KR20160024463A - Pharmaceutical composition comprising compounds for inhibiting differentiation and formation of osteoclast - Google Patents

Abstract

The present invention relates to a pharmaceutical composition containing a compound or a pharmaceutically acceptable salt thereof, wherein the compound inhibits osteoclast production and differentiation into osteoclast. The pharmaceutical composition containing, as an active ingredient, the compound or the pharmaceutically acceptable salt thereof can be used as a treating agent for metabolic bone diseases such as periodontal diseases, inflammatory abnormal resorption of alve olar bone, inflammatory bone resorption diseases, bone metastatic cancer, solid cancer bony metastases, musculoskeletal complications caused by solid cancer bony metastasis, hypercalcemia due to malignant tumor, multiple myeloma, primary bone tumor, osteoporosis, rheumatoid arthritis, degenerative arthritis, and Paget′s disease.

Description

[0001] The present invention relates to a pharmaceutical composition containing compounds having osteoclast differentiation and production inhibitory effects,

The present invention relates to a pharmaceutical composition comprising a compound having an osteoclastogenesis inhibitory effect and an osteoclast differentiation inhibitory effect or a pharmaceutically acceptable salt thereof, wherein the compound of the present invention or a pharmaceutically acceptable salt thereof is effective The pharmaceutical composition containing the pharmaceutical composition of the present invention can be used for the treatment of periodontal disease, inflammatory alveolar bone disorder, inflammatory bone resorption disease, bone metastatic cancer, solid tumor bone metastasis, musculoskeletal complications due to solid tumor metastasis, Can be used as a therapeutic agent for bone metabolic diseases such as multiple myeloma, primary bone tumor, osteoporosis, rheumatoid arthritis, degenerative arthritis, and Paget's disease.

The bone is the hardest tissue that forms the skeleton of a person. It functions to support the soft tissue and body weight of the human body and surrounds the internal organs to protect the internal organs from external impact. It is also an important part of the body that not only supports the muscles or organs but also stores calcium or other essential minerals, such as phosphorus or magnesium, in the body. Therefore, the bone of the grown adult maintains the balance while dynamically and continuously repeating the generation and absorption process of removing the old bone and replacing it with the new bone. This is called bone remodeling, and bone formation Osteoblasts and osteoclasts that destroy bone are involved. Osteoblasts maintain bone homeostasis by regulating the differentiation of osteoclasts responsible for bone resorption through the secretion of RANKL (receptor activator of nuclear factor-κB ligand) and its inducible receptor, OPG (osteoprotegerin) . More specifically, when RANKL binds to RANK, which is an osteoclast precursor cell surface receptor, osteoclast precursor cells mature into osteoclasts and bone resorption occurs. When OPG binds to RANKL, binding between RANKL and RANK is blocked, .

The osteoclast formed from the precursor cells absorbs or destroys the old bone and releases a small amount of calcium accumulated in the bone into the blood stream to maintain the body function. Such metabolism is regulated by physical effects, hormonal system and local factors. Various bone diseases occur when bone absorption exceeds bone formation and bone mass falls below the limit due to various factors.

Osteoporosis is a condition in which the bone mass is reduced to a certain level and the microstructure of the bone tissue is degenerated to continuously increase the risk of fracture. It is a state in which the minerals (especially calcium) and the matrix constituting the bone are decreased, And the osteoclastic action occurs in a state where the osteoclast action is increased.

Osteoarthritis is the most common form of arthritis involving bone and cartilage, and inflammatory reactions overexpress osteoclast differentiation. This increases the expression of bone resorption activating factors such as IL-1, TNF-α, and MMP, leading to bone loss and cartilage damage.

Bone metastasis of cancer cells frequently occurs in breast cancer, prostate cancer or multiple myeloma. Especially, osteolytic metastasis is known to be caused by activation of osteoclast cells such as breast cancer cells. Activated osteoclasts destroy the balance of the microenvironment around the bone, resulting in bone-related diseases such as osteolysis and resulting pathological fractures, leukopenia, bone malformations, hypercalcemia, pain and neuropsychiatric syndrome.

A representative bone disease treatment agent currently used is Novartis's zoledronic acid, which treats bone metastasis and osteoporosis in early breast cancer patients. Zoledronic acid is a bisphosphonate-based compound that is modified to pyrophosphate, which is used for corrosion prevention of metal pipes.

From the pharmacokinetic point of view, pyrophosphate having a structure of P-O-P is easily hydrolyzed to a phosphate, whereas bisphosphonate has a relatively long half-life because it has a P-C-P structure and is not hydrolyzed. Bisphosphonates are used to treat osteoporosis, which inhibits osteoclasts and reduces bone resorption. Most bisphosphonates are made from intravenous use because they are very hydrophilic in structure and poorly absorbed. Bisphosphonate attaches to the bony surface where bone formation is active and inhibits osteoclast maturation and prevents bone resorption. It also inhibits osteoclast migration to sites where bone resorption takes place and reduces the production of cytokines that promote bone resorption. In addition, it inhibits tumor cell invasion in bone matrix or induces tumor cell death.

Bisphosphonates induced osteonecrosis of the jaws (BIONJ) is the most serious problem other than stroke and atrial fibrillation, which are side effects of bisphosphonates. BIONJ is a disease in which the jaw and root are exposed to the outside due to necrosis of the jaw. Bone tissue maintains its homeostasis through repetitive bone resorption and formation processes (ie, Bone Remodeling), where osteoclasts are first activated, resulting in bone resorption, where osteoblast cells are activated to form bone. Inhibition of osteoclast maturation by bisphosphonate inhibits bone resorption and osteoblast-induced osteogenesis does not result in regeneration of bone, resulting in accumulation of micro-injuries and fractures. Bisphosphonate not only exposes the root gum by damaging the mucous membrane of the gum, but also has a greater effect of bisphosphonate, especially in the case of jaws with frequent masticatory movements, since it has a tenfold higher bone turnover rate than normal bone. In addition, the vasopressin action of bisphosphonate prevents the supply of sufficient blood flow to increase the possibility of BIONJ induction.

In addition, calcium supplementation, which is widely used as a bone disease treatment agent, inhibits the secretion of parathyroid hormone and prevents bone loss due to bone resorption. However, it is known that individual differences in bone mass maintenance are severe. In the case of hormone therapy using estrogen or calcitonin, And decreased the incidence of rectal cancer, and side effects such as breast cancer, myocardial infarction, and venous thrombosis have been reported.

Accordingly, the present inventors have completed the present invention by studying the inventive compound for inhibiting osteoclastogenesis and osteoclast differentiation while studying a therapeutic agent for bone metabolic diseases.

And that the compound strongly inhibits osteoclast formation and osteoclast differentiation, thereby completing the present invention.

Korean Laid-Open Patent Publication No. 2013-0060775; Korean Registered Patent No. 740,565; Korean Patent No. 718,490; Korean Patent Publication No. 2010-0113417.

Yamaguchi A. et al., Tanpakushitsu Kakusan Koso., 50 (6Suppl): 664-669 (2005); Takayanagi H. Nat Rev Immunol. 2007 Apr; 7 (4): 292-304; Cohen-Solal M. et al., Therapie., 58: 391-393 (2003); Theill LE. Et al., Annu Rev Immunol., 20: 795-823 (2002); Wagner EF. et al., Curr Opin Genet. Dev., 11: 527-532 (2001); William J. et al., Nature., 423: 337342 (2003); Kozlow W. et al., J Mammary Gland Biol Neoplasia., 10 (2): 169-180 (2005); Boyde A. et al., Scan Electron Microsc., 4: 1537-1554 (1986); Roodman GD., N Engl J Med., 350: 1655-1664 (2004); Iqbal MM., South Med. J., 93 (1): 2-18 (2000); Nelson, H. D. et al., JAMA, 288: 872-881, 2002; Lemay, A., J. Obstet. Bynaecol. Can., 24: 711-7152-3.

The present invention provides a pharmaceutical composition for preventing or treating bone metabolic diseases by providing a pharmaceutical composition containing as an active ingredient a compound that exhibits osteoclastogenesis inhibition and osteoclast differentiation inhibitory activity and a pharmaceutically acceptable salt thereof I want to.

The present invention solves this problem by providing a compound of formula (Ia) or (Ib) or a pharmaceutically acceptable salt thereof:

(Ia)

(Ib)

In this formula,

R ₁ , R ₂ , R ₃ And R ₄ are each independently hydrogen, hydroxy or methoxy.

The present invention relates to a pharmaceutical composition comprising a compound having an effect of inhibiting osteoclastogenesis and inhibiting osteoclast differentiation, or a pharmaceutically acceptable salt thereof, wherein the compound of the present invention, or a pharmaceutically acceptable salt thereof, It has excellent effect of inhibiting cell formation and inhibiting osteoclast differentiation. Accordingly, the pharmaceutical composition according to the present invention is useful as a pharmaceutical composition for treating periodontal disease, inflammatory alveolar bone disease, inflammatory bone resorption disease, bone metastatic cancer, solid tumor metastasis, musculoskeletal complications due to solid tumor metastasis, Metabolic diseases such as multiple myeloma, primary bone tumors, osteoporosis, rheumatoid arthritis, degenerative arthritis, and Paget's disease.

Figure 1 shows the results of cytotoxicity measurements of cyanidin-3-glucoside, cyanidin chloride and coumarinin chloride at various concentrations.
FIG. 2 shows the effect of cyanidin-3-glucoside, cyanidin chloride and coumarinin chloride on osteoclast differentiation. BMMs were treated with M-CSF and RANKL to induce osteoclast differentiation, And the results of measurement of cell differentiation ability are shown.
FIG. 3 shows the results of real-time PCR measurement of the expression of differentiation markers expressed in the osteoclast differentiation process to reaffirm that cyanidin-3-glucoside inhibits the differentiation of BMMs, which are precursor cells, into osteoclasts will be.
FIG. 4 shows the results of measuring the activity of MAPK (ERK, JNK, p38) and Iκbα over time by pretreating cyanidin-3-glucoside with BMM and treating RANKL with time.
FIG. 5 shows the effect of cyanidin-3-glucoside on the expression of c-Fos and NFATc1, important transcription factors in osteoclast differentiation, by inducing differentiation by treating BMMs with M-CSF and RANKL, 3-glucoside was treated, and the results are shown.

The present invention relates to a pharmaceutical composition comprising a compound having an osteoclastogenesis inhibitory effect and an osteoclast differentiation inhibitory effect, or a pharmaceutically acceptable salt thereof. More particularly, the present invention relates to a pharmaceutical composition comprising a compound of the formula (Ia) or Or a pharmaceutically acceptable salt thereof as an active ingredient. The present invention also provides a pharmaceutical composition for preventing or treating bone metabolic diseases,

(Ia)

(Ib)

In this formula,

R ₁ , R ₂ , R ₃ And R ₄ are each independently hydrogen, hydroxy or methoxy.

In an embodiment in accordance with the invention, in formula Ia, R ₁ , R ₂ , R ₃ And R < ₄ > are both hydroxy; R ₁ , R ₂ and R ₄ may be hydroxy and R ₃ may be hydrogen.

In one embodiment according to the present invention, in formula Ib, R ₁ , R ₂ and R ₃ are both hydroxy (delphinidin-3-glucoside); R ₁ and R ₂ are each hydroxy and R ₃ is hydrogen (cyanidin-3-glucoside); Or R < ₁ > is methoxy, R < _{2 &} And R ₃ is a compound that is hydroxy (petunidin-3-glucoside).

In one embodiment according to the present invention, in formula Ib, R ₁ and R ₂ are each hydroxy and R ₃ is hydrogen.

In one aspect of the invention, the bone metabolic disease is selected from the group consisting of periodontal disease, inflammatory alveolar bone disorder, inflammatory bone resorption disease, bone metastatic cancer, solid tumor metastasis, musculoskeletal complications due to solid tumor metastasis, And may be any disease selected from the group consisting of hypercalcemia, multiple myeloma, primary bone tumor, osteoporosis, rheumatoid arthritis, degenerative arthritis, Paget's disease, and more specifically, periodontal disease, Inflammatory alveolar bone disorder, or inflammatory bone resorption disease. However, the present invention is not limited thereto, and may include all diseases that can be prevented or treated due to osteoclast generation and osteoclast differentiation inhibitory activity.

When the compound is used in the form of a pharmaceutically acceptable salt, an acceptable salt can be selected when it is used as a prophylactic or therapeutic agent for bone metabolic diseases in a salt present relative to an active ingredient.

Such salts include, but are not limited to, acid addition salts formed with pharmaceutically acceptable free acids. Acid addition salts include those derived from inorganic acids such as hydrochloric acid, nitric acid, phosphoric acid, sulfuric acid, hydrobromic acid, hydroiodic acid, nitrous acid or phosphorous acid, and aliphatic mono- and dicarboxylates, phenyl-substituted alkanoates, hydroxyalkanoates, Dioleate, aromatic acid, aliphatic and aromatic sulfonic acids. Such pharmaceutically innocuous salts include, but are not limited to, sulfate, pyrosulfate, bisulfate, sulfite, bisulfite, nitrate, phosphate, monohydrogenphosphate, dihydrogenphosphate, metaphosphate, pyrophosphate chloride, bromide, Butyrate, caprate, heptanoate, propiolate, oxalate, malonate, succinate, succinate, maleic anhydride, maleic anhydride, , Sebacate, fumarate, maleate, butyne-1,4-dioate, hexane-1,6-dioate, benzoate, chlorobenzoate, methylbenzoate, dinitrobenzoate, hydroxybenzoate, Methoxybenzoate, phthalate, terephthalate, benzenesulfonate, toluene sulfonate, chlorobenzene But are not limited to, phenates, xylenesulfonates, phenylacetates, phenylpropionates, phenylbutyrates, citrates, lactates, hydroxybutyrates, glycolates, maleates, tartrates, methanesulfonates, propanesulfonates, naphthalene- Naphthalene-2-sulfonate, or mandelate.

The acid addition salts according to the invention may be prepared by conventional methods, for example by dissolving the compounds in an excess of an aqueous acid solution and precipitating the salts using a water-miscible organic solvent such as methanol, ethanol, acetone or acetonitrile .

In addition, the compounds of the present invention can be used in the form of a pharmaceutically acceptable metal salt using a base. The alkali metal or alkaline earth metal salt is obtained, for example, by dissolving the compound in an excess amount of an alkali metal hydroxide or an alkaline earth metal hydroxide solution, filtering the insoluble compound salt, and evaporating and drying the filtrate. At this time, it is preferable for the metal salt to produce sodium, potassium or calcium salt. In addition, the corresponding silver salt is obtained by reacting an alkali metal or alkaline earth metal salt with a suitable silver salt (e.g., silver nitrate).

The pharmaceutical composition of the present invention may include a pharmaceutically acceptable carrier. The composition comprising a pharmaceutically acceptable carrier may be of various forms, whether oral or parenteral. In the case of formulation, a diluent or excipient such as a filler, an extender, a binder, a wetting agent, a disintegrant, or a surfactant is usually used. Solid formulations for oral administration include tablets, pills, powders, granules, capsules, and the like, which may contain at least one excipient such as starch, calcium carbonate, sucrose or lactose, gelatin, . In addition to simple excipients, lubricants such as magnesium stearate, talc, and the like may also be used. Liquid preparations for oral administration include suspensions, solutions, emulsions, syrups and the like. Various excipients such as wetting agents, sweeteners, fragrances, preservatives and the like may be included in addition to water and liquid paraffin, which are simple diluents commonly used. have. Formulations for parenteral administration include sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, and suppositories. Propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like can be used as the non-aqueous solvent and suspension agent. Examples of the suppository base include witepsol, macrogol, tween 61, cacao paper, laurin, glycerogelatin and the like.

In one aspect according to the present invention, the compound of formula (Ia) or (Ib) or a pharmaceutically acceptable salt thereof may be used in a drug delivery system for the prevention, amelioration or treatment of bone metabolic diseases have. For example, a compound of the formula (Ia) or (Ib) or a pharmaceutically acceptable salt thereof may be used for the purpose of preventing, ameliorating or treating a bone metabolic disease by loading or coating the sustained-release drug delivery system. More specifically, It can be used as a therapeutic agent for periodontal disease.

The pharmaceutical composition may be in the form of tablets, pills, powders, granules, capsules, suspensions, solutions, emulsions, syrups, sterilized aqueous solutions, non-aqueous solvents, suspensions, emulsions, freeze- It can have one formulation.

The composition of the present invention is administered in a pharmaceutically effective amount.

The term "pharmaceutically effective amount " as used herein means an amount sufficient to treat a disease at a reasonable benefit / risk ratio applicable to medical treatment, and the effective dose level will vary depending on the species and severity, age, sex, , Sensitivity to the drug, time of administration, route of administration and rate of release, duration of treatment, factors including co-administered drugs, and other factors well known in the medical arts. However, for a desired effect, the compound of the present invention or a pharmaceutically acceptable salt thereof may be administered at 1 to 200 mg / kg, preferably 10 to 100 mg / kg, per day.

The composition of the present invention may be administered as an individual therapeutic agent or in combination with other therapeutic agents for bone metabolic diseases, and may be administered sequentially or simultaneously with conventional therapeutic agents. And can be administered singly or multiply. It is important to take into account all of the above factors and to administer the amount in which the maximum effect can be obtained in a minimal amount without adverse effect, and can be easily determined by those skilled in the art.

The term "individual" in the present invention means all animals, including humans, who have already developed or are capable of developing a disease that can be prevented or treated through osteoclast generation and / or differentiation inhibitory activity, By administering the composition to a subject, the disease can be effectively prevented and treated.

The route of administration of the composition may be administered via any conventional route so long as it can reach the target tissue. The composition of the present invention may be administered intraperitoneally, intravenously, intramuscularly, subcutaneously, intradermally, orally, intranasally, intrapulmonarily, or rectally, though it is not intended to be limited thereto. The composition may also be administered by any device capable of transferring the active agent to the target cell.

The present invention also There is provided a food composition for preventing or ameliorating a metabolic disease comprising a compound of the formula (Ia) or (Ib) or a salt thereof as an active ingredient:

(Ia)

(Ib)

In this formula,

R ₁ , R ₂ , R ₃ And R ₄ are each independently hydrogen, hydroxy or methoxy.

In an embodiment in accordance with the invention, in formula Ia, R ₁ , R ₂ , R ₃ And R < ₄ > are both hydroxy; R ₁ , R ₂ and R ₄ may be hydroxy and R ₃ may be hydrogen.

In one aspect according to the present invention, in Formula Ib, R_One, R₂ And R₃Are all hydroxy (delphinidin-3-glucoside); R_One And R₂Are each hydroxy and R₃Is hydrogen (cyanidin-3-glucoside); Or R_OneIs methoxy, R₂ And R₃May be a compound (petunidin-3-glucoside).

In one embodiment according to the present invention, in formula Ib, R ₁ and R ₂ are each hydroxy and R ₃ is hydrogen.

The present invention also There is provided a health functional food composition for preventing or ameliorating a metabolic disease comprising a compound of the formula (Ia) or (Ib) or a salt thereof as an active ingredient:

(Ia)

(Ib)

Wherein R ₁ , R ₂ , R ₃ And R ₄ are each independently hydrogen, hydroxy or methoxy.

In an embodiment in accordance with the invention, in formula Ia, R ₁ , R ₂ , R ₃ And R < ₄ > are both hydroxy; R ₁ , R ₂ and R ₄ may be hydroxy and R ₃ may be hydrogen.

In one embodiment according to the present invention, in formula Ib, R ₁ , R ₂ and R ₃ are both hydroxy (delphinidin-3-glucoside); R ₁ and R ₂ are each hydroxy and R ₃ is hydrogen (cyanidin-3-glucoside); Or R < ₁ > is methoxy, R < _{2 &} And R ₃ is a compound that is hydroxy (petunidin-3-glucoside).

In one embodiment according to the present invention, in formula Ib, R ₁ and R ₂ are each hydroxy and R ₃ is hydrogen.

In one aspect of the invention, the bone metabolic disease is selected from the group consisting of periodontal disease, inflammatory alveolar bone disorder, inflammatory bone resorption disease, bone metastatic cancer, solid tumor metastasis, musculoskeletal complications due to solid tumor metastasis, And may be any disease selected from the group consisting of hypercalcemia, multiple myeloma, primary bone tumor, osteoporosis, rheumatoid arthritis, degenerative arthritis, Paget's disease, and more specifically, periodontal disease, Inflammatory alveolar bone disease or inflammatory bone resorption disease, but the present invention is not limited thereto, and it may include all diseases that can be prevented or improved due to osteoclast generation and osteoclast differentiation inhibitory activity.

The food or health functional food according to the present invention comprises a compound of the formula (Ia) or (Ib) or salt thereof in the form of a suitable food supplementary additive.

The term "food-aid additive " in the present invention means a component which can be added to foods in a supplementary manner, and it can be appropriately selected and used by those skilled in the art as added to produce food or health functional food of each formulation. Examples of food-aid additives include flavors such as various nutrients, vitamins, minerals (electrolytes), synthetic flavors and natural flavors, colorants and fillers, pectic acid and its salts, alginic acid and its salts, organic acids, , a pH adjusting agent, a stabilizer, a preservative, a glycerin, an alcohol, and a carbonating agent used in a carbonated drink. However, the types of the food auxiliary additives of the present invention are not limited by these examples.

The term "health functional food" in the present invention refers to a food prepared and processed in the form of tablets, capsules, powders, granules, liquids and circles by using raw materials and components having useful functions in the human body. Here, the term "functionality" means that the structure and function of the human body are controlled to obtain nutritional effects or effects useful for health use such as physiological actions. The health functional food of the present invention can be prepared by a method commonly used in the art and can be prepared by adding raw materials and ingredients that are conventionally added in the art. In addition, unlike general medicines, there is an advantage that there are no side effects that may occur when a medicine is taken for a long time by using food as a raw material, and it is excellent in portability, and the health functional food of the present invention is an auxiliary agent for prevention or improvement of bone metabolic diseases It is possible to ingest.

The amount of the active ingredient to be mixed can be suitably determined according to the intended use (prevention, health or therapeutic treatment). Generally, the compound of the formula (I) of the present invention or a salt thereof is added in an amount of 1 to 10% by weight, preferably 5 to 10% by weight, of the raw material composition. However, in the case of long-term ingestion intended for health and hygiene purposes or for the purpose of controlling health, the amount can also be used in the above-mentioned range.

There is no particular limitation on the kind of the food. Examples of foods to which the above substances can be added include drinks, tea, drinks, alcoholic drinks and vitamin complexes, meat, sausages, bread, chocolates, candies, snacks, confectionery, pizza, ramen noodles, Dairy products, various soups, etc., and includes all the health foods in the ordinary sense.

The health food composition of the present invention may contain various flavors or natural carbohydrates as an additional ingredient as well as ordinary foods. The above-mentioned natural carbohydrates are sugar saccharides such as monosaccharides such as glucose and fructose, disaccharides such as maltose and sucrose, polysaccharides such as dextrin and cyclodextrin, and xylitol, sorbitol and erythritol. Examples of sweeteners include natural sweeteners such as tau martin and stevia extract, synthetic sweeteners such as saccharin and aspartame, and the like. The ratio of the natural carbohydrate is generally about 0.01 to 0.04 g, preferably about 0.02 to 0.03 g per 100 g of the composition of the present invention.

Hereinafter, the present invention will be described in more detail with reference to Examples and Experimental Examples. The following examples and experimental examples are provided to aid understanding of the present invention and are not intended to limit the scope of the present invention thereto.

<Materials and Methods>

Experimental animal

Mice used for osteoclast differentiation and other experiments were 6-8 weeks old C57BL / 6J and purchased from Orient Bio Inc. (SeungNam, Korea).

Experimental reagent

Cyanidin-3-glucoside (C3G) was purchased from Polyphenols Laboratories AS ( www.polyphenols.com ) in Norway and dissolved in 0.1% DMSO (dimethyl sulfoxide) solution after the purchase. Cyanidin chloride and coumarinin chloride were purchased from Sigma (cat # 79457 and cat # 52976, respectively). Cell culture medium, fetal bovine serum (FBS), and materials required for cell culture were purchased from Hyclone (USA). Recombinant RANKL (Receptor activator of nuclear factor kappa-B ligand) isolated from Escherichia coli was provided by Professor Kim, Nano-Sung of Chonnam National University Medical School, and recombinant M-CSF (macrophage colony-stimulating factor) . Antibodies specific for p-ERK, p-JNK, p-p38, p-IκBα, ERK, JNK, p38 and IκBα were purchased from Cell Signaling Technology, USA. Antibodies against NFATc1 and c-Fos were purchased from Santa Cruz Biotechnology, USA. All chemicals used in the experiments were purchased from Sigma, USA.

BMMs  (Precursor cells) preparation Preparation of BMMs  ( 전구체 cells of osteoclast ))

Bone marrow cells were isolated from the femur of 6-8 week old male C57BL / 6J mice and cultured in α-MEM (α-minimal essential medium) medium containing 10% FBS and antibiotics (penicillin + streptomycin) / ml of macrophage colony-stimulating factor (M-CSF) was added to the cells and incubated for 3 days in an incubator at 37 ° C and 5% CO ₂ . After 3 days, the floating cells were removed and the cells attached to the culture dish were separated by treatment with trypsin. These cells were used as bone marrow-derived macrophages (BMMs) and precursor cells of osteoclasts.

Statistical Analysis Statistical Analysis )

Experimental results in the following experimental examples were analyzed using Student's two-tailed t-test and expressed as mean ± SD. P <0.05 was considered statistically significant. All experiments were repeated at least 3 times, and representative results were shown.

Experimental Example  1. Cytotoxicity measurement ( Cell viability assay )

To determine the effect of C3G on osteoclast proliferation, cytotoxicity assay was performed. Using the EZ-Cytox Enhanced Cell Viability Assay Kit purchased from Itsbio Korea, the cytotoxicity of cyanidin-3-glucoside in osteoclast proliferation was measured according to the manufacturer's instructions Respectively.

BMMs used as osteoclast precursor cells were injected at a concentration of 1 × 10 ⁴ cells / well in a 96-well culture plate and cultured for 3 days at various concentrations of C3G in the presence of M-CSF and RANKL Reference). Or 100 μM of C3G in the presence of M-CSF and RANKL was cultured for 4 days and the viability was measured daily (see FIG. 1b). The treated cells were treated with EZ-Cytox reagent and cultured at 37 ° C for 4 hours. After 4 hours, absorbance was measured at 450 nm using an ELISA reader (Sunrise (TM), Tecan, Switzerland). The cytotoxicity results were expressed as a percentage of the absorbance of the control (0 uM C3G) in the sample.

As a result of measuring the cytotoxicity by treating C3G at various concentrations for 3 days, the cell proliferation was almost the same as the control group not treated with C3G at the concentration up to 200 uM used in the experiment (FIG. 1A).

In addition, when cells were treated with 100 uM of C3G for 4 days, the cell proliferation during the incubation period did not show a significant difference when compared with the control without C3G treatment (Fig. 1B). Therefore, it can be seen that C3G having a concentration of 200 μM or less has no cytotoxicity.

As a result of the cytotoxicity measurement test of the cyanidin chloride compound in the same manner as described above, cytotoxicity was hardly observed, and the results are as shown in Fig. 1C and Fig. 1D.

Experimental Example  2. In vitro  Osteoclast differentiation measurement

To investigate the effect of C3G on osteoclast differentiation, osteoclast differentiation was measured by treatment with C3G. Bone marrow cells were separated from the tibiae and femur of 6-8 weeks old mice and then treated with M-CSF (30 ng / ml) in α-MEM medium containing 10% FBS for 3 days . Three days later, bone marrow cell-derived macrophages (BMMs), which were attached cells, were recovered and used as precursor cells of osteoclasts. BMMs were cultured for 4 days in media containing M-CSF (30 ng / ml) and RANKL (100 ng / ml) to produce osteoclasts. Every 3 days, we replaced it with a new badge with M-CSF and RANKL. Depending on the experiment, 100 μM of C3G was also treated during this incubation period. The cultured cells were fixed with 10% formalin solution and TRAP (tartrate resistant acidic phosphatase) activity was measured by TRAP solution assay by adding p NPP (para-nitrophenyl phosphate, Sigma, USA) solution. The activity was measured by measuring the absorbance at 405 nm using an ELISA reader. The TRAP solution assay can measure the total TRAP activity of monocytes, mononuclear cells, di- and multinuclear osteoclasts expressing TRAP present on a culture plate. The cells were then rinsed with distilled water and the osteoclasts were stained with TRAP staining solution for direct osteoclast staining and observed with a microscope. TRAP ⁺ multinuclear cells with more than 3 nuclei were regarded as adult osteoclasts and their number was measured under a microscope.

To investigate the effect of C3G on osteoclast differentiation, BMMs were treated with M-CSF (30 ng / ml) and RANKL (100 ng / ml) to induce differentiation into osteoclasts, followed by TRAP solution assay and TRAP staining (TRAP ⁺ multinuclear osteoclast, MNCs) in the control group treated with only M-CSF and RANKL. However, as shown in FIGS. 2A and 2B, TRAP-positive multinuclear osteoclasts (MNCs) In the C3G treated group, the number of TRAP-positive polynuclear osteoclasts was decreased depending on the C3G concentration. As a result of TRAP solution assay for measuring total TRAP activity, it was found that the total TRAP activity was decreased depending on the C3G concentration as in the TRAP staining (Fig. 2C). These results demonstrate that C3G inhibits the differentiation of BMMs into TRAP - positive osteoclasts and suppresses the production of TRAP - positive polynuclear osteoclasts.

The effect of cyanidin chloride and coumarinin chloride on osteoclast differentiation was measured in the same manner as above, and the results of the experiment are shown in Figs. 2d to 2i.

Experimental Example  3. Real-time quantitative polymerase chain reaction Real - time quantitative PCR  polymerase chain reaction ))

To investigate the effect of C3G on the expression of differentiation marker genes in osteoclasts, real-time PCR was performed. BMMs were cultured for 4 days with or without C3G treatment under conditions with M-CSF and RANKL. As shown in the experimental results, total RNA (total RNA) was extracted from the cells of each culture date by treatment with Trizol solution (Invitrogen, USA). CDNA (complementary DNA) was synthesized using a random primer and Maxima reverse transcriptase (Thermo Scientific, IL, USA) according to the manufacturer's instructions. The synthesized cDNA was mixed with the dataQuest SYBR Green qPCR master mix (Affymetrix, USA) and real-time PCR was performed using the StepOnePlus ™ Real-Time PCR Systems (Applied Biosystems, USA). MRNA levels were normalized using GAPDH (Glyceraldehyde 3-phosphate dehydrogenase). The primer sequences used in this experiment are shown in Table 1. (Table 1. Nucleotide sequences of primers used in real-time PCR).

In order to confirm that C3G inhibits the differentiation of BMMs, which are precursor cells, into osteoclasts, the expression of differentiation markers expressed in osteoclast differentiation was measured by real-time PCR. As shown in Fig. 3, Acp5 The expression levels of CpG , Trap , Oscar , Atp6v0d2 , Tm7sf4 ( Dc - stamp ), Cathepsin K ( CtsK ) and Nfatc1 genes were significantly decreased in the C3G treated group compared to the control group not treated with C3G. These results confirm the results of FIG. 2 that C3G inhibits osteoclast differentiation.

Experimental Example  4. Western Blot  analysis

To investigate the effect of C3G on signal transduction mechanism of osteoclast differentiation, proteins were extracted and subjected to western blot analysis using a specific antibody.

First, 100 μM of C3G was preincubated in BMMs√ (1 × 10 ⁶ cells / well, 6 well culture plate) treated with M-CSF (30 ng / ml) and RANKL (100 ng / ml) , 5 min, 15 min, 30 min, and 60 min to measure the activity of MAPKs (mitogen-activated protein kinases). The cells were washed with phosphate buffered saline (PBS), and then treated with 1 mM phenylmethylsulfonyl fluoride (PMSF), a protease-inhibitor cocktail (Roche, Germany) and a phosphate inhibitor tablet (RIPA) buffer (25 mM Tris-HCl, pH 7.6, 150 mM NaCl, 1% NP-40, 1% sodium deoxycholate, 0.1 mM) supplemented with phosphatase inhibitor tablets (Thermo Scientific, USA) % SDS). The cell extracts (30 μg) were then subjected to SDS gel electrophoresis (SDS-PAGE) and attached to PVDF membranes (Amersham Hybond-P, GE-Healthcare Life Science, USA). Western blot analysis was then performed using specific antibodies to p-ERK, p-JNK, p-p38, p-Iκbα, ERK, JNK, p38, Iκbα and Actin.

The expression of c-Fos and NFATc1, the major transcription factors of osteoclast differentiation, was measured by Western blot analysis using each specific antibody.

In general, when osteoclasts are differentiated, MAPK signal is activated by RANKL and Iκbα activity is increased. To investigate the mechanism of C3G-induced osteoclast differentiation, intracellular signaling by RANKL treatment was investigated. Specifically, BMG was preincubated with C3G (100 μM) for 2 hours and treated with RANKL (100 ng / ml) to measure the activity of MAPK and Iκbα over time. As a result, C3G In the untreated control group, phosphorylation of all MAPKs (ERK, JNK, p38) was increased within 5 minutes and activated. However, in the experimental group treated with C3G, activation level of ERK, JNK, and p38 was decreased compared to the control group. However, the activity of Iκbα was observed to be activated in both control and experimental groups. These results indicate that C3G inhibits osteoclastogenesis by inhibiting MAPKs signaling in osteoclast differentiation induced by RANKL.

We investigated the effect of C3G on the expression of c-Fos and NFATc1, the most important transcription factors in osteoclast differentiation. More specifically, BMGs were treated with M-CSF and RANKL, mu M). As a result, as shown in FIG. 5A, the expression level of c-Fos increased from 6 hours after the RANKL treatment in the control group, and showed a maximum expression level after 24 hours. However, the expression of c-Fos in the C3G-treated experimental group was significantly lower than that of the control group. In addition, the expression level of NFATc1 was also decreased in the C3G-treated experimental group compared to the control group (FIG. 5B). These results demonstrate that C3G strongly inhibits the expression of c-Fos and NFATc1, the major transcription factors of osteoclast differentiation, and inhibits the expression of c-Fos and NFATc1 by inhibition of MAPKs activation I could.

Claims (10)

A pharmaceutical composition for preventing or treating bone metabolic diseases, comprising a compound of the formula (Ia) or (Ib) or a pharmaceutically acceptable salt thereof as an active ingredient:
(Ia)

(Ib)

In this formula,
R ₁ , R ₂ , R ₃ And R ₄ are each independently hydrogen, hydroxy or methoxy.
The method according to claim 1,
In the case of compounds of formula (Ia)
R ₁ , R ₂ , R ₃ And R &lt; ₄ &gt; are both hydroxy;
A pharmaceutical composition for preventing or treating bone metabolic diseases, wherein R ₁ , R ₂ and R ₄ are hydroxy and R ₃ is hydrogen.
The method according to claim 1,
In the case of a compound of formula (Ib)
R ₁ , R ₂ and R ₃ are both hydroxy;
R ₁ and R ₂ are each hydroxy and R ₃ is hydrogen; or
R ₁ is methoxy, R ₂ And R ₃ is hydroxy characterized in that the, bone metabolic disorders, preventing or treating pharmaceutical composition.
The method according to claim 1,
In the case of a compound of formula (Ib)
R ₁ and R ₂ are each hydroxy, and R ₃ is hydrogen.
The method according to claim 1,
Bone metabolic diseases include periodontal disease, inflammatory alveolar bone disease, inflammatory bone resorption disease, bone metastatic cancer, solid tumor metastasis, musculoskeletal complications due to solid tumor metastasis, hypercalcemia due to malignant tumors, multiple myeloma, primary osteoarthritis, osteoporosis, rheumatoid arthritis, degenerative arthritis, and Paget's disease. &lt; Desc / Clms Page number 19 &gt;
A food composition for preventing or ameliorating a metabolic disease comprising a compound of the formula (Ia) or (Ib) or a salt thereof as an active ingredient:
(Ia)

(Ib)

In this formula,
R ₁ , R ₂ , R ₃ And R ₄ are each independently hydrogen, hydroxy or methoxy.
The method according to claim 6,
In the case of a compound of formula (Ia)
R ₁ , R ₂ , R ₃ And R &lt; ₄ &gt; are both hydroxy;
R ₁ , R ₂ and R ₄ are hydroxy, and R ₃ is hydrogen.
The method according to claim 6,
In the case of a compound of formula (Ib)
R ₁ , R ₂ and R ₃ are both hydroxy;
R ₁ and R ₂ are each hydroxy and R ₃ is hydrogen; or
R ₁ is methoxy, R ₂ And R &lt; ₃ &gt; is hydroxy.
The method according to claim 6,
In the case of a compound of formula (Ib)
R ₁ and R ₂ are each hydroxy, and R ₃ is hydrogen.
The method according to claim 6,
Bone metabolic diseases include periodontal disease, inflammatory alveolar bone disease, inflammatory bone resorption disease, bone metastatic cancer, solid tumor metastasis, musculoskeletal complications due to solid tumor metastasis, hypercalcemia due to malignant tumors, multiple myeloma, primary bone tumors, osteoporosis, rheumatoid arthritis, degenerative arthritis, and Paget's disease. &lt; Desc / Clms Page number 19 &gt;

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