JPH05132426A - Agent for promoting formation of bone tissue - Google Patents

Abstract

PURPOSE:To provide an agent for promoting the formation of bone tissue, containing a cell growth factor such as fibroblast growth factor and epidermal growth factor, effective for promoting the bone formation in the bone of hematherm animal and useful as an agent for treating bone-decreasing diseases, osteoporosis, etc. CONSTITUTION:The objective agent for promoting the formation of bone tissue, effective for promoting the proliferation of osteoblast and the bone formation in the bone tissue of hematherm animal and useful as a preventive and therapeutic agent for bone-decreasing diseases can be prepared by using a factor belonging to the family of fibroblast growth factor (FGF), epidermal growth factor (EGF), its mutein, etc., having a molecular weight of 5,000-25,000 and forming the factor in the form of pharmaceutical preparation with pharmacologically permissible additives such as an excipient (e.g. carboxymethyl cellulose and sodium alginate), a diluting agent (e.g. physiological saline solution), an oily solvent (e.g. olive oil), a dissolution assistant (e.g. sodium salicylate), an isotonic agent (e.g. glucose) and a stabilizer (e.g. human serum albumin).

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to an agent for promoting bone formation in bone of warm-blooded animals, which contains cell growth factor.

[0002]

2. Description of the Related Art Osteoporosis is a pathological condition in which bone mass is morbidly decreased, and clinical symptoms such as atrophy of height, round back, and compression fracture of the spine and fracture of limbs are exhibited. In developed countries, the number of patients with osteoporosis is increasing with the aging of the population, and the threat is increasing. Bone loss, which is the cause of osteoporosis, is caused by various physiological abnormalities such as glaucoma, especially estrogen deficiency due to menopause,
It is also known to occur due to calcitonin deficiency, growth hormone deficiency, hyperparathyroidism, hyperthyroidism, etc., diseases (rheumatoid arthritis and diabetes), abnormal nutrition and metabolism (calcium, vitamin D, vitamin C, Alternatively, vitamin K deficiency, excessive intake of protein, phosphorus, or sodium), exercise quantity, and inheritance and race differences are also involved. It also occurs as a symptom of a side effect (Cushing's syndrome) of a corticosteroid used for treating various intractable diseases. In the bone tissue of grown animals, bone melting and digestion by osteoclasts (so-called bone resorption) and bone formation by osteoblasts proceed in a balanced state, so that the bone tissue is constantly renewed (reformed). Modeling). Due to various causes of osteoporosis, the balance between bone resorption and bone formation is lost, and the amount of bone resorption exceeds the amount of bone formation.
It can be considered that bone mass continues to decrease. Conventionally,
As therapeutic agents, estrogen, calcitonin, active vitamin D, ipriflavone, etc. have been administered.

[0003]

[Problems to be Solved by the Invention] Osteoporosis is a disease caused by a decrease in bone mass.
A drug having an inhibitory effect on bone mass loss or an effect of increasing bone mass may be administered. However, all of the above-mentioned therapeutic agents currently used have a main effect of suppressing bone resorption, and there is no therapeutic agent having an effect of promoting bone formation (an effect of increasing bone mass). Therefore, even if these drugs are administered to an osteoporosis patient for a long period of time, the effect is often only delayed in the progression of symptoms, and these drugs have almost no therapeutic effect particularly in advanced osteoporosis accompanied by fracture. In order to treat osteoporosis, it has been desired to develop a drug exhibiting a strong bone mass increasing action or osteogenesis promoting action with short-term administration.

[0004]

In view of the above circumstances, the present inventors have conducted extensive studies and as a result, found that cell growth factor has a strong bone formation promoting action in bone in the living body, and based on this, As a result of further research, the present invention has been completed.

The present invention relates to (1) an osteogenesis promoter in bone of warm-blooded animals, which contains a cell growth factor, and (2)
A method for promoting bone formation in bone, which comprises administering a preparation containing a cell growth factor to a warm-blooded animal.

The cell growth factor has a molecular weight of 500.
The thing which is 0-25000 is mentioned. Examples of the cell growth factor used in the present invention include fibroblast growth factor (F
GF family member, nerve growth factor (NG)
F) -1, NGF-2 / NT-3, epidermal growth factor (EG
F), platelet-derived growth factor (PDGF), bone morphogenetic protein (BMP) -1, BMP-2A, BMP-2B, BM
P-3, insulin-like growth factor (IGF) -I, IG
F-II, colony stimulating factor (CSF), erythropoietin (EPO), thrombopoietin, β2 microglobulin (β2-m), interleukin (IL) -1, IL
-2, IL-3, IL-4, IL-5, IL-6, cartilage-derived factor (CDF), transforming growth factor (T
GF) -α, TGF-β, TGF-γ2, osteoinductive factor (OIF), tumor angiogenic factor (TAF), insulin, transferrin, fibronectin, laminin,
Examples thereof include chondronectin, hydronectin, hepatocyte growth factor (HGF), leukemia cell-derived growth factor (LGF) and macrophage growth factor (MGF). In addition, these cell growth factors may be natural types or muteins thereof. Among these, those belonging to the FGF family are particularly preferable.

Basic FGF (hereinafter referred to as b
It may be abbreviated as FGF. ) Or an acidic one (hereinafter, may be abbreviated as aFGF).
Also, int-2, hst-1, k-FGF, FGF-5, F
Any one that broadly belongs to the FGF family such as GF-6 may be used. Also, these muteins may be used.
Examples of the FGF include those derived from mammals. Examples of the mammal include humans, monkeys, pigs, cows, sheep, horses and the like. Examples of the FGF include those extracted from various organs whose existence has already been clarified, such as the brain and pituitary gland. It may also be manufactured by a genetic engineering method. As the bFGF, recombinant D
Those obtained by NA technology are listed (Fuess.
Letters, Vol. 213, pp. 189-194 (1987)
); European Patent Application Publication No. 237,966). In the present specification, recombinant human basic FGF may be abbreviated as “rhbFGF”.

In the present invention, FGF muteins can also be used. The FGF mutein is originally a mutated amino acid sequence of the original peptide or protein. Therefore, the mutation includes addition of amino acids, deletion of constituent amino acids, and substitution with other amino acids. The addition of the amino acid is at least 1
Examples of amino acids added are individual amino acids. Examples of the deletion of the constituent amino acids include those lacking at least one FGF constituent amino acid. Examples of the substitution with another amino include substitution of at least one FGF-constituting amino acid with another amino acid. As at least one amino acid in a mutein in which at least one amino acid is added to FGF,
It does not include methionine, which is due to the initiation codon used when expressing the peptide, or the signal peptide. The number of amino acids added is at least 1, but any number may be used as long as the characteristics of FGF are not lost. More preferably, homology with FGF is recognized, and part or all of the amino acid sequences of proteins showing similar activity can be mentioned. FGF
Of the mutein in which at least one FGF-constituting amino acid is deleted, any number may be used as long as the characteristics of FGF are not lost.
The number of at least one FGF-constituting amino acid before substitution in a mutein in which at least one FGF-constituting amino acid of FGF is substituted with another amino acid is:
Any number may be used as long as the characteristics of FGF are not lost. The most preferable substituted mutein is one in which at least one constituent amino acid, cysteine, is replaced with serine. In the substitution, two or more substitutions may be performed at the same time. In particular, it is preferable that 2 or 3 constituent amino acids be substituted. The mutein may be a combination of two or three of the above additions, deletions and substitutions. Further, the mutein may have a sugar chain-binding site introduced therein. The mutein is, for example, a European patent application (hereinafter, may be abbreviated as EP) publication No. 281,822, 326,907.
No. 394,951. As the mutein, a mutein in which at least one human basic FGF-constituting amino acid is replaced with another amino acid is preferable. Especially positions 70 and 8 of human bFGF
Human bFGF mutein CS23 in which Cys at position 8 is replaced with Ser is preferable (see EP-281,822).

As the aFGF, Biochemical and
Biophysical Research Communications, 138 , 61
1-617 (1986). Furthermore, examples of the aFGF mutein include a deletion type mutein. For example, aFGF
90 of any of the 140 amino acid sequences of
A deletion type mutein consisting of 133 polypeptide chains is exemplified. More preferably, the amino acid sequence of aFGF is composed of any continuous 120 to 131 polypeptide chains. Specific examples of the aFGF mutein include those described in EP Publication No. 420,222. As the above hst-1, Proc.
Natl. Acad. Sci. USA, 84 , 2980 (198)
7), Proc. Natl. Acad. Sci. USA, 84 , 7305.
(1987), Molecular and Cellular Biology, 8 ,
The proteins disclosed in 2933 (1988) can be mentioned. Furthermore, examples of hst-1 muteins include deletion muteins thereof, and examples thereof include hst-
1 has a deletion of at least one amino acid among the 175 constituent amino acids, and more preferably a mutein in which any 1 to 47 consecutive amino acids of the hst-1 constituent amino acids are deleted. And more preferably any consecutive 1 of the constituent amino acids of hst-1.
To muteins in which 1 to 27 amino acids are deleted, and more preferably muteins in which any consecutive 1 to 27 amino acids among the constituent amino acids of hst-1 are deleted. Specific examples of the hst-1 mutein include those described in EP Publication No. 421,455. As the EGF, for example, one obtained by a recombinant technique can be used, and a specific production method thereof includes, for example, the method described in EP Publication No. 326,046.

By applying the present invention, bone formation in the bone of warm-blooded animals is promoted. More specifically, the intraosseous osteogenesis promoting agent of the present invention containing a cell growth factor may be used in long bones such as femurs, sternum and muscular bone depending on the administration route, dose and administration period. In irregular bones such as flat bone and vertebra, osteoblast proliferation, calcified cancellous bone proliferation, bone matrix increase, and bone mineral increase are prominent. Also, the toxicity of cell growth factors is low. Therefore, the prophylactic / therapeutic agent of the present invention can be used as a prophylactic / therapeutic agent for osteopenic diseases such as osteoporosis. Examples of the bone disease in the present invention include congenital bone disease and acquired bone disease, and examples thereof include osteopenic disease, osteoporosis, osteopenia, osteodystrophy, osteogenesis imperfecta, hypercalcemia. And the like. In particular,
The prophylactic / therapeutic agent for bone diseases of the present invention brings about the proliferation of osteoblasts, the formation of new bone and the increase of bone mineral content in various long bones, flat bones and irregular bones of the whole body, resulting in bone formation. Since it reinforces the tissue, it is particularly effective in the treatment of bone diseases such as osteoporosis accompanied by loss of bone itself and cavitation of bone tissue.

The preventive / therapeutic agent for bone diseases used in the present invention may be used as it is, or together with other pharmacologically acceptable carriers, excipients and diluents, as a pharmaceutical composition (eg, injection,
As tablets, capsules, solutions, ointments, warm-blooded animals (eg, humans, mice, rats, hamsters, rabbits,
It can be safely administered parenterally or orally to dogs, cats, cows, sheep, pigs, horses), especially mammals, for the purpose of treating those bone diseases. In particular, parenteral administration is preferred. The formulation is preferably in the form of, for example, an injection, a solution for use in injection administration, or a lyophilized product. Further, a sustained release agent can be prepared according to the purpose.

[0012] In formulating as a pharmaceutical composition,
According to known pharmaceutical manufacturing methods, if necessary, pharmaceutically acceptable additives, diluents, excipients and the like are used. For example, in the case of an aqueous solution for injection, a solvent such as an aqueous solvent (eg, distilled water), a water-soluble solvent (eg, physiological saline, Ringer's solution), an oily solvent (eg, sesame oil, olive oil),
Alternatively, if desired, a solubilizing agent (eg, sodium salicylate, sodium acetate), a buffer solution (eg, sodium citrate, glycerin), an isotonicity agent (eg, glucose, invert sugar), a stabilizer (eg, human serum albumin, Polyethylene glycol), a preservative (eg, benzyl alcohol, phenol), a soothing agent (eg, benzalkonium chloride, procaine hydrochloride) and the like are used by conventional means. Further, for example, in order to prepare a solid injection preparation, a diluent (eg, distilled water, physiological saline, glucose), an excipient (eg, carboxymethylcellulose (CMC), sodium alginate), a preservative (eg, benzyl alcohol,
A solid injection preparation can be produced by a conventional method by mixing benzalkonium chloride, phenol), a soothing agent (glucose, calcium gluconate, procaine hydrochloride) and the like. Further, upon formulation, monosaccharides such as glucose, amino acids, various salts, human serum albumin, etc. may be added. In addition, isotonic agents, pH adjusting agents, soothing agents, preservatives, etc. may be added. A stable and effective formulation can be prepared.

The dosage of the preventive / therapeutic agent for bone diseases of the present invention is such that the cell growth factor is about 1 to 1000 pg / ml, more preferably about 1 as the amount distributed in the body fluid of the living body.
It is preferable to administer an amount of 0 to 500 pg / ml. As the dose, the dose when administered is about 0.
1 to 1000 μg / kg, more preferably 1 to 300 μg
An amount which is / kg. To administer the prophylactic / therapeutic agent for bone diseases of the present invention parenterally, for example, intravenous administration,
Subcutaneous administration, intramuscular administration, intramedullary cavity administration, intraosseous administration, transmucosal administration and the like can be mentioned. Examples of routes for transmucosal administration include nasal, buccal and rectal routes. The prophylactic / therapeutic agent of the present invention is preferably administered so that the drug is systemically present in a warm-blooded animal body. Thus, for example, it is preferably administered to the cardiovascular system. In particular, intravenous administration is preferred. The administration may be performed once a day or intermittently, for example, about once a week or about twice a week. In addition, it may be formed into a sustained-release preparation for administration, and examples of the sustained-release preparation include microcapsules and implants. In particular, what was molded into a sustained release formulation
It is preferable that the effect of the main drug is exerted for a long time by implanting under the skin. Further, a method of embedding or injecting a sustained-release agent molded into the bone marrow cavity or bone tissue directly to exert the effect of the main drug over a long period of time can be preferably used.

Since calcium is required for bone formation when the preparation of the present invention is administered, a calcium agent may be used in combination. In addition, a bone resorption inhibitor [eg, active vitamin D ₂ , active vitamin D ₃ , calcitonin, estrogen, ipriflavone (Osten (trade name)), etc.] may be used in combination.

Administration of the prophylactic / therapeutic agent of the present invention causes osteoblast proliferation and bone formation in the endosteum of the diaphysis and metaphysis, but the bone tissue extends toward the periosteum side (outside) of the bone. Does not happen. That is, the prophylactic / therapeutic agent of the present invention promotes the growth of calcified cancellous bone only in the endosteum. Therefore, the bone matrix increases toward the bone marrow cavity. Increased calcified cancellous bone in the diaphysis increases the thickness of the diaphyseal cortex. In addition, the thickness of the trabecular bone in the cancellous bone of the metaphysis increases. Increased cortical bone thickness in the diaphysis and increased trabecular bone trabecular thickness increase skeletal strength. In this way, it can be used for the prevention and treatment of dose-reducing diseases such as osteoporosis, osteopenia, and hypercalcemia. The prophylactic / therapeutic agent of the present invention, which promotes bone formation in bone and does not promote periosteum (outer) bone formation, is capable of producing bone without causing inconvenience such as pain and skeletal deformation due to extension of bone tissue toward the outside of bone. Since the amount can be increased, it is suitable for preventive treatment of osteopenic diseases. As described above, the action of conventionally applied drugs against osteopenic diseases is intended to reduce bone resorption by antagonizing the mechanism of remodelinb. Conventionally applied drugs reduce bone resorption by osteoclasts and the effect of increasing bone mass, if at all, is indirect and only slow. On the other hand, the promoter containing the cell growth factor of the present invention directly promotes osteoblast proliferation and bone formation without going through the mechanism of remodelinb, so that the effect is direct and rapid. ..

Recombinant human basic FGF mutein CS23 (hereinafter referred to as rhb
It may be abbreviated as FGF mutein CS23. ) Is
Examples 7 and 2 of EP Publication 281,822
4, Biochemical and Biophysical Research Communications Volume 151 Volume 701-70
It is produced by the method described on page 8 (1988). That is, the rhbFGF mutein CS23 was transformed into the transformant Escherichia coli MM294 / pTB762 (IFO 1
4613, FERM BP-1645), and manufactured and purified by the above-mentioned method. Transformant mentioned above
E. coli MM294 / pTB762 has been deposited with the Institute for Fermentation (IFO) and the Institute for Microbial Technology (FRI), Ministry of International Trade and Industry. The deposit number and the deposit date are shown in the following [Table 1]. Regarding deposits to the FRI, domestic deposits were initially made and FERM P
The deposit number indicated by the number is attached, the deposit is switched to the deposit under the Budapest Treaty, the deposit number indicated by the FERMBP number is attached, and the deposit is stored in the Institute (FRI).

[Table 1]

Human bF used in the examples described below
GF is a transformant Escherichia coli K12MM294 / pTB6 described in EP Publication No. 237,966.
69 (IFO 14532, FERMBP-1281)
Is produced by the method described in the examples of the publication. Further, human aFGF used in Examples described later is a transformant Escherichia coli MM294 (DE3) / described in EP Publication No. 406,738.
pLysS, pTB975 (IFO 14936, FER
MBP-2599) and the method described in Example 1 of the publication. The hst-1 mutein N27 used in Examples described later is a transformant Escherichia c described in EP Publication No. 421,455.
oli MM294 (DE3) / pLysS, pTB1051
(IFO 14952, FERM BP-2621), and manufactured by the method described in the example of the publication. The EGF used in the examples described below is
It is manufactured by the method described in EP Publication No. 326,046. That is, the EGF
llus brevis H102 (FERM BP-1087 (US Pat. No. 4,946,789)) and transformant Bacillus.
brevis 47-5 (pNU200-EGF31) (IF
O 14729, FERM BP-1772) -isolated plasmid pNU200-EGF31 introduced transformant Bacillus brevis H102 (pNU200-
It was produced by the method described in Examples 1, 2, 3 (ii), 4 and 5 of EP Publication No. 326,046 using EGF31).

The present invention will be described in more detail below with reference to Reference Examples and Examples. These are merely examples and do not limit the present invention in any way. Reference Example 1 Bone Growth or Bone Formation Measurement Method A region from the distal end 5 mm to 10 mm of the femur or tibia was cut out in a ring shape to prepare a sample. Subsequently, an X-ray photograph of the sagittal section of the sample was taken with a soft X-ray photographic device (Softex Co., CSM-2 type). From this X-ray photograph, using an image analysis device (Pierce 555 type), the cross-sectional area of the specimen (A)
Then, the bone marrow cavity area (B) was calculated, and the B value was subtracted from the A value to calculate the cortical bone area, which was expressed as a percentage of A.

Example 1 Effect of rhbFGF mutein CS23 on bone formation in rats 0.01 and 0.03 of rhbFGF mutein CS23 dissolved in 50 mM citrate buffer were used in 4 male and 4 female rats (Jcl: Wistar system) in each group. , 0.1 or 0.3 mg / kg / day was intravenously administered from 6 weeks old for 2 weeks. Immediately after the end of administration, the animals were killed, the femur, sternum and ribs were fixed with 10% formalin, decalcified and embedded in paraffin, and sliced in the longitudinal direction to obtain 4 micron sections. After staining these sections with hematoxylin-eosin (HE), histopathological examination was performed to examine the effect on bone tissue. Physiological saline was administered to the control group in the same manner as the administration group. As a result, in all femurs, sternums and ribs, osteoblast proliferation and bone matrix increase were observed at doses of 0.1 mg / kg or more in a dose-dependent manner.
In the femur, this finding was found on the bone marrow cavity side of the epiphyseal cartilage (so-called growth zone) and the bone marrow cavity side of the diaphysis (see [Fig. 1], [Fig. 2], [Fig. 3], [Fig. 4]), In the sternum and ribs, it was found on the bone marrow cavity side in the central part of the bone. Details of FIGS. 1 to 4 are shown below. [Fig. 1]: Femur, Jcl: Wistar rat, female 8 weeks old, control group. HE stain, × 8.5, no abnormalities. [Fig.2]: Femur, Jcl: Wistar rat, 8 weeks old female, rh
bFGF mutein CS23, 0.3 mg / kg group. H-E stain, x 8.5, shows marked formation of new bone in the bone marrow cavity. [Fig. 3]: Femur, Jcl: Wistar rat, female 8 weeks old, control group. HE stain, × 85, no abnormality. [Figure 4]: Femur, Jcl: Wistar rat, female 8 weeks old, rh
bFGF mutein CS23, 0.3 mg / kg group. H-E stain, x 8.5, shows marked formation of new bone in the bone marrow cavity. No bone formation is observed on the periosteum side (arrow).

In the 0.3 mg / kg group, the same finding was observed on the bone marrow cavity side of the rib at the costal costal cartilage joint. In all of these bones, bone formation was observed only on the bone marrow cavity side of the bone, and not on the outer side (periosteal side) of the bone. In addition,
Osteoclast hyperplasia was not seen at any dose. In addition, an image was observed in which osteoblasts proliferated and endochondral ossification was promoted also in the epiphyses of the 0.3 mg / kg administration group (see [Fig. 5] and [Fig. 6]). Details of FIGS. 5 and 6 are shown below. [FIG. 5]: Sternum, Jcl: Wistar rat, female 8 weeks old, control group. HE stain, × 170, no abnormalities. [Figure 6]: Sternum, Jcl: Wistar rat, female 8 weeks old, rhb
FGF mutein CS23, 0.3 mg / kg group. H / E staining, hypertrophy of x170 osteoblasts (arrow), hyperplasia and formation of new bone. According to the method described in Reference Example 1, the cortical bone area (%) was obtained for the femoral specimens in the 0.3 mg / kg administration group (results are shown in [Table 2]). The cortical bone area (%) in the 0.3 mg / kg administration group showed a statistically significant increase as compared with that in the physiological saline administration group.

[Table 2] ──────────────────────────── Number of groups Cortical bone area (%) ────────── ─────────────────── Physiological saline administration group 4 59.6 ± 2.4 rhbFGF mutein CS23 4 70.2 ± 2.1 * 0.3 mg / kg administration group ──────────────────────────── *: The value between the two groups is statistically significant at the 1% level. Show. (Average ± SE)

Example 2 Bone formation promoting action of rhbFGF in rats 0.02 M T was added to 4 female rats (Jcl: Wistar system) in each group.
0.1% of human basic fibroblast growth factor (rhbFGF) dissolved in ris-HCl-1M NaCl buffer (pH 7.4).
03, 0.1 or 0.3 mg / kg / day was intravenously administered from 6 weeks old for 2 weeks. One day after the final administration, the animals were killed, the femur, the sternum and the ribs were fixed with 10% formalin, decalcified, embedded in paraffin, and sliced in the long axis direction to obtain 4 micron sections. These sections were used for hematoxylin and eosin (H
-After staining with E), histopathological examination was performed to examine the effect on bone tissue. 0.02M Tris- for the control group
The HCl-1M NaCl buffer (pH 7.4) was administered in the same manner as in the administration group. As a result, proliferation of osteoblasts and formation of new bone were observed at a dose-dependent intensity at 0.03 mg / kg or more in the femur and 0.1 mg / kg or more in the sternum. This finding shows that in the femur, the bone marrow cavity side of the epiphyseal cartilage (so-called growth zone)
And in the medullary cavity side of the diaphysis (see [Fig. 7] and [Fig. 8]), and in the sternum in the central portion of the bone on the medullary cavity side. No bone formation was observed on the periosteum side. [Fig. 7],
Details of FIG. 8 are shown below. [FIG. 7]: Femur, Jcl: Wistar rat, female 8 weeks old, control group, HE staining × 85, no abnormal place. [Figure 8]: Femur, Jcl: Wistar rat, female 8 weeks old, rh
bFGF 0.3 mg / kg group, HE stain × 85, showing formation of new bone in bone marrow cavity. No bone formation is observed on the periosteum side (arrow). According to the method described in Reference Example 1, the cortical bone area (%) was obtained for the femoral specimens in the 0.3 mg / kg administration group (results are shown in [Table 3]). The cortical bone area (%) in the 0.3 mg / kg administration group showed a statistically significant increase as compared with that in the Tris-hydrochloric acid buffer administration group.

[Table 3] ─────────────────────────────── Group number of cortical bone area (%) ────── ───────────────────────── 0.02M Tris-HCl buffer administration group 4 43.6 ± 0.9 rhbFGF 0.3 mg / kg administration group 4 52.0 ± 1.3 * ─────────────────────────────── *: The value between the two groups is 1% Indicates that the level is statistically significant. (Average ± SE)

Example 3 Bone formation promoting action of rhaFGF in rats Tris-HCl was added to 4 female rats (Jcl: Wistar strain) in each group.
0.03 of human acidic fibroblast growth factor (rhaFGF) dissolved in 0.9M NaCl buffer (pH 7.4),
0.1 or 0.3 mg / kg / day was intravenously administered from 6 weeks old for 2 weeks. One day after the final administration, the animals were killed and the femur,
The sternum and ribs were fixed with 10% formalin, decalcified, embedded in paraffin, and sliced in the longitudinal direction to obtain 4 micron sections. These sections were used for hematoxylin and eosin (H
After staining with E), histopathological examination was performed to examine the effect on bone tissue. The control group was Tris-HCl-0.9.
The MNaCl buffer solution (pH 7.4) was administered in the same manner as in the administration group. As a result, in the femur and sternum, osteoblast proliferation and new bone formation were observed at a dose-dependent strength at 0.03 mg / kg or higher. This finding was found on the bone marrow cavity side of the epiphyseal cartilage (so-called growth zone) and the bone marrow cavity side of the diaphysis in the femur (see [Fig. 9] and [Fig. 10]), and in the sternum the bone marrow cavity side at the center of the bone Was recognized by. No bone formation was observed on the periosteum side. Details of FIGS. 9 and 10 are shown below. [FIG. 9]: Femur, Jcl: Wistar rat, female 8 weeks old, control group, HE staining × 85, no abnormalities were observed. [Fig. 10]: Femur, Jcl: Wistar rat, female 8 weeks old,
The rhaFGF 0.3 mg / kg group, HE stain x85, and formation of new bone in the bone marrow cavity are shown. Bone matrix growth is not observed on the periosteum side (arrow). According to the method described in Reference Example 1, the cortical bone area (%) of the 0.1 mg / kg-administered femoral specimen was determined (the results are shown in [Table 4]). The cortical bone area (%) in the 0.1 mg / kg administration group showed a statistically significant increase as compared with that in the Tris-hydrochloric acid buffer administration group.

[Table 4] ─────────────────────────────── Group number of cortical bone area (%) ────── ───────────────────────── 0.02M Tris-HCl buffer administration group 4 43.9 ± 0.6 rhaFGF 0.1 mg / kg administration group 4 48.7 ± 1.8 * ─────────────────────────────── *: The value between the two groups is 1% Indicates that the level is statistically significant. (Average ± SE)

Example 4 Bone formation promoting action of hst-1 mutein N27 in rats 0.01 M T in 4 female rats (Jcl: Wistar system) in each group
Recombinant human heparin-binding secretory tron dissolved in ris-HCl-0.2M NaCl buffer (pH 7.4)
forming protein-1 (hst-1) mutein N27 0.0
3, 0.1 or 0.3 mg / kg / day was intravenously administered from 6 weeks old for 2 weeks. One day after the final administration, the animals were killed, the femur, sternum and ribs were fixed with 10% formalin, decalcified, embedded in paraffin, and sliced in the longitudinal direction to obtain 4 micron sections. These sections were used for hematoxylin and eosin (H
-After staining with E), histopathological examination was performed to examine the effect on bone tissue. 0.01M Tris- for the control group
HCI-0.2M NaCl buffer (pH 7.4) was administered in the same manner as in the dosing group. As a result, the thighbone and sternum have a value of 0.
At doses of 0.3 mg / kg or more and on ribs at 0.1 mg / kg or more, osteoblast proliferation and new bone formation were observed with a dose-dependent intensity. This finding was found in the femur on the marrow cavity side of the epiphyseal cartilage (so-called growth zone) and on the marrow cavity side of the diaphysis (Fig. 11).
(See FIG. 12)), but in the sternum and ribs, it was found on the bone marrow cavity side of the central part of the bone. No bone formation was observed on the periosteum side. Details of FIGS. 11 and 12 are shown below. [Fig. 11]: Femur, Jcl: Wistar rat, female 8 weeks old,
Control group, HE stain × 85, no abnormalities. [Fig. 12]: Femur, Jcl: Wistar rat, female 8 weeks old,
hst-1 mutein N27, 0.3 mg / kg group, HE stain ×
85, shows the formation of new bone in the bone marrow cavity. No bone formation is observed on the periosteum side (arrow). According to the method described in Reference Example 1, the cortical bone area (%) was determined for the femoral specimens in the 0.1 mg / kg administration group (results are shown in [Table 5]). The cortical bone area (%) in the 0.1 mg / kg administration group showed a statistically significant increase as compared with that in the Tris-hydrochloric acid buffer administration group.

[Table 5] ─────────────────────────────── Group number of cortical bone area (%) ────── ───────────────────────── 0.01M Tris-HCl buffer administration group 4 46.4 ± 1.2 hst-1 mutein N27 0.1 mg / kg administration group 4 52.9 ± 2.3 * ─────────────────────────────── *: Value between two groups Is statistically significant at the 1% level. (Average ± SE)

Example 5 Effect of EGF on bone formation in rats 0.04, 0.1 of epidermal growth factor (EGF) dissolved in ammonium acetate buffer was used in 4 female rats (Jcl: Wistar system) in each group. Or 0.3 mg / kg / day from 6 weeks old to 2
It was administered intravenously for a week. Kill the animals one day after the last dose,
The femur, sternum, and ribs were fixed with 10% formalin, decalcified, embedded in paraffin, and sliced in the longitudinal direction to obtain 4 micron sections. After staining these sections with hematoxylin-eosin (HE), histopathological examination was performed to examine the effect on bone tissue. An ammonium acetate buffer solution (pH 6.8) was administered to the control group in the same manner as the administration group. As a result, osteoblast proliferation and new bone formation were observed at doses of 0.1 mg / kg or more in the femur in a dose-dependent manner. This finding was found on the marrow cavity side of the epiphyseal cartilage of the femur (so-called growth zone) and on the marrow cavity side of the diaphysis (see [Fig. 13] and [Fig. 14]). No bone formation is observed on the periosteum side. [Fig. 13],
Details of FIG. 14 are shown below. [FIG. 13]: Femur, Jcl: Wistar rat, female 8 weeks old,
Control group, HE stain × 85, no abnormalities. [FIG. 14]: Femur, Jcl: Wistar rat, female 8 weeks old,
EGF 0.3 mg / kg group, HE stain × 85, formation of new bone in bone marrow cavity are shown. No bone formation is observed on the periosteum side (arrow). According to the method described in Reference Example 1, the cortical bone area (%) of the tibial specimen in the 0.3 mg / kg administration group was determined (the results are shown in [Table 6]). The cortical bone area (%) in the 0.3 mg / kg administration group showed a statistically significant increase as compared with that in the ammonium acetate buffer administration group.

[Table 6] ─────────────────────────────── Group number of cortical bone area (%) ────── ───────────────────────── Ammonium acetate buffer administration group 4 57.0 ± 2.0 EGF 0.3 mg / kg administration group 4 66.1 ± 1.3 * ─────────────────────────────── *: Statistics at the level of 1% between the two groups It is shown to be statistically significant. (Average ± SE)

Example 6 Effect of rhbFGF mutein CS23 on calcified cancellous bone formation in rats 0.1 mg / mg of rhbFGF mutein CS23 dissolved in 50 mM citrate buffer in 5 male rats (Jcl: Wistar system)
kg / day was intravenously administered from 6 weeks old for 2 weeks. 5 in control group
The animals received 50 mM citrate buffer as in the dosing group. After the administration, the animals were exsanguinated and killed, and tibias were collected.
It was immersed and fixed in 0% ethanol. The tibia proximal part cut with a bone cutter was stained with Vila Nueva Bone (VB), dehydrated, and then embedded in methyl methacrylate resin. From the resin-embedded tibia, a slice of a frontal plane of 250 microns was cut out using a bone slicer, and further polished to 70 microns using a polishing machine. A contact microradiograph (CMR) was prepared from this polished sample using a soft X-ray photography system. The film was developed with D-19, stopped and fixed, air-dried, and then sealed with Canadian balsam to prepare a light microscope sample. Further, the polished sample was further polished to 50 microns and sealed, and then a light microscope sample was prepared. As a result, in the microscopic observation of the polished specimen,
When 0.1 mg / kg was administered, a partial thickening of the growth plate was observed in the tibia proximal part, and a significant increase in cancellous bone was observed in the bone marrow from the metaphysis to the diaphysis (Figs. 15 and 16). reference). Observation of CMR specimens revealed a clear shadow image in most of the regenerated cancellous bone, which revealed that the regenerated cancellous bone was calcified bone (see [Fig. 17] and [Fig. 18]). .. Details of FIGS. 15 to 18 are shown below. [FIG. 15]: Tibia, Jcl: Wistar rat, male 8 weeks old, control group. VB stain, x 8.5. Abnormal place. [FIG. 16]: Tibia, Jcl: Wistar rat, male 8 weeks old, rh
bFGF mutein CS23, 0.1 mg / kg group. VB staining,
8.5. There is significant cancellous bone hyperplasia within the metaphysis and diaphysis and partial thickening of the proximal growth plate. [FIG. 17]: Tibia, Jcl: Wistar rat, male 8 weeks old, control group. CMR, x 7.5. Abnormal place. [FIG. 18]: Tibia, Jcl: Wistar rat, male 8 weeks old, rh
bFGF mutein CS23, 0.1 mg / kg group. CMR, ×
7.5. A marked increase in calcified cancellous bone is seen from the metaphysis to the diaphysis.

Example 7 Osteogenesis-promoting effect of rhbFGF mutein CS23 in aged rats 0.3 mg / kg / kg of rhbFGF mutein CS23 dissolved in 50 mM citrate buffer was used in 4 male and 4 female rats (Jcl: Wistar system) in each group. The day was intravenously administered for 2 weeks from the age of 71 weeks. Immediately after the administration, the animal was killed, and the femur, sternum, fibula and lumbar vertebra were fixed with 10% formalin, decalcified, embedded in paraffin, and sliced in the long axis direction to obtain 4 micron sections. These sections were used for hematoxylin and eosin (H
After staining with E), histopathological examination was performed to examine the effect on bone tissue. As a control group, 50 mM citrate buffer solution (pH 7.0) was administered in the same manner as the administration group. As a result, osteoblast proliferation and new bone formation were observed with dose-dependent intensity in all bones. This finding was found on the marrow cavity side of the epiphyseal cartilage and the medullary cavity side of the diaphysis in the femur (FIGS. 19 and 20) and the fibula, and on the medullary cavity side of the central parts of the sternum and the lumbar vertebra. No bone formation was observed on the periosteum side (arrow). [Fig. 1
9] and [FIG. 20] are shown in detail below. [FIG. 19]: Femur, Jcl: Wistar rat, female 73 weeks old, control group. HE stain, × 45, no abnormalities. [FIG. 20]: Femur, Jcl: Wistar rat, female 73 weeks old, rhbFGF mutein CS23, 0.3 mg / kg group, H
-E staining, x45, shows the formation of new bone in the bone marrow cavity. No bone formation is observed on the periosteum side (arrow). From the above, the result that rhbFGF mutein CS23 significantly forms new bone in aged rats indicates that the growth factor causes significant bone formation regardless of the age of the animal. Therefore, the growth factor is suitable as a prophylactic / therapeutic agent for osteopenic diseases often seen in the elderly.

Example 8 Production of Injection Solution An aqueous solution (pH 7.4) containing 0.5 mg of rhbFGF mutein CS23; 10 mg of sucrose; 15 mg of sodium citrate per ml was prepared to obtain a stable injection solution.

[0028]

INDUSTRIAL APPLICABILITY The osteogenesis promoter in the bones of warm-blooded animals containing the cell growth factor of the present invention promotes osteoblast proliferation and osteogenesis in vertebrate bone tissue. Bone formation occurs inside the bone so that abnormal deformation of the bone does not occur.
Therefore, the prophylactic / therapeutic agent of the present invention is useful as a prophylactic / therapeutic agent for osteopenic diseases.

[0026]

[Brief description of drawings]

FIG. 1 is a micrograph of the morphology of an organism, showing a histological image of a femur of a control group, which was obtained in Example 1 and in which no drug was administered.

FIG. 2 is a micrograph of the morphology of an organism, showing a histological image of the femur of the rhbFGF mutein CS23-administered group obtained in Example 1.

FIG. 3 is a micrograph of the morphology of an organism, showing a histological image of the femur of a control group without drug administration, which was obtained in Example 1.

FIG. 4 is a micrograph of the morphology of an organism, showing a histological image of the femur of the rhbFGF mutein CS23-administered group obtained in Example 1.

FIG. 5 is a micrograph of the morphology of the organism, showing the histology of the femur of the control group without drug administration obtained in Example 1.

FIG. 6 is a micrograph of the morphology of an organism, showing a histological image of the femur of the rhbFGF mutein CS23-administered group obtained in Example 1.

FIG. 7 is a micrograph of the morphology of the organism, showing the histology of the femur of the control group without drug administration obtained in Example 2.

FIG. 8 is a micrograph of the morphology of an organism, showing a histological image of the femur of the rhbFGF-administered group obtained in Example 2.

FIG. 9 is a micrograph of the morphology of the organism, showing the histology of the femur of the control group without drug administration obtained in Example 3.

FIG. 10 is a micrograph of the morphology of an organism, showing a histological image of the femur of the rhaFGF-administered group obtained in Example 3.

FIG. 11 is a micrograph of the morphology of the organism, showing the histology of the femur of the control group without drug administration obtained in Example 4.

FIG. 12 is a micrograph of the morphology of an organism, showing the histology of the femur of the hst-1 mutein N27-administered group obtained in Example 4.

FIG. 13 is a micrograph of the morphology of the organism, showing the histology of the femur of the control group without drug administration obtained in Example 5.

FIG. 14 is a micrograph of the morphology of an organism, showing a histological image of the femur of the EGF-administered group obtained in Example 5.

FIG. 15 is a micrograph of the morphology of the organism, showing the histology of the tibia of the control group without drug administration, obtained in Example 6.

FIG. 16 is a micrograph of the morphology of an organism showing a histological image of the tibia of the rhbFGF mutein CS23-administered group obtained in Example 6.

FIG. 17 is a CMR of the tibia of a control group obtained in Example 6 without drug administration.

FIG. 18 is a CMR of the tibia of the rhbFGF mutein CS23-administered group obtained in Example 6.

FIG. 19 is a micrograph of the morphology of the organism, showing the histology of the femur of the control group without drug administration obtained in Example 7.

FIG. 20 is a micrograph of the morphology of an organism, showing a histological image of the femur of the rhbFGF mutein CS23-administered group obtained in Example 7.

Claims (11)

[Claims] 1. An osteogenesis promoter in bone of a warm-blooded animal, which contains a cell growth factor. 2. The promoter according to claim 1, which is used for osteopenic diseases. 3. The bone tissue formation promoting agent according to claim 1, which is for osteoporosis. 4. The cell growth factor has a molecular weight of about 5,000 to about 25.
The accelerator according to claim 1, which is 000. 5. The cell growth factor is fibroblast growth factor (FG).
The promoter according to claim 1, which is a factor belonging to F) family. 6. The promoter according to claim 5, wherein the factor belonging to the FGF family is human basic FGF or its mutein. 7. The promoter according to claim 6, wherein the mutein is a mutein in which at least one human basic FGF-constituting amino acid is replaced with another amino acid. 8. The cell growth factor is epidermal growth factor (EG
The promoter according to claim 1, which is F) or a mutein thereof. 9. The promoter according to claim 1, wherein the cell growth factor is hst-1 or its mutein. 10. The accelerator according to claim 1, which further contains calcium. 11. A method for promoting bone formation in bone, which comprises administering a preparation containing a cell growth factor to a warm-blooded animal.