WO2010041716A1 - 腱骨移行部組織または靭帯骨移行部組織の再生促進剤 - Google Patents
腱骨移行部組織または靭帯骨移行部組織の再生促進剤 Download PDFInfo
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- WO2010041716A1 WO2010041716A1 PCT/JP2009/067570 JP2009067570W WO2010041716A1 WO 2010041716 A1 WO2010041716 A1 WO 2010041716A1 JP 2009067570 W JP2009067570 W JP 2009067570W WO 2010041716 A1 WO2010041716 A1 WO 2010041716A1
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/16—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
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- 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
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- 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
- A61P19/04—Drugs for skeletal disorders for non-specific disorders of the connective tissue
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/475—Growth factors; Growth regulators
- C07K14/4753—Hepatocyte growth factor; Scatter factor; Tumor cytotoxic factor II
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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
Definitions
- the present invention relates to a regeneration promoter for tendon bone transition tissue or ligament bone transition tissue.
- the anterior cruciate ligament (ACL) and the posterior cruciate ligament (PCL) are tissues that connect the femur and tibia at the knee joint, so when the ACL or PCL ruptures The stability of the knee joint cannot be maintained. Since ligaments cannot be sewn when they tear, ligament reconstruction is often attempted to repair or replace damaged ligaments. For example, in an ACL reconstruction operation in which a damaged ACL is reconstructed by replacing it with a ligament graft, a patella tendon with a bone block attached thereto has been conventionally used as the graft to be transplanted. In such ACL reconstruction, since bones function as ligaments when they stick together, there is an advantage that rehabilitation can be done quickly, but there is a problem that postoperative pain and muscle strength are reduced.
- ACL reconstruction using a tendon such as a hamstring (knee flexor tendon, semi-tendonoid tendon, thin muscle tendon) which is a part of a tendon of a muscle that bends the knee has been reported (for example, non- (See Patent Document 1). More specifically, the surgical procedure involves the formation of a bone tunnel (through hole) at the upper end of the tibia and the lower end of the femur, placing one end of the graft tendon within the bone tunnel of the femur, The other end is placed in the bone tunnel of the tibia.
- a tendon such as a hamstring (knee flexor tendon, semi-tendonoid tendon, thin muscle tendon) which is a part of a tendon of a muscle that bends the knee has been reported (for example, non- (See Patent Document 1). More specifically, the surgical procedure involves the formation of a bone tunnel (through hole) at the upper end of the tibia and the
- the graft tendon thus extends between the femur and tibia so that the graft tendon works to restore the normal function of the knee joint with substantially the same function as the original ACL.
- ACL reconstruction is to reconstruct normal ACL function and kinematics of the knee joint, there is sufficient tendon bone transition tissue at the part where the bone surface in the bone tunnel contacts the grafted tendon. It needs to be regenerated with strength. For this reason, there exists a problem that it takes time until a graft tendon functions as a ligament.
- Examples of the drug that promotes regeneration of the tendon transitional tissue include BMP-2 (Bone Morphogenetic Protein-2: Non-patent document 2), TGF- ⁇ 1 (Transforming Growth Factor- ⁇ 1: Non-patent document 3), and the like. Although known, it has not yet been put into practical use as a drug that promotes regeneration of tendon bone transition tissue.
- HGF hepatocyte growth factor
- HGF protein hepatocyte growth factor
- Non-Patent Document 6 does not describe or suggest such regeneration of the complicated tendon bone transition tissue.
- HGF protein has been reported to suppress the expression of TGF- ⁇ 1, which is known as a drug that promotes regeneration of the aforementioned tendon transitional tissue (Non-patent Document 7).
- An object of the present invention is to provide a drug that promotes regeneration of a tendon bone transition tissue or a ligament bone transition tissue.
- the present invention relates to a contact site between a transplanted tendon or ligament piece and bone after reconstruction of a ligament such as an ACL, or a gap between a transplanted tendon or ligament piece and bone, or a ligament bone transition tissue.
- the object is to provide a drug that promotes regeneration.
- the present inventors have found that the HGF protein has a regeneration promoting action on the tendon bone transition tissue or ligament bone transition tissue, thereby completing the present invention. It was.
- the present invention relates to a regeneration promoter for tendon bone transition tissue or ligament bone transition tissue.
- An agent for promoting regeneration of a tendon bone transition tissue or a ligament bone transition tissue comprising the following (1) or (2) as an active ingredient: (1) The following (1-a) or (1-b) or (1-c), (1-a) HGF (Hepatocyte Growth Factor) protein, (1-b) a partial peptide of HGF protein, which has a regeneration promoting action on tendon bone transition tissue or ligament bone transition tissue, (1-c) a salt of (1-a) or (1-b), (2) DNA comprising the following (2-a) or (2-b) or (2-c), (2-a) DNA encoding HGF protein, (2-b) a DNA encoding a peptide that is a partial peptide of the HGF protein and has an action of promoting regeneration of a tendon bone transition tissue or a ligament bone transition tissue; (2-c) Hybridizing under stringent conditions with DNA comprising a base sequence complementary to the DNA of (2-a) or (2-b), and tendon bone transition tissue or ligament bone transition portion DNA encoding
- HGF protein is the following (1-d) or (1-e): (1-d) a protein comprising the amino acid sequence represented by SEQ ID NO: 3 or 4, (1-e) a protein comprising an amino acid sequence substantially the same as the amino acid sequence represented by SEQ ID NO: 3 or 4, and having a regeneration promoting effect on a tendon bone transition tissue or a ligament bone transition tissue.
- the protein of (1-e) is “having at least 85% identity with the amino acid sequence represented by SEQ ID NO: 3 or 4, and promoting regeneration of tendon bone transition tissue or ligament bone transition tissue. A protein having an action ”.
- 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 [1] ]
- HSV-1 vector Sendai virus envelope vector
- HVJ-E Sendai virus envelope vector
- adenovirus vector or adeno-associated virus vector [1]
- the regeneration promoter according to any one of [4] and [5].
- the present invention also relates to the use of the HGF protein or DNA encoding the same.
- (1) or (2) which is used in a method for promoting regeneration of a tendon bone transition tissue or a ligament bone transition tissue: (1) The following (1-a) or (1-b) or (1-c), (1-a) HGF protein, (1-b) a partial peptide of HGF protein, which has a regeneration promoting action on tendon bone transition tissue or ligament bone transition tissue, (1-c) a salt of (1-a) or (1-b), (2) DNA comprising the following (2-a) or (2-b) or (2-c), (2-a) DNA encoding HGF protein, (2-b) a DNA encoding a peptide that is a partial peptide of the HGF protein and has an action of promoting regeneration of a tendon bone transition tissue or a ligament bone transition tissue; (2-c) Hybridizing under stringent conditions with DNA comprising a base sequence complementary to the DNA of (2-a) or (2-b), and tendon bone transition tissue or ligament bone transition portion DNA encoding a protein or peptide having
- a method for promoting regeneration of a tendon bone transition tissue or a ligament bone transition region tissue wherein the following (1) or (2) is administered to a patient with a tissue injury of a tendon bone transition region or a ligament bone transition region: (1) The following (1-a) or (1-b) or (1-c), (1-a) HGF protein, (1-b) a partial peptide of HGF protein, which has a regeneration promoting action on tendon bone transition tissue or ligament bone transition tissue, (1-c) a salt of (1-a) or (1-b), (2) DNA comprising the following (2-a) or (2-b) or (2-c), (2-a) DNA encoding HGF protein, (2-b) a DNA encoding a peptide that is a partial peptide of the HGF protein and has an action of promoting regeneration of a tendon bone transition tissue or a ligament bone transition tissue; (2-c) Hybridizing under stringent conditions with DNA comprising a base sequence complementary to the DNA of (2-a) or (2-b),
- the regeneration promoter of the tendon bone transition tissue or ligament bone transition site tissue of the present invention is a surface where the grafted tendon or ligament piece separated from the bone and the bone contact, or the gap between the graft tendon or ligament piece and the bone, Regeneration of the tendon transition tissue or the ligament bone transition tissue can be promoted. For this reason, adhesion (adhesion, fusion) between a tendon and a bone or a ligament and a bone separated from a bone can be promoted due to damage or tearing.
- ligament reconstruction of limb joints eg, knee, foot, elbow, shoulder, etc.
- MPFL medialelopatelofemoral ligament
- a ulnar ligament reconstruction or a rotator cuff repair etc.
- HGF protein is a protein that has been identified as a potent mitogen (mytogen) for mature hepatocytes, is what is referred to as hepatocyte growth factor (Hepatocyte Growth Factor) (Non (See Patent Documents 4 and 5).
- Hepatocyte Growth Factor hepatocyte Growth Factor
- SF scatter factor
- TCF Tumor Cytotoxic Factor
- the “HGF protein” used in the present invention is a known substance, and any substance prepared by various methods can be used as long as it has been purified to the extent that it can be used as a medicine.
- the HGF protein can be obtained, for example, by culturing primary cultured cells or cell lines that produce HGF protein, and separating and purifying from the culture supernatant.
- 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.
- 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 an 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, Asn at position 561. And a sugar chain is added to 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.
- the HGF protein targeted by the present invention has an action of promoting regeneration of a tendon bone transition tissue or a ligament bone transition tissue, one or a plurality of (inclusive of a plurality of amino acids) in the amino acid sequence shown in SEQ ID NO: 3 or 4 above.
- one or a plurality of (inclusive of a plurality of amino acids) in the amino acid sequence shown in SEQ ID NO: 3 or 4 above For example, 2 to 35, preferably 2 to 20, more preferably 2 to 10; the same shall apply hereinafter) amino acids may be deleted, substituted, inserted or added, and similarly sugar chains May be deleted, substituted, inserted or added.
- 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 has at least 85% identity with the amino acid sequence shown in SEQ ID NO: 3 or 4 as long as it has a regeneration promoting action on the tendon bone transition tissue or ligament bone transition tissue. You may have.
- 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 95.7% and 96.4% identity with the amino acid sequence shown in SEQ ID NO: 3 or 4, respectively
- the amino acid sequence shown in SEQ ID NO: 6 is It has 95.1% and 95.7% identity with the amino acid sequence shown in SEQ ID NO: 3 or 4, 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.
- the HGF protein targeted in the present invention has amino acids 1 to 31 in each of the amino acid sequences shown in SEQ ID NOs: 3 and 4 as long as it has a regeneration promoting action on the tendon bone transition tissue or ligament bone transition tissue.
- a signal sequence consisting of a region, it may have a signal sequence of another protein. Examples of such signal sequences include signal sequences of human serum albumin, interferon, human amylase and the like.
- the HGF protein targeted in the present invention comprises a base sequence complementary to the base sequence shown in SEQ ID NO: 1 or 2 as long as it has a regeneration promoting effect on the tendon bone transition tissue or ligament bone transition tissue. It may be a protein produced from a cell having DNA that hybridizes with DNA under stringent conditions.
- 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 FM3A2 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, and the like are common.
- the HGF protein has a regeneration promoting action on the tendon bone transition tissue or the ligament bone transition tissue can be evaluated as follows. For example, at least the bone and the tendon or ligament separated from the bone are fixed with a ligament fixture or the like. The target HGF protein is allowed to act on this according to the method described in Examples and the like described later. As a result, if, for example, more effective regeneration of the tendon bone transition tissue or ligament bone transition tissue is observed when the HGF protein is allowed to act compared to the case where nothing acts (control), the HGF It is determined that the protein has a regeneration promoting effect on the tendon bone transition tissue or the ligament bone transition tissue. *
- 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 to delete sugar chains, and sugar chains are not added.
- the amino acid sequence at the sugar chain addition site is mutated, or those in which the amino acid sequence is mutated so that the sugar chain is added to a site different from the natural sugar chain addition site.
- HGF protein specifically, for example, a sugar chain against AccessionHNo.AccessNP_001010932 human HGF registered in NCBI database (NCBI-GenBank Flat File Release 164.0; the same applies in the following).
- the sugar chain was prevented from being added by substituting Asn at position 289 for Gln, Asn at position 397 for Gln, Thr for position 471 for Gly, Asn for position 561 for Gln and Asn for position 648 for Gln.
- HGF protein [Fukuta K et al., Biochemical Journal, 388, 555-562 (2005)].
- 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). Also good.
- 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 used in the present invention has a carboxyl group or a carboxylate other than the C-terminus
- the HGF protein used in the present invention also includes those in which the carboxyl group or carboxylate is amidated or esterified. It is.
- the ester in this case include the aforementioned C-terminal ester.
- the amino group of the N-terminal methionine residue in the protein is a protecting group (for example, a C 1-6 acyl such as a C 2-6 alkanoyl group such as formyl group and acetyl).
- a protecting group for example, a C 1-6 acyl such as a C 2-6 alkanoyl group such as formyl group and acetyl.
- a glutamyl group produced by cleavage of the N-terminal side in vivo, pyroglutamine oxidation, reactive groups on the side chains of amino acids in the molecule for example, —OH, —SH, An amino group, an imidazolyl group, an indolyl group, a guanidino group, etc.
- an appropriate protecting group for example, a C 1-6 acyl group such as a C 2-6 alkanoyl group such as formyl group, acetyl, etc.
- a complex protein such as a so-called glycoprotein to which a sugar chain is bound is also included.
- HGF protein used in the present invention those derived from humans described above are preferably used when applied to humans, but mammals other than humans (for example, monkeys, cows, horses, pigs, sheep, dogs, cats) , Rat, mouse, rabbit, hamster, guinea pig, chimpanzee, etc.).
- HGF proteins examples include mouse-derived HGF proteins (eg, Accession No. AAB31855, NP_034557, BAA01065, BAA01064, etc.) registered in the NCBI database and the like, rat-derived HGF proteins (eg, Accession No. NP_058713, etc.), 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 When used as an active ingredient of the regeneration accelerator of the present invention, they may be purified to such an extent that they can be used as pharmaceuticals, and so long as they are prepared by various methods. Can do.
- the purification method is not limited, and examples thereof include column chromatography using heparin / sepharose or hydroxyapatite.
- the HGF protein may be used alone, or a mixed protein with various proteins as long as the regeneration promoting action of the tendon bone transition tissue or ligament bone transition tissue is not impaired. Can be used as
- HGF protein partial peptide used in the present invention and tendon transition tissue or ligament bone transition tissue A peptide having a regeneration promoting activity (hereinafter sometimes abbreviated as HGF partial peptide) is a partial peptide of the HGF protein, and similar to the HGF protein, promotes regeneration of the transitional tissue of the tendon or ligament bone Any one having at least an action may be used.
- the number of amino acids of the HGF partial peptide is a peptide containing an amino acid sequence of at least about 20 or more, preferably about 50 or more, more preferably about 100 or more of the constituent amino acid sequences of the HGF protein. Etc. are preferred.
- such an HGF partial peptide includes an amino acid sequence from the 32nd amino acid to the 210th amino acid from the N-terminal side of the human HGF amino acid sequence represented by SEQ ID NO: 3 (the N-terminal of HGF).
- a peptide represented by a hairpin loop to the first kringle domain) or an amino acid sequence from the 32nd amino acid to the 288th amino acid from the N-terminal side of the human HGF amino acid sequence represented by SEQ ID NO: 3 (N of HGF) Peptides represented by the sequence from the terminal hairpin loop to the second kringle domain are preferred.
- the HGF partial peptide of the present invention includes a partial peptide having at least about 80% homology with the amino acid sequence of the aforementioned HGF partial peptide, preferably a partial peptide having about 90% homology, more preferably A partial peptide having a homology of about 95% or more and having a regeneration promoting action on at least the same tendon bone or ligament bone transition tissue as the aforementioned HGF protein is also included.
- the C-terminus is carboxyl group (—COOH), carboxylate [—COOM (M is as defined above)], amide (—CONH 2 ) or ester (—COOR; R is 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 the Gln produced by cleaving 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 also be evaluated in the same manner as described above as “having a regeneration promoting effect on the tendon bone transition tissue or ligament bone transition tissue”.
- the HGF partial peptide of the present invention 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 target peptide can be produced by eliminating the group.
- Known condensation methods and protecting group removal include, for example, M. Bodanszky and M. A.
- the partial peptide of the HGF protein can be purified and isolated by combining ordinary purification methods such as solvent extraction, distillation, column chromatography, liquid chromatography, crystallization or recrystallization.
- HGF protein or partial peptide thereof may be in a free (free form) or in the form of a salt.
- the salt of the HGF protein or its partial peptide used in the present invention include physiologically acceptable salts with acids or bases, and physiologically acceptable acid addition salts are particularly preferable.
- salts examples 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, benzenesulfonic acid, and the like.
- the HGF protein or HGF partial peptide of the present invention when obtained in a free form, it can be converted into an appropriate salt by a known method, and conversely, when obtained as a salt, by a known method. Can be converted to free form.
- DNA encoding HGF protein refers to DNA capable of expressing the aforementioned HGF protein.
- DNA containing DNA encoding HGF protein include, for example, Nature, 342, 440 (1989); Patent No. 2777678; Biochem. Biophys. Res. Commun., 1989, 163, p. 967-973. Proc. Natl. Acad. Sci. USA, 1991, Vol. 88 (16), p.7001-7005, etc., for example, Accession No. M69718, M73240, AC004960, AY246560 in GenBank / EMBL / DDBJ Preferred examples include DNA encoding human-derived HGF protein registered as M29145 or M73240.
- the DNA encoding the HGF protein used in the present invention is preferably a human-derived DNA as described above when applied to humans, but mammals other than humans (eg monkeys, cows, horses, pigs, sheep) DNA encoding HGF protein derived from dogs, cats, rats, mice, rabbits, hamsters, guinea pigs, chimpanzees, etc.).
- DNA encoding such an HGF protein examples include, for example, DNA encoding mouse-derived HGF protein (for example, Accession No. S71816, NM_010427, D10213, D10212, etc.) registered in the NCBI database and the like, rat-derived HGF, etc. DNA encoding a protein (eg, Accession No. NM_017017), DNA encoding a bovine-derived HGF protein (eg, Accession No. NM_001031751, AB110822, etc.), DNA encoding a cat-derived HGF protein (eg, Accession No.
- NM_001009830 DNA encoding dog-derived HGF protein (eg AccessionAccessNo. NM_001002964, AB090353 etc.) or DNA encoding chimpanzee-derived HGF protein (eg Accession No. XM_519174), but not limited thereto.
- dog-derived HGF protein eg AccessionAccessNo. NM_001002964, AB090353 etc.
- DNA encoding chimpanzee-derived HGF protein eg Accession No. XM_519174
- DNA having the base sequence represented by SEQ ID NO: 1 or 2 is preferably mentioned.
- the base sequence represented by SEQ ID NO: 1 corresponds to the base sequence located at positions 73 to 2259 of the base sequence of Accession No. M60718, and the DNA comprising the base sequence is the amino acid represented by SEQ ID NO: 3 It corresponds to DNA encoding HGF protein consisting of sequence.
- 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.
- 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 66th to 2237 of the base sequence of Accession No. M73240, and the DNA comprising the base sequence comprises the amino acid sequence represented by SEQ ID NO: 4. It corresponds to DNA encoding HGF protein.
- 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 is not limited to the DNA described above, and the HGF protein of the present invention can be used as long as it is a DNA encoding a protein having an action of promoting regeneration of the tendon bone transition tissue or ligament bone transition tissue. It can be used as encoding DNA.
- “having a regeneration promoting effect on the tendon bone transition tissue or ligament bone transition tissue” can be evaluated by the same method as described above.
- Examples thereof include DNA encoding a protein having a sequence and promoting the regeneration of a tendon bone transition tissue or a ligament bone transition tissue.
- DNA encoding the HGF protein can be easily obtained, for example, by 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: hereafter abbreviated as Molecular Cloning 3rd Edition)) be able to.
- 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.
- ⁇ phage incorporating such cDNA is infected and cultured in 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 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.
- the DNA encoding the HGF protein is administered to the patient in the form of a recombinant expression vector in which the DNA is incorporated.
- 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.
- the regeneration promoter for tendon bone transition tissue or ligament bone transition tissue of the present invention is DNA that hybridizes under stringent conditions with DNA comprising a base sequence complementary to the DNA encoding the HGF protein.
- DNA encoding a protein having a regeneration promoting action on the tendon bone transition tissue or the ligament bone transition tissue may be included.
- DNA examples include DNA that hybridizes under stringent conditions with DNA having a base sequence complementary to the DNA having the base sequence represented by SEQ ID NO: 1 or 2, and Preferable examples include DNA encoding a protein having an action of promoting regeneration of ligament bone transition tissue.
- DNA that hybridizes under stringent conditions with DNA comprising a base sequence complementary to the DNA encoding the HGF protein, or with DNA comprising a base sequence complementary to the base sequence represented by SEQ ID NO: 1 or 2 Is, for example, colony hybridization using as a probe a DNA comprising a base sequence complementary to the DNA encoding the HGF protein or a partial sequence of a DNA comprising a base sequence complementary to the base sequence represented by SEQ ID NO: 1 or 2.
- the filter is washed under a condition of about 65 ° C. using an SSC solution having a concentration of about 0.1 to 2 times (the composition of the SSC solution having a concentration of 1 consists of 150 mM sodium chloride and 15 mM sodium citrate).
- the DNA which can be identified by this can be mentioned. Stringent conditions are the same in the following.
- the DNA that hybridizes under such stringent conditions is about 80% or more, preferably 85% or more, more preferably about 90% or more, particularly preferably with the DNA encoding the above-mentioned HGF protein.
- Examples thereof include DNA having a base sequence having a homology of about 95% or more. More specifically, for example, as DNA that hybridizes under stringent conditions with DNA consisting of a base sequence complementary to the base sequence represented by SEQ ID NO: 1 or 2, it is represented by SEQ ID NO: 1 or 2.
- Hybridization can be performed according to a known method, for example, the method described in the above-mentioned Molecular Cloning 3rd edition. Moreover, when using a commercially available library, it can carry out according to the method as described in an attached instruction manual.
- DNA encoding a peptide that is a partial peptide of HGF protein and has a promoting action on regeneration of tendon bone transition tissue or ligament bone transition tissue The regeneration promoter for tendon bone transition tissue or ligament bone transition tissue according to the present invention is a partial peptide of the HGF protein and, like the HGF protein, promotes regeneration of the tendon transition tissue or ligament bone transition tissue.
- DNA encoding a peptide having an action may be included.
- “having a regeneration promoting effect on the tendon bone transition tissue or ligament bone transition tissue” can be evaluated in the same manner as described above.
- Such DNA may be any DNA as long as it has a nucleotide sequence encoding the above-mentioned partial peptide and encodes a peptide having a regeneration promoting action on the tendon bone transition tissue or ligament bone transition tissue. May be.
- Specific examples of such a DNA include, for example, a DNA having a partial base sequence of a DNA having the base sequence represented by SEQ ID NO: 1 or 2, and, like the HGF protein, Examples thereof include DNA encoding a peptide having an action of promoting regeneration of tissue or ligament bone transition site tissue.
- 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).
- Such DNA is not limited to those described above, and is about 80% of the above-mentioned HGF protein partial peptide, which encodes a peptide having a regeneration promoting effect on the tendon bone transition tissue or ligament bone transition tissue. % Or more, preferably about 85% or more, more preferably about 90% or more, particularly preferably about 95% or more of the base sequence, and at least of tendon bone transition tissue or ligament bone transition tissue Examples thereof include DNA encoding a peptide having a regeneration 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.
- genomic DNA containing a peptide that is a partial peptide of the HGF protein and encodes a peptide having a regeneration promoting effect on the tendon bone transition tissue or ligament bone transition tissue
- genomic DNA, genomic DNA library, cell or Preferred examples include tissue-derived cDNA, cell or tissue-derived cDNA library, or synthetic DNA.
- vector into which the genomic DNA fragment is cloned into the library include bacteriophage, plasmid, cosmid, or phagemid.
- RNA encoding a peptide that is a partial peptide of the HGF protein and has an action of promoting regeneration of the tendon bone transition tissue or ligament bone transition tissue can express the HGF protein by reverse transcriptase. If possible, it can be used in the present invention.
- 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.
- a DNA encoding a peptide having an action of promoting regeneration of a tendon bone transition tissue or a ligament bone transition tissue The regeneration promoter for tendon bone transition tissue or ligament bone transition tissue according to the present invention is a partial peptide of the aforementioned HGF protein, which has a regeneration promoting effect on tendon bone transition tissue or ligament bone transition tissue DNA that hybridizes under stringent conditions with DNA consisting of a base sequence complementary to DNA encoding DNA, and that encodes a peptide having a regeneration promoting effect on tendon bone transition tissue or ligament bone transition tissue Etc. may be included.
- Such DNA includes a base sequence having a homology of about 80% or more, preferably about 85% or more, preferably about 90% or more, more preferably about 95% or more with the DNA encoding the HGF partial peptide. And DNA having a DNA encoding a peptide having a regeneration promoting action on the tendon bone transition tissue or ligament bone transition tissue.
- a DNA for example, a DNA that hybridizes under stringent conditions with a DNA having a base sequence complementary to a DNA having a partial base sequence of the DNA having the base sequence represented by SEQ ID NO: 1 or 2.
- action of a tendon bone transition part tissue or a ligament bone transition part tissue is illustrated. More specifically, such DNA includes about 80% or more, preferably about 85% or more, more preferably about about 80% 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 nucleotide sequence having a homology of 90% or more, particularly preferably about 95% or more, and having a DNA encoding a peptide having a regeneration promoting action on tendon bone transition tissue or ligament bone transition tissue. .
- tendon bone tissue or ligament bone transition tissue can usually be described as a tissue that mediates between skeletal muscle and bone, and “ligament” is usually a tissue that mediates between bone and bone I can explain. However, in the present invention, “tendon” may be referred to as including a ligament that mediates between bones.
- the tendon in the present invention includes a graft tendon (hereinafter also referred to as “transplant tendon”), and the ligament in the present invention includes a graft ligament (hereinafter also referred to as “transplant ligament”). Including.
- the transplant tendon may be an autograft tendon or an allograft tendon.
- the autograft tendons used may typically be taken from hamstrings, patella tendons, femoral flexor tendons or the like. Commonly used allograft tendons are taken from cadaver sources and may be hamstrings, patella tendons, femoral flexor tendons, Achilles tendons, or shin or elbow tendons. Further, a synthetic (or artificial) transplanted tendon or a xenograft tendon may be used, or a hybrid transplanted tendon of the self-transplanted tendon (or allograft tendon) and a synthetic transplanted tendon or the like. The graft tendon can be used by being folded in multiple.
- the transplant ligament may also be an autograft ligament or an allograft ligament.
- the autograft ligament used may typically be taken from the intestinal shin ligament or the like.
- Commonly used allograft ligaments are taken from a cadaver source and include the intestinal shin ligament, medial collateral ligament (MCL), lateral collateral ligament (LCL), anterior cruciate ligament (ACL), posterior cruciate ligament (PCL), lateral It may be a ligament, a triangular ligament, a tibial ligament, a clavicular clavicular ligament, or a femoral head ligament.
- a synthetic (or artificial) graft ligament or a xenograft ligament may be used, or a hybrid graft ligament including the autograft ligament (or allograft ligament) and a synthetic graft ligament or the like.
- the graft ligament can be used by being folded in multiple.
- the “tendon bone transition portion” or “ligament bone transition portion” is not particularly limited as long as it is a site where the tendon or the ligament and the bone are attached (or fixed or fused).
- ligament reconstruction The site
- the bone tunnel is drilled using, for example, a drill guide and a drill.
- the ligament reconstruction include ligament reconstruction in the knee joint, ankle joint, elbow joint, wrist joint, shoulder joint, and the like.
- ACL reconstruction surgery PCL reconstruction surgery in the knee joint
- MPFL reconstruction for recurrent ligament reconstruction or recurrent patella dislocation recurrent ligament reconstruction or recurrent patella dislocation
- lateral ankle ligament reconstruction recurrent patella dislocation
- ulnar ligament reconstruction of the elbow joint or rotator cuff repair of the shoulder joint.
- a bone tunnel is drilled at the upper end (proximal portion) of the tibia and the lower end of the femur.
- the graft tendon is passed through the bone tunnel and secured to extend between the tibia and femur with substantially the same function as the original ACL.
- the transitional tissue of the tendon bone is regenerated, so that the graft tendon functions as a ligament.
- the normal functioning of the knee joint can be restored. Thereby, a ligament can be re-formed between the tibia and the femur.
- the fixing can be performed using, for example, a bone screw (an interference fit screw) or a similar fastener, a ligament fixing device, a suture thread (for example, a nylon thread, a silk thread, or the like).
- a bone screw an interference fit screw
- a ligament fixing device for example, a suture thread, for example, a nylon thread, a silk thread, or the like.
- Fasteners, ligament anchors or sutures can be used alone or in combination.
- the ligament fixing device include a stainless steel washer (made by Zimmer Co., Ltd.) and an end button (made by Smith & Neffer Endoscopy Co., Ltd.).
- the tendon transition part or ligament bone transition part has complicated anatomical characteristics, and includes, for example, a collagen fiber layer including penetrating fibers.
- Regeneration can also be expressed as “reconstruction”, and refers to the re-creation of the tendon bone transition tissue or ligament bone transition tissue.
- ligament reconstruction transplanted tendons (or transplanted tendons) All the states in which the ligament) is attached (fixed, fused) to the bone with sufficient strength and can function physiologically in vivo.
- Regeneration of the tendon transition tissue or ligament bone transition tissue is usually performed by the following processes (1) to (5): (1) A process in which a non-directional granulation tissue is formed between a graft tendon or a graft ligament and a bone, (2) The process by which collagen fibers are formed between the graft tendon or graft ligament and the bone, (3) The process in which collagen fibers become directional in the bone direction, (4) The process of appearance of a sharpy like fiber entering the bone tissue from the graft tendon or graft ligament, (5) The process of penetrating fibers matures, including. The same applies to regeneration other than ligament reconstruction.
- Tendon bone transition tissue or ligament bone transition tissue regeneration promoting agent comprises (a) HGF depending on the type of active ingredient as shown below. It can be classified into a regeneration accelerator having a protein / partial peptide as an active ingredient and (b) a regeneration accelerator having an HGF gene as an active ingredient.
- HGF protein / partial peptide Regeneration promoter containing 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 salt thereof (Hereinafter, these may be referred to as “HGF protein / partial peptide”) as an active ingredient.
- HGF gene Regenerative promoter containing HGF gene as active ingredient DNA encoding HGF protein described in (4) above, (5) DNA hybridizing with the DNA under stringent conditions, and (6) A regeneration promoter comprising as an active ingredient DNA encoding an HGF partial peptide, and (7) DNA that hybridizes with the DNA under stringent conditions (hereinafter collectively referred to as “HGF gene”).
- the administration form, administration method, dosage and the like are the above-mentioned “HGF protein / partial peptide”. There may be a difference between a certain case and the case of the “HGF gene” described above.
- the dosage form, administration method, dosage and the like of the regeneration promoter of the present invention can be appropriately designed and changed according to the type of the active ingredient.
- (A) Regeneration accelerator comprising HGF protein / partial peptide as active ingredient
- the regeneration accelerator of (a) can take various preparation forms such as a liquid agent and a solid agent, but generally HGF protein, HGF
- the 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 can be adjusted to generally about 0.0002 to 0.5 w / v%, preferably about 0.001 to 0.2 w / v% with respect to the whole injection solution. 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 by conventional means on formulations.
- the additive added to the propellant may be any additive as long as it is generally used for inhalation preparations.
- a solvent, a preservative, a stabilizer, an isotonic agent, a 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 HGF protein content 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 / partial peptide used in the present invention can be made into 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.
- polysaccharides such as starch, dextran or chitosan
- proteins such as collagen or gelatin
- Polyamino acids such as polyglutamic acid, polylysine, polyleucine, polyalanine or polymethionine
- polyester such as fumaric acid / polyethylene glycol / vinyl pyrrolidone copolymer
- polyorthoester polyalkylcyanoacrylic acid such as polymethyl- ⁇ -cyanoacrylic acid
- polycarbonate such as polyethylene carbonate or polypropylene carbonate .
- 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 / w%, about 0.01 to 30 w / w with respect to the biodegradable polymer. % Is preferred.
- an injection or a spray is locally applied to the site where the grafted tendon or ligament piece contacts the bone (interface), the gap between the grafted tendon or ligament piece and the bone, or a peripheral site (direct injection or spray). It is preferable to spray (or spray), or to apply (implant) a sustained-release preparation (depot) locally to the interface or its peripheral site.
- the dosage is appropriately selected according to the dosage form, the degree of disease, age, etc. Usually, it is contained once per time in the regeneration promoter for tendon bone transition tissue or ligament bone transition tissue of the present invention.
- the amount of HGF protein to be added is 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.
- the administration interval may be from once a day to once every several months.
- a sustained-release preparation depot
- continuous administration using a sustained-release pump once every several months. But you can.
- Specific administration methods include, for example, a method in which a recombinant expression vector incorporating an HGF gene or the like is applied locally (local injection) to an interface or a surrounding tissue (eg, bone, muscle, etc.).
- Expression vectors include naked plasmids, or detoxified retroviruses, adenoviruses, adeno-associated viruses, herpes viruses (type I herpes simplex virus, etc.), vaccinia viruses, poxviruses, polioviruses, symbis viruses, Sendai viruses, SV40.
- a DNA virus such as an immunodeficiency virus (HIV) or an RNA virus can be used, but the present invention is not limited thereto.
- 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., J. Gen. Virol. 1998, 79, p.3019-3026; Palmer JA, et al., J. Virol., 2000, 74 Vol., P. 5604-5618; Lilley CE, et al., J. Virol., 2001, Vol. 75, p.4343-4356).
- the HVJ-E vector can be produced, for example, by the method described in USP 6,913,923.
- 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 substance include a film-forming polymer.
- the film-forming polymer examples include carboxymethyl cellulose, cellulose acetate phthalate, ethyl cellulose, alginic acid or a salt thereof, gelatin, gelatin / gum arabic, nitrocellulose, polyvinyl alcohol, hydroxypropyl cellulose, polylactic acid, polyglycolic acid, lactic acid-glycol.
- examples thereof include acid copolymers, 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.
- the regeneration promoter of the present invention is applicable to mammals other than humans (eg, monkeys, cows, horses, pigs, sheep, dogs, cats, rats, mice, rabbits, hamsters, guinea pigs, chimpanzees, etc.). it can.
- the regeneration promoting agent of the present invention preferably has a site (interface) where the transplanted tendon and bone or the transplanted ligament and bone contact after surgery when ligament reconstruction or the like is applied. It can be used in the periphery. And the regeneration promoter for tendon bone transition tissue or ligament bone transition tissue according to the present invention regenerates the tendon transition tissue or ligament bone transition tissue in the gap between the interface or the graft tendon or graft ligament and the bone. Can do.
- Example 1 Method of effect of HGF protein on tissue regeneration of tendon bone transition in a rabbit autograft tendon model : Japanese white rabbit (2.5-3.0 kg), mitazolam (Dolmicum: manufactured by Astellas) 3 mg and medetomidine hydrochloride (Dmitor: Japan) After intramuscular injection of 1 mg of Zenyaku Co., Ltd., anesthesia was performed by continuous intravenous administration of about 260 mg / hour of pentobarbital sodium (Somnopentyl: manufactured by Kyoritsu Pharmaceutical Co., Ltd.). The hind limbs were depilated and disinfected, and the following surgery was performed on each hind limb under clean operation.
- the skin on the front surface of the hind limb was incised by about 5 cm, and an extension digitorum longus (hereinafter abbreviated as EDL) tendon attached to the femoral condyle was cut off at the origin (attachment) of the femur.
- EDL extension digitorum longus
- the outer surface of the tibia was developed, and a through-hole (hereinafter referred to as “bone tunnel”) was created from the outer surface of the proximal part of the tibia toward the inner surface using a drill with a diameter of 2.5 mm. As shown in FIG.
- the free end of the EDL tendon (transplanted tendon) cut off first is pulled into the bone tunnel from the outer surface of the tibia and pulled out to the inner surface of the tibia, and a stainless steel washer (Zimmer Co., Ltd.) and 3- It was fixed using 0 nylon yarn (manufactured by Bear Medic Co., Ltd.).
- the limb position at the time of fixation was the intermediate position of the ankle joint.
- the bone tunnel into which the transplanted tendon was drawn was thoroughly washed with physiological saline (manufactured by Otsuka Pharmaceutical Co., Ltd.).
- cancellous bone containing 10 ⁇ L of 10 ⁇ g of HGF protein dissolved in 10 ⁇ L of physiological saline was transplanted to the contact surface and gap between the transplanted tendon and bone tunnel of the right hind limb (HGF administration group).
- HGF protein consisting of the amino acid sequence shown in SEQ ID NO: 6 was used as the HGF protein.
- a cancellous bone containing 10 ⁇ L of physiological saline was transplanted into the contact surface and gap between the transplanted tendon and the bone tunnel in the left hind limb (control group).
- the cancellous bone used when the bone tunnel was prepared was used (0.05 g). After transplanting the cancellous bone, the wound was sutured and closed. After the operation, the rabbit was returned to the cage and freely moved without restraining or fixing the limbs.
- FIG. 2 shows an optical micrograph of 8 weeks after the operation.
- the HGF administration group (right figure A)
- collagen fibers and penetrating fibers having a direction toward the bone direction appear in the interface as compared with the control group (left figure B).
- the penetrating fibers are thick and mature.
- the organization score of the interface is shown in Table 1. As apparent from Table 1, it was found that the formation (regeneration) of the tendon transitional tissue was promoted at the interface in the HGF administration group at an early stage after the operation.
- Biomechanical examination was performed by comparing the rupture strength of the tendon transition tissue formed by the graft tendon attached to the bone tunnel.
- the breaking strength was calculated from the following equation by measuring the maximum load (Newton; N) when the graft tendon was pulled out from the bone tunnel and as the maximum load for a bone tunnel length of 1 mm.
- Breaking strength (N / mm) Maximum load / Bone tunnel length
- the maximum load is determined by fixing the extracted tissue tibia to the tensile tester using a tensile tester (Instron, model 4482), It was grasped and pulled in the direction of the long axis of the bone tunnel, and was used as a load when the graft tendon was pulled out from the bone tunnel. Excluded were those where the grafted tendon was torn before being pulled out of the bone tunnel.
- Table 2 shows the breaking strength at 2 weeks and 6 weeks after the operation. At any time point, the breaking strength of the HGF administration group was higher than that of the control group.
- Example 2 Effect of HGF protein on tendon bone transition tissue regeneration in rabbit autograft tendon model
- an HGF protein consisting of the amino acid sequence shown in SEQ ID NO: 4 was used in the same manner as in Example 1 above. The experiment was conducted. As a result, as in Example 1, the HGF protein was recognized to promote regeneration of tendon bone transition tissue or ligament bone transition tissue.
- 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 regeneration promoter for tendon or ligament bone transition tissue of the present invention is useful as a medical drug for promoting regeneration of the tendon or ligament bone transition tissue.
- FIG. 1 is a diagram showing an EDL tendon (left diagram) in a rabbit limb joint in Example 1 and a state (right diagram) in which the EDL tendon is implanted into a bone.
- FIG. 2 is a view showing an optical micrograph of the tendon transitional tissue at 8 weeks after the EDL tendon transplantation in Example 1.
- A shows an HE-stained image of the HGF administration group
- B shows an HE-stained image of the control group.
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Abstract
Description
(1)下記(1-a)もしくは(1-b)または(1-c)、
(1-a)HGF(Hepatocyte Growth Factor)蛋白質、
(1-b)HGF蛋白質の部分ペプチドであって、腱骨移行部組織または靭帯骨移行部組織の再生促進作用を有するペプチド、
(1-c)(1-a)または(1-b)の塩、
(2)下記(2-a)もしくは(2-b)または(2-c)を含むDNA、
(2-a)HGF蛋白質をコードするDNA、
(2-b)HGF蛋白質の部分ペプチドであって、腱骨移行部組織または靭帯骨移行部組織の再生促進作用を有するペプチドをコードするDNA、
(2-c)前記(2-a)または(2-b)のDNAと相補的な塩基配列からなるDNAとストリンジェントな条件下でハイブリダイズし、かつ腱骨移行部組織または靭帯骨移行部組織の再生促進作用を有する蛋白質またはペプチドをコードするDNA。
(1-a)HGF蛋白質、
(1-b)HGF蛋白質の部分ペプチドであって、腱骨移行部組織または靭帯骨移行部組織の再生促進作用を有するペプチド、
(1-c)(1-a)または(1-b)の塩。
(1-d)配列番号3または4で表されるアミノ酸配列からなる蛋白質、
(1-e)配列番号3または4で表されるアミノ酸配列と実質的に同一のアミノ酸配列を含む蛋白質であって、腱骨移行組織または靭帯骨移行組織の再生促進作用を有する蛋白質。
(2-a)HGF蛋白質をコードするDNA、
(2-b)HGF蛋白質の部分ペプチドであって、腱骨移行部組織または靭帯骨移行部組織の再生促進作用を有するペプチドをコードするDNA、
(2-c)前記(2-a)または(2-b)のDNAと相補的な塩基配列からなるDNAとストリンジェントな条件下でハイブリダイズし、かつ腱骨移行部組織または靭帯骨移行部組織の再生促進作用を有する蛋白質もしくはペプチドをコードするDNA。
(2-d)配列番号1または2で表される塩基配列からなるDNA、
(2-e)配列番号1または2で表される塩基配列と相補的な塩基配列からなるDNAとストリンジェントな条件下でハイブリダイズし、かつ腱骨移行部組織または靭帯骨移行部組織の再生促進作用を有する蛋白質をコードするDNA。
(1)下記(1-a)もしくは(1-b)または(1-c)、
(1-a)HGF蛋白質、
(1-b)HGF蛋白質の部分ペプチドであって、腱骨移行部組織または靭帯骨移行部組織の再生促進作用を有するペプチド、
(1-c)(1-a)または(1-b)の塩、
(2)下記(2-a)もしくは(2-b)または(2-c)を含むDNA、
(2-a)HGF蛋白質をコードするDNA、
(2-b)HGF蛋白質の部分ペプチドであって、腱骨移行部組織または靭帯骨移行部組織の再生促進作用を有するペプチドをコードするDNA、
(2-c)前記(2-a)または(2-b)のDNAと相補的な塩基配列からなるDNAとストリンジェントな条件下でハイブリダイズし、かつ腱骨移行部組織または靭帯骨移行部組織の再生促進作用を有する蛋白質またはペプチドをコードするDNA。
(1)下記(1-a)もしくは(1-b)または(1-c)、
(1-a)HGF蛋白質、
(1-b)HGF蛋白質の部分ペプチドであって、腱骨移行部組織または靭帯骨移行部組織の再生促進作用を有するペプチド、
(1-c)(1-a)または(1-b)の塩、
(2)下記(2-a)もしくは(2-b)または(2-c)を含むDNA、
(2-a)HGF蛋白質をコードするDNA、
(2-b)HGF蛋白質の部分ペプチドであって、腱骨移行部組織または靭帯骨移行部組織の再生促進作用を有するペプチドをコードするDNA、
(2-c)前記(2-a)または(2-b)のDNAと相補的な塩基配列からなるDNAとストリンジェントな条件下でハイブリダイズし、かつ腱骨移行部組織または靭帯骨移行部組織の再生促進作用を有する蛋白質またはペプチドをコードするDNA。
(1)下記(1-a)もしくは(1-b)または(1-c)、
(1-a)HGF蛋白質、
(1-b)HGF蛋白質の部分ペプチドであって、腱骨移行部組織または靭帯骨移行部組織の再生促進作用を有するペプチド、
(1-c)(1-a)または(1-b)の塩、
(2)下記(2-a)もしくは(2-b)または(2-c)を含むDNA、
(2-a)HGF蛋白質をコードするDNA、
(2-b)HGF蛋白質の部分ペプチドであって、腱骨移行部組織または靭帯骨移行部組織の再生促進作用を有するペプチドをコードするDNA、
(2-c)前記(2-a)または(2-b)のDNAと相補的な塩基配列からなるDNAとストリンジェントな条件下でハイブリダイズし、かつ腱骨移行部組織または靭帯骨移行部組織の再生促進作用を有する蛋白質またはペプチドをコードするDNA。
「HGF蛋白質」は、前述するように、成熟肝細胞に対する強力なマイトゲン(mytogen)として同定された蛋白質であり、肝細胞増殖因子(Hepatocyte Growth Factor)と称されているものである(非特許文献4、5など参照)。その名称はHGF以外にSF(scatter factor)、TCF(Tumor Cytotoxic Factor)等が使用されている。
本発明で用いる、HGF蛋白質の部分ペプチドであって腱骨移行部組織または靭帯骨移行部組織の再生促進作用を有するペプチド(以下、HGF部分ペプチドと略記する場合がある)としては、前記HGF蛋白質の部分ペプチドであって、前記HGF蛋白質と同様に腱骨または靭帯骨移行部組織の再生促進作用を少なくとも有するものであればいずれのものであってもよい。本発明において、HGF部分ペプチドのアミノ酸の数は、前記のHGF蛋白質の構成アミノ酸配列のうち少なくとも約20個以上、好ましくは約50個以上、より好ましくは約100個以上のアミノ酸配列を含有するペプチド等が好ましい。具体的には、例えば、このようなHGF部分ペプチドとしては、配列番号3で表されるヒトHGFアミノ酸配列のN末端側から32番目のアミノ酸から210番目のアミノ酸までのアミノ酸配列(HGFのN末端ヘアピンループから第1クリングルドメインまでの配列)で示されるペプチドや、配列番号3で表されるヒトHGFアミノ酸配列のN末端側から32番目のアミノ酸から288番目のアミノ酸までのアミノ酸配列(HGFのN末端ヘアピンループから第2クリングルドメインまでの配列)で示されるペプチド等が好ましく挙げられる。
本発明のHGF蛋白質またはその部分ペプチドはフリー(遊離体)の状態であっても、また塩の形態を有していてもよい。本発明に用いられるHGF蛋白質またはその部分ペプチドの塩としては、酸または塩基との生理学的に許容される塩が挙げられ、とりわけ生理学的に許容される酸付加塩が好ましい。この様な塩としては、例えば、無機酸(例えば、塩酸、リン酸、臭化水素酸、硫酸等)との塩、あるいは有機酸(例えば、酢酸、ギ酸、プロピオン酸、フマル酸、マレイン酸、コハク酸、酒石酸、クエン酸、リンゴ酸、蓚酸、安息香酸、メタンスルホン酸、ベンゼンスルホン酸等)との塩等が挙げられる。
本発明において「HGF蛋白質をコードするDNA」とは、前述のHGF蛋白質を発現し得るDNAをいう。HGF蛋白質をコードするDNAを含むDNAとしては、例えば、Nature,342,440(1989);特許第2777678号公報;Biochem. Biophys. Res. Commun., 1989年,第163巻,p.967-973;Proc. Natl. Acad. Sci. U.S.A.,1991年,第88巻(16号),p.7001-7005等に記載され、例えば、GenBank/EMBL/DDBJにAccession No. M69718、M73240、AC004960、AY246560、M29145またはM73240等として登録されているヒト由来のHGF蛋白質をコードするDNA等が好ましく挙げられる。
本発明の腱骨移行部組織または靭帯骨移行部組織の再生促進剤には、前記HGF蛋白質をコードするDNAと相補的な塩基配列からなるDNAとストリンジェントな条件下でハイブリダイズするDNAであって、前記HGF蛋白質と同様に、腱骨移行部組織または靭帯骨移行部組織の再生促進作用を有する蛋白質をコードするDNA等が包含されていてもよい。このようなDNAとしては、配列番号1または2で表わされる塩基配列を有するDNAと相補的な塩基配列からなるDNAとストリンジェントな条件下でハイブリダイズするDNAであって、腱骨移行部組織または靭帯骨移行部組織の再生促進作用を有する蛋白質をコードするDNA等が好ましく挙げられる。
本発明の腱骨移行部組織または靭帯骨移行部組織の再生促進剤には、前記HGF蛋白質の部分ペプチドであって前記HGF蛋白質と同様に腱骨移行部組織または靭帯骨移行部組織の再生促進作用を有するペプチドをコードするDNAが包含されていてもよい。ここでも、「腱骨移行部組織または靭帯骨移行部組織の再生促進作用を有する」とは、前述と同様に評価することができる。
本発明の腱骨移行部組織または靭帯骨移行部組織の再生促進剤には、前述のHGF蛋白質の部分ペプチドであって、腱骨移行部組織または靭帯骨移行部組織の再生促進作用を有するペプチドをコードするDNAと相補的な塩基配列からなるDNAとストリンジェントな条件下でハイブリダイズするDNAであって、腱骨移行部組織または靭帯骨移行部組織の再生促進作用を有するペプチドをコードするDNA等が包含されていてもよい。
「腱」は、通常、骨格筋と骨とを仲介する組織として説明でき、また「靭帯」は、通常、骨と骨とを仲介する組織として説明できる。但し、本発明において「腱」は、骨と骨とを仲介する靭帯を包含して称する場合もある。また、本発明における腱は、移植片の腱(以下、「移植腱」という場合もある)を含み、本発明における靭帯は、移植片の靭帯(以下、「移植靭帯」という場合もある)を含む。
(1)移植腱または移植靭帯と骨との間に方向性のない肉芽組織が形成される過程、
(2)移植腱または移植靭帯と骨との間にコラーゲン線維が形成される過程、
(3)コラーゲン線維が骨方向に方向性を有するようになる過程、
(4)移植腱または移植靭帯から骨組織のなかに入り込む貫通線維(sharpey like fiber)が出現する過程、
(5)貫通線維が成熟する過程、
を含む。靭帯再建術以外における再生についても同様に説明される。
本発明の腱骨移行部組織または靭帯骨移行部組織の再生促進剤は、下記に示すように有効成分の種類によって、(a)HGF蛋白質/部分ペプチドを有効成分とする再生促進剤と、(b)HGF遺伝子を有効成分とする再生促進剤に分類することができる。
上記(2)で説明するHGF蛋白質、(3)で説明するHGF蛋白質の部分ペプチド(HGF部分ペプチド)、またはこれらの少なくとも一方の塩(これらを以下、「HGF蛋白質/部分ペプチド」と称する場合がある)を有効成分として含有する再生促進剤。
上記(4)で説明するHGF蛋白質をコードするDNA、(5)当該DNAとストリンジェントな条件下でハイブリダイズするDNA、(6)で説明するHGF部分ペプチドをコードするDNA、(7)当該DNAとストリンジェントな条件でハイブリダイズするDNA(以下、これらを「HGF遺伝子」と総称する)を有効成分として含有する再生促進剤。
当該(a)の再生促進剤は、例えば液剤や固形剤等の種々の製剤形態をとりうるが、一般的にはHGF蛋白質、HGF部分ペプチド、またはそれらの塩を慣用の担体と共に注射剤、噴射剤、徐放性製剤(例えば、デポ剤)等の形態に製剤化されるのが好ましい。ここで注射剤や噴射剤は、水性製剤または油性製剤のいずれでもよい。
HGF遺伝子を患者に投与する場合には、常法、例えば別冊実験医学,遺伝子治療の基礎技術,羊土社,1996、別冊実験医学,遺伝子導入&発現解析実験法,羊土社,1997、日本遺伝子治療学会編遺伝子治療開発研究ハンドブック、エヌ・ティー・エス,1999等に記載の方法に従って、行うことが好ましい。
ウサギ自家移植腱モデルにおける腱骨移行部組織再生に対するHGF蛋白質の効果
方法:日本白色家兎(2.5~3.0kg)にミタゾラム(ドルミカム:アステラス株式会社製)3mgと塩酸メデトミジン(ドミトール:日本全薬株式会社製)1mgを筋注後に、ペントバルビタールナトリウム(ソムノペンチル:共立製薬株式会社製)約260mg/時間を持続静脈内投与することにより麻酔を施した。後肢を除毛、消毒し、清潔操作下に両後肢各々において次の手術を行った。まず、後肢前面の皮膚を約5cm切開し、大腿骨外顆に付着する長趾伸筋(extension digitorum longus;以下、EDLと略記する。)腱を大腿骨起始(付着)部で切離した。次いで、脛骨の外側面を展開し、直径2.5mmのドリルを用いて、脛骨近位部外側面から内側面に向けて貫通孔(以下、これを「骨トンネル」という)を作製した。先に切離したEDL腱(移植腱)の遊離端を、図1に示すように、脛骨外側面から骨トンネル内に引き込み、脛骨内側面に引き抜き、ステンレス製ワッシャー(ジンマー株式会社製)と3-0ナイロン糸(株式会社ベアーメディック社製)を用いて固定した。固定時の肢位は足関節中間位とした。移植腱を引き込んだ骨トンネル内を充分に生理食塩液(大塚製薬株式会社製)で洗浄した。次いで、右後肢の移植腱と骨トンネルとの接触面及び間隙に、HGF蛋白質10μgを10μLの生理食塩水に溶解したもの10μLを含ませた海綿骨を移植した(HGF投与群)。なお、ここでHGF蛋白質として、配列番号6に示されるアミノ酸配列からなるHGF蛋白質を用いた。左後肢の移植腱と骨トンネルとの接触面及び間隙に、生理食塩水10μLを含ませた海綿骨を移植した(コントロール群)。海綿骨は骨トンネル作製時に、採取したものを用いた(0.05g)。海綿骨を移植後、創を縫合し、閉鎖した。術後、ウサギをケージに戻し、四肢の拘束や固定等せずに自由に運動させた。
摘出した組織を、10%(v/v)ホルマリン液で2日間保存後に生理食塩水で洗浄し、80%(v/v)メチルアルコールに2日間浸漬した。次いで、ブランクリュクロ液を用いて、7日間かけて脱灰を行った。脱灰後、脛骨の骨トンネル長軸に沿って切片を作成した。作成した切片を、ヘマトキシリン-エオジン(HE)染色及びマッソントリクローム染色を実施した。染色された切片を光学顕微鏡で観察し、以下の基準でスコア化した。
-:インターフェース(移植腱と骨トンネルとの接触面及び間隙)に、新しい組織の形成が認められない。
++:インターフェースに、骨方向に向かって方向性を持つコラーゲン線維が現れる
+++:インターフェースの方向性を持つコラーゲン線維の間に貫通線維が現れる
++++:貫通線維が太く成熟する。
生体力学的検討は、移植腱が骨トンネルに付着して形成された腱骨移行部組織の破断強度を比較することにより実施した。破断強度は、骨トンネルから移植腱が引き抜かれるときの最大荷重(ニュートン;N)を測定し、骨トンネル長1mmに対する最大荷重として、以下の式から算出した。
最大荷重は、引張試験機(インストロン社製、モデル4482)を用いて、摘出した組織の脛骨を引張試験機に固定した後、EDLを把持して骨トンネル長軸方向に引張り、骨トンネルから移植腱が引き抜かれるときの荷重とした。なお、移植腱が骨トンネルから引き抜かれる前に断裂したものは、除外した。
ウサギ自家移植腱モデルにおける腱骨移行部組織再生に対するHGF蛋白質の効果
上記で使用したHGF蛋白質に代えて、配列番号4に示されるアミノ酸配列からなるHGF蛋白質を用いて、上記実施例1と同様に実験を行った。その結果、実施例1と同様に、HGF蛋白質に腱骨移行部組織または靭帯骨移行部組織の再生促進作用が認められた。
生理食塩水100mL中に、HGF蛋白質lmg、マンニトール1g及びポリソルベー卜80を10mg含む溶液を無菌的に調製し、これを1mLずつバイアルに分注した後、凍結乾燥して密封することにより、凍結乾燥製剤の形態を有する再生促進剤を得た。
0.02Mリン酸緩衝液(0.15M NaCl及び0.01%ポリソルベート80含有、pH7.4)100mL中に、HGF蛋白質(配列番号6)1mg及びヒト血清アルブミン100mgを含む水溶液を無菌的に調製し、これを1mLずつバイアルに分注した後、凍結乾燥して密封することにより、凍結乾燥製剤の形態を有する再生促進剤を得た。
乳酸-グリコール酸共重合体(乳酸/グリコール酸=50/50,重量平均分子量=10,000;和光純薬工業株式会社製)1.9gをジクロロメタン3.0mLに溶解する。この有機溶媒液に、HGF蛋白質の凍結乾燥粉末100mgを添加し、ミキサーミル(株式会社レッチェ)を用いて微粒化し、HGF分散液を調製する。この分散液を0.1w/v%ポリビニルアルコール水溶液800mLに添加し、ホモミキサーを用いて撹拌・乳化する。室温で3時間撹拌してジクロロメタンを揮散させた後、遠心分離(約2,000rpm)することによりマイクロカプセルを分取する。次いで蒸留水400mLを用いて2回洗浄後、D-マンニトール0.2gを添加し凍結乾燥する。凍結乾燥後、更に残留溶媒を除去するため、40℃下で3日間真空乾燥してHGF蛋白質を含有する徐放性マイクロカプセルを得る(生体内分解性高分子に対するHGFの配合比率:5.3w/w%)。
乳酸-グリコール酸共重合体(乳酸/グリコール酸=50/50,重量平均分子量=10,000;和光純薬工業株式会社製)1.89gと酸化亜鉛10mgとをジクロロメタン3.0mLに溶解する。この有機溶媒液に、HGF蛋白質の凍結乾燥粉末100mgを添加し、ミキサーミル(株式会社レッチェ)を用いて微粒化し、HGF分散液を調製する。この分散液を0.1w/v%ポリビニルアルコール水溶液800mLに添加し、ホモミキサーを用いて撹拌・乳化する。室温で3時間撹拌してジクロロメタンを揮散させた後、遠心分離(約2,000rpm)することによりマイクロカプセルを分取する。次いで蒸留水400mLを用いて2回洗浄後、D-マンニトール0.2gを添加し凍結乾燥する。凍結乾燥後、更に残留溶媒を除去するため、40℃で3日間真空乾燥してHGF蛋白質を含有する徐放性マイクロカプセルを得る(生体内分解性高分子に対するHGFの配合比率:5.3w/w%)。
乳酸-グリコール酸共重合体(乳酸/グリコール酸=75/25,重量平均分子量=15,000;和光純薬工業株式会社製)1.7gをジクロロメタン2.7mLに溶解する。この有機溶媒液に、HGF蛋白質の凍結乾燥粉末300mgを添加し、ミキサーミル(株式会社レッチェ)を用いて微粒化し、HGF分散液を調製する。この分散液を0.1w/v%ポリビニルアルコール水溶液800mLに添加し、ホモミキサーを用いて撹拌・乳化する。室温で3時間撹拌してジクロロメタンを揮散させた後、遠心分離(約2,000rpm)することによりマイクロカプセルを分取する。次いで蒸留水400mLを用いて2回洗浄後、D-マンニトール0.2gを添加し凍結乾燥する。凍結乾燥後、更に残留溶媒を除去するため、40℃で3日間真空乾燥して、HGF蛋白質を含有する徐放性マイクロカプセルを得る(生体内分解性高分子に対するHGFの配合比率:17.6w/w%)。
乳酸-グリコール酸共重合体(乳酸/グリコール酸=75/25,重量平均分子量=15,000;和光純薬工業株式会社製)1.69gと酸化亜鉛10mgとをジクロロメタン2.7mLに溶解する。この有機溶媒液に、HGF蛋白質の凍結乾燥粉末300mgを添加し、ミキサーミル(株式会社レッチェ)を用いて微粒化し、HGF分散液を調製する。この分散液を0.1w/v%ポリビニルアルコール水溶液800mLに添加し、ホモミキサーを用いて撹拌・乳化する。室温で3時間撹拌してジクロロメタンを揮散させた後、遠心分離(約2,000rpm)することによりマイクロカプセルを分取する。次いで蒸留水400mLを用いて2回洗浄後、D-マンニトール0.2gを添加し凍結乾燥する。凍結乾燥後、更に残留溶媒を除去するため、40℃で3日間真空乾燥して、HGF蛋白質を含有する徐放性マイクロカプセルを得る(生体内分解性高分子に対するHGFの配合比率:17.8w/w%)。
DL-乳酸重合体(乳酸/グリコール酸=100/0,重量平均分子量=5,000;和光純薬工業株式会社製)5gを塩化メチレン50mLに溶解し、10w/v%の溶液を調製する。次いで、この溶液にHGF蛋白質の凍結乾燥粉末2.5mgを添加する。これを別に40℃に加温しておいた0.5w/v%キトサン水溶液に加え、ホモミキサーを用いて1000rpmの撹拌速度で撹拌し乳化する。得られる乳化液を室温で更に3時間撹拌して塩化メチレンを蒸散させ、次いで、遠心分離(約2,000rpm)して生成したマイクロスフィアを集め、これを予め40℃に加温しておいた蒸留水を用いて5回洗浄し、次いで室温で減圧乾燥し、HGFを含有するマイクロスフィアを得る(生体内分解性高分子に対するHGFの配合比率:0.05w/w%)。
乳酸-グリコール酸共重合体(乳酸/グリコール酸=75/25,重量平均分子量=5,000;和光純薬工業株式会社製)10gを塩化メチレン:エタノール(4:1)200mLに溶解し、5w/v%の溶液を調製する。次いで、この溶液にHGF蛋白質の凍結乾燥粉末2.5mgを添加する。これを、別に40℃に加温しておいた1w/v%ゼラチン水溶液の中に、500rpmの速度でホモミキサーを用いて攪拌しながら、少量ずつ加え乳化する。得られる乳化液を室温で更に3時間撹拌して塩化メチレンとエタノールを蒸散させ、次いで、遠心分離(約2,000rpm)して生成したマイクロスフィアを集める。回収したマイクロスフィアを、予め40℃に加温しておいた蒸留水を用いて5回洗浄し、室温で減圧乾燥し、HGF蛋白質を含有するマイクロスフィアを得る(生体内分解性高分子に対するHGFの配合比率:0.025w/w%)。
2w/v%HGF含有水溶液0.2mLと2w/v%アテロコラーゲンのリン酸緩衝液溶液2mLを混合した後、凍結乾燥を行う。なお、ここでHGF含有水溶液は、実験例1に記載する方法で調製することができる。得られた凍結乾燥品は、液体窒素を用いて低温で粉砕した後、金型にいれて圧縮成型し、円柱状のHGF含有徐放性製剤を得る(生体内分解性高分子に対するHGFの配合比率:10w/w%)。
0.01w/v%HGF含有水溶液100mLと2w/v%コラーゲン水溶液50gを均一に混合攪拌し、凍結乾燥する。その後、液体窒素を用いて低温粉砕する。これを棒状に圧縮成型し、HGF蛋白質を含有する徐放性製剤を得る(生体内分解性高分子に対するHGFの配合比率:1w/w%)。
HGF蛋白質1mgを、2w/v%アテロコラーゲン溶液2mLに溶解した後、凍結乾燥を行う。得られた凍結乾燥物を粉砕した後、円柱状に圧縮成型し、HGF蛋白質を含有する徐放性製剤を得る(生体内分解性高分子に対するHGFの配合比率:2.5質量%)。
ヒアルロナンのナトリウム塩(極限粘度数45000cc/g)0.58gを20mLの水と混合し、膨潤させる。次にこの混合物に、2N水酸化ナトリウム2mLを加え、撹拌して均一な溶液とする。これに、2.4mLの水に0.10gのジビニルスルホンを加えて撹拌して調製した溶液を加え混合物とし、その混合物を70分放置し、得られるゲルをバイオトリス緩衝液(リン酸塩緩衝の0.15M NaCl,pH約7.2)の223mL中に入れ、3時間膨潤させる。次に膨潤させたゲルに1mLの2N HClを加える。1時間後に、0.6mLの2N HClを加え、16時間放置した。0.35mLの2N HClを加え、膨潤ゲルを緩衝液中3日間ゆっくり撹拌する。均一な粘弾性の柔らかなゲルが得られ、これを0.15M NaClで5日間透析する。このゲルを、緩衝食塩水中の1w/v%HGFと混合して、HGF蛋白質の最終濃度を0.25w/v%とし、HGF含有製剤を得る(生体内分解性高分子に対するHGFの配合比率:約25w/v%)。
5 骨トンネル
10 ステンレス製ワッシャー
12 長趾伸筋(EDL)
14 脛骨外側面
16 脛骨内側面
18 脛骨近位部
20 脛骨
22 大腿骨外顆
24 腓骨
Claims (10)
- 下記(1)または(2)を有効成分として含有することを特徴とする腱骨移行部組織または靭帯骨移行部組織の再生促進剤:
(1)下記(1-a)もしくは(1-b)または(1-c)、
(1-a)HGF(Hepatocyte Growth Factor)蛋白質、
(1-b)HGF蛋白質の部分ペプチドであって、腱骨移行部組織または靭帯骨移行部組織の再生促進作用を有するペプチド、
(1-c)(1-a)または(1-b)の塩、
(2)下記(2-a)もしくは(2-b)または(2-c)を含むDNA、
(2-a)HGF蛋白質をコードするDNA、
(2-b)HGF蛋白質の部分ペプチドであって、腱骨移行部組織または靭帯骨移行部組織の再生促進作用を有するペプチドをコードするDNA、
(2-c)前記(2-a)または(2-b)のDNAと相補的な塩基配列からなるDNAとストリンジェントな条件下でハイブリダイズし、かつ腱骨移行部組織または靭帯骨移行部組織の再生促進作用を有する蛋白質またはペプチドをコードするDNA。 - 有効成分が、下記(1-a)もしくは(1-b)または(1-c)であることを特徴とする請求項1に記載の再生促進剤:
(1-a)HGF蛋白質、
(1-b)HGF蛋白質の部分ペプチドであって、腱骨移行部組織または靭帯骨移行部組織の再生促進作用を有するペプチド、
(1-c)(1-a)または(1-b)の塩。 - HGF蛋白質が、下記(1-d)または(1-e)であることを特徴とする請求項1に記載の再生促進剤:
(1-d)配列番号3または4で表されるアミノ酸配列からなる蛋白質、
(1-e)配列番号3または4で表されるアミノ酸配列と少なくとも85%の同一性を有するアミノ酸配列を有し、腱骨移行部組織または靭帯骨移行部組織の再生促進作用を有する蛋白質。 - 有効成分が、下記(2-a)もしくは(2-b)または(2-c)を含むDNAであることを特徴とする請求項1に記載の再生促進剤:
(2-a)HGF蛋白質をコードするDNA、
(2-b)HGF蛋白質の部分ペプチドであって、腱骨移行部組織または靭帯骨移行部組織の再生促進作用を有するペプチドをコードするDNA、
(2-c)前記(2-a)または(2-b)のDNAと相補的な塩基配列からなるDNAとストリンジェントな条件下でハイブリダイズし、かつ腱骨移行部組織または靭帯骨移行部組織の再生促進作用を有する蛋白質もしくはペプチドをコードするDNA。 - HGF蛋白質をコードするDNAが、下記(2-d)または(2-e)であることを特徴とする請求項1に記載の再生促進剤:
(2-d)配列番号1または2で表される塩基配列からなるDNA、
(2-e)配列番号1または2で表される塩基配列と相補的な塩基配列からなるDNAとストリンジェントな条件下でハイブリダイズし、かつ腱骨移行部組織または靭帯骨移行部組織の再生促進作用を有する蛋白質をコードするDNA。 - DNAが、I型単純ヘルペスウイルス(HSV-1)ベクター、センダイウイルス・エンベロープ(HVJ-E)ベクター、アデノウイルスベクターまたはアデノ随伴ウイルスベクターに組み込まれていることを特徴とする請求項1に記載の再生促進剤。
- 局所適用形態を有する請求項1に記載の再生促進剤。
- 腱骨移行部組織または靭帯骨移行部組織の再生促進剤の製造の為の、下記(1)または(2)の使用:
(1)下記(1-a)もしくは(1-b)または(1-c)、
(1-a)HGF蛋白質、
(1-b)HGF蛋白質の部分ペプチドであって、腱骨移行部組織または靭帯骨移行部組織の再生促進作用を有するペプチド、
(1-c)(1-a)または(1-b)の塩、
(2)下記(2-a)もしくは(2-b)または(2-c)を含むDNA、
(2-a)HGF蛋白質をコードするDNA、
(2-b)HGF蛋白質の部分ペプチドであって、腱骨移行部組織または靭帯骨移行部組織の再生促進作用を有するペプチドをコードするDNA、
(2-c)前記(2-a)または(2-b)のDNAと相補的な塩基配列からなるDNAとストリンジェントな条件下でハイブリダイズし、かつ腱骨移行部組織または靭帯骨移行部組織の再生促進作用を有する蛋白質またはペプチドをコードするDNA。 - 腱骨移行部組織または靭帯骨移行部組織の再生を促進する方法に使用される、下記(1)または(2):
(1)下記(1-a)もしくは(1-b)または(1-c)、
(1-a)HGF蛋白質、
(1-b)HGF蛋白質の部分ペプチドであって、腱骨移行部組織または靭帯骨移行部組織の再生促進作用を有するペプチド、
(1-c)(1-a)または(1-b)の塩、
(2)下記(2-a)もしくは(2-b)または(2-c)を含むDNA、
(2-a)HGF蛋白質をコードするDNA、
(2-b)HGF蛋白質の部分ペプチドであって、腱骨移行部組織または靭帯骨移行部組織の再生促進作用を有するペプチドをコードするDNA、
(2-c)前記(2-a)または(2-b)のDNAと相補的な塩基配列からなるDNAとストリンジェントな条件下でハイブリダイズし、かつ腱骨移行部組織または靭帯骨移行部組織の再生促進作用を有する蛋白質またはペプチドをコードするDNA。 - 腱骨移行部組織または靭帯骨移行部組織損傷患者に、下記(1)または(2)を投与することを特徴とする腱骨移行部組織または靭帯骨移行部組織の再生促進方法:
(1)下記(1-a)もしくは(1-b)または(1-c)、
(1-a)HGF蛋白質、
(1-b)HGF蛋白質の部分ペプチドであって、腱骨移行部組織または靭帯骨移行部組織の再生促進作用を有するペプチド、
(1-c)(1-a)または(1-b)の塩、
(2)下記(2-a)もしくは(2-b)または(2-c)を含むDNA、
(2-a)HGF蛋白質をコードするDNA、
(2-b)HGF蛋白質の部分ペプチドであって、腱骨移行部組織または靭帯骨移行部組織の再生促進作用を有するペプチドをコードするDNA、
(2-c)前記(2-a)または(2-b)のDNAと相補的な塩基配列からなるDNAとストリンジェントな条件下でハイブリダイズし、かつ腱骨移行部組織または靭帯骨移行部組織の再生促進作用を有する蛋白質またはペプチドをコードするDNA。
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Also Published As
Publication number | Publication date |
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EP2351574B1 (en) | 2016-08-24 |
US20110312887A1 (en) | 2011-12-22 |
US8927493B2 (en) | 2015-01-06 |
EP2351574A1 (en) | 2011-08-03 |
EP2351574A4 (en) | 2012-03-28 |
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