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WO2010147109A1 - Inducteur d'angiogenèse comprenant de la gélatine génétiquement modifiée et un facteur de croissance de fibroblastes basiques - Google Patents

Inducteur d'angiogenèse comprenant de la gélatine génétiquement modifiée et un facteur de croissance de fibroblastes basiques Download PDF

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Publication number
WO2010147109A1
WO2010147109A1 PCT/JP2010/060101 JP2010060101W WO2010147109A1 WO 2010147109 A1 WO2010147109 A1 WO 2010147109A1 JP 2010060101 W JP2010060101 W JP 2010060101W WO 2010147109 A1 WO2010147109 A1 WO 2010147109A1
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amino acid
angiogenesis
acid sequence
genetically modified
gly
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Japanese (ja)
Inventor
一隆 荻原
乃梨子 石川
章二 大屋
哲男 平等
健太郎 中村
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Fujifilm Corp
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Fujifilm Corp
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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
    • C07K14/78—Connective tissue peptides, e.g. collagen, elastin, laminin, fibronectin, vitronectin or cold insoluble globulin [CIG]
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/02—Drugs for skeletal disorders for joint disorders, e.g. arthritis, arthrosis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/08—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P27/00—Drugs for disorders of the senses
    • A61P27/16—Otologicals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/06—Antiarrhythmics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/08—Vasodilators for multiple indications
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
    • 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 factor [FGF]
    • C07K14/503—Fibroblast growth factor [FGF] basic FGF [bFGF]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the present invention relates to an angiogenesis inducer using genetically modified gelatin and basic fibroblast growth factor.
  • Angiogenesis mainly represents a phenomenon in which new blood vessels are formed from existing blood vessels.
  • angiogenesis there are angiogenesis related to angiogenesis in the embryonic period, endometrium and corpus luteum formation, wound healing and the like. Utilizing an angiogenic event for treatment is practiced as an angiogenic therapy.
  • the importance of angiogenesis has been clarified as a therapeutic method for wound healing, ischemic diseases, etc., and in treatments widely called regenerative medicine such as organ regeneration, cell transplantation, and natural healing effects. Because of the therapeutic effect of angiogenesis itself, or because angiogenesis enhances the therapeutic effect, therapeutic agents targeting angiogenesis have been developed.
  • bFGF basic fibroblast growth factor
  • bFGF vascular endothelial growth factor
  • the growth factor is expensive, and since the risk of canceration is expected because it actively induces blood vessels, the growth factor is pointed out. It is preferable that the effect is exerted in as little amount as possible, and therefore, it is required to apply bFGF to a site where angiogenesis is required efficiently.
  • Patent Document 3 discloses a bFGF preparation using a naturally-derived cross-linked gelatin gel such as pig or bovine, but gelatin itself is a carrier for containing bFGF and reduces the amount of bFGF used. It is not used.
  • gelatin is cross-linked, and bFGF is contained in the gelatin gel to regenerate blood vessels.
  • Patent Document 3 relies on bFGF only for angiogenesis, and gelatin is used only as a base material for sustained release. Therefore, there is a drawback that a large amount of bFGF is required for angiogenesis.
  • the present invention has been made to solve the problem of providing an angiogenesis-inducing agent that is safe for a living body and excellent in bioadhesiveness and that can reduce the amount of bFGF used.
  • the present inventors have induced vascular endothelial cells with bFGF by using genetically modified gelatin having high affinity with vascular endothelial cells, and the vascular endothelial cells with genetically modified gelatin. It was found that angiogenesis can be induced with a smaller amount of bFGF used by positively adhering to the present invention, and the present invention has been completed.
  • an angiogenesis-inducing agent comprising genetically modified gelatin having an amino acid sequence derived from a partial amino acid sequence of collagen and basic fibroblast growth factor as active ingredients.
  • the genetically modified gelatin has a repetition of a sequence represented by Gly-XY, which is characteristic of collagen (X and Y each independently represents one of amino acids) (a plurality of Gly-XY are the same)
  • the molecular weight is 2 KDa or more and 100 KDa or less.
  • the genetically modified gelatin has a repetition of a sequence represented by Gly-XY, which is characteristic of collagen (X and Y each independently represents one of amino acids) (a plurality of Gly-XY are the same)
  • the molecular weight is 10 KDa or more and 90 KDa or less.
  • the genetically modified gelatin has a repetition of a sequence represented by Gly-XY, which is characteristic of collagen (X and Y each independently represents one of amino acids) (a plurality of Gly-XYs are the same) Or may be different), including two or more cell adhesion signals per molecule.
  • the cell adhesion signal is an amino acid sequence represented by Arg-Gly-Asp.
  • the amino acid sequence of the recombinant gelatin does not include serine and threonine.
  • the amino acid sequence of the genetically modified gelatin does not include serine, threonine, asparagine, tyrosine, and cysteine.
  • the amino acid sequence of the recombinant gelatin does not include the amino acid sequence represented by Asp-Arg-Gly-Asp.
  • the genetically modified gelatin is Formula: A-[(Gly-XY) n ] m -B (In the formula, A represents an arbitrary amino acid or amino acid sequence, B represents an arbitrary amino acid or amino acid sequence, n Xs independently represent any of the amino acids, and n Ys each independently represent an amino acid. N represents an integer of 3 to 100, and m represents an integer of 2 to 10. Note that n Gly-XY may be the same or different.
  • the genetically modified gelatin is Formula: Gly-Ala-Pro-[(Gly-XY) 63 ] 3 -Gly (In the formula, 63 X's each independently represent any amino acid, and 63 Y's each independently represent any amino acid. The n Gly-XY may be the same or different. Good.) Indicated by Preferably, the genetically modified gelatin is (1) an amino acid sequence represented by SEQ ID NO: 1, or (2) an amino acid sequence having an angiogenic action having 80% or more homology with the amino acid sequence represented by SEQ ID NO: 1. Have
  • the genetically modified gelatin is cross-linked.
  • crosslinking is performed with aldehydes, condensing agents, or enzymes.
  • the angiogenesis inducer of the present invention induces angiogenesis by accumulating at an angiogenesis site.
  • the angiogenesis further comprises administering a recombinant gelatin having an amino acid sequence derived from a partial amino acid sequence of collagen and a basic fibroblast growth factor to a subject in need of induction of angiogenesis.
  • a method of inducing is provided.
  • a recombinant gelatin having an amino acid sequence derived from a partial amino acid sequence of collagen and a basic fibroblast growth factor for the production of an angiogenesis inducer.
  • the angiogenesis-inducing agent of the present invention is characterized by containing genetically modified gelatin, it has no risk of canceration, is safe for the living body, and has excellent bioadhesiveness. Moreover, according to the angiogenesis inducer of the present invention, it is possible to reduce the amount of bFGF used.
  • FIG. 1 shows the results of measuring the amount of hemoglobin contained in blood in order to quantify the angiogenic effect.
  • FIG. 2 shows the results of the HUVEC cell adhesion test.
  • FIG. 3 shows the results of the HUVEC cell adhesion test.
  • FIG. 4 shows HUVEC cell photographs on plates coated with various proteins.
  • FIG. 5 shows a comparison of the area of one HUVEC cell on a plate coated with various proteins.
  • FIG. 6 shows inhibition of HUVEC cell adhesion by anti- ⁇ V antibody.
  • a genetically modified gelatin having an amino acid sequence derived from a partial amino acid sequence of collagen can be used, for example, those described in EP1014176A2, US6992172, WO2004-85473, WO2008 / 103041, etc. However, it is not limited to these.
  • Preferred as the genetically modified gelatin used in the present invention is the genetically modified gelatin of the following embodiment.
  • the genetically modified gelatin used in the present invention is excellent in biocompatibility due to the inherent performance of natural gelatin, and is not naturally derived.
  • the genetically modified gelatin used in the present invention is more uniform than natural ones and the sequence is determined, the strength and degradability can be precisely designed with less blur due to cross-linking described later. It is.
  • the molecular weight of the genetically modified gelatin used in the present invention is preferably from 2 to 100 KDa. More preferably, it is 2.5 to 95 KDa. More preferably, it is 5 to KDa. Most preferably, it is 10 KDa or more and 90 KDa or less.
  • the genetically modified gelatin used in the present invention preferably has a repeating sequence represented by Gly-XY characteristic of collagen.
  • the plurality of Gly-X-Ys may be the same or different.
  • Gly-XY Gly represents glycine
  • X and Y represent any amino acid (preferably any amino acid other than glycine).
  • the GXY sequence characteristic of collagen is a very specific partial structure in the amino acid composition and sequence of gelatin / collagen compared to other proteins. In this part, glycine accounts for about one third of the whole, and in the amino acid sequence, it is one in three repeats. Glycine is the simplest amino acid, has few constraints on the arrangement of molecular chains, and greatly contributes to the regeneration of the helix structure upon gelation.
  • the amino acids represented by X and Y are rich in imino acids (proline, oxyproline), and preferably account for 10% to 45% of the total.
  • 80% or more of the sequence, more preferably 95% or more, and most preferably 99% or more of the amino acids are GXY repeating structures.
  • General gelatin has 1: 1 polar amino acids, both charged and uncharged.
  • the polar amino acid specifically means cysteine, aspartic acid, glutamic acid, histidine, lysine, asparagine, glutamine, serine, threonine, tyrosine, arginine, and among these polar uncharged amino acids are cysteine, asparagine, glutamine, serine. , Threonine, tyrosine.
  • the proportion of polar amino acids is 10 to 40%, preferably 20 to 30%, of all the constituent amino acids.
  • the ratio of the uncharged amino acid in the polar amino acid is 5% or more and less than 20%, preferably less than 10%. Furthermore, it is preferable that any one amino acid among serine, threonine, asparagine, tyrosine and cysteine is not included in the sequence, preferably two or more amino acids.
  • the minimum amino acid sequence that acts as a cell adhesion signal in a polypeptide is known (for example, “Pathophysiology”, Vol. 9, No. 7 (1990), page 527, published by Nagai Publishing Co., Ltd.).
  • the genetically modified gelatin used in the present invention preferably has two or more of these cell adhesion signals in one molecule.
  • IKVAV sequence, LRE sequence, DGEA sequence, and HAV sequence are preferable, RGD sequence, YIGSR sequence, PDSGR sequence, LGTIPG sequence, IKVAV sequence, and HAV sequence, and particularly preferably RGD sequence.
  • RGD sequences an ERGD sequence is preferred.
  • the number of amino acids between RGDs is not uniform between 0 and 100, preferably between 25 and 60.
  • the content of the minimum amino acid sequence is preferably 3 to 50, more preferably 4 to 30, and particularly preferably 5 to 20 per protein molecule from the viewpoint of cell adhesion and proliferation. Most preferably, it is 12.
  • the ratio of the RGD motif to the total number of amino acids is preferably at least 0.4%.
  • each stretch of 350 amino acids has at least 1 stretch.
  • it contains two RGD motifs.
  • the ratio of RGD motif to the total number of amino acids is more preferably at least 0.6%, more preferably at least 0.8%, more preferably at least 1.0%, more preferably at least 1.2%. And most preferably at least 1.5%.
  • the number of RGD motifs in the genetically modified gelatin is preferably at least 4, more preferably 6, more preferably 8, more preferably 12 or more and 16 or less per 250 amino acids.
  • a ratio of 0.4% of the RGD motif corresponds to at least one RGD sequence per 250 amino acids. Since the number of RGD motifs is an integer, a gelatin of 251 amino acids must contain at least two RGD sequences to meet the 0.4% feature.
  • the recombinant gelatin of the present invention comprises at least 2 RGD sequences per 250 amino acids, more preferably comprises at least 3 RGD sequences per 250 amino acids, more preferably at least 4 per 250 amino acids. Contains one RGD sequence.
  • the genetically modified gelatin of the present invention it contains at least 4 RGD motifs, preferably 6, more preferably 8, more preferably 12 or more and 16 or less. Further, the genetically modified gelatin may be partially hydrolyzed.
  • the genetically modified gelatin used in the present invention preferably has a repeating structure of A-[(Gly-XY) n ] m -B.
  • m is preferably 2 to 10, and preferably 3 to 5.
  • n is preferably 3 to 100, more preferably 15 to 70, and most preferably 50 to 65.
  • the naturally occurring collagen referred to here may be any naturally occurring collagen, but is preferably type I, type II, type III, type IV, and type V. More preferred are type I, type II and type III.
  • the collagen origin is preferably human, cow, pig, mouse, rat. More preferably, it is a human.
  • the isoelectric point of the genetically modified gelatin used in the present invention is preferably 5 to 10, more preferably 6 to 10, and further preferably 7 to 9.5.
  • the genetically modified gelatin is not deaminated.
  • the genetically modified gelatin does not have procollagen and procollagen.
  • the genetically modified gelatin has no telopeptide.
  • the genetically modified gelatin is a substantially pure collagen material prepared with a nucleic acid encoding natural collagen.
  • the genetically modified gelatin used in the present invention (1) the amino acid sequence of SEQ ID NO: 1; or (2) 80% or more (more preferably 90% or more, most preferably 95% or more) of homology with the amino acid sequence of SEQ ID NO: 1, An amino acid sequence having an angiogenic effect; A genetically modified gelatin having
  • the genetically modified gelatin used in the present invention can be produced by a genetic recombination technique known to those skilled in the art, for example, according to the method described in EP1014176A2, US6992172, WO2004-85473, WO2008 / 103041, and the like. Specifically, a gene encoding the amino acid sequence of a predetermined recombinant gelatin is obtained, and this is incorporated into an expression vector to produce a recombinant expression vector, which is then introduced into a suitable host to produce a transformant. To do. By culturing the obtained transformant in an appropriate medium, genetically modified gelatin is produced.
  • the genetically modified gelatin used in the present invention is prepared by recovering the genetically modified gelatin produced from the culture. be able to.
  • the performance of the genetically modified gelatin alone is insufficient, it may be mixed with other materials or combined.
  • different types of genetically modified gelatin other biopolymers or synthetic polymers may be mixed.
  • the biopolymer include polysaccharides, polypeptides, proteins, nucleic acids, antibodies and the like. Preferred are polysaccharides, polypeptides, and proteins.
  • polysaccharides, polypeptides, and proteins include collagen, gelatin, albumin, fibroin, and casein. Furthermore, these may be partially chemically modified as necessary. For example, hyaluronic acid ethyl ester may be used.
  • polysaccharide examples include glycosaminoglycans represented by hyaluronic acid and heparin, chitin, and chitosan.
  • polyamino acids examples include poly- ⁇ -glutamic acid.
  • the genetically modified gelatin used in the present invention can be chemically modified depending on the application.
  • Chemical modifications include the introduction of low molecular weight compounds or various polymers (biopolymers (sugars, proteins), synthetic polymers, polyamides) into the carboxyl group or amino group of the side chain of genetically modified gelatin, Examples include cross-linking between gelatins.
  • Examples of the introduction of the low molecular weight compound into the genetically modified gelatin include a carbodiimide-based condensing agent.
  • the crosslinking agent used in the present invention is not particularly limited as long as the present invention can be carried out, and may be a chemical crosslinking agent or an enzyme.
  • the chemical cross-linking agent include formaldehyde, glutaraldehyde, carbodiimide, cyanamide and the like. Preferred are formaldehyde and glutaraldehyde.
  • examples of cross-linking of genetically modified gelatin include light irradiation on gelatin into which a photoreactive group has been introduced, or light irradiation in the presence of a photosensitizer.
  • the photoreactive group include a cinnamyl group, a coumarin group, a dithiocarbamyl group, a xanthene dye, and camphorquinone.
  • the enzyme When performing cross-linking by an enzyme, the enzyme is not particularly limited as long as it has a cross-linking action between genetically modified gelatin chains.
  • trans-glutaminase and laccase most preferably trans-glutaminase is used for cross-linking. it can.
  • a specific example of a protein that is enzymatically cross-linked with transglutaminase is not particularly limited as long as it has a lysine residue and a glutamine residue.
  • the transglutaminase may be derived from a mammal or may be derived from a microorganism. Specifically, transglutaminase derived from a mammal that has been marketed as an Ajinomoto Co., Ltd.
  • Human-derived blood coagulation factors Factor XIIIa, Haematologic Technologies, Inc.
  • Factor XIIIa Haematologic Technologies, Inc.
  • guinea pig liver-derived transglutaminase goat-derived transglutaminase
  • rabbit-derived transglutaminase manufactured by Oriental Yeast Co., Ltd., Upstate USA Inc., Biodesign International, etc. Etc.
  • the cross-linking of genetically modified gelatin has two processes: a process of mixing a genetically modified gelatin solution and a cross-linking agent and a process of reacting these uniform solutions.
  • the mixing temperature when the genetically modified gelatin is treated with the crosslinking agent is not particularly limited as long as the solution can be uniformly stirred, but is preferably 0 ° C. to 40 ° C., more preferably 0 ° C. to 30 ° C. More preferably, it is 3 ° C to 25 ° C, more preferably 3 ° C to 15 ° C, still more preferably 3 ° C to 10 ° C, and particularly preferably 3 ° C to 7 ° C.
  • the reaction temperature is not particularly limited as long as the crosslinking proceeds, but is substantially 0 ° C. to 60 ° C., more preferably 0 ° C. to 40 ° C. in view of denaturation and degradation of the recombinant gelatin.
  • the temperature is preferably 3 ° C to 25 ° C, more preferably 3 ° C to 15 ° C, still more preferably 3 ° C to 10 ° C, and particularly preferably 3 ° C to 7 ° C.
  • basic fibroblast growth factor (bFGF) is used in combination with genetically modified gelatin.
  • bFGF basic fibroblast growth factor
  • the form of bFGF used in the present invention is not particularly limited, and may be naturally derived bFGF or genetically modified bFGF.
  • genetically modified bFGF for example, those commercially available as Trafermin (Fiblast) (Kaken Pharmaceutical Co., Ltd.) may be used.
  • a drug can be encapsulated as desired.
  • the drug is a physiologically active ingredient.
  • Specific examples include transdermal absorption agents, topical therapeutic agents, oral therapeutic agents, cosmetic ingredients, and supplement ingredients.
  • Specific examples of the drug include anti-inflammatory agents, antibacterial agents, antibiotic agents, immunosuppressive agents, antioxidant agents, anticancer agents, vitamins, nucleic acids, and antibodies. Particularly preferred are anti-inflammatory agents.
  • As the anti-inflammatory agent either steroidal or non-steroidal may be used.
  • anti-inflammatory agents include, for example, aspirin, acetaminophen, phenachicene, indomethacin, diclofenac sodium, piroxicam, fenoprofen calcium, ibuprofen, chlorpheniramine maleate, diflunisal, dexamethasone sodium phosphate, paclitaxel, docetaxel, 5 -Fluorouracil, topotensin, cisplatin, rapamycin, tacrolimus, cyclosporine.
  • vitamins both water-soluble and fat-soluble are used.
  • Specific examples of the vitamin include vitamin A, vitamin B group, vitamin C, vitamin D group, vitamin E, and vitamin K, for example.
  • Specific drugs have been listed above, but as long as the genetically modified gelatin used in the present invention is used, the present invention is not limited to the drugs listed above.
  • an angiogenesis is achieved by administering a genetically modified gelatin having an amino acid sequence derived from the above-described partial amino acid sequence of collagen to a subject (for example, a mammal such as a human) in need of angiogenesis induction. Can be induced.
  • the angiogenesis-inducing agent of the present invention can be appropriately determined in dosage, usage, and dosage form according to the intended use.
  • the angiogenesis-inducing agent of the present invention may be directly administered to a target site in a living body, or distilled water for injection, physiological saline for injection, pH 5-8 buffer (phosphate system, citric acid) It may be suspended in a liquid excipient such as an aqueous solvent such as a system and administered by injection, coating, or the like. Further, it may be applied after mixing with an appropriate excipient to form an ointment, gel or cream.
  • the administration form of the angiogenesis inducer of the present invention may be oral or parenteral (for example, intravenous administration, intramuscular administration, subcutaneous administration, intradermal administration, etc.).
  • parenteral for example, intravenous administration, intramuscular administration, subcutaneous administration, intradermal administration, etc.
  • direct injection into the heart muscle from the intraventricular lumen using a catheter, or catheter in the stenosis or occlusion part in the coronary artery It is also possible to release or apply them locally using.
  • Examples of the dosage form include orally administered drugs such as tablets, powders, capsules, granules, extracts and syrups, or injections (for example, intravenous injections, intramuscular injections, subcutaneous injections, intradermal injections). Etc.) and the like.
  • the form of the angiogenesis-inducing agent of the present invention is not particularly defined, and examples thereof include sponges, films, nonwoven fabrics, fibers (tubes), particles, meshes and the like.
  • the formulation of the angiogenesis-inducing agent of the present invention can be performed according to methods known to those skilled in the art.
  • the pharmaceutical carrier when the pharmaceutical carrier is a liquid, it can be dissolved or dispersed, and when the pharmaceutical carrier is a powder, it can be mixed or adsorbed.
  • pharmaceutically acceptable additives for example, preservatives, stabilizers, antioxidants, excipients, binders, disintegrants, wetting agents, lubricants, coloring agents, fragrances
  • Flavoring agents skin coats, suspending agents, emulsifiers, solubilizers, buffering agents, tonicity agents, plasticizers, surfactants or soothing agents, and the like.
  • the dose of the genetically modified gelatin is not particularly limited, but is, for example, 1 to 100 mg, preferably 1 to 50 mg per 1 cm 2 of the surface area of the organism to be administered.
  • the dose of bFGF is not particularly limited, and is, for example, 1 ⁇ g to 1 mg, preferably 1 ⁇ g to 100 ⁇ g, per 1 cm 2 of the surface area of the organism to be administered.
  • Examples of the target diseases of the angiogenesis-inducing agent of the present invention include ischemic diseases, cell / tissue regeneration therapy, cell transplantation treatment, diabetic skin ulcer, hearing loss, heart disease, acute coronary syndrome, acute myocardial infarction, unstable angina Disease, promotion of fracture healing, pseudo joint, bone union failure, nerve regeneration treatment, and the like.
  • CBE3 described below was prepared as a genetically modified gelatin (described in WO2008-103041).
  • the amino acid sequence of CBE3 does not include serine, threonine, asparagine, tyrosine and cysteine.
  • CBE3 has an ERGD sequence. Isoelectric point: 9.34
  • Amino acid sequence (SEQ ID NO: 1 in the sequence listing) (same as SEQ ID NO: 3 in WO2008 / 103041, except that X at the end is corrected to “P”)
  • GAP GAPGLQGAPGLQGMPGERGAAGLPGPKGERGDAGPKGADGAPGAPGLQGMPGERGAAGLPGPKGERGDAGPKGADGAPGKDGVRGLAGPIGPPGERGAAGLPGPKGERGDAGPKGADGAPGKDGVRGLAGPIGPPGPAGAPGAPGLQGMPGERGAAGLPGAPG
  • the resulting gel was quenched at ⁇ 50 ° C. and lyophilized at ⁇ 10 ° C. for 48 hours.
  • the water content of the gel was measured from the change in the weight of the crosslinked R-Gel before and after the swelling treatment with PBS for 24 hours at 4 ° C., and was 95.4%.
  • the obtained gel was cut out at 5 mg.
  • 50 ⁇ l of PBS containing 35 ⁇ g of bFGF was dropped, and left overnight at 4 ° C. to impregnate 35 ⁇ g of bFGF into the cross-linked R-Gel to prepare a bFGF-containing R-Gel preparation.
  • the 35 ⁇ g bFGF-containing crosslinked R-Gel preparation prepared above was implanted subcutaneously in the back of the mouse. Separately, as a control group, 50 ⁇ l of a PBS solution containing 35 ⁇ g of bFGF was subcutaneously administered. As another control group, 5 mg of bFGF-impregnated PI-5 gel (medgel) impregnated with 50 ⁇ l of 35 ⁇ g of bFGF in PBS was subcutaneously implanted. Three days after the administration, the skin of the mouse was peeled off, and the preparation embedding and the PBS solution administration site were observed.
  • the state of the tissue around the administration site was the same as in the untreated group, and no gross change was observed.
  • the bFGF-impregnated PI-5 gel-administered group almost no angiogenesis image was observed as in the untreated group.
  • the tissue around the preparation implantation site was also visually red, and an angiogenic effect that was clearly one of bFGF was confirmed.
  • the bFGF-impregnated PI-5 gel administration group has not been able to efficiently adhere vascular endothelial cells induced with bFGF.
  • vascular endothelial cells induced with bFGF can be positively adhered to exert an angiogenic effect.
  • the amount of hemoglobin contained in the blood was measured.
  • a hemoglobin B-Test Wako kit (Wako Pure Chemical Industries) was used for the measurement.
  • 5 mg of the above-prepared 35 ⁇ g bFGF-containing cross-linked R-Gel preparation was implanted subcutaneously in the back of the mouse.
  • 50 ⁇ l of PBS solution was subcutaneously administered.
  • 5 mg of bFGF-impregnated PI-5 gel (medgel) impregnated with 50 ⁇ l of 35 ⁇ g of bFGF in PBS was subcutaneously implanted.
  • the skin of the mouse was peeled off, and the surrounding tissue of 1.5 cm ⁇ 1.5 cm was taken out mainly from the site where the preparation was embedded and the PBS solution was administered, and placed in a tube.
  • the surrounding tissue was finely cut in a tube, and 300 ⁇ l of an extract (10 mM Tris, 1 mM EDTA, pH 7.8) was added.
  • the mixture was stirred overnight at 4 ° C. with a rotator, and then the supernatant was separated by centrifugation. Using the supernatant, the amount of hemoglobin was measured according to the protocol of the kit.
  • HUVEC was cultured using endothelial cell basic medium-2 (serum-free) (EBM TM- 2) and endothelial cell medium kit-2 (2% FBS) (EGM TM- 2 BulletKit TM ) (Takara Bio Inc.) .
  • EBM TM- 2 endothelial cell basic medium-2
  • EBM TM- 2 BulletKit TM endothelial cell medium kit-2
  • An EDTA-containing 0.25% trypsin solution was used at the time of passage and cell detachment.
  • HUVEC grown to a sufficient amount in a T-75 flask was peeled from the bottom of the flask, and the supernatant was removed by centrifugation. Thereafter, the cells were washed with endothelial cell culture medium-2 containing the above endothelial cell culture medium kit-2, and the supernatant was again removed by centrifugation.
  • 0.1% BSA was added to endothelial cell culture medium-2 without
  • Fibrogen pig skin-derived gelatin
  • PSK cow bone-derived gelatin
  • a plate coated with G1917P was prepared, and R-Gel was dissolved in PBS (phosphate buffer) at a concentration of 1 mg / mL to prepare an R-Gel solution.
  • Fibronectin was dissolved at a concentration of 1 mg / mL to prepare a fibronectin solution
  • Fibrogen was dissolved at a concentration of 1 mg / mL in PBS (phosphate buffer) to prepare a fibrogen solution.
  • PSK was dissolved at a concentration of 1 mg / mL to prepare a PSK solution.
  • G1917P was dissolved at a concentration of 1 mg / mL in PBS (phosphate buffer solution) to prepare a G1917P solution. Dilute with PBS and use for plate addition.
  • ⁇ Non-treated 96-well plate (IWAKI) was used for the plate.
  • a solution obtained by diluting the above lysate with PBS was added to a non-treated 96-well plate at 50 ⁇ L / well so that the protein concentration was 0.02, 0.1, 0.2, 2.0 ⁇ g / well. Thereafter, incubation was performed at 37 ° C. for 2 hours, and after removing the solution, 100 ⁇ L of PBS was added to all wells and washed to remove PBS (washing step). The washing step was performed 3 times. This resulted in coating plates with different coating proteins and coating concentrations.
  • DNA assay was used.
  • 100 ⁇ L of SDS solution (20 mg of SDS dissolved in 100 mL of 1 ⁇ SSC solution: 1 ⁇ SSC solution is 17.999 g NaCl and 8.823 g Na 3 Citrate 2 L ultrapure 2) and leave at 37 ° C. for 1 hour.
  • Transfer the total amount of each individual solution to a 96-well black plate (Non-treated) and add 100 ⁇ L of Hoechst solution (20 ⁇ L of Hoechst 33258 and 20 mL of 1 ⁇ SSC solution) to all wells.
  • the fluorescence intensity was measured with a reader.
  • the plate reader used was Gemini EM (Molecular Devices), and the fluorescence intensity was measured at an excitation wavelength of 355 nm and a measurement wavelength of 460 nm.
  • a calibration curve was prepared from a suspension of HUVEC cells with the number of cells adjusted.
  • FIG. 2 and FIG. 3 The results of the obtained cell adhesion test (DNA assay) are shown in FIG. 2 and FIG. As a result, R-Gel showed better cell adhesion to HUVEC than fibronectin, fibrogen, PSK and G1917P. Moreover, the state of cell adhesion on the R-Gel coating plate, cell adhesion on the Fibrogen coating plate, cell adhesion on the PSK coating plate, and cell adhesion on the G1917P coating plate is shown in FIG. In the R-Gel coated plate, it can be visually confirmed that the number of adherent cells is large. At the same time, from this photograph, the area of one individually attached cell was determined with the software ImageJ. The result is shown in FIG. This indicates that R-Gel has a significantly larger cell area than Fibrogen, PSK, and G1917P, so there is a stronger bond between R-Gel and HUVEC than the others. I found out.
  • the coating concentration was 0.2 ⁇ g / well, and the experiment was conducted using an R-Gel coating plate and a fibronectin coating plate.
  • the prepared HUVEC cells were incubated with a sufficient concentration of anti-human ⁇ V monoclonal antibody (MAB1980: CHEMICON) at 37 ° C for 30 minutes, and the same amount of PBS was added and incubated at 37 ° C for 30 minutes, respectively. It indicated as antibody-treated HUVEC and untreated HUVEC.
  • Cell seeding was performed by adding a solution prepared so that the antibody-treated HUVEC or untreated HUVEC was 1 million cells / mL to the plate at 100 ⁇ L / well.
  • the cell adhesion time was 1 hour at 37 ° C. as in (2) above. Quantification of the number of cells was also performed by DNA assay in the same manner as in (2) above.

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Abstract

L'invention porte sur un inducteur de l'angiogenèse, sûr pour des organismes vivants, ayant d'excellentes propriétés bio-adhésives et pouvant réduire la quantité de bFGF à utiliser. L'inducteur d'angiogenèse comprend, en tant qu'ingrédients actifs, une gélatine génétiquement modifiée ayant une séquence d'acide aminé issue d'une séquence partielle d'acide aminé pour le collagène et un facteur de croissance des fibroblastes basiques.
PCT/JP2010/060101 2009-06-15 2010-06-15 Inducteur d'angiogenèse comprenant de la gélatine génétiquement modifiée et un facteur de croissance de fibroblastes basiques Ceased WO2010147109A1 (fr)

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Cited By (11)

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US20130071441A1 (en) * 2011-08-31 2013-03-21 Fujifilm Corporation Cell construct for cell transplantation and cell aggregate for cell transplantation
EP2578246A2 (fr) 2011-09-29 2013-04-10 Fujifilm Corporation Structure pour la migration de cellules endothéliales vasculaires
WO2013137268A1 (fr) 2012-03-12 2013-09-19 富士フイルム株式会社 Procédé de fabrication d'un matériau de réparation des tissus
WO2014141877A1 (fr) 2013-03-12 2014-09-18 富士フイルム株式会社 Matériau de réparation de tissu
US10682842B2 (en) 2015-09-11 2020-06-16 Fujifilm Corporation Method for producing gelatin structure, and gelatin structure production system
WO2022202938A1 (fr) 2021-03-26 2022-09-29 富士フイルム株式会社 Contenant de lyophilisation
WO2022202632A1 (fr) 2021-03-24 2022-09-29 富士フイルム株式会社 Dispositif de traitement thermique et procédé de fabrication d'un objet de traitement thermique
WO2022202633A1 (fr) 2021-03-26 2022-09-29 富士フイルム株式会社 Dispositif d'alimentation en poudre
WO2022210204A1 (fr) 2021-03-31 2022-10-06 富士フイルム株式会社 Procédé de broyage, procédé de fabrication de bloc polymère et dispositif de broyage
WO2023053710A1 (fr) 2021-09-30 2023-04-06 富士フイルム株式会社 Kit de matériau de restauration de tissu et procédé de restauration de tissu
WO2023188492A1 (fr) 2022-03-30 2023-10-05 富士フイルム株式会社 Matériau de restauration de tissu et procédé de production de matériau de restauration de tissu

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US6992172B1 (en) * 1999-11-12 2006-01-31 Fibrogen, Inc. Recombinant gelatins
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Cited By (14)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US9211266B2 (en) * 2011-08-31 2015-12-15 Fujifilm Corporation Cell construct for cell transplantation and cell aggregate for cell transplantation
US20130071441A1 (en) * 2011-08-31 2013-03-21 Fujifilm Corporation Cell construct for cell transplantation and cell aggregate for cell transplantation
US20150202344A1 (en) * 2011-08-31 2015-07-23 Fujifilm Corporation Cell construct for cell transplantation and cell aggregate for cell transplantation
EP2578246A2 (fr) 2011-09-29 2013-04-10 Fujifilm Corporation Structure pour la migration de cellules endothéliales vasculaires
EP2578246A3 (fr) * 2011-09-29 2015-01-14 Fujifilm Corporation Structure pour la migration de cellules endothéliales vasculaires
WO2013137268A1 (fr) 2012-03-12 2013-09-19 富士フイルム株式会社 Procédé de fabrication d'un matériau de réparation des tissus
WO2014141877A1 (fr) 2013-03-12 2014-09-18 富士フイルム株式会社 Matériau de réparation de tissu
US10682842B2 (en) 2015-09-11 2020-06-16 Fujifilm Corporation Method for producing gelatin structure, and gelatin structure production system
WO2022202632A1 (fr) 2021-03-24 2022-09-29 富士フイルム株式会社 Dispositif de traitement thermique et procédé de fabrication d'un objet de traitement thermique
WO2022202938A1 (fr) 2021-03-26 2022-09-29 富士フイルム株式会社 Contenant de lyophilisation
WO2022202633A1 (fr) 2021-03-26 2022-09-29 富士フイルム株式会社 Dispositif d'alimentation en poudre
WO2022210204A1 (fr) 2021-03-31 2022-10-06 富士フイルム株式会社 Procédé de broyage, procédé de fabrication de bloc polymère et dispositif de broyage
WO2023053710A1 (fr) 2021-09-30 2023-04-06 富士フイルム株式会社 Kit de matériau de restauration de tissu et procédé de restauration de tissu
WO2023188492A1 (fr) 2022-03-30 2023-10-05 富士フイルム株式会社 Matériau de restauration de tissu et procédé de production de matériau de restauration de tissu

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