JPH04178334A - Therapeutic agent for osteopetrosis - Google Patents

Abstract

PURPOSE:To obtain the subject therapeutic agent, containing a human monocyte- macrophage colony stimulating factor (M-CSF) as an active ingredient, having action on increase in osteoclastic cells and continuously administrable to patients for a long period without any side effects. CONSTITUTION:A therapeutic agent containing M-CSF isolated from human urine, a culture solution of a cell capable of producing the M-CSF or a culture solution of a genetic recombinant cell capable of expressing the M-CSF as an active ingredient. The M-CSF is a glycoprotein of homo-dimers composed of the same subunits and has 70000-90000 daltons molecular weight measured by an SDS-polyacrylamide gel electrophoresis with 35000-45000 daltons molecular weight of subunits prepared by dissociation with a reducing agent and making biological activity disappear. The aforementioned subunit protein is constructed from 214 amino acids (the M-CSF derived from the human urine) or 223 amino acids (genetic recombinant M-CSF).

Description

[Detailed Description of the Invention] [Industrial Application Field] The present invention relates to a therapeutic agent for osteopetrosis containing human monocyte-macrophage colony stimulating factor (hereinafter referred to as M-C8F) as an active ingredient. The present invention relates to a drug that is highly effective in treating osteopetrosis by stimulating the proliferation of osteoclasts through the action of human monocyte-macrophage colony-stimulating factor on osteoclasts.

[Prior Art] Colony stimulating factor (hereinafter abbreviated as C3F) is a hematopoietic factor that stimulates the differentiation and proliferation of hematopoietic stem cells present in mammalian hematopoietic tissue, such as bone marrow, and most C3Fs are composed of glycoproteins. ing. Conventionally, factors that act on monocyte-macrophage stem cells (M-C8F or C3F-1)
, a factor acting on granulocyte-monocytic stem cells (GM-CS
F) Factor that acts on granulocytic stem cells (G-CSF)
Four types of factors are known: , and a factor [Multi-CSF or interleukin-3 (IL-3)] that acts on pluripotent stem cells common to granulocytes, monocytes, erythrocytes, and megakaryocytes.

Among them, M-CSF, one of the factors that acts on monocyte-macrophage stem cells, was first isolated from human urine by the applicant and others (Patent No. 1.304.140. In this invention, M-C3F It has been proven that there are no side effects and that it can be administered continuously for a long period of time), and subsequently, the applicant et al. developed a cancer immune adjuvant (Japanese Patent Application Laid-Open No. 193227/1999), a therapeutic agent for thrombocytopenia (Japanese Patent Application Laid-Open No. 1-2 (17244)). Publication No.)
, therapeutic agent for hematopoietic diseases (Patent Application No. 1-2213224),
Anti-malignant tumor agent (Japanese Patent Application Laid-open No. 2-2254111), therapeutic agent for malignant tumor (Japanese Patent Application No. 1-856512), therapeutic agent for hyperlipidemia (Japanese Patent Application No. 1-117373), therapeutic agent for myelodysplastic syndrome Applications such as (Japanese Patent Application No. 1-150087) have been filed.

Bone metabolism consists of two processes: bone formation and bone resorption. That is, old bone is resorbed by osteoclasts, while new bone is formed by osteoclasts (this is called bone tissue glue modeling).

Bone metabolic abnormalities are caused by abnormalities in the number of cells present in bone tissue and their activity. There are three types of cells in bone: osteoblasts, osteocytes, and osteocytes, and from mesenchymal cells to preosteoblasts → osteoblasts → osteocytes, and from mesenchymal cells to preosteoclasts →
Differentiate and develop like osteoclasts.

Premarble shoulder is a genetic disease characterized by a tendency to fracture, impaired hematopoiesis, and cranial nerve symptoms, and is a congenital bone disorder with increased trabeculae and calcification of cartilage, which often occur in long bones due to abnormal bone metabolism. There is hyperplasia, and anemia and decreased osteoclasts due to decreased bone marrow cavity are observed.

Traditionally, the mainstay of treatment for this disease has been symptomatic treatment.
Antibiotics are administered for infections, blood transfusions and cortisone are administered for anemia, and surgical decompression is used for nerve compression. On the other hand, parathyroid hormone, growth hormone, thyroid hormone, etc. are known as drugs that may increase the number of osteoclasts or enhance their action, but these drugs have side effects and cannot be used continuously for long periods of time. Cannot be administered to patients.

As mentioned above, it has been known that M-C3F acts on bone marrow stem cells and has the effect of promoting differentiation and induction into monocytes and macrophages, but it is not known that M-C3F has an osteoclast proliferation effect. It is not described in any literature and is not known in the past.

[Problems to be Solved by the Invention] In view of the above-mentioned current state of treatment for osteoporosis, there has been a long-awaited development of a drug that has no side effects and can be continuously administered to patients over a long period of time.

The present inventors have completed the present invention by conducting intensive research on developing the use of M-CSF, and by discovering that M-C3F has the effect of increasing osteoclasts.

[Means for Solving the Problems] The present invention provides a therapeutic agent for marble shoulder, characterized in that it contains human monocyte-macrophage colony-stimulating factor as an active ingredient; A marble shoulder treatment agent that acts on cells and stimulates the proliferation of osteoclasts; human monocyte-macrophage colony-stimulating factor is added to human urine, a culture medium of human monocyte-macrophage colony-stimulating factor-producing cells, or human monocyte-macrophage colony-stimulating factor. A therapeutic agent for marble front shoulder obtained from a culture medium of genetically modified cells capable of expressing a monocyte-macrophage colony stimulating factor; a marble front shoulder characterized in that the human monocyte-macrophage colony stimulating factor has the following physicochemical properties: Relating to therapeutic agents.

a) A homodimer consisting of subunits with the same molecular weight, the molecular weight measured by sodium dodecyl sulfate polyacrylamide gel electrophoresis is 701,000 to 90,000 Daltons, and biological activity is lost when dissociated with a reducing agent. The molecular weight measured by sodium dodecyl sulfate polyacrylamide gel electrophoresis for the subunit was 35,100.
0 to 45,000 daltons b) Subunit amino acid sequence The subunit protein constituting the homodimer is derived from human urine, consisting of the following 214 amino acids, or genetically modified protein consisting of 223 amino acids. Glo7Glu-Val-3++-Glu-T7+-C is a human monocyte macrophage colony stimulating factor
ys-3++-Hi+-Mel-11e-G17-5e
r-Gly-His-Leu-Gl++-5er-Le
u-Gln-Aip-Leu-11e-^sp-3et
-Gln-Met-Glu-Tbr-3e+-Cyl-
Gln-Ile-Tbr-Phe-Glu-Phe-V
al-^+p-Gln-Glu-Gln-Leu-L7
s-Aip-Pro-Va 1-Cyl-Ty+-Le
u-L7+-Ly+-Ala-Phe-Leu-Leu
-Va 1-Gln-Aip-11e-Me t-G
l u-As p-Tbr-Me I-A r g-P
b t-A rg-person+p-Asn-Th+-P+o-
^5n-Ala711e-Ala-11e-11al-
Gln-Leu-Gln-Glu-Leu-3++-L
eu-^B-Leu-Ly+-3++-C7+-Phe
-Thr-L7+-Aip-Ty+-Glu-Glu-
Hi +-A+p-L7s-Ala-Cy+4al-A
ip-Tbr-Pbe-Ty "-Glu-Tbr-P+
o-Leu-Jn-Lev-Leu-Gl u-Lys
-Va 1-LH-^+n-YakPhe-A+n-G
lo-Tbr-Lys-person+n-Leu-Leu-As
p-LH-^5p-T+p-A+n-11e-Phe-
Se+-L7s-Asn-Cyl-A+n-Ain-3
e+-Phe-Ala-Glu-C7+-Se+-3e
t-Gln-Aip-Val-Val-Tbr-Lys
-Pro-Aip-Cys-Ain-C7+-Leo-
T7+-Pro-Lys-Ala-11e-Pro-S
e+-3et-Aip-Pro-Ala-Se+-Va
l-3++-Pro-His-Gln-Pro-Leu
-Ala-Pro-3et-Met-Ala-Pro-
Va 1-Ala-Gly-Leu-Tbr-T+p-
Glu-Human+p-3++-Glu-Gly-Th+-G
lu-Gly-5et-3e+-Leu-Leu-Pr
o-Gly-Glu-Gln-Pro-Leu-His
-Tbr-Val-Asp-Pro- or Glu-Glu4al-5et-Glu-Ty+-C7
s-5e+-1(is-Mef-11e-Gly-3e
+-G17-His-Leu-Gln-Se+-Leu
-Gln-AB-Leu-Ile-^+p-3e+-G
ln-Mel-Glu-Tbr-3et-Cys-Gl
n-11e-Tbr-Phe-Glu-Phe-Val
-Aip-Gln-Glu-Gln-Leu-Lys-
Aip-Pro-Va 1-Cys-T7+-Leu-
Lys-L7i-Ala-Ph+-L+u-Leu4a
l-Gln-Asp-11t-MeiGlu-Aip-
Tbr-Met-^+g-Phe-A+g-Asp-A
+n-Tbr-Pro-Ain-Ala-lle-Al
a-11e4al-Gln-Leu-Gln-Glu-
Leu-3e+-Leu-Arg-L+u-LH-3e
+-Cys-Ph+-Tbr-Lys-Asp-Ty+
-Glu-Glu-His-Aip-Lys-Ala-
Cys-Val-A; g-Tbr-Phe-Ty+-G
lu-Tbr-Pro-Leu-Gln-Leo-Le
, u-Glu-LH4al-Lys-Asn4al-P
he-Ain-Glu-Tbr-L7+-Asn-Le
u-Leu-Asp-Lys-Aip-T+p-Asn
-11e-Phe-3e+-L7i-Ain-Cys-
Ain-Asn-3et-Phe-Ala-Glo-C
H-3er-3et-Gln-Aip-Va14al-
Tbr-L7+-Pro-Aip-Cys-^+n-C
ys-Leu-Ty+-P+o-Lys-Ala-11
e-Pro-3etzu [-All-PIO-Ala
-Se+-Val-Se+-? o-)1i+-Gln-
Pro-Leu-Ala-Pro-Ser-Mef-A
la-Pro-Val-^1a-Gly-Leu-Th
+-T+p-Glu-Aip-3e+-Glu-Gly
-Tbr-Glu-Gly-3et-5et-Leu-
Leu-Pta-Gll-Gla-Gln-Pta-L
eu-His-Thr-Val-Aip-Pro-Gl
y-3e+-Ala-Lys-Gln-Arg-Pro
-Pro-Arg- M-CSF of the present invention (Wong, G, G, e
tal; 5 science, vol. 235, p. 1504, 1987) is a known C8F-1 (Kawasa
ki, E, S,, et al, 5cienc
e% Vol. 230, p. 291, 1985), two polypeptides containing sugar chains are disulfide bonded, and are composed of a biologically active homodimer, with a molecular weight of 70,000 to 90,000. 000 Daltons and C
The molecular weight is larger than that of 3F-1. Furthermore, the polypeptide of the subunit constituting M-CSF is 214 or 2
It consists of 23 amino acids and has a molecular weight of 2L400 Daltons, which is 14.000 to 17.0 that of C3F-1.
Greater than 00. Also, if the amino acid sequence of the subunit is compared with C3F-1, it is 149 from the first position of the N-terminus.
The amino acid sequences from position 150 to position 214 or 223 are the same, but the amino acid sequences from position 150 to position 214 or 223 are
Unlike that predicted from F-1 cDNA, C3F
-1 gene was not encoded. Therefore, M-
C3F was found to be a separate factor from the known C3F-1. Moreover, the above-mentioned known glycoprotein, GM-CSF
(Wong, G, Gel al, 5science
, vol. 228, p. 810, 1985) in terms of biological activity and physical and chemical properties.

The M-C3F of the present invention can be isolated from human urine by chemical methods, or from the culture solution of M-C3F-producing cells or the culture solution of genetically modified cells capable of expressing M-C3F. You can also do that. An example of isolation from human urine by chemical methods (Nobuya Yanai et al.: Basic and Clinical Studies, Vol. 22,
2000 pages, 1988) is as follows.

Urine from a healthy person is adjusted to a pH range of 8.0 to 9.0, viscous substances in the urine are precipitated and removed, and the supernatant is purified to a pH of 1.
Concentrate and desalt using an ultrafiltration membrane that passes between 0.000 and 50.000 daltons. Concentrate to at least 200 times [protein concentration of 1% (weight/volume. The same applies hereinafter unless otherwise specified)] and then adjust the pH to 6.5 to 7.
5 and heat at 60° C. for 10 hours to inactivate the virus), the generated precipitate is removed by centrifugation, and the active ingredient is adsorbed onto an anion exchanger (for example, DEAE-cellulose, etc.).

Then 0.05-0.1M buffer (p) 16.5-7
．． 5) and then wash the ion exchanger with 02-0.4M
The active ingredient is eluted with a buffer solution (p'H 6,5-75). Concentrate the eluate with an ultrafiltration membrane as necessary to obtain a concentration of 1 to 4M
with a gel filtration agent [e.g., Sephacryl S-300 (registered trademark, manufactured by Pharmacia), etc.] equilibrated with a buffer solution (pH 6.5 to 7.5) containing salts (e.g., ammonium sulfate, common salt, etc.). Gel filtration, molecular weight range 70
,000 to 150,000 daltons is collected.

The fractions were then treated with hydrophobic affinities [e.g., phenyl sepharose (
Registered trademark. (manufactured by Pharmacia), etc.], and
Elute with 1.0 M salt-containing buffer (pH 6.5-7.5). The eluate was concentrated with an ultrafiltration membrane, gel-filtered with a high-performance liquid gel filtration column [for example, TSKG-3000SW (manufactured by Toyo Soda Co., Ltd.)], and the molecular weight range was 70.00.
Collect the fraction from 0 to 150.000 daltons. The fractions were concentrated again and diluted with 0.1% trifluoroacetic acid solution (pH 1
~2)) and adsorbed onto a reverse phase column for high performance liquid chromatography [e.g., Ii-Pore 214TP (registered trademark, manufactured by Vydac)], and then adsorbed on a solvent containing a 0.1% trifluoroacetic acid solution (e.g., , acetonitrile,
The active fraction is collected and lyophilized.

This active fraction is a pure substance containing M-C8F with a specific activity of 1.times.10@8 units/.multidot..protein, and has, for example, the following physical and chemical properties.

a) Isoelectric point Isoelectric point (pl) measured by polyacrylamide gel isoelectric focusing method and sucrose density gradient isoelectric focusing method
) are 31-37.

b) Constituent monosaccharides of the sugar chain The constituent monosaccharides of the sugar chain identified by hydrolysis and analysis by high performance liquid chromatography are mannose, galactose, N-acetyl lactosamine, and N-acetylneuraminic acid.

C) Circular Dichroism Spectrum The far-ultraviolet CD spectrum produced by the circular dichroism dispersion needle has minimum peaks at wavelengths of 208 nm and 222 nm, respectively, and contains an α-helical structure.

d) Thermal stability No loss of biological activity when heated at 60±0.5°C for 60 minutes.

e) Infrared absorption spectrum wave numbers 1680an-', 1200an-', and ll3
Intensity absorption at 0an-', wave number 1540an-', 143
0 cm-', and 1070C! Moderate absorption is shown at n-'.

A method for isolating M-C3F from the culture medium of genetically modified cells capable of expressing M-C3F (Japanese Patent Application No. 1-117372)
Examples of the above are as follows.

Anti-M-C obtained by immunizing a rabbit with M-C8F purified from the culture medium of M-C3F genetically modified cells (CHO cells)
Dialyze the 3F antibody in 0.1M phosphate buffer (pH 7.0) and adjust the concentration. The antibody solution was previously mixed with distilled water and 0
, Add formyl-cellulofine washed with 1M phosphate buffer, stir at room temperature, add sodium cyanoborohydride, stir further, formyl-cellulofine and anti-M-C3F antibody are combined, and the antibody binding support is obtained. Prepare the body. Next, the mixture is washed with a 0.2i1J HCl buffer, a Tris buffer containing sodium cyanoborohydride is added, and the mixture is stirred at room temperature to inactivate unreacted groups. Afterwards, the antibody-bound support was diluted with 0.0.
Wash thoroughly with 2MIJ acid buffer.

The culture solution of genetically modified cells (CHO cells) capable of expressing M-C8F was concentrated using a ultraconcentrator, desalted, and treated with DEA.
Adsorb to E-cellulose, remove non-adsorbed contaminants, elute with 0.3M aqueous solution of sodium chloride, adjust the concentration by adding sodium chloride to the eluate,
Prepare a solution containing 8F. This M-C3F-containing solution is added to the antibody-bound support, stirred at 106C or less, and subjected to batch chromatography. Filter with a glass filter and collect the antibody-bound support at 0.5M
The antibody binding support was thoroughly washed with 002M phosphate buffer containing sodium chloride, and then washed with 0.2M acetate buffer (p
Add H2,5) and stir at 10°C to elute M-CSF. The pH of the eluate is adjusted to 70, and the eluate is concentrated and desalted using an ultrafiltration membrane to obtain the M-C3F fraction. This fraction was subjected to high performance liquid chromatography using a reverse phase column with a linear concentration gradient of acetonitrile 0 to 100% (pH 2,0) containing 0.1 trifluoroacetic acid, M-C3F was collected, lyophilized, and M-CSF get.

The M-C3F obtained as described above was mixed with a buffer solution (pl (6,5-7) containing human serum albumin and sugars as stabilizers.
5) is added and dissolved, sterile filtered, and sterilely freeze-dried to produce a premarthrosis treatment agent.

In addition to human serum albumin and saccharides, it can also be used in combination with pharmacologically acceptable ingredients, and even with drugs conventionally used for the treatment of marbled shoulders.

As is clear from the examples described later, the thus produced therapeutic agent for premarthrosis is at least 30 μg/m
Contains M-C8F at a concentration of 1.

Next, the present invention will be explained in detail by showing test examples.

Test Example 1 This test was conducted to examine the effects and effective dosage of M-C3F.

(1) Preparation of sample A therapeutic agent for premarthrosis was prepared by the same method as in Example 1.

(2) Test method An 11-day-old mouse model of marble anterior shoulder pathology.
111011 mouse (with typical marble disease pathology,
40 animals (with extremely low numbers of osteoclasts and peritoneal macrophages) were divided into 5 groups and treated with the therapeutic agent for pre-petrosis of the present invention at 0 (control group), 1 μg (group 1), 2. 5μ
g (group 2), 5 μg (group 3), and 75 μg (group 4).
group) in two or three divided doses by subcutaneous administration.
It was administered for 4 days.

Mouse peripheral blood was collected from the eye cavity after anesthesia, and the number of nucleated cells was counted using a hemocytometer.

The number of differentiated cells is May-G+unwald/Gi
It was measured by emsa staining.

The tibia and femur were fixed in 0.1 M cacodylate buffer (pH 7.4) containing 2% paraformaldehyde and 2% glutaraldehyde for 2 hours at 4°C. After washing away the buffer, the specimens were dehydrated with ethanol, embedded in JB-4 medium, and sections were prepared. , 4 micron thick sections were stained with tolidine blue to determine the general condition of the tissue and 1aNI212-usistint.
Acid phospholase (hereinafter referred to as TRACP) activity was observed using an optical microscope.

Other specimens were decalcified with 5% EDT^(pi(7,21), then fixed with 1% osmic acid, and treated with Epon8.
12, and ultrathin sections were stained and observed with an electron microscope.

(3) Test results The results of this test were as shown in Table 1 and Figures 1 to 3. FIGS. 1 and 3 are optical micrographs of the control group and group 3 mouse tibias, respectively, and FIG. 3 is an electron micrograph of the third group mouse tibias. In FIG. 2, arrows and arrowheads indicate Tl1ACP-positive osteoclasts and TRACP-positive mononuclear cells, respectively.

(Margins below) As is clear from Table 1, the number of monocytes in the peripheral blood of the mice in Groups 3 and 4 increased significantly, but there was no significant difference between these groups.

Furthermore, it was observed that the lesion in the tibia of the group 3 mice in FIG. 2 was significantly improved compared to the premarble shoulder lesion in the tibia of the control group mice in FIG. 1. In other words, in Fig. 1, there is no increase in trabeculae, decrease in medullary cavities, and no presence of TRACP-positive cells, which are markers of osteoclasts, whereas in Fig. 2, most of the trabeculae are composed of marrow. replaced by the cavity, T
RACP-positive osteoclasts (arrow) were distributed along the remaining trabecular surface, and TRACP-positive mononuclear cells (arrowhead) were observed within the medullary cavity.

Furthermore, as is clear from Fig. 3, in the tibiae of group 3 mice, giant cells with typical osteoclast features such as well-developed wrinkled borders, transparent areas, and lysosomal structure appeared. was recognized. Optical microscopic observation and electron microscopic observation of the tibiae of the second group of mice showed that the lesions were improved compared to those of the control group and the first group, although not as markedly as those of the third group. These results indicate that M-C3F promotes the differentiation and proliferation of osteoclasts, accelerates the bone resorption process, restores normal bone metabolism, and is effective in treating premarble shoulder.

Since this effect was observed at a dose of at least 25 μg per day, it is estimated that this corresponds to at least 100 μg/kg body weight per day in humans when calculated from the average body weight of mice. Furthermore, M-C3F is usually administered via intravenous, intraarterial, intramuscular, subcutaneous, intraperitoneal, or other routes of administration.

Test Example 2 (Toxicity) M-C3F prepared by the same method as Example 2 was used to determine acute toxicity using the method of Richard et al. (J.

Pha+maco1. Exp, Them, , 9
0, p. 99, 1949) in C3TBL male mice. The results are shown in Table 2.

Table 2 Administration method LD. r unit/ki (g/kw
) Intraperitoneal administration 1X10' (4) Intravenous administration 5X10' (2) Subcutaneous administration
lXl0'' (4) Next, the present invention will be explained in further detail by showing examples, but the present invention is not limited to the following examples.

Example 1 Anti-M-C9F antibody obtained by immunizing rabbits with purified recombinant M-CSF was added to 0.1M phosphate buffer (pH 7,
0), the concentration was adjusted to 20 mg/ml, the antibody solution was added to 200 ml of 100 g of formylcellulofine, which had been previously washed with distilled water and O, IMIJ acid buffer, and stirred at room temperature for 2 hours. 700 ml of sodium cyanoborochloride was added, and the mixture was further stirred for 16 hours to bind formylcellulofine and anti-M-C3F antibody to prepare an antibody-bound support. Afterwards, the mixture was washed with 0.2 M Tris-HCl buffer, and 200 ml of Tris buffer containing 500 μl of sodium cyanoborohydride was added, and the mixture was stirred at room temperature for 4 hours to inactivate unreacted groups and remove the antibody-bound support. It was thoroughly washed with 0.02M phosphate buffer containing 0.5M sodium chloride. This antibody-bound support has 32
．． It was bound to the anti-M-C8F antibody of 6.

Concentrate and desalt the culture solution + OL of genetically modified cells capable of expressing M-C8F (CHO cells) using an ultrafiltration concentrator,
DEAE- adsorbed on cellulose, removed unadsorbed contaminants, eluted with 0.3M aqueous sodium chloride solution, added sodium chloride to the eluate to adjust the concentration to 0.5M, and contained M-C3F. A solution was prepared. This M-C3
The specific activity of F was 3×10 6 units/■.

This M-C3F-containing solution (500 ml in total) was added to 100 g of the above antibody-bound support, stirred overnight at 10° C. or lower, and subjected to batch chromatography. After stirring, filter through a glass filter, collect the antibody-bound support, and add 112M containing 0.5M sodium chloride.
The antibody-bound support was thoroughly washed with J acid buffer.

After washing 0.2M acetate buffer (pH 2, 5) 500 m
The mixture was stirred for 1 hour using IQ'C, and M-C8F was eluted. The pi of the eluate was adjusted to 7.0 and concentrated and desalted using an ultrafiltration membrane to obtain the M-C3F fraction. This fraction was filtered into Hi-Pou+214TP (Vydac, diameter 2.
High performance liquid chromatography with a linear concentration gradient of 0-100% acetonitrile (pl(2,0)) containing 01 trifluoroacetic acid on a reverse phase column of 2x25);
The M-C3F was collected and lyophilized to obtain about 20 μm of M-C8F. The specific activity of the obtained M-CSF was 1.9×108
Unit/■, purity by 5DS-PAGE method is 98
% or more.

0 containing 5.5 g of mannitol in the obtained M-C3F,
Phosphorus buffer containing 15M sodium chloride (pH 7,2) 5
50 ml was added to dissolve M-C3F, sterilized using a filtration sterilization device (manufactured by Millibore) equipped with a nitrocellulose-based sterile filtration membrane, and sterilized by dry heat at 180°C for 2 hours. 1 ml of the solution was aseptically filled into vials, lyophilized aseptically, and the vials were sealed to obtain 500 bottles of the marble anterior shoulder treatment agent.

Example 2 Healthy human urine 1[)OOA' was adjusted to pH 11.5, the precipitate was filtered out, and the molecular weight cut off was 50. Concentrate and desalt using ON Dalton's ultrafiltration membrane (manufactured by Amicon Co., Ltd. I (I [l Centrifugation (5
, 0O (l x g for 30 minutes) to remove the precipitate. Next 11. l] Mix with DEAE-cellulose equilibrated with 2M phosphate buffer (pH 7,2) and adsorb onto this, and add O, 112M phosphate buffer (pH 7,2) and O,
[15M bolus added G, 02M phosphate buffer (pH 7,
2), then Q, 25M salt addition 0.02
Elution was performed with M phosphate buffer (pH 7,2), and the eluate was concentrated using an ultrafiltration membrane (manufactured by Amicon, HIOPIO). Sephacryl S-300 (registered trademark).

Column (diameter 20an) packed with
.

The concentrated solution was gel-filtered with a 1M ammonium sulfate-added buffer (pH 7,2) using a 1M ammonium sulfate-added buffer (pH 7,2), and a fraction with a molecular weight range of 70,000 to 150,000 daltons was filtered using the above-mentioned 1M ammonium sulfate-added buffer (pH 7,2). Phenyl Sepharose 4B (registered trademark, manufactured by Pharmacia) column (diameter 10an, length 21Jan) equilibrated with 2)
), then Q, ! Elution was performed with iM ammonium sulfate loading buffer (pH 7.2). The eluate was concentrated using an ultrafiltration membrane (manufactured by Amicon, HIQPIO), and the TS
KG-30005W (manufactured by Toyo Soda Co., Ltd.) column (diameter 2
．． 5an, length 60an) and subjected to high performance liquid chromatography using a molecular weight range of 70.000 to 150.
000 Dalton fraction was obtained. This fraction was concentrated again using an ultrafiltration membrane (manufactured by Amicon, HIOPIO), and Hi-P
ore 214TP (registered trademark. Manufactured by Vydac)
Reversed phase column (diameter 2.5 an.

High performance liquid chromatography with a linear gradient of acetonitrile 1 to IO [IX CpH 2,0) containing 0.1% trifluoroacetic acid with length 60) and M-C8
F was eluted, the eluate was lyophilized, and M-C5F was approximately 4
I got it. This process was repeated to obtain about 40 ml of M-C8F. Hereinafter, in the same manner as in Example 1, the marble anterior shoulder treatment agent 50
I got 0 pieces.

Example 3 Sephacryl S-300 (registered trademark, manufactured by Pharmacia) was added to TSKG-3000SW (manufactured by Toyo Soda), and Phenyl Sepharose 4B (registered trademark, manufactured by Pharmacia) was added to TSK Phenyl-5PW (manufactured by Toyo Soda). 500 bottles of a marble anterior shoulder treatment agent were obtained in the same manner as in Example 2, except for the following changes and the purification by high performance liquid chromatography.

[Effect of the invention] The effects achieved by the present invention are as follows.

(1) Osteoclast proliferation is promoted by the osteopetrosis therapeutic agent of the present invention.

(2) The therapeutic agent for osteopetrosis of the present invention is effective as a fundamental therapeutic agent for osteopetrosis.

(3) The therapeutic agent for osteopetrosis of the present invention has the advantage of fewer side effects than conventionally known osteoclast proliferation agents.

[Brief explanation of the drawing]

Figures 1 to 3 are photographs showing the morphology of living things.
The figure is a micrograph of the op10p mouse tibia, and the second
The figure is a photomicrograph of oplop 7 mouse tibia after administration of M-C3F;
0p is an electron micrograph of mouse tibia tissue.

Claims (4)

[Claims] (1) A therapeutic agent for osteopetrosis characterized by containing human monocyte-macrophage colony stimulating factor as an active ingredient. (2) Claim that the human monocyte-macrophage colony stimulating factor acts on osteoclasts to stimulate osteoclast proliferation (
1) The therapeutic agent for osteopetrosis described above. (3) Human monocyte-macrophage colony stimulating factor
Claim (1) or claim 1, which is obtained from human urine, a culture solution of human monocyte-macrophage colony-stimulating factor-producing cells, or a culture solution of genetically modified cells capable of expressing human monocyte-macrophage colony-stimulating factor. The therapeutic agent for osteopetrosis according to item (2). (4) The therapeutic agent for osteopetrosis according to claims (1) to (3), wherein the human monocyte-macrophage colony stimulating factor has the following physicochemical properties. a) A homodimeric glycoprotein consisting of subunits with the same molecular weight and a molecular weight of 70,000 to 90,00 as measured by sodium dodecyl sulfate polyacrylamide gel electrophoresis.
00 daltons, and the molecular weight of the subunit, which has been dissociated with a reducing agent to eliminate its biological activity, is 35,000 to 45,000 daltons as measured by sodium dodecyl sulfate polyacrylamide gel electrophoresis; b) of the subunit; The subunit protein constituting the amino acid sequence homodimer is human urine-derived protein consisting of 214 amino acids as shown below, or recombinant human monocyte macrophage colony stimulating factor consisting of 223 amino acids [gene sequence is Yes] [There is a gene sequence]