該生理活性ぺプ チドに由来しない少なくとも 1つのアミノ酸を含む、組成物を提供する。この組成物は 、本発明のペプチドを少なくとも 1種類以上含んでおり、その種類ごとにぺプチダー ゼによる耐性、代謝などが異なることが見出され、組み合わせ投与することによって 徐放効果があることが確認された。このようなアイデアで徐放効果を達成したのは、本 発明者らの知る限り、これまでになぐ従って、本発明は、顕著な効果を奏するといえ る。  In another aspect, the present invention relates to a composition comprising at least two peptides comprising the amino acid sequence of a bioactive peptide, wherein at least one of the peptides comprises at least one amino acid not derived from the bioactive peptide. A composition comprising: This composition contains at least one kind of the peptide of the present invention, and it has been found that the peptidase resistance, metabolism, etc. are different for each kind, and that the combination administration has a sustained release effect. confirmed. As far as the inventors of the present invention have achieved the sustained release effect based on such an idea, the present invention has never been so far. Therefore, it can be said that the present invention has a remarkable effect.
[0163] 1つの実施形態において、本発明の組成物に含まれるべきペプチドのうちアミノ酸 の部分は、アミノ酸数が異なる少なくとも 2種が存在し、好ましくは少なくとも 3種存在 し、さらに好ましくは、少なくとも 4種、 5種、 6種、 7種、 8種、 9種、 10種、それ以上で あることが理解される。  [0163] In one embodiment, the amino acid portion of the peptide to be included in the composition of the present invention has at least two types of amino acids having different numbers of amino acids, preferably at least three types, and more preferably at least three types. It is understood that there are 4, 5, 6, 7, 8, 9, 10, and more.
[0164] 別の実施形態において、本発明の組成物に含まれるべきペプチドのうちアミノ酸の 部分は、アミノ酸が含まれる位置が異なる少なくとも 2種が存在し、好ましくは少なくと も 3種存在し、さらに好ましくは、少なくとも 4種、 5種、 6種、 7種、 8種、 9種、 10種、そ れ以上であることが理解される。  [0164] In another embodiment, the amino acid portion of the peptide to be included in the composition of the present invention includes at least two types of amino acids differing in the position at which the amino acid is included, and preferably includes at least three types of amino acids, It is understood that more preferably at least 4, 5, 6, 7, 8, 9, 9, 10 or more.
[0165] 他の好ま 、実施形態にぉ 、て、本発明の組成物にぉ 、て含まれるべきペプチド は、少なくとも 2種、より好ましくは、少なくとも 3種組成物中に存在する。このように多 種類含まれるときは、ペプチドのうち少なくとも 1種は、天然の配列の生理活性べプチ ドであってもよい。 3種含ませることによって、 2種において達成されていた効果よりさ らに顕著な効果 (たとえば、 3倍〜 5倍またはそれ以上)の徐放効果を達成したことが 本発明において確認された。従って、本発明は、 1種よりも 2種、 2種よりも 3種、ある いはそれ以上の種類のペプチドを含ませることが顕著に有利であり得る。 [0165] In another preferred embodiment, the peptide to be included in the composition of the present invention. Is present in at least two, more preferably at least three compositions. When such multiple types are included, at least one of the peptides may be a bioactive peptide having a natural sequence. It has been confirmed in the present invention that the inclusion of three types achieves a sustained release effect that is more remarkable (for example, three to five times or more) than the effect achieved in the two types. Thus, it may be significantly advantageous for the present invention to include more than two, more than two, or more peptides.
[0166] 本発明の組成物において含まれるべきペプチドは、本明細書において上述した任 意の形態を採り得ることが理解される。  [0166] It is understood that the peptide to be included in the composition of the present invention can take any of the forms described herein above.
[0167] 好ましい実施形態では、 2種類以上の生理活性ペプチドに由来するペプチドが組 成物中に含まれていてもよい。このような組成物は、 2種以上の生理活性効果を達成 しょうとするときに望ましい形態である。あるいは、 2種以上の生理活性ペプチドの相 乗効果を達成するために本発明の組成物が使用され得る。あるいは、 2種以上の生 理活性ペプチドの作用時期をコントロールするような組成物を構成することも可能で ある。  [0167] In a preferred embodiment, a peptide derived from two or more types of bioactive peptides may be contained in the composition. Such compositions are a desirable form when trying to achieve more than one bioactive effect. Alternatively, the compositions of the present invention can be used to achieve a synergistic effect of two or more bioactive peptides. Alternatively, it is also possible to construct a composition that controls the time of action of two or more physiologically active peptides.
[0168] 従って、本発明の組成物は、さらに徐放成分などの他の成分を含んでいてもよいこ とが理解される。  [0168] Therefore, it is understood that the composition of the present invention may further contain other components such as a sustained release component.
[0169] 別の局面において、本発明は、生理活性ペプチドを、徐放させる方法であって、 A) 生理活性ペプチドと、該生理活性ペプチドに由来しない少なくとも 1つのアミノ酸とを 含む、ペプチドを、徐放が企図される被検体に投与する工程、を包含する、方法を提 供する。ここで使用されるペプチドは、本明細書において上述したような任意の形態 を使用することができることが理解される。また、投与および被検体についてもまた、 本明細書において記載される任意の形態を採り得ることが理解される。  [0169] In another aspect, the present invention provides a method for sustained release of a bioactive peptide, comprising: A) a peptide comprising a bioactive peptide and at least one amino acid not derived from the bioactive peptide; Administering to a subject for which a sustained release is contemplated. It is understood that the peptides used herein can use any form as described herein above. It is also understood that the administration and the subject can also take any of the forms described herein.
[0170] 他の局面において、本発明は、生理活性ペプチドを、徐放させる方法であって、 A) 生理活性ペプチドと、該生理活性ペプチドに由来しない少なくとも 1つのアミノ酸とを 含む、ペプチドを含む組成物を、徐放が企図される被検体に投与する工程であって 、該組成物は、該ペプチド中の該アミノ酸が複数種類存在する、工程、を包含する、 方法を提供する。ここで使用される組成物は、本明細書において上述したような任意 の形態を使用することができることが理解される。また、投与および被検体についても また、本明細書にぉ 、て記載される任意の形態を採り得ることが理解される。 [0170] In another aspect, the present invention relates to a method for sustained release of a bioactive peptide, comprising: A) a peptide comprising a bioactive peptide and at least one amino acid not derived from the bioactive peptide. Administering a composition to a subject for which sustained release is intended, wherein the composition comprises a plurality of the amino acids in the peptide. It is understood that the compositions used herein can use any form as described herein above. Also for administration and subject It is understood that any form described in this specification can be adopted.
[0171] 別の局面において、本発明は、生理活性ペプチドと、該生理活性ペプチドに由来 しない少なくとも 1つのアミノ酸とを含む、ペプチドの、生理活性作用を発揮させる徐 放処方物の製造のための、使用を提供する。ここで使用されるペプチドおよび除法 処方物の製造の方法は、本明細書において上述される任意の形態を使用することが できることが理解される。  [0171] In another aspect, the present invention relates to a method for producing a sustained-release formulation for producing a bioactive effect, comprising a bioactive peptide and at least one amino acid not derived from the bioactive peptide. Provide, use. It is understood that the methods of making the peptides and exclusion formulations used herein can employ any of the forms described herein above.
[0172] 以上、本発明を、理解の容易のために好ましい実施形態を示して説明してきた。以 下に、実施例に基づいて本発明を説明するが、上述の説明および以下の実施例は 、例示の目的のみに提供され、本発明を限定する目的で提供したのではない。従つ て、本発明の範囲は、本明細書に具体的に記載された実施形態にも実施例にも限 定されず、特許請求の範囲によってのみ限定されることが理解されるべきである。 実施例  As described above, the present invention has been described with reference to the preferred embodiments for easy understanding. Hereinafter, the present invention will be described based on examples. However, the above description and the following examples are provided for illustrative purposes only, and are not provided for limiting the present invention. Therefore, it should be understood that the scope of the present invention is not limited to the embodiments or examples specifically described herein, but only by the appended claims. . Example
[0173] 以下、本発明を実施例に基づきより具体的に説明する。  Hereinafter, the present invention will be described more specifically with reference to examples.
[0174] (実施例 1 ペプチドの合成）  (Example 1 Synthesis of Peptide)
Fmocィ匕'手による t¾タカ率固ネ目法 (K.Nokihara, et al., Innovation and Perspectives in Solid-Phase Synthesis 1992, ed., R. Epton, Intercept Limited, Andover,  Fmoc-Daily's t¾taka ratio fixation method (K. Nokihara, et al., Innovation and Perspectives in Solid-Phase Synthesis 1992, ed., R. Epton, Intercept Limited, Andover,
UK, 445-448, 1992, Design andApplications of a Novel Simultaneous Multiple Solid-Phase Peptide Synthesizer;軒原清史、有機合成哲学協会誌、 52,347-358, 1994,高効率ペプチド合成：多種品目同時自動合成とペプチドライブラリー）に基づ き、ペプチド自動合成機を用いて種々のアミノ酸配列を有するペプチドの代表例を 合成した。  UK, 445-448, 1992, Design and Applications of a Novel Simultaneous Multiple Solid-Phase Peptide Synthesizer; Kiyoshi Fumihara, Journal of the Society of Synthetic Organic Philosophy, 52,347-358, 1994 Rally), representative examples of peptides having various amino acid sequences were synthesized using an automatic peptide synthesizer.
合成したペプチドのアミノ酸配列を以下に示す。  The amino acid sequence of the synthesized peptide is shown below.
[0175] 配列番号 1： SVVYGLR [0175] SEQ ID NO: 1: SVVYGLR
配列番号 2： GGGGGG SVVYGLR (本明細書にぉ 、て G6とも!/、う） 配列番号 3： SVVYGLR - GGGGGG  SEQ ID NO: 2: GGGGGG SVVYGLR (both in this specification and G6!) SEQ ID NO: 3: SVVYGLR-GGGGGG
配列番号 4： GGG - SVVYGLR - GGG (本明細書にぉ 、て WGとも!/、う） 配列番号 5： GGG SVVYGLR  SEQ ID NO: 4: GGG-SVVYGLR-GGG (both in this specification and WG! / SEQ ID NO: 5) GGG-SVVYGLR
配列番号 6： SVVYGLR -GGG 得られたペプチドを、液体クロマトグラフィー結合質量スペクトル (LCMS)システムで 検定し、高純度であることを確認した (単一成分、質量理論値と一致)。 SEQ ID NO: 6: SVVYGLR -GGG The resulting peptide was assayed on a liquid chromatography coupled mass spectrometry (LCMS) system and confirmed to be highly pure (single component, consistent with mass theory).
[0176] このような手順に従って、以下の実施例において使用されるペプチドを作製した。 [0176] According to such a procedure, peptides used in the following Examples were produced.
[0177] [0177]
(実施例 2 合成ペプチドの生理活性（=血管新生作用）の確認）  (Example 2 Confirmation of physiological activity (= angiogenic action) of synthetic peptide)
実施例 1で合成した本発明のペプチドの存在下でラットの血管内皮細胞を三次元 口 ^しプ 。  Rat vascular endothelial cells were three-dimensionally opened in the presence of the peptide of the present invention synthesized in Example 1.
[0178] 合成したペプチドは、配列番号 1、 2および 4のものについて、単独、およびそれら の任意の 2種類の組み合わせならびに 3種類すベてを含む組み合わせを含むものを 使用した。  [0178] As the synthesized peptides, those containing SEQ ID NOS: 1, 2 and 4 were used singly or in combination of any two of them and combinations containing all three kinds.
[0179] この操作は具体的には次のようにして行った。細胞は transformed rat lung e ndothelial cells (TRLEC細胞）を用いた。 10ん gZmlの濃度のペプチド溶液混和 コラーゲン I層中に TRLEC細胞を播種し、 14日間の炭酸ガスインキュベータ一中で 培養した。コントロールは、因子なし（一）および従来より血管新生因子として知られて Vヽるタンパク質 VEGF ( + )で行った。  [0179] This operation was specifically performed as follows. The cells used were transformed rat lung endothelial cells (TRLEC cells). TRLEC cells were seeded in the collagen I layer mixed with a peptide solution having a concentration of 10 gZml, and cultured in a carbon dioxide incubator for 14 days. Controls were performed without factor (1) and with the protein VEGF (+), previously known as an angiogenic factor.
[0180] 14日後に培養細胞を顕微鏡で観察したところ、コントロールはまったく管腔を形成 しな力つた。本発明のペプチドおよび VEGFは管腔を形成し、この管腔を囲包する 細胞同士が接着されていた。さらに、倍率 7000倍で電子顕微鏡で管腔部分を観察 したところ、管腔の内壁にマイクロピライ (微細細胞突起）が複数個形成されているこ とが認められた。さらに、倍率 15000倍で、管腔を取り囲む血管内皮細胞同士の接 合部分を観察したところ、内皮細胞同士が堅固に結合されている領域、すなわち、タ イトジャンクション (密着結合）が認められた。これらの所見は内皮細胞が極性を獲得 して、管腔を形成したことを示す。なお、極性というのは細胞が頭と尻尾のような機能 分担する部分をもつ性質で、内皮細胞を通常培養していても極性はなぐしたがって 管腔も作らないので、このペプチドで誘導されたことを示している。管腔形成長さは有 意に本発明のペプチドの方が VEGFより優れていた。これらの結果から、本発明の ペプチドには血管内皮培養細胞力 構成される組織中に、細胞同士を接着させてそ れらの間に管腔を形成する作用（生体内では、この管腔が血管になる）があることが 確認された。その模式図は、図 1に示す。 [0180] After 14 days, the cultured cells were observed under a microscope, and 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. Further, when the lumen was observed with an electron microscope at a magnification of 7000 times, it was confirmed that a plurality of micropirai (fine cell projections) were formed on the inner wall of the lumen. Further, at a magnification of 15,000, the junction of the vascular endothelial cells surrounding the lumen was observed. As a result, a region where the endothelial cells were tightly bonded, that is, a tight junction was observed. These findings indicate that the endothelial cells acquired polarity and formed a lumen. Polarity is a property of cells having a part that shares functions such as head and tail.Even if endothelial cells are normally cultured, they lose polarity and do not form a lumen, so it was induced by this peptide. Is shown. The peptide of the present invention was significantly superior in tube formation length to VEGF. From these results, the peptide of the present invention adheres cells to each other in a tissue composed of vascular endothelial cultured cells. It has been confirmed that there is an action of forming a lumen between them (in a living body, this lumen becomes a blood vessel). The schematic diagram is shown in FIG.
[0181]  [0181]
(実施例 3 DASアツセィによる in vivoでの生理活性 (血管新生作用）の確認） 細胞培養液として用いられる DMEM (ダルベッコ修飾イーグル培地）中に lOngZ mlの濃度のプチドを溶解した溶液を得た。直径 0. 45mmの円筒の上下を Millipore フィルター (Millipore, USA)で塞 、で構成されるマイクロセルをマウス（BALBZc AnNCrj、 6週齢、メス；日本クレア）の背部に埋め込んだ後、マイクロセル内に上記 本発明のペプチド等のペプチド溶液や VEGF溶液を注入した。また、対照として、ぺ プチドを含まな 、リン酸緩衝液 (PBS)単独も注入した (コントロール)。 5日後に、マイ クロセル周辺の組織の様子を顕微鏡で観察した。その模式図は、図 2に示す。 Example 3 Confirmation of In Vivo Physiological Activity (Angiogenic Action) by DAS Atssay A solution was prepared by dissolving a peptide at a concentration of lOngZ ml in DMEM (Dulbecco's modified Eagle medium) used as a cell culture medium. Diameter 0. 45 mm Millipore filter the upper and lower cylinder (Millipore, USA) busy at, in the constructed microcell mice (BALBZc AnNCrj, 6 weeks old, female; CLEA Japan) after filling the back of microcells A peptide solution such as the peptide of the present invention and a VEGF solution were injected therein. As a control, phosphate buffer (PBS) alone containing no peptide was also injected (control). Five days later, the state of the tissue around the microcell was observed with a microscope. The schematic diagram is shown in FIG.
[0182] ペプチド SWYGLR (配列番号 1)単独を用いた場合の血管新生の様子を図 3に 示す。 6G単独の場合の血管新生の様子（5日目）を図 4に示す。 WG単独の場合の 血管新生の様子（5日目）を図 5に示す。ペプチド SWYGLRと WGとの組み合わせ の場合の血管新生の様子（5日目）を図 6に示す。 G6とペプチド SWYGLRとの組 み合わせの場合の血管新生の様子（5日目）を図 7に示す。 G6と WGとの組み合わ せの場合の血管新生の様子（5日目）を図 8に示す。 3つの組み合わせの血管新生 の様子（5日目）を図 9に示す。図 10には、 10日目の G6と W10との組み合わせの血 管新生の様子を示す。図 11には、 10日目の 3つの組み合わせの場合の血管新生の 様子を示す。 [0182] Fig. 3 shows the state of angiogenesis when peptide SWYGLR (SEQ ID NO: 1) alone was used. Fig. 4 shows the state of angiogenesis in the case of 6G alone (day 5). Figure 5 shows the state of angiogenesis (day 5) in the case of WG alone. FIG. 6 shows the state of angiogenesis (day 5) in the case of the combination of peptide SWYGLR and WG. Figure 7 shows the state of angiogenesis (day 5) in the case of the combination of G6 and the peptide SWYGLR. Figure 8 shows the state of angiogenesis in the combination of G6 and WG (day 5). Figure 9 shows the angiogenesis of the three combinations (day 5). FIG. 10 shows the angiogenesis of the combination of G6 and W10 on day 10. FIG. 11 shows the state of angiogenesis for the three combinations on day 10.
[0183] 結果から明らかなように、 3つの組み合わせの効果が持続していることが明らかにな つた。また、 5日目の結果から明らかなように、どのような形態でグリシンのようなァミノ 酸が結合していても、徐放効果があるようであること分力つた（5日目のみの評価を提 示する予定です)。  [0183] As is clear from the results, it was clear that the effects of the three combinations were sustained. In addition, as is clear from the results on the fifth day, it was confirmed that the release of the amino acid, such as glycine, in any form seems to have a sustained release effect (evaluation only on the fifth day). Will be provided).
[0184] また、 10日目につ 、ては、 3つの組み合わせが最も高 、効果を示して 、た。これに ついで、 WGよりも G6の方が徐放効果が高いようであることが分力つた。この理由とし ては、 1つ、 2つ程度のグリシンの付加では、生理活性ペプチドの活性がそのまま発 揮される可能性が高いことが挙げられるがそれらに限定されない。従って、 3つ以上 のアミノ酸が付カ卩 (好ましくは 6つ程度以上）されることが好まし 、ようである。 [0184] On the 10th day, the combination of the three was the most effective and showed the highest effect. Subsequent to this, G6 seemed to have a better sustained release effect than WG. The reason for this is that the addition of about one or two glycines has a high possibility that the activity of the physiologically active peptide is directly exerted, but is not limited thereto. Therefore, three or more It seems that the amino acids are preferably added (preferably about 6 or more).
[0185] 図 3〜： L 1に示した写真からも明らかなように、徐放構造にした場合でも、 SWYGL Rの 3つの効果である、血管新生および血管ネットワーク形成能が発揮されていること が明らかになった。これは、血管が新生され、新生された血管が新生し、網状となり始 めている様子が顕著であることからも明らかである。従って、本発明のペプチドは、複 数の活性を有するペプチドにおいて、その複数の活性を保持させるのに有効であり 得ることが明らかになった。 [0185] Fig. 3-: As is clear from the photograph shown in L1, even when the sustained release structure is used, the three effects of SWYGL R, namely, the angiogenesis and vascular network forming ability are exhibited. Was revealed. This is also evident from the remarkable appearance of the newly formed blood vessels, and the newly formed blood vessels are beginning to renew and begin to reticulate. Therefore, it became clear that the peptide of the present invention can be effective in retaining a plurality of activities in a peptide having a plurality of activities.
[0186] 実施例 1にお!/、て合成したペプチドの血管新生数にっ 、て計数した結果を以下に 示す。血管新生数については、 5日目よりも 10日目のほうが亢進されていた。特に、 WGよりも G6の方が徐放効果が高いようであることが分力つた。特に、 3つの組み合 わせでの徐放効果が高いことが明らかになった。 [0186] The results of counting the number of angiogenesis of the peptide synthesized in Example 1 are shown below. The number of angiogenesis was enhanced on day 10 rather than on day 5. In particular, G6 seemed to have a higher sustained release effect than WG. In particular, it became clear that the sustained release effect of the three combinations was high.
[0187] 実施例 1にお!/、て合成したペプチドの血管新生の長さにつ 、て計数した結果を以 下に示す。新生血管 1本あたりの平均ピクセル数を、実体顕微鏡 (ォリンパス、 SZX1 2、 Japan)にて観察した。得られた画像を Photoshop (登録商標）（Adobe、 Japan) にて読み取り。ピクセルで計数した。スコアは、以下のとおりとした。 [0187] The results of counting the length of angiogenesis of the peptide synthesized in Example 1 are shown below. The average number of pixels per new blood vessel was observed with a stereoscopic microscope (Olympus, SZX12, Japan). The obtained image was read by Photoshop (registered trademark) (Adobe, Japan). Counted in pixels. The scores were as follows.
[0188] スコア 1:100未満 [0188] Score less than 1: 100
スコア 2:100以上 125未満  Score 2: 100 or more and less than 125
スコア 3:125以上 150未満  Score 3: 125 or more and less than 150
スコア 4:150以上 175未満  Score 4: 150 or more and less than 175
スコア 5:175以上 200未満  Score 5: 175 or more and less than 200
スコア 6:200以上  Score 6: 200 or more
血管新生の長さに対する効果についても、 5日目よりも 10日目のほうが亢進されて いた。特に、 WGよりも G6の方が徐放効果が高いようであることが分力つた。特に、 3 つの組み合わせでの徐放効果が高いことが明らかになった。  The effect on the length of angiogenesis was also enhanced on day 10 rather than on day 5. In particular, G6 seemed to have a higher sustained release effect than WG. In particular, it became clear that the sustained release effect of the three combinations was high.
[0189] 次に、上述のように、計算した [0189] Next, calculation was performed as described above.
これらの結果について、血管ネットワーク評価について、評価したものを図 12に示 す。ネットワーク形成能については、組織での血管新生の状態を実体顕微鏡 (ォリン パス、 SZX12、 Japan)にて観察し、得られた画像を Photoshop (登録商標）（Adob e、 Japan)にて読み取りネットワーク能を以下のようにスコア化した。 Figure 12 shows the results of the evaluation of the vascular network evaluation. Regarding the ability to form a network, the state of angiogenesis in a tissue was observed with a stereoscopic microscope (Olympus, SZX12, Japan), and the obtained image was analyzed using Photoshop (registered trademark) (Adob e, Japan), the reading network ability was scored as follows.
[0190] Nwl：ネットワーク形成前記で血管新生は認められるが、各新生血管は単独の状 fe。 Nwl: Network formation Angiogenesis is observed as described above, but each new blood vessel is a single fe.
[0191] Nw2 :ネットワーク形成中期であり、各新生血管同士は、はしご上の側枝が係った 状態。  [0191] Nw2: In the middle stage of network formation, each new blood vessel is in a state in which the side branch on the ladder is engaged.
[0192] Nw3 :ネットワーク完成期であり、はしご上側枝がさらに側枝を出した状態。  [0192] Nw3: The network is in its final stage, and the ladder upper branch has further extended the side branch.
[0193] Nw4 :ネットワーク成熟期であり、広範囲に新生血管叢を示す状態。 [0193] Nw4: Network maturation stage, showing a wide range of neovascular plexus.
[0194] ネットワーク形成能に対する効果についても、 5日目よりも 10日目のほうが亢進され ていた。特に、 WGよりも G6の方が徐放効果が高いようであることが分力つた。特に、 3つの組み合わせでの徐放効果が高いことが明らかになった。 [0194] The effect on network-forming ability was enhanced on day 10 rather than on day 5. In particular, G6 seemed to have a higher sustained release effect than WG. In particular, it was revealed that the sustained release effect of the three combinations was high.
[0195] このように、本発明による、アミノ酸付加および複数の種類のペプチドの含有によつ て、顕著に徐放効果が達成された。 [0195] Thus, a remarkable sustained release effect was achieved by the addition of amino acids and the inclusion of plural types of peptides according to the present invention.
[0196]  [0196]
(実施例 4：他の生理活性ペプチド (ラミニン由来血管新生阻害ペプチド) ) 次に、血管新生阻害作用を有する生理活性ペプチド (配列番号 7)およびそのアミ ノ酸付カ卩による改変体を、実施例 1に準じて作製した。作製したものは以下のとおりで ある。  (Example 4: Another bioactive peptide (laminin-derived angiogenesis inhibitory peptide)) Next, a bioactive peptide having an angiogenesis inhibitory action (SEQ ID NO: 7) and a modified product thereof using amino acid-adhesive caspase were prepared. It was produced according to Example 1. The fabricated ones are as follows.
[0197]  [0197]
合成したペプチドのアミノ酸配列を以下に示す。  The amino acid sequence of the synthesized peptide is shown below.
[0198] 配列番号 7 :天然型 YIGSR [0198] SEQ ID NO: 7: Native YIGSR
配列番号 8 : GGGGGG— YIGSR  SEQ ID NO: 8: GGGGGG— YIGSR
配列番号 9： YIGSR-GGGGGG  SEQ ID NO: 9: YIGSR-GGGGGG
配列番号 10 : GGG— YIGSR— GGG  SEQ ID NO: 10: GGG— YIGSR— GGG
配列番号 11： GGG -YIGSR  SEQ ID NO: 11: GGG -YIGSR
配列番号 12 : YIGSR— GGG これらのペプチドについて、血管新生アツセィ（実施例 2および 3に記載される）を用 いて活性をアツセィしたところ、 5日目と 10日目とでは、 10日目に、血管新生阻害効 果がアミノ酸付加のものの方が効果が高くなつていることが明らかになる。 SEQ ID NO: 12: YIGSR—GGG These peptides were assayed for activity using an angiogenesis assay (described in Examples 2 and 3). Angiogenesis inhibitory effect It is clear that the effect of the amino acid-added fruits is higher.
[0199]  [0199]
このように、本発明による、アミノ酸付加および複数の種類のペプチドの含有によつ て、顕著に徐放効果が達成される。  Thus, a remarkable sustained release effect is achieved by the addition of amino acids and the inclusion of plural types of peptides according to the present invention.
[0200]  [0200]
(実施例 5：他のアミノ酸の付加）  (Example 5: Addition of other amino acids)
次に、グリシンとは別のアミノ酸を用いて、徐放効果を確認した。実施例 1に準じて、 以下の合成ペプチドを合成した。生理活性ペプチドは、実施例 1と同じものを用いた 。コントロールとして、チロシンがフエ-ルァラニンになった改変体も試験した。  Next, the sustained release effect was confirmed using an amino acid different from glycine. The following synthetic peptides were synthesized according to Example 1. The same bioactive peptide as in Example 1 was used. As a control, a variant in which tyrosine was changed to phenalanine was also tested.
[0201] 配列番号 1： SVVYGLR [0201] SEQ ID NO: 1: SVVYGLR
配列番号 13： SVVFGLR  SEQ ID NO: 13: SVVFGLR
配列番号 14： AAAAAA SWYGLR  SEQ ID NO: 14: AAAAAA SWYGLR
配列番号 15： SVVYGLR AAAAAA  SEQ ID NO: 15: SVVYGLR AAAAAA
配列番号 16： AAA SWYGLR AAA  SEQ ID NO: 16: AAA SWYGLR AAA
配列番号 17： AAA SWYGLR  SEQ ID NO: 17: AAA SWYGLR
配列番号 18： SVVYGLR -AAAo  SEQ ID NO: 18: SVVYGLR -AAAo
[0202] (実施例 6：生体代用材料との併用） [0202] (Example 6: Combination with biosubstitute material)
本発明のペプチドは、徐放効果が増強されることから、人工骨等の生体代用材料、 人工臓器の体内埋め込みおよび臓器の修復に用いることができる。人工骨の代表で あるヒドロキシアパタイトで代用骨を作製する。  Since the sustained release effect is enhanced, the peptide of the present invention can be used for biosubstitute materials such as artificial bones, for implanting artificial organs in the body, and for repairing organs. Produce a bone substitute using hydroxyapatite, a representative of artificial bone.
[0203] キャリアタンパク質として、動物由来コラーゲンを部分加水分解し、アレルゲンの部 分を除去したゼラチンである FreAlagin AD タイプ (宮城化学工業株式会社製、 分子量 2000〜20000)を用いた。 FreAlagin AD タイプは、臨床での使用が認 められているものである。 [0203] As a carrier protein, FreAlagin AD type (manufactured by Miyagi Chemical Industry Co., Ltd., molecular weight 2,000 to 20,000), 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.
[0204] 代用骨を、以下のようにしてゼラチン結合ペプチドに付着させる。簡略には、 FreAl agin AD (キャリアタンパク質） 100 mg を 2. 5 mL の MilliQ 水に溶解した。 本発明のペプチド 1. 20- 1. 32 mg を 0. 1 M リン酸緩衝液（ρΗ 7. 0) 1 m L に溶解し，キャリアタンパク質に氷冷下でカ卩えた。ダルタルアルデヒド（25%溶液 を 0. 1 M リン酸緩衝液 (pH 7. 0)で 10倍に希釈し、 0. 15 mL を得られた混 合物に滴下 (4°C)、反応混合物を 4°Cで 3〜4時間撹絆した。これにより、徐放効果 が達成されたペプチドの N末端のァミノ基と、キャリアタンパク質のァミノ基が共有結 合される。これを、骨の代わりに移植することによって、その周囲に血管ネットワークを 形成することが可能となる。 [0204] The bone substitute is attached to the gelatin-binding peptide as follows. Briefly, 100 mg of FreAlagin AD (carrier protein) was dissolved in 2.5 mL of MilliQ water. 1.20-1.32 mg of the peptide of the present invention was dissolved in 1 mL of 0.1 M phosphate buffer (ρΗ7.0), and added to the carrier protein under ice-cooling. Daltaraldehyde (25% solution Was diluted 10-fold with 0.1 M phosphate buffer (pH 7.0), 0.15 mL was added dropwise to the resulting mixture (4 ° C), and the reaction mixture was added at 3 ° C at 3 ° C. Stirred for 4 hours. As a result, the amino group at the N-terminus of the peptide having achieved the sustained release effect and the amino group of the carrier protein are covalently bonded. By implanting this in place of bone, it becomes possible to form a vascular network around it.
[0205]  [0205]
(実施例 7：他の生理活性ペプチド (ラミニン由来血管新生ペプチド) )  (Example 7: Other bioactive peptides (laminin-derived angiogenic peptides))
次に、血管新生作用を有する生理活性ペプチド (配列番号 19)およびもう一つの生 理活性ペプチド (配列番号 20)、ならびにそれらのアミノ酸付カ卩による改変体を、実 施例 1に準じて作製した。作製したものは以下のとおりである。合成したペプチドのァ ミノ酸配列を以下に示す。  Next, a physiologically active peptide having an angiogenic action (SEQ ID NO: 19) and another physiologically active peptide (SEQ ID NO: 20), and their modified forms with amino acid-added syrup were prepared according to Example 1. did. The produced ones are as follows. The amino acid sequence of the synthesized peptide is shown below.
[0206] 配列番号 19：天然型 SIKVAV [0206] SEQ ID NO: 19: native SIKVAV
配列番号 27： GGGGGG SIKVAV  SEQ ID NO: 27: GGGGGG SIKVAV
配列番号 28： SIKVAV -GGGGGG  SEQ ID NO: 28: SIKVAV -GGGGGG
配列番号 29： GGG SIKVAV GGG  SEQ ID NO: 29: GGG SIKVAV GGG
配列番号 30： GGG SIKVAV  SEQ ID NO: 30: GGG SIKVAV
配列番号 31： SIKVAV - GGG  SEQ ID NO: 31: SIKVAV-GGG
配列番号 20：天然型 IKVAV  SEQ ID NO: 20: Natural type IKVAV
配列番号 32： GGGGGG -IKVAV  SEQ ID NO: 32: GGGGGG -IKVAV
配列番号 33： IKVAV— GGGGGG  SEQ ID NO: 33: IKVAV—GGGGGG
配列番号 34： GGG - IKVAV -GGG  SEQ ID NO: 34: GGG-IKVAV -GGG
配列番号 35： GGG IKVAV  SEQ ID NO: 35: GGG IKVAV
配列番号 36： IKVAV -GGG  SEQ ID NO: 36: IKVAV -GGG
これらのペプチドについて、血管新生アツセィ（実施例 2および 3に記載される）を用 いて活性をアツセィしたところ、 5日目と 10日目とでは、 10日目に、血管新生効果が アミノ酸付加のものの方が効果が高くなつていることが明らかになる。また、複数種類 混ぜた場合、単数種類の場合よりも徐放効果があることが確認される。  When these peptides were assayed for activity using an angiogenesis assay (described in Examples 2 and 3), on days 5 and 10, on day 10, the angiogenic effect showed an amino acid addition. It becomes clear that things are more effective. In addition, it is confirmed that when multiple types are mixed, a sustained release effect is obtained as compared with the case of a single type.
[0207] このように、本発明による、アミノ酸付加および複数の種類のペプチドの含有によつ て、別の血管新生ペプチドにお!/、ても顕著に徐放効果が達成される。 [0207] As described above, according to the present invention, the addition of amino acids and the inclusion of plural types of peptides Thus, even with another angiogenic peptide, a remarkable sustained release effect is achieved.
[0208] (実施例 8：他の生理活性ペプチド (骨再生ペプチド) ) (Example 8: Other bioactive peptides (bone regeneration peptides))
次に、骨再生作用を有する生理活性ペプチド (配列番号 22)およびそのアミノ酸付 加による改変体を、実施例 1に準じて作製した。作製したものは以下のとおりである。 合成したペプチドのアミノ酸配列を以下に示す。  Next, a physiologically active peptide having a bone regeneration action (SEQ ID NO: 22) and a variant obtained by adding an amino acid thereto were produced according to Example 1. The produced ones are as follows. The amino acid sequence of the synthesized peptide is shown below.
[0209] 配列番号 22：天然型 ALKRQGRTLYGFGG [0209] SEQ ID NO: 22: native ALKRQGRTLYGFGG
配列番号 37： GGGGGG - ALKRQGRTLYGFGG  SEQ ID NO: 37: GGGGGG-ALKRQGRTLYGFGG
配列番号 38： ALKRQGRTLYGFGG GGGGGG  SEQ ID NO: 38: ALKRQGRTLYGFGG GGGGGG
配列番号 39： GGG - ALKRQGRTLYGFGG GGG  SEQ ID NO: 39: GGG-ALKRQGRTLYGFGG GGG
配列番号 40： GGG - ALKRQGRTLYGFGG  SEQ ID NO: 40: GGG-ALKRQGRTLYGFGG
配列番号 41： ALKRQGRTLYGFGG - GGG  SEQ ID NO: 41: ALKRQGRTLYGFGG-GGG
これらのペプチドについて、骨形成アツセィ（EMBO 1992;11:1867-1873を参照） を用いて活性をアツセィしたところ、 5日目と 10日目とでは、 10日目に、骨形成効果 がアミノ酸付加のものの方が効果が高くなつていることが明らかになる。また、複数種 類混ぜた場合、単数種類の場合よりも徐放効果があることが確認される。  The activity of these peptides was determined using the bone formation assay (see EMBO 1992; 11: 1867-1873). It becomes clear that those are more effective. In addition, it is confirmed that when multiple types are mixed, a sustained release effect is obtained as compared with the case of a single type.
[0210] このように、本発明による、アミノ酸付加および複数の種類のペプチドの含有によつ て、骨形成ペプチドにおいても顕著に徐放効果が達成される。 [0210] As described above, by the addition of amino acids and the inclusion of a plurality of types of peptides according to the present invention, a remarkable sustained release effect can be achieved even for osteogenic peptides.
[0211] (実施例 9 :他のアミノ酸の付加） (Example 9: Addition of other amino acids)
次に、グリシンとは別のアミノ酸を用いて、徐放効果を確認した。実施例 1に準じて、 以下の合成ペプチドを合成した。生理活性ペプチドは、実施例 1と同じものを用いた  Next, the sustained release effect was confirmed using an amino acid different from glycine. The following synthetic peptides were synthesized according to Example 1. The same bioactive peptide was used as in Example 1.
[0212] 配列番号 1： SVVYGLR [0212] SEQ ID NO: 1: SVVYGLR
配列番号 42： GLGLGL - SWYGLR  SEQ ID NO: 42: GLGLGL-SWYGLR
配列番号 43： SVVYGLR - GLGLGL  SEQ ID NO: 43: SVVYGLR-GLGLGL
配列番号 44： GLG SWYGLR -GLG  SEQ ID NO: 44: GLG SWYGLR -GLG
配列番号 45： GLG - SWYGLR  SEQ ID NO: 45: GLG-SWYGLR
配列番号 46： SVVYGLR -GLG  SEQ ID NO: 46: SVVYGLR -GLG
配列番号 19 : SIKVAV 配列番号 47： GLGLGL - SIKVAV SEQ ID NO: 19: SIKVAV SEQ ID NO: 47: GLGLGL-SIKVAV
配列番号 48： SIKVAV - GLGLGL  SEQ ID NO: 48: SIKVAV-GLGLGL
配列番号 49： GLG - SIKVAV -GLG  SEQ ID NO: 49: GLG-SIKVAV -GLG
配列番号 50： GLG SIKVAV  SEQ ID NO: 50: GLG SIKVAV
配列番号 51： SIKVAV - GLG  SEQ ID NO: 51: SIKVAV-GLG
配列番号 20 :IKVAV  SEQ ID NO: 20: IKVAV
配列番号 52： GLGLGL -IKVAV  SEQ ID NO: 52: GLGLGL -IKVAV
配列番号 53： IKVAV- GLGLGL  SEQ ID NO: 53: IKVAV-GLGLGL
配列番号 54： GLG-IKVAV-GLG  SEQ ID NO: 54: GLG-IKVAV-GLG
配列番号 55： GLG IKVAV  SEQ ID NO: 55: GLG IKVAV
配列番号 56： IKVAV -GLG  SEQ ID NO: 56: IKVAV-GLG
このように、本発明による、他の種類のアミノ酸付カ卩および複数の種類のペプチドの 含有によって、骨形成ペプチドにお!、ても顕著に徐放効果が達成される。  As described above, the osteogenic peptide can achieve a remarkably sustained release effect by the inclusion of other kinds of amino acid-added syrup and plural kinds of peptides according to the present invention.
[0213] 以上のように、本発明の好ましい実施形態を用いて本発明を例示してきた力 本発 明は、この実施形態に限定して解釈されるべきものではない。本発明は、特許請求 の範囲によってのみその範囲が解釈されるべきであることが理解される。当業者は、 本発明の具体的な好ましい実施形態の記載から、本発明の記載および技術常識に 基づいて等価な範囲を実施することができることが理解される。本明細書において引 用した特許、特許出願および文献は、その内容自体が具体的に本明細書に記載さ れているのと同様にその内容が本明細書に対する参考として援用されるべきであるこ とが理解される。 [0213] As described above, the present invention, which has exemplified the present invention using the preferred embodiment of the present invention, should not be construed as being limited to this embodiment. It is understood that the scope of the present invention should be construed only by the appended claims. It is understood that those skilled in the art can implement an equivalent range based on the description of the present invention and common technical knowledge from the description of the specific preferred embodiments of the present invention. Patents, patent applications, and references cited herein should be incorporated by reference in their entirety, as if the content itself were specifically described herein. Is understood.
産業上の利用可能性  Industrial applicability
[0214] 本発明は、生理活性ペプチドを利用する任意の (獣医学、医薬、医療、歯学など） において有用である。特に、本発明は、生活習慣病 (例えば、心筋梗塞、脳梗塞、閉 塞性大動脈硬化症などの虚血性疾患など）の治療にも有用である。本発明は、長期 効果を一回の投与で達成することから、医薬分野において顕著な効果を達成するこ とが理解される。 [0214] The present invention is useful in any (veterinary, pharmaceutical, medical, dentistry, etc.) utilizing a bioactive peptide. In particular, the present invention is also useful for treating lifestyle-related diseases (eg, myocardial infarction, cerebral infarction, ischemic diseases such as obstructive aortic sclerosis). It is understood that the present invention achieves a remarkable effect in the field of medicine because it achieves a long-term effect with a single administration.

Claims

請求の範囲  The scope of the claims
[I] 生理活性ペプチドと、該生理活性ペプチドに由来しない少なくとも 1つのアミノ酸とを 含む、ペプチド。  [I] A peptide comprising a bioactive peptide and at least one amino acid not derived from the bioactive peptide.
[2] 前記生理活性ペプチドは、ュビキチンによって認識されな 、、請求項 1に記載のぺ プチド。  [2] The peptide according to claim 1, wherein the bioactive peptide is not recognized by ubiquitin.
[3] 前記生理活性ペプチドは、分子量 30kDa以下である、請求項 1に記載のペプチド。  [3] The peptide according to claim 1, wherein the bioactive peptide has a molecular weight of 30 kDa or less.
[4] 前記生理活性ペプチドは、分子量 20kDa以下である、請求項 1に記載のペプチド。 [4] The peptide according to claim 1, wherein the bioactive peptide has a molecular weight of 20 kDa or less.
[5] 前記生理活性ペプチドは、分子量 lOkDa以下である、請求項 1に記載のペプチド。 [5] The peptide according to claim 1, wherein the bioactive peptide has a molecular weight of 10 kDa or less.
[6] 前記生理活性ペプチドは、 300アミノ酸長以下のペプチドまたはその改変体である、 請求項 1に記載のペプチド。 [6] The peptide according to claim 1, wherein the bioactive peptide is a peptide having a length of 300 amino acids or less or a variant thereof.
[7] 前記生理活性ペプチドは、 200アミノ酸長以下のペプチドまたはその改変体である、 請求項 1に記載のペプチド。 [7] The peptide according to claim 1, wherein the bioactive peptide is a peptide having a length of 200 amino acids or less or a variant thereof.
[8] 前記生理活性ペプチドは、 100アミノ酸長以下のペプチドまたはその改変体である、 請求項 1に記載のペプチド。 [8] The peptide according to claim 1, wherein the bioactive peptide is a peptide having a length of 100 amino acids or less or a variant thereof.
[9] 前記アミノ酸は、少なくとも 1つのグリシンを含む、請求項 1に記載のペプチド。 [9] The peptide according to claim 1, wherein the amino acid includes at least one glycine.
[10] 前記アミノ酸は、前記生理活性ペプチドの C末端および N末端力もなる群より選択さ れる少なくとも 1つに結合される、請求項 1に記載のペプチド。 [10] The peptide according to claim 1, wherein the amino acid is bound to at least one selected from the group consisting of C-terminal and N-terminal forces of the bioactive peptide.
[II] 前記アミノ酸は、前記生理活性ペプチドの C末端および N末端の両方に結合される、 請求項 1に記載のペプチド。  [II] The peptide according to claim 1, wherein the amino acid is bound to both the C-terminus and the N-terminus of the bioactive peptide.
[12] 前記アミノ酸は、複数のグリシンを含む、請求項 1に記載のペプチド。  [12] The peptide according to claim 1, wherein the amino acid includes a plurality of glycines.
[13] 前記アミノ酸は、グリシンを 3〜： L0個含む、請求項 1に記載のペプチド。  [13] The peptide according to claim 1, wherein the amino acid contains 3 to L0 glycines.
[14] 前記アミノ酸は、前記生理活性ペプチドの天然の配列とは異なる配列を含む、請求 項 1に記載のペプチド。  [14] The peptide according to claim 1, wherein the amino acid comprises a sequence different from a natural sequence of the bioactive peptide.
[15] 前記アミノ酸は、前記生理活性ペプチドのリーダー配列の天然の配列とは異なる配 列を含む、請求項 1に記載のペプチド。 [15] The peptide according to claim 1, wherein the amino acid comprises a sequence different from a natural sequence of a leader sequence of the bioactive peptide.
[16] 前記生理活性ペプチドは、血管新生、血管新生阻害、骨再生、抗炎症作用 (インテ ダリン阻害)、糖尿病治療、鎮痛作用、抗痴呆からなる群より選択される効果を有する[16] The bioactive peptide has an effect selected from the group consisting of angiogenesis, angiogenesis inhibition, bone regeneration, anti-inflammatory action (intedalin inhibition), diabetes treatment, analgesic action, and anti-dementia.
、請求項 1に記載のペプチド。 The peptide according to claim 1.
[17] 前記生理活性ペプチドは、ペプチド SVVYGLR、ペプチド SIKVAV、ペプチド 'IKV AV (ラミニン由来ペプチド）、ペプチド YIGSR (ラミニン由来ペプチド）、ペプチド AL KRQGRTLYGFGG (骨原性増殖ペプチド； OGP)、ペプチド GDGRHDL (インテ グリン阻害剤）、ペプチド FVNQHLCGSHL VEAL YLVCHERGFFYTPKTGIV EQCCTSICSLYQLENYCN (インスリン）、ペプチド YGGFM (Met—エンケファリ ン）、 YGGFL (Leu—エンケフアリン）、ペプチド MLARMLNTTI SACFLSLLAF[17] The bioactive peptides include peptide SVVYGLR, peptide SIKVAV, peptide 'IKVAV (laminin-derived peptide), peptide YIGSR (laminin-derived peptide), peptide AL KRQGRTLYGFGG (osteoproliferative peptide; OGP), peptide GDGRHDL (integrity). Glin inhibitor), peptide FVNQHLCGSHL VEAL YLVCHERGFFYTPKTGIV EQCCTSICSLYQLENYCN (insulin), peptide YGGFM (Met—Enkephalin), YGGFL (Leu—Enkephalin), peptide MLARMLNTTI SACFLSLLAF
SSACYFQNCP RGGKRAISDM ELRQCLPCGP GGKGRCFGPS IC CADELGCF VGTAEALRCQ EENYLPSPCQ SGQKPCGSGG RCAA VGICCS DESCVAEPEC HDGFFRLTRA REPSNATQLD GPARALL LRL VQLAGTRESV DSAKPRVY (バソプレシン）またはその改変体である、 請求項 1に記載のペプチド。 SSACYFQNCP RGGKRAISDM ELRQCLPCGP GGKGRCFGPS IC CADELGCF VGTAEALRCQ EENYLPSPCQ SGQKPCGSGG RCAA VGICCS DESCVAEPEC HDGFFRLTRA REPSNATQLD GPARALL LRL VQLAGTRESV DSAKPRVY (Vasopressin)
[18] 前記生理活性ペプチドは、所望されな!、活性を有しな!/、、請求項 1に記載のぺプチ ド、。  [18] The peptide according to claim 1, wherein the bioactive peptide has no desired activity and no activity.
[19] 前記生理活性ペプチドは、ペプチド自体で存在する場合、生体内半減期が、 2日以 下である、請求項 1に記載のペプチド。  [19] The peptide according to claim 1, wherein the physiologically active peptide has an in vivo half-life of 2 days or less when the peptide itself is present.
[20] 請求項 1に記載のペプチドを含む、薬学的組成物。 [20] A pharmaceutical composition comprising the peptide according to claim 1.
[21] さらなる徐放成分をさらに含む、請求項 20に記載の薬学的組成物。 [21] The pharmaceutical composition according to claim 20, further comprising an additional sustained release component.
[22] 生理活性ペプチドのアミノ酸配列を含む少なくとも 2種のペプチド含む組成物であつ て、該ペプチドの少なくとも 1種は、該生理活性ペプチドに由来しない少なくとも 1つ のアミノ酸を含む、組成物。 [22] A composition comprising at least two kinds of peptides containing the amino acid sequence of a bioactive peptide, wherein at least one kind of the peptide contains at least one amino acid not derived from the bioactive peptide.
[23] 前記ペプチドは、少なくとも 2種力 前記アミノ酸を含み、該アミノ酸の部分は、ァミノ 酸数が異なる少なくとも 2種が存在する、請求項 22に記載の組成物。 [23] The composition according to claim 22, wherein the peptide comprises at least two kinds of amino acids, and the amino acid portion includes at least two kinds of amino acids having different numbers of amino acids.
[24] 前記ペプチドは、少なくとも 2種力 前記アミノ酸を含み、該アミノ酸の部分は、ァミノ 酸が含まれる位置が異なる少なくとも 2種が存在する、請求項 22に記載の組成物。 [24] The composition according to claim 22, wherein the peptide includes at least two kinds of amino acids, and the amino acid portion includes at least two kinds of amino acids having different positions at which amino acids are included.
[25] 前記ペプチドは、少なくとも 3種が存在する、請求項 22に記載の組成物。 [25] The composition according to claim 22, wherein at least three kinds of the peptides are present.
[26] 前記生理活性ペプチドは、ュビキチンによって認識されな 、、請求項 22に記載の組 成物。 26. The composition according to claim 22, wherein the bioactive peptide is not recognized by ubiquitin.
[27] 前記生理活性ペプチドは、分子量 30kDa以下である、請求項 22に記載の組成物。 [27] The composition according to claim 22, wherein the bioactive peptide has a molecular weight of 30 kDa or less.
[28] 前記生理活性ペプチドは、分子量 20kDa以下である、請求項 22に記載の組成物。 [28] The composition according to claim 22, wherein the bioactive peptide has a molecular weight of 20 kDa or less.
[29] 前記生理活性ペプチドは、分子量 lOkDa以下である、請求項 22に記載の組成物。 29. The composition according to claim 22, wherein the bioactive peptide has a molecular weight of 10 kDa or less.
[30] 前記生理活性ペプチドは、 300アミノ酸長以下のペプチドまたはその改変体である、 請求項 22に記載の組成物。 [30] The composition according to claim 22, wherein the bioactive peptide is a peptide having a length of 300 amino acids or less or a variant thereof.
[31] 前記生理活性ペプチドは、 200アミノ酸長以下のペプチドまたはその改変体である、 請求項 22に記載の組成物。 [31] The composition according to claim 22, wherein the bioactive peptide is a peptide having a length of 200 amino acids or less or a variant thereof.
[32] 前記生理活性ペプチドは、 100アミノ酸長以下のペプチドまたはその改変体である、 請求項 22に記載の組成物。 32. The composition according to claim 22, wherein the physiologically active peptide is a peptide having a length of 100 amino acids or less or a variant thereof.
[33] 前記アミノ酸は、少なくとも 1つのグリシンを含む、請求項 22に記載の組成物。 [33] The composition of claim 22, wherein the amino acid comprises at least one glycine.
[34] 前記アミノ酸は、前記生理活性ペプチドの C末端および N末端力もなる群より選択さ れる少なくとも 1つに結合される、請求項 22に記載の組成物。 [34] The composition according to claim 22, wherein the amino acid is bound to at least one selected from the group consisting of C-terminal and N-terminal forces of the bioactive peptide.
[35] 前記アミノ酸は、前記生理活性ペプチドの C末端および N末端の両方に結合される、 請求項 22に記載の組成物。 [35] The composition according to claim 22, wherein the amino acid is bound to both the C-terminus and the N-terminus of the bioactive peptide.
[36] 前記アミノ酸は、複数のグリシンを含む、請求項 22に記載の組成物。 [36] The composition according to claim 22, wherein the amino acid comprises a plurality of glycines.
[37] 前記アミノ酸は、グリシンを 3〜： L0個含む、請求項 22に記載の組成物。 [37] The composition according to claim 22, wherein the amino acid comprises 3 to L0 glycines.
[38] 前記アミノ酸は、グリシンを 3〜6個含む、請求項 22に記載の組成物。 [38] The composition according to claim 22, wherein the amino acid contains 3 to 6 glycines.
[39] 前記アミノ酸は、前記生理活性ペプチドの天然の配列とは異なる配列を含む、請求 項 22に記載の組成物。 [39] The composition according to claim 22, wherein the amino acid comprises a sequence different from a natural sequence of the bioactive peptide.
[40] 前記アミノ酸は、前記生理活性ペプチドのリーダー配列の天然の配列とは異なる配 列を含む、請求項 22に記載の組成物。 [40] The composition according to claim 22, wherein the amino acid comprises a sequence different from a natural sequence of a leader sequence of the bioactive peptide.
[41] 前記組成物は、少なくとも 1種の天然の配列を有する生理活性ペプチドを含む、請求 項 22に記載の組成物。 [41] The composition according to claim 22, wherein the composition comprises a bioactive peptide having at least one natural sequence.
[42] 前記生理活性ペプチドは、血管新生、血管新生阻害、骨再生、抗炎症作用 (インテ ダリン阻害)、糖尿病治療、鎮痛作用、抗痴呆からなる群より選択される効果を有する 、請求項 22に記載の組成物。  42. The physiologically active peptide has an effect selected from the group consisting of angiogenesis, angiogenesis inhibition, bone regeneration, anti-inflammatory action (intedalin inhibition), diabetes treatment, analgesic action, and anti-dementia. A composition according to claim 1.
[43] 前記生理活性ペプチドは、ペプチド SVVYGLR、ペプチド SIKVAV、ペプチド ΊΚν AV (ラミニン由来ペプチド）、ペプチド YIGSR (ラミニン由来ペプチド）、ペプチド AL KRQGRTLYGFGG (骨原性増殖ペプチド； OGP)、ペプチド GDGRHDL (インテ グリン阻害剤）、ペプチド FVNQHLCGSHL VEAL YLVCHERGFFYTPKTGIV EQCCTSICSLYQLENYCN (インスリン）、ペプチド YGGFM (Met—エンケファリ ン）、ペプチド YGGFL (Leu—エンケフアリン）、ペプチド MLARMLNTTI SACF LSLLAF SSACYFQNCP RGGKRAISDM ELRQCLPCGP GGKGRCF GPS ICCADELGCF VGTAEALRCQ EENYLPSPCQ SGQKPCGSGG RCAAVGICCS DESCVAEPEC HDGFFRLTRA REPSNATQLD GP ARALLLRL VQLAGTRESV DSAKPRVY (バソプレシン）またはその改変体 である、請求項 22に記載の組成物。 [43] The bioactive peptides include peptide SVVYGLR, peptide SIKVAV, peptide ΊΚν AV (laminin-derived peptide), peptide YIGSR (laminin-derived peptide), peptide AL KRQGRTLYGFGG (osteoproliferative peptide; OGP), peptide GDGRHDL (integrity). Glynn inhibitors), peptide FVNQHLCGSHL VEAL YLVCHERGFFYTPKTGIV EQCCTSICSLYQLENYCN (insulin), peptide YGGFM (Met- Enkefari down), peptide YGGFL (Leu- Enkefuarin), peptide MLARMLNTTI SACF LSLLAF SSACYFQNCP RGGKRAISDM ELRQCLPCGP GGKGRCF GPS ICCADELGCF VGTAEALRCQ EENYLPSPCQ SGQKPCGSGG RCAAVGICCS DESCVAEPEC HDGFFRLTRA REPSNATQLD GP ARALLLRL 23. The composition according to claim 22, which is VQLAGTRESV DSAKPRVY (vasopressin) or a variant thereof.
[44] 前記生理活性ペプチドは、所望されな!、活性を有しな ヽ、請求項 22に記載の組成 物。 [44] The composition according to claim 22, wherein the bioactive peptide is not desired and has no activity.
[45] 前記生理活性ペプチドは、ペプチド自体で存在する場合、生体内半減期が、 2日以 下である、請求項 22に記載の組成物。  [45] The composition according to claim 22, wherein the physiologically active peptide has a half-life in vivo of 2 days or less when the peptide itself is present.
[46] 前記生理活性ペプチドは、 2種類以上が存在する、請求項 22に記載の組成物。 46. The composition according to claim 22, wherein two or more types of the physiologically active peptide are present.
[47] さらなる徐放成分をさらに含む、請求項 22に記載の組成物。 [47] The composition of claim 22, further comprising an additional sustained release component.
[48] 生理活性ペプチドを、徐放させる方法であって、 [48] A method for sustained release of a physiologically active peptide,
A)生理活性ペプチドと、該生理活性ペプチドに由来しない少なくとも 1つのアミノ酸 とを含む、ペプチドを、徐放が企図される被検体に投与する工程、  A) a step of administering a peptide comprising a bioactive peptide and at least one amino acid not derived from the bioactive peptide to a subject for which sustained release is intended;
を包含する、方法。  A method comprising:
[49] 生理活性ペプチドを、徐放させる方法であって、 [49] A method for sustained release of a physiologically active peptide,
A)生理活性ペプチドと、該生理活性ペプチドに由来しない少なくとも 1つのアミノ酸 とを含む、ペプチドを含む組成物を、徐放が企図される被検体に投与する工程であ つて、該組成物は、該ペプチド中の該アミノ酸が複数種類存在する、工程、 を包含する、方法。  A) a step of administering a peptide-containing composition comprising a physiologically active peptide and at least one amino acid not derived from the physiologically active peptide to a subject intended for sustained release, wherein the composition comprises: A plurality of the amino acids in the peptide.
[50] 生理活性ペプチドと、該生理活性ペプチドに由来しない少なくとも 1つのアミノ酸とを 含む、ペプチドの、生理活性作用を発揮させる徐放処方物の製造のための、使用。  [50] Use of a peptide containing a bioactive peptide and at least one amino acid not derived from the bioactive peptide for the production of a sustained-release formulation that exerts a bioactive action.
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WO2018014091A1 (en) * 2016-07-22 2018-01-25 University Of Utah Research Foundation Insulin analogs
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CN113366013A (en) * 2018-12-03 2021-09-07 株式会社海培普研究所 Novel compound and angiogenesis agent comprising same
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