WO2010008023A1 - Bone elongation promoter - Google Patents

Abstract

The object aims to promote the bone elongation in an interbone gap after osteotomy or in bone lengthening.  Disclosed is a bone elongation promoter characterized by comprising, as an active ingredient: HGF protein, a partial peptide thereof having substantially the same bone elongation promoting activity as that of HGF protein, or a salt of HGF protein or the partial peptide thereof; or DNA comprising DNA which encodes HGF protein, DNA comprising DNA which encodes a partial peptide of HGF protein having substantially the same bone elongation promoting activity as that of HGF protein, or DNA comprising DNA which can hybridize with DNA comprising a nucleotide sequence complementary to any one of the foregoing two DNA molecules under stringent conditions and encodes a protein or peptide having substantially the same bone elongation promoting activity as that of HGF protein.

Description

Bone elongation promoter

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.

For short stature patients such as short stature and dwarfism, or for patients with large difference in length of upper limbs or lower limbs due to accidents or surgery etc., osteotomy or bone extension for the purpose of correction or correction Is done. However, in osteotomy, the length of bone that can be extended by a single operation is limited, and repeated osteotomy is required to extend the bone to the required length. For this reason, the burden on the patient is very large. 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. In this case, it takes about half a year to one year from the start of bone extension to the end of treatment, and during that time, not only must the external fixator be kept on, but the possibility of re-fracture is also high. large. 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. In particular, complex fractures, fractures, and the like have bone defects and may cause short limbs or deformation, for example.

For these reasons, in the field of orthopedics, there is a strong demand for drugs for promoting bone extension in osteotomy, bone extension and fracture treatment. As agents that promote bone healing, for example, basic fibroblast growth factor (bFGF), bone morphogenetic protein (BMP), and the like are known (for example, see Patent Documents 1 and 2).

On the other hand, hepatocyte growth factor (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. In Patent Document 3, HGF is exemplified as one of drugs that improve recovery of the sternum after sternotomy. However, no effect has been demonstrated. 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.

Japanese Patent Laid-Open No. 5-124975 (corresponding US Pat. No. 6,833,354) JP-T 2000-502336 (corresponding US Pat. No. 5,854,207, etc.) JP-T 2003-510289 (corresponding US Pat. No. 6732738)

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)”). Specifically, 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. Objective. 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.

As a result of intensive studies to solve the above-mentioned problems, the present inventors, as shown in the experimental examples to be described later, have a hepatocyte growth factor (hereinafter referred to as “HGF protein”, the protein) This gene is also referred to as “HGF gene”), and has been found to have an effect of promoting bone elongation in the above-mentioned osteotomy gap, thereby completing the present invention.

That is, the present invention has the embodiments described below.
(I) 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.

(I-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-5) The bone elongation promoter described in any of (I-1) to (I-4) having a local administration form.

(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.

(II) 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.

(II-4) The bone elongation promoter described in any of (II-1) to (II-3) having a local administration form.

(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.

(III) Use of 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 ID NO: 3 or 4.

(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.

(IV) HGF protein or DNA for promoting bone elongation
(IV-1) The following (1-a) to (1-g) to promote bone extension in the osteotomy gap of patients undergoing osteotomy or osteotomy, fracture patients, or patients undergoing osteoplasty Or a protein or peptide according to any one of
(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 ID NO: 3 or 4.

(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 elongation promoting action, having at least 85% identity with the amino acid sequence shown in SEQ ID NO: 3 or 4.

(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. To demonstrate. 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.

More specific effects include the following.

】 It is said that 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. However, according to 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.

Further, according to 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. Usually, in 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. In addition, there is a possibility of re-fracture in the meantime, and the burden on the patient is great. If the treatment period can be shortened by the bone elongation promoter of the present invention, the burden on the patient will be greatly reduced. Furthermore, 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.

Further, according to 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.

In the above osteotomy, osteogenesis and fracture treatment, 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. FIG. 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.

(1) Explanation of terms The abbreviations such as base sequences (nucleotide sequences) and nucleic acids in the present specification are defined by IUPAC-IUB [IUPAc-IUB communication on Biological Nomenclature, Eur. J. Biochem., 138; 9 ( 1984)], “Guidelines for the preparation of specifications including base sequences or amino acid sequences” (edited by the Patent Office) and conventional symbols in the field.

In this specification, “gene” refers to a regulatory region, a coding region, an exon, and an intron without distinction unless otherwise specified. In addition, the term “gene” in the present specification means not only DNA but also mRNA which is a transcript thereof.

In this specification, “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.

In the present invention, the term “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. Here, it is sufficient that 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. Note that “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.

In the present invention, the “bone cross section” refers to a surface (bone cut surface) from which bone has been cut. In addition, “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.

In the present invention, 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.

In the present invention, 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.

Also, “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. For example, Ilizarov method etc. can be illustrated as a treatment method.

In the present invention, 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.

(2) HGF protein “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. Alternatively, 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.

In the present invention, the HGF protein is preferably a protein produced from a gene encoding human-derived HGF (hHGF). As such a gene encoding hHGF, a DNA having a base sequence represented by SEQ ID NO: 1 or 2 is preferable.

As such an 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. Incidentally, the natural HGF protein is a glycoprotein. For example, 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.

The 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.

In addition, as long as the HGF protein targeted in the present invention has a bone elongation promoting action, one or a plurality (for example, 2 to 35, preferably 2 to 20) of the amino acid sequence shown in SEQ ID NO: 3 or 4 above. More preferably 2 to 10 amino acids) 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 γ-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.

Furthermore, 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. Here, “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.

In addition, as an 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.

In addition, as long as the HGF protein targeted in the present invention has a bone elongation promoting action, 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. Examples of such signal sequences include signal sequences of human serum albumin, interferon, human amylase and the like.

In addition, 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

Here, 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 As 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. Further, 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.

As long as the HGF protein targeted by the present invention has a bone elongation promoting action, 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. Examples of 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. In addition, those in which 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. As such 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. HGF protein in which sugar chains are prevented from being added by replacing Thr at position 471 with Gly, Asn at position 561 with Gln, and Asn at position 648 with Gln [Fukuta K et al., Biochemical Journal, 388, 555- 562 (2005)].

In the HGF protein targeted by the present invention, 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. Here, 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 α-naphthyl; C _7- such as phenyl-C _1-2 alkyl groups such as benzyl and phenethyl or α-naphthyl-C _1-2 alkyl groups such as α-naphthylmethyl; _{A 14} aralkyl group and a C _2-6 alkanoylmethyl group such as acetyloxymethyl, pivaloyloxymethyl and the like are used. In addition, 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. . Examples of the ester in this case include the aforementioned C-terminal ester.

Furthermore, in the HGF protein targeted by the present invention, 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.). A group protected by an acyl group, the N-terminal side cleaved in vivo and a glutamyl group produced by pyroglutamine oxidation, a reactive group on the side chain of an amino acid in the molecule (eg, —OH, —SH) , 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). Or a complex protein such as a so-called glycoprotein to which a sugar chain is bound.

In addition, as for 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. Examples of such 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, 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).

These 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. Particularly preferred are physiologically acceptable acid addition salts. Examples of such 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.

Further, in the bone elongation promoter of the present invention, 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.

(2) Partial peptide of HGF protein 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.

Specifically, 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). A peptide consisting of the amino acid sequence from the 32nd amino acid to the 288th amino acid of the amino acid sequence represented by SEQ ID NO: 3 (sequence from the N-terminal hairpin loop of HGF to the second kringle domain). Can be listed. Further, 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.

In the HGF partial peptide of the present invention, 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. Furthermore, in the HGF partial peptide, 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. And those in which 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. As 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. Bodanszky and MAOndetti, Peptide Synthesis, Interscience Publishers, New York (1066), Schroeder and Luebke, The Peptide. ), Academic Press, New York (1065), and the like. After the reaction, 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.

When 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. Here, examples of the salt of the HGF partial peptide include physiologically acceptable salts with an acid or a base, like the HGF protein.

(3) DNA encoding HGF protein
In the present invention, “DNA encoding HGF protein” refers to DNA capable of expressing the aforementioned HGF protein. Examples of 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.).

Examples of such DNA 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.).

As a specific example of DNA encoding the HGF protein, for example, DNA consisting of the base sequence represented by SEQ ID NO: 1 or 2 is preferably mentioned. Here, 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. It corresponds to DNA encoding the HGF protein consisting of In the DNA recombination technique, 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. M73240, and 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. Here, 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 “.

Here, 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. In addition, a cDNA library prepared from a cell line expressing HGF protein and tissue material according to a conventional method can also be used. A λ 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.

In the present invention, 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. Examples of 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.

As described in (b) of (5) Bone Elongation Promoter described later, 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. To be administered. Such 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. Or DNA viruses or RNA viruses, such as immunodeficiency virus (HIV), are mentioned. Among them, 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.

(4) 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.

More specifically, 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.

As such 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.

Further, in the present invention, 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. Can do. Examples of 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.

(5) Bone elongation promoter The 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.

(A) 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.

(B) 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.

(A) 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. Here, the injection or propellant may be either an aqueous preparation or an oily preparation.

In the case of an aqueous injection, according to a known method, for example, an aqueous solvent (water for injection, purified water, etc.) is added to 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) , Hydroxypropylcellulose, polyvinyl alcohol, polyethylene glycol, etc.), stabilizers (sucrose, sodium bisulfite, sodium thiosulfate, sodium edetate, sodium citrate, ascorbic acid, dibutylhydroxytoluene, etc.) or pH adjusters ( By dissolving HGF protein or HGF partial peptide in a solution to which hydrochloric acid, sodium hydroxide, phosphoric acid, acetic acid, etc.) are appropriately added, and then sterilizing by filtration with a filter, etc., and then filling in an aseptic container Can be prepared.

Further, 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. Good. In the case of an oily injection, for example, sesame oil or soybean oil is used as the oily solvent, and benzyl benzoate or benzyl alcohol may be blended as a solubilizing agent. The prepared injection solution is usually filled in an appropriate ampoule or vial. 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. When the HGF protein or HGF partial peptide is produced as a spray, the additive blended in the spray may be any additive generally used in inhalation preparations, for example, In addition to the propellant, the above-mentioned solvent, preservative, stabilizer, tonicity agent, pH adjuster and the like can be blended. Examples of the propellant include a liquefied gas propellant or a compressed gas. Examples of 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. Examples of the compressed gas 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. By using the HGF protein or HGF partial peptide as 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. 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; polylactic acid, polyglycolic acid, lactic acid-glycolic acid copolymer; polycaprolactone, poly-β-hydroxybutyric acid, polymalic acid, polyanhydride Or polyester such as fumaric acid / polyethylene glycol / vinyl pyrrolidone copolymer; polyorthoester or polyalkylcyanoacrylic acid such as polymethyl-α-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. When a lactic acid-glycolic acid copolymer is used, the composition ratio (lactic acid / glycolic acid) (mol%) varies depending on the sustained release period. For example, 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. For example, 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.

As an administration method, 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. Usually, the content of the HGF protein or HGF partial peptide contained in the bone elongation promoter of the present invention is usually 0. .1 μg to 500 mg, preferably 1 μg to 50 mg, more preferably 10 μg to 25 mg. In addition, 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.

(B) When the HGF gene, which is a bone elongation promoter containing the HGF gene as an active ingredient , is administered to a patient, 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.). .

Here, as 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). Among them, 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. As the 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. For example, in the case of an injection, 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.

In the case of producing a microcapsule, for example, 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 μm. Examples of the coating material 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.

For example, in osteotomy, 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. In the osteotomy, 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). According to 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.

Favorable examples of osteogenesis include Ilizarov method. In the bone extension, after cutting the bone, 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. As described above, 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. When 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. According to the bone extension, the bone can be extended to a total of about 10 cm.

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

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.

As described above, 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.

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.

Hereinafter, the present invention will be described using experimental examples and formulation examples, but the present invention is not limited thereto.

[Experimental Example 1]
Effect of HGF protein on bone elongation in rabbit osteotomy model (1) Experimental method Nippon White Rabbit (2.5-3.0kg), Ketamine Hydrochloride (Daiichi Sankyo Propharma Co., Ltd .; 35mg / kg) and Xylazine (Bayer Yakuhin Co., Ltd .; 5 mg / kg) was administered subcutaneously, followed by intravenous anesthesia with pentobarbital sodium (Abbott Laboratories; 40-50 mg / kg), and the skin in the anterior center of the right tibia Was incised longitudinally. The soft tissue and fascia around the periosteum were then removed and the rabbit tibia was cut with a bone saw. The cut tibia was pulled apart at an interval of 5 mm to create a 5 mm bone gap (gap) in the tibia. Next, as shown in FIG. 1, 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). 4) Inserted and fixed with a one-sided external fixator.

Immediately after mounting the external fixator and once a day from the next day for 1 week (total 8 times), 10 μg of HGF protein is dissolved in 10 μL of physiological saline in the bone gap (gap between the cut tibias). The prepared HGF-containing aqueous solution was injected transcutaneously (HGF administration group). Here, the HGF protein consisting of the amino acid sequence shown in SEQ ID NO: 6 was used as the HGF protein. As a control, 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).

In the 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).

(2) Experimental results The results are shown in 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). . In 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), whereas 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.

[Experimental example 2]
Effect of HGF protein on bone elongation in rabbit osteotomy model The same experiment as described above was performed using the HGF protein consisting of the amino acid sequence shown in SEQ ID NO: 4 instead of the HGF protein used above. As a result, as in Experimental Example 1, the HGF protein was found to promote bone elongation and bone fusion at the bone cut surface.

Hereinafter, formulation examples of the bone elongation promoter of the present invention will be described. In the preparation examples, 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.

[Formulation Example 1]
A solution containing 1 mg of HGF protein, 1 g of mannitol and 10 mg of polysorbate 80 is aseptically prepared in 100 mL of physiological saline, dispensed 1 mL each into a vial, freeze-dried and sealed, and then freeze-dried. A bone elongation promoter having the form of the preparation was obtained.

[Formulation Example 2]
An aqueous solution containing 1 mg of HGF protein (SEQ ID NO: 6) and 100 mg of human serum albumin in 100 mL of 0.02 M phosphate buffer (containing 0.15 M NaCl and 0.01% polysorbate 80, pH 7.4) was prepared aseptically. Was dispensed into vials in an amount of 1 mL, and freeze-dried and sealed to obtain a bone elongation promoter having the form of a freeze-dried preparation.

[Formulation Example 3]
1.9 g of lactic acid-glycolic acid copolymer (lactic acid / glycolic acid = 50/50, weight average molecular weight = 10,000; manufactured by Wako Pure Chemical Industries, Ltd.) is dissolved in 3.0 mL of dichloromethane. To this organic solvent solution, 100 mg of lyophilized powder of HGF protein is added and atomized using a mixer mill (Lecce Inc.) to prepare an HGF dispersion. This dispersion is added to 800 mL of 0.1 w / v% polyvinyl alcohol aqueous solution, and stirred and emulsified using a homomixer. After stirring at room temperature for 3 hours to volatilize dichloromethane, 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%).

[Formulation Example 4]
1.89 g of lactic acid-glycolic acid copolymer (lactic acid / glycolic acid = 50/50, weight average molecular weight = 10,000; manufactured by Wako Pure Chemical Industries, Ltd.) and 10 mg of zinc oxide are dissolved in 3.0 mL of dichloromethane. To this organic solvent solution, 100 mg of lyophilized powder of HGF protein is added and atomized using a mixer mill (Lecce Inc.) to prepare an HGF dispersion. This dispersion is added to 800 mL of 0.1 w / v% polyvinyl alcohol aqueous solution, and stirred and emulsified using a homomixer. After stirring at room temperature for 3 hours to volatilize dichloromethane, 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%).

[Formulation Example 5]
1.7 g of lactic acid-glycolic acid copolymer (lactic acid / glycolic acid = 75/25, weight average molecular weight = 15,000; manufactured by Wako Pure Chemical Industries, Ltd.) is dissolved in 2.7 mL of dichloromethane. To this organic solvent solution, 300 mg of lyophilized powder of HGF protein is added and atomized using a mixer mill (Lecce) to prepare an HGF dispersion. This dispersion is added to 800 mL of 0.1 w / v% polyvinyl alcohol aqueous solution, and stirred and emulsified using a homomixer. After stirring at room temperature for 3 hours to volatilize dichloromethane, 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%).

[Formulation Example 6]
1.69 g of lactic acid-glycolic acid copolymer (lactic acid / glycolic acid = 75/25, weight average molecular weight = 15,000; manufactured by Wako Pure Chemical Industries, Ltd.) and 10 mg of zinc oxide are dissolved in 2.7 mL of dichloromethane. To this organic solvent solution, 300 mg of lyophilized powder of HGF protein is added and atomized using a mixer mill (Lecce) to prepare an HGF dispersion. This dispersion is added to 800 mL of 0.1 w / v% polyvinyl alcohol aqueous solution, and stirred and emulsified using a homomixer. After stirring at room temperature for 3 hours to volatilize dichloromethane, 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%).

[Formulation Example 7]
5 g of DL-lactic acid polymer (lactic acid / glycolic acid = 100/0, weight average molecular weight = 5,000; manufactured by Wako Pure Chemical Industries, Ltd.) is dissolved in 50 mL of methylene chloride to prepare a 10 w / v% solution. Next, 2.5 mg of lyophilized powder of HGF protein is added to this solution. This is added to a 0.5 w / v% chitosan aqueous solution that has been heated to 40 ° C., and stirred and emulsified at a stirring speed of 1000 rpm using a homomixer. 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%).

[Formulation Example 8]
10 g of lactic acid-glycolic acid copolymer (lactic acid / glycolic acid = 75/25, weight average molecular weight = 5,000; manufactured by Wako Pure Chemical Industries, Ltd.) is dissolved in 200 mL of methylene chloride: ethanol (4: 1), and 5 w / v. % Solution is prepared. Next, 2.5 mg of lyophilized powder of HGF protein is added to this solution. This is emulsified by adding little by little into a 1 w / v% gelatin aqueous solution heated to 40 ° C. while stirring with a homomixer at a speed of 500 rpm. 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%).

[Formulation Example 9]
After mixing 0.2 mL of an aqueous solution containing 2 w / v% HGF and 2 mL of a phosphate buffer solution of 2 w / v% atelocollagen, lyophilization is performed. Here, the 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%).

[Formulation Example 10]
100 mL of a 0.01 w / v% HGF-containing aqueous solution and 50 g of a 2 w / v% collagen aqueous solution are uniformly mixed and stirred, and lyophilized. Thereafter, it is pulverized at low temperature using liquid nitrogen. This is compression-molded into a rod shape to obtain a sustained-release preparation containing HGF protein (HGF compounding ratio with respect to biodegradable polymer: 1 w / w%).

[Formulation Example 11]
1 mg of HGF protein 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. Next, 1 mL of 2N HCl is added to the swollen gel. After 1 hour, 0.6 mL of 2N HCl was added and left for 16 hours. Add 0.35 mL of 2N HCl and stir the swollen gel slowly in buffer for 3 days. A uniform viscoelastic soft gel is obtained, which is dialyzed against 0.15 M NaCl for 5 days. This gel is mixed with 1 w / v% HGF in buffered saline to obtain a final concentration of HGF protein of 0.25 w / v% to obtain an HGF-containing preparation (ratio of HGF to biodegradable polymer: About 25 w / v%).

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.

Claims (15)

1. An agent for promoting bone elongation in a bone cutting gap, comprising at least one of the following (1-a) to (1-c) as an active ingredient:
   (1-a) HGF (Hepatocyte Growth Factor) 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).
2. The bone elongation promoter according to claim 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 protein having a bone elongation-promoting action produced by a cell having 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 .
3. The bone elongation promoter according to claim 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.
4. The bone elongation promoter according to claim 3, wherein the protein described in (1-g) above is a protein consisting of the amino acid sequence represented by SEQ ID NO: 5 or 6.
5. A bone elongation promoting agent in a bone cutting gap, comprising as an active ingredient at least one DNA of any one of the following (2-a) to (2-c):
   (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.
6. The bone elongation promoter according to claim 5, 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.
7. A DNA selected from the group consisting of (2-a) to (2-c) according to claim 5 and (2-d) and (2-e) according to claim 6 is a type I herpes simplex virus An agent for promoting bone elongation, which is incorporated into an (HSV-1) vector, Sendai virus envelope (HVJ-E) vector, adenovirus vector or adeno-associated virus vector.
8. The bone elongation promoter according to claim 1 or 5, which has a local administration form.
9. The bone elongation promoter according to claim 1 or 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.
10. Use of the protein or peptide described in any of (1-a) to (1-g) below for the production of a bone elongation promoter:
    (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 ID NO: 3 or 4.
11. 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.
12. Any one of the following (1-a) to (1-g) to promote bone extension in the bone cutting gap of patients who have undergone osteotomy or osteogenesis, fracture patients, or patients who have undergone osteoplasty Protein or peptide to:
    (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 protein having a bone elongation-promoting action produced by a cell having 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 ,
    (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.
13. Any of the following (2-a) to (2-e) for promoting bone extension in the bone cutting gap of a patient undergoing osteotomy or osteotomy, fracture patient, or osteoplasty patient DNA to:
    (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.
14. The protein or peptide described in any one of the following (1-a) to (1-g) is added to a bone cutting gap or a bone cutting gap of an osteotomy or osteotomy patient, a fracture patient, or an osteoplasty patient. A method for promoting bone elongation, characterized by administration to the periphery thereof:
    (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 protein having a bone elongation-promoting action produced by a cell having 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 ,
    (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.
15. The protein or peptide described in any one of the following (2-a) to (2-e) is added to a bone cutting gap or a bone cutting gap of an osteotomy or osteotomy patient, a fracture patient, or an osteoplasty patient. A method for promoting bone elongation, characterized by administration to the periphery thereof:
    (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.

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