KR20210149498A - Pharmaceutical composition for prevention or treatment of bone disease containing Flunarizine or pharmaceutically acceptable salts thereof as an active ingredient - Google Patents

Abstract

The present invention relates to a pharmaceutical composition for the prevention or treatment of bone disease containing flunarizine or a pharmaceutically acceptable salt thereof as an active component, wherein the composition inhibits important factors related to osteoclast differentiation, so that there is an effect of inhibiting bone resorption by inhibiting the differentiation of osteoclasts, and thus can be usefully used in preclinical and clinical studies. In addition, since it is possible to treat bone-related diseases caused by imbalance of osteoclasts, the pharmaceutical composition can be widely used for developing an agent for preventing or treating bone-related diseases.

Description

Pharmaceutical composition for the prevention or treatment of bone disease containing flunarizine or a pharmaceutically acceptable salt thereof as an active ingredient {Pharmaceutical composition for prevention or treatment of bone disease containing Flunarizine or pharmaceutically acceptable salts thereof as an active ingredient}

The present invention relates to a pharmaceutical composition for preventing or treating bone disease containing flunarizine or a pharmaceutically acceptable salt thereof as an active ingredient.

Osteoporosis, a representative metabolic bone disease, is a disease in which the bone marrow becomes thinner due to a decrease in calcification of the bone tissue, and as a result, the bone marrow cavity is widened. It is easy to fracture even on impact.

Osteoporosis is characterized by a decrease in bone mass and abnormalities in the microstructure. As aging progresses, the balance between resorption of old bone and the formation of new bone is broken, and bone remodeling is not performed smoothly, resulting in sloppy bones. , increasing the risk of breaking or breaking. Such bone mass is affected by several factors, such as genetic factors, nutritional intake, hormonal changes, and differences in exercise and lifestyle. It is a major cause of bone loss.

On the other hand, although there are individual differences, in general, bone mass is the highest at the age of 14 to 18, and decreases by about 1% per year in old age. In particular, in the case of women, bone loss continuously progresses after the age of 30, and when they reach menopause, bone loss progresses rapidly due to hormonal changes. That is, the estrogen concentration rapidly decreases upon reaching menopause. At this time, as by IL-7 (interleukin-7), a large amount of B-lymphocytes are produced and B-cell precursors (pre-B) in the bone marrow (bone marrow). cells) accumulate, and this increases the amount of IL-6, which increases the activity of osteoclasts, resulting in a decrease in bone mass.

As such, osteoporosis is an unavoidable symptom for the elderly, particularly postmenopausal women, although there is a difference in degree, and interest in osteoporosis and its therapeutic agents is gradually increasing in developed countries as the population ages. In addition, around the world, a market of about 130 billion dollars is formed related to the treatment of bone diseases and is expected to increase further in the future.

In Korea, the prevalence of osteoporosis is rapidly increasing as life expectancy approaches 80 years of age. reported to have This suggests that osteoporosis is a more common disease than diabetes or cardiovascular disease, and that osteoporosis is a very important health problem when estimating the pain and cost of treatment for patients suffering from fractures.

So far, several substances have been developed for the treatment of osteoporosis. Among them, estrogen, which is most often used as a treatment for osteoporosis, has a disadvantage in that its practical efficacy has not yet been verified, and it has to be taken continuously for life. Alendronate also has problems in that its efficacy is not clear, absorption in the digestive tract is slow, and it causes inflammation in the stomach and esophageal mucosa. Calcium preparations are known to be effective with few side effects, but they are a preventive agent rather than a therapeutic agent. In addition, vitamin D preparations such as calcitonin are known, but their efficacy and side effects have not been sufficiently studied. Accordingly, there is a need for a new therapeutic agent for metabolic bone disease with fewer side effects and excellent effects.

Osteoclasts are large multinucleated cells that destroy or absorb bone tissue that becomes unnecessary in the process of bone growth in vertebrates, and are cells differentiated from osteoclast precursors. Osteoclast progenitor cells are differentiated into osteoclasts in the presence of M-CSF and receptor activator of nuclear factor kappa-Β ligand (RANKL), and through fusion, they form multinucleated osteoclasts. Osteoclasts bind to bone through αvβ3 integrin and create an acidic environment, while secreting various collagenases and proteases to cause bone resorption. Inhibition of these osteoclasts can be an effective method for the treatment of bone disease.

On the other hand, flunarizine is a T-type calcium blocker, known as a calcium channel blocker that blocks calcium channels, and is a substance that has been approved by the US FDA and is mainly used as a treatment for migraine. Several studies have also reported that flunarizine inhibits other types of calcium channels, including L-type calcium channels, N-type calcium channels, and inositol 1,4,5-triphosphate (IP3) receptors. However, the osteoclast differentiation-regulating effect and the use of the present invention for the treatment of bone diseases have not been known to date.

Accordingly, the present inventors identified that flunarizine inhibits bone resorption by osteoclasts and inhibits osteoclast formation while searching for substances that inhibit osteoclast activity to find new substances for treating bone diseases, By confirming that flunarizine can be usefully used as a pharmaceutical composition for treating bone disease, the present invention has been completed.

It is an object of the present invention to provide a novel drug capable of preventing or treating bone diseases caused by excessive number or activity of osteoclasts, such as osteoporosis and rheumatoid arthritis.

In order to achieve the above object,

The present invention provides a pharmaceutical composition for preventing or treating bone disease containing flunarizine or a pharmaceutically acceptable salt thereof as an active ingredient,

In addition, the present invention provides a health functional food for preventing or improving bone disease containing flunarizine or a pharmaceutically acceptable salt thereof as an active ingredient.

The pharmaceutical composition for the prevention or treatment of bone diseases containing flunarizine or a pharmaceutically acceptable salt thereof of the present invention as an active ingredient suppresses important factors related to osteoclast differentiation, thereby inhibiting the differentiation of osteoclasts. It has an effect of inhibiting absorption and can be usefully used in preclinical and clinical studies. In addition, since it can treat bone-related diseases caused by imbalance of osteoclasts, it can be widely used for preventing or developing therapeutic agents for bone-related diseases.

1 is a diagram showing the concentration and time-dependent effects of flunarizine, a composition of the present invention, on the differentiation of RANKL-treated osteoclast progenitor cells (BMMs) according to the concentration of flunarizine.
1A is a photograph of TRAP staining of osteoclasts cultured with RANKL (50 ng/ml) and M-CSF (40 ng/ml) for 3 or 4 days in the presence of various concentrations of flunarizine (scale bar 100 μm) .
1B is a graph of quantitative analysis of the number of TRAP-stained multinucleated osteoclasts (counting TRAP-positive multinucleated cells (MNCs) containing more than 3 nuclei).
Figure 1c is a graph showing the cytotoxicity analysis (M: M-CSF / M + R: M-CSF and RANKL) by concentration of flunarizine.
2 is a diagram showing the effect of flunarizine of the present invention on the expression of NFATc1 and target genes in RANKL-treated osteoclast progenitor cells (BMMs).
Figure 2a is a diagram showing the results of immunoblotting (immunoblotting) confirming the effect of each concentration of flunarizine on the expression of NFATc1.
Figure 2b is a diagram showing the results of immunoblotting (immunoblotting) confirming the time-dependent effect of flunarizine on the expression of NFATc1.
Figure 2c is a quantitative analysis graph showing the mRNA expression change of NFATc1 and its target genes according to flunarizine treatment quantified through RT-PCR.
3 is a diagram using fura-2 fluorescence to determine whether flunarizine affects the calcium vibration of osteoclasts.
The left figure of FIG. 3A shows a fura-2 image of BMM cells, and the right graph shows traces of changes in the fura-2 fluorescence ratio in single cells of each experiment.
3B is a graph quantifying cells exhibiting calcium oscillations.
4 is a diagram confirming the regulation of ^{Ca +-related signal transduction system by flunarizine.}
4A is a diagram showing the results of immunoblotting using antibodies against p-CaMKIV, CaMKIV, p-CREB and CREB in cell lysates.
Figure 4b is a diagram illustrating the quantification of the mRNA level of the target gene of c-fos by real-time PCR by incubating cells with RANKL in the presence of 6 μM flunarizine for 12 hours.
Figure 4c is a diagram showing the results of immunoblotting (immunoblotting) using an antibody against c-fos in the cell lysate.
Figure 4d shows that RAW264.7 cells were transfected with 0.45 mg of pAP-1-Luc (AP-1 reporter plasmid) and 0.15 mg of pRL-SV40 (control) for 24 hours and the lysates of the cells were analyzed by double luciferase. It is a figure measured using the system.
Figure 4e is a diagram illustrating the quantification of the mRNA level of the target gene of PGC1β by real-time PCR by incubating cells with RANKL in the presence of 6 μM flunarizine for 72 hours.
Figure 4f is a diagram showing the results of immunoblotting (immunoblotting) using the antibody against PGC1β in the cell lysate.
5 is a diagram illustrating treatment with ionomycin, an intracellular calcium-increasing compound, in order to determine whether flunarizine inhibits osteoclast differentiation by blocking calcium signal transduction.
Figure 5a is a diagram examined by an optical microscope after fixing the cells and performing TRAP staining (scale bar 200 μM).
FIG. 5B is a graph quantified by counting TRAP-positive MNCs containing 10 or more nuclei in FIG. 5A .
6 is a diagram illustrating the measurement of actinling and absorption functions in flunarizine-treated differentiated osteoclasts.
FIG. 6a is a diagram illustrating BMM cells treated with RANKL for 3 days, followed by addition of 6 μM flunarizine to differentiated osteoclasts for 24 hours and observed with an optical microscope over time (scale bar, 100 μm).
Figure 6b is a view observed under an optical microscope after fixing the cells and staining with TRAP (scale bar, 400 μm).
FIG. 6c is a graph quantifying TRAP-positive multinucleated cells (MNCs) in FIG. 6b.
FIG. 6d is a diagram illustrating BMM cells observed under a fluorescence microscope after culturing on glass in the presence of RANKL for 3 days and then exposing them to 6 μM flunarizine for 12 hours (scale bar, 200 μm).
FIG. 6e is a diagram visualized by staining resorption pits with hematoxylin after BMM cells were cultured on a dentine disc in the presence of RANKL for 3 days and then exposed to 6 μM flunarizine on day 0 or 4;
6F is a graph in which the pit area in FIG. 6E is digitized.
7 is a diagram confirming the down-regulation of signals required for osteoclast maturation and function by flunarizine.
Figure 7a shows that BMM cells were cultured with RANKL for 3 days, then differentiated osteoclasts were cultured with 6 μM flunarizine for 24 hours, and then cell lysates were prepared using antibodies specific for integrin β3, p130Cas and c-Src. It is a diagram analyzing immunoblotting.
7B is a diagram illustrating quantification of mRNA levels of genes involved in osteoclast maturation and function by real-time PCR.
8 is a view confirming the inhibitory effect of LPS or OVX-induced bone destruction by flunarizine.
Figure 8a is a diagram showing a photographic image of the skull stained with TRAP and hematoxylin.
Figure 8b is a diagram showing a cross-sectional photographic image of the skull stained with TRAP.
8c is a graph quantifying osteoclasts stained with bone cavity and TRAP.
8D is a diagram showing a micro-CT image of a femur of a mouse that has undergone simulated surgery or ovariectomy (OVX).
FIG. 8E is a histologically analyzed graph of the femur of FIG. 8D (BMD, bone mineral density; BV/TV, bone mass as a fraction of total bone mass; Tb.N, trabecular number; Tb.Th, trabecular thickness; Tb.Sp; , fiber separation; BS/BV, bone surface as a volume ratio; BS/TV, bone surface density; Tb.pf, trabecular pattern coefficient).

Hereinafter, the present invention will be described in detail.

Flunarizine, which the present invention contains as an active ingredient, has a structure represented by the following Chemical Formula 1:

The present invention provides a pharmaceutical composition for the prevention or treatment of bone diseases, characterized in that it contains the compound represented by Formula 1, or a pharmaceutically acceptable salt thereof, as an active ingredient.

In addition, flunarizine of the present invention is characterized in that the concentration of 0.1 to 10 μM. The flunarizine composition having a concentration of less than 0.1 μM has a problem in that the treatment efficiency of bone disease is lowered, and when it is 10 μM or more, there is a problem that toxicity may occur to cells. In this aspect, the concentration of the pharmaceutical composition containing flunarizine as an active ingredient may be preferably 2 to 8 μM, more preferably 6 μM.

In addition, the composition is NFATc1, Acp5, Mmp9, Ctsk, CalcrC, Dcstamp, c-FOS, p-CaMKIV, p-CREB, PGC1β, Dock5, Src, Atp6v0d2 and Itgb3 any one or more expression or activity selected from the group consisting of can reduce

In the composition, Acp5, Mmp9, Ctsk, CalcrC and Dcstamp are NFATc1 target genes, and flunarizine can inhibit NFATc1 gene expression, and Acp5, Mmp9, Ctsk, CalcrC and Dcstamp can also inhibit gene expression.

In addition, the composition may inhibit bone damage caused by inflammation or oophorectomy. The inflammation can be induced by injecting LPS (Lipopolysaccharide) into a mouse model, which is an inflammation-inducing substance.

In addition, the composition may suppress calcium oscillation of osteoclasts.

The calcium oscillation can be inhibited by flunarizine, which can lead to down-regulation of p-CaMKIV, p-CREB and c-Fos, and the protein expression of PGC1β and mRNA levels of PGC1β target genes can also be down-regulated. have.

In addition, bone diseases to which the composition of the present invention can be applied include growth retardation, fracture, osteoporosis due to excessive bone resorption of osteoclasts, rheumatoid arthritis, periodontal disease, Paget It is preferable that at least one selected from the group consisting of disease (Paget disease) and metastatic bone cancers, but is not limited thereto.

In a specific experimental example of the present invention, it was confirmed that flunarizine represented by Chemical Formula 1 effectively inhibits the differentiation of osteoclast progenitor cells (BMMs) by RANKL (see FIG. 1 ). Also, when flunarizine was treated, mRNA expression of NFATc1 protein, a transcription factor most important in osteoclast differentiation, and its target genes, Acp5, Mmp9, Ctsk, CalcrC and Dcstamp genes, was suppressed (see FIG. 2 ).

In addition, it was confirmed that calcium oscillation of osteoclasts was significantly suppressed in the presence of flunarizine (see FIG. 3), and it was confirmed that the expression of c-Fos protein and mRNA, known as upstream regulators of NFATc1, was significantly suppressed. (See FIG. 4), it was confirmed that phosphorylation of CaMKIV, CREB, etc. is inhibited (see FIG. 4).

In addition, flunarizine inhibits the formation of actin rings and bone resorption by osteoclasts in the dentine disc (see FIG. 6), suppresses the end of osteoclastogenesis (see FIG. 7), and osteoclast maturation and function It was confirmed that the mRNA levels of Dcstamp, Dock5, MMP9, Ctsk, Src, Atp6v0d2 and Itgb3 genes, which play an important role in the (see FIG. 8).

Therefore, the composition containing flunarizine of the present invention as an active ingredient can be usefully used for the prevention or treatment of bone diseases.

In addition, the present invention includes not only the compound represented by Formula 1, but also a pharmaceutically acceptable salt thereof, and possible solvates, hydrates, racemates, or stereoisomers prepared therefrom.

The present invention can be used in the form of the compound represented by Formula 1 or a pharmaceutically acceptable salt thereof, and as the salt, an acid addition salt formed by a pharmaceutically acceptable free acid is useful. Acid addition salts include 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, hydroxy alkanoates and alkanes. It is obtained from non-toxic organic acids such as doates, aromatic acids, aliphatic and aromatic sulfonic acids. Such pharmaceutically non-toxic salts include sulfate, pyrosulfate, bisulfate, sulfite, bisulfite, nitrate, phosphate, monohydrogen phosphate, dihydrogen phosphate, metaphosphate, pyrophosphate chloride, bromide, ioda. Id, fluoride, acetate, propionate, decanoate, caprylate, acrylate, formate, isobutyrate, caprate, heptanoate, propiolate, oxalate, malonate, succinate, suberate , sebacate, fumarate, maleate, butyne-1,4-dioate, hexane-1,6-dioate, benzoate, chlorobenzoate, methylbenzoate, dinitrobenzoate, hydroxybenzoate, methylbenzoate oxybenzoate, phthalate, terephthalate, benzenesulfonate, toluenesulfonate, chlorobenzenesulfonate, xylenesulfonate, phenylacetate, phenylpropionate, phenylbutyrate, citrate, lactate, hydroxybutyrate, glycolate, malate, tartrate, methanesulfonate, propanesulfonate, naphthalene-1-sulfonate, naphthalene-2-sulfonate or mandelate.

The acid addition salt according to the present invention is prepared by a conventional method, for example, by dissolving the compound represented by Formula 1 in an excess aqueous acid solution, and dissolving the salt in a water-miscible organic solvent such as methanol, ethanol, acetone or acetonitrile. It can be prepared by precipitation using In addition, it may be prepared by heating the same amount of the compound represented by the formula (1) and an aqueous acid solution or alcohol, then evaporating the mixture to dryness, or suction filtration of the precipitated salt.

In addition, a pharmaceutically acceptable metal salt can be prepared using a base. The alkali metal or alkaline earth metal salt is obtained, for example, by dissolving the compound in an excess alkali metal hydroxide or alkaline earth metal hydroxide solution, filtering the undissolved compound salt, and evaporating and drying the filtrate. In this case, it is pharmaceutically suitable to prepare a sodium, potassium or calcium salt as the metal salt. The corresponding silver salt is also obtained by reacting an alkali metal or alkaline earth metal salt with a suitable negative salt (eg silver nitrate).

When formulating the composition, it is usually prepared using a diluent or excipient such as a filler, an extender, a binder, a wetting agent, a disintegrant, and a surfactant.

Solid preparations for oral administration include tablets, pills, powders, granules, capsules, troches, etc., and these solid preparations include at least one excipient in one or more compounds represented by Formula 1 of the present invention, for example, It is prepared by mixing starch, calcium carbonate, sucrose or lactose or gelatin. In addition to simple excipients, lubricants such as magnesium stearate talc are also used. Liquid formulations for oral administration include suspensions, solutions, emulsions, or syrups. In addition to commonly used simple diluents such as water and liquid paraffin, various excipients such as wetting agents, sweeteners, fragrances, and preservatives may be included. can

Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solutions, suspension solutions, emulsions, lyophilized formulations, suppositories, and the like.

Non-aqueous solvents and suspensions may include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate. As the base of the suppository, witepsol, macrogol, tween 81, cacao butter, taurine paper, glycerol, gelatin, etc. may be used.

The composition according to the present invention is administered in a pharmaceutically effective amount. In the present invention, "pharmaceutically effective amount" means an amount sufficient to treat a disease at a reasonable benefit/risk ratio applicable to medical treatment, and the effective dose level is the type, severity, and drug activity of the patient. , can be determined according to factors including sensitivity to drug, administration time, administration route and excretion rate, duration of treatment, concurrent drugs, and other factors well known in the medical field. The composition of the present invention may be administered as an individual therapeutic agent or in combination with other therapeutic agents, may be administered sequentially or simultaneously with conventional therapeutic agents, and may be administered singly or multiple times. In consideration of all of the above factors, it is important to administer an amount that can obtain the maximum effect with a minimum amount without side effects, which can be easily determined by those skilled in the art.

Specifically, the effective amount of the compound according to the present invention may vary depending on the age, sex, and weight of the patient, and in general, 0.1 mg to 100 mg per kg body weight, preferably 0.5 mg to 10 mg per kg body weight, is administered daily or every other day Or, it can be administered in divided doses 1 to 3 times a day. However, since the administration route, the severity of the disease, sex, weight, age, etc. may increase or decrease, the dosage does not limit the scope of the present invention in any way.

The composition of the present invention may be used alone or in combination with methods using surgery, radiation therapy, hormone therapy, chemotherapy, and biological response modifiers.

In addition, the present invention provides a health functional food composition for preventing or improving bone disease containing flunarizine represented by Formula 1 or a pharmaceutically acceptable salt thereof as an active ingredient.

In addition, flunarizine of the present invention is characterized in that the concentration of 0.1 to 10 μM. The flunarizine composition having a concentration of less than 0.1 μM has a problem in that the treatment efficiency of bone disease is lowered, and when it is 10 μM or more, there is a problem that toxicity may occur to cells. In this aspect, the concentration of the health functional food composition containing flunarizine as an active ingredient may be preferably 2 to 8 μM, more preferably 6 μM.

In addition, the composition is NFATc1, Acp5, Mmp9, Ctsk, CalcrC, Dcstamp, c-FOS, p-CaMKIV, p-CREB, PGC1β, Dock5, Src, Atp6v0d2 and Itgb3 any one or more expression or activity selected from the group consisting of can reduce

In the composition, Acp5, Mmp9, Ctsk, CalcrC and Dcstamp are NFATc1 target genes, and flunarizine can inhibit NFATc1 gene expression, and Acp5, Mmp9, Ctsk, CalcrC and Dcstamp can also inhibit gene expression.

In addition, the composition may inhibit bone damage caused by inflammation or oophorectomy.

The inflammation can be induced by injecting LPS (Lipopolysaccharide) into a mouse model, which is an inflammation-inducing substance, and bone damage by ovariectomy can be performed using an OVX-induced model of postmenopausal osteoporosis.

In addition, the composition may suppress calcium oscillation of osteoclasts.

The calcium oscillation can be inhibited by flunarizine, which can lead to down-regulation of p-CaMKIV, p-CREB and c-Fos, and the protein expression of PGC1β and mRNA levels of PGC1β target genes can also be down-regulated. have.

In addition, bone diseases to which the composition of the present invention can be applied include growth retardation, fracture, osteoporosis due to excessive bone resorption of osteoclasts, rheumatoid arthritis, periodontal disease, Paget It is preferable that at least one selected from the group consisting of disease (Paget disease) and metastatic bone cancers, but is not limited thereto.

"Health functional food" as used herein refers to a nutrient that is easily deficient in daily meals or manufactured using raw materials or ingredients (functional raw materials) having useful functions in the human body, and maintains normal functions of the human body or promotes health through physiological function activation. It refers to food that maintains and improves food safety, but is not limited thereto and is not used in the meaning of excluding health food in the ordinary sense.

The composition of the present invention may be added to food as it is or used together with other food or food ingredients, and may be appropriately used according to a conventional method. The mixing amount of the active ingredient may be appropriately determined depending on the purpose of its use (for prevention or improvement). In general, the amount of the compound in the health functional food may be added in an amount of 0.01 to 90 parts by weight based on the total weight of the food. However, during long-term ingestion for health and hygiene or health control purposes, the amount may be less than the above range, and since there is no problem in terms of safety, the active ingredient may be used in an amount greater than or equal to the above range.

The health functional beverage composition of the present invention is not particularly limited in other ingredients except for containing the composition as an essential ingredient in the indicated ratio, and may contain various flavoring agents or natural carbohydrates as additional ingredients like a conventional beverage. Examples of the above-mentioned natural carbohydrates include monosaccharides such as glucose, fructose and the like; disaccharides such as maltose, sucrose and the like; and polysaccharides, for example, conventional sugars such as dextrin and cyclodextrin, and sugar alcohols such as xylitol, sorbitol, and erythritol. As flavoring agents other than those described above, natural flavoring agents {thaumatin, stevia extract (eg, rebaudioside A, glycyrrhizin, etc.)} and synthetic flavoring agents (saccharin, aspartame, etc.) can be advantageously used have. The proportion of the natural carbohydrate is generally about 1 to 20 g, preferably about 5 to 12 g per 100 g of the composition of the present invention.

In addition to the above, the composition of the present invention includes various nutrients, vitamins, minerals (electrolytes), flavoring agents such as synthetic and natural flavoring agents, coloring agents and thickeners (cheese, chocolate, etc.), pectic acid and its salts, alginic acid and its salts, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohols, carbonation agents used in carbonated beverages, and the like. In addition, the composition of the present invention may contain natural fruit juice and pulp for the production of fruit juice beverages and vegetable beverages.

These components may be used independently or in combination. The proportion of these additives is not critical, but is generally selected in the range of 0.1 to about 20 parts by weight per 100 parts by weight of the composition of the present invention.

Hereinafter, the present invention will be described in detail by way of experimental examples.

However, the following experimental examples only illustrate the present invention, and the content of the present invention is not limited by the following experimental examples.

<Example 1> Experimental preparation

1-1. Reagent and Antibody Preparation

A DNA fragment encoding soluble mouse RANKL (amino acids 157 to 316) was cloned into the salI and NotI sites of pET-28b (Novagen). A DNA fragment encoding mature human M-CSF (amino acids 33 to 190) was cloned into the NdeI and BamHI sites of pET-11a (Novagen).

RANKL and M-CSF proteins were expressed in Escherichia coli BL21(DE3) by isopropyl-β-D-thiogalactopyranoside induction. His-tagged RANKL was purified using anion exchange and Ni-chelating column chromatography, and M-CSF was obtained by anion exchange and hydrophobic column chromatography from refolding of insoluble protein. Flunarizine (FN) dihydrochloride was purchased from AK sci. Flunarizine was prepared as a 28.64 mg/ml (60 mM) stock in DMSO and stored at -80°C. Ionomycin was purchased from Agscientific, Fura-2 AM from Molecular probe, Pluronic F-127 from Invitrogen, and lipopolysaccharide (LPS) from Sigma.

Rabbit polyclonal antibodies to β-actin, PGC1β and phospho-CaMKIV were purchased from Abcam.

Rabbit polyclonal antibodies against phospho-JNK, phospho-ERK, phospho-p38 and phospho-CREB were purchased from Cell Signaling Technology. Rabbit polyclonal antibodies to JNK1, p38 and c-Fos, and mouse polyclonal antibodies to ERK2, NFATc1, integrins β3, 5 CaMKIV and CREB were purchased from Santa Cruz Biotechnology, Inc.

1-2. Preparation of osteoclast progenitor cells (bone marrow-derived macrophage)

Bone marrow-derived macrophage (BMM) was prepared from the femur and tibia of 6-8 week old C57BL/6 male mice.

Specifically, bone marrow cells were isolated from bone marrow using α-minimum essential medium (α-MEM) containing 10% fetal bovine serum (FBS), 100 U/ml penicillin, and 100 g/ml streptomycin. marrow cavity) and harvested by centrifugation at 800 rpm at room temperature. The cell pellet was then resuspended in the same medium. After incubation at 37°C for 1 day, non-adherent cells were harvested and incubated in Gey's solution for 10 minutes to remove red blood cells. After clarification by centrifugation, cells were cultured in α-MEM containing 10% FBS, 100 U/ml penicillin and 100 g/ml streptomycin and 40 ng/ml recombinant human M-CSF. After 3 days, adherent cells were used as osteoclast progenitors for osteoclast generation.

<Experimental Example 1> Inhibition of osteoclast differentiation of flunarizine

The present inventors performed an experiment on BMM cells in order to confirm the inhibitory effect of flunarizine on osteoclast differentiation.

Specifically, 40 ng/ml of macrophage-colony stimulating factor (M-CSF) and 50 ng/ml of RANKL (Receptor activator of nuclear) in BMM cells in the presence of various concentrations (0, 2, 4, 6 and 8 μM) of flunarizine factor kappa-Β ligand) for 3 or 4 days to differentiate into osteoclasts, and after PBS washing treatment, fixation with 4% paraformaldehyde, leukocyte acid phosphatase cytochemistry kit (SigmaAldrich), followed by TRAP staining , The number of TRAP-stained osteoclasts with 3 or more multinuclear cells was analyzed under a microscope.

As a result, osteoclast differentiation was significantly inhibited from 4 μM flunarizine when treated with RANKL for 3 days to differentiate into osteoclasts (FIG. 1a), and osteoclasts from 6 μM flunarizine when treated for 4 days Differentiation was significantly inhibited (Fig. 1b).

In addition, in order to exclude that the osteoclast differentiation inhibitory effect of flunarizine is due to cytotoxicity, cytotoxicity was analyzed with the EASY Cytox (WST-1) assay kit. Add 10 μl of 2-(4-iodophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium monosodium salt (WST-1) reagent (Roche Applied Science) to each BMM cell. After incubation at 37 °C for 2-4 hours, absorbance at 450 nm was measured.

As a result, it was confirmed that when flunarizine at various concentrations (0, 2, 4, 6 and 8 μM) was treated for 3 days, cytotoxicity by flunarizine did not appear up to a concentration of 8 μM ( FIG. 1c ). This shows that the inhibitory effect on osteoclast differentiation of flunarizine is not due to apoptosis.

<Experimental Example 2> Inhibition of NFATc1 expression by flunarizine

To confirm the osteoclast differentiation inhibitory effect of flunarizine, BMM cells were treated with 50ng/ml of RANKL for 2 days in the presence of flunarizine at various concentrations (0, 2, 4, 6 and 8 μM) to differentiate them into osteoclasts. In order to confirm the expression of NFATc1 protein, which is the most important transcription factor known in the differentiation of osteoclasts, an immunoblot using an antibody against NFATc1 was performed.

Specifically, the protein was separated using a 10% polyacrylamide gel, and the separated protein was transferred to nitrocellulose membranes. All non-specific binding sites on the membrane were blocked using 5% skim milk dissolved in TBST (0.05% Tween-20 in Tris-buffered saline, pH 7.4). Primary antibodies (mouse monoclonal antibodies, Santa Cruz Biotechnology Inc) were treated at 4°C for 14 hours, and then secondary antibodies were treated at room temperature for 1 hour and analyzed by West save Up (Ab Frontier).

As a result, it was confirmed that the NFATc1 protein was significantly inhibited from the flunarizine concentration of 6 μM ( FIG. 2A ), and the inhibitory effect continued until 4 days ( FIG. 2B ).

Also, mRNA expression changes of NFATc1 and its target genes Acp5, Mmp9, Ctsk, CalcrC and Dcstamp genes were confirmed by real time-PCR.

Specifically, for real-time-PCR, total RNA was extracted using Trizol reagent (Invitrogen) and quantitatively analyzed at absorbance at 260 nm. Total RNA (2㎍) containing 0.5㎍ of oligo(dT) primers After incubation at 70° C. for 5 minutes to make 15 μl, it was immediately cooled on ice. The first strand of cDNA was synthesized in a final volume of 25 μl containing 200 units of M-MLV reverse transcriptase (Promega), 24 units of ribonuclease inhibitor and 0.25 mM each dNTP, at 42°C for 1 hour and at 70°C for 10 minutes. ABI 7300 real time in the final 20 μl volume including 0.8 μl of diluted cDNA template (1:2.5), 10 μl of 2X SYBR Green PCR Master Mix (M Biotech) and 1 μl of each gene-specific primer (Genotech) 10 μM It was amplified using a PCR System (Applied Biosystems), and amplification was tested by performing 40 cycles at 50° C. for 2 minutes, 95° C. for 2 minutes, then 95° C. for 15 seconds, and 60° C. for 1 minute. The sequences of the primers used in the experiment are shown in Table 1 below.

Real-time PCR primer sequence and amplicon size Gene Primer (sense & antisence) SEQ ID NO: Amplicon
size (bp) NFC1 5'-CTTCAGCTGGAGGACACC-3'
5'-CCAATGAACAGCTGTAGCG-3' SEQ ID NO: 1
SEQ ID NO: 2 79 ctsk 5'-ACCACTGCCTTCCAATACG-3'
5'-CGTGGGCGTTATACATACAAC-3' SEQ ID NO: 3
SEQ ID NO: 4 99 Mmp9 5'-AGACGACATAGACGGCATC-3'
5'-TGCTGTCGGCTGTGGTTC-3' SEQ ID NO: 5
SEQ ID NO: 6 97 Acp5 5'-CCAGCGACAAGAGGTTCC-3'
5'-AGAGACGTTGCCAAGGTGAT-3' SEQ ID NO: 7
SEQ ID NO: 8 113 Calcr 5'-TGATGACTCTCAGGACAATG-3'
5'-ACTGGATCAATTCTGTAGGAG-3' SEQ ID NO: 9
SEQ ID NO: 10 87 dcstamp 5'-TTATGTGTTTCCACGAAGCCCTA-3'
5'-ACAGAAGAGAGCAGGGCAACG-3' SEQ ID NO: 11
SEQ ID NO: 12 141 Dock5 5'-TGGTGACACAGGGACAGTGG-3'
5'-CACCCCAACTATGCACGTGG-3 SEQ ID NO: 13
SEQ ID NO: 14 168 cFos 5'-GAGAAACGGAGAATCCGAAG-3'
5'-GAGAAACGGAGAATCCGAAG-3' SEQ ID NO: 15
SEQ ID NO: 16 97 Sod2 5'-ATTAACGCGCAGATCATGCA-3'
5′-TGTCCCCCACCATTGAACTT-3 SEQ ID NO: 17
SEQ ID NO: 18 161 Cox2 5'-GATCATAAGCGAGGACCTG-3'
5'-GTCTGTCCAGAGTTTCACC-3' SEQ ID NO: 19
SEQ ID NO: 20 85 itgb3 5'-GGAGTGGCTGATCCAGATGT-3'
5′-TCTGACCATCTTCCCTGTCC-3′ SEQ ID NO: 21
SEQ ID NO: 22 138 ATP6v0d2 5'-CAGAGATGGAAGCTGT-3'
5'-TGCCAAATGAGTTCAG-3' SEQ ID NO: 23
SEQ ID NO: 24 282 Ppargc1b 5'-CTCCAGGCAGGTTCAACCC-3'
5'-GGGCCAGAAGTTCCCTTAGG-3' SEQ ID NO: 25
SEQ ID NO: 26 83 Mt-cyb 5'-GCCACCTTGACCCGATTCT-3'
5'-TTGCTAGGGCCGCGATAAT-3 SEQ ID NO: 27
SEQ ID NO: 28 64 Mt-nd4 5'-CATCACTCCTATTCTGCCTAGCAA-3'
5'-TCCTCGGGCCATGATTATAGTAC-3' SEQ ID NO: 29
SEQ ID NO: 30 74 Mt-co1 5'-TTTTCAGGCTTCACCCTAGATGA-3'
5'-GAAGAATGTTATGTTTACTCCTACGAATATG-3' SEQ ID NO: 31
SEQ ID NO: 32 81 Mt-co3 5'-CGGAAGTATTTTTCTTTGCAGGAT-3'
5'-CAGCAGCCTCCTAGATCATGTG-3' SEQ ID NO: 33
SEQ ID NO: 34 82 b-actin 5'-ACCCTAAGGCCAACCGTG-3'
5'-GCCTGGATGGCTACGTAC-3' SEQ ID NO: 35
SEQ ID NO: 36 81 Ctsk, Cathepsin K; Mmp9, matirx metalloprotease 9; Acp5, acid phosphatase 5, tartrat-resistant; CR, Calcitonim receptor; DC-STAMP, Dendritic cell-specific transmembrane protein; Dock5, dedicator of cytokinesis 5; SOD2, superoxide dismutase 2; COX2, cyclooxygenase 2; PGC-1βperoxisome proliferator-activated receptor-γ coactivator 1β; ND4, NADH dehydrogenase subunit 4; COX1, cytochrome c oxidase subunit 1;

As a result, it was confirmed that the mRNA expression of NFATc1 and its target genes Acp5, Mmp9, Ctsk, CalcrC and Dcstamp genes was significantly suppressed when 50 ng/ml RANKL was treated in the presence of 6 μM flunarizine for 2 days (Fig. 2c). ).

<Experimental Example 3> Attenuation of RANFL-stimulated Ca+ signal transduction and downstream pathways in osteoclast progenitor cells by flunarizine

Calcium oscillation induced by RANKL plays an important role in osteoclast differentiation. Therefore, we investigated whether flunarizine affects calcium oscillations in osteoclasts.

Specifically, BMM cells were plated on a cover slip glass at the bottom of a 12-well plate and incubated with 40 ng/ml M-CSF and 50 ng/ml RANKL for 48 hours, and the cells were incubated with or without 6 μM flunarizine. and treated for 48 hours. Cells were then loaded with 5 mM Fura-2 AM and 5 μM Pluronic F12 at room temperature for 30 min at RT with regular buffers: 10 mM HEPES, 140 mM NaCl, 10 mM glucose, 5 mM KCl, 1 mM CaCl and 1 mM MgCl ₂ , pH 7.4. Cytoplasmic calcium oscillations of cells were measured using a fluorescence microscope (Axio Observer A1; Zeiss) at excitation wavelengths of 340 and 380 nm and emission wavelengths of 510 nm. Fluctuation-positive cells were defined as cells exhibiting at least 3 spontaneous events (>0.05 in Fura-2 ratio) during a 600 sec observation period under basal conditions.

In FIG. 3, the left side shows the fura-2 image of the cells, and the right graph shows the traces of changes in the fura-2 fluorescence ratio in a single cell of each experiment (FIG. 3a). Calcium oscillations were significantly inhibited by 6 μM flunarizine compared to controls without flunarizine ( FIG. 3b ). Impaired calcium oscillations by flunarizine lead to downregulation of p-CaMKIV, p-CREB and c-Fos, which play important roles in osteoclast differentiation ( FIGS. 4a to 4d ). In addition, protein expression of PGC1β and mRNA levels of target genes of PGC1β were also down-regulated when cells were incubated with flunarizine ( FIGS. 4E and 4F ).

In order to determine whether flunarizine inhibits osteoclast differentiation by blocking calcium signal transduction, the effect of flunarizine on osteoclasts was rescued by treatment with an intracellular calcium-increasing compound, ionomycin. (Fig. 5). These results show that flunarizine inhibits the differentiation of osteoclasts by attenuating calcium oscillations, and that disrupted calcium signaling causes downregulation of related signals in osteoclasts.

<Experimental Example 4> Inhibition of osteoclast formation and absorption function of flunarizine

In order to discover how flunarizine regulates osteoclast formation and function, an experiment was performed to measure actinling reduction and bone resorption function in flunarizine-treated differentiated osteoclasts.

Specifically, after the BMM cells were differentiated into osteoclasts by incubation with RANKL for 3 days, 6 μM flunarizine was added to the differentiated osteoclasts for 12 hours, 24 hours or 48 hours. Then, to stain actinling, BMM cells were fixed with a 3.74% formaldehyde solution in PBS, permeabilized with 0.1% Triton X-100, and incubated with Alexa Fluor 488-phalloidin (Invitrogen) for 20 minutes. After washing with PBS, cells were incubated with 4',6-diamidino-3-phenylindole (Roche) for 2 minutes and photographed under a fluorescence microscope. And for the measurement of bone resorption, BMM cells were plated on a dentine disc (Immunodiagnostic Systems, Ltd.) and treated with 40ng/ml M-CSF and 50ng/ml RANKL. Cells were completely removed from the dentine disc through abrasion with a cotton tip, and the dentine disc was stained with hematoxylin. Photographs of the resorption pits were taken with an optical microscope at a magnification of ×100, and their areas were measured through Image-Pro Plus 4.5 software (Media Cybernetics).

As a result, the actin ring of the osteoclasts treated with planarizin disappeared, whereas the actin ring of the control osteoclasts became thicker and larger (FIG. 6a). The disappeared actinling and reduced osteoclasts by planarizin were confirmed by TRAP staining (FIG. 6b), TRAP-positive cell counting (FIG. 6c) and actinling staining (FIG. 6d). To determine whether flunarizine inhibits the uptake function of osteoclasts, BMMs were differentiated into osteoclasts with RANKL on dentine discs in the presence or absence of flunarizine (Figure 6e), and flunarizine inhibits the uptake function of osteoclasts. was confirmed to inhibit (Fig. 6f).

<Experimental Example 5> Late inhibition of osteoclastogenesis of flunarizine

At the end of osteoclastogenesis, osteoclasts form multinucleated cells by cell-cell fusion and migration, and develop actin rings to form a sealing zone where bone resorption occurs. Thus, repressing genes involved in cell migration or fusion or forming actin rings have significant effects on osteoclast maturation or function. In order to determine whether flunarizine reduces the expression of osteoclast-related genes, an experiment was performed using the same real-time PCR method as in Experimental Example 2, and in this case, the Primer of Table 1 was used. .

Osteoclasts adhere to the bone surface by downstream pathways including integrin receptors and src and p130cas, which are down-regulated by flunarizine (Fig. 7a). Flunarizine also plays an important role in osteoclast maturation and function, including Dcstamp that promotes cell-cell fusion, Dock5 that regulates migration, MMP9 and Ctsk (Cathepsin K) that are involved in bone resorption, as well as Src, Atp6v0d2 and Itgb3. reduced the mRNA level of the gene (Fig. 7b).

<Experimental Example 6> Inhibition of bone damage due to inflammation and ovariectomy by flunarizine

To confirm the inhibitory effect of flunarizine on bone damage induced by inflammation in an animal model, LPS (Lipopolysaccharide, 12.5 mg/kg body weight), an inflammation-inducing substance, was injected twice a day into the mouse skull. At this time, vehicle (10% DMAC + 10% Tween 80 + 80% distilled water) or flunarizine (10mg/kg) were administered together, and 5 days after the first injection, the skull was fixed with 4% paraformaldehyde. , was observed after TRAP staining by washing with PBS. In addition, descaling was performed with 0.5M ethylenediaminetetraacetic acid for 7 days, and after making a paraffin block, it was cut and stained with TRAP and Hematoxylin, and then observed.

As a result, it was confirmed that there were many TRAP-stained parts in the skull treated with LPS only, but the TRAP-stained part was significantly reduced in the skulls treated with flunarizine ( FIGS. 8a and 8b ). In addition, as a result of quantitative analysis, it was confirmed that the number of bone cavities and osteoclasts increased by LPS treatment was significantly reduced by flunarizine (FIG. 8c). This suggests that flunarizine can prevent inflammation-induced bone disease.

In addition, the therapeutic potential of flunarizine was confirmed using an OVX-induced model of postmenopausal osteoporosis.

Specifically, ovariectomy (OVX) and microcomputed tomography (μCT) were performed as described above. Female 12-week-old mice underwent sham surgery or OVX, and were injected intraperitoneally with flunarizine (10 mg/kg) 6 times per week. Mice were euthanized 3 weeks after surgery, and femurs were evaluated using µCT.

As a result, in the OVX-induced model, bone mineral density, bone surface, bone number and bone volume were decreased, and bone pattern factor and bone separation were increased by OVX. Some of these effects of OVX were significantly reversed by flunarizine treatment ( FIGS. 8d and 8e ), indicating that flunarizine has therapeutic potential to treat pathological bone diseases such as osteoporosis.

<110> Ewha University - Industry Collaboration Foundation <120> Pharmaceutical composition for prevention or treatment of bone disease containing Flunarizine or pharmaceutically acceptable salts thereof as an active ingredient <130> 2020P-03-017 <160> 36 <170> KoPatentIn 3.0 <210> 1 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> Nfatc1 sense primer <400> 1 cttcagctgg aggacacc 18 <210> 2 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Nfatc1 antisense primer <400> 2 ccaatgaaca gctgtagcg 19 <210> 3 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Ctsk sense primer <400> 3 accactgcct tccaatacg 19 <210> 4 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Ctsk antisense primer <400> 4 cgtggcgtta tacatacaac 20 <210> 5 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Mmp9 sense primer <400> 5 agacgacata gacggcatc 19 <210> 6 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> Mmp9 antisense primer <400> 6 tgctgtcggc tgtggttc 18 <210> 7 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> Acp5 sense primer <400> 7 ccagcgacaa gaggttcc 18 <210> 8 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Acp5 antisense primer <400> 8 agagacgttg ccaaggtgat 20 <210> 9 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Calcr sense primer <400> 9 tgatgactct caggacaatg 20 <210> 10 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Calcr antisense primer <400> 10 actggatcaa tctgtaggag 20 <210> 11 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Dcstamp sense primer <400> 11 ttatgtgttt ccacgaagcc cta 23 <210> 12 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Dcstamp antisense primer <400> 12 acagaagaga gcagggcaac g 21 <210> 13 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Dock5 sense primer <400> 13 tggtgacaca gggacagtgg 20 <210> 14 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Dock5 antisense primer <400> 14 caccccaact atgcacgtgg 20 <210> 15 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> cFos sense primer <400> 15 gagaaacgga gaatccgaag 20 <210> 16 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> cFos antisense primer <400> 16 gagaaacgga gaatccgaag 20 <210> 17 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Sod2 sense primer <400> 17 attaacgcgc agatcatgca 20 <210> 18 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Sod2 antisense primer <400> 18 tgtccccccac cattgaactt 20 <210> 19 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Cox2 sense primer <400> 19 gatcataagc gaggacctg 19 <210> 20 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Cox2 antisense primer <400> 20 gtctgtccag agtttcacc 19 <210> 21 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Itgb3 sense primer <400> 21 ggagtggctg atccagatgt 20 <210> 22 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Itgb3 antisense primer <400> 22 tctgaccatc ttccctgtcc 20 <210> 23 <211> 16 <212> DNA <213> Artificial Sequence <220> <223> Atp6v0d2 sense primer <400> 23 cagagatgga agctgt 16 <210> 24 <211> 16 <212> DNA <213> Artificial Sequence <220> <223> Atp6v0d2 antisense primer <400> 24 tgccaaatga gttcag 16 <210> 25 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Ppargc1b sense primer <400> 25 ctccaggcag gttcaaccc 19 <210> 26 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Ppargc1b antisense primer <400> 26 gggccagaag ttcccttagg 20 <210> 27 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Mt-cyb sense primer <400> 27 gccaccttga cccgattct 19 <210> 28 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Mt-cyb antisense primer <400> 28 ttgctagggc cgcgataat 19 <210> 29 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Mt-nd4 sense primer <400> 29 catcactcct attctgccta gcaa 24 <210> 30 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Mt-nd4 antisense primer <400> 30 tcctcgggcc atgattatag tac 23 <210> 31 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Mt-co1 sense primer <400> 31 ttttcaggct tcaccctaga tga 23 <210> 32 <211> 31 <212> DNA <213> Artificial Sequence <220> <223> Mt-co1 antisense primer <400> 32 gaagaatgtt atgtttactc ctacgaatat g 31 <210> 33 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Mt-co3 sense primer <400> 33 cggaagtatt tttctttgca ggat 24 <210> 34 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Mt-co3 antisense primer <400> 34 cagcagcctc ctagatcatg tg 22 <210> 35 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> b-actin sense primer <400> 35 accctaaggc caaccgtg 18 <210> 36 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> b-actin antisense primer <400> 36 gcctggatgg ctacgtac 18

Claims (13)

A pharmaceutical composition for the prevention or treatment of bone disease, characterized in that it contains flunarizine represented by the following formula (1) or a pharmaceutically acceptable salt thereof as an active ingredient:
[Formula 1]

.
According to claim 1,
The flunarizine is a pharmaceutical composition for preventing or treating bone disease, characterized in that the concentration of 0.1 to 10 μM.
According to claim 1,
The composition exhibits at least one expression or activity selected from the group consisting of NFATc1, Acp5, Mmp9, Ctsk, CalcrC, Dcstamp, c-FOS, p-CaMKIV, p-CREB, PGC1β, Dock5, Src, Atp6v0d2 and Itgb3. A pharmaceutical composition for the prevention or treatment of bone disease, characterized in that it reduces.
According to claim 1,
The composition is a pharmaceutical composition for preventing or treating bone disease, characterized in that it suppresses bone damage caused by inflammation.
According to claim 1,
The composition is characterized in that suppressing calcium oscillation of osteoclasts, a pharmaceutical composition for the prevention or treatment of bone disease.
According to claim 1,
The composition is characterized in that inhibiting osteoclastogenesis (osteoclastogenesis), a pharmaceutical composition for the prevention or treatment of bone disease.
8. The method of claim 7,
The osteoclast generation is characterized in that the osteoclast generation late stage (late stage), a pharmaceutical composition for the prevention or treatment of bone disease.
According to claim 1,
The bone diseases include growth retardation, fractures, osteoporosis due to excessive bone resorption of osteoclasts, rheumatoid arthritis, periodontal disease, Paget disease, and metastatic bone cancer (metastatic). Bone cancers), characterized in that any one or more selected from the group consisting of, a pharmaceutical composition for the prevention or treatment of bone diseases.
A health functional food composition for the prevention or improvement of bone diseases, characterized in that it contains flunarizine or a pharmaceutically acceptable salt thereof represented by the following formula (1) as an active ingredient:
[Formula 1]

.
10. The method of claim 9,
The flunarizine is a health functional food composition for the prevention or improvement of bone disease, characterized in that the concentration of 0.1 to 10μM.
10. The method of claim 9,
The composition exhibits at least one expression or activity selected from the group consisting of NFATc1, Acp5, Mmp9, Ctsk, CalcrC, Dcstamp, c-FOS, p-CaMKIV, p-CREB, PGC1β, Dock5, Src, Atp6v0d2 and Itgb3. A health functional food composition for the prevention or improvement of bone disease, characterized in that it reduces.
10. The method of claim 9,
The composition is a health functional food composition for preventing or improving bone disease, characterized in that it suppresses bone damage caused by inflammation.
10. The method of claim 9,
The bone diseases include growth retardation, fractures, osteoporosis due to excessive bone resorption of osteoclasts, rheumatoid arthritis, periodontal disease, Paget disease, and metastatic bone cancer (metastatic). Bone cancers), characterized in that any one or more selected from the group consisting of, a health functional food composition for the prevention or improvement of bone diseases.

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