JPH07149659A - Therapeutic agent for bone injury - Google Patents

Abstract

PURPOSE:To obtain a therapeutic agent for various bone injuries including bone coloboma and fracture, comprising a BUF as an active ingredient. CONSTITUTION:This therapeutic agent for various bone injuries contains at least one component selected from BUF-3 (action A), BUF-4 (actibin AB) and BUF-5 (actibin B) as an active ingredient. BUFs promote formation of callus in a process of fracture cure and are useful for treating bone coloboma. A dose is 1ng to 5mg per adult and administered once to several times. The therapeutic agent for various bone injuries is capable of shortening treatment period of fracture diseases and improving strength of repaired bone. The therapeutic agent can be pharmaceutically manufactured into injection, tablet, capsule, etc., and mixed with polybutyric acid, polyglycol, etc., and pharmaceutically manufactured into a sustained release administrative agents. BUFs are obtained by culturing a cell of an eucaryote transformed with a plasmid containing a gene encoding BUFs in a culture solution.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a therapeutic agent for bone damage.
More specifically, BUFs (in the present invention, BUFs are BUFs
F-3, BUF-4 and BUF-5) as an active ingredient.

[0002]

2. Description of the Related Art Usually, bone union after a fracture requires a long period of 2 weeks or more, and in a femoral neck fracture, the average union time is as long as 12 weeks. Especially in the fracture of the elderly,
In general, bone union may take a long time and sufficient bone strength may not be obtained.In such a case, the patient becomes "bedridden" due to generalized weakening of the bone and deterioration of general condition associated with a long-term bed. Not a few. In addition, delay of bone union and union failure often occur due to other causes. However, in the treatment of bone fractures, the present condition is to rely on the natural healing ability of the living body except for reduction, fixation, infection defense, etc. of the fractured part, and the aim was to positively and completely heal the fracture. Pharmacological treatment is not usually given. However, if necessary, nutritional treatment such as calcium administration, estrogen,
Hormone treatments such as active vitamin D have been carried out, but there is a demand for the development of a drug capable of further accelerating fracture healing and increasing the strength of fused bones.

[0003]

In view of such circumstances, the present invention provides a new bone capable of shortening the healing period of various bone damage diseases including bone defects and fractures and improving the strength of repaired bone. It is intended to provide a therapeutic agent for damage.

[0004]

As a result of intensive studies to achieve the above object, the present inventors have found that BUFs are excellent therapeutic agents for bone damage, and have completed the present invention. It was That is, the present invention is a therapeutic agent for bone damage containing one or more components of BUF-3, BUF-4 and BUF-5 as an active ingredient.

BUF-3, BUF-4 and BUF-5 are activin A, activin AB and activin B, respectively.
Also called. The structure is such that BUF-3 is monomer A.
, BUF-4 has a heterodimer structure of monomers A and B, and BUF-5 has a homodimer structure of monomer B. The amino acid sequences of the monomers A and B are already known, but just in case, the amino acid sequence of the monomer A is shown in SEQ ID NO: 1 in the sequence listing, and the amino acid sequence of the monomer B is shown in the sequence listing. SEQ ID NO: 2 of each is shown. Well, B
UF-3 is a polypeptide derived from human leukemia cells, which was initially purified by the present inventors using the differentiation-inducing action on mouse friend leukemia cells F5-5 as an index (Japanese Patent Laid-Open Publication No. 6-58242).
2-240700). After that, BUF-4 to BUF-3
And BUFs added with BUF-5 have been developed, and BUFs have a blood glucose lowering action (see JP-A-2-88526, JP-A-63-255236) and anemia treatment effect (JP-A-2. -108627), and that it has a therapeutic effect on thrombocytopenia (JP-A-4-36084).
0), it has an action of promoting hair growth and hair nourishment (JP-A-3-27)
5615) and the fact that it has an egg-dividing action on fertilized eggs (Japanese Patent Laid-Open No. 173805/1993). Also, B
Whether UFs have been shown to be useful in the treatment of osteoporosis (JP-A-63-277629, JP-A-2-108626) is useful in treating bone damage such as fractures. Has not been reported at all.

In the present invention, it is considered that BUFs promote the formation of callus during the healing process of bone fracture and are effective for the treatment of bone damage. The dose of BUFs may be 1 ng to 5 mg per adult, usually once or multiple times. Of course, since the dose varies depending on the condition of the patient, the weight of the patient and other factors recognized by those skilled in the art, it is not necessary to strictly adhere to the above dose, and the dose may be flexibly determined.
In addition, the above doses are applicable to the case where only one kind selected from BUFs is administered and the case where two or more kinds are administered in combination. Furthermore, the therapeutic agent for bone damage of the present invention may be administered systemically (intravenous administration, intramuscular administration, subcutaneous administration, etc.) or locally to bone injury.

Formulation of the BUFs used in the present invention is carried out by a usual method. When preparing an injection, the BUFs as the main ingredient may be adjusted with a pH adjusting agent, a buffer, a stabilizer, if necessary.
An injection may be prepared by adding a preservative or the like. It is also possible to formulate into a dosage form such as a tablet or a capsule, and further to mix with polylactic acid, polyglycol or the like into a sustained release dosage form.

The therapeutic agent for bone damage of the present invention is not only for humans but also for domestic animals, competitive animals such as horses, companion animals including dogs and cats, captive wild animals and other bone fractures. It may be applied to the treatment of bone damage. In this case, the dosage form, dose and route of administration are basically the same as those for humans.

For the production of BUFs, a gene encoding BUFs, that is, a monomer A gene for BUF-3 (activin A), BUF-4 (activin AB) is used.
For the monomers A and B genes, BUF-5
For (activin B), a method may be used in which eukaryotic cells transformed with a plasmid containing the gene of monomer B are cultured in a culture medium to produce BUFs in the culture medium. (JP-A-63-258
0, see JP-A-2-108626). Alternatively, a method of causing human or animal-derived cells such as monocytic leukemia cells to produce BUFs in the presence of a stimulant such as phorbol ester may be used (Murata, M. et al .: Pro
c. Natl. Acad. Sci. USA. 1988, 85, 2434-2438). Purification of the BUFs thus produced is carried out according to a conventional method for purifying a polypeptide. For example, a crude polypeptide preparation can be obtained by concentrating the culture solution by an ultrafiltration method, salting out the polypeptide from this concentrated solution, performing dialysis and then performing ion exchange chromatography using an anion exchanger.
Contaminant proteins can be removed from this crude product by a hydrophobic chromatography method or a chromatofocusing method, and further purified by reverse layer high performance liquid chromatography.

The indications for the therapeutic agent for bone damage of the present invention include:
The following A to E are exemplified. That is, A) various traumatic fractures, B) various fatigue fractures, C) pathological fractures, D) cases in which healing of the fractures is prolonged due to various causes, or cases in which union is present, and pseudounion is formed. Case,
And E) Jaw bone damage due to alveolar pyorrhea. The C) pathological fracture is subdivided into Eri as follows. B) Fractures associated with osteoporosis (including micro-fractures within the prosthesis), and those associated with osteoporosis are primary osteoporosis (senile, postmenopausal, juvenile) and secondary osteoporosis (hyperthyroidism, Cushing's syndrome). "Including those caused by steroid administration", acromegaly, hypogonadism "hypopituitary dysfunction, Klinefelter syndrome, Turner syndrome", osteogenesis imperfecta, hypophosphatasia, homocystinuria,
Immobilized osteoporosis, diabetes). B) Fractures associated with osteomalacia, c) bone fractures associated with malignant tumors, d) bone fractures associated with multiple myeloma, and e) bone fractures associated with congenital osteogenesis imperfections, f)
Fractures associated with bone cysts, g) Fractures associated with purulent osteomyelitis,
H) Fractures associated with marble disease and i) Fractures associated with nutritional disorders.

The therapeutic agent for bone damage of the present invention includes other therapeutic agents for bone diseases (calcium agents, calcitonin, estrogen, ipriflavone, active vitamin D, etc.), anti-inflammatory agents, agents administered to prevent infection, etc. Also, it may be used in combination with other drugs. In addition, the agent of the present invention is a bone morphogenetic protein (BMP) -1, BMP-2, BMP-3, BMP-4, BMP-5, BMP-6, BMP-
7, transforming growth factor (TGF) -β1, TGF-β
2, other TGF-β3, fibroblast growth factor (FGF) and other factors that promote bone formation may be used in combination.

The embodiments of the present invention will be described below.
The present invention is not limited to the embodiments.

Example 1 Seven-week-old SD type female rats (6 rats) were fractured to the fibula on both sides, one side was the side to which BUFs were administered, and the BUF administration solution was locally administered to the fracture site, and the other side was The control administration liquid was locally administered as the control administration side. Acetate buffer containing BUF-3 (400 μg / ml) as a BUF administration solution (1
4.9 mM, pH 5) was prepared, and an acetate buffer (14.9 mM, pH 5) was used as a control administration solution. The specific procedure is shown below. Each animal was anesthetized with ether, the skin and muscle layers of the left and right lower limbs were incised, the fibula was exposed, and the central part of the fibula was cut with scissors. The cut fibula was reduced with tweezers, and 25 μl of the BUF-3 administration solution (containing 10 μg of BUF-3) and 25 μl of the control administration solution were dropped on each administration side using a pipette. After suturing the skin, 10,000 U of penicillin G potassium (0.1 ml, manufactured by Toyo Brewery) was subcutaneously administered. After that, 1 every day
For 10 days, 25 μl of the BUF-3 administration solution and 25 μl of the control administration solution were administered to the fracture site on each administration side using a microsyringe. On the 11th day after the fracture, the fibula on both sides of each animal was excised and the weight of the fibula was measured. The callus weight was determined by subtracting the weight of the fibula of each untreated rat of the same age from the weight of each fibula.

As shown in Table 1, the callus weight on the BUF-3 administration side increased by about 20% as compared with the control administration side.

[0015]

[Table 1]

(Example 2) It was examined whether BUFs promote fracture healing in aged rats with delayed fracture healing. Four
Bilateral fibula of 0-week-old SD type female rats (5 animals) was fractured, and one side of each animal was the BUFs administration side and the contralateral side was the control administration side. Preparation of the BUFs administration liquid was performed in the same manner as in Example 1, and 25 μl (10 μL) of the BUF-3 administration liquid was added to the BUFs administration side.
25 μl of the control administration solution was administered to the fracture administration site on the control administration side. The operation for cutting the fibula was performed in the same manner as in Example 1, and each administration liquid was dropped on each administration side using a pipette at the fibula cutting portion during the operation. For 13 days thereafter, each administration solution was administered to the fractured site once daily to each administration side using a microsyringe. On day 14 after the fracture, the fibula on both sides of each animal was excised and the weight and volume were measured. The volume of the fibula was determined by measuring the buoyancy with respect to water with an electronic balance. The callus weight and callus volume were determined by subtracting the weight and the volume of the fibula and the volume of the fibula of the same week-old rat, respectively.

As shown in Table 2, the callus weight and callus volume were increased by 57% and 64% on the BUF-3 administration side and the control administration side, respectively.

[0018]

[Table 2]

(Example 3) Whether or not systemic administration of BUFs promotes bone fracture healing was examined using ovariectomized rats. Bilateral ovaries of 7 6-week-old SD female rats were extracted under ether anesthesia, and 10,000 U of penicillin G potassium (0.1 ml) was subcutaneously administered after extraction. Five days after the ovariectomy, a left fibula cutting operation was performed in the same manner as in Example 1. Rats with fractured fibula were divided into a BUF-3 administration group (3 animals) and a control administration group (4 animals). The BUF-3 administration solution and the control administration solution were prepared in the same manner as in Example 1. The animals in the BUF-3 administration group had BUF once a day for 11 days from the fibula fracture day.
-3 10 μg (25 μl) was intramuscularly administered to the buttocks. Similarly, the animals of the control group were intramuscularly administered with the control administration solution (25 μl) to the buttocks. On day 11 after the fracture, the fibula on both sides of each rat was taken out, and the fibula weight and the fibula volume were measured by the same method as in Example 2. The fibula weight and fibula volume on the non-fracture side (right side) were subtracted from the fracture side (left side) to obtain the callus weight and callus volume.

As shown in Table 3, the callus weight was increased by 44% and the callus volume was increased by 52% in the BUF-3 administration group as compared with the control group.

[0021]

[Table 3]

(Example 4) It was examined whether or not BUFs promote fracture healing in terms of bone strength. Bilateral fibula of 10-month-old female SD rats (17 rats) was fractured, and one side of each animal was the BUFs administration side and the contralateral side was the control administration side. BU
Preparation of the F group administration liquid was performed in the same manner as in Example 1, and 25 μl of BUF-3 administration liquid (10 μg of BUF-3 was prepared on the BUFs administration side.
25 μl of the control administration liquid was administered locally to the fracture on the control administration side. The operation for cutting the fibula was performed in the same manner as in Example 1, and each administration liquid was dropped on each administration side using a pipette at the fibula cutting portion during the operation. After that, each administration liquid was administered to the fractured site once daily to each administration side using a microsyringe. The fibula on both sides of each animal was excised on the 14th or 21st day after the fracture, and the bone strength was measured using a bone rheometer.

As shown in Table 4, on days 14 and 2 of fracture
The bone strength on the 1st day increased on the BUF-3 administration side as compared to the control administration side.

[0024]

[Table 4]

[0025]

[Effect] The therapeutic agent for bone damage containing the BUFs of the present invention as an active ingredient can shorten the healing period of bone damage diseases such as bone defects and fractures due to various causes and improve the strength of repaired bone. It can be said to be an extremely useful drug.

[0026]

[Sequence list]

SEQ ID NO: 1 Sequence length: 116 Sequence type: Amino acid Topology :: Linear Sequence type: Peptide sequence Gly Leu Glu Cys Asp Gly Lys Val Asn Ile Cys Cys Lys Lys Gln Phe 1 5 10 15 Phe Val Ser Phe Lys Asp Ile Gly Trp Asn Asp Trp Ile Ile Ala Pro 20 25 30 Ser Gly Tyr His Ala Asn Tyr Cys Glu Gly Glu Cys Pro Ser His Ile 35 40 45 Ala Gly Thr Ser Gly Ser Ser Leu Ser Phe His Ser Thr Val Ile Asn 50 55 60 His Tyr Arg Met Arg Gly His Ser Pro Phe Ala Asn Leu Lys Ser Cys 65 70 75 80 Cys Val Pro Thr Lys Leu Arg Pro Met Ser Met Leu Tyr Tyr Asp Asp 85 90 95 Gly Gln Asn Ile Ile Lys Lys Asp Ile Gln Asn Met Ile Val Glu Glu 100 105 110 Cys Gly Cys Ser 115

SEQ ID NO: 2 Sequence length: 115 Sequence type: Amino acid Topology :: Linear Sequence type: Peptide sequence Gly Leu Glu Cys Asp Gly Arg Thr Asn Leu Cys Cys Arg Gln Gln Phe 1 5 10 15 Phe Ile Asp Phe Arg Leu Ile Gly Trp Asn Asp Trp Ile Ile Ala Pro 20 25 30 Thr Gly Tyr Tyr Gly Asn Tyr Cys Glu Gly Ser Cys Pro Ala Tyr Leu 35 40 45 Ala Gly Val Pro Gly Ser Ala Ser Ser Phe His Thr Ala Val Val Asn 50 55 60 Gln Tyr Arg Met Arg Gly Leu Asn Pro Gly Thr Val Asn Ser Cys Cys 65 70 75 80 Ile Pro Thr Lys Leu Ser Thr Met Ser Met Leu Tyr Phe Asp Asp Glu 85 90 95 Tyr Asn Ile Val Lys Arg Asp Val Pro Asn Met Ile Val Glu Glu Cys 100 105 110 Gly Cys Ala 115

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁶ Identification code Internal reference number FI technical display area A61K 37/02 ADT

Claims (1)

[Claims] 1. BUF-3, BUF-4 and BUF-5
A therapeutic agent for bone damage, which comprises one or more of the following as an active ingredient.