JPH1029950A - Accelerator of osteogenesis - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain an accelerator of osteogenesis useful for treatment for osteoporosis or other bone disease by including midkine. SOLUTION: This accelerator of osteogenesis includes midkine, being a growth factor bondable to heparin reactive to retinoic acid and having activities such as survival acceleration of neurocyte and sthenia of a fibrinogenolysis system in a vascular endothelial cell, as an active ingredient. Further, the midkine combined with at least one factor concerning osteogenesis, e.g. growth simulator derived from blood platelet, enhances a growth promoting effect and is used for a treatment or prevention for a disease accompanying lowering of a bone strength or a fracture. The accelerator is administered in a daily dose of 0.1-1,000mg in one or several divided portions and preferably by an intravenous, hypodermic or intramuscular injection in a form of an aqueous solution or mixed with pharmaceutically permissible carrier.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a midkine (mi
dkine: hereinafter referred to as “MK”) as an active ingredient, and an agent for treating and / or preventing a disease or fracture accompanied by a decrease in bone strength, comprising MK as an active ingredient.

[0002]

2. Description of the Related Art Bone not only serves as a pillar of a living body and protects internal organs, but also forms blood cells, metabolizes minerals such as calcium and phosphorus, and maintains the homeostasis of minerals in the living body. I am carrying it.

[0003] Bone tissue is constantly turning over even after its growth, and a part of the existing bone is absorbed by osteoclasts, which are bone resorbing cells. Cells form new bone. Through the process of bone remodeling (bone modification), bone morphology and bone mass are maintained as a whole.

The functional unit of this bone remodeling is "basi
c multicellular unit (BMU) "(P
arfitt AM, Calif. Tissue Int. 36, 37-45, 198
4) Each stage is regulated and controlled in a complex network created by many regulatory factors.

[0005] Regulators involved in bone remodeling include systemic regulators such as parathyroid hormone (PT).
H), vitamin D metabolites, calcitonin, sex hormones, and other hormones. Local regulatory factors include insulin-like growth factor IGF, transforming growth factor-β (TGF-
β), platelet-derived growth factor (PDGF), β ₂ -microglobulin, various growth factors such as fibroblast growth factor (FGF), osteoinductive protein (BMP), prostaglandin E ₂ (P
GE ₂ ), interleukins (IL-1, 4, 6) and the like.

[0006] Bone growth, maintenance and repair in bone remodeling relies on a balance between the rate of bone formation and the rate of bone resorption. When this balance is lost and bone resorption exceeds bone formation, bone mass is reduced, resulting in osteoporosis and other bone disorders.

[0007] Accordingly, it is considered that osteoporosis and other bone diseases can be treated or prevented by activating bone formation or suppressing bone resorption. Drugs used for osteoporosis are those that promote osteoblast activity and promote bone formation (bone formation promoting agent), those that suppress bone resorption by osteoclasts (bone resorption inhibitor), and bone turnover. Activating (bone metabolism activating agent). Among the main drugs currently used in Japan, calcitonin, ipriflavone,
Estrogen is a bone resorption inhibitor, an active vitamin
D is considered to be both a bone metabolism activator and a bone formation promoter.

[0008]

All of the above-mentioned existing osteoporosis treatment agents are suspected to have a certain effect of increasing bone mass. In addition, since osteoclasts and osteoblasts secrete local factors to each other and affect their respective differentiation and activation, suppression of osteoclast activity by bone resorption inhibitors will result in inactivation of osteoblasts. It is highly likely that they cause bone turnover and slow down bone turnover. For these reasons, development of a novel highly effective therapeutic agent for osteoporosis has been desired.

An object of the present invention is to provide a novel bone formation promoting agent useful for osteoporosis and other bone diseases.
Still another object of the present invention is to provide a novel bone formation promoting agent that, when used in combination with at least one factor involved in bone formation, synergistically or additively promotes bone formation.

[0010]

SUMMARY OF THE INVENTION MK is a retinoic acid-reactive heparin-binding growth factor (Kadomatsu, K. eta).
l., Biochem. Biophys. Res.Commun., 151, 1312-131
8, 1988), promoting the survival of nerve cells and enhancing the fibrinolytic system in vascular endothelial cells. In mouse embryos,
It has been reported that MK is expressed in cartilage and tooth buds.
The present inventors have focused on such biological activity of MK, and cultured mammalian osteoblasts in the presence of MK, promoted the growth of osteoblasts, when combined with MK and PDGF Found that the growth promoting effect was further enhanced, and completed the present invention. That is, the present invention specifically includes the inventions described in the respective claims.

Hereinafter, the present invention will be described in detail.

[0012]

DETAILED DESCRIPTION OF THE INVENTION Bone tissue is a complex tissue made up of highly differentiated osteoblasts, osteoclasts, and osteocytes. The bone tissue organ culture system that can comprehensively evaluate bone metabolism is the most advanced field in the area of tissue culture,
It is difficult to elucidate the functional activity of each bone cell and the mechanism of action between them. Further, even if an attempt is made to examine factors involved in bone formation, it is impossible to clarify which factors act on which cells.

[0013] Various attempts have been made to separate and culture each cell group from bone tissue. Luben et al. Sequentially digested bone fragments with collagenase and separately cultured the cells released from the bone fragments at each stage, thereby culturing a group of cells that were rich in osteoclasts and osteoblast-like cells, respectively. Method was established (RA Luben, Endocrinology, 99,5
26, 1996). However, this primary culture system retains the function of each normal cell to some extent, but has a problem in the uniformity of each cell. Therefore, as a result of a demand for a cell line that retains the characteristics of bone cells, several cell lines have been established at present. Representative examples include ROS 17/2 and UMR106 derived from rat osteosarcoma, Saos-2 derived from human sarcoma, and MC3T3-E1 derived from mouse calvaria.

Osteoblasts are mononuclear cells derived from mesenchymal stem cells, preosteoblasts, osteoblasts, and osteocytes embedded in bone matrix and intraosseous bone that covers the bone surface. Differentiates into membrane cells. The proliferated and differentiated osteoblasts produce bone matrix (osteoid) and fill the bone resorption fossa. Under the control of osteoblasts, the osteoid matures, calcifies and becomes bone. Preosteoblasts and osteoblasts not only produce such bone matrix, but also insulin-like growth factors (IGF-I, -II), β ₂ -microglobulin,
Osteoinductive protein (BMP), transforming growth factor-β (TGF-β), IL-1, TNF-α, prostaglandin
It secretes various local factors such as E ₂ (PGE ₂ ) and is thought to regulate bone formation by an autocrine / paracrine mechanism. In addition, osteoblasts are parathyroid hormone (PT
H), epidermal growth factor (EGF), activated vitamin [1α, 2
5 (OH) ₂ D ₃ ], estrogen, TNF-α, PGE ₂ , IL-1 and other receptors that control the proliferation and differentiation of osteoblasts themselves and differentiate osteoclasts・ It is considered that activation is controlled. To date, at least a dozen or so local factors have been identified that regulate the osteoblast mechanism, many of which have been extracted from bone matrix.

[0015] Based on the above, it is considered that a substance that promotes the proliferation of preosteoblasts and osteoblasts in mammals promotes bone formation, and is a disease accompanied by a decrease in bone strength, particularly osteoporosis and renal dystrophy. It is expected to be effective in the prevention and treatment of osteodystrophy, osteomalacia, Pasette's disease, hyperparathyroidism, periodontal disease, etc., and is also effective in promoting the healing of fractures where normal healing does not occur Sex can be expected. In addition, a substance that enhances alkaline phosphatase activity (ALPase activity), which is the most important phenotypic trait of osteoblasts, promotes osteoblast differentiation. It is considered to be effective for treatment. As the bone cells that can be used for ALPase activity measurement, for example, the established cell lines described above can be used.

The MK of the present invention promotes the growth of osteoblasts derived from rats at a concentration of, for example, 20 ng / ml, and furthermore, when combined with a specific factor involved in bone formation, for example, PDGF, increases the proliferation of osteoblasts. Can be synergistically promoted. PDGF is
It does not affect osteoblast functions such as collagen synthesis ability, but promotes osteoblast proliferation (Graves DT et al.,
Centrella M. et al., Endocrinology 125, 13, 198
6) and has a bone resorption promoting effect.

[0017] It should be noted that those skilled in the art are aware of the combination of MK with at least one of systemic and local factors involved in bone formation or at least one of growth factors (collectively, factors involved in bone formation). It is possible to promote the enhancement of osteoblasts and osteoblast proliferative activity, and easily select an optimal combination for bone formation by ordinary experiments. For example,
The ALPase activity can be measured using an established cell line of bone cells, and the optimal combination of MK for promoting bone formation and a factor involved in bone formation can also be selected. Therefore, such combinations are also included in the present invention. Here, in the present invention, MK
Factors to be tested in combination with are divided into 1) systemic hormones and 2) growth factors and cytokines. Systemic hormones include PTH, 1α, 25 (OH) ₂ D ₃ , calcitonin, sex hormones, glucocorticoids, thyroid hormones, insulin, and the like. Growth factors and cytokines include IL-1 (α, β ), IL-3, IL-6, IL-4, GM-CS
F, M-CSF, IFN-α, TGF-β, BMP, TGF-α, EGF, IGF-
I, IGF-II, PGE ₂ , TNF-α, β ₂ -microglobulin, FGF
(Acidic, basic), PDGF and the like.

The MK of the present invention is derived from humans (JP-A-6-21777).
8), mouse-derived (Kadomatsu, K. etal., Biochem. Bio
phys. Res. Commun., 151, 1312-1318, 1988), or any of mammals such as rats. The MK of the present invention also includes derivatives or analogs in which some amino acids of MK or partial peptides of MK that express biological activity of MK are substituted or deleted. Furthermore, the MK of the present invention includes glucosylated MK and non-glycosylated MK.

MK which is an active ingredient of the bone formation promoting agent of the present invention
When administered to patients suffering from osteoporosis or other bone diseases, the dosage is generally 0.1 to 1000 mg per day, depending on the patient's sex, weight, symptoms, etc., and is administered in one or several doses can do. The dosage form is preferably administered as an aqueous solution or as a mixture with a pharmaceutically acceptable carrier by intravenous, subcutaneous, or intramuscular injection.

[0020]

Examples of the present invention will be described below. Example 1
Human MK having the amino acid sequence of SEQ ID NO: 3 of Japanese Patent Application No. 7-255354 was used as MK used in Nos. 3 to 3.

Example 1 Effect of MK on osteoblasts Primary cultured cells of rat fetal calvaria-derived osteoblasts (Rat ca
lvaria derived osteoblast-rich cells). Under a stereoscopic microscope, the connective tissue was peeled off from the calvaria of the newborn rat (1 to 2 days after birth) with tweezers, leaving only bone. Cell treatment solution (0.1% collagenase (Sigma: 0.15U
/ mg), 0.05% trypsin (GIBCO BRL), 4 mM EDTA) was repeated 12 times in total. First, in the first six times,
After treatment with the cell treatment solution for 20 minutes, the mixture was centrifuged and the supernatant was discarded. This operation requires sediment. New cell treatment solution was added to the sediment. This operation with the cell treatment solution was repeated 6 times (in this operation, the sediment was always left and not discarded. Up to this point, the supernatant was discarded because fibroblasts were mainly digested). After further treatment for 20 minutes, the mixture was centrifuged. The supernatant was collected here (the supernatant is empirically known to be rich in osteoblasts). A fresh cell treatment solution was added to the sediment. This operation was repeated six times while collecting the supernatant while leaving the sediment. All cells (six times) collected by centrifugation were collected. The collected cells for 6 times were suspended in a culture solution 10% FCS / MEM (modified Eagle's medium). The cells were collected by centrifugation again. The cells were suspended in an appropriate amount of 10% FCS / MEM and counted. Cells were seeded on culture dishes at two different cell concentrations. 1) 1 ml was spread on a 24-well culture dish at a cell concentration of 5 × 10 ⁴ / ml. 2) 1 ml was spread on a 24-well culture dish at a cell concentration of 1 × 10 ⁵ / ml. Both were cultured at 37 ° C., 5% carbon dioxide concentration and 100% wet condition. The next day, the culture solution (1
0% FCS / MEM) was replaced. After 3 to 4 days, the medium was replaced with a serum-free culture solution (F12 / DMEM, ITS) and MK was added. 8 hours later,
³ H-thymidine was added at 2 mCi / well. ³ H- thymidine 14-16 hours after the addition was measured ³ H- thymidine incorporation into the cells. The results are summarized in a graph in FIG. MK was added to primary osteoblast cells at a concentration of 20 ng / ml,
When culturing is continued for about 24 hours, 5 × 10 ⁴ cells or 1 × 10 ⁵
Initiation of culture with either of the primary culture cell numbers promoted cell proliferation by about 2.5 times.

Example 2 Effects of MK and PD on rat osteoblasts
Effect of GF (platelet-derived growth factor) Primary culture of rat fetal calvaria-derived osteoblasts (Rat ca
Cells were prepared in the same manner as in Example 1 using lvaria derived osteoblast-rich cells). The number of cells was started by spreading 1 ml of the cells at a cell concentration of 1 × 10 ⁵ / ml in a 24-well culture dish. 37 ℃, 5
The cells were cultured in a 100% humid condition with a concentration of 100% carbon dioxide. The next day, the culture solution (10% FCS / MEM) was replaced. After 3 to 4 days, replace with a serum-free culture medium (F12 / DMEM, ITS) and
(20ng / ml) only, PDGF (20ng / ml) (human PDGF (BTI))
Only four groups of controls, both with and without both MK and PDGF (20 ng / ml each), were prepared. Eight hours later, ³ H-thymidine was added at 2 mCi / well. Add ³ H-thymidine and add 14
After 1616 hours, the uptake of ³ H-thymidine into cells was measured. The results are summarized in a graph in FIG. The effect on rat osteoblasts was higher than that of MK alone when PDGF
The effect was enhanced when used together. It has been suggested that MK acts on cells in cooperation with certain growth factors (or cytokines) and may synergistically promote their proliferation.

Example 3 Expression of MK in Bone and Chondrogenesis In order to elucidate the function of MK in bone and cartilage, a) during mouse extremity limb formation, and b) mouse fracture healing
The expression of MK was examined immunohistologically.

A) Regarding the mouse embryo limb formation process, first, the forelimbs of the mouse embryo (embryos 10 to 16 days) were fixed with 4% paraformaldehyde to prepare paraffin specimens. Mouse MK
Immunohistological staining was carried out using a polyclonal antibody against.

B) Regarding the examination during the healing process of a mouse fracture, first, a 0.2 mm intramedullary nail was inserted into the tibia of a mouse (10 to 12 weeks old), and the mouse was fractured. On days 4, 7, 14, and 28 after the fracture, the target mice were sacrificed sequentially. The required part was fixed with 4% paraformaldehyde. Next, it was decalcified with 10% EDTA. Finally, paraffin specimens were prepared. Each paraffin specimen was immunohistologically stained using a polyclonal antibody against mouse MK. From the results of each staining, in a), MK expression was observed in chondrocytes. In b), expression of MK was observed in the cells of the periosteum proliferating on the fourth day. From day 7 onward, MK expression was observed in chondrocytes, particularly strong in proliferating chondrocytes,
Hypertrophic chondrocytes had only a small expression. The expression of MK was also observed in cells in the new fibrous bone after 14 days. As described above, MK expression was observed in both the process of cartilage formation in development and endochondral ossification in fracture healing,
Was suggested to be involved in bone and cartilage formation.

[0026]

According to the present invention, MK exhibits a proliferative activity on osteoblasts, and when MK is combined with an osteogenic factor such as PDGF, the proliferative activity on osteoblasts is synergistically increased. It was revealed that it could be enhanced. Further MK
Since is especially observed in proliferating chondrocytes in the early stage of osteoblasting, it can be expected to be effective as a therapeutic and / or preventive agent for diseases or fractures accompanied by a decrease in bone strength.

[Brief description of the drawings]

FIG. 1 shows the results of measuring the amount of ³ H-thymidine incorporated into cells using primary cultured cells of rat fetal calvaria-derived osteoblasts.

FIG. 2 is a diagram showing the results of measuring the amount of ³ H-thymidine incorporated into cells using primary cultured cells of rat fetal calvaria-derived osteoblasts.

 ──────────────────────────────────────────────────続 き Continuing on the front page (72) Inventor Hisashi Iwata 65, Tsurumai-cho, Showa-ku, Nagoya-shi, Aichi Prefecture (72) Takashi Muramatsu 2845, Kuroiishi, Ohira-ji, Ojiro, Tenpaku-ku, Nagoya-shi, Aichi 35

Claims (6)

[Claims] 1. An osteogenesis promoter comprising midkine as an active ingredient. 2. An osteogenesis promoter comprising, as an active ingredient, a combination of midkine and at least one factor involved in bone formation. 3. The bone formation promoting agent according to claim 2, wherein the factor involved in bone formation is platelet-derived growth factor (PDGF). 4. A therapeutic and / or prophylactic agent for a disease or fracture accompanied by a decrease in bone strength, comprising midkine as an active ingredient. 5. The therapeutic and / or prophylactic agent according to claim 4, further comprising at least one factor involved in bone formation. 6. The therapeutic and / or prophylactic agent according to claim 5, wherein the factor involved in bone formation is platelet-derived growth factor (PDGF).