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WO2010008023A1 - Promoteur d'élongation osseuse - Google Patents

Promoteur d'élongation osseuse Download PDF

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Publication number
WO2010008023A1
WO2010008023A1 PCT/JP2009/062816 JP2009062816W WO2010008023A1 WO 2010008023 A1 WO2010008023 A1 WO 2010008023A1 JP 2009062816 W JP2009062816 W JP 2009062816W WO 2010008023 A1 WO2010008023 A1 WO 2010008023A1
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Prior art keywords
dna
bone
protein
hgf
seq
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English (en)
Japanese (ja)
Inventor
勝郎 富田
弘行 土屋
秀憲 松原
邦夫 松本
敬吾 花田
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Kringle Pharma Inc
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Kringle Pharma Inc
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A61K48/005—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'active' part of the composition delivered, i.e. the nucleic acid delivered
    • 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
    • A61K38/1833—Hepatocyte growth factor; Scatter factor; Tumor cytotoxic factor II
    • 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

Definitions

  • the present invention relates to a bone elongation promoter. Specifically, the present invention relates to a drug that can achieve bone extension from at least one bone section to the opposite bone section in a gap formed between bone sections in a shorter period of time. More specifically, the present invention enables shortening of the treatment period after osteotomy, bone extension or fracture by promoting bone extension in the bone gap caused by osteotomy, bone extension or fracture. It is related to the drug.
  • Bone extension is a treatment method in which after extending a bone to be extended, a special instrument, for example, an external fixator, is attached and gradually extended to a target length over time.
  • Fracture is a disorder that can occur due to an accident or the like, and its healing often requires a relatively long period of time.
  • complex fractures, fractures, and the like have bone defects and may cause short limbs or deformation, for example.
  • bFGF basic fibroblast growth factor
  • BMP bone morphogenetic protein
  • HGF hepatocyte growth factor
  • Patent Document 3 HGF is a protein that was first identified as a powerful mitogen for mature hepatocytes, and its gene was cloned in 1989 (see Non-Patent Documents 1 and 2). ). Thereafter, it has been reported that various tissues have various effects such as angiogenesis, cell differentiation, proliferation, and anti-apoptosis.
  • HGF is exemplified as one of drugs that improve recovery of the sternum after sternotomy.
  • the sternotomy is a procedure in which the incision surface of the sternum is closed by suturing, and it is usually recognized that there is almost no gap between the cut surfaces of the sternum. That is, Patent Document 3 only describes the effect when the cut surface and the cut surface of the sternum are fused, and what is the effect when there is a gap between the cut surface and the cut surface? Is also not described.
  • the present invention relates to a bone elongation promoting agent, and more specifically, in a gap formed in a bone cutting part (hereinafter also referred to as “bone cutting gap” or “bone gap”), at least one bone cross section and the other facing it.
  • An object of the present invention is to provide a drug that promotes bone extension to a bone cross section (hereinafter referred to as “bone extension in a bone cutting gap (or bone gap)”).
  • the present invention provides a drug that is effectively used to promote bone extension in a bone cutting gap in a treatment process such as osteotomy, bone extension, or osteopathy after fracture.
  • Another object of the present invention is to shorten the treatment period of osteotomy or osteogenesis or the treatment period after fracture by using this drug.
  • HGF protein hepatocyte growth factor
  • Bone elongation promoter containing HGF protein as an active ingredient (I-1) Bone comprising at least one of the following (1-a) to (1-c) as an active ingredient Bone elongation promoter in the cutting gap: (1-a) HGF protein, (1-b) a peptide that is a partial peptide of HGF protein and has an effect of promoting bone elongation, (1-c) A salt of (1-a) or (1-b).
  • (I-2) The bone elongation promoter according to (I-1), wherein the HGF protein is the following (1-d) or (1-e): (1-d) a protein produced by a cell having a DNA comprising the base sequence represented by SEQ ID NO: 1 or 2, (1-e) a bone elongation-promoting action produced by cells having DNA that hybridizes under stringent conditions with a DNA comprising a base sequence complementary to the base sequence shown in SEQ ID NO: 1 or 2 Having protein.
  • (I-3) The bone elongation promoter described in (I-1), wherein the HGF protein is the following (1-f) or (1-g): (1-f) a protein comprising the amino acid sequence shown in SEQ ID NO: 3 or 4, (1-g) A protein having a bone elongation promoting action, having at least 85% identity with the amino acid sequence shown in SEQ ID NO: 3 or 4.
  • the protein described in (1-d) or (1-g) above is a protein consisting of the amino acid sequence represented by SEQ ID NO: 5 or 6, (I-2) or (I-3) The bone elongation promoter described in 1.
  • (I-6) The bone according to any one of (I-1) to (I-5), which has a dosage form for treating at least one of the opposing bone cross sections in a bone cutting gap exceeding 3 mm. Elongation promoter.
  • Bone elongation promoter containing DNA encoding HGF protein as an active ingredient (II-1) containing at least one of the following DNAs (2-a) to (2-c) as an active ingredient Bone elongation promoting agent in bone cutting gap characterized by: (2-a) DNA encoding HGF protein, (2-b) DNA encoding a peptide that is a partial peptide of HGF protein and has a bone elongation promoting action, (2-c) a protein or peptide that hybridizes with a DNA comprising a base sequence complementary to the DNA of (2-a) or (2-b) under stringent conditions and has a bone elongation promoting action. DNA to encode.
  • (II-2) The bone elongation promoter described in (II-1), wherein the DNA encoding the HGF protein is the following (2-d) or (2-e): (2-d) DNA comprising the base sequence shown in SEQ ID NO: 1 or 2, (2-e) DNA that hybridizes under stringent conditions with a DNA consisting of a base sequence complementary to the base sequence shown in SEQ ID NO: 1 or 2 and encodes a protein having a bone elongation promoting action.
  • (II-3) selected from the group consisting of (2-a) to (2-c) described in (II-1) and (2-d) and (2-e) described in (II-2)
  • a bone elongation promoter wherein the DNA is incorporated into a type I herpes simplex virus (HSV-1) vector, Sendai virus envelope (HVJ-E) vector, adenovirus vector or adeno-associated virus vector.
  • HSV-1 vector herpes simplex virus
  • HVJ-E Sendai virus envelope
  • adenovirus vector or adeno-associated virus vector.
  • (II-5) The bone according to any one of (II-1) to (II-4), which has a dosage form for treating at least one of the opposing bone cross sections in a bone cutting gap exceeding 3 mm. Elongation promoter.
  • HGF protein for production of bone elongation promoter (III-1) Protein described in any of (1-a) to (1-g) below for production of bone elongation promoter Or use of peptides: (1-a) HGF protein, (1-b) a peptide that is a partial peptide of HGF protein and has an effect of promoting bone elongation, (1-c) a salt of (1-a) or (1-b), (1-d) a protein produced by a cell having a DNA comprising the base sequence represented by SEQ ID NO: 1 or 2, (1-e) a bone elongation-promoting action produced by cells having DNA that hybridizes under stringent conditions with a DNA comprising a base sequence complementary to the base sequence shown in SEQ ID NO: 1 or 2 Protein, (1-f) a protein comprising the amino acid sequence shown in SEQ ID NO: 3 or 4, (1-g) A protein having a bone elongation promoting action, having at least 85% identity with the amino acid sequence shown in SEQ
  • (III-2) Use of the DNA described in any of (2-a) to (2-e) below for the production of a bone elongation promoter: (2-a) DNA encoding HGF protein, (2-b) DNA encoding a peptide that is a partial peptide of HGF protein and has a bone elongation promoting action, (2-c) a protein or peptide that hybridizes with a DNA comprising a base sequence complementary to the DNA of (2-a) or (2-b) under stringent conditions and has a bone elongation promoting action.
  • DNA to encode (2-d) DNA comprising the base sequence shown in SEQ ID NO: 1 or 2, (2-e) DNA that hybridizes under stringent conditions with a DNA consisting of a base sequence complementary to the base sequence shown in SEQ ID NO: 1 or 2 and encodes a protein having a bone elongation promoting action.
  • HGF protein or DNA for promoting bone elongation (IV) HGF protein or DNA for promoting bone elongation (IV-1)
  • (IV-2) Any of the following (2-a) to (2-e) to promote bone extension in the osteotomy or osteotomy patient, fracture patient, or osteotomy gap DNA described in: (2-a) DNA encoding HGF protein, (2-b) DNA encoding a peptide that is a partial peptide of HGF protein and has a bone elongation promoting action, (2-c) a protein or peptide that hybridizes with a DNA comprising a base sequence complementary to the DNA of (2-a) or (2-b) under stringent conditions and has a bone elongation promoting action.
  • DNA to encode (2-d) DNA comprising the base sequence shown in SEQ ID NO: 1 or 2, (2-e) DNA that hybridizes under stringent conditions with a DNA consisting of a base sequence complementary to the base sequence shown in SEQ ID NO: 1 or 2 and encodes a protein having a bone elongation promoting action.
  • (V) Bone elongation promotion method (V-1) A protein or peptide described in any of (1-a) to (1-g) below is treated with osteotomy or bone extension surgery patient, fracture patient or bone A method for promoting bone extension characterized by administering to an osteotomy patient at or near the bone cutting gap: (1-a) HGF protein, (1-b) a peptide that is a partial peptide of HGF protein and has an effect of promoting bone elongation, (1-c) a salt of (1-a) or (1-b), (1-d) a protein produced by a cell having a DNA comprising the base sequence represented by SEQ ID NO: 1 or 2, (1-e) a bone elongation-promoting action produced by cells having DNA that hybridizes under stringent conditions with a DNA comprising a base sequence complementary to the base sequence shown in SEQ ID NO: 1 or 2 Protein, (1-f) a protein comprising the amino acid sequence shown in SEQ ID NO: 3 or 4, (1-g) A protein having a bone
  • V-2 A protein or peptide described in any of the following (2-a) to (2-e) is used in a patient who has undergone osteotomy or bone extension, a fracture patient, or a patient who has undergone osteoarthroplasty.
  • a method for promoting bone elongation comprising administering to a bone cutting gap site or its periphery: (2-a) DNA encoding HGF protein, (2-b) DNA encoding a peptide that is a partial peptide of HGF protein and has a bone elongation promoting action, (2-c) a protein or peptide that hybridizes with a DNA comprising a base sequence complementary to the DNA of (2-a) or (2-b) under stringent conditions and has a bone elongation promoting action.
  • DNA to encode (2-d) DNA comprising the base sequence shown in SEQ ID NO: 1 or 2, (2-e) DNA that hybridizes under stringent conditions with a DNA consisting of a base sequence complementary to the base sequence shown in SEQ ID NO: 1 or 2 and encodes a protein having a bone elongation promoting action.
  • the bone elongation promoting agent of the present invention has an effect of promoting bone elongation in a gap between a bone cross section and an opposing bone cross section, that is, a bone gap (bone cutting gap) between the bone cross sections facing each other in a bone cutting portion.
  • the bone elongation promoting agent of the present invention has a superior bone elongation effect in, for example, bone lengthening in osteotomy or bone lengthening for limbs shortened due to short stature or accident etc., or bone lengthening after osteopathy of fracture To demonstrate.
  • the length of bone extension that can be performed by one osteotomy is about 3 mm. For this reason, in order to extend the bone by about 3 cm, it is usually necessary to repeat 10 osteotomy operations.
  • the bone elongation promoting agent of the present invention the bone can be stretched to a length exceeding 3 mm by one osteotomy, so the number of osteotomy procedures can be reduced. For example, when the bone is elongated by about 5 mm by one osteotomy using the bone elongation promoter of the present invention, the number of repetitions of osteotomy necessary to finally extend the bone by about 3 cm is six times, The number of treatments can be greatly reduced. This greatly reduces the burden on the patient and shortens the treatment period.
  • the bone elongation promoter of the present invention it is possible to promote bone elongation in bone distraction surgery and accelerate the formation of bone tissue and bone regeneration, thereby shortening the treatment period.
  • bone extension it takes about six months to one year from the start of bone extension to the end of treatment, and an external fixator must be worn during that period.
  • an external fixator there is a possibility of re-fracture in the meantime, and the burden on the patient is great.
  • the treatment period can be shortened by the bone elongation promoter of the present invention, the burden on the patient will be greatly reduced.
  • the shortening of the treatment period can also reduce the risk of complications such as bacterial infection and neurovascular disorders in the external fixator screw or the steel wire insertion portion to be worn, and thus the risk can be reduced.
  • the bone elongation promoting agent of the present invention in the treatment of fracture, even when the bone is shortened or lost due to, for example, a complicated fracture or a fractured fracture, a gap is created between the bones. Since osteopathy can be performed, it is possible to avoid that the length of the limb is shortened or deformed after the operation. Further, since the bone extension in the bone gap can be promoted, the treatment period can be shortened.
  • the shortening of the treatment period not only reduces the physical burden on the patient, but also increases the cost of treatment for the patient and their family members.
  • the burden can be reduced and the national medical cost burden can be reduced.
  • FIG. A is a view showing a mounting mode of the one-side external fixator in Experimental Example 1.
  • FIG. B is a view showing a one-side external fixator attached to a rabbit foot.
  • FIG. C is a view showing an X-ray image of a rabbit foot to which a one-side external fixator is attached.
  • Fig. A shows a roentgenogram of the tibial cut from the control group immediately after the tibial cut (Op.) To 10 weeks after the operation (1w, 2w, 3w, 4w, 6w, 8w, 10w) in Experimental Example 1 It is a figure which shows an upper stage: a front image, a lower stage: a side image.
  • B shows X-ray images of the tibial section from the immediately after tibial amputation (Op.) To the fifth week after surgery (1w, 2w, 3w, 4w, 5w) in the HGF administration group (upper: It is a figure which shows a front image, a lower stage: a side image.
  • gene refers to a regulatory region, a coding region, an exon, and an intron without distinction unless otherwise specified.
  • gene in the present specification means not only DNA but also mRNA which is a transcript thereof.
  • DNA has double-stranded DNA including human genomic DNA, single-stranded DNA including cDNA and synthetic DNA (sense strand), and a sequence complementary to the sense strand, unless otherwise specified. Both single-stranded DNA (antisense strand) and fragments thereof are included.
  • bone extension or “bone extension in the bone cutting gap” refers to a bone gap formed by bone cutting (bone cutting gap) from one bone cross-section to the other bone cross-section facing this. Means stretching the bone.
  • the bone elongation occurs at least from one bone cross-section toward the other bone cross-section, but preferably both bone cross-sections, that is, both bone cross-sections face each other. This occurs toward the other bone cross section.
  • bone extension includes that the bone tissue is extended in a smooth state with the same thickness or thickness as the bones on both sides in the cut portion of the bone, and the bone is regenerated. Bone extension also includes callus formation in the bone gap.
  • the “bone cross section” refers to a surface (bone cut surface) from which bone has been cut.
  • bone cutting is not limited to crossing and longitudinal cutting, but includes all the states in which bones are cut off.
  • the cause of bone cutting is not particularly limited, and includes artificial bone cutting by surgery (for example, osteotomy and bone extension), or bone cutting caused by an accident such as a fracture.
  • the “bone cutting gap (or bone gap)” refers to a gap (gap, defect, gap) at the bone cutting portion.
  • the width of the gap is not limited, but is preferably a gap exceeding 3 mm. More preferably, the gap is more than 3 mm and within 10 mm.
  • the cause of the bone cutting gap is not particularly limited, and includes bone cutting gap after osteotomy, bone cutting gap in bone extension, bone cutting gap in fracture, bone cutting gap after osteopathy in fracture, etc. included.
  • the “osteotomy” means that after cutting a bone of a hand or a foot, the cut surface of the bone is fixed with a predetermined gap, and the bone is regenerated to extend the bone and join the cut surfaces.
  • the technique to do By repeating this treatment multiple times, the bone can be extended to the required length. For example, when the length of the left and right limbs is different due to short stature or trauma etc., there is a technique in which the bones of the limbs are cut and moved to a predetermined length and stretched.
  • the cut bone is extended from its cut surface by regeneration and is fixed by intramedullary nail fixation or the like until the bone gap is sufficiently or completely eliminated. That is, in the bone cutting gap formed by the above procedure, the bone is regenerated and extended from the cut surface of the bone, the cut surfaces of the extended bone are fused, the bone gap is filled, and the bone is reconstructed.
  • bone extension means that after cutting the bone, the bones on both sides of the cut are fixed with a movable fixing tool, and the bone cutting gap is gradually widened by pulling them apart about 0.5 to 1 mm every day. This is a procedure to extend the bone by repeating this until the required length.
  • Ilizarov method etc. can be illustrated as a treatment method.
  • the “bone extension promoting action” refers to an action of promoting bone extension in the osteotomy gap occurring in the above osteotomy or osteogenesis, or a fracture or osteopath. Evaluation of whether or not a test substance has an effect of promoting bone elongation is performed, for example, when a test substance is administered (test group) and when a test substance is not administered (control group) according to the method described in Experimental Example 1. This can be done by comparing the speed of bone extension in the bone cutting gap. When the test group has a faster bone elongation than the control group, it can be determined that the test substance has a bone elongation promoting effect.
  • HGF protein is a protein identified as a powerful mitogen for mature hepatocytes, as described above, and is called a hepatocyte growth factor. Yes (see Non-Patent Documents 1 and 2). In addition to HGF, SF (scatter factor), TCF (Tumor cytotoxic factor), etc. are used.
  • the HGF protein can be obtained by, for example, culturing primary cultured cells or established cells that produce HGF protein, separating and purifying from the culture supernatant and the like.
  • the gene encoding the HGF protein is incorporated into an appropriate vector by genetic engineering techniques, and this is inserted into an appropriate host cell for transformation, and the desired recombinant HGF protein is obtained from the culture supernatant of the transformant. It can also be obtained by separating. (See, for example, JP-A-5-111382, Biochem. Biophys. Res. Comm., 1989, 163, p. 967).
  • the host cell is not particularly limited, and various host cells conventionally used in genetic engineering techniques such as Escherichia coli, yeast or animal cells can be used.
  • the HGF protein is preferably a protein produced from a gene encoding human-derived HGF (hHGF).
  • hHGF human-derived HGF
  • a DNA having a base sequence represented by SEQ ID NO: 1 or 2 is preferable.
  • HGF protein specifically, the HGF protein of SEQ ID NO: 3 or 5 produced by a cell into which DNA comprising the nucleotide sequence shown in SEQ ID NO: 1 has been introduced by recombinant DNA technology, is also represented by SEQ ID NO: 2. Mention may be made of the HGF protein of SEQ ID NO: 4 or 6 produced by a cell into which DNA having the nucleotide sequence shown is introduced.
  • the HGF proteins shown in SEQ ID NOs: 3 to 6 are all human-derived natural HGF proteins and have mitogenic activity and motogenic activity as HGF.
  • Such HGF protein is registered in the NCBI database (NCBI-GenBank Flat File Release 164.0) or the like as, for example, Accession No.P14210 (SEQ ID NO: 3) or Accession No.NP 001010932 (SEQ ID NO: 4).
  • the HGF protein having the amino acid sequence represented by SEQ ID NO: 4 is a 5-amino acid deficient HGF from which 5 amino acid residues located at positions 161 to 165 of the amino acid sequence represented by SEQ ID NO: 3 have been deleted. It is a protein.
  • the natural HGF protein is a glycoprotein.
  • the HGF protein represented by Accession No.NP 001010932 (SEQ ID NO: 4) is Asn at position 289, Asn at position 397, Thr at position 471, Thr at position 561.
  • a sugar chain is added to Asn and Asn at position 648.
  • amino acid sequences shown in SEQ ID NOs: 5 and 6 are amino acid sequences of mature proteins obtained by cleaving the 31st amino acid region (signal sequence) from the N-terminus in SEQ ID NOs: 3 and 4.
  • one or a plurality for example, 2 to 35, preferably 2 to 20
  • Such HGF protein can be produced by well-known technical means such as genetic engineering techniques and site-directed mutagenesis.
  • the amino acid to be inserted, the amino acid to be substituted, and the amino acid to be added may be non-natural amino acids other than 20 kinds of natural amino acids.
  • the unnatural amino acid may be any compound as long as it has an amino group and a carboxyl group, and examples thereof include ⁇ -aminobutyric acid.
  • the amino acid sequence shown in SEQ ID NO: 4 is a 5-amino acid deficient HGF protein in which five amino acid residues are deleted from the amino acid sequence shown in SEQ ID NO: 3, as described above.
  • the HGF protein targeted in the present invention may have at least 85% identity with the amino acid sequence shown in SEQ ID NO: 3 or 4 as long as it has a bone elongation promoting action.
  • Preferred is a protein having 90% or more, more preferably 95% or more identity with the amino acid sequence shown in SEQ ID NO: 3 or 4.
  • the amino acid sequence shown in SEQ ID NO: 5 has 96% and 95.6% identity with the amino acid sequence shown in SEQ ID NO: 3, respectively, and the amino acid sequence shown in SEQ ID NO: 6 is shown in SEQ ID NO: 4. It has 96% and 95.7% identity with the amino acid sequence shown, respectively.
  • identity means the degree of coincidence of amino acid residues constituting each sequence among the sequences by comparing the primary structures (amino acid sequences) of the proteins.
  • HGF protein comprising an amino acid sequence having high identity with the amino acid sequence shown in SEQ ID NO: 3 or 4
  • other humans such as Accession No. BAA14348 or Accession No. AAC71655 registered in the NCBI database. Derived HGF can be mentioned.
  • each of the amino acid sequences shown in SEQ ID NOs: 3 and 4 is replaced with a signal sequence consisting of the 1st to 31st amino acid regions, It may have a protein signal sequence.
  • signal sequences include signal sequences of human serum albumin, interferon, human amylase and the like.
  • the HGF protein targeted in the present invention is a DNA that hybridizes under stringent conditions with a DNA comprising a base sequence complementary to the base sequence shown in SEQ ID NO: 1 or 2 as long as it has a bone elongation promoting action. It may be a protein produced from a cell having
  • stringent conditions include hybridization at about 65 ° C. in the presence of about 0.7 to 1 M sodium chloride, and then about 0.1 to 2 times the concentration of the SSC solution (the composition of the 1 time concentration of the SSC solution). Can be mentioned that is washed at about 65 ° C. using 150 mM sodium chloride and 15 mM sodium citrate).
  • a gene encoding an HGF protein specifically, a DNA comprising the base sequence shown in SEQ ID NO: 1 or 2, or a DNA that hybridizes with a DNA comprising a base sequence complementary to the DNA under stringent conditions
  • a method for producing the HGF protein of the present invention using cells for example, primary cultured cells or established cells having these DNAs are cultured, and the target HGF protein is isolated and purified from the obtained culture supernatant.
  • a method can be mentioned.
  • the above DNA is incorporated into an appropriate vector by genetic engineering techniques, transformed by inserting it into an appropriate host cell, and the desired HGF protein (recombinant protein) is obtained from the culture supernatant of this transformant. For example, see JP-A-5-111382, JP-A-11-1499, Biochem.chemBiophys. Res. Commun. 1989, Vol. 163, p.967. ).
  • the host cell is not particularly limited, and various host cells conventionally used in genetic engineering techniques such as E. coli, yeast or animal cells can be used. Since the natural HGF protein is a glycoprotein, animal cells are preferably used as host cells when producing glycoproteins in the same manner. Examples of animal cells include CHO cells, COS cells, mouse L cells, mouse C127 cells, mouse FM3A cells, and the like. Transfer of expression vectors into animal cells is performed by transfection, microinjection or the like, among which the calcium phosphate method is the most common. Culture of animal cells transformed by transfer can be carried out by suspension culture or adherent culture by a conventional method. As the medium, MEM, RPMI 1640, etc. are common.
  • the presence or absence of glycosylation and the number of glycosylation are not particularly limited. That is, the number of naturally occurring numbers (one to plural) may be HGF proteins that have been deleted, substituted, inserted or added.
  • HGF proteins in which sugar chains are deleted, substituted, inserted or added include those in which sugar chains added to natural HGF proteins are treated with enzymes or the like to delete sugar chains, and sugar chains are not added.
  • HGF protein specifically, for example, Accession No. NP_001010932 human HGF registered in the NCBI database, the 289-position Asn of the glycosylation site is Gln, and the 397-position Asn is Gln.
  • the C-terminus is any of a carboxyl group (—COOH), a carboxylate [—COOM (M represents a metal)], an amide (—CONH 2 ), or an ester (—COOR). May be.
  • R in the ester is, for example, a C 1-6 alkyl group such as methyl, ethyl, n-propyl, isopropyl or n-butyl; for example, a C 3-8 cycloalkyl group such as cyclopentyl, cyclohexyl; C 6-12 aryl groups such as ⁇ -naphthyl; C 7- such as phenyl-C 1-2 alkyl groups such as benzyl and phenethyl or ⁇ -naphthyl-C 1-2 alkyl groups such as ⁇ -naphthylmethyl; A 14 aralkyl group and a C 2-6 alkanoylmethyl group such as acetyloxymethyl, pivaloyloxymethyl and the like are used.
  • a C 1-6 alkyl group such as methyl, ethyl, n-propyl, isopropyl or n-butyl
  • the HGF protein targeted by the present invention includes a carboxyl group or carboxylate other than the C-terminus, and the carboxyl group or carboxylate further amidated or esterified.
  • the ester in this case include the aforementioned C-terminal ester.
  • the amino group of the N-terminal methionine residue is a protective group (for example, a C 1-6 such as a C 2-6 alkanoyl group such as formyl group, acetyl, etc.).
  • An amino group, an imidazolyl group, an indolyl group, a guanidino group, etc.) protected with an appropriate protecting group for example, a C 1-6 acyl group such as a C 2-6 alkanoyl group such as formyl group or acetyl.
  • a complex protein such as a so-called glycoprotein to which a sugar chain is bound.
  • HGF protein when it applies to a human, the above-mentioned thing derived from a human is used suitably, but mammals other than a human (for example, a monkey, a cow, a horse, a pig, a sheep, a dog, a cat, a rat, a mouse, Rabbit, hamster, guinea pig, chimpanzee, etc.) may be used.
  • HGF proteins include mouse-derived HGF proteins (eg, Accession No. AAB31855, NP_034557, BAA01065, BAA01064, etc.) registered in the NCBI database, etc., rat-derived HGF proteins (eg, Accession No.
  • NP_058713 Bovine-derived HGF protein (eg, Accession No.NP_001026921, BAD02475, etc.), cat-derived HGF protein (eg, Accession No.NP_001009830, BAC10545, BAB21499, etc.), dog-derived HGF protein (eg, Accession No.NP_001002964, BAC57560, etc.) or chimpanzee-derived HGF Examples thereof include, but are not limited to, proteins (for example, Accession No. XP 519174).
  • HGF proteins need only be purified to the extent that they can be used as pharmaceuticals when used as the active ingredient of the bone elongation promoter of the present invention, and so long as they are prepared by various methods. be able to.
  • the purification method is not limited, and examples thereof include column chromatography using heparin / sepharose or hydroxyapatite.
  • HGF protein can also be used in the form of a physiologically acceptable salt with acid or base.
  • physiologically acceptable acid addition salts include salts with inorganic acids (eg, hydrochloric acid, phosphoric acid, hydrobromic acid, sulfuric acid, etc.), or organic acids (eg, acetic acid, formic acid, propionic acid, fumaric acid, maleic acid, And salts with succinic acid, tartaric acid, citric acid, malic acid, succinic acid, benzoic acid, methanesulfonic acid, benzenesulfonic acid, and the like.
  • inorganic acids eg, hydrochloric acid, phosphoric acid, hydrobromic acid, sulfuric acid, etc.
  • organic acids eg, acetic acid, formic acid, propionic acid, fumaric acid, maleic acid
  • succinic acid tartaric acid, citric acid, malic acid, succinic acid, benzoic acid, methanesulfonic acid, benzen
  • the HGF protein may be used alone or as a mixed protein with various proteins as long as the bone elongation promoting action is not impaired.
  • HGF partial peptide The partial peptide of HGF protein targeted by the present invention (hereinafter also referred to as "HGF partial peptide”) is the above-mentioned partial peptide of HGF protein, and at least has a bone elongation promoting action. What is necessary is just to have.
  • the number of amino acids constituting the HGF partial peptide is at least about 20 or more, preferably about 50 or more, more preferably about 100 or more of the amino acids constituting the HGF protein.
  • a peptide having a sequence is preferred.
  • such an HGF partial peptide includes an amino acid sequence from the 32nd amino acid to the 210th amino acid of the amino acid sequence represented by SEQ ID NO: 3 (from the N-terminal hairpin loop of HGF to the first kringle domain).
  • the HGF partial peptide of the present invention has a peptide having at least about 80% identity, preferably a peptide having about 90% identity, more preferably about 95%, with the amino acid sequence of the HGF partial peptide.
  • Peptides having the above identity and having at least a bone elongation promoting action are also included.
  • the C-terminus is carboxyl group (—COOH), carboxylate [—COOM (M is as defined above)], amide (—CONH 2 ) or ester (—COOR; R is as defined above. Any of the above may be used.
  • the amino group of the methionine residue at the N-terminal is protected with a protecting group, and Gln produced by cleavage of the N-terminal side in vivo is pyroglutamine oxidized, as in the HGF protein.
  • a substituent on the side chain of an amino acid in the molecule is protected with an appropriate protecting group, or a complex peptide such as a so-called glycopeptide to which a sugar chain is bound.
  • the HGF partial peptide can be produced according to a known peptide synthesis method or by cleaving the HGF protein with an appropriate peptidase.
  • a peptide synthesis method for example, either a solid phase synthesis method or a liquid phase synthesis method may be used. That is, a partial peptide or amino acid that may have a protecting group capable of constituting an HGF protein is condensed with a remaining part that may have a protecting group, and when the product has a protecting group, The desired HGF peptide can be produced by removing the group.
  • Known condensation methods and elimination of protecting groups include, for example, M.
  • the HGF partial peptide can be purified and isolated by a combination of usual purification methods such as solvent extraction, distillation, column chromatography, liquid chromatography, crystallization or recrystallization.
  • the partial peptide obtained by the above method is a free form, it can be converted into an appropriate salt by a known method. Conversely, when it is obtained as a salt, it can be converted into a free form by a known method. Can do.
  • the salt of the HGF partial peptide include physiologically acceptable salts with an acid or a base, like the HGF protein.
  • DNA encoding HGF protein refers to DNA capable of expressing the aforementioned HGF protein.
  • DNA encoding the HGF protein include Nature, 342, 440 (1989); Japanese Patent No. 2777678; Japanese Patent Laid-Open No. 11-1499; Biochem. Biophys, Res. Commun., 1989, 163, p.967-973; Proc. Natl. Acad. Sci. USA, 1991, Vol. 88 (No. 16), p.7001-7005, etc., for example, Accession No. M60718 in GeneBank / EMBL / DDBJ, A preferred example is DNA encoding a human-derived HGF protein registered as M73240, AC004960, AY246560, M29145, M73240 or the like.
  • the DNA encoding the HGF protein used in the present invention is preferably the above-mentioned human-derived DNA when applied to humans, but mammals other than humans (for example, monkeys, cows, horses, pigs, sheep) DNA encoding HGF protein derived from dogs, cats, rats, mice, rabbits, hamsters, guinea pigs, chimpanzees, etc.).
  • DNA examples include, for example, DNA encoding mouse-derived HGF protein (for example, Accession No. S71816, NH_010427, D10213, D10212, etc.) registered in the NCBI database, etc., DNA encoding rat-derived HGF protein (Eg, Accession No.NM_017017), DNA encoding bovine-derived HGF protein (eg, Accession No.NM_001031751, AB110822, etc.), DNA encoding feline-derived HGF protein (eg, Accession No.NM_001009830, AB080187, AB04046610, etc.), dogs Examples include, but are not limited to, DNAs encoding HGF proteins derived from DNA (for example, Accession® No. NM_001002964, AB090353, etc.) or DNAs encoding HGF proteins derived from chimpanzees (for example, Accession® No. XM_519174, etc.).
  • DNA encoding the HGF protein for example, DNA consisting of the base sequence represented by SEQ ID NO: 1 or 2 is preferably mentioned.
  • the nucleotide sequence represented by SEQ ID NO: 1 corresponds to the nucleotide sequence located at positions 73 to 2259 of the nucleotide sequence of Accession No. M60718, and the DNA comprising the nucleotide sequence is the amino acid sequence represented by SEQ ID NO: 3.
  • the HGF protein (SEQ ID NO: 3) expressed and produced in the cell is cleaved from the signal sequence when secreted outside the cell, and matured from the amino acid sequence shown in SEQ ID NO: 5. It becomes HGF protein. Therefore, the DNA consisting of the base sequence shown in SEQ ID NO: 1 also corresponds to the DNA encoding (producing) the HGF protein consisting of the amino acid sequence shown in SEQ ID NO: 5.
  • the base sequence represented by SEQ ID NO: 2 corresponds to the base sequence located at Nos. 66 to 2237 of the base sequence of Accession No.
  • the DNA corresponding to the base sequence is the amino acid sequence represented by SEQ ID NO: 4. It corresponds to DNA encoding the HGF protein consisting of Such a HGF protein (SEQ ID NO: 4) is also a mature HGF protein consisting of the amino acid sequence shown in SEQ ID NO: 6 by cleaving the signal sequence when secreted outside the cell in the DNA recombination technique. Therefore, the DNA consisting of the base sequence shown in SEQ ID NO: 2 corresponds to the DNA encoding (producing) the HGF protein consisting of the amino acid sequence shown in SEQ ID NO: 6.
  • the DNA encoding the HGF protein targeted by the present invention is not limited to those described above, and hybridizes with DNA comprising a base sequence complementary to the DNA under stringent conditions and has a bone elongation promoting action. Also included are DNA encoding proteins. Specifically, the above DNA hybridizes under stringent conditions with a DNA comprising a base sequence complementary to the base sequence represented by SEQ ID NO: 1 or 2.
  • stringent conditions include hybridization at about 65 ° C. in the presence of about 0.7 to 1 M sodium chloride, and then about 0.1 to 2 times the concentration of the SSC solution (the composition of the 1 time concentration of the SSC solution). Can be mentioned that is washed at about 65 ° C. using 150 mM sodium chloride and 15 mM sodium citrate).
  • Such DNA is not limited, but is about 85% or more, preferably about 90% or more in the DNA encoding the HGF protein, preferably the DNA consisting of the base sequence shown in base number 1 or 2 and the base sequence. More preferably, DNA encoding a protein having a base sequence having a homology of about 95% or more and having at least a bone elongation promoting action is exemplified.
  • the DNA encoding the HGF protein can be easily obtained by using, for example, a normal hybridization method or PCR method using a cDNA library having the DNA. Specifically, the DNA can be obtained, for example, by molecular cloning (Molecular Cloning, A Laboratory Manual, Third Edition, J. Sambrook et al., Cold Spring Harbor Lab. Press, 2001: It can be done with reference to basic documents such as “.
  • examples of the cDNA library having DNA encoding the HGF protein include human-derived liver cDNA library, spleen cDNA library, placenta cDNA library and the like. These libraries can be obtained commercially from Clontech.
  • a cDNA library prepared from a cell line expressing HGF protein and tissue material according to a conventional method can also be used. A ⁇ phage incorporating such a cDNA is cultured and infected with E. coli according to the method described in “Molecular Cloning 3rd Edition”, and the formed plaque is deduced from the partial amino acid sequence of the HGF protein.
  • DNA encoding the target HGF protein can be obtained by performing a plaque hybridization method using an oligonucleotide prepared from the base sequence as a probe, or by performing a PCR method.
  • the RNA encoding the HGF protein can also be used in the present invention as long as it can express the aforementioned HGF protein by reverse transcriptase.
  • the RNA include RNA obtained by preparing an mRNA fraction from cells or tissues and amplified by RT-PCR. The RNA can also be obtained by known means.
  • the DNA encoding the HGF protein is in the form of a recombinant expression vector in which the DNA is incorporated, and the bone gap site or its surrounding tissue.
  • expression vectors include naked plasmids, detoxified retroviruses, adenoviruses, adeno-associated viruses, herpes viruses (type I herpes simplex virus, etc.), vaccinia viruses, poxviruses, polioviruses, symbis viruses, Sendai viruses, SV40.
  • DNA viruses or RNA viruses, such as immunodeficiency virus (HIV) are mentioned.
  • type I herpes simplex virus (HSV-1) vector, Sendai virus envelope (HVJ-E) vector, adenovirus vector, adeno-associated virus (AAV) vector and the like are preferable.
  • DNA encoding a partial peptide of HGF protein As long as the DNA encoding the partial peptide (HGF partial peptide) of the HGF protein targeted by the present invention has a base sequence encoding the above-mentioned HGF partial peptide and encodes a peptide having an elongation promoting action, It can be anything. Specifically, as such DNA, for example, DNA having a partial base sequence of DNA having the base sequence represented by SEQ ID NO: 1 or 2, and encoding a peptide having a bone elongation promoting action Etc.
  • such DNA includes, for example, the 94th to 630th base sequences of the human HGF base sequence represented by SEQ ID NO: 1 (from the N-terminal hairpin loop of HGF to the first kringle. DNA having a peptide encoding a peptide up to the domain) and the 94th to 864th base sequences of the human HGF base sequence represented by SEQ ID NO: 1 (from the N-terminal hairpin loop of HGF to the second kringle domain) Preferred examples include DNA having a DNA encoding the above peptide).
  • the DNA encoding the HGF partial peptide targeted by the present invention includes a DNA comprising a base sequence complementary to the above-mentioned partial peptide of the HGF protein and encoding a peptide having a bone elongation promoting action, and a string. DNA that hybridizes under a gentle condition and that encodes a peptide having a bone elongation promoting action is included.
  • Such a DNA has a base sequence having a homology of about 85% or more, preferably about 90% or more, more preferably about 95% or more with a DNA encoding an HGF partial peptide, and has a bone elongation promoting action.
  • Examples include DNA encoding a peptide.
  • DNA specifically, for example, it hybridizes under stringent conditions with DNA having a base sequence complementary to DNA having a partial base sequence of DNA consisting of the base sequence represented by SEQ ID NO: 1 or 2. And DNA encoding a peptide having a bone elongation promoting action. More specifically, such DNA includes about 80% or more, preferably about 90% or more, more preferably about 90% or more of DNA encoding a partial base sequence of DNA having the base sequence represented by SEQ ID NO: 1 or 2. Examples thereof include DNA having a base sequence having a homology of 95% or more and having a DNA encoding a peptide having a bone elongation promoting action.
  • the DNA can be easily obtained by, for example, a normal hybridization method or PCR method, and the DNA is specifically obtained with reference to the basic document such as the Molecular Cloning 3rd Edition. Can do.
  • an RNA encoding a peptide that is a partial peptide of the HGF protein and has a bone elongation promoting action can be used in the present invention as long as it can express the HGF protein by reverse transcriptase.
  • the RNA include RNA obtained by preparing an mRNA fraction from a cell or tissue and amplified by RT-PCR, and are within the scope of the present invention.
  • the RNA can also be obtained by known means.
  • Bone elongation promoter of the present invention comprises, as shown below, (a) a bone elongation promoter comprising an HGF protein / partial peptide as an active ingredient, and (b) HGF, depending on the type of active ingredient. It can be classified as a bone elongation promoter containing a gene as an active ingredient.
  • Bone elongation promoter comprising HGF protein / partial peptide as active ingredient HGF protein described in (2) above, partial peptide of HGF protein (HGF partial peptide) described in (3), or at least one of these A bone elongation promoter containing salt as an active ingredient.
  • Bone elongation promoter containing HGF gene as active ingredient DNA containing HGF protein or HGF partial peptide described in (4) above (hereinafter collectively referred to as “HGF gene”) is contained as an active ingredient Bone elongation promoter.
  • the dosage form, administration method, dosage and the like of the bone elongation promoter of the present invention can be appropriately designed and changed according to the type of the active ingredient.
  • Bone elongation promoting agent comprising HGF protein / partial peptide as active ingredient
  • the bone elongation promoting agent of (a) can take various preparation forms such as liquids and solids, but generally HGF protein
  • the HGF partial peptide or a salt thereof is preferably formulated into a form of an injection, a propellant, a sustained-release preparation (for example, a depot) and the like together with a conventional carrier.
  • the injection or propellant may be either an aqueous preparation or an oily preparation.
  • an aqueous solvent water for injection, purified water, etc.
  • a pharmaceutically acceptable additive such as an isotonic agent (sodium chloride, potassium chloride, glycerin, mannitol, Sorbitol, boric acid, borax, glucose, propylene glycol, etc.), buffer (phosphate buffer, acetate buffer, borate buffer, carbonate buffer, citrate buffer, Tris buffer, glutamate buffer, epsilon) Aminocaproic acid buffer, etc.), preservative (methyl paraoxybenzoate, ethyl paraoxybenzoate, propyl paraoxybenzoate, butyl paraoxybenzoate, chlorobutanol, benzyl alcohol, benzalkonium chloride, sodium dehydroacetate, sodium edetate, boro Acid, borax, etc.), thickener (hydroxyethylcellulose) , Hydroxypropy
  • an isotonic agent sodium chloride, potassium chloride, glycerin,
  • a suitable solubilizing agent such as alcohol (ethanol etc.), polyalcohol (propylene glycol, polyethylene glycol etc.) or nonionic surfactant (polysorbate 80, polyoxyethylene hydrogenated castor oil 50 etc.) may be further blended.
  • alcohol ethanol etc.
  • polyalcohol propylene glycol, polyethylene glycol etc.
  • nonionic surfactant polysorbate 80, polyoxyethylene hydrogenated castor oil 50 etc.
  • polysorbate 80 polyoxyethylene hydrogenated castor oil 50 etc.
  • the HGF protein content in the injection is not limited, but is usually about 0.0002 to 0.5 w / v%, preferably about 0.001 to 0.2 w / v% with respect to 100 w / v% of the whole injection solution. Can be adjusted. It should be noted that liquid preparations such as injections are preferably stored after removing moisture by freeze storage or freeze drying. The freeze-dried preparation is used by adding distilled water for injection at the time of use and re-dissolving it.
  • Sprays can also be prepared using conventional means on formulations.
  • the additive blended in the spray may be any additive generally used in inhalation preparations, for example,
  • the above-mentioned solvent, preservative, stabilizer, tonicity agent, pH adjuster and the like can be blended.
  • the propellant include a liquefied gas propellant or a compressed gas.
  • the liquefied gas propellant include fluorinated hydrocarbons (alternative chlorofluorocarbons such as HCFC22, HCFC-123, HCFC-134a, and HCFC142), liquefied petroleum, dimethyl ether, and the like.
  • the compressed gas examples include soluble gas (carbon dioxide gas, nitrous oxide gas, etc.) or insoluble gas (nitrogen gas, etc.).
  • the content of the HGF protein or HGF partial peptide in the propellant can be usually adjusted to about 0.0002 to 5 w / v%, preferably about 0.001 to 2 w / v% based on the entire propellant.
  • the HGF protein or HGF partial peptide can be used as a sustained-release preparation (for example, a depot) together with a biodegradable polymer.
  • a sustained-release preparation for example, a depot
  • effects such as reduction in the number of administrations, sustained action and reduction in side effects can be expected.
  • the sustained-release preparation can be produced according to a known method.
  • the biodegradable polymer used in the sustained-release preparation can be appropriately selected from known biodegradable polymers.
  • biodegradable polymers for example, polysaccharides such as starch, dextran or chitosan; proteins such as collagen or gelatin
  • Polyamino acids such as polyglutamic acid, polylysine, polyleucine, polyalanine or polymethionine
  • polycaprolactone poly- ⁇ -hydroxybutyric acid, polymalic acid, polyanhydride
  • polyester such as fumaric acid / polyethylene glycol / vinyl pyrrolidone copolymer
  • polyorthoester or polyalkylcyanoacrylic acid such as polymethyl- ⁇ -cyanoacrylic acid
  • polycarbonate such as polyethylene carbonate or polypropylene carbonate It is done.
  • polyester polylactic acid or lactic acid-glycolic acid copolymer is preferable, and polylactic acid or lactic acid-glycolic acid copolymer is more preferable.
  • the composition ratio (lactic acid / glycolic acid) (mol%) varies depending on the sustained release period.
  • the sustained release period is about 2 to 3 months, preferably about 2 weeks. In the case of 1 month, about 100/0 to 50/50 is preferable.
  • the weight average molecular weight of the polylactic acid or lactic acid-glycolic acid copolymer is generally preferably about 5,000 to 20,000.
  • Polylactic acid or lactic acid-glycolic acid copolymer can be produced according to a known production method, for example, a production method described in JP-A No. 61-28521.
  • the mixing ratio of the biodegradable polymer and the HGF protein is not particularly limited.
  • the HGF protein is usually about 0.001 to 50 w / v%, about 0.01 to 30 w / v with respect to the biodegradable polymer. % Is preferred.
  • an injection or a spray is locally applied (direct injection or spraying) to the bone gap site or its peripheral site, or a sustained-release preparation (depot) is locally applied to the bone gap site or its peripheral site. It is preferable to apply (embed).
  • the dose is appropriately selected according to the dosage form, the degree of disease, age, etc.
  • the content of the HGF protein or HGF partial peptide contained in the bone elongation promoter of the present invention is usually 0. .1 ⁇ g to 500 mg, preferably 1 ⁇ g to 50 mg, more preferably 10 ⁇ g to 25 mg.
  • the number of administrations is appropriately selected depending on the dosage form, the degree of disease, age, etc., and can be administered once or continuously at a certain interval. In the case of continuous administration, the administration interval may be from once a day to once every several months. For example, in the case of administration using a sustained-release preparation (depot) or continuous administration using a sustained-release pump, once every several months. But you can.
  • HGF gene which is a bone elongation promoter containing the HGF gene as an active ingredient
  • conventional methods such as separate experimental medicine, basic techniques of gene therapy, Yodosha, 1996, separate experimental medicine, gene It is preferably carried out according to the method described in the introduction & expression analysis experiment method, Yodosha, 1997, gene therapy development research handbook edited by the Japanese Society for Gene Therapy, NTS, 1999 and the like.
  • Specific administration methods include, for example, a method in which a recombinant expression vector in which an HGF gene is incorporated is locally applied (local injection) to a bone gap site or a surrounding tissue (for example, bone, muscle, etc.). .
  • an expression vector a naked plasmid, a detoxified retrovirus, an adenovirus, an adeno-associated virus, a herpes virus (type I herpes simplex virus, etc.), a vaccinia virus, a pox virus, a poliovirus, a simbis virus, a Sendai virus
  • examples include, but are not limited to, DNA viruses or RNA viruses such as SV40 or immunodeficiency virus (HIV).
  • type I herpes simplex virus (HSV-1) vector, Sendai virus envelope (HVJ-E) vector, adenovirus vector, adeno-associated virus (AAV) vector and the like are preferable.
  • HSV-1 vectors include a non-replicating HSV-1 (HSV1764 / HSV1764 / HSV1764 / 4- / pR19) vector (Coffin RS, et al., GenJ.Gen.Virol. 1998, Vol. 79, p.3019-3026; Palmer JA et al., J. Virol., 2000, Vol. 74 , P. 5604-5618; Lilley CE, et al., J. Virol., 2001, Vol. 75, p.4343-4356).
  • the HVJ-E vector can be produced by the method described in USP 6913923, for example.
  • HVJ-E vector for example, GenomONE-Neo EX HVJ Envelope Transfection Kit (manufactured by Cosmo Bio Inc.) can be preferably used.
  • AAV vectors belong to non-pathogenic viruses, are highly safe, and can efficiently introduce genes into cells.
  • AAV-2, AAV-4, AAV-5 etc. are mentioned as an AAV vector.
  • These HSV-1 vectors, HVJ-E vectors or AAV vectors can safely express the target gene for a long period of time.
  • the vector used in the present invention is particularly preferably an HSV-1 vector, an HVJ-E vector or an AAV vector that enables safe and long-term expression.
  • Formulation forms for administering the HGF gene to a patient include various known preparation forms suitable for each of the above administration forms, such as injections, sprays, sustained-release preparations (depot preparations), microcapsules, etc. Can be taken. Injections, sprays and sustained-release preparations (depots) can be prepared in the same manner as in the case of the aforementioned HGF protein.
  • the gene transfer vector is usually about 1 ⁇ 10 5 to 1 ⁇ 10 12 pfu / mL, preferably about 1 ⁇ 10 6 to 1 ⁇ 10. Can be adjusted to 11 pfu / mL.
  • a host cell into which an expression plasmid containing an HGF gene is introduced as a core substance is used as a coating substance according to a known method (for example, a coacervation method, an interfacial polymerization method or a double nozzle method).
  • the microcapsules can be produced as fine particles having a diameter of about 1 to 500, preferably about 100 to 400 ⁇ m.
  • the coating material examples include carboxymethylcellulose, cellulose acetate phthalate, ethylcellulose, alginic acid or a salt thereof, gelatin, gelatin gum arabic, nitrocellulose, polyvinyl alcohol, hydroxypropylcellulose, polylactic acid, polyglycolic acid, lactic acid-glycolic acid copolymer And film-forming polymers such as chitosan-alginate, cellulose sulfate-poly (dimethyldiallyl) ammonium chloride, hydroxyethyl methacrylate-methyl methacrylate, chitosan-carboxymethylcellulose, alginate-polylysine-alginate, and the like.
  • the content and dosage of the HGF gene in these preparations can be appropriately adjusted depending on the disease to be treated, the age, weight, etc. of the patient.
  • the dose varies depending on the type of HGF gene transfer vector, but is usually 1 ⁇ 10 6 pfu to 1 ⁇ 10 12 pfu, preferably 1 ⁇ 10 7 pfu to 2 ⁇ 10 11 pfu in terms of HGF gene transfer vector. More preferably, 1.5 ⁇ 10 7 pfu to 1.5 ⁇ 10 11 pfu is preferably administered once every several days to several months.
  • Such a bone elongation promoting agent of the present invention can be used, for example, at a postoperative bone cutting gap site or its peripheral part when osteotomy, osteotomy or osteopathy in a fracture is performed. Then, according to the bone elongation promoting agent of the present invention, as shown in Experimental Examples 1 and 2, it is possible to effectively promote bone elongation in the postoperative bone cutting gap.
  • the length that can be extended by one treatment is usually about 3 mm, and the bone can be stretched to the required length by repeating the treatment several times.
  • the length that can be extended by one treatment is equal to the length of the bone cutting gap (the length between the bone cross section after the osteotomy and the bone cross section).
  • the bone elongation promoting agent of the present invention it is possible to promote the elongation of bone in the bone cutting gap, so that the bone cutting gap that can be formed by a single treatment can have a length exceeding about 3 mm (that is, The length exceeding about 3 mm can be extended by one treatment.)
  • the upper limit of the bone cutting gap (length that can be extended by one treatment) can be 10 mm.
  • the thickness is preferably 9 mm, more preferably 8 mm, still more preferably 7 mm, still more preferably 6 mm, and particularly preferably 5 mm.
  • osteogenesis include Ilizarov method.
  • the bones on both sides thereof are separated by about 0.5 to 1 mm every day, and this can be repeated to the required length to extend the bone.
  • the cut bone and the bone are connected with a special external fixator.
  • Bone separation can be performed using a device such as an external fixator.
  • the external fixator is preferably completely fixed when the bone is pulled away to the required length.
  • a bone gap is formed in a portion (extended portion) from which the bone is separated.
  • the bone is extended from the cut surface of the bone to the bone gap, the extended bone cut surfaces are fused, the bone gap is filled, and the bone is reconstructed.
  • the external fixator is removed and treatment is considered complete.
  • the final length between bone cross sections produced by bone distraction may correspond to the total length that causes the bone to extend.
  • the bone extension the bone can be extended to a total of about 10 cm.
  • the gap when forming a bone cutting gap after osteopathy in a fracture, when using the bone elongation promoter of the present invention in combination, the gap can be set in a range exceeding 3 mm for the above reasons.
  • the upper limit of the length is about 10 mm, preferably 9 mm, more preferably 8 mm, still more preferably 7 mm, even more preferably 6 mm, and particularly preferably 5 mm.
  • the bone cross section after osteopathy may be left in a fractured state, or the bone at the fractured part may be further trimmed by osteotomy or the like. Fractures include simple fractures, complex fractures, fractured fractures and the like. As described above, in the bone cutting gap formed in this way, the bone is extended from the cut surface of the bone, the cut surfaces of the extended bone are fused, the bone gap is filled, and the bone is reconstructed.
  • the bone elongation promoter of the present invention can be applied not only to humans but also to mammals other than humans (eg monkeys, cows, horses, pigs, sheep, dogs, cats, rats, mice, rabbits, hamsters, guinea pigs, chimpanzees, etc.) Applicable.
  • mammals other than humans eg monkeys, cows, horses, pigs, sheep, dogs, cats, rats, mice, rabbits, hamsters, guinea pigs, chimpanzees, etc.
  • the bone elongation promoting agent of the present invention can be used at a post-operative bone gap site or its peripheral part when, for example, osteotomy, osteogenesis, or osteopathy in a fracture is applied. . Then, the bone elongation promoting agent of the present invention promotes bone elongation from at least one, preferably both bone sections, toward the bone gap between the bone sections generated from one bone section and the opposite bone section.
  • the bone cut surfaces can be fused with each other in a shorter time and effectively, the bone gap can be filled, and the bone can be reconstructed. Examples of the application of the bone elongation promoter of the present invention include the following.
  • osteotomy or osteogenesis When osteotomy or osteogenesis is performed, for example, when the limb bones such as short stature, poor height increase, dwarfism, etc. are stretched; patients with large leg length differences due to congenital malformations, accidents, or surgery, etc. When the limb of the limb is shortened; When the bone of a patient who has suffered from bone deformation due to an accident or the like has been deformed; and when the bone used for autologous or allogeneic transplantation is extracted Restoration; craniofacial bone lengthening in cases such as micromaxillary disease (small mandibular disorder), microcephaly, cruzon disease, or maxillary undergrowth due to cleft lip and palate, etc. is indicated. Examples of fractures include various traumatic fractures; fatigue fractures; osteopathy that requires correction or correction of bone length, shape, etc. in fractures associated with osteoporosis, osteomalacia, osteogenesis imperfecta, marble disease, etc. Is applied.
  • the cut tibia was pulled apart at an interval of 5 mm to create a 5 mm bone gap (gap) in the tibia.
  • two short pins made by Stryker
  • each having a diameter of 2 mm are provided on each of the separated tibias so as to be perpendicular (perforation is made in the direction perpendicular to the bones) (total amount).
  • HGF protein is dissolved in 10 ⁇ L of physiological saline in the bone gap (gap between the cut tibias).
  • the prepared HGF-containing aqueous solution was injected transcutaneously (HGF administration group).
  • HGF protein consisting of the amino acid sequence shown in SEQ ID NO: 6 was used as the HGF protein.
  • physiological saline containing no HGF protein was used instead of the HGF-containing aqueous solution, and the same was injected into the bone gap (control group).
  • the condition of the bone gap was 10 weeks after surgery and every other week (11 times in total) for the control group.
  • X-rays were taken and evaluated with X-ray images (total 6 times).
  • FIG. Fig. A shows an X-ray image of the bone gap of the control group (the upper part is a front image, the lower part is a side image), and Fig. B shows an X-ray image of the bone gap part of the HGF administration group (the upper part is a front image and the lower part is a side image).
  • the control group to which no HGF protein was administered bone extension and fusion in the bone gap were incomplete, and the bone formed in the bone gap was greatly deformed (FIG. A)
  • HGF administration In the group bones were normally formed from the tibias on both sides in the bone gap and bone formation was promoted, and bone fusion was promoted and bone fusion was promoted at the bone cut surface.
  • any of the HGF proteins having the amino acid sequences shown in SEQ ID NOs: 3 to 6 can be used as the HGF protein.
  • the microcapsules are separated by centrifugation (about 2,000 rpm). Next, after washing twice with 400 mL of distilled water, 0.2 g of D-mannitol is added and freeze-dried. After lyophilization, in order to further remove the residual solvent, vacuum-dried at 40 ° C. for 3 days to obtain sustained-release microcapsules containing HGF protein (HGF-to-biodegradable polymer content ratio: 5.3 w) / W%).
  • the microcapsules are separated by centrifugation (about 2,000 rpm). Next, after washing twice with 400 mL of distilled water, 0.2 g of D-mannitol is added and freeze-dried. After lyophilization, in order to further remove the residual solvent, vacuum-dried at 40 ° C. for 3 days to obtain sustained-release microcapsules containing HGF protein (HGF-to-biodegradable polymer blending ratio: 5.3 w / w%).
  • the microcapsules are separated by centrifugation (about 2,000 rpm). Next, after washing twice with 400 mL of distilled water, 0.2 g of D-mannitol is added and freeze-dried. After lyophilization, in order to further remove residual solvent, vacuum drying is performed at 40 ° C. for 3 days to obtain a sustained-release microcapsule containing HGF protein (HGF blending ratio with respect to biodegradable polymer: 17.6 w) / W%).
  • the microcapsules are separated by centrifugation (about 2,000 rpm). Next, after washing twice with 400 mL of distilled water, 0.2 g of D-mannitol is added and freeze-dried. After lyophilization, in order to further remove the residual solvent, vacuum drying is performed at 40 ° C. for 3 days to obtain a sustained release microcapsule containing HGF protein (HGF blending ratio with respect to biodegradable polymer: 17.8 w) / W%).
  • the resulting emulsion was further stirred at room temperature for 3 hours to evaporate the methylene chloride, and then the microspheres produced by centrifugation (about 2,000 rpm) were collected and preheated to 40 ° C. Washing 5 times with distilled water, and then drying under reduced pressure at room temperature to obtain microspheres containing HGF (HGF content relative to biodegradable polymer: 0.05 w / w%).
  • the resulting emulsion is stirred at room temperature for an additional 3 hours to evaporate methylene chloride and ethanol, and then centrifuged (approximately 2,000 rpm) to collect the resulting microspheres.
  • the collected microspheres are washed 5 times with distilled water preheated to 40 ° C. and dried under reduced pressure at room temperature to obtain microspheres containing HGF protein (HGF against biodegradable polymer).
  • the mixing ratio 0.025 w / w%).
  • HGF-containing aqueous solution can be prepared by the method described in Experimental Example 1.
  • the obtained freeze-dried product is pulverized at low temperature using liquid nitrogen, and then compression-molded in a mold to obtain a cylindrical HGF-containing sustained release preparation (formulation of HGF with biodegradable polymer) (Ratio: 10 w / w%).
  • HGF protein 1 mg is dissolved in 2 mL of 2 w / v% atelocollagen solution and then freeze-dried.
  • the obtained freeze-dried product is pulverized and then compression-molded into a cylindrical shape to obtain a sustained-release preparation containing HGF protein (HGF content relative to biodegradable polymer: 2.5% by mass).
  • [Formulation Example 12] 0.58 g of sodium salt of hyaluronan (intrinsic viscosity 45000 cc / g) is mixed with 20 mL of water and swollen. Next, 2 mL of 2N sodium hydroxide is added to the mixture and stirred to obtain a homogeneous solution. To this, a solution prepared by adding 0.10 g of divinylsulfone to 2.4 mL of water and stirring is added to form a mixture. The mixture is allowed to stand for 70 minutes, and the resulting gel is put into a biotris buffer (phosphate buffer). Of 0.15 M NaCl, pH about 7.2) and swell for 3 hours.
  • phosphate buffer phosphate buffer
  • the bone elongation-promoting agent of the present invention is useful as a medical drug that promotes bone elongation and bone formation after osteotomy or osteogenesis, or after fracture or osteoarthroplasty.

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Abstract

L'objectif est de favoriser l'élongation osseuse dans l'espace inter-os après une ostéotomie ou lors de l'allongement d'un os. L'invention porte sur un promoteur d'élongation osseuse caractérisé en ce qu'il comprend, en tant qu'ingrédient actif, une protéine HGF, un peptide partiel de celle-ci ayant sensiblement la même activité favorisant l'élongation osseuse que celle de la protéine HGF, ou un sel de la protéine HGF ou du peptide partiel de celle-ci; ou de l'ADN comprenant l'ADN qui code pour la protéine HGF, l'ADN comprenant l'ADN qui code pour un peptide partiel de la protéine HGF ayant sensiblement la même activité favorisant l'élongation osseuse que celle de la protéine HGF, ou de l'ADN comprenant de l'ADN qui peut s'hybrider avec l'ADN comprenant une séquence nucléotidique complémentaire de l'une quelconque des deux molécules d'ADN précédentes dans des conditions rigoureuses et qui code pour une protéine ou un peptide ayant sensiblement la même activité favorisant l'élongation osseuse que celle de la protéine HGF.
PCT/JP2009/062816 2008-07-18 2009-07-15 Promoteur d'élongation osseuse Ceased WO2010008023A1 (fr)

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JPPCT/JP2008/063021 2008-07-18

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JP2017507123A (ja) * 2014-01-27 2017-03-16 ベイジン ストマトロジー ホスピタル，キャピタル メディカル ユニバーシティ 歯原性幹細胞および遺伝的に改変された歯原性幹細胞の使用
CN106714823A (zh) * 2014-09-10 2017-05-24 克霖固鲁制药股份有限公司 适合神经系统疾病的治疗的hgf制剂

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CN106714823A (zh) * 2014-09-10 2017-05-24 克霖固鲁制药股份有限公司 适合神经系统疾病的治疗的hgf制剂

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