WO2006135027A1 - Agent for treatment of wound and/or promotion of wound healing - Google Patents

Agent for treatment of wound and/or promotion of wound healing Download PDF

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Publication number
WO2006135027A1
WO2006135027A1 PCT/JP2006/312072 JP2006312072W WO2006135027A1 WO 2006135027 A1 WO2006135027 A1 WO 2006135027A1 JP 2006312072 W JP2006312072 W JP 2006312072W WO 2006135027 A1 WO2006135027 A1 WO 2006135027A1
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Prior art keywords
growth factor
wound healing
wound
fibroblast growth
seq
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PCT/JP2006/312072
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French (fr)
Japanese (ja)
Inventor
Toru Imamura
Akiko Kuramochi
Mitsuko Kawano
Yuuko Oda
Masahiro Asada
Masashi Suzuki
Junko Oki
Nozomi Tsujino
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National Institute Of Advanced Industrial Science And Technology
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Publication of WO2006135027A1 publication Critical patent/WO2006135027A1/en

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    • 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
    • C07K14/475—Growth factors; Growth regulators
    • C07K14/50—Fibroblast growth factors [FGF]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/18—Growth factors; Growth regulators
    • A61K38/1825—Fibroblast growth factor [FGF]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A61P17/02—Drugs for dermatological disorders for treating wounds, ulcers, burns, scars, keloids, or the like
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6872—Intracellular protein regulatory factors and their receptors, e.g. including ion channels
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/158—Expression markers
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/475—Assays involving growth factors
    • G01N2333/50—Fibroblast growth factors [FGF]
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2500/00—Screening for compounds of potential therapeutic value

Definitions

  • the present invention relates to a wound treatment agent containing a component that promotes skin regeneration, a wound healing promoter, and a method for screening a substance effective for wound treatment and / or promotion of wound healing in an aging individual.
  • FGF fibroblast growth factor
  • Patent Document 1 discloses a wound healing agent containing FGF1.
  • wound healing agents containing FGF2 are currently marketed.
  • the gene products of FGF1 and FGF2 do not increase as a biological response in the wound healing phase, and the wound healing effect is a result of artificially using these growth factors.
  • FGF2 cannot directly promote the proliferation of skin epidermal cells. Therefore, treating a wound with FGF1 or FGF2 may not cause a perfect healing response equivalent to physiological healing.
  • Non-Patent Documents 2 to 18 listed above suggest that FGF plays an important role in the proliferation and differentiation of skin cells.
  • the knowledge of how the FGF group is involved in wound healing remains unclear.
  • Non-Patent Document 1 Ornitz DM, Itoh N: Fibroblast growth factors.
  • Genome Biol2 REVIE WS3005, 2001
  • Non-Patent Document 2 Bai XY, Miao D, Goltzman D & Karaplis AC 2003 Theautosomal domi nant hypophosphatemic rickets R176Q mutation in fibroblast growthfactor 23 resists proteolytic cleavage and enhances in vivo biological potency.Journal of Biological C hemistry 278 9843-9849.
  • Patent Document 3 Beyer TA, Werner S, Dickson C & Grose R 2003 Fibroblast growth factor 22 and its potentialrole during skin development and repair. ExperimentalCell Research 287 228-236.
  • Non-Patent Document 4 Bikfalvi A, Klein S, Pintucci G, Rifkin DB 1997 Biologicalroles of fibro blast growth factor- 2. Endocrinology Review 18 26-45.
  • Non-Patent Document 5 Gao Z, Flaitz CM & Mackenzie IC 1996 Expression ofkeratinocyte growth factor in periapical lesions. Journal of Dentistry Research 75 1658- lb 3.
  • Non-Patent Document 6 GuoL, Degenstein L & Fuchs E 1996 Keratinocyte growth factor is re quired forhair development but not for wound healing. Genesand Development 10 lb
  • Non-Special Terms 7 Igarashi M, Finch PW & Aaronson SA 1998 Characterization of recom binant human fibroblast growth factor (FGF) -10 reveals functionalsimilarities with k eratinocyte growth factor (FGF-7). Journal of Biological Chemistry 27313230-1323 5.
  • Non-Special Reference 8 Jameson J, Ugarte K, Chen N, Yacm P, Fuchs E, Boismenu R & Havra n WL 2002 A role for skin gd T cells in wound repair. Science 296 747-749.
  • Non-Patent Document 9 KawanoM, Suzuki S, Suzuki M, Oki J & Imamura T 2004 Bulge- and b asallayer-specific expression of nbroblast growth factor 13 (FHF-2) in mouse skin. Journal of Investigative Dermatology 122 1084-1090 .
  • Non-Patent Document 10 Kaw Hos M, Kuramochi- Komi A, Asada M, Suzuki M, Oki J, Jiang J & Imamura T 2005 Comprehensive analysis of FGF and FGFR expression in s ⁇ : FGF 18 is highly expressed m hair follicles and capable of inducing anagen from telogen st agehair follicles. Journal of Investigative Dermatology 124 877-885.
  • Non-patent literature ll Marchese C, Chedid M, Dirsch OR, Csaky KG, Santanelli F, Latini C, LaRochelle WJ, Torrisi MR & Aaronson SA 1995 Modulation of keratinocyte gro wth factor and its receptor inreepithelializing human skin.Journal of Experimental Medicine 1821369-1376.
  • Non-Patent Document 12 Martin P, Hopkinson-Woolley J & McCluskey J 1992 Growthfactors and cutaneous wound repair-Progress in Growth Factor Research 425-44.
  • Non-Patent Document 13 MartinP 1997 Wound healing ⁇ aiming for perfect skin regeneration. Science 27675-81.
  • Non-Patent Document 14 OrtegaS, Ittmann M, Tsang SH, Ehrlich M & Basilico C 1998 Neuro nal defects anadelayed wound healing in mice lacking fibroblast growth factor-2. Proceedings of National Academy of bciencesUSA 95 5672-5677.
  • Non-Patent Document 15 Paddock HN, Schultz GS & Mast BA 2003 Methods in remark ithelializa tion.A porcine model ofpartial-thickness wounds.Methods in Molecular Medicine 78 17-36.
  • Non-Patent Document 16 Singer AJ & Clark RA 1999 Mechanism of disease: cutaneous wound healing.New Englandjournal of Medicine 341 738-746.
  • Non-Patent Document 17 Steiling H & Werner S 2003 Fibroblast growth factors: key players in epithelial morphogenesis, repair and cytoprotection.Current Opinion in Biotechnolo gy 14 533-537.
  • Non-Patent Document 18 WernerS, Smola H, Liao X, Longaker MT, Krieg T, Hofschneider P H & Williams LT1994 The function of KGF in epithelial morphogenesis and woundre -epithelialisation. Science 266819-822.
  • Patent Document 1 Japanese Translation of Special Publication 2004-501203
  • an object of the present invention is to elucidate the wound healing action that occurs as a biological reaction and to provide a novel wound healing agent and / or wound healing promoter. It is another object of the present invention to provide a wound healing agent and / or a wound healing promoter effective in an aging individual whose wound healing is delayed.
  • the present inventors belong to the FGF family using young animals and aging animals in order to identify FGF genes that play an important role in skin wound healing.
  • a comprehensive search for the expression of all 22 genes at each stage of healthy skin and wound healing 21 days after wound creation, 2, 4, 7, 15, 21 days).
  • the high expression and function of the FGF gene which was not known, have been found.
  • the level of FGF7, 10 and 22 mRNA expression was higher in healthy skin, while the levels of FGF7 and 10 increased 2- to 3.5-fold over time after wounding.
  • FGF9, 16, 18 and 23 mRNA expression levels were moderate or low in healthy skin S, and increased 2-3-fold after wounding.
  • the mRNA expression level of the FGF23 gene reached a very high level in the early stage of wound healing.
  • Transforming growth factor 1 ⁇ hereinafter referred to as TGF-beta
  • HGF hepatocyte growth factor
  • the present inventors have compared the wound healing response in young animals and aging animals, so that the expression level of most of these FGF genes is higher in aging animals with delayed wound healing than in young animals. I found that it was low.
  • the present inventors have also found that the protein of FGF23 is selectively present in the local area where the wound is being regenerated.
  • the present inventors conducted a detailed functional analysis on FGF23 and found that FGF23 has a function of promoting the proliferation of fibroblasts, which are considered to be important for wound healing. Furthermore, the present inventors have found that the early gene Fos, which is induced to induce cell proliferation, migration, and differentiation, is expressed at a high level at the FGF23 expression site. The present invention has been completed based on such novel findings.
  • the gist of the present invention is as follows.
  • Wound treatment containing as an active ingredient the full length or partial peptide of at least one fibroblast growth factor selected from the group consisting of fibroblast growth factor 9, 11, 16, 18 and 23 And / or agents for promoting wound healing.
  • a nucleic acid encoding a full-length or partial peptide of at least one fibroblast growth factor selected from the group consisting of fibroblast growth factor 9, 11, 16, 18, and 23 is used as an active ingredient. Containing wound treatment and Z or wound healing promotion agent.
  • An agent for treating wounds and / or promoting wound healing of an aging individual comprising a full-length or partial peptide as an active ingredient.
  • An agent for treating wounds and / or promoting wound healing in an aging individual comprising a nucleic acid encoding a full-length or partial peptide as an active ingredient.
  • a wound treatment agent and / or a wound healing promoter can be provided.
  • the screening method of the present invention can screen for substances that are effective in treating wounds and / or promoting wound healing in aging individuals.
  • FIG. 1A is a diagram showing expression profiles of FGF member genes in each stage of wound healing.
  • FIG. 1B shows the expression profiles of FGF member genes at each stage of wound healing.
  • FIG. 2 is a characteristic diagram showing expression profiles of genes belonging to the FGFR family at each stage of wound healing.
  • FIG. 3 is a photograph showing the results of a tissue immunostaining experiment (stained with an anti-FGF23 antibody). The localization of FGF23 protein during the wound healing process examined by immunostaining is shown. Left side: healthy skin (upper epidermis, dermis and subcutaneous fat visible below) right side: healing skin ("force, scab" is on top) FGF23 protein strength indicated by red signal It can be seen that it is specifically expressed only in the local area of the wound and the immediate vicinity of the healing process.
  • FIG. 4 is a characteristic diagram showing the results of DNA synthesis assembly.
  • NIH3T3 cells are cultured in the presence of the FGF23 protein at the concentration shown on the X axis, and the number of cells measured using TetraColor ONE reagent (Seikagaku Corporation) after a certain time is shown on the Y axis.
  • FIG. 5 is a photograph showing the results of a tissue immunostaining experiment (stained with an anti-Fos antibody).
  • the present invention relates to a wound treatment comprising at least one full-length or partial peptide of fibroblast growth factor selected from the group consisting of fibroblast growth factor 9, 11, 16, 18 and 23 as an active ingredient and Z or A drug for promoting wound healing is provided.
  • the present invention also provides a nucleic acid encoding a full-length or partial peptide of at least one fibroblast growth factor selected from the group consisting of fibroblast growth factors 9, 11, 16, 18, and 23. Provides wound healing and Z or wound healing promoting agents that are contained as active ingredients.
  • FGF23 One of the active ingredients that may be contained in the drug of the present invention is FGF23.
  • FGF23 for example, human-derived FGF23 having the amino acid sequence shown in SEQ ID NO: 2 can be used.
  • usable FGF23 is not limited to human origin, for example, other mammals FGF23 derived from fish, FGF23 derived from fish, and the like can be used. Other mammals may include, but are not limited to, mice, rats, chickens, turkeys, tusks, pigs, hidges, and rabbits.
  • a probe prepared based on the nucleotide sequence of human-derived FGF23 shown in SEQ ID NO: 1 is used in accordance with a standard method, a gene encoding FGF23 other than humans other than human can be isolated.
  • nucleotide sequence and amino acid sequence of human-derived FGF23 are shown in SEQ ID NOs: 1 and 2, respectively.
  • the nucleotide sequence of mouse FGF23 mRNA and the amino acid sequence encoded by it are shown in SEQ ID NOs: 3 and 4, respectively.
  • the nucleotide sequence of rat FGF23 mRNA and the amino acid sequence encoded by it are shown in SEQ ID NOs: 5 and 6, respectively.
  • the base sequence of mRNA of zebrafish-derived FGF23 and the amino acid sequence encoded by it are shown in SEQ ID NOs: 7 and 8, respectively.
  • FGF23 has very high homology in mammals, and the function of FG F23 in mammals is almost the same. I can understand. It has been reported that FGF23 regulates the maintenance of phosphate concentration in the body, and its abnormality is thought to cause rickets. The function of FGF23 in wound healing is completely unknown.
  • FGF23 may be a wild type or a mutant. Variants may be found in nature or may be artificially created. Examples of wild-type FGF23 include those having the amino acid sequences shown in SEQ ID NOs: 2, 4, 6, and 8. Mutant FG F23 consists of an amino acid sequence in which one or several amino acids have been deleted, substituted and added from the amino acid sequences shown in SEQ ID NOs: 2, 4, 6 and 8, and is used for wound healing and / or wound healing. Mention may be made of proteins that have healing promoting effects. Here, examples of the several amino acids include 2 to 54, more preferably 2 to 27. FGF23 is known to be cleaved between 179 R and 180 S in vivo.
  • the wound healing promoting effect is lost. It can be used as an active ingredient of the drug of the present invention.
  • the drug of the present invention may contain the above-mentioned partial fragment of FGF23 as an active ingredient.
  • the partial fragment of FGF23 include a polypeptide consisting of the 25th to 251st amino acid sequences in SEQ ID NO: 2, or a polypeptide consisting of the 25th to 179th amino acid sequences in SEQ ID NO: 2. That is, even a polypeptide consisting of the 25th to 251st amino acid sequences has a high probability of binding to the receptor and heparin and promoting the healing of wounds.
  • a polypeptide consisting of amino acid sequences 42 to 159 is most preferably used as a partial fragment of FGF23.
  • the partial fragment of FGF23 may be a peptide having a length of about 3-50 amino acids consisting of any part of the 25th to 251st amino acid sequences as long as it has a wound healing and / or wound healing promoting action.
  • peptides consisting of amino acids exceeding this numerical range are also included in the partial fragment of FGF23 in the present invention.
  • the full length and partial peptides of FGF23 defined as described above have an action of promoting wound healing. The details of this action will be described later in Examples, but the cells are cultured in a medium containing the full length of FGF23 and a partial peptide, and DNA synthesis and cell number increase in skin fibroblasts by the full length and partial peptide of FGF23. This can be confirmed by examining the induction ability of
  • Other active ingredients that may be contained in the drug of the present invention include full-length or partial peptides of FGF9, 11, 16, and 18.
  • the nucleotide sequences of mRNAs of FGF9, 11, 16 and 18 are shown in SEQ ID NOs: 11 (human-derived FGF9), 15 (human-derived FGF11), 17 (human-derived FGF16), 19 (human-derived FGF18) and 21 (human-derived FGF18 ).
  • the amino acid sequences encoded by the mRNAs of FGF9, 11, 16, and 18 are SEQ ID NOs: 12 (human-derived FGF9), 16 (human-derived FGF11), 18 (human-derived FG F16), 20 (chicken-derived FGF18) and 22 respectively.
  • the full-length or partial peptide of at least one fibroblast growth factor selected from the group consisting of FGF9, 11, 16, 18, and 23 is, for example, a solution adapted to skin application, a tarim , Ointments, gels, lotions, shampoos or aerosols, and are provided as a medicament for wound treatment and / or promotion of wound healing. This drug is particularly effective when applied to aging individuals.
  • the wound healing and Z or wound healing promoting agent of the present invention comprises an active ingredient (eg, FGF9, 11, 16, 18, 23) together with a pharmaceutically acceptable carrier adapted for topical administration. Etc.) is administered in the form of a pharmaceutical composition.
  • active ingredient eg, FGF9, 11, 16, 18, 23
  • a pharmaceutically acceptable carrier adapted for topical administration. Etc.
  • the proportion of active ingredient to carrier can vary between:! And 90% by weight.
  • the drug of the present invention further contains a phenone.
  • the agent of the present invention may contain other purified or recombinant-derived protein growth factors well known in the art, and increase the wound healing promoting effect by the active ingredient contained in the agent of the present invention.
  • the growth factor to be enhanced is not particularly limited, and epidermal growth factor (EGF), platelet-derived growth factor (PDGF), transforming growth factor-a (TGF-chicken), transforming growth factor- ⁇ (TGF- ⁇ ), Transforming growth factor-factor belonging to the ⁇ superfamily, bone morphogenetic factor (BMP), insulin-like growth factor (IGF) (eg, IGF-I, II, etc.) and vascular endothelial growth factor.
  • EGF epidermal growth factor
  • PDGF platelet-derived growth factor
  • TGF-chicken transforming growth factor-a
  • TGF- ⁇ Transforming growth factor-factor belonging to the ⁇ superfamily
  • bone morphogenetic factor BMP
  • IGF insulin-like growth factor
  • the drug of the present invention may contain one of these other purified or
  • PDGF, hPDGF and methods for producing them are described in US Pat. No. 4,350,687, Nichiya No. 320, pp. 695-699 (1986), EMBO Journal No. 3, No. 921- 928 (1984), EMBO Journal 3rd, 2963-2967 (1984), which can be referred to.
  • TGF- ⁇ , hTGF- ⁇ and methods for producing them are described in European Patent Application Publication No. 154,434, Cell No. 38, pages 287-297 (1984), You can refer to it.
  • TGF-J3, hTGF-iS and their production methods are described in US Pat. No. 4,774,228, US Pat. No. 4,774,322, DNA No. 7, pages 1-8 (1988), Journal “Ob” Biologicano. Chemistry 262, pp. 12127-12131 (1987) and can be referred to.
  • IGF-I, hIGF_I and methods for producing them are described in International Publication No. WO / 88/03409, European Patent Application Publication No. 264,074, and European Patent Application Publication No. 219,814. can do.
  • IGF-II, hIGF-IV and their production methods are described in European Patent Application Publication No. 280,460 and can be referred to.
  • the agent of the present invention may contain other wound treatment agents, wound healing promoters and the like well known in the art.
  • Other wound treatment agents and wound healing promoters are not particularly limited, and examples thereof include popidone pod, popidone sucrose, and fiblast spray.
  • the drug of the present invention may contain one of these other wound healing agents and wound healing promoters, or may contain more than one kind.
  • the pharmaceutically acceptable carrier adapted for topical administration is not particularly limited, but is a paste such as a hydrophilic petrolatum or an ointment such as polyethylene glycol ointment, a rubber such as xantham.
  • Solution such as alcohol, aqueous or buffer, gel such as aluminum hydroxide or sodium alginate gel, albumin such as human or animal albumin, collagen such as human or animal collagen, alkylcellulose, hydroxyalkylcellulose And cellulose such as alkyl hydroxyalkyl cellulose, methinoresenorelose, hydroxyethinoresenorelose, canoleoxy methinorescenellose, hydride
  • Examples thereof include polymers such as Pluronic TM polyol exemplified by 127; tetronic such as Tetronic 1508, and alginate such as sodium alginate.
  • the dose varies depending on the patient's age, sex, weight and symptoms, therapeutic effect, administration method, treatment time, type of active ingredient contained in the pharmaceutical composition, etc.
  • Patient (weight 60 kg) per person, in the range of about 0.01 to about 100 / ig / day m 2 , preferably about 0.01 to about 100 / ig / day m 2 in terms of the amount of active ingredient Good.
  • the frequency of administration is not particularly limited, but is preferably 1 to 2 times / day.
  • the agent of the present invention comprises, as an active ingredient, a nucleic acid encoding at least one full-length fibroblast growth factor or a partial peptide selected from the group consisting of FGF9, 11, 16, 18, and 23. It may be a wound healing and Z or wound healing promoting agent.
  • the nucleic acid encoding the full length or partial peptide of at least one fibroblast growth factor selected from the group consisting of FG F9, 11, 16, 18, and 23 is cDNA and is incorporated into an expression vector. Is preferred. That is, the agent of the present invention can be used for gene therapy using the above expression vector.
  • the expression vector is a force S having a sequence such as a promoter for realizing expression in animal cells, and is not particularly limited. Examples of expression vectors include, but are not limited to, plasmid vectors and viral vectors.
  • cDNA encoding a full-length or partial peptide of at least one fibroblast growth factor selected from the group consisting of FGF9, 11, 16, 18, and 23, is incorporated into a viral vector, and the patient is infected with a virus (detoxified) having the recombinant viral vector.
  • a virus detoxified
  • at least one full-length polypeptide and / or partial polypeptide of fibroblast growth factor selected from the group consisting of fibroblast growth factor 9, 11, 16, 18, and 23 is produced, and wound healing Power S to promote S
  • Gene transfer methods using viral vectors include, for example, retrovirus, adenovirus, adeno-associated virus, herpes virus, vaccinia virus, box virus, poliovirus, simbis virus and other DNA viruses or RNA viruses, TR4 Alternatively, a method of introducing a DNA encoding a mutant TR4 can be mentioned. Of these, retro Particularly preferred are methods using viruses, adenoviruses, adeno-associated viruses, and vaccinia viruses.
  • Non-viral gene transfer methods include a method in which an expression plasmid is directly administered intramuscularly (DNA vaccine method), a ribosome method, a ribofuctin method, a microinduction method, a calcium phosphate method, an electoral position method, etc.
  • DNA vaccine method a method in which an expression plasmid is directly administered intramuscularly
  • ribosome method a method in which an expression plasmid is directly administered intramuscularly
  • a ribosome method a ribofuctin method
  • microinduction method a microinduction method
  • calcium phosphate method a calcium phosphate method
  • electoral position method etc.
  • the DNA vaccine method and the ribosome method are preferred.
  • a drug for gene therapy when administered by an in vivo method, it is administered by an appropriate administration route such as intravenous, arterial, subcutaneous, intradermal, intramuscular, etc., depending on the disease, symptoms and the like.
  • the drug for gene therapy is generally considered to be an injection or the like.
  • a conventional carrier may be added if necessary.
  • a ribosome or membrane fusion liposome Sendai virus-ribosome, etc.
  • it can be prepared as a ribosome preparation such as a suspension, a freezing agent, or a centrifugal concentrated freezing agent.
  • an expression vector incorporating cDNA encoding a full-length or partial peptide of at least one fibroblast growth factor selected from the group consisting of FGF9, 11, 16, 18, and 23 is administered to humans, Patient (body weight 60 kg) —per person, 0.01-100 mg, preferably 0.1-10 mg.
  • the amount of cDNA encoding the full length or partial peptide of the desired fibroblast growth factor contained in the vector to be administered is 0.001 to 10 mg, preferably 0, per adult patient (body weight 60 kg). ⁇
  • a single dose of 005 to 0.1 mg is recommended. Administration may be repeated at an appropriate frequency until a therapeutic effect is observed.
  • the present invention monitors gene expression during wound healing processes in living bodies with different aging states, compares gene expression levels between young individuals and aging individuals, and compared to young individuals. Effective in wound therapy and Z or wound healing promotion in aging individuals, including determining that products of genes with low expression levels in individuals are effective in wound treatment and / or wound healing promotion in aging individuals
  • a method for screening a substance is provided.
  • young and aged experimental animal individuals are used and their wound healing responses are compared. Examples of experimental animals include mice, rats, chickens, turkeys, ushi, pigs, hidges, and rabbits, excluding humans.
  • the age of young and aging is not limited in any way, but for example, mice are about 8 weeks old as young individuals and about 35 weeks old as individuals. These age differences may be larger or smaller.
  • the expression of various genes is monitored during the wound healing process of experimental animals.
  • the type of gene is not limited at all.
  • the expression of 22 FGF genes and 4 FGF receptor genes are monitored.
  • the expression of the FGF gene in the experimental animal can be analyzed, for example, by using a conventional method such as tissue staining or ELISA using the FGF23 antibody, or the amount of FGF23 gene mRNA in the experimental animal can be determined by quantitative reverse transcription PCR or When analyzed by the Northern Plot method, etc., it is possible to monitor using any method.
  • the expression level of the FGF23 gene in wound healing in an aging animal individual may be reduced compared to the expression level of FGF23 gene in wound healing in a young animal individual.
  • a candidate substance that may have a function of treating wounds and promoting wound healing in an aging animal individual by supplementing the product of the gene.
  • FGF7, 9, 10, 11, 16, 18, 22, 23, TGF-beta, HGF, etc. are candidate substances (see the examples below).
  • the substance screened in this way can be used alone in the clinical application, but it can be used as a pharmaceutical composition by blending with a pharmaceutically acceptable carrier. You can also use it.
  • the ratio of the active ingredient to the carrier at this time can be varied between 1 to 90% by weight.
  • powerful drugs can be administered in various forms, and examples of their administration forms include parenteral administration such as solutions, creams, ointments, gels, lotions, shampoos or aerosols.
  • the dose can be appropriately selected depending on symptoms, age, body weight and the like.
  • a nucleic acid encoding the full-length or partial peptide of the screened substance may be used for wound therapy and / or gene therapy for promoting wound healing in an aging individual.
  • the expression vector, gene transfer method, dosage, administration method and the like are the same as described above.
  • the present invention provides a full-length or partial peptide of at least one growth factor selected from the group consisting of FGF7, 9, 10, 11, 16, 18, 22, 22 and 23, TGF-beta, and HGF. It provides a drug for treating wounds and / or promoting wound healing in aging individuals that is contained as an active ingredient.
  • the present invention relates to a full-length or partial peptide of at least one growth factor selected from the group consisting of FGF7, 9, 10, 11, 16, 18, 22, and 23, TGF-beta, and HGF force.
  • a drug for treating wounds and promoting Z or wound healing in an aging individual containing the encoded nucleic acid as an active ingredient is provided.
  • Examples of active ingredients that may be contained in the drug of the present invention include FGF7, 9, 10, 11, 16, 18, 22 and 23, TGF_beta, and full-length or partial peptides of HGF.
  • the base sequences of FGF7, 9, 10, 11, 16, 18, 22, and 23, TGF-beta and HGF mRNA are shown in SEQ ID NOs: 9 (human-derived FGF7), 11 (human-derived FGF9), 13 (human-derived FGF10 ), 15 (human-derived FGF11), 17 (human-derived FGF16), 19 (human-derived FGF18), 21 (human-derived FGF18), 23 (chicken-derived FGF22), 1 (chicken-derived FGF23), 3 (mouse Origin FGF23), 5 (rat-derived FGF23), 7 (zebrafish-derived FGF23), 25 (human-derived TGF_beta), 27 (human-derived HGF) and 29 (human-derived HGF).
  • TGF7, 9, 10, 11, 16, 18, 22, and 23, TGF-beta and HGF mRNA are SEQ ID NOs: 10 (human-derived FGF7), 12 (human-derived FGF9), 14 (human-derived) (FGF10), 16 (human-derived FGF11), 18 (human-derived FGF16), 20 (human-derived FGF18), 22 (human-derived FGF18), 24 (human-derived FGF22), 2 (human-derived FGF23), 4 (mouse-derived FGF23 ), 6 (rat-derived FGF23), 8 (zebrafish-derived FGF23), 26 (human-derived TGF-beta), 28 (human-derived HGF) and 30 (human-derived HGF).
  • the FGF family in a mouse skin full-thickness wound healing model The expression profile of the gene to which it belongs was examined.
  • Hairless male mice 25 hrs (7 weeks old and 33 weeks old) obtained from Nippon SLC Co., Ltd. for standard wound healing and experimental water healing experiments After acclimating for 1 week for 7-week-old mice and for 2 weeks for 33-week-old mice, euthanize 5 mice and isolate the skin samples on the back. The rest of the 20 mice were cut under a Nembutal anesthesia, the dorsal skin was cut into a circle with a diameter of 8 mm at 4 locations, and full-thickness wounds were created.
  • Total RNA was prepared from skin samples prepared as described above using Isogen (Nippon Gene Co., Ltd.) according to the manufacturer's instructions. Next, mRNA was purified from the total RNA using 01igotex-dT30 Super mRNA purification kit (Takara Bio Inc.). The purified mRNA sample (lOOng) was in a saddle type, and reverse transcription reaction was performed in a total volume of 20 ml using Superscript II (Gibco BRL) using oligo (dT) 12-18 as a primer.
  • a specific primer set was designed so that each FGF mRNA level could be analyzed by real-time PCR (Table 1). When quantifying mRNA for each FGF, a cDNA fragment is required as a copy number control. The same primer set was used when cloning this cDNA fragment.
  • PCR amplification kit A product obtained by diluting the above-mentioned reverse transcription reaction product (RT mixture) 10-fold with distilled water was used as a PCR amplification kit.
  • PHi polymerase (Stratagene) was used according to the manufacturer's instructions.
  • a part of the PCR amplification reaction mixture was used for electrophoresis using a 2.0% agarose gel to confirm the size of the growth product.
  • the PCR product was ligated to pCR-Bluntn-Better (Invitrogen) and used for transformation of E. coli.
  • the nucleotide sequence of the recombinant plasmid was confirmed using a BigDye terminator cycle sequencing kit and ABI PRISM 310 Genetic Analyzer (Applied Biosystems).
  • Real-time PCR amplification was performed with 2 ml of the RT mixture diluted 1:10 in a bowl, Light Cycler (Roche Diagnostics).
  • the plasmid DNA corresponding to each FGF was used as a copy number control.
  • Cyber Green was used for quantification of DNA amplified by Light Cy cler.
  • the Light Cycler reaction was performed at the optimal annealing temperature, extension time, and acquisition temperature. Primer specificity was first verified using whole mouse cDNA as a background sequence. Primer specificity and reaction conditions were further experimentally verified by cloning amplified fragments into plasmid DNA to verify these sequences.
  • the melting point temperature of the amplification products was measured at the end of the individual experiment, and it was proved that they were uniform at the melting point temperature.
  • the product was analyzed by 2.0% agarose gel electrophoresis, confirming that the DNA fragment of known size was accurately amplified by the reaction.
  • This experimental system ensures specific quantification of individual genes.
  • the number of housekeeping gene copies was evaluated by the same method to confirm that these amounts were within the expected range.
  • FIG. 1A Expression profile of FGF and FGFR family members, TGF-beta, HGF
  • FIG. IB Expression profile of FGF and FGFR family members, TGF-beta, HGF
  • FIG. 2 The mRNA expression levels of each FGF and FGFR at each time point of wound healing are shown in FIG. 1A, FIG. IB and FIG. 2, respectively.
  • FGFR2 mRNA is lower than that of FGFR1 or 3, including the Illb subclass, which is specific to epithelial cells ( Figure 2), and the expression level of FGFR4 is the lowest among the four types of receptors. (Fig. 2).
  • TGF-beta mRNA and HGF mRNA As for TGF-beta mRNA and HGF mRNA, the expression level in healthy skin was high, but it was revealed that the expression level was further increased during the wound healing process.
  • Example 1 since the expression of FGF23 mRNA in skin was newly clarified, in this example, the distribution of FGF23 protein was measured by immunohistochemical staining using a specific antibody.
  • Anti-FGF23 antibody was prepared as follows. Considering the secondary structure, hydrophilicity, phase isomerism with other proteins, etc. from the primary structure of FGF23, a partial peptide that is considered to be highly antigenic and specific is predicted, and this is chemically synthesized and purified. . This peptide was covalently bound to mosianin protein (KLH) to a keyhole limpet and injected multiple times with an adjuvant to a rabbit to obtain serum containing antibodies. After assessing antibody titers by ELISA (de The specificity was confirmed by immunoblotting.
  • KLH mosianin protein
  • Immunohistochemical staining was performed by staining a paraffin-embedded tissue section with an anti-FGF23 antibody. Skin of young mice during the wound healing process (4th day after wound creation) was fixed in formalin, then wrapped in paraffin, and 4 ⁇ m thick sections were prepared. This was pasted on a slide glass and deparaffinized. Subsequently, the antigen was activated by boiling in 0.01 M citrate (pH 6.0) for 2 minutes and incubated with anti-FGF23 antibody. After washing, the cells were incubated with Piotin-yagi anti-rabbit IgG and washed. This was incubated with avidinized peroxidase and developed with an aminoethylcarbazole reagent which is a substrate for peroxidase.
  • Fig. 3 shows the results of immunohistochemical staining.
  • FGF23 protein is present at a high level, and the skin around the wound and the bottom, especially granulation tissue, where cell migration and proliferation are actively occurring (Fig. 3; purple signal).
  • Fig. 3 purple signal
  • no signal was generated (not shown).
  • Mouse fibroblasts (NIH3T3) (obtained from ATCC) were subcultured in a growth medium (DMEM basal medium supplemented with 10% pup sera) and used for [ 3 H] -thymidine uptake assay.
  • the cells were trypsinized and dispensed into 24 well plates (Sumilon) at a density of 5 ⁇ 10 3 cells / well to 24 walls (HFDP cells) and maintained at 37 ° C. with growth medium.
  • the growth medium is replaced with DMEM basal medium containing 0.5% thiacol-adsorbed serum, and the cells are cultured for 48 hours, followed by the prescribed concentration in the presence of 5 ⁇ g / ml heparin. Stimulated with FGF.
  • Immunohistological staining was performed by staining a paraffin-embedded tissue section with an anti-c-fos antibody (Calbiochem-Novabiochem) in the same manner as in Example 2. Skin tissue sections were prepared from the skin of young mice during the wound healing process (4 days after wound creation).
  • FIG. Figure 5 shows the results of staining a skin section of the wound healing process (4 days after wound creation) with anti-c-fos antibody.
  • the region strongly stained by the anti-c-fos antibody was found to be highly similar to the expression site of the FGF23 protein observed in Example 2.
  • the agent of the present invention can be used for wound healing and / or promotion of wound treatment.
  • the agent of the present invention is effective for wound healing and promotion of Z or wound treatment in aging individuals.
  • a substance effective for wound healing and Z or wound treatment promotion in an aging individual can be searched.
  • SEQ ID NO: 1 shows the nucleotide sequence of mRNA of human-derived FGF23. ⁇ SEQ ID 2>
  • SEQ ID NO: 2 shows the amino acid sequence encoded by the mRNA of human-derived FGF23.
  • SEQ ID NO: 3 shows the nucleotide sequence of mRNA of mouse-derived FGF23.
  • SEQ ID NO: 4 shows the amino acid sequence encoded by the mRNA of mouse-derived FGF23.
  • SEQ ID NO: 5 shows the nucleotide sequence of mRNA of rat-derived FGF23.
  • SEQ ID NO: 6 shows the amino acid sequence encoded by rat FGF23 mRNA.
  • SEQ ID NO: 7 shows the nucleotide sequence of mRNA of zebrafish-derived FGF23.
  • SEQ ID NO: 8 shows an amino acid sequence encoded by mRNA of FGF23 derived from zebrafish.
  • SEQ ID NO: 9 shows the nucleotide sequence of human FGF7 mRNA.
  • SEQ ID NO: 10 shows the amino acid sequence encoded by mRNA of human-derived FGF7.
  • SEQ ID NO: 11 shows the nucleotide sequence of mRNA of human-derived FGF9.
  • SEQ ID NO: 12 shows the amino acid sequence encoded by mRNA of human-derived FGF9.
  • SEQ ID NO: 13 shows the nucleotide sequence of mRNA of human-derived FGF10.
  • SEQ ID NO: 14 shows the amino acid sequence encoded by the mRNA of human-derived FGF10.
  • SEQ ID NO: 15 shows the nucleotide sequence of mRNA of human-derived FGF11.
  • SEQ ID NO: 16 shows the amino acid sequence encoded by the mRNA of human-derived FGF11.
  • SEQ ID NO: 17 shows the nucleotide sequence of mRNA of human-derived FGF16.
  • SEQ ID NO: 18 shows the amino acid sequence encoded by the mRNA of human-derived FGF16.
  • SEQ ID NO: 19 shows the nucleotide sequence of mRNA of human-derived FGF18 (variant 1).
  • SEQ ID NO: 20 represents the amino acid sequence encoded by human FGF18 (variant 1) mRNA; SEQ ID NO: 21>
  • SEQ ID NO: 21 shows the nucleotide sequence of mRNA of human-derived FGF18 (variant 2).
  • SEQ ID NO: 22 shows the amino acid sequence encoded by human FGF18 (variant 2) mRNA
  • SEQ ID NO: 23 shows the nucleotide sequence of mRNA of human-derived FGF22.
  • SEQ ID NO: 24 shows the amino acid sequence encoded by the mRNA of human-derived FGF22.
  • SEQ ID NO: 25 shows the nucleotide sequence of mRNA of human-derived TGF_beta.
  • SEQ ID NO: 26 shows the amino acid sequence encoded by the mRNA of human-derived TGF_beta. ⁇ SEQ ID NO: 27>
  • SEQ ID NO: 27 shows the nucleotide sequence of mRNA of human-derived HGF (variant 1).
  • SEQ ID NO: 28> shows the amino acid sequence encoded by mRNA of human-derived HGF (variant 1).
  • SEQ ID NO: 29 shows the nucleotide sequence of mRNA of human-derived HGF (variant 3).
  • SEQ ID NO: 30 shows the amino acid sequence encoded by mRNA of human-derived HGF (variant 3). ⁇ SEQ ID NO: 3:! To 74>
  • SEQ ID NO: 3:! -74 shows the nucleotide sequence of the primer.

Abstract

An agent for treatment of a wound and/or promotion of the healing of a wound comprising a full-length or partial peptide of at least one fibroblast growth factor selected from the group consisting of fibroblast growth factors 9, 11, 16, 18 and 23 as an active ingredient; and a method for screening a substance which is effective for the treatment of a wound and/or the promotion of the healing of a wound in an aged individual, the method comprising monitoring the expression of genes during the course of wound healing process in living bodies with different aging states, comparing the quantities of the expressed genes between younger individuals and elder individuals, and determining an expression product of a gene of which quantity is lower in the younger individuals compared to that in the elder individuals as a substance effective for the treatment of a wound and/or the promotion of the healing of a wound in the elder individuals.

Description

明 細 書  Specification
創傷治療及び Z又は創傷治癒促進のための薬剤  Drugs for wound treatment and promoting Z or wound healing
技術分野  Technical field
[0001] 本発明は、皮膚の再生を促進する成分を含有する創傷治療剤、創傷治癒促進剤 並びに加齢個体の創傷治療及び/又は創傷治癒促進に効果がある物質のスクリー ニング方法に関する。  [0001] The present invention relates to a wound treatment agent containing a component that promotes skin regeneration, a wound healing promoter, and a method for screening a substance effective for wound treatment and / or promotion of wound healing in an aging individual.
背景技術  Background art
[0002] 繊維芽細胞増殖因子（以下 FGFと称する）といった様々なポリペプチド増殖因子が 皮膚組織において発現していることが知られている。マウス及びヒトにおいては、 FGF は 22種類の異なる遺伝子によってコードされる（非特許文献 1)。特に、 FGF1、 FGF2 、 FGF5、 FGF7、 FGF10、 FGF13、 FGF18及び FGF22は、皮膚細胞及び毛包細胞に ぉレ、て発現してレ、ることが報告されてレ、る（非特許文献 2〜： 18参照）。  [0002] It is known that various polypeptide growth factors such as fibroblast growth factor (hereinafter referred to as FGF) are expressed in skin tissue. In mice and humans, FGF is encoded by 22 different genes (Non-patent Document 1). In particular, it has been reported that FGF1, FGF2, FGF5, FGF7, FGF10, FGF13, FGF18 and FGF22 are expressed and expressed in skin cells and hair follicle cells (Non-Patent Documents 2 to 4). : 18).
[0003] また、特表 2004-501203号公報（特許文献 1)には、 FGF1を含む創傷治癒剤が開 示されている。また、 FGF2を含む創傷治癒剤は現在市販されている。し力しながら、 FGF1や FGF2の遺伝子産物は、創傷治癒の局面で生体の反応として増加するわけ ではなぐその創傷治癒効果は人為的にこれら増殖因子を使用した結果として起こる ものである。また、 FGF2は皮膚表皮細胞の増殖を直接促進することはできなレ、。よつ て、 FGF1や FGF2を用いて、創傷を治療することは、生理的治癒と同等の完璧な治 癒反応を引き起こせなレ、可能性がある。  [0003] Also, Japanese Patent Publication No. 2004-501203 (Patent Document 1) discloses a wound healing agent containing FGF1. In addition, wound healing agents containing FGF2 are currently marketed. However, the gene products of FGF1 and FGF2 do not increase as a biological response in the wound healing phase, and the wound healing effect is a result of artificially using these growth factors. In addition, FGF2 cannot directly promote the proliferation of skin epidermal cells. Therefore, treating a wound with FGF1 or FGF2 may not cause a perfect healing response equivalent to physiological healing.
[0004] 上掲した非特許文献 2〜： 18を含む他の文献から、 FGFが皮膚細胞の増殖及び分 化に重要な役割を担っていることが示唆される。し力、しながら、創傷治癒に対して、 F GF群がどのように関与するのかといつた知見は依然として不明のままである。また、 若齢では速やかに治癒する創傷が、加齢時には遅延することが経験的に知られてい る力 その現象における FGF群の関与は不明のままである。  [0004] Other documents including Non-Patent Documents 2 to 18 listed above suggest that FGF plays an important role in the proliferation and differentiation of skin cells. However, the knowledge of how the FGF group is involved in wound healing remains unclear. In addition, it has been empirically known that wounds that heal quickly at a young age are delayed at aging. The involvement of the FGF group in this phenomenon remains unclear.
[0005] 非特許文献 1： Ornitz DM, Itoh N: Fibroblast growth factors. Genome Biol2:REVIE WS3005, 2001  [0005] Non-Patent Document 1: Ornitz DM, Itoh N: Fibroblast growth factors. Genome Biol2: REVIE WS3005, 2001
非特許文献 2 : Bai XY, Miao D, Goltzman D & Karaplis AC 2003 Theautosomal domi nant hypophosphatemic rickets R176Q mutation in fibroblast growthfactor 23 resists proteolytic cleavage and enhances in vivo biological potency. Journal of Biological C hemistry 278 9843-9849. Non-Patent Document 2: Bai XY, Miao D, Goltzman D & Karaplis AC 2003 Theautosomal domi nant hypophosphatemic rickets R176Q mutation in fibroblast growthfactor 23 resists proteolytic cleavage and enhances in vivo biological potency.Journal of Biological C hemistry 278 9843-9849.
特許文献 3 : Beyer TA， Werner S， Dickson C & Grose R 2003 Fibroblast growth fa ctor 22 and its potentialrole during skin development and repair. ExperimentalCell R esearch 287 228-236.  Patent Document 3: Beyer TA, Werner S, Dickson C & Grose R 2003 Fibroblast growth factor 22 and its potentialrole during skin development and repair. ExperimentalCell Research 287 228-236.
非特許文献 4 : Bikfalvi A， Klein S， Pintucci G， Rifkin DB 1997 Biologicalroles of fibro blast growth factor- 2. EndocrinologyReview 18 26-45. Non-Patent Document 4: Bikfalvi A, Klein S, Pintucci G, Rifkin DB 1997 Biologicalroles of fibro blast growth factor- 2. Endocrinology Review 18 26-45.
非特許文献 5 : Gao Z， Flaitz CM & Mackenzie IC 1996 Expression ofkeratinocyte gr owth factor in periapical lesions. Journal of Dentistry Research 75 1658- lbり 3. Non-Patent Document 5: Gao Z, Flaitz CM & Mackenzie IC 1996 Expression ofkeratinocyte growth factor in periapical lesions. Journal of Dentistry Research 75 1658- lb 3.
非特許文献 6 : GuoL， Degenstein L & Fuchs E 1996 Keratinocyte growth factor is re quired forhair development but not for wound healing. Genesand Development 10 lbNon-Patent Document 6: GuoL, Degenstein L & Fuchs E 1996 Keratinocyte growth factor is re quired forhair development but not for wound healing. Genesand Development 10 lb
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非特言午文献 7 : Igarashi M， Finch PW & Aaronson SA 1998 Characterizationof recom binant human fibroblast growth factor (FGF) -10 reveals functionalsimilarities with k eratinocyte growth factor (FGF-7). Journal of Biological Chemistry 27313230-1323 5. Non-Special Terms 7: Igarashi M, Finch PW & Aaronson SA 1998 Characterization of recom binant human fibroblast growth factor (FGF) -10 reveals functionalsimilarities with k eratinocyte growth factor (FGF-7). Journal of Biological Chemistry 27313230-1323 5.
非特言午文献 8 : Jameson J, Ugarte K， Chen N， Yacm P, Fuchs E， Boismenu R& Havra n WL 2002 A role for skin gd T cells in wound repair. Science 296 747-749. Non-Special Reference 8: Jameson J, Ugarte K, Chen N, Yacm P, Fuchs E, Boismenu R & Havra n WL 2002 A role for skin gd T cells in wound repair. Science 296 747-749.
非特許文献 9 : KawanoM， Suzuki S， Suzuki M, Oki J & Imamura T 2004 Bulge- and b asallayer-specific expression of nbroblast growth factor 13 (FHF-2) in mouse skin. Jo urnal of Investigative Dermatology 122 1084 - 1090. Non-Patent Document 9: KawanoM, Suzuki S, Suzuki M, Oki J & Imamura T 2004 Bulge- and b asallayer-specific expression of nbroblast growth factor 13 (FHF-2) in mouse skin. Journal of Investigative Dermatology 122 1084-1090 .
非特許文献 10 : Kaw細 M， Kuramochi-Komi A， Asada M， Suzuki M， Oki J, Jiang J & Imamura T 2005Comprehensive analysis of FGF and FGFR expression in s謹： FGF 18 is highlyexpressed m hair follicles and capable of inducing anagen from telogen st agehair follicles. Journal of InvestigativeDermatology 124 877-885. Non-Patent Document 10: Kaw Hos M, Kuramochi-Komi A, Asada M, Suzuki M, Oki J, Jiang J & Imamura T 2005 Comprehensive analysis of FGF and FGFR expression in s 謹: FGF 18 is highly expressed m hair follicles and capable of inducing anagen from telogen st agehair follicles. Journal of Investigative Dermatology 124 877-885.
非特許文献 l l : Marchese C， Chedid M， Dirsch OR, Csaky KG, Santanelli F，Latini C， LaRochelle WJ, Torrisi MR & Aaronson SA 1995 Modulation of keratinocyte gro wth factor and its receptor inreepithelializing human skin. Journal ofExperimental M edicine 1821369-1376. Non-patent literature ll: Marchese C, Chedid M, Dirsch OR, Csaky KG, Santanelli F, Latini C, LaRochelle WJ, Torrisi MR & Aaronson SA 1995 Modulation of keratinocyte gro wth factor and its receptor inreepithelializing human skin.Journal of Experimental Medicine 1821369-1376.
非特許文献 12 : Martin P, Hopkinson-Woolley J & McCluskey J 1992 Growthfactors and cutaneous wound repair-Progress in Growth Factor Research 425-44. Non-Patent Document 12: Martin P, Hopkinson-Woolley J & McCluskey J 1992 Growthfactors and cutaneous wound repair-Progress in Growth Factor Research 425-44.
非特許文献 13 : MartinP 1997 Wound healing― aiming for perfect skin regeneration. Science 27675-81. Non-Patent Document 13: MartinP 1997 Wound healing― aiming for perfect skin regeneration. Science 27675-81.
非特許文献 14 : OrtegaS, Ittmann M， Tsang SH， Ehrlich M & Basilico C 1998 Neuro nal defects anadelayed wound healing in mice lacking fibroblast growth factor-2. Pro ceedings of National Academy of bciencesUSA 95 5672-5677. Non-Patent Document 14: OrtegaS, Ittmann M, Tsang SH, Ehrlich M & Basilico C 1998 Neuro nal defects anadelayed wound healing in mice lacking fibroblast growth factor-2. Proceedings of National Academy of bciencesUSA 95 5672-5677.
非特許文献 15 : Paddock HN, Schultz GS & Mast BA 2003 Methods in re印 ithelializa tion. A porcine model ofpartial-thickness wounds. Methods inMolecular Medicine 78 17-36. Non-Patent Document 15: Paddock HN, Schultz GS & Mast BA 2003 Methods in remark ithelializa tion.A porcine model ofpartial-thickness wounds.Methods in Molecular Medicine 78 17-36.
非特許文献 16 : Singer AJ & Clark RA 1999 Mechanism of disease:cutaneous wound healing. New Englandjournal of Medicine 341 738-746. Non-Patent Document 16: Singer AJ & Clark RA 1999 Mechanism of disease: cutaneous wound healing.New Englandjournal of Medicine 341 738-746.
非特許文献 17 : Steiling H & Werner S 2003 Fibroblast growth factors :key players in epithelial morphogenesis, repair and cytoprotection. Current Opinion in Biotechnolo gy 14 533-537. Non-Patent Document 17: Steiling H & Werner S 2003 Fibroblast growth factors: key players in epithelial morphogenesis, repair and cytoprotection.Current Opinion in Biotechnolo gy 14 533-537.
非特許文献 18 : WernerS, Smola H, Liao X, Longaker MT, Krieg T, Hofschneider P H & Williams LT1994 The function of KGF in epithelial morphogenesis and woundre -epithelialisation. Science 266819-822. Non-Patent Document 18: WernerS, Smola H, Liao X, Longaker MT, Krieg T, Hofschneider P H & Williams LT1994 The function of KGF in epithelial morphogenesis and woundre -epithelialisation. Science 266819-822.
特許文献 1：特表 2004-501203号公報 Patent Document 1: Japanese Translation of Special Publication 2004-501203
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
そこで、本発明は、生体反応として起こる創傷治癒作用を解明し、新規な創傷治療 剤及び/又は創傷治癒促進剤を提供することを目的としている。さらに、創傷治癒が 遅延する加齢個体において有効な創傷治療剤及び/又は創傷治癒促進剤を提供 することを目的としている。  Accordingly, an object of the present invention is to elucidate the wound healing action that occurs as a biological reaction and to provide a novel wound healing agent and / or wound healing promoter. It is another object of the present invention to provide a wound healing agent and / or a wound healing promoter effective in an aging individual whose wound healing is delayed.
課題を解決するための手段 [0007] 上述した目的を達成するため本発明者らは、皮膚の創傷治癒に重要な働きをする FGF遺伝子を特定するために、若齢動物と加齢動物を用いて、 FGFファミリーに属す る 22遺伝子全ての発現を、健康皮膚、及び創傷治癒の各ステージ (創傷作成後 2、 4 、 7、 15、 21日目までの 21日間）で網羅的に検索したところ、これまで皮膚でその機能 の知られていなかった FGF遺伝子の高発現と機能を見いだすに至った。例えば、若 齢個体では、 FGF7, 10及び 22遺伝子の mRNA発現レベルが健康皮膚では高ぐそ のうち FGF7及び 10のレベルは創傷後の時間経過中に 2〜3.5倍増加した。 FGF9, 16 ， 18及び 23の mRNA発現レベルは健康皮膚では中程度あるいは低かった力 S、創傷後 に 2〜33倍増加した。特に、 FGF23遺伝子の mRNA発現レベルは創傷治癒の初期に 極めて高いレベルに到達した。また、トランスフォーミング増殖因子一 β (以下 TGF-b etaと称する）及び肝細胞増殖因子（以下 HGFと称する)も健康皮膚で mRNAの発現レ ベルが高ぐ創傷治癒中にアップレギュレートされた。さらに本発明者等は、若齢動 物と加齢動物における創傷治癒反応を比較することにより、これらの FGF遺伝子のほ とんどの発現レベル力 創傷治癒が遅延する加齢動物では若齢動物よりも低レ、こと を発見した。 Means for solving the problem [0007] In order to achieve the above-mentioned object, the present inventors belong to the FGF family using young animals and aging animals in order to identify FGF genes that play an important role in skin wound healing. A comprehensive search for the expression of all 22 genes at each stage of healthy skin and wound healing (21 days after wound creation, 2, 4, 7, 15, 21 days). The high expression and function of the FGF gene, which was not known, have been found. For example, among young individuals, the level of FGF7, 10 and 22 mRNA expression was higher in healthy skin, while the levels of FGF7 and 10 increased 2- to 3.5-fold over time after wounding. FGF9, 16, 18 and 23 mRNA expression levels were moderate or low in healthy skin S, and increased 2-3-fold after wounding. In particular, the mRNA expression level of the FGF23 gene reached a very high level in the early stage of wound healing. Transforming growth factor 1β (hereinafter referred to as TGF-beta) and hepatocyte growth factor (hereinafter referred to as HGF) were also up-regulated during wound healing with high mRNA expression levels in healthy skin. Furthermore, the present inventors have compared the wound healing response in young animals and aging animals, so that the expression level of most of these FGF genes is higher in aging animals with delayed wound healing than in young animals. I found that it was low.
[0008] また、本発明者等は、 FGF23のタンパク質が、創傷を受けて再生中の局所に選択 的に存在していることも見出した。  [0008] Further, the present inventors have also found that the protein of FGF23 is selectively present in the local area where the wound is being regenerated.
[0009] また、本発明者等は、 FGF23に関する詳細な機能解析を行ったところ、 FGF23には 創傷治癒に重要と考えられる繊維芽細胞の増殖を促進する機能があることを見出し た。さらに、本発明者等は、 FGF23の発現部位において、細胞の増殖や遊走、分化 の刺激時に発現誘導される初期遺伝子 Fosが高いレベルで発現していることを見出 した。本発明は、このような新規な知見に基づいて完成されたものである。  [0009] Further, the present inventors conducted a detailed functional analysis on FGF23 and found that FGF23 has a function of promoting the proliferation of fibroblasts, which are considered to be important for wound healing. Furthermore, the present inventors have found that the early gene Fos, which is induced to induce cell proliferation, migration, and differentiation, is expressed at a high level at the FGF23 expression site. The present invention has been completed based on such novel findings.
[0010] 本発明の要旨は以下の通りである。  [0010] The gist of the present invention is as follows.
[0011] (1)繊維芽細胞増殖因子 9、 11、 16、 18及び 23からなる群より選択される少なくとも 1種の繊維芽細胞増殖因子の全長又は部分ペプチドを有効成分として含有する創 傷治療及び/又は創傷治癒促進のための薬剤。  [0011] (1) Wound treatment containing as an active ingredient the full length or partial peptide of at least one fibroblast growth factor selected from the group consisting of fibroblast growth factor 9, 11, 16, 18 and 23 And / or agents for promoting wound healing.
[0012] (2)繊維芽細胞増殖因子が野生型である（1)記載の薬剤。  [0012] (2) The drug according to (1), wherein the fibroblast growth factor is a wild type.
[0013] (3)繊維芽細胞増殖因子が変異体である（1)記載の薬剤。 [0014] (4)他のタンパク質増殖因子、創傷治癒剤及び/又は創傷治癒促進剤を更に含むこ とを特徴とする（1)〜（3)のいずれかに記載の薬剤。 [0013] (3) The drug according to (1), wherein the fibroblast growth factor is a mutant. [0014] (4) The drug according to any one of (1) to (3), further comprising another protein growth factor, a wound healing agent and / or a wound healing promoter.
[0015] (5)繊維芽細胞増殖因子 9、 11、 16、 18及び 23からなる群より選択される少なくとも 1種の繊維芽細胞増殖因子の全長又は部分ペプチドをコードする核酸を有効成分と して含有する創傷治療及び Z又は創傷治癒促進のための薬剤。  [0015] (5) A nucleic acid encoding a full-length or partial peptide of at least one fibroblast growth factor selected from the group consisting of fibroblast growth factor 9, 11, 16, 18, and 23 is used as an active ingredient. Containing wound treatment and Z or wound healing promotion agent.
[0016] (6)繊維芽細胞増殖因子の全長又は部分ペプチドをコードする核酸が cDNAであり、 かつ発現ベクターに組み込まれてレ、る（5)記載の薬剤。  [0016] (6) The agent according to (5), wherein the nucleic acid encoding the full-length or partial peptide of fibroblast growth factor is cDNA and incorporated into an expression vector.
[0017] (7)加齢状態の異なる生体の創傷治癒過程における遺伝子の発現をモニターし、若 齢個体と加齢個体との間で遺伝子の発現量を比較し、若齢個体と比べて加齢個体 での発現量が低い遺伝子の産物を加齢個体の創傷治療及び/又は創傷治癒促進 に効果があると判定することを含む、加齢個体の創傷治療及び Z又は創傷治癒促進 に効果がある物質をスクリーニングする方法。  [0017] (7) The expression of genes during wound healing processes in living bodies with different aging conditions is monitored, and the expression level of genes is compared between young individuals and aging individuals. It is effective for wound treatment of aging individuals and for promoting Z or wound healing, including determining that the product of a gene whose expression level is low in the aging individuals is effective for wound treatment and / or promotion of wound healing of the aging individual. A method of screening a substance.
[0018] (8)繊維芽細胞増殖因子 7、 9、 10、 11、 16、 18、 22及び 23、トランスフォーミング 増殖因子 並びに肝細胞増殖因子からなる群より選択される少なくとも 1種の増殖 因子の全長又は部分ペプチドを有効成分として含有する加齢個体の創傷治療及び /又は創傷治癒促進のための薬剤。  [0018] (8) of at least one growth factor selected from the group consisting of fibroblast growth factor 7, 9, 10, 11, 16, 18, 22, and 23, transforming growth factor and hepatocyte growth factor An agent for treating wounds and / or promoting wound healing of an aging individual comprising a full-length or partial peptide as an active ingredient.
[0019] (9)繊維芽細胞増殖因子 7、 9、 10、 11、 16、 18、 22及び 23、トランスフォーミング 増殖因子 並びに肝細胞増殖因子からなる群より選択される少なくとも 1種の増殖 因子の全長又は部分ペプチドをコードする核酸を有効成分として含有する加齢個体 の創傷治療及び/又は創傷治癒促進のための薬剤。  [0019] (9) of at least one growth factor selected from the group consisting of fibroblast growth factor 7, 9, 10, 11, 16, 18, 22, and 23, transforming growth factor and hepatocyte growth factor An agent for treating wounds and / or promoting wound healing in an aging individual comprising a nucleic acid encoding a full-length or partial peptide as an active ingredient.
発明の効果  The invention's effect
[0020] 本発明により、創傷治療剤及び/又は創傷治癒促進剤を提供することができる。ま た、加齢個体で有効な創傷治療剤及び Z又は創傷治癒促進剤を提供することがで きる。本発明の創傷治療剤及び Z又は創傷治癒促進剤を用いれば、創傷治癒の促 進及び/又は創傷の治療をより効果的に行うことができる。  [0020] According to the present invention, a wound treatment agent and / or a wound healing promoter can be provided. In addition, it is possible to provide a wound healing agent and Z or wound healing promoter effective in an aging individual. By using the wound therapeutic agent and Z or wound healing promoter of the present invention, it is possible to promote wound healing and / or treat wounds more effectively.
[0021] また、本発明のスクリーニング方法により、加齢個体の創傷治療及び/又は創傷治 癒促進に効果がある物質をスクリーニングすることができる。  [0021] Furthermore, the screening method of the present invention can screen for substances that are effective in treating wounds and / or promoting wound healing in aging individuals.
図面の簡単な説明 [0022] [図 1A]創傷治癒の各段階における FGF各メンバー遺伝子の発現プロファイルを示す 図である。 Brief Description of Drawings [0022] FIG. 1A is a diagram showing expression profiles of FGF member genes in each stage of wound healing.
[図 1B]創傷治癒の各段階における FGF各メンバー遺伝子の発現プロファイルを示す 図である。  FIG. 1B shows the expression profiles of FGF member genes at each stage of wound healing.
[図 2]傷治癒の各段階における FGFRファミリーに属する遺伝子の発現プロファイルを 示す特性図である。  FIG. 2 is a characteristic diagram showing expression profiles of genes belonging to the FGFR family at each stage of wound healing.
[図 3]組織免疫染色実験 (抗 FGF23抗体で染色）の結果を示す写真である。免疫染 色で調べた創傷治癒過程での FGF23タンパク質の局在が示されてレ、る。図左側：健 常皮膚 (上が表皮、その下に真皮、皮下脂肪が見える）図右側:治癒過程の皮膚（「 力、さぶた」が上に載っている）赤いシグナルで示される FGF23タンパク質力 治癒過 程の創傷とごく近傍という局所にのみ特異的に発現していることがわかる。  FIG. 3 is a photograph showing the results of a tissue immunostaining experiment (stained with an anti-FGF23 antibody). The localization of FGF23 protein during the wound healing process examined by immunostaining is shown. Left side: healthy skin (upper epidermis, dermis and subcutaneous fat visible below) right side: healing skin ("force, scab" is on top) FGF23 protein strength indicated by red signal It can be seen that it is specifically expressed only in the local area of the wound and the immediate vicinity of the healing process.
[図 4]DNA合成アツセィの結果を示す特性図である。 X軸に示す濃度の FGF23タンパ ク質共存下で NIH3T3細胞を培養し、一定時間後に TetraColor ONE試薬（生化学ェ 業株式会社)を用いて測定した細胞数を Y軸に表す。  FIG. 4 is a characteristic diagram showing the results of DNA synthesis assembly. NIH3T3 cells are cultured in the presence of the FGF23 protein at the concentration shown on the X axis, and the number of cells measured using TetraColor ONE reagent (Seikagaku Corporation) after a certain time is shown on the Y axis.
[図 5]組織免疫染色実験 (抗 Fos抗体で染色）の結果を示す写真である。  FIG. 5 is a photograph showing the results of a tissue immunostaining experiment (stained with an anti-Fos antibody).
発明を実施するための最良の形態  BEST MODE FOR CARRYING OUT THE INVENTION
[0023] 以下、本発明を詳細に説明する。  [0023] Hereinafter, the present invention will be described in detail.
[0024] 1.創傷治療及び/又は創傷治癒促進のための薬剤  [0024] 1. Drug for wound treatment and / or promotion of wound healing
本発明は、繊維芽細胞増殖因子 9、 11、 16、 18及び 23からなる群より選択される 少なくとも 1種の繊維芽細胞増殖因子の全長又は部分ペプチドを有効成分として含 有する創傷治療及び Z又は創傷治癒促進のための薬剤を提供する。  The present invention relates to a wound treatment comprising at least one full-length or partial peptide of fibroblast growth factor selected from the group consisting of fibroblast growth factor 9, 11, 16, 18 and 23 as an active ingredient and Z or A drug for promoting wound healing is provided.
[0025] また、本発明は、繊維芽細胞増殖因子 9、 11、 16、 18及び 23からなる群より選択さ れる少なくとも 1種の繊維芽細胞増殖因子の全長又は部分ペプチドをコードする核 酸を有効成分として含有する創傷治療及び Z又は創傷治癒促進のための薬剤を提 供する。  [0025] The present invention also provides a nucleic acid encoding a full-length or partial peptide of at least one fibroblast growth factor selected from the group consisting of fibroblast growth factors 9, 11, 16, 18, and 23. Provides wound healing and Z or wound healing promoting agents that are contained as active ingredients.
[0026] 本発明の薬剤に含まれてもよい有効成分の一つとして、 FGF23がある。 FGF23は、 例えば配列番号 2に示すアミノ酸配列からなるヒト由来の FGF23を使用することができ る。また、使用可能な FGF23としては、ヒト由来に限定されず、例えば、他の哺乳動物 由来の FGF23、魚類由来の FGF23などを使用することができる。他の哺乳動物として は、マウス、ラット、ニヮトリ、七面鳥、ゥシ、ブタ、ヒッジ及びゥサギ等を挙げることがで きるが、これらに限定されなレ、。例えば、配列番号 1に示すヒト由来 FGF23の塩基配 列に基づいて作製したプローブを定法に従って用いれば、ヒトを除く他の哺乳動物 力 FGF23をコードする遺伝子を単離することができる。 [0026] One of the active ingredients that may be contained in the drug of the present invention is FGF23. As FGF23, for example, human-derived FGF23 having the amino acid sequence shown in SEQ ID NO: 2 can be used. In addition, usable FGF23 is not limited to human origin, for example, other mammals FGF23 derived from fish, FGF23 derived from fish, and the like can be used. Other mammals may include, but are not limited to, mice, rats, chickens, turkeys, tusks, pigs, hidges, and rabbits. For example, if a probe prepared based on the nucleotide sequence of human-derived FGF23 shown in SEQ ID NO: 1 is used in accordance with a standard method, a gene encoding FGF23 other than humans other than human can be isolated.
[0027] ヒト由来 FGF23の塩基配列及びアミノ酸配列をそれぞれ配列番号 1及び 2に示す。  [0027] The nucleotide sequence and amino acid sequence of human-derived FGF23 are shown in SEQ ID NOs: 1 and 2, respectively.
マウス由来 FGF23の mRNAの塩基配列及びそれがコードするアミノ酸配列をそれぞ れ配列番号 3及び 4に示す。ラット由来 FGF23の mRNAの塩基配列及びそれがコード するアミノ酸配列をそれぞれ配列番号 5及び 6に示す。ゼブラフィッシュ由来 FGF23 の mRNAの塩基配列及びそれがコードするアミノ酸配列をそれぞれ配列番号 7及び 8 に示す。これら配列番号 2、 4、 6及び 8に示すアミノ酸配列を比較すると判るように、 哺乳動物において FGF23は非常に高い相同性を有しており、哺乳動物における FG F23の機能はほぼ同様であると理解することができる。 FGF23は体内リン酸濃度の維 持を調節していることが報告されており、その異常はくる病の原因となると考えられて いる力 FGF23の創傷治癒における機能については、全く知られていない。  The nucleotide sequence of mouse FGF23 mRNA and the amino acid sequence encoded by it are shown in SEQ ID NOs: 3 and 4, respectively. The nucleotide sequence of rat FGF23 mRNA and the amino acid sequence encoded by it are shown in SEQ ID NOs: 5 and 6, respectively. The base sequence of mRNA of zebrafish-derived FGF23 and the amino acid sequence encoded by it are shown in SEQ ID NOs: 7 and 8, respectively. As can be seen by comparing the amino acid sequences shown in SEQ ID NOs: 2, 4, 6, and 8, FGF23 has very high homology in mammals, and the function of FG F23 in mammals is almost the same. I can understand. It has been reported that FGF23 regulates the maintenance of phosphate concentration in the body, and its abnormality is thought to cause rickets. The function of FGF23 in wound healing is completely unknown.
[0028] FGF23は野生型であっても、変異体であってもよい。変異体は天然に見出されるも のであっても、人工的に作製されたものであってもよい。野生型 FGF23としては、配列 番号 2、 4、 6及び 8に示すアミノ酸配列を有するものを挙げることができる。変異体 FG F23としては、配列番号 2、 4、 6及び 8に示すアミノ酸配列のうち、 1又は数個のァミノ 酸が欠失、置換及び付加されたアミノ酸配列からなり、創傷治癒及び/又は創傷治 癒促進作用があるタンパク質を挙げることができる。ここで、数個のアミノ酸とは、例え ば 2〜54個、より好ましくは 2〜27個を例示することができる。生体内で FGF23は 179 番 Rと 180番 Sの間で切断されることが公知である力 176番 Rまたは 179番 Rのミューテ ーシヨンが起こると、体内リン酸塩の排泄調節活性が異常昂進することが知られてい る (Bai XY, Miao D, Goltzman D & Karaplis Aし 2003 The autosomal dominant hypo phosphatemic rickets R176Q mutation in fibroblast growth factor 23 resists proteoly tic cleavage and enhances in vivo biological potency. Journal of Biological Chemistry 278 9843-9849.) また、配列番号 2に示すアミノ酸配列における 42〜159番目の領 域は、様々な FGFに共通性の高いコア部分であると考えられる。したがって、配列番 号 2に示すアミノ酸配列における 42〜159番目の領域を除く領域であれば、 1又は数 個のアミノ酸が欠失、置換及び付加されたとしても、創傷治癒促進作用を欠損するこ となぐ本発明の薬剤の有効成分として使用することができる。 [0028] FGF23 may be a wild type or a mutant. Variants may be found in nature or may be artificially created. Examples of wild-type FGF23 include those having the amino acid sequences shown in SEQ ID NOs: 2, 4, 6, and 8. Mutant FG F23 consists of an amino acid sequence in which one or several amino acids have been deleted, substituted and added from the amino acid sequences shown in SEQ ID NOs: 2, 4, 6 and 8, and is used for wound healing and / or wound healing. Mention may be made of proteins that have healing promoting effects. Here, examples of the several amino acids include 2 to 54, more preferably 2 to 27. FGF23 is known to be cleaved between 179 R and 180 S in vivo. When a 176 R or 179 R mutation occurs, the excretion regulating activity of phosphate in the body is abnormally promoted. (Bai XY, Miao D, Goltzman D & Karaplis A 2003 The autosomal dominant hypo phosphatemic rickets R176Q mutation in fibroblast growth factor 23 resists proteoly tic cleavage and enhances in vivo biological potency.Journal of Biological Chemistry 278 9843 -9849.) The 42nd to 159th regions in the amino acid sequence shown in SEQ ID NO: 2 The region is considered to be a core part highly common to various FGFs. Therefore, in the region excluding the 42nd to 159th regions in the amino acid sequence shown in SEQ ID NO: 2, even if one or several amino acids are deleted, substituted, or added, the wound healing promoting effect is lost. It can be used as an active ingredient of the drug of the present invention.
[0029] また、本発明の薬剤は、上述した FGF23の部分断片を有効成分として含有してもよ レ、。 FGF23の部分断片としては、例えば配列番号 2における 25〜251番目のアミノ酸 配列からなるポリペプチド、または、配列番号 2における 25〜179番目のアミノ酸配列 力、らなるポリペプチドを挙げることができる。すなわち、 25〜251番目のアミノ酸配列か らなるポリペプチドであっても、受容体とへパリンに結合し、創傷の治癒を促進する作 用を有している蓋然性が高レ、。また、 FGF23の部分断片としては、最も好ましくは 42 〜159番目のアミノ酸配列からなるポリペプチドを使用することができる。なお、配列 番号 4、 6及び 8に示すアミノ酸配列においても、上記の範囲に相当するアミノ酸配列 力 なるポリペプチドであっても、本発明の薬剤の有効成分として使用することができ る。さらに、 FGF23の部分断片としては、創傷治癒及び/又は創傷治癒促進作用が あるなら、 25〜251番目のアミノ酸配列のうちいずれの部分からなる長さ 3-50アミノ酸 程度のペプチドであっても良い。ただし、この数値範囲を超えるアミノ酸からなるぺプ チドであつても、本発明における FGF23の部分断片に含まれる。  [0029] The drug of the present invention may contain the above-mentioned partial fragment of FGF23 as an active ingredient. Examples of the partial fragment of FGF23 include a polypeptide consisting of the 25th to 251st amino acid sequences in SEQ ID NO: 2, or a polypeptide consisting of the 25th to 179th amino acid sequences in SEQ ID NO: 2. That is, even a polypeptide consisting of the 25th to 251st amino acid sequences has a high probability of binding to the receptor and heparin and promoting the healing of wounds. Moreover, as a partial fragment of FGF23, a polypeptide consisting of amino acid sequences 42 to 159 is most preferably used. Even in the amino acid sequences shown in SEQ ID NOs: 4, 6 and 8, even polypeptides having amino acid sequence strengths corresponding to the above ranges can be used as the active ingredient of the drug of the present invention. Further, the partial fragment of FGF23 may be a peptide having a length of about 3-50 amino acids consisting of any part of the 25th to 251st amino acid sequences as long as it has a wound healing and / or wound healing promoting action. . However, peptides consisting of amino acids exceeding this numerical range are also included in the partial fragment of FGF23 in the present invention.
[0030] 上述したように定義された FGF23の全長及び部分ペプチドは、創傷治癒を促進す る作用を有する。この作用は、詳細を実施例にて後述するが、 FGF23の全長及び部 分ペプチドを含む培地にて細胞を培養し、 FGF23の全長及び部分ペプチドによる皮 膚繊維芽細胞における DNA合成や細胞数増加の誘導能を調べることで確認すること ができる。  [0030] The full length and partial peptides of FGF23 defined as described above have an action of promoting wound healing. The details of this action will be described later in Examples, but the cells are cultured in a medium containing the full length of FGF23 and a partial peptide, and DNA synthesis and cell number increase in skin fibroblasts by the full length and partial peptide of FGF23. This can be confirmed by examining the induction ability of
[0031] 本発明の薬剤に含まれてもよい他の有効成分として、 FGF9, 11, 16及び 18の全長 又は部分ペプチドがある。 FGF9, 11， 16及び 18の mRNAの塩基配列をそれぞれ配列 番号 11(ヒト由来 FGF9)、 15(ヒト由来 FGF11)、 17(ヒト由来 FGF16)、 19(ヒト由来 FGF1 8)及び 21(ヒト由来 FGF18)に示す。 FGF9, 11， 16及び 18の mRNAがコードするァミノ 酸配列をそれぞれ配列番号 12(ヒト由来 FGF9)、 16(ヒト由来 FGF11)、 18(ヒト由来 FG F16)、 20(ヒ卜由来 FGF18)及び 22(ヒ卜由来 FGF18)こ示す。 [0032] FGF9, 11, 16, 18及び 23からなる群より選択される少なくとも 1種の繊維芽細胞増殖 因子の全長又は部分ペプチドは、例えば、皮膚適用向けに適合化された溶液、タリ ーム、軟膏、ゲル、ローション、シャンプー又はエアゾールといった形態で製剤化され 、創傷治療及び/又は創傷治癒促進のための薬剤として提供される。この薬剤は、 加齢個体への適用が特に効果的である。 [0031] Other active ingredients that may be contained in the drug of the present invention include full-length or partial peptides of FGF9, 11, 16, and 18. The nucleotide sequences of mRNAs of FGF9, 11, 16 and 18 are shown in SEQ ID NOs: 11 (human-derived FGF9), 15 (human-derived FGF11), 17 (human-derived FGF16), 19 (human-derived FGF18) and 21 (human-derived FGF18 ). The amino acid sequences encoded by the mRNAs of FGF9, 11, 16, and 18 are SEQ ID NOs: 12 (human-derived FGF9), 16 (human-derived FGF11), 18 (human-derived FG F16), 20 (chicken-derived FGF18) and 22 respectively. (Bird-derived FGF18) [0032] The full-length or partial peptide of at least one fibroblast growth factor selected from the group consisting of FGF9, 11, 16, 18, and 23 is, for example, a solution adapted to skin application, a tarim , Ointments, gels, lotions, shampoos or aerosols, and are provided as a medicament for wound treatment and / or promotion of wound healing. This drug is particularly effective when applied to aging individuals.
[0033] 本発明の創傷治療及び Z又は創傷治癒促進のための薬剤は、局所投与用に適合 化された薬学的に許容される担体と共に有効成分 (例えば、 FGF9, 11， 16, 18， 23な ど）を含んだ医薬組成物の形で投与される。有効成分の担体に対する割合は:!〜 90 重量％の間で変動され得る。  [0033] The wound healing and Z or wound healing promoting agent of the present invention comprises an active ingredient (eg, FGF9, 11, 16, 18, 23) together with a pharmaceutically acceptable carrier adapted for topical administration. Etc.) is administered in the form of a pharmaceutical composition. The proportion of active ingredient to carrier can vary between:! And 90% by weight.
[0034] 本発明の薬剤は、さらにへノ^ンを含有することが好ましい場合もある。  [0034] In some cases, it is preferable that the drug of the present invention further contains a phenone.
[0035] また、本発明の薬剤は、当業界で周知の他の精製又は組換え由来タンパク質増殖 因子を含有していても良い、本発明の薬剤に含まれる有効成分による創傷治癒促進 効果を増加又は増強させる増殖因子としては特に限定されず、表皮増殖因子 (EGF )、血小板由来増殖因子 (PDGF)、トランスフォーミング増殖因子- a (TGF-ひ）、トラ ンスフォーミング増殖因子- β (TGF- β )、トランスフォーミング増殖因子- βスーパー ファミリーに属する因子、骨形成因子 (BMP)、インシュリン様増殖因子 (IGF) (例えば 、 IGF-I, IIなど）及び血管内皮細胞増殖因子の群を挙げることができる（ファン'ブラ ント及びクラウスナー，バイオテクノロジー，第 6卷，第 25-30頁， 1988年）。本発明の 薬剤は、これらの他の精製又は組換え由来タンパク質増殖因子のうち 1種類を含む ものであってもよレ、が、複数種を含むものであってもよレ、。  [0035] In addition, the agent of the present invention may contain other purified or recombinant-derived protein growth factors well known in the art, and increase the wound healing promoting effect by the active ingredient contained in the agent of the present invention. The growth factor to be enhanced is not particularly limited, and epidermal growth factor (EGF), platelet-derived growth factor (PDGF), transforming growth factor-a (TGF-chicken), transforming growth factor-β (TGF-β ), Transforming growth factor-factor belonging to the β superfamily, bone morphogenetic factor (BMP), insulin-like growth factor (IGF) (eg, IGF-I, II, etc.) and vascular endothelial growth factor. Yes (Phant Brant and Klausner, Biotechnology, Vol. 6, pp. 25-30, 1988). The drug of the present invention may contain one of these other purified or recombinantly derived protein growth factors, or may contain more than one.
[0036] EGF、 hEGF及びこれらの製造方法にっレ、ては、米国特許第 3,914,824号、米国特 許第 3,948,875号、米国特許第 4，528，186号、国際公開第 WO/85/00369号パンフレ ット、プロシーデイング .ォブ .ナショナル .ァカデミ一.ォブ .サイエンス USAの第 80卷 、第 7461-7465頁（1983年）に記載されており、参照することができる。  [0036] According to EGF, hEGF and methods for producing them, US Patent No. 3,914,824, US Patent No. 3,948,875, US Patent No. 4,528,186, International Publication No. WO / 85/00369 Pamphlet, Proc. Of Ob. National Academy of Sciences, USA 80, 7461-7465 (1983), which can be referred to.
[0037] PDGF、 hPDGF及びこれらの製造方法については、米国特許第 4,350, 687号、ネー チヤ一の第 320卷，第 695-699頁（1986年）、 EMBOジャーナルの第 3卷，第 921-928 頁（1984年）、 EMBOジャーナルの第 3卷，第 2963-2967頁（1984年）に記載されており 、参照することができる。 [0038] TGF- α、 hTGF- α及びこれらの製造方法については、欧州特許出願公開第 154,4 34号、セルの第 38卷，第 287-297頁（1984年）に記載されており、参照することができ る。 TGF- J3、 hTGF- iS及びこれらの製造方法については、米国特許第 4,774,228号 、米国特許第 4,774,322号、 DNAの第 7卷，第 1-8頁（1988年）、ジャーナル'ォブ 'バ ィォロジカノぃケミストリーの第 262卷，第 12127-12131頁（1987年）に記載されており、 参照することができる。 [0037] PDGF, hPDGF and methods for producing them are described in US Pat. No. 4,350,687, Nichiya No. 320, pp. 695-699 (1986), EMBO Journal No. 3, No. 921- 928 (1984), EMBO Journal 3rd, 2963-2967 (1984), which can be referred to. [0038] TGF-α, hTGF-α and methods for producing them are described in European Patent Application Publication No. 154,434, Cell No. 38, pages 287-297 (1984), You can refer to it. TGF-J3, hTGF-iS and their production methods are described in US Pat. No. 4,774,228, US Pat. No. 4,774,322, DNA No. 7, pages 1-8 (1988), Journal “Ob” Biologicano. Chemistry 262, pp. 12127-12131 (1987) and can be referred to.
[0039] IGF-I、 hIGF_I及びこれらの製造方法については、国際公開第 WO/88/03409号パ ンフレット、欧州特許出願公開第 264,074号、欧州特許出願公開第 219,814号に記載 されており、参照することができる。 IGF-II, hIGF-Π及び及びこれらの製造方法につ いては、欧州特許出願公開第 280,460号に記載されており、参照することができる。  [0039] IGF-I, hIGF_I and methods for producing them are described in International Publication No. WO / 88/03409, European Patent Application Publication No. 264,074, and European Patent Application Publication No. 219,814. can do. IGF-II, hIGF-IV and their production methods are described in European Patent Application Publication No. 280,460 and can be referred to.
[0040] さらに、本発明の薬剤は、当業界で周知の他の創傷治療剤、創傷治癒促進剤など を含んでもよい。他の創傷治療剤及び創傷治癒促進剤としては、特に限定されず、 ポピドンョード、ポピドンョード白糖、フィブラストスプレーなどを挙げることができる。 本発明の薬剤は、これらの他の創傷治療剤及び創傷治癒促進剤のうち 1種類を含む ものであってもよレ、が、複数種を含むものであってもよレ、。  [0040] Furthermore, the agent of the present invention may contain other wound treatment agents, wound healing promoters and the like well known in the art. Other wound treatment agents and wound healing promoters are not particularly limited, and examples thereof include popidone pod, popidone sucrose, and fiblast spray. The drug of the present invention may contain one of these other wound healing agents and wound healing promoters, or may contain more than one kind.
[0041] さらにまた、局所投与用に適合化された薬学的に許容される担体としては、特に限 定されないが、親水ワセリン又はポリエチレングリコール軟膏のような軟膏、キサンゴ ムのようなゴム等のペースト、アルコール、水性又は緩衝液のような溶液、水酸化アル ミニゥム又はアルギン酸ナトリウムゲルのようなゲル、ヒト又は動物アルブミンのような アルブミン、ヒト又は動物コラーゲンのようなコラーゲン、アルキルセルロース、ヒドロキ シアルキルセルロース及びアルキルヒドロキシアルキルセルロースのようなセルロース 、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、カノレボキシメチノレセノレロース、ヒド  [0041] Furthermore, the pharmaceutically acceptable carrier adapted for topical administration is not particularly limited, but is a paste such as a hydrophilic petrolatum or an ointment such as polyethylene glycol ointment, a rubber such as xantham. Solution such as alcohol, aqueous or buffer, gel such as aluminum hydroxide or sodium alginate gel, albumin such as human or animal albumin, collagen such as human or animal collagen, alkylcellulose, hydroxyalkylcellulose And cellulose such as alkyl hydroxyalkyl cellulose, methinoresenorelose, hydroxyethinoresenorelose, canoleoxy methinorescenellose, hydride
127で例示されるプル口ニック（Pluronic (商標））ポリオ—ルのようなポリマ—；テトロニ ック 1508のようなテトロニック（tetronic)、アルギン酸ナトリウムのようなアルギン酸塩 を挙げることができる。 Examples thereof include polymers such as Pluronic ™ polyol exemplified by 127; tetronic such as Tetronic 1508, and alginate such as sodium alginate.
[0042] 投与量は、患者の年齢、性別、体重および症状、治療効果、投与方法、処理時間 、あるいは医薬組成物に含まれる有効成分の種類などにより異なるが、例えば、成人 患者（体重 60kg)—人あたり、有効成分の量に換算して、約 0.01〜約 100 /i g/日ん m2 、好ましくは、約 0.01〜約 100 /i g/日ん m2の範囲で投与するとよい。投与回数は特に 制限されないが、好ましくは 1〜2回/日である。 [0042] The dose varies depending on the patient's age, sex, weight and symptoms, therapeutic effect, administration method, treatment time, type of active ingredient contained in the pharmaceutical composition, etc. Patient (weight 60 kg) —per person, in the range of about 0.01 to about 100 / ig / day m 2 , preferably about 0.01 to about 100 / ig / day m 2 in terms of the amount of active ingredient Good. The frequency of administration is not particularly limited, but is preferably 1 to 2 times / day.
[0043] また、本発明の薬剤は、 FGF9, 11, 16， 18及び 23からなる群より選択される少なくと も 1種の繊維芽細胞増殖因子の全長又は部分ペプチドをコードする核酸を有効成分 として含有する創傷治療及び Z又は創傷治癒促進のための薬剤であってもよい。 FG F9, 11， 16, 18及び 23からなる群より選択される少なくとも 1種の繊維芽細胞増殖因子 の全長又は部分ペプチドをコードする核酸が cDNAであり、かつ発現ベクターに組み 込まれていることが好ましい。すなわち、本発明の薬剤は、上記発現ベクターを用い た遺伝子治療に使用することができる。発現ベクターとしては、動物細胞において発 現を実現するためのプロモーターなどの配列を備える力 S、特に限定されるものではな レ、。発現ベクターとしては、例えば、プラスミドベクター、ウィルスベクターなどが利用 可能である力 これらに限定されない。 [0043] Further, the agent of the present invention comprises, as an active ingredient, a nucleic acid encoding at least one full-length fibroblast growth factor or a partial peptide selected from the group consisting of FGF9, 11, 16, 18, and 23. It may be a wound healing and Z or wound healing promoting agent. The nucleic acid encoding the full length or partial peptide of at least one fibroblast growth factor selected from the group consisting of FG F9, 11, 16, 18, and 23 is cDNA and is incorporated into an expression vector. Is preferred. That is, the agent of the present invention can be used for gene therapy using the above expression vector. The expression vector is a force S having a sequence such as a promoter for realizing expression in animal cells, and is not particularly limited. Examples of expression vectors include, but are not limited to, plasmid vectors and viral vectors.
[0044] より具体的には、遺伝子治療においては、 FGF9, 11, 16, 18及び 23からなる群より 選択される少なくとも 1種の繊維芽細胞増殖因子の全長又は部分ペプチドをコード する cDNAを、例えばウィルスベクターに組み込んで、該組換えウィルスベクターを有 するウィルス (無毒化されたもの）を患者に感染させる。患者体内では繊維芽細胞増 殖因子 9、 11、 16、 18及び 23からなる群より選択される少なくとも 1種の繊維芽細胞 増殖因子の全長ポリペプチド及び/又は部分ポリペプチドが産生され、創傷治癒を促 進すること力 Sできる。 [0044] More specifically, in gene therapy, cDNA encoding a full-length or partial peptide of at least one fibroblast growth factor selected from the group consisting of FGF9, 11, 16, 18, and 23, For example, it is incorporated into a viral vector, and the patient is infected with a virus (detoxified) having the recombinant viral vector. In the patient, at least one full-length polypeptide and / or partial polypeptide of fibroblast growth factor selected from the group consisting of fibroblast growth factor 9, 11, 16, 18, and 23 is produced, and wound healing Power S to promote S
[0045] 遺伝子治療用の薬剤を細胞内に導入する方法としては、ウィルスベクターを利用し た遺伝子導入方法、非ウィルス性の遺伝子導入方法（日経サイエンス， 1994年 4月号 , 20-45頁、実験医学増刊， 12(15X1994)、実験医学別冊「遺伝子治療の基礎技術」 ,羊土社 (1996))のいずれの方法も適用することができる。  [0045] As a method for introducing a gene therapy drug into a cell, a gene introduction method using a viral vector, a non-viral gene introduction method (Nikkei Science, April 1994, pp. 20-45, Experimental medicine special edition, 12 (15X1994), experimental medicine separate volume "Basic technology of gene therapy", Yodosha (1996)) can be applied.
[0046] ウィルスベクターによる遺伝子導入方法としては、例えばレトロウイルス、アデノウィ ノレス、アデノ関連ウィルス、ヘルぺスウィルス、ワクシニアウィルス、ボックスウィルス、 ポリオウイルス、シンビスウィルス等の DNAウィルス又は RNAウィルスに、 TR4あるいは 変異 TR4をコードする DNAを組み込んで導入する方法が挙げられる。このうち、レトロ ウィルス、アデノウイルス、アデノ関連ウィルス、ワクシニアウィルスを用いた方法が、 特に好ましい。非ウィルス性の遺伝子導入方法としては、発現プラスミドを直接筋肉 内に投与する方法（DNAワクチン法）、リボソーム法、リボフヱクチン法、マイクロインジ ヱクシヨン法、リン酸カルシウム法、エレクト口ポレーシヨン法等が挙げられ、特に DNA ワクチン法、リボソーム法が好ましい。 [0046] Gene transfer methods using viral vectors include, for example, retrovirus, adenovirus, adeno-associated virus, herpes virus, vaccinia virus, box virus, poliovirus, simbis virus and other DNA viruses or RNA viruses, TR4 Alternatively, a method of introducing a DNA encoding a mutant TR4 can be mentioned. Of these, retro Particularly preferred are methods using viruses, adenoviruses, adeno-associated viruses, and vaccinia viruses. Non-viral gene transfer methods include a method in which an expression plasmid is directly administered intramuscularly (DNA vaccine method), a ribosome method, a ribofuctin method, a microinduction method, a calcium phosphate method, an electoral position method, etc. The DNA vaccine method and the ribosome method are preferred.
[0047] また遺伝子治療用の薬剤を実際に医薬として作用させるには、 DNAを直接体内に 導入するインビボ法及びヒトからある種の細胞を取り出し体外で DNAを該細胞に導入 し、その細胞を体内に戻すエタスビボがある（日経サイエンス， 1994年 4月号， 20-45 頁、月刊薬事， 36(1)， 23-48(1994)、実験医学増刊， 12(15)(1994))。  [0047] In addition, in order to cause a drug for gene therapy to actually act as a medicine, an in vivo method in which DNA is directly introduced into the body and a certain cell from a human is taken out, and DNA is introduced into the cell outside the body. There is etas vivo that is returned to the body (Nikkei Science, April 1994, 20-45 pages, Monthly Pharmaceutical Affairs, 36 (1), 23-48 (1994), Experimental Medicine Extra Number, 12 (15) (1994)).
[0048] 例えば、遺伝子治療用の薬剤がインビボ法により投与される場合は、疾患、症状等 に応じ、静脈、動脈、皮下、皮内、筋肉内等、適当な投与経路により投与される。また インビボ法により投与する場合は、遺伝子治療用の薬剤は一般的には注射剤等とさ れる力 必要に応じて慣用の担体を加えてもよい。また、リボソーム又は膜融合リポソ ーム（センダイウィルス—リボソーム等）の形態にした場合は、懸濁剤、凍結剤、遠心 分離濃縮凍結剤等のリボソーム製剤とすることができる。  [0048] For example, when a drug for gene therapy is administered by an in vivo method, it is administered by an appropriate administration route such as intravenous, arterial, subcutaneous, intradermal, intramuscular, etc., depending on the disease, symptoms and the like. When administered by an in vivo method, the drug for gene therapy is generally considered to be an injection or the like. A conventional carrier may be added if necessary. In the case of a ribosome or membrane fusion liposome (Sendai virus-ribosome, etc.), it can be prepared as a ribosome preparation such as a suspension, a freezing agent, or a centrifugal concentrated freezing agent.
[0049] FGF9, 11, 16， 18及び 23からなる群より選択される少なくとも 1種の繊維芽細胞増殖 因子の全長又は部分ペプチドをコードする cDNAを組み込んだ発現ベクターをヒトに 投与する場合、成人患者（体重 60kg)—人あたり、 0.01〜100 mg、好ましくは 0.1〜10 mgの 1回量で投与するとよい。投与されるベクターに含まれる所望の繊維芽細胞増 殖因子の全長又は部分ペプチドをコードする cDNAの量としては、成人患者（体重 60 kg)—人あたり、 0·001〜10 mg、好ましくは 0·005〜0.1 mgの 1回量で投与するとよい。 投与は、治療効果が認められるまで、適当な頻度で繰り返すとよい。  [0049] When an expression vector incorporating cDNA encoding a full-length or partial peptide of at least one fibroblast growth factor selected from the group consisting of FGF9, 11, 16, 18, and 23 is administered to humans, Patient (body weight 60 kg) —per person, 0.01-100 mg, preferably 0.1-10 mg. The amount of cDNA encoding the full length or partial peptide of the desired fibroblast growth factor contained in the vector to be administered is 0.001 to 10 mg, preferably 0, per adult patient (body weight 60 kg). · A single dose of 005 to 0.1 mg is recommended. Administration may be repeated at an appropriate frequency until a therapeutic effect is observed.
[0050] 2.スクリーニング方法  [0050] 2. Screening method
本発明は、加齢状態の異なる生体の創傷治癒過程における遺伝子の発現をモニ ターし、若齢個体と加齢個体との間で遺伝子の発現量を比較し、若齢個体と比べて 加齢個体での発現量が低い遺伝子の産物を加齢個体の創傷治療及び/又は創傷 治癒促進に効果があると判定することを含む、加齢個体の創傷治療及び Z又は創傷 治癒促進に効果がある物質をスクリーニングする方法を提供する。 [0051] 本発明のスクリーニング方法では、若齢及び加齢の実験動物個体を用い、その創 傷治癒反応を比較する。実験動物としては、ヒトを除ぐ例えば、マウス、ラット、ニヮト リ、七面鳥、ゥシ、ブタ、ヒッジ及びゥサギ等を挙げることができる。若齢及び加齢の程 度としては、何ら限定されないが、例えば、マウスでは若齢個体として 8週齢程度、加 齢個体として 35週齢程度が挙げられる。これらの年齢差はさらに大きくてもよいし、小 さくてもよい。 The present invention monitors gene expression during wound healing processes in living bodies with different aging states, compares gene expression levels between young individuals and aging individuals, and compared to young individuals. Effective in wound therapy and Z or wound healing promotion in aging individuals, including determining that products of genes with low expression levels in individuals are effective in wound treatment and / or wound healing promotion in aging individuals A method for screening a substance is provided. [0051] In the screening method of the present invention, young and aged experimental animal individuals are used and their wound healing responses are compared. Examples of experimental animals include mice, rats, chickens, turkeys, ushi, pigs, hidges, and rabbits, excluding humans. The age of young and aging is not limited in any way, but for example, mice are about 8 weeks old as young individuals and about 35 weeks old as individuals. These age differences may be larger or smaller.
[0052] 次に、本発明のスクリーニング方法では、実験動物の創傷治癒過程における様々 な遺伝子の発現をモニターする。遺伝子の種類としては、何ら限定されないが、例え ば、 22種の FGF遺伝子及び 4種の FGF受容体遺伝子の発現をモニターする。実験動 物における FGF遺伝子の発現は、例えば、 FGF23抗体を用いた組織染色や ELISA等 の常法を用いて解析するか、あるいは実験動物内における FGF23遺伝子の mRNA量 を定量的逆転写 PCR法やノーザンプロット法等により解析するといつた方法によりモ 二ターすること力 Sできる。  [0052] Next, in the screening method of the present invention, the expression of various genes is monitored during the wound healing process of experimental animals. The type of gene is not limited at all. For example, the expression of 22 FGF genes and 4 FGF receptor genes are monitored. The expression of the FGF gene in the experimental animal can be analyzed, for example, by using a conventional method such as tissue staining or ELISA using the FGF23 antibody, or the amount of FGF23 gene mRNA in the experimental animal can be determined by quantitative reverse transcription PCR or When analyzed by the Northern Plot method, etc., it is possible to monitor using any method.
[0053] これらいずれかの解析により、例えば、若齢動物個体での創傷治癒における FGF2 3遺伝子の発現量と比べて、加齢動物個体での創傷治癒における FGF23遺伝子の 発現量が低減していれば、当該遺伝子の産物を補給することによって加齢動物個体 での創傷の治療、創傷治癒促進といった機能を有する可能性がある候補物質である と判断できる。この方法により、 FGF7, 9， 10, 11， 16, 18， 22， 23, TGF- beta, HGFな どが候補物質であると判断できた (後述の実施例を参照のこと)。  [0053] By any of these analyses, for example, the expression level of the FGF23 gene in wound healing in an aging animal individual may be reduced compared to the expression level of FGF23 gene in wound healing in a young animal individual. For example, it can be determined that a candidate substance that may have a function of treating wounds and promoting wound healing in an aging animal individual by supplementing the product of the gene. By this method, it was possible to determine that FGF7, 9, 10, 11, 16, 18, 22, 23, TGF-beta, HGF, etc. are candidate substances (see the examples below).
[0054] このようにスクリーニングされた物質は、臨床へ応用するに際し、上記有効成分を単 独で用いることも可能であるが、薬学的に許容され得る担体と配合して医薬組成物と して用レ、ることもできる。この時の有効成分の担体に対する割合は、 1〜90重量％の 間で変動され得る。また、力かる薬剤は種々の形態で投与することができ、それらの 投与形態としては、溶液、クリーム、軟膏、ゲル、ローション、シャンプー又はエアゾー ルといった非経口投与を挙げることができる。また、その投与量は、症状、年齢、体重 等によって適宜選択することができる。  [0054] The substance screened in this way can be used alone in the clinical application, but it can be used as a pharmaceutical composition by blending with a pharmaceutically acceptable carrier. You can also use it. The ratio of the active ingredient to the carrier at this time can be varied between 1 to 90% by weight. In addition, powerful drugs can be administered in various forms, and examples of their administration forms include parenteral administration such as solutions, creams, ointments, gels, lotions, shampoos or aerosols. The dose can be appropriately selected depending on symptoms, age, body weight and the like.
[0055] あるいはまた、スクリーニングされた物質の全長又は部分ペプチドをコードする核酸 を加齢個体の創傷治療及び/又は創傷治癒促進のための遺伝子治療に用いてもよ ぐ発現ベクター、遺伝子導入法、投与量、投与方法などは上述したものと同様であ る。 [0055] Alternatively, a nucleic acid encoding the full-length or partial peptide of the screened substance may be used for wound therapy and / or gene therapy for promoting wound healing in an aging individual. The expression vector, gene transfer method, dosage, administration method and the like are the same as described above.
[0056] 従って、本発明は、 FGF7, 9, 10， 11, 16, 18， 22及び 23, TGF-beta並びに HGFか らなる群より選択される少なくとも 1種の増殖因子の全長又は部分ペプチドを有効成 分として含有する加齢個体の創傷治療及び/又は創傷治癒促進のための薬剤を提 供する。  [0056] Accordingly, the present invention provides a full-length or partial peptide of at least one growth factor selected from the group consisting of FGF7, 9, 10, 11, 16, 18, 22, 22 and 23, TGF-beta, and HGF. It provides a drug for treating wounds and / or promoting wound healing in aging individuals that is contained as an active ingredient.
[0057] さらにまた、本発明は、 FGF7, 9, 10， 11, 16, 18， 22及び 23, TGF- beta並びに HGF 力 なる群より選択される少なくとも 1種の増殖因子の全長又は部分ペプチドをコード する核酸を有効成分として含有する加齢個体の創傷治療及び Z又は創傷治癒促進 のための薬剤を提供する。  [0057] Furthermore, the present invention relates to a full-length or partial peptide of at least one growth factor selected from the group consisting of FGF7, 9, 10, 11, 16, 18, 22, and 23, TGF-beta, and HGF force. Provided is a drug for treating wounds and promoting Z or wound healing in an aging individual containing the encoded nucleic acid as an active ingredient.
[0058] 本発明の薬剤に含まれてもよい有効成分として、 FGF7, 9， 10, 11， 16， 18, 22及び 2 3、 TGF_beta並びに HGFの全長又は部分ペプチドがある。 FGF7, 9， 10, 11, 16， 18, 22及び 23、 TGF-beta並びに HGFの mRNAの塩基配列をそれぞれ配列番号 9(ヒト由 来 FGF7)、 11(ヒト由来 FGF9)、 13(ヒト由来 FGF10)、 15(ヒト由来 FGF11)、 17(ヒト由来 FGF16)、 19(ヒト由来 FGF18)、 21(ヒト由来 FGF18)、 23(ヒ卜由来 FGF22)、 1(ヒ卜由来 F GF23)、 3(マウス由来 FGF23)、 5(ラット由来 FGF23)、 7(ゼブラフィッシュ由来 FGF23)、 25(ヒト由来 TGF_beta)、 27(ヒト由来 HGF)及び 29(ヒト由来 HGF)に示す。 FGF7, 9， 10 ， 11, 16, 18， 22及び 23、 TGF-beta並びに HGFの mRNAがコードするアミノ酸配列を それぞれ配列番号 10(ヒト由来 FGF7)、 12(ヒト由来 FGF9)、 14(ヒト由来 FGF10)、 16( ヒト由来 FGF11)、 18(ヒト由来 FGF16)、 20(ヒト由来 FGF18)、 22(ヒト由来 FGF18)、 24( ヒト由来 FGF22)、 2 (ヒト由来 FGF23)、 4 (マウス由来 FGF23)、 6 (ラット由来 FGF23)、 8 (ゼブラフィッシュ由来 FGF23)、 26 (ヒト由来 TGF- beta)、 28(ヒト由来 HGF)及び 30( ヒト由来 HGF)に示す。  [0058] Examples of active ingredients that may be contained in the drug of the present invention include FGF7, 9, 10, 11, 16, 18, 22 and 23, TGF_beta, and full-length or partial peptides of HGF. The base sequences of FGF7, 9, 10, 11, 16, 18, 22, and 23, TGF-beta and HGF mRNA are shown in SEQ ID NOs: 9 (human-derived FGF7), 11 (human-derived FGF9), 13 (human-derived FGF10 ), 15 (human-derived FGF11), 17 (human-derived FGF16), 19 (human-derived FGF18), 21 (human-derived FGF18), 23 (chicken-derived FGF22), 1 (chicken-derived FGF23), 3 (mouse Origin FGF23), 5 (rat-derived FGF23), 7 (zebrafish-derived FGF23), 25 (human-derived TGF_beta), 27 (human-derived HGF) and 29 (human-derived HGF). The amino acid sequences encoded by FGF7, 9, 10, 11, 16, 18, 22, and 23, TGF-beta and HGF mRNA are SEQ ID NOs: 10 (human-derived FGF7), 12 (human-derived FGF9), 14 (human-derived) (FGF10), 16 (human-derived FGF11), 18 (human-derived FGF16), 20 (human-derived FGF18), 22 (human-derived FGF18), 24 (human-derived FGF22), 2 (human-derived FGF23), 4 (mouse-derived FGF23 ), 6 (rat-derived FGF23), 8 (zebrafish-derived FGF23), 26 (human-derived TGF-beta), 28 (human-derived HGF) and 30 (human-derived HGF).
実施例  Example
[0059] 以下、実施例を用いて本発明をより詳細に説明するが、本発明の技術的範囲は以 下の実施例に限定されるものではない。  [0059] Hereinafter, the present invention will be described in more detail with reference to examples. However, the technical scope of the present invention is not limited to the following examples.
[0060] 〔実施例 1〕 [Example 1]
本実施例では、マウスの皮膚の全層欠損創傷治癒モデルにおける FGFファミリーに 属する遺伝子の発現プロファイルを検討した。 In this example, the FGF family in a mouse skin full-thickness wound healing model The expression profile of the gene to which it belongs was examined.
[0061] <材料と方法 >  [0061] <Materials and methods>
マウスおよび皮膚サンプルの調製  Preparation of mouse and skin samples
皮膚の全層欠損創傷治癒の実験用としてへアレス雄マウス (hr 25匹（7週齢及び 3 3週齢)を日本エスエルシー株式会社から入手し、標準的な実験動物用飼料および 水を任意に摂取させて飼育した。 7週齢マウスについては 1週間、 33週齢マウスにつ いては 2週間馴化させた後、マウス 5匹を安楽死させ、背部の皮膚サンプルを分離し、 「0日（休止期）」サンプルとした。残りのマウス 20匹については、ネンブタール麻酔下 で背部皮膚を 4力所について、直径 8mmの円形に切り取り、全層欠損創傷を作成し た。そして快復後、異なる時期、すなわち創傷作成後 2日、 4日、 7日、 15日、 21日を 選択し、マウスを安楽死させ、創傷部を含む 8mmx8mmの正方形の全層皮膚サンプ ル (表皮、毛幹、毛包、皮脂腺、皮下脂肪、皮膚筋及び血管を含む)を採取した。全 層皮膚サンプルは、後述する mRNA分離、 in situハイブリダィゼーシヨン及び免疫組 織染色に使用した。なお、これらの実験は基本的に同一試験計画によって 3回実施 して結果の再現性を確証した。  Hairless male mice (25 hrs (7 weeks old and 33 weeks old) obtained from Nippon SLC Co., Ltd. for standard wound healing and experimental water healing experiments After acclimating for 1 week for 7-week-old mice and for 2 weeks for 33-week-old mice, euthanize 5 mice and isolate the skin samples on the back. The rest of the 20 mice were cut under a Nembutal anesthesia, the dorsal skin was cut into a circle with a diameter of 8 mm at 4 locations, and full-thickness wounds were created. Select the timing, i.e., 2, 4, 7, 15, 21 days after wound creation, euthanize the mouse, and 8 mm x 8 mm square full thickness skin sample (epidermis, hair shaft, hair, including wound) Sac, sebaceous glands, subcutaneous fat, skin muscles and blood vessels). The full-thickness skin samples were used for mRNA separation, in situ hybridization, and immunohistochemical staining, which will be described later, and these experiments were basically performed three times with the same test plan to ensure the reproducibility of the results. Confirmed.
免疫組織染色用の皮膚サンプルは、最初に 10%ホルムアルデヒドで室温 24時間固 定し、標準的な方法によってパラフィンで包理した。  Skin samples for immunohistochemical staining were first fixed with 10% formaldehyde for 24 hours at room temperature and embedded in paraffin by standard methods.
[0062] mRNA調製および逆転写  [0062] mRNA preparation and reverse transcription
トータル RNAは、上述したように準備した皮膚サンプルから Isogen (株式会社ニッポ ンジーン)を製造業者の取扱説明書に従って使用して調製した。次に、 01igotex-dT3 0 Super mRNA精製キット（タカラバイオ株式会社）を用いて、トータル RNAから mRNA を精製した。精製した mRNAサンプル（lOOng)を铸型とし、プライマとして〇ligo(dT)12 -18を用いた Superscript II (Gibco BRL)によって全量 20mlで逆転写反応を行った。  Total RNA was prepared from skin samples prepared as described above using Isogen (Nippon Gene Co., Ltd.) according to the manufacturer's instructions. Next, mRNA was purified from the total RNA using 01igotex-dT30 Super mRNA purification kit (Takara Bio Inc.). The purified mRNA sample (lOOng) was in a saddle type, and reverse transcription reaction was performed in a total volume of 20 ml using Superscript II (Gibco BRL) using oligo (dT) 12-18 as a primer.
[0063] PCRプライマの設訐および FGF cDNAのクローニング  [0063] Design of PCR primers and cloning of FGF cDNA
各 FGFの mRNA量をリアルタイム PCRによって分析できるように、特異的なプライマ セットを設計した（表 1)。各 FGFの mRNAを定量する際、コピー数対照として cDNAフ ラグメントが必要となる。この cDNAフラグメントをクローニングする際、同じプライマセッ トを使用した。
Figure imgf000017_0001
Figure imgf000017_0002
A specific primer set was designed so that each FGF mRNA level could be analyzed by real-time PCR (Table 1). When quantifying mRNA for each FGF, a cDNA fragment is required as a copy number control. The same primer set was used when cloning this cDNA fragment.
Figure imgf000017_0001
Figure imgf000017_0002
〔〕〔 上述した逆転写反応の反応産物（RT混合物）を蒸留水で 10倍希釈したものを PCR 増幅の铸型とした。 PCR増幅には、 PHiポリメラーゼ（Stratagene社製）を製造業者の取 扱説明書に従って使用した。そして、 PCR増幅反応混合液の一部を 2.0%ァガロース ゲルを用いた電気泳動に使用し、増殖産物の大きさを確証した。また、各 FGFに相当 するそれぞれの cDNAをクローニングするため、 PCR産物を pCR-Bluntn-ΤΟΡΟベタ ター（Invitrogen社製）に連結して E.coliの形質転換に使用した。リコンビナントプラスミ ドのヌクレオチド配列は BigDyeターミネータサイクルシークェンシングキット及び ABI P RISM 310 Genetic Analyzer (Applied Biosystems社製）を用いて確認した。 [] [ A product obtained by diluting the above-mentioned reverse transcription reaction product (RT mixture) 10-fold with distilled water was used as a PCR amplification kit. For PCR amplification, PHi polymerase (Stratagene) was used according to the manufacturer's instructions. A part of the PCR amplification reaction mixture was used for electrophoresis using a 2.0% agarose gel to confirm the size of the growth product. In addition, in order to clone each cDNA corresponding to each FGF, the PCR product was ligated to pCR-Bluntn-Better (Invitrogen) and used for transformation of E. coli. The nucleotide sequence of the recombinant plasmid was confirmed using a BigDye terminator cycle sequencing kit and ABI PRISM 310 Genetic Analyzer (Applied Biosystems).
[0065] リアルタイム PCRによる mRNAコピー数の定量 [0065] Quantification of mRNA copy number by real-time PCR
リアルタイム PCR増幅は、 1:10に希釈した RT混合物 2mlを铸型として用レ、、 Light Cy cler (Roche Diagnostics社製）によって実施した。各 FGFの絶対コピー数を得るため、 各 FGFに対応するプラスミツド DNAをコピー数対照としてそれぞれ使用した。 Light Cy clerによって増幅した DNAの定量には、 Cyber Greenを使用した。特異性の高いプラ イマとともに、最適なアニーリング温度、伸長時間、および取得温度で Light Cycler反 応を実施した。プライマの特異性はバックグラウンド用の配列としてマウスの全 cDNA を用いて最初に確証した。プライマの特異性および反応条件はプラスミド DNAに増幅 フラグメントをクローニングすることによって、さらに実験的に実証してこれらの配列を 検証した。  Real-time PCR amplification was performed with 2 ml of the RT mixture diluted 1:10 in a bowl, Light Cycler (Roche Diagnostics). In order to obtain the absolute copy number of each FGF, the plasmid DNA corresponding to each FGF was used as a copy number control. Cyber Green was used for quantification of DNA amplified by Light Cy cler. Along with highly specific primers, the Light Cycler reaction was performed at the optimal annealing temperature, extension time, and acquisition temperature. Primer specificity was first verified using whole mouse cDNA as a background sequence. Primer specificity and reaction conditions were further experimentally verified by cloning amplified fragments into plasmid DNA to verify these sequences.
さらに個別実験の最後に増幅産物の融点温度を測定してこれらが融点温度で均一 であることが実証された。定量実験後、産物を 2.0%ァガロースゲル電気泳動で分析 し、反応によって既知サイズの DNAフラグメントが正確に増幅されていることが確証さ れた。この実験系によって個別遺伝子の特異的な定量が保証される。 mRNAの評価 では、 Light Cyclerによる同一実験に標的 cDNA配列を有する DNAの連続希釈サン プノレを含め、これらの測定から個別サンプノレと関連する mRNAの絶対コピー数を算出 した。またハウスキーピング遺伝子のコピー数を同じ方法で評価してこれらの量が予 測範囲内であることを確認した。 Furthermore, the melting point temperature of the amplification products was measured at the end of the individual experiment, and it was proved that they were uniform at the melting point temperature. After the quantification experiment, the product was analyzed by 2.0% agarose gel electrophoresis, confirming that the DNA fragment of known size was accurately amplified by the reaction. This experimental system ensures specific quantification of individual genes. the evaluation of the mRNA, Light Cycler including serial dilutions San Punore of DNA having a target cDNA sequence in the same experiments by, and calculates the absolute copy number of m RNA associated From these measurements the individual Sanpunore. In addition, the number of housekeeping gene copies was evaluated by the same method to confirm that these amounts were within the expected range.
[0066] ぐ結果 > [0066] Results>
FGF及び FGFRファミリメンバー、 TGF- beta、 HGFの発現プロファイル 創傷治癒の各時点における各 FGFおよび FGFRの mRNA発現量をそれぞれ図 1A、 図 IB及び図 2に示した。 Expression profile of FGF and FGFR family members, TGF-beta, HGF The mRNA expression levels of each FGF and FGFR at each time point of wound healing are shown in FIG. 1A, FIG. IB and FIG. 2, respectively.
図 1Aから、創傷治癒の過程で発現量が高まる遺伝子は Fgf-5、 -7、 -9、 -10、 -11、 _ 16、 -18及び- 23であることが判明した。本実施例では、健康な皮膚で発現することが 既に知られているこれら遺伝子、すなわち FGF1、 _2、 -5、 -7、 -10、 -13及び- 22に加 えて、健康な皮膚ではほとんど発現がみられなレ、 FGF23 mRNAも創傷治癒の過程で は高い量で発現しており、 FGF23 mRNA量が創傷作成後 2日目ないし 4日目にピー クを示す（図 1A)ことが明らかになった。また、図 1Aからは、加齢個体において、上記 の創傷治癒の過程で発現量が高まる遺伝子の多くが、若齢個体よりも発現レベルが 低下することが明らかとなった。  From FIG. 1A, it was found that the genes whose expression levels increase during the wound healing are Fgf-5, -7, -9, -10, -11, _16, -18 and -23. In this example, in addition to these genes already known to be expressed in healthy skin, ie, FGF1, _2, -5, -7, -10, -13 and -22, it is almost expressed in healthy skin. In the wound healing process, FGF23 mRNA is also expressed at high levels, and it is clear that FGF23 mRNA levels show a peak on the second to fourth days after wound creation (Fig. 1A). became. In addition, from FIG. 1A, it has been clarified that in many aging individuals, the expression level of many genes whose expression levels are increased in the above wound healing process is lower than that in young individuals.
FGFRに関しては、 FGFR1及び FGFR3の mRNAともに多量に発現されたが（図 2)、 前者では最も強い発現力 ¾日目及び 15日目に現れ、後者では創傷作成後の発現昂 進はみられなかった。 FGFR2の mRNA発現量は、上皮細胞に特異的である Illbサブク ラスも含めて FGFR1又は 3の mRNAよりも少なく（図 2)、 FGFR4の mRNA発現量は 4種 類の受容体の中で最も低かった（図 2)。  Regarding FGFR, both FGFR1 and FGFR3 mRNAs were expressed in large amounts (Fig. 2), but the former showed the strongest expression on the ¾th and 15th days, and the latter did not promote expression after wound creation. It was. The expression level of FGFR2 mRNA is lower than that of FGFR1 or 3, including the Illb subclass, which is specific to epithelial cells (Figure 2), and the expression level of FGFR4 is the lowest among the four types of receptors. (Fig. 2).
TGF-beta mRNA, HGF mRNAに関しては、健康な皮膚での発現レベルも高いが、 創傷治癒の過程で、さらに発現量が高まることが明らかになった。  As for TGF-beta mRNA and HGF mRNA, the expression level in healthy skin was high, but it was revealed that the expression level was further increased during the wound healing process.
[0067] 〔実施例 2〕 [Example 2]
実施例 1において、皮膚における FGF23 mRNAの発現が新規に明らかになつたた め、本実施例では、特異的抗体を用いて免疫組織染色により FGF23タンパク質の分 布を測定した。  In Example 1, since the expression of FGF23 mRNA in skin was newly clarified, in this example, the distribution of FGF23 protein was measured by immunohistochemical staining using a specific antibody.
[0068] <材料と方法 > [0068] <Materials and methods>
鍾  鍾
抗 FGF23抗体は以下のように作成した。 FGF23の一次構造から、二次構造、親水 性、他のタンパク質との相異性などを勘案して、抗原性と特異性が高いと考えられる 部分ペプチドを予測し、これを化学合成して精製した。このペプチドを、キーホールリ ンペットへモシァニンタンパク質（KLH)と共有結合させ、ゥサギにアジュバントと共に 複数回注射し、抗体を含む血清を得た。抗体のタイターを ELISA法で評価した後（デ ータは示さない）、ィムノブロッテイングにより特異性を確認した。 Anti-FGF23 antibody was prepared as follows. Considering the secondary structure, hydrophilicity, phase isomerism with other proteins, etc. from the primary structure of FGF23, a partial peptide that is considered to be highly antigenic and specific is predicted, and this is chemically synthesized and purified. . This peptide was covalently bound to mosianin protein (KLH) to a keyhole limpet and injected multiple times with an adjuvant to a rabbit to obtain serum containing antibodies. After assessing antibody titers by ELISA (de The specificity was confirmed by immunoblotting.
免疫組織染色はパラフィン包坦組織切片を抗 FGF23抗体で染色することにより行つ た。若齢マウスの創傷治癒過程 (創傷作成後 4日目）の皮膚をホルマリン固定後、パ ラフィンに包坦し、 4マイクロメートルの厚さの切片を作成した。これをスライドグラス上 に貼り付け、脱パラフィン処理を行った。その後、 0.01Mクェン酸 (pH6.0)中で 2分間煮 沸して抗原活性化を行い、抗 FGF23抗体とインキュベートした。洗浄後、ピオチンィ匕 ャギ抗ゥサギ IgGとインキュベートし、洗浄した。これをアビジン化パーォキシデースと インキュベートし、パーォキシデースの基質であるアミノエチルカルバゾール試薬で 発色した。  Immunohistochemical staining was performed by staining a paraffin-embedded tissue section with an anti-FGF23 antibody. Skin of young mice during the wound healing process (4th day after wound creation) was fixed in formalin, then wrapped in paraffin, and 4 μm thick sections were prepared. This was pasted on a slide glass and deparaffinized. Subsequently, the antigen was activated by boiling in 0.01 M citrate (pH 6.0) for 2 minutes and incubated with anti-FGF23 antibody. After washing, the cells were incubated with Piotin-yagi anti-rabbit IgG and washed. This was incubated with avidinized peroxidase and developed with an aminoethylcarbazole reagent which is a substrate for peroxidase.
ぐ結果 >  Results>
免疫組織染色の結果を図 3に示す。図 3に示すように、創傷治癒過程 4日目におけ る皮膚では、 FGF23タンパク質は高いレベルで存在し、細胞の遊走や増殖が盛んに 起こっている創の周囲及び底部の皮膚、特に肉芽組織にのみ発現されることを認め た（図 3 ;紫色のシグナル）。一方、 FGF23 mRNAがほとんど発現していない健常皮膚 ではシグナルを生じな力 た（図は示さない）。  Fig. 3 shows the results of immunohistochemical staining. As shown in Figure 3, in the skin on the 4th day of the wound healing process, FGF23 protein is present at a high level, and the skin around the wound and the bottom, especially granulation tissue, where cell migration and proliferation are actively occurring (Fig. 3; purple signal). On the other hand, in healthy skin where FGF23 mRNA was hardly expressed, no signal was generated (not shown).
[0069] 〔実施例 3〕 [Example 3]
本実施例では、 FGF23の皮膚における機能を評価するため、線維芽細胞（NIH3T3 細胞）について、 FGF23による細胞増殖促進効果を調べた。  In this example, in order to evaluate the function of FGF23 in the skin, the effect of FGF23 on cell proliferation was examined for fibroblasts (NIH3T3 cells).
[0070] <材料と方法 > [0070] <Materials and methods>
細朐増殖アツセィ  Sassy breeding Atsey
マウス線維芽細胞（NIH3T3) (ATCCから入手）は、生育培地（10%仔ゥシ血清をカロ えた DMEM基礎培地）で継代培養して [3H] -チミジン取り込みアツセィに使用した。 細胞はトリプシン処理して 24wellプレート（Sumilon社製）に、密度が 5 X 103細胞/ゥェ ルで 24ゥヱル (HFDP細胞）に分注して生育培地で 37°Cに維持した。次の日に生育培 地を、 0.5%チヤコール吸着子ゥシ血清を含む DMEM基礎培地で置き換え、細胞を 48 時間培養してから、 5 μ g/mlのへパリンの共存下で、所定の濃度の FGFで刺激した。 Mouse fibroblasts (NIH3T3) (obtained from ATCC) were subcultured in a growth medium (DMEM basal medium supplemented with 10% pup sera) and used for [ 3 H] -thymidine uptake assay. The cells were trypsinized and dispensed into 24 well plates (Sumilon) at a density of 5 × 10 3 cells / well to 24 walls (HFDP cells) and maintained at 37 ° C. with growth medium. On the following day, the growth medium is replaced with DMEM basal medium containing 0.5% thiacol-adsorbed serum, and the cells are cultured for 48 hours, followed by the prescribed concentration in the presence of 5 μg / ml heparin. Stimulated with FGF.
FGFを加えてから 24時間後に再び FGFをカ卩え、さらに 24時間後に細胞を採取して、 細胞数を計測した。 <結果 > 24 hours after the addition of FGF, FGF was picked up again, and another 24 hours later, the cells were collected and the number of cells was counted. <Result>
結果を図 4に示す。図 4から判るように、適切な濃度（1及び 3 ng/ml)の FGF23によつ て、細胞数の増加が誘導されていた。  The results are shown in Fig. 4. As can be seen from FIG. 4, an increase in cell number was induced by FGF23 at appropriate concentrations (1 and 3 ng / ml).
この結果から、 FGF23の全長及び/又は部分ペプチドは、皮膚の創傷治癒の一過 程である繊維芽細胞の増殖を促進する作用があることが実証された。  From these results, it was demonstrated that the full length and / or partial peptide of FGF23 has an action of promoting the proliferation of fibroblasts, which is a process of skin wound healing.
[0071] 〔実施例 4〕 [Example 4]
本実施例では、 FGF23の in vivoでの影響を調べるため、細胞の増殖や分化のシグ ナル伝達の結果として起こる初期遺伝子 c_f0Sの発現が誘導されているかどうかを創 傷治癒の過程にあるマウスで試験した。 In this embodiment, in order to examine the effect of the in vivo of FGF23, there whether the expression of the early genes c _f 0S consequent to signal transduction of cell proliferation and differentiation is induced in the process of wound wound healing Tested in mice.
[0072] <材料と方法 > [0072] <Materials and methods>
鍾  鍾
免疫組織染色は、実施例 2と同様の操作によりパラフィン包埋組織切片を抗 c-fos 抗体（Calbiochem-Novabiochem社）で染色することにより行った。皮膚組織切片は、 若齢マウスの創傷治癒過程 (創傷作成後 4日目）の皮膚から作成した。  Immunohistological staining was performed by staining a paraffin-embedded tissue section with an anti-c-fos antibody (Calbiochem-Novabiochem) in the same manner as in Example 2. Skin tissue sections were prepared from the skin of young mice during the wound healing process (4 days after wound creation).
<結果 >  <Result>
結果を図 5に示す。図 5は創傷治癒過程 (創傷作成後 4日目）の皮膚切片を抗 c-fos 抗体で染色した結果である。抗 c-fos抗体によって強く染まる領域は、実施例 2で観 察された FGF23タンパク質の発現部位と類似性が高いことが分かった。  The results are shown in FIG. Figure 5 shows the results of staining a skin section of the wound healing process (4 days after wound creation) with anti-c-fos antibody. The region strongly stained by the anti-c-fos antibody was found to be highly similar to the expression site of the FGF23 protein observed in Example 2.
この結果から、 FGF23タンパク質の発現によって、細胞の増殖や分化、遊走につな 力 ¾c-fosの発現が誘導されてレ、ると考えられることが in vivoにおレ、て示された。 産業上の利用可能性 From this result, it was shown in vivo that the expression of FGF23 protein is thought to induce the expression of c- fos, a force that leads to cell proliferation, differentiation and migration. Industrial applicability
[0073] 本発明の薬剤は、創傷治癒及び/又は創傷治療促進のために利用できる。特に、 本発明の薬剤は、加齢個体の創傷治癒及び Z又は創傷治療促進に有効である。 [0073] The agent of the present invention can be used for wound healing and / or promotion of wound treatment. In particular, the agent of the present invention is effective for wound healing and promotion of Z or wound treatment in aging individuals.
[0074] また、本発明のスクリーニング方法により、加齢個体における創傷治癒及び Z又は 創傷治療促進に効果のある物質を探索することができる。 [0074] Further, by the screening method of the present invention, a substance effective for wound healing and Z or wound treatment promotion in an aging individual can be searched.
配列表フリーテキスト  Sequence listing free text
[0075] <配列番号 1 > [0075] <SEQ ID NO: 1>
配列番号 1は、ヒト由来 FGF23の mRNAのヌクレオチド配列を示す。 <配列番号 2 > SEQ ID NO: 1 shows the nucleotide sequence of mRNA of human-derived FGF23. <SEQ ID 2>
配列番号 2は、ヒト由来 FGF23の mRNAがコードするアミノ酸配列を示す。  SEQ ID NO: 2 shows the amino acid sequence encoded by the mRNA of human-derived FGF23.
<配列番号 3 > <SEQ ID NO: 3>
配列番号 3は、マウス由来 FGF23の mRNAのヌクレオチド配列を示す。  SEQ ID NO: 3 shows the nucleotide sequence of mRNA of mouse-derived FGF23.
<配列番号 4 > <SEQ ID NO: 4 >
配列番号 4は、マウス由来 FGF23の mRNAがコードするアミノ酸配列を示す。 ぐ配列番号 5 >  SEQ ID NO: 4 shows the amino acid sequence encoded by the mRNA of mouse-derived FGF23. SEQ ID NO: 5>
配列番号 5は、ラット由来 FGF23の mRNAのヌクレオチド配列を示す。  SEQ ID NO: 5 shows the nucleotide sequence of mRNA of rat-derived FGF23.
ぐ配列番号 6 > SEQ ID NO: 6>
配列番号 6は、ラット由来 FGF23の mRNAがコードするアミノ酸配列を示す。  SEQ ID NO: 6 shows the amino acid sequence encoded by rat FGF23 mRNA.
<配列番号 7 > <SEQ ID NO: 7 >
配列番号 7は、ゼブラフィッシュ由来 FGF23の mRNAのヌクレオチド配列を示す。 <配列番号 8 >  SEQ ID NO: 7 shows the nucleotide sequence of mRNA of zebrafish-derived FGF23. <SEQ ID NO: 8>
配列番号 8は、ゼブラフィッシュ由来 FGF23の mRNAがコードするアミノ酸配列を示 す。  SEQ ID NO: 8 shows an amino acid sequence encoded by mRNA of FGF23 derived from zebrafish.
<配列番号 9 >  <SEQ ID NO: 9>
配列番号 9は、ヒト由来 FGF7の mRNAのヌクレオチド配列を示す。  SEQ ID NO: 9 shows the nucleotide sequence of human FGF7 mRNA.
<配列番号 10 > <SEQ ID NO: 10>
配列番号 10は、ヒト由来 FGF7の mRNAがコードするアミノ酸配列を示す。  SEQ ID NO: 10 shows the amino acid sequence encoded by mRNA of human-derived FGF7.
<配列番号 11 > <SEQ ID NO: 11>
配列番号 11は、ヒト由来 FGF9の mRNAのヌクレオチド配列を示す。  SEQ ID NO: 11 shows the nucleotide sequence of mRNA of human-derived FGF9.
<配列番号 12 > <SEQ ID NO: 12>
配列番号 12は、ヒト由来 FGF9の mRNAがコードするアミノ酸配列を示す。  SEQ ID NO: 12 shows the amino acid sequence encoded by mRNA of human-derived FGF9.
<配列番号 13 > <SEQ ID NO: 13>
配列番号 13は、ヒト由来 FGF10の mRNAのヌクレオチド配列を示す。  SEQ ID NO: 13 shows the nucleotide sequence of mRNA of human-derived FGF10.
<配列番号 14 > <SEQ ID NO: 14>
配列番号 14は、ヒト由来 FGF10の mRNAがコードするアミノ酸配列を示す。  SEQ ID NO: 14 shows the amino acid sequence encoded by the mRNA of human-derived FGF10.
<配列番号 15 > 配列番号 15は、ヒト由来 FGF11の mRNAのヌクレオチド配列を示す。 <SEQ ID NO: 15> SEQ ID NO: 15 shows the nucleotide sequence of mRNA of human-derived FGF11.
<配列番号 16 > <SEQ ID NO: 16>
配列番号 16は、ヒト由来 FGF11の mRNAがコードするアミノ酸配列を示す。  SEQ ID NO: 16 shows the amino acid sequence encoded by the mRNA of human-derived FGF11.
<配列番号 17 > <SEQ ID NO: 17>
配列番号 17は、ヒト由来 FGF16の mRNAのヌクレオチド配列を示す。  SEQ ID NO: 17 shows the nucleotide sequence of mRNA of human-derived FGF16.
<配列番号 18 > <SEQ ID NO: 18>
配列番号 18は、ヒト由来 FGF16の mRNAがコードするアミノ酸配列を示す。  SEQ ID NO: 18 shows the amino acid sequence encoded by the mRNA of human-derived FGF16.
<配列番号 19 > <SEQ ID NO: 19>
配列番号 19は、ヒト由来 FGF18(variant 1)の mRNAのヌクレオチド配列を示す。 <配列番号 20 >  SEQ ID NO: 19 shows the nucleotide sequence of mRNA of human-derived FGF18 (variant 1). <SEQ ID NO: 20>
配列番号 20は、ヒト由来 FGF18(variant 1)の mRNAがコードするアミノ酸配列を示す く配列番号 21 >  SEQ ID NO: 20 represents the amino acid sequence encoded by human FGF18 (variant 1) mRNA; SEQ ID NO: 21>
配列番号 21は、ヒト由来 FGF18(variant 2)の mRNAのヌクレオチド配列を示す。 <配列番号 22 >  SEQ ID NO: 21 shows the nucleotide sequence of mRNA of human-derived FGF18 (variant 2). <SEQ ID NO: 22>
配列番号 22は、ヒト由来 FGF18(variant 2)の mRNAがコードするアミノ酸配列を示す  SEQ ID NO: 22 shows the amino acid sequence encoded by human FGF18 (variant 2) mRNA
<配列番号 23 > <SEQ ID NO: 23>
配列番号 23は、ヒト由来 FGF22の mRNAのヌクレオチド配列を示す。  SEQ ID NO: 23 shows the nucleotide sequence of mRNA of human-derived FGF22.
<配列番号 24 > <SEQ ID NO: 24>
配列番号 24は、ヒト由来 FGF22の mRNAがコードするアミノ酸配列を示す。  SEQ ID NO: 24 shows the amino acid sequence encoded by the mRNA of human-derived FGF22.
<配列番号 25 > <SEQ ID NO: 25>
配列番号 25は、ヒト由来 TGF_betaの mRNAのヌクレオチド配列を示す。  SEQ ID NO: 25 shows the nucleotide sequence of mRNA of human-derived TGF_beta.
<配列番号 26 > <SEQ ID NO: 26>
配列番号 26は、ヒト由来 TGF_betaの mRNAがコードするアミノ酸配列を示す。 <配列番号 27 >  SEQ ID NO: 26 shows the amino acid sequence encoded by the mRNA of human-derived TGF_beta. <SEQ ID NO: 27>
配列番号 27は、ヒト由来 HGF(variant 1)の mRNAのヌクレオチド配列を示す。 <配列番号 28 > 配列番号 28は、ヒト由来 HGF(variant 1)の mRNAがコードするアミノ酸配列を示す。 <配列番号 29 > SEQ ID NO: 27 shows the nucleotide sequence of mRNA of human-derived HGF (variant 1). <SEQ ID NO: 28> SEQ ID NO: 28 shows the amino acid sequence encoded by mRNA of human-derived HGF (variant 1). <SEQ ID NO: 29>
配列番号 29は、ヒト由来 HGF(variant 3)の mRNAのヌクレオチド配列を示す。  SEQ ID NO: 29 shows the nucleotide sequence of mRNA of human-derived HGF (variant 3).
<配列番号 30 > <SEQ ID NO: 30>
配列番号 30は、ヒト由来 HGF(variant 3)の mRNAがコードするアミノ酸配列を示す。 <配列番号 3：!〜 74 >  SEQ ID NO: 30 shows the amino acid sequence encoded by mRNA of human-derived HGF (variant 3). <SEQ ID NO: 3:! To 74>
配列番号 3：!〜 74は、プライマーのヌクレオチド配列を示す。  SEQ ID NO: 3:! -74 shows the nucleotide sequence of the primer.

Claims

請求の範囲 The scope of the claims
[1] 繊維芽細胞増殖因子 9、 11、 16、 18及び 23からなる群より選択される少なくとも 1 種の繊維芽細胞増殖因子の全長又は部分ペプチドを有効成分として含有する創傷 治療及び/又は創傷治癒促進のための薬剤。  [1] Wound treatment and / or wound containing, as an active ingredient, the full length or partial peptide of at least one fibroblast growth factor selected from the group consisting of fibroblast growth factor 9, 11, 16, 18 and 23 Drugs for promoting healing.
[2] 繊維芽細胞増殖因子が野生型である請求項 1記載の薬剤。  [2] The agent according to claim 1, wherein the fibroblast growth factor is a wild type.
[3] 繊維芽細胞増殖因子が変異体である請求項 1記載の薬剤。  [3] The drug according to claim 1, wherein the fibroblast growth factor is a mutant.
[4] 他のタンパク質増殖因子、創傷治癒剤及び/又は創傷治癒促進剤を更に含むこと を特徴とする請求項：!〜 3のいずれかに記載の薬剤。  [4] The drug according to any one of [1] to [3], further comprising another protein growth factor, a wound healing agent and / or a wound healing promoter.
[5] 繊維芽細胞増殖因子 9、 11、 16、 18及び 23からなる群より選択される少なくとも 1 種の繊維芽細胞増殖因子の全長又は部分ペプチドをコードする核酸を有効成分と して含有する創傷治療及び/又は創傷治癒促進のための薬剤。  [5] Contains as an active ingredient a nucleic acid encoding the full-length or partial peptide of at least one fibroblast growth factor selected from the group consisting of fibroblast growth factor 9, 11, 16, 18 and 23 Agents for wound treatment and / or promotion of wound healing.
[6] 繊維芽細胞増殖因子の全長又は部分ペプチドをコードする核酸が cDNAであり、か つ発現ベクターに組み込まれている請求項 5記載の薬剤。  [6] The agent according to claim 5, wherein the nucleic acid encoding the full-length or partial peptide of fibroblast growth factor is cDNA and is incorporated into an expression vector.
[7] 加齢状態の異なる生体の創傷治癒過程における遺伝子の発現をモニターし、若齢 個体と加齢個体との間で遺伝子の発現量を比較し、若齢個体と比べて加齢個体で の発現量が低い遺伝子の産物を加齢個体の創傷治療及び/又は創傷治癒促進に 効果があると判定することを含む、加齢個体の創傷治療及び/又は創傷治癒促進に 効果がある物質をスクリーニングする方法。  [7] The expression of genes during wound healing processes in living bodies with different aging conditions is monitored, and the expression level of genes is compared between young individuals and aging individuals. A substance having an effect on wound treatment and / or promotion of wound healing in an aging individual, including determining that a gene product having a low expression level is effective in wound treatment and / or promotion of wound healing in an aging individual How to screen.
[8] 繊維芽細胞増殖因子 7、 9、 10、 11、 16、 18、 22及び 23、トランスフォーミング増 殖因子一 /3並びに肝細胞増殖因子からなる群より選択される少なくとも 1種の増殖因 子の全長又は部分ペプチドを有効成分として含有する加齢個体の創傷治療及び Z 又は創傷治癒促進のための薬剤。  [8] At least one growth factor selected from the group consisting of fibroblast growth factor 7, 9, 10, 11, 16, 18, 22, and 23, transforming growth factor 1/3, and hepatocyte growth factor A drug for treating wounds and promoting Z or wound healing of an aging individual containing the full length or partial peptide of a child as an active ingredient.
[9] 繊維芽細胞増殖因子 7、 9、  [9] Fibroblast growth factor 7, 9,
10、 Ten,
11、 16、 18、 22及び 23、トランスフォーミング増 殖因子一 /3並びに肝細胞増殖因子からなる群より選択される少なくとも 1種の増殖因 子の全長又は部分ペプチドをコードする核酸を有効成分として含有する加齢個体の 創傷治療及び/又は創傷治癒促進のための薬剤。 11, 16, 18, 22, and 23, a nucleic acid encoding the full length or partial peptide of at least one growth factor selected from the group consisting of transforming growth factor 1/3 and hepatocyte growth factor as an active ingredient A drug for treating wounds and / or promoting wound healing in aging individuals.
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