JPH07242564A - Bone formation accelerator - Google Patents

Abstract

PURPOSE:To obtain a bone formation accelerator which contains the nerve growth factor as an active ingredient and is suitably useful in prevention and treatment for osteopathies such as dysosteogenesls because the differentiation of preosteoblasts into osteoblasts is promoted and the synthesis of collagen as a major protein for bone matrix is activated. CONSTITUTION:The object bone formation accelerator contains, as an active ingredient, the nerve growth factor originating from a variety of mammalians including human. The dose of the active ingredient is 0.1 to 1,000mg/day in once to several times. It is preferred that the accelerator is administered intravenously, subcutaneously or intramuscularly in the form of an aqueous solution or in a mixture with a pharmaceutically permissible support.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a bone formation promoter containing nerve growth factor (NGF) as an active ingredient, which can be applied to the prevention and treatment of bone diseases such as osteoporosis and other bone formation disorders. .

[0002]

2. Description of the Related Art Osteoporosis, which frequently occurs in elderly people or post-menopausal women, causes the loss of bone proteins and calcium due to the imbalance of bone formation and bone resorption toward bone resorption, resulting in the formation of voids in the bone. It is an illness that makes you feel like you have entered. This disease is not only painful, but bones in such a state are very fragile and the frequency of fractures increases, which is a serious problem especially in elderly patients. Current,
Calcitonin that relieves pain, ipriflavone that suppresses bone resorption, vitamin D3 that enhances absorption of calcium from the intestinal tract, or calcium preparations are used, but none have shown satisfactory results in clinical practice. .

On the other hand, nerve growth factor (NGF) is a substance that promotes nerve growth and has been isolated from many species of animals, and its structure has been clarified. This NGF is 116
It is a dimer in which two polypeptides each consisting of ˜118 amino acids are bound, and it is known that they are most contained in the mouse submandibular gland. It is known that NGF has extremely high homology between mammals, and that NGF works effectively even among different species by its neurite outgrowth activity. However, it is completely unknown that NGF promotes bone formation.

[0004]

[Problems to be Solved by the Invention] Postmenopausal osteoporosis is
Since it is considered that estrogen deficiency causes abnormally high bone resorption, a bone resorption inhibitor has been expected and studied as a therapeutic drug for osteoporosis. However, osteoclasts that perform bone resorption and osteoblasts that perform bone formation mutually secrete local factors, which influence each other's differentiation and activation. Therefore, suppressing osteoclasts is highly likely to cause inactivation of osteoblasts and blunted bone turnover. Therefore, it is considered desirable to use a substance that activates osteoblasts, which are cells that make bones, and promotes bone formation as a therapeutic agent for osteoporosis.

[0005]

Means for Solving the Problems As a result of searching for an osteogenesis promoting active substance, the present inventors have found that nerve growth factor (NGF)
Found that they promote alkaline phosphatase activity, which is a marker of preosteoblast differentiation, and further promote collagen synthesis, and completed the present invention.

The NGF used in the present invention is of human origin, as well as mouse, rat, guinea pig, rabbit,
Any animal-derived animal such as cow and snake may be used. For example, from the mouse submandibular gland, Bocchini,
V. et al. [Proc. Natl. Acad. Sci. USA, 64: 787-794]
(1969)] and methods such as Mobley, WC [Biochemistry15;
5543-5551, (1976)] for easy isolation and purification. In addition, it can be isolated from snake venom or guinea pig prostate. Also, NGF derived from various mammals including human can be produced by using a genetic engineering technique. NGF may be a partial peptide of nerve growth factor that expresses nerve growth factor activity.

Further, in the present invention, a mutein in which a part of amino acids of NGF or a partial peptide of NGF expressing NGF activity is substituted or deleted can be used as an active ingredient, and this aspect is also within the scope of the present invention. .

The dose of nerve growth factor or partial peptide, which is the active ingredient of the osteogenesis promoting agent of the present invention, depends on the sex of the patient,
Although it depends on the age, body weight, symptom, etc., it is generally 0.1 to 1000 mg per day, which can be administered in 1 to several divided doses. This drug can be used as an aqueous solution, or
It is preferable to administer it by intravenous, subcutaneous, or intramuscular injection by mixing with a pharmaceutically acceptable carrier.

In order to produce an injection, the active ingredient is optionally added with hydrochloric acid, sodium hydroxide, emulsion, sodium lactate,
Dissolve in distilled water for injection with pH adjusting agents such as sodium monohydrogen phosphate and sodium dihydrogen phosphate, isotonic agents such as sodium chloride and glucose, and sterilize and filter into ampoules, or further mannitol and dextrin. Alternatively, cyclodextrin, gelatin, etc. may be added and freeze-dried under vacuum to prepare a solution-type injection before use. Further, lecithin, polysorbate 80, polyoxyethylene hydrogenated castor oil and the like may be added to the active ingredient and emulsified in water to give an emulsion for injection.

Preparations other than injectable preparations can be produced by known preparation methods, for example, the method described in the Japanese Pharmacopoeia, 10th Edition General Rules for Preparation or a method with appropriate improvements.

[0011]

EXAMPLES The present invention will be described in detail below with reference to examples. However, the present invention is not limited to these examples.

Example 1 1 ml of alpha MEM (Flow) prepared in a 24-well plate
laboratories) medium (10% newborn bovine serum; ICN Bio
medikals) and MC3T3-E1 cells at 1 × 10 ⁴ cells / ml, and the cells were grown at 37 ° C. and 5% CO ₂ for 4 days.
It was cultured for 8 hours. After removing the medium, the same medium was newly added, and mouse beta NGF was further added at 2 ng / ml. This was cultivated in the above-mentioned carbon dioxide gas culturing device, and the culturing was continued every 3 days while exchanging the medium with the same medium containing NGF. It was measured by the method.

(Measurement of alkaline phosphatase activity)
After removing the medium and washing the cells with PBS (-), 0.05% T
A 2 mM MgCl ₂ solution of riton X (Wako Pure Chemical Industries, Ltd.) was added at 500 microliters per well to suspend the cells, which were transferred to a centrifuge tube, sonicated for 30 seconds, and then centrifuged. The alkaline phosphatase activity of the centrifugation supernatant was
The measurement was performed using an alkaline phospha B test Wako measurement kit (Wako Pure Chemical Industries) according to the method of Lowry et al. (J. Biol. Chem., 193, 265-273, 1951). The activity of cells to which NGF was added was shown in FIG. 1, with the cells without NGF as a control.

Example 2 1 ml of alpha MEM (Flow) prepared in a 24-well plate
laboratories) medium (10% newborn bovine serum; ICN Bio
medikals) and MC3T3-E1 cells at 1 × 10 ⁴ cells / ml, and mouse beta NG.
The cells to which F was added at 2 ng / ml and the cells to which F was not added were cultured at 37 ° C. under 5% CO ₂ for 6 days. After removing the medium, 100 microliters of 0.5 M per well
After adding an acetic acid solution and allowing it to stand for 5 minutes, the amount of extracted collagen was measured using a collagen assay kit (manufactured by SIRCOL). The results are shown in Fig. 2.

From the above results, it was revealed that NGF differentiates preosteoblasts into osteoblasts and further promotes bone collagen synthesis. Therefore, this activity of NGF as a bone morphogenetic factor can be effectively used for the prevention and treatment of bone diseases such as osteoporosis and other bone dysplasia.

[0016]

INDUSTRIAL APPLICABILITY The osteogenesis promoting agent of the present invention promotes the differentiation of preosteoblasts into osteoblasts and activates the synthesis of collagen which is a major protein of bone matrix. Therefore, it can be used as a prophylactic and therapeutic drug for bone diseases such as osteoporosis and the like which show bone hypoplasia.

[Brief description of drawings]

FIG. 1 is a graph showing alkaline phosphatase activity of preosteoblasts in Example 1.

FIG. 2 is a graph showing the amount of collagen synthesis by preosteoblasts in Example 2.

Claims (1)

[Claims] 1. An osteogenesis promoter containing nerve growth factor as an active ingredient.