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Inhibition of osteoclast formation

An osteocytic, neonatal technique for fungal immunomodulatory proteins

Inactive Publication Date: 2017-03-15
YEASTERN BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

None of these studies showed that FIP ​​is useful for the prevention or treatment of bone loss-related diseases

Method used

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  • Inhibition of osteoclast formation
  • Inhibition of osteoclast formation
  • Inhibition of osteoclast formation

Examples

Experimental program
Comparison scheme
Effect test

example 1

[0066] Example 1: Preparation of FIPs

[0067] The FIP-gts of SEQ ID NO: 1 was recombinantly expressed in S. cerevisiae host cells as described in WO2005 / 040375A1. Cells expressing FIP-gts were disrupted, centrifuged, and the supernatant was passed through a filter and molecular sieve to obtain proteins between 10 kDa and 100 kDa. The filtrate was further analyzed by FPLC to 75 column (GE Healthcare) for purification. The purity was over 95% as determined by FPLC.

[0068] FIP-vvo (with SEQ ID NO: 3) and FIP-gmi (with SEQ ID NO: 6) were prepared using the same method described in the present application, and the proteins thus prepared were found to have a purity of over 95%.

example 2

[0069] Example 2: FIP is not toxic to preosteogenic cells

[0070] RAW 264.7 cells, a murine leukemia monocyte / macrophage cell line, were purchased from the Bioresource Conservation and Research Center of the Food Industry Development Institute (Hsinchu, Taiwan). RAW 264.7 cells were divided into 1×10 5 The density of cells / well hole was implanted in 24-well plate culture dish, and in the presence of various concentrations of FIP-gts (2.5-20 μg / ml) prepared in Example 1, cultured in supplemented with Dulbecco's Modified Eagle Medium (DMEM; Sigma Chemical Co., St. Louis, MO, USA) in 10% heat-inactivated calf serum (FBS; Gibco, NY, USA) for 5 days. The supernatant was removed and 25 μl of 2.5 mg / ml MTT (3-[4,5-lutidine-2-yl]-2 , 5-diphenyltetrazolium bromide; Sigma Chemical Co., St. Louis, Missouri, USA). The plates were incubated at 37°C for 1 hour to allow conversion of MTT to water-insoluble formazan crystals. Subsequently, the supernatant was removed and dimethyl sulfoxi...

example 3

[0073] Example 3: Inhibitory Effect of FIP on Osteocytogenesis

[0074] RAW 264.7 cells were suspended in DMEM supplemented with 10% heat-deactivated calf serum, and 1 × 10 5 Cells / well were seeded into 24-well plate culture dishes, and then cultured to the next day. On day 1, the medium was removed and α-minimal basal medium (α-MEM; GIBCO LifeTechnologies, Grand Island, NY, USA) was added to the cell culture with or without 2.5-10 g / ml of FIP-gts and 100 ng / ml of RANKL (PeproTech Inc, Rocky Hill, NJ, USA). On day 5, α-MEM was removed and cell cultures were washed twice with PBS.

[0075] Then, relying on the fact that only mature osteoclasts secrete tartrate-resistant acid phosphatase (TRAP), a TRAP activity assay was used to evaluate the degree of proosteocyte differentiation induced by RANKL. In this assay, cell cultures in each well were treated with 200 microliters of 0.1% Triton X-100, and the cell cultures were washed once with PBS. Next, 100 microliters of TRAP so...

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Abstract

The present invention generally relates to the novel pharmaceutical use of fungal immunoregulatory proteins. More specifically, the present invention relates to the use of fungal immunoregulatory proteins and compositions containing fungal immunoregulatory proteins to inhibit osteoblast neovascularization, inhibit bone resorption, and prevent and treat disorders associated with bone loss.

Description

technical field [0001] The present invention generally relates to novel medical uses of fungal immunomodulatory proteins. More specifically, the present invention relates to the use of fungal immunomodulatory proteins and compositions containing fungal immunomodulatory proteins to inhibit osteoclast regeneration, inhibit bone resorption, and prevent and treat bone loss-related diseases. Background technique [0002] Ganoderma plant (Ganoderma) is a rare and precious Chinese herbal medicine. It has been commonly known as "Ganoderma" in China for more than 5,000 years. Various species of Ganoderma lucidum include: red ganoderma (G. lucidum; red), tree tongue ganoderma (G. applanatum; brown), pine cedar ganoderma (G. tsugae; reddish brown), sweet ganoderma (G. sinense; black ) and Ganoderma lucidum (G. oregonense; dark brown). [0003] It is known that Ganoderma lucidum has anti-allergic (Chen H.Y et al., J.Med.Mycol.1992; 33:505-512), liver protection (Lin J.M.et al., Am J C...

Claims

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Application Information

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IPC IPC(8): A61K38/16A61P19/08
CPCA61K38/168
Inventor 黄仁勇陈子智
Owner YEASTERN BIOTECH
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