JP2605835B2 - Osteoporosis treatment - Google Patents

Description

The present invention relates to a therapeutic agent for porous bone. More specifically, polypeptides BUF-4 and BUF-5 (hereinafter BUF-4 and BUF-5)
) As an active ingredient.

2. Description of the Related Art In recent years, the number of osteoporosis (also called osteoporosis) patients has increased in Japan with the increase in the elderly population, and this has become a major social problem. Currently, the number of affected patients is estimated to be about 4 million, but osteoporosis is a disease in which bone mass is reduced and skeletal tissue is atrophied. This disease is a very dangerous disease in which the trabecular bone becomes thin and the bone becomes brittle due to the decrease in bone mass, and as a result, the vertebrae, femur, tibia and the like are fractured.

This disease can be broadly divided into two types: osteoporosis in menopause seen in postmenopausal women and senile osteoporosis seen in elderly men and women.

As the therapeutic agents for osteoporosis, suppressing a conventional osteolysis calcitonin, female hormone (especially administered to postmenopausal women), active vitamin D ₃ and anabolic Suraroido is used.

However, these treatments stop bone loss,
Fundamental treatment of increasing bone mass has not been achieved.

In addition, when vitamin D ₃ is used, there is a problem that some side effects are present, such as appearance of hypercalciuria.

Therefore, at present, it is desired to develop a therapeutic agent for osteoporosis, which has a small number of side effects, suppresses a decrease in bone mass, and further increases the bone mass.

SUMMARY OF THE INVENTION Accordingly, the object of the present invention is to find a factor effective in fundamental treatment of osteoporosis from various proteins produced by human cells, with few side effects, and to obtain a novel osteoporosis. It is to provide a therapeutic agent.

Means for Solving the Problems In order to solve the above-mentioned problems, the present inventors have searched various human cell products for an osteogenesis promoting agent, and found that human malignant monocyte cells were identified as a specific differentiation inducer. BUF-4 produced by culturing in the presence of
And BUF-5 have an effect of promoting the proliferation of human bone marrow cells, and have found that this effect can fundamentally treat osteoporosis, and have completed the present invention. That is, the therapeutic agent for osteoporosis of the present invention comprises at least one of the polypeptides BUF-4 and BUF-5 having the following physicochemical properties:
It is characterized by containing more than one kind of substance as an active ingredient.

(1) Physicochemical properties of BUF-4 (a) Structure: monomer A (see FIG. 1) and monomer B
(See FIG. 2) Heterodimer (b) Molecular weight: 16 ± 1 kd for both monomer A and monomer B
(SDS-electrophoresis in the presence of 1.0% mercaptoethanol) 25 ± 1 kd as heterodimer (SDS-electrophoresis in the absence of mercaptoethanol) (c) Isoelectric point: pI 7.3 ± 0.5 (isoelectric point (Electrophoresis method) (d) pH stability: stable in the range of pH 2.0 to 10.0 (e) Thermal stability: stable by heating at 65 ° C for 60 minutes (f) Organic solvent stability: low alcohol, acetonitrile Stable (g) Protease resistance: completely inactivated by pronase treatment (h) Amino acid sequence: The amino acid sequence of monomer A is the first
In the figure, the amino acid sequence of monomer B is shown in FIG.

(2) Physicochemical properties of BUF-5 (a) Structure: homodimer structure of monomer B (b) Molecular weight: 16 ± 1 kd as monomer (in the presence of 1.0 mercaptoethanol, SDS-electrophoresis) 25 as homodimer ± 1kd (SDS-electrophoresis in the absence of mercaptoethanol) (e) Isoelectric point: pI 7.3 ± 0.5 (isoelectric focusing) (d) pH stability: pH 2.0 to 10.0 Stable (e) Thermal stability: stable by heating at 65 ° C for 60 minutes (f) Organic solvent stability: stable against lower alcohol and acetonitrile (g) Protease resistance: completely inactivated by pronase treatment.

(H) Amino acid sequence: The amino acid sequence of monomer B is the second
Shown in the figure.

If BUF-4 and 5 according to the present invention have an action even if they do not have the exact same amino acid sequence as the amino acid sequence shown in FIGS. 1 and 2, the substance is contained in BUF-4 and 5 according to the present invention. Is done.

That is, a substance having a structure in which one or more amino acids in the amino acid sequence shown in FIG. 1 or FIG. 2 are substituted with another amino acid, and in the sequence, one or more amino acids are located at the N-terminal or C-terminal. A substance having an added structure, and furthermore, one N-terminal or C-terminal from the sequence.
One or more amino acids are deleted and a substance having a structure consisting of a continuous amino acid sequence is also a BUF of the present invention.
-4 and 5. It has been reported that BUF-4 has a follicle stimulating hormone secreting action (Vale, W.,
River, J., Vaughan, J., McClintock, R., Corrigan, A., Woo,
W., Karr, D. and Spiess, J. (1986) Nature 321,776-77.
7) BUF-4 is also called Activin, but the name BUF-4 will be used in the present invention.

On the other hand, BUF-5 is a substance disclosed in JP-A-63-119679.

As described above, BUF-4 and BUF-5 are already known to have an action such as a follicle stimulating hormone releasing action, but no report has been made on the osteogenic action as in the present invention.

By the way, BUF-4 and BUF-5 of the present invention have a calcifying or osteogenic effect on the osteoblast-like cell line MC3T3, and show no toxicity on cultured mouse and human cells. It is considered to be effective in treating osteoporosis in humans.

Since the therapeutic agent for osteoporosis of the present invention contains at least one or more substances of BUF-4 and BUF-5 as active ingredients, it may contain the above-mentioned active ingredient alone or two or more kinds. May be contained in combination.

The therapeutic agent for osteoporosis of the present invention is mainly administered parenterally (intravenously, subcutaneously, intramuscularly). The dose of the active ingredient varies depending on the symptoms, but when only one of BUF-4 or BUF-5 is used alone, any one of the substances is generally used in a dose range of 0.05 to 50 mg per adult in general. It may be administered once or several times.

Also, when BUF-4 and BUF-5 are administered in combination, the efficacy of both is almost equal, so that 0.05 mg to 50 mg per adult is usually used.
It may be administered once or in several divided doses in the mg dose range. Thus, the daily dose is about 0.05-200 mg.
Of course, the dose varies depending on the condition of the patient, the weight of the patient, and other factors recognized by those skilled in the art. Therefore, it is not necessary to strictly adhere to the above dose, and the dose may be determined flexibly.

Formulation of BUF-4 and BUF-5 used in the present invention is carried out by an ordinary method, and is mainly used as an injection, but is also formulated into a dosage form such as a capsule or a tablet. When preparing an injection, a pH adjuster, a buffer, a stabilizing agent, a preservative, etc. are added as necessary to the main drug BUF-4 and BUF-5, and an intravenous, subcutaneous, or intramuscular injection is prepared by a conventional method. And it is sufficient.
When an oral preparation is prepared, excipients are added to BUF-4 and BUF-5, and if necessary, a binder, a disintegrant, a coloring agent and the like are added to obtain tablets, capsules and the like by a conventional method.

Next, a method for producing BUF-4 and BUF-5 having a bone formation promoting action will be described below.

Production of BUF-4 and BUF-5 is performed by a recombinant DNA method.

Methods for producing BUF-4 include eukaryotes transformed with a plasmid containing the gene encoding BUF-4, ie, monomer A and monomer B, and BUF-5.
Is produced by culturing eukaryotic cells transformed with a gene encoding BUF-5, that is, a plasmid containing monomer B, in a culture medium, and culturing BUF-
4 or BUF-5 is manufactured (JP-A-63-119679).

Now, the BUF-4 or BUF-5 produced in this way
Is purified according to a general polypeptide purification method. For example, the culture solution is concentrated by an ultrafiltration method, the polypeptide is salted out from the concentrated solution, dialyzed, and then subjected to ion exchange chromatography using an anion exchanger to obtain a crude polypeptide preparation. Most of the crude protein is removed from the crude sample by hydrophobic chromatography or chromatofocusing. Further, by combining the two, the purification ratio can be further improved. The thus purified preparation was subjected to reverse phase high performance liquid chromatography (HPLC) or FPLC (Pharmacia Fast Protein Peptide) equipped with a Super Rose or MonoQHR5 / 5 column.
It can be purified by high performance gel filtration using a Polynucleotide Liquid Chromatography system or ion exchange chromatography.

Further, in addition to the above-described general purification method for polypeptides, purification using a purification method utilizing an organic solvent containing a predetermined concentration of an organic acid developed by the present inventors (Japanese Patent Application No. 63-131268). It does not matter.

The safety of the drug of the present invention has been confirmed in toxicity tests with animals.

Effect of the present invention The osteoporous therapeutic agent comprising BUF-4 and BUF-5 according to the present invention as an active ingredient is different from conventional therapeutic agents such as active vitamin D ₃ and calcitonin. It is effective for the treatment of fundamental osteoporosis because it has an action of promoting osteoporosis.

Since BUF-4 and BUF-5 are proteins derived from humans, they have low antigenicity and are unlikely to cause allergy, so that they can be used for a long time.

Further osteoporosis therapeutic agents of the invention also has the advantage that I different from the activated vitamin D _3, does not cause the course side effects such as hypercalciuria.

 Hereinafter, the present invention will be described specifically with reference to Examples.

Example The osteoblast-like cell line MC3T3-E1 (H. Kodama et al. Jpn. JO
ral Biol. 23 : 899-901 (1981)) was spliced into 2 × 10 ⁵ cells in 1 ml of α-MEM medium containing 10% fetal bovine serum, and seeded in a 24-well multiwell plate. . 37 ℃, 5%
Leave in the presence of CO ₂ , gently remove the medium after 2 days,
1 ml of the same fresh medium was added. Thereafter, the medium was replaced similarly every two days. On day 12, 10 ^-11 to 10 ^-9 M b
4 or 1 ml of α-MEM medium (containing 1% fetal calf serum, 20 mM HEPES, 2.9 mM PO ₄ ) containing BUF-5, and 2 days later, 100,000 cpm of group A was replaced with ⁴⁵ ml. Group B contains 1 μCi of [ ³ H] TdR in 1 ml of the same medium containing CaCl ₂
After replacing with 1 ml, the mixture was allowed to stand at 37 ° C. and 5% CO _{2 for} 2 days, and then washed twice with a phosphate buffer saline. Subsequently, in group A, the cells were transferred to a vial for liquid scintillation counter, 0.1 ml of 60% HClO ₄ and 0.2 ml of 30% H ₂ O ₂ were added, and the mixture was heated at 70 ° C. for 1 hour, and then methylcellosolve was added thereto for radioactivity. Was measured. In group B, 10% TCA was added to the cells, and the cells were washed twice, washed with ethanol ether (3: 1), dissolved in 250 µ of NaOH, and measured for radioactivity. In each case, no BUF-4 and BUF-5 were used as controls.

From the results of group A, as shown in FIG. 3, ⁴⁵ Ca accumulation to the substrate layer by MC3T3E1 cells increased in a dose-dependent by BUF-4 and BUF-5, about the administration of 10 ^-9 M 50 % Increased. On the other hand, from the results of the group B, as shown in FIG.
³ H] thymidine was incorporated into DNA by 10 ^-11 M BUF-4 and
80% with BUF-5, plus ^10-9 M BUF-4 and BUF-5
To 60%.

[Brief description of the drawings]

FIG. 1 shows the amino acid sequence of monomer A. FIG. 2 shows the amino acid sequence of monomer B. FIG. 3 shows that C of BUF-4 and BUF-5 against MC3T3-E1 cells.
a Shows the effect of accumulation. Figure 4 shows the influence of ^[3 H] TdR incorporation for MC3T3-E1 cells BUF-4 and BUF-5.

Claims (1)

(57) [Claims] An agent for treating osteoporosis, comprising as an active ingredient at least one substance selected from the group consisting of polypeptides BUF-4 and BUF-5.