JPH0948738A - Bone formation promoter - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain a bone formation promoter capable of promoting proliferation, differentiation and formation of osteoblast and useful for treating and preventing bone amount-decreasing diseases such as osteoporosis. SOLUTION: This promoter contains an osteoblast proliferation promoting factor (bOGF-II) and an insulin-like growth factor I (IGF-I) as active ingredients. A blend ratio of bOGF-II to IGF-I is 1,000:1 to 1:100. The promoter is more effectively used as a treating agent for various bone methabolism-related diseases such as osteoblast by combinedly using bOGF-II and IGF-I. BOGF-II can efficiently be isolated in high yield from culture medium of human fibroblast. Production of bOGF-II from the raw material is carried out by ordinary method generally used for separation of a protein like substance from the biological substance. OGF(Osteoblast Growth Factor) activity as an index for bone formation promoting action is measured by examining increase of cell numbers and intake promotion of<3> H(tritium)-thimidine using osteoblast strain or normal osteoblast as a target cell. IGF-I is a single chain peptide composed of 70 amino acids and having 7,649 molecular weight.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to an osteogenesis promoter having an excellent osteogenic action. The osteogenesis promoting agent of the present invention promotes proliferation, differentiation or activation of osteoblasts, and is useful for treating or preventing osteopenia and other osteopenia.

[0002]

BACKGROUND OF THE INVENTION Human bones constantly undergo resorption and remodeling. The cells that play a central role in this process are osteoblasts that are responsible for bone formation and osteoclasts that are responsible for bone resorption. Osteoporosis is a typical example of a disease caused by abnormal bone metabolism that these cells are in charge of. This disease is caused by the fact that bone resorption by osteoclasts outweighs bone formation by osteoblasts. Although the mechanism of development of this disease has not been completely clarified yet, it is a disease that causes bone pain and causes fractures due to weakening of the bone. With the increase in the elderly population, the occurrence of this disease, which causes the occurrence of bedridden elderly people due to fractures, has become a social problem, and the development of a therapeutic drug therefor is urgently needed. Such bone loss due to abnormal bone metabolism is expected to be treated by promoting bone formation, suppressing bone resorption, or improving the balance between them.

As described above, bone formation is expected to be promoted by proliferation, differentiation, or activation of osteoblasts responsible for bone formation. In recent years, interest in bioactive proteins (cytokines) having such activities has been increasing, and intensive research has been conducted. As a cytokine that promotes proliferation or differentiation of osteoblasts, fibroblast growth factor (FGF: Rodan SB et a
l., Endocrinology vol. 121, p1917, 1987) family, insulin-like growth factor-I
factor-I; IGF-I: Hock JM et al., Endocri
nology vol. 122, p254, 1988), insulin-like growth factor-II (IGF-II: McCarthy T. et al., Endocri.
nology vol. 124, p301, 1989), Activin A (Activ
in A; Centrella M. et al., Mol. Cell. Biol. vol.
11, p250, 1991), transforming growth factor-β
(transforming growth factor-β; Noda M., The Bon
e, vol. 2, p29, 1988, Marie, PJ et al., J. Bon
e Mineral Res., vol. 4, pS-276, 1989), Vasculotropin; Biochem. Biophys. Res. C
ommun. vol. 199, p380, 1994), and ectopic bone morphogenic protein (BMP: BMP-2; Yamag.
uchi, A. et al., J. Cell Biol. vol. 113, p682,199
1, OP-1; Sampath TK et al., J. Biol. Chem. Vo
l. 267, p20532, 1992, Knutsen R. et al., Bioche
m. Biophys. Res. Commun. vol. 194, p1352, 1993) and other cytokines have been reported. These cytokines are expected to be agents for improving osteopenia by promoting bone formation, and insulin-like growth factor-I
Some of the above cytokines, such as (IGF-I) and ectopic bone morphogenetic protein family cytokines, have been clinically tested as bone metabolism improving agents. Calcitonin, which is a cytokine that suppresses bone resorption, is already on the market as a therapeutic drug for osteoporosis and a pain relieving drug.

IGF-I is a single-chain peptide having a structure similar to insulin and composed of 70 amino acids and having a molecular weight of 7649, and is also called somatomedin. I
The main producing organ of GF-I is considered to be the liver, and the blood concentration in normal humans is about 200 ng / ml. It is known that its blood concentration has a positive correlation with growth hormone (Growth Hormone). Most of IGF-I exists in the blood in an inactivated form by binding with a binding protein (IGF-binding protein), and IGF-I existing in a free form is 1% or less. As for the biological activity of this cytokine, it has been reported that it exerts many biological actions reported for insulin, such as promoting glucose oxidation in adipocytes and promoting glucose uptake into lipids (Luft.
R. et al., Advances in Metabolic Disorders vol.
8, Academic Press, New York, 1975). Also, IGF-
I is myotube formation of myotubes (Ewton D.
Z. et al., J. Cell. Physiol., Vol. 120, p263, 198
4) Development of glial cells (oligodendroglia) (McMorris
FA et al., Proc. Natl. Acad. Sci. USA, vol. 83,
p822, 1986) and the like are also reported to be promoted. In addition, IGF-I was added to the proliferation of fibroblasts (Zapf J. et al.,
Eur. J. Biochem, vol. 87, p285, 1978 and Rechler
(MM et al., Eur. J. Biochem, vol. 82, p5, 1978)
And proliferation of chondrocytes and osteoblasts (Luft R. et al. Eds., Ad
vances in Metabolic Disorders, vol. 8, Academic Pr
ess, New York, 1975 and Yukio Kato et al., Biochemistry, vo
l. 33, p389, 1982).
In addition to the action of IGF-I on osteoblasts, the ability to promote glycogen synthesis (Schmidt Ch. Et al., Calcif.
Tissue Int., Vol. 35, p578, 1983) has also been reported.
Such various biological activities have been reported in IGF-I, and at present, neurodegenerative diseases (peripheral neuropathies), amyotrophic lateral sclerosis, non-insulin-dependent diabetes mellitus, improved nutrition and metabolism, macula Development as a pharmaceutical is underway for degeneration, wound healing, osteoporosis and the like.
In addition, this IGF-I has already been put on the market in Japan as a drug intended for insulin receptor dysfunction and the like.

Currently, active vitamin D is clinically used as a drug for treating bone-related diseases and shortening the treatment period.
_3. Calcitonin and its derivatives, hormone preparations such as estradiol, ipriflavone or calcium preparations are used. However, the therapeutic methods using these are not satisfactory in terms of their effects and therapeutic results, and the development of new therapeutic agents to replace them has been desired. As described above, bone metabolism is regulated by the balance between bone formation and bone resorption, and cytokines that promote the proliferation of osteoblasts are expected to be therapeutic agents for osteopenia and other osteopenia. In view of such a situation, the present inventors extensively sought an osteoblast growth promoting factor, and as a result of intensive research, found a novel osteoblast growth promoting factor (bOGF-II), and further increasing the concentration of the protein of the present invention. Accumulation of these substances has led to the establishment of an efficient purification method (Japanese Patent Application No. 6-168984).
issue). Furthermore, the present inventors have examined means for further promoting the bone formation by this bOGF-II.

[0006]

DISCLOSURE OF THE INVENTION The present invention relates to bOGF-
It is an object of the present invention to provide an osteogenesis promoter which can further promote the osteoblast proliferation effect of II and can effectively treat or prevent osteopenia and other osteopenia.

[0007]

[Means for Solving the Problems] In order to solve the above-mentioned problems, the present inventors have developed an osteoblast growth promoting factor, particularly bOG.
A search was made for substances that further promote bone formation by F-II. As a result, it was found that the administration of bOGF-II in combination with IGF-I promotes osteogenesis more effectively than the administration of bOGF-II or IGF-I alone, and thus the present invention is realized. It came to eggplant. That is,
The present invention relates to an osteogenesis promoter containing osteoblast growth factor and insulin-like growth factor-I as active ingredients. Specifically, bOGF-II and IGF-I are used as osteoblast growth factors.
And an osteogenesis promoting agent comprising a combination of More specifically, the weight ratio of bOGF-II and IGF-I is from 1000: 1.
The present invention relates to an osteogenesis promoter having a ratio of 1: 100. Hereinafter, the present invention will be described in detail.

The osteoblast growth factor used in the present invention, particularly bOGF-II, is efficiently produced from the culture solution of human fibroblasts,
It can be isolated in high yield. BOG from this raw material
The production of F-II is carried out in the same manner as a general method commonly used for separating proteinaceous substances from biological substances, and the desired bOGF
-It can be carried out according to various purification operations utilizing the physical and chemical properties of II. The OGF (Osteoblast Growth Factor) activity, which is an index of the osteogenesis promoting action of the present invention, uses an osteoblast cell line or a normal osteoblast cell as a target cell to increase the number of cells and uptake of ³ H (tritium) -thymidine. It can be measured by testing acceleration. Mouse osteoblast cell line MC3T3-E1 is a preferred target cell.
(J. Oral. Biol. 23, 899-901, 1981 and J. Cell. Bio
l.96, 191-198, 1983) can be used. This cell line is responsive to vitamin D ₃ and parathyroid hormone,
It is also a cell line that has been reported to be calcified in vitro in a process similar to that in vivo. For the activity measurement, preferably, a serum-free medium is used to test the promotion of ³ H-thymidine uptake, whereby highly sensitive and accurate measurement can be performed.

In addition, IGF-I, as described above,
It is a single-chain peptide having a molecular weight of 7649 composed of 70 amino acids and is a known substance.

An osteoblast growth promoting factor used in the present invention,
In particular, bOGF-II and IGF-I are more effectively used as a therapeutic agent for various bone metabolism-related diseases such as osteoporosis when used in combination. The pharmaceutical composition therefor may contain bOGF-II and IGF-I related to the present invention as active ingredients, and may contain other pharmaceutically acceptable carriers. This composition is safely administered orally or parenterally to humans and animals as a pharmaceutical composition. The form of the pharmaceutical composition includes an injectable composition,
Examples include infusion compositions, suppositories, nasal agents, sublingual agents, transdermal absorption tapes and the like. In the case of an injectable composition, it is a mixture of a pharmacologically effective amount of bOGF-II and IGF-I related to the present invention and a pharmaceutically acceptable carrier, in which amino acids, sugars, cellulose derivatives, It is also possible to use excipients and / or activators which are generally added to injectable compositions such as and other organic and / or inorganic compounds.
When preparing an injection using bOGF-II and IGF-I related to the present invention and these excipients and / or activators, if necessary, a pH adjuster, a buffer, a stabilizer , A solubilizer, etc. are added to prepare various injections by a conventional method. The method of administration of these injections may be any of intravenous injection, subcutaneous injection, intramuscular injection and arterial injection.

The osteogenesis promoter of the present invention contains bOGF-II and IGF-I in its composition, and its weight ratio is 1000: 1 to 1: 100. The weight ratio is preferably 100: 1 to 1:10, and particularly preferably 100: 1 to 1: 1. As a method of administering this pharmaceutical composition, (a) a method of administering IGF-I after administering bOGF-II in various diseases associated with a decrease in bone mass or a decrease in bone strength. (b) In various diseases similar to the above, bOG after administration of IGF-I
A method of administering F-II. (c) In various diseases similar to the above, a method of administering a pharmacologically and pharmaceutically acceptable preparation containing IGF-I and bOGF-II can be mentioned.
The osteogenesis promoter of the present invention is prepared according to such an administration method. That is, bOGF-II and IGF-I may each be prepared as a single preparation, and the two agents may be used in combination,
Further, two components of bOGF-II and IGF-I may be contained in one drug. The osteogenesis promoter of the present invention may be used in combination with other therapeutic agents for abnormal bone metabolism. Furthermore, the composition of the present invention may be administered in combination with various drugs that cause a decrease in bone mass or a decrease in bone strength. As bOGF-II and IGF-I to be administered, human bO
It is particularly preferred to use GF-II and human IGF-I.

Hereinafter, the present invention will be described in more detail with reference to examples, but these are merely examples and the present invention is not limited thereto.

BEST MODE FOR CARRYING OUT THE INVENTION

Example 1 Preparation of bOGF-II bOGF-II was purified from human fetal lung fibroblast IMR-90 culture medium according to the method described in Japanese Patent Application No. 6-168984. Briefly, human fetal lung fibroblast / IMR-
90 (ATCC, CCL 186) was adhered to an alumina ceramic piece in a roller bottle and cultured to prepare a culture solution. or,
Purification of bOGF-II from this culture solution was performed by sequentially performing the following series of steps. (1) Heparin Sepharose
CL-6B column chromatography, (2) HiLoad-Q / FF column chromatography, (3) HiLoad-S / HP column chromatography, (4) Heparin 5PW column chromatography (first run), (5) Heparin 5PW column Chromatography (2nd time), (6) Heparin 5PW column chromatography (3rd time). These steps will be described in more detail.

(1) Heparin-Sepharose CL-6B column chromatography IMR-90 culture solution was filtered with a filter, and then 10 mM T
Heparin-Sepharose equilibrated with ris-HCl, pH 7.5
It was applied to a CL-6B column (5 x 4.1 cm). 10 mM Tris-HCl, pH
The column was washed with 7.5, 10 mM Tris-HCl containing 2M NaCl,
Elution was performed at pH 7.5 to obtain a heparin-Sepharose CL-6B adsorbed fraction.

(2) HiLoad-Q / FF column chromatography This heparin / sepharose adsorption fraction was diluted with 10 mM Tris-HCl,
After dialysis against pH 7.5, CHA becomes 0.1%
Anion exchange column (HiLoad-Q / FF, 2.6 × 10 cm, Pharmacia) equilibrated with 50 mM Tris-HCl, pH 7.5 containing 0.1% CHAPS after allowing PS to stand overnight at 4 ° C. Then, the non-adsorbed fraction was obtained.

(3) HiLoad-S / HP Column Chromatography This HiLoad-Q non-adsorbed fraction was added to 50 m containing 0.1% CHAPS.
Cation exchange column (H) equilibrated with M Tris-HCl, pH 7.5
iLoad-S / HP, Pharmacia). 0.1% CHAPS
After washing with 50 mM Tris-HCl, pH 7.5 containing 100 mM, elution was carried out with a linear gradient of NaCl to 1 M for 100 minutes for preparative separation. OGF activity was measured using each fraction. O
Three peaks with GF activity (peak 1; bOGF-
I, peak 2; bOGF-II, peak 3; bOGF-II
I got

(4) Affinity column (heparin-5P
W) Chromatography Peak 2 (bOGF-II) fraction containing 0.1% CHAPS 5
0.1% CHAPS after 3-fold dilution with 0 mM Tris-HCl, pH 7.5
Affinity column equilibrated with 50 mM Tris-HCl, pH 7.5 (Heparin-5PW, 0.8 × 7.5 cm, Tosoh)
I went to 50 mM Tris-HCl, pH 7.5 containing 0.1% CHAPS
After washing with, elution was performed with a linear gradient of NaCl to 2 M for 60 minutes, and after fractionation, the OGF activity was measured using each fraction.

Fractions with high OGF activity were diluted 3-fold with 50 mM Tris-HCl, pH 7.5 containing 0.1% CHAPS, and then 0.1% CH
It was applied to an affinity column (heparin-5PW, 0.8 × 7.5 cm, Toso Co.) equilibrated with 50 mM Tris-HCl, pH 7.5 containing APS. 50 mM Tris-HCl, pH containing 0.1% CHAPS, pH
After washing with 7.5, elution was performed with a linear gradient of NaCl to 2 M for 60 minutes, and fractionation was performed. OGF using each fraction
The activity was measured. The fraction with high OGF activity is 0.1
After diluting with 50 mM Tris-HCl, pH 7.5 containing% CHAPS,
It was applied to an affinity column (heparin-5PW, 0.5 × 7.5 cm, Tosoh) equilibrated with 50 mM Tris-HCl, pH 7.5 containing 0.1% CHAPS. After washing with 50 mM Tris-HCl, pH 7.5 containing 0.2M NaCl and 0.1% CHAPS, NaCl was added for 60 minutes.
Was eluted with a linear gradient from 0 M to 0.8 M, and fractionated. The protein concentration of purified bOGF-II prepared by these methods was determined by the Lowry method using BSA (bovine serum albumin) as a standard.

[0018]

Example 2 Measurement of Osteoblast Growth-Promoting Activity (OGF Activity) OGF activity was measured by DNA of mouse osteoblast cell line MC3T3-E1.
It was performed by measuring the synthesis promoting activity. Add 50 μl of sample diluted in α-MEM medium without nucleic acid (Gibco BRL) containing 0.2% BSA to 96-well microplate, and add MC
3T3-E1 cells were seeded in 2x10 ³ cells / 50 μl α-MEM medium,
The cells were cultured at 37 ° C for 5 to 20 hours in 5% CO ₂ . After culturing, 10 μl / well of ³ H-thymidine (TRK686, Amersham) diluted to 0.1 mCi / ml with 10 mM phosphate buffered saline was added, and after further culturing for 2 hours, ³ H incorporated into cells -
The radioactivity of thymidin was measured with a matrix β counter (Packard). The obtained radioactivity (cpm) was defined as OGF activity.

[0019]

Example 3 Production of a combination preparation of bOGF-II and IGF-I bOGF-II 200 ng IGF-I 20 ng human serum albumin 100 mg The above composition was mixed with 10 mM phosphate buffered saline (PBS),
The solution was dissolved in pH7.2 to prepare a total volume of 20 ml, and after sterilization, 2 ml each was dispensed into vials, which was freeze-dried and then sealed.

BOGF-II 200 ng IGF-I 2 μg Tween 80 2 mg sorbitol 4 g The above composition was mixed with 10 mM phosphate buffered saline (PBS),
The solution was dissolved in pH7.2 to prepare a total volume of 20 ml, and after sterilization, 2 ml each was dispensed into vials, which was freeze-dried and then sealed.

BOGF-II 2 μg IGF-I 2 μg glycine 2 g human serum albumin 50 mg The above composition was mixed with 10 mM phosphate buffered saline (PBS),
The solution was dissolved in pH7.2 to prepare a total volume of 20 ml, and after sterilization, 2 ml each was dispensed into vials, which was freeze-dried and then sealed. The preparations obtained by these methods are used as injections such as intravenous injection.

[0022]

Example 4 Osteoblast expansion by combination of bOGF-II and IGF-I
Breeding promotion test a) bOGF-II and IGF-I were tested for their osteoblast proliferation promoting activity. bOGF-II was used after purification by the above method and determination of protein concentration. IGF
-I as a recombinant human IGF-I (Intergen
Company) was used. First, 0, 1, 10 or 100 ng / ml IG
The effect of bOGF-II in the presence of FI was tested. That is,
In the presence of these different concentrations of IGF-I, bOGF-II
Was serially diluted 2-fold from 100 ng / ml, and the osteoblast proliferation-promoting activity when IGF-I and bOGF-II were combined and coexisted according to the method described above was tested. The results are shown in FIG.

In the presence of 1 ng / ml IGF-I, bOGF
The apparent specific activity of -II increased about 10-fold. Also, 10ng
In the presence of / ml IGF-I, the apparent specific activity of bOGF-II increased about 20-fold. The effect of bOGF-II in the presence of 100 ng / ml IGF-I was almost the same as that in the presence of 10 ng / ml IGF-I. In addition, the maximum effect of bOGF-II in promoting the proliferation of osteoblasts in the presence of 1-100 ng / ml IGF-I was increased about 2-fold.

B) Next, 0, 1, 10 or 100 ng / ml
The effect of IGF-I was tested in the presence of bOGF-II. That is, in the presence of these different concentrations of bOGF-II, IGF
The concentration of -I was serially diluted 2-fold from 100 ng / ml, and the effects of IGF-I and bOGF-II were tested according to the method described above. The results are shown in FIG.

Under the conditions used in the test, IGF-I alone did not find any growth-promoting effect on mouse osteoblastic cell line MC3T3-E1 cells. In the presence of 1 ng / ml bOGF-II, promotion of osteoblast proliferation was observed at about 1 ng / ml or more of IGF-I, and this action reached a plateau at about 3 ng / ml IGF-I. About 0.1 in the presence of 10 ng / ml bOGF-II.
IGF-I above ng / ml promoted osteoblast proliferation, and this action reached a plateau at about 1 ng / ml IGF-I. In the presence of 100 ng / ml bOGF-II, about 0.1 ng / ml
It was observed that IGF-I in a concentration range of about 10 ng / ml promoted proliferation of osteoblasts. From these results, bOGF-II
And bGF-II compared to the administration of IGF-I alone
It was found that the combined use of and IGF-I more effectively promotes bone formation.

[0026]

INDUSTRIAL APPLICABILITY An osteoblast growth promoting factor, particularly bOGF-
The combination of II and IGF-I as an osteogenesis promoter is effective in promoting osteogenesis more effectively and in treating or preventing osteopenia such as osteoporosis.

[Brief description of drawings]

FIG. 1 bO in the presence of 0, 1, 10, 100 ng / ml IGF-I
It shows a growth promoting effect on mouse osteoblastic cell lines tested by changing the concentration of GF-II. The test was performed in triplicate, and the result shows the average value.

FIG. 2: I in the presence of 0, 1, 10, 100 ng / ml bOGF-II
It shows the effect of promoting the proliferation of mouse osteoblastic cell lines tested by changing the concentration of GF-I. The test was performed in triplicate, and the result shows the average value.

 ─────────────────────────────────────────────────── ─── Continuation of front page (72) Inventor Kyoji Yamaguchi 702-1 Shimamachi, Omiya-shi, Saitama Lions Garden 1-524 Higashi-Omiya (72) Inventor Kazuki Yano 578-15 Ishibashi, Ishibashi-cho, Shimotsuga-gun, Tochigi Nishiura Heights 3-1 (72) Inventor Fumie Kobayashi 3777-4 Shimookamoto, Kawauchi-cho, Kawachi-gun, Tochigi Prefecture 72 (72) Koji Higashio 1769-10 Yamada, Kawagoe-shi, Saitama Prefecture

Claims (3)

[Claims] 1. An osteogenesis promoter comprising osteoblast growth factor and insulin-like growth factor-I as active ingredients. 2. The osteogenesis promoting agent according to claim 1, wherein the osteoblast growth factor is osteoblast growth promoting factor (bOGF-II). 3. The osteogenesis promoter according to claim 1, wherein the mixing ratio of the osteoblast growth factor and the insulin-like growth factor-I is 1000: 1 to 1: 100 by weight.