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A novel bone mass increasing agent

A bone mass and drug technology, applied in the field of enhancing bone formation, can solve the problem of unexplained mechanism and so on

Inactive Publication Date: 2010-07-07
ORIENTAL YEAST
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0010] Meanwhile, the mechanism by which bone formation signals are transmitted to osteoblasts in response to the transmission of bone resorption signals has not been elucidated

Method used

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  • A novel bone mass increasing agent
  • A novel bone mass increasing agent
  • A novel bone mass increasing agent

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0154] Example 1: Differentiation of human mesenchymal stem cells

[0155] Reagent

[0156] Synthetic peptides were used as experimental reagents. Synthetic peptide D is a peptide having the amino acid sequence shown in SEQ ID NO: 7, and is a cyclic peptide consisting of 9 amino acids and containing 2 cysteine ​​residues bonded to each other by a disulfide bond. Synthetic peptide D has been reported to bind to RANKL (Aoki et al., JClin Invest 116:1525, 2006). As a control peptide, a synthetic peptide lacking the above-mentioned functions was used.

[0157] Cultured cells

[0158] Human mesenchymal stem cells were purchased from Cambrex Corporation. Maintenance medium produced by Lonza was used for subculture.

[0159] Differentiation of human mesenchymal stem cells

[0160] Human mesenchymal stem cells were seeded into 96-well plates (1×10 3 cells / well) (Nunc) and 48-well plates (2.4×10 3 cells / well) (IWAKI). After 24 hours, the culture supernatant was removed from ea...

Embodiment 2

[0165] Example 2: Calcification of human mesenchymal stem cells

[0166] ALP staining

[0167] During differentiation, peptide D was added at a concentration of 300 μM. On day 7, cells were fixed with 10% neutral buffered formalin solution, followed by refixation with acetone / ethanol solution.

[0168] A staining solution (500 µL) prepared according to the following composition was added to the cells, which were further incubated at 37°C for 10 minutes, washed with water, and dried.

[0169] (composition of staining solution)

[0170] Naphthol AS-MX Phosphate (SIGMA): 5mg

[0171] N,N-Dimethylformamide (Wako): 0.5 mL

[0172] 0.1M Tris-HCl (pH 8.5): 50mL

[0173] Fast blue half salt (SIGMA): 30mg

[0174] Alizarin red S staining

[0175] On day 21 after differentiation, the cells were washed with PBS and fixed with 10% neutral buffered formalin solution.

[0176] After removing the fixative, the cells were washed with water. 1% Alizarin Red S staining solution (Nacala...

Embodiment 3

[0178] Example 3: Differentiation of mouse osteoblast precursor cell line (MC3T3-E1)

[0179] Cultured cells

[0180] Mouse osteoblast precursor cell line MC3T3-E1 (subclone number 4) cells were purchased from ATCC.

[0181] Mouse osteoblast precursor cells (MC3T3-E1)

[0182] Cells mixed with 10% FBS+αMEM (GIBCO) were seeded into 96-well plates (8×10 3 cells / well) and 48-well plate (2×10 4 cells / well). After 48 hours, the culture supernatant of each plate was replaced with 10% FBS+αMEM containing 5 mM β-glycerophosphate (SIGMA) and 10 μg / mL sodium ascorbate (SIGMA). Then, replace with new medium every 3 or 4 days. As a control, MC3T3-E1 was cultured using 10% FBS+αMEM as a maintenance medium. Once the medium was changed, peptides were added to the plates at a concentration of 300 [mu]M. According to the method described in Example 1, ALP activity measurement and Alizarin Red S staining were performed on the 7th day and the 21st day.

[0183] Differentiation of mouse o...

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Abstract

Disclosed is a bone formation enhancing agent comprising an RANKL-acting molecule which can enhance the differentiation, maturation or calcification of an osteoblast or a cell capable of being differentiated into an osteoblast. Specifically disclosed is a pharmaceutical composition for the treatment or prevention of a metabolic bone disease accompanied by the decrease in bone mass, which comprises, as an active ingredient, a compound which can act on an RANKL located on an osteoblast or a cell capable of being differentiated into an osteoblast to accelerate the differentiation, proliferation,maturation or calcification of the osteoblast or the cell capable of being differentiated into an osteoblast.

Description

technical field [0001] The present invention relates to a method for enhancing bone formation (osteogenesis), that is, by administering an effective amount of osteoblasts or cells capable of differentiating into osteoblasts (such as osteoblast precursor cells, mesenchymal stem cells, stromal cells, and osteoblasts) muscle cells), thereby enhancing the differentiation and maturation of said cells. [0002] In addition, the present invention relates to pharmaceutical compositions for stimulating bone formation. [0003] Furthermore, the present invention relates to a screening method for a substance acting on RANKL and used for signaling, a substance obtained by such a screening method, and a pharmaceutical composition containing the obtained substance. Background technique [0004] Bone is an active organ that undergoes continuous bone remodeling and maintains serum calcium concentration through repeated formation and resorption / destruction in bone morphogenesis. In general...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K45/00A61K38/00A61P19/02A61P19/08A61P19/10C07K14/705C12Q1/02G01N33/15G01N33/50
CPCA61K38/1793A61K39/39541C07K2317/76A61K38/45A61K38/1875G01N33/5047C07K16/2875A61K45/06A61P19/00A61P19/02A61P19/08A61P19/10A61K2300/00
Inventor 保田尚孝古屋优里子田口祐介
Owner ORIENTAL YEAST
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