Osteoclast differentiation inhibitor and postmenopausal osteoporosis prevention / improvement agent

Abstract

To provide an osteoclast differentiation inhibitor and a postmenopausal osteoporosis preventive / improver containing 10-hydroxy-2-decenoic acid as an active ingredient.SOLUTION: The inventors have completed this invention by finding out that 10-hydroxy-2-decenoic acid has the action of inhibiting osteoclast differentiation.SELECTED DRAWING: Figure 7

Description

Technical Field

[0001] The present invention relates to an osteoclast differentiation inhibitor and a preventive / ameliorative agent for postmenopausal osteoporosis, which contain 10-hydroxy-2-decenoic acid as an active ingredient.

Background Art

[0002] Bone is an important organ that supports the body and is also a metabolic organ such as calcium necessary for maintaining life. Bone homeostasis is maintained by the balance between bone resorption by osteoclasts and bone formation by osteoblasts, and the disruption of this balance causes various bone diseases. Osteoporosis is one of the bone diseases. Osteoporosis is classified into idiopathic osteoporosis, postmenopausal osteoporosis, degenerative or senile osteoporosis, secondary osteoporosis, etc. Idiopathic osteoporosis develops in childhood or adolescence, although the amount of hormones and vitamins in the body is normal and there is no obvious cause. Postmenopausal osteoporosis is caused by a decrease in estrogen, which induces mobilization and enhanced response of osteoclast precursors, resulting in increased bone resorption. Degenerative or senile osteoporosis is associated with a decrease in the number and activity of osteoblasts and is not primarily associated with enhanced osteoclast activity. It is thought to be caused by a decrease in vitamin D synthesis necessary for calcium absorption. Secondary osteoporosis accounts for less than 5% of all osteoporosis, and its causes include endocrine diseases, drug induction, etc.

[0003] In particular, postmenopausal osteoporosis is common in elderly women. It induces mobilization and enhanced response of osteoclast precursors, resulting in increased bone resorption.

[0004] As preventive and treatment methods for osteoporosis, administration of calcium, active vitamin D, selective estrogen receptor modulators, bisphosphonates, anti-RANKL (Receptor activator of NF-κB ligand) antibodies, etc. are carried out.

[0005] Furthermore, it has been reported that royal jelly enhances the expression of the procollagen 1α1 gene, which is a differentiation marker of osteoblasts, and promotes bone formation (Patent Document 1). In addition, 10-hydroxy-2-decenoic acid contained in royal jelly is known to have estrogenic effects (Patent Document 2). As causes of osteoporosis due to estrogen deficiency, RANKL, OPG (osteoprotegerin), and Sema3A derived from osteocytes and the like are regulated in expression by estrogen, and appropriate expression thereof is considered to be involved in maintaining bone mass (Non-Patent Document 1). In contrast, in the present invention, 10-hydroxy-2-decenoic acid directly binds to FFAR4 of osteoclasts, thereby suppressing the activation of NF-κB signal, and the mechanism of action is different from the estrogenic effect.

Prior Art Documents

Patent Documents

[0006]

Patent Document 1

Patent Document 2

Non-Patent Documents

[0007]

Non-Patent Document 1

Summary of the Invention

Problems to be Solved by the Invention

[0008] The present invention provides an inhibitor of osteoclast differentiation and a preventive / ameliorating agent for postmenopausal osteoporosis, which contain 10-hydroxy-2-decenoic acid as an active ingredient.

Means for Solving the Problems

[0009] As a result of intensive studies, the present inventors have found that 10-hydroxy-2-decenoic acid has an effect of suppressing osteoclast differentiation, and have thus completed the present invention. That is, the present invention comprises the following: 1. A differentiating inhibitor of osteoclast precursors into osteoclasts, with 10-hydroxy-2-decenoic acid as the active ingredient. 2. A preventive and ameliorating agent for postmenopausal osteoporosis, with 10-hydroxy-2-decenoic acid as the active ingredient. 3. A food composition for inhibiting the differentiation of osteoclast precursors into osteoclasts, characterized by having 10-hydroxy-2-decenoic acid as the active ingredient and having an inhibitory effect on the differentiation of osteoclast precursors into osteoclasts. 4. A food composition for preventing and ameliorating postmenopausal osteoporosis, characterized by having 10-hydroxy-2-decenoic acid as the active ingredient and having a preventive and ameliorating effect on postmenopausal osteoporosis. 5. A differentiating inhibitor of osteoclast precursors into osteoclasts, with 10-hydroxy-2-decenoic acid as the active ingredient, wherein the direct binding of 10-hydroxy-2-decenoic acid to FFAR4 of osteoclasts suppresses the activation of the NF-κB signal. 6. A preventive and ameliorating agent for postmenopausal osteoporosis, with 10-hydroxy-2-decenoic acid as the active ingredient, wherein the direct binding of 10-hydroxy-2-decenoic acid to FFAR4 of osteoclasts suppresses the activation of the NF-κB signal. 7. A food composition for inhibiting the differentiation of osteoclast precursors into osteoclasts, with 10-hydroxy-2-decenoic acid as the active ingredient, wherein the direct binding of 10-hydroxy-2-decenoic acid to FFAR4 of osteoclasts suppresses the activation of the NF-κB signal and has an inhibitory effect on the differentiation of osteoclast precursors into osteoclasts. 8. A food composition for preventing and ameliorating postmenopausal osteoporosis, with 10-hydroxy-2-decenoic acid as the active ingredient, wherein the direct binding of 10-hydroxy-2-decenoic acid to FFAR4 of osteoclasts suppresses the activation of the NF-κB signal and has a preventive and ameliorating effect on postmenopausal osteoporosis.

Effects of the Invention

[0010] According to the present invention, by suppressing osteoclast differentiation, an osteoclast differentiation inhibitor, and further a preventive and ameliorative agent for postmenopausal osteoporosis can be provided.

Brief Description of the Drawings

[0011]

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Mode for Carrying Out the Invention

[0012] In the present invention, 10-hydroxy-2-decenoic acid has been found as a participating component that suppresses osteoclast differentiation. Furthermore, postmenopausal osteoporosis is caused by inducing mobilization and hyperreactivity of osteoclast precursors, resulting in increased bone resorption. Therefore, 10-hydroxy-2-decenoic acid can also be used as a preventive and ameliorating agent for postmenopausal osteoporosis by suppressing the differentiation of osteoclast precursors into osteoclasts.

[0013] One aspect of the present invention relates to an inhibitor of differentiation of osteoclast precursors into osteoclasts having 10-hydroxy-2-decenoic acid as an active ingredient. In this specification, the inhibition of differentiation of osteoclast precursors into osteoclasts is also simply referred to as osteoclast differentiation inhibition. Another aspect of the present invention relates to a preventive and ameliorating agent for postmenopausal osteoporosis having 10-hydroxy-2-decenoic acid as an active ingredient. One aspect of the present invention relates to a food composition, which is characterized by having 10-hydroxy-2-decenoic acid as an active ingredient and having an inhibitory effect on the differentiation of osteoclast precursors into osteoclasts, and relates to a food composition for inhibiting the differentiation of osteoclast precursors into osteoclasts. Also, it relates to a food composition for preventing and ameliorating postmenopausal osteoporosis, which is characterized by having 10-hydroxy-2-decenoic acid as an active ingredient and having a preventive and ameliorating effect on postmenopausal osteoporosis.

[0014] The agent of the present invention can be produced by mixing the active ingredient, 10-hydroxy-2-decenoic acid, with one or more pharmaceutically acceptable carriers and by any method well known in the technical field of pharmaceutics.

[0015] As the pharmacologically acceptable carrier, various organic or inorganic carrier substances commonly used as formulation materials are used. Specific examples include excipients, lubricants, binders, disintegrants in solid formulations, solvents, solubilizers, suspending agents, isotonic agents, buffers, soothing agents, etc. in liquid formulations. When formulating, formulation additives such as preservatives, antioxidants, coloring agents, sweetening agents, etc. may be used as necessary.

[0016] Examples of pharmacologically acceptable additives include excipients such as sucrose, starch, mannitol, sorbitol, lactose, glucose, cellulose, talc, calcium phosphate, calcium carbonate, etc., binders such as cellulose, methylcellulose, hydroxypropylcellulose, polypropylpyrrolidone, gelatin, gum arabic, polyethylene glycol, sucrose, starch, etc., disintegrants such as starch, carmellose, hydroxypropyl starch, sodium - glycol - starch, sodium hydrogen carbonate, calcium phosphate, calcium citrate, etc., lubricants such as magnesium stearate, talc, sodium lauryl sulfate, etc., flavoring agents such as citric acid, menthol, diammonium glycyrrhizinate, glycine, orange oil, etc., preservatives such as sodium benzoate, sodium bisulfite, methyl paraben, propyl paraben, etc., stabilizers such as citric acid, sodium citrate, acetic acid, etc., suspending agents such as methylcellulose, povidone, aluminum stearate, etc., dispersants such as surfactants, diluents such as water, physiological saline, orange juice, etc., base waxes such as cacao butter, polyethylene glycol, white kerosene, etc., but are not limited thereto.

[0017] The administration target of the agent of the present invention is usually a mammal. Examples of mammals include rodents such as mice, rats, hamsters, guinea pigs, experimental animals such as rabbits, livestock such as pigs, cows, goats, horses, sheep, minks, pets such as dogs, cats, primates such as humans, monkeys, cynomolgus monkeys, rhesus monkeys, marmosets, orangutans, chimpanzees, etc., but are not limited thereto. The mammal is preferably a primate (such as a human) or a rodent (such as a mouse).

[0018] 10-Hydroxy-2-decenoic acid is a component unique to royal jelly and is known not to exist in other foods. Royal jelly is a milky white paste-like substance secreted by the hypopharyngeal glands of young worker bees, and its components are rich in amino acids including essential amino acids, constituting high-quality proteins. Furthermore, it contains trace components such as vitamins, minerals, and carbohydrates. For example, among vitamins, vitamin B1, vitamin B2, vitamin B6, niacin, pantothenic acid, vitamin A, vitamin C, vitamin E, etc. can be mentioned. Among minerals, potassium, magnesium, calcium, copper, iron, phosphorus, etc. can be mentioned. Among carbohydrates, glucose, fructose, etc. can be mentioned. Furthermore, it contains acetylcholine-like substances, organic acids, fatty acids, etc.

[0019] There are no restrictions on the origin or production method of 10-hydroxy-2-decenoic acid, and it may be a natural product or a synthetic product. Furthermore, partially purified or highly purified products obtained by fractionation, etc. may also be used. As a natural product, royal jelly can be used, and examples include raw royal jelly, dried royal jelly, and adjusted royal jelly. Also, the countries of origin of royal jelly can include, for example, Japan, China, Taiwan, Thailand, Brazil, European countries, Oceanian countries, the United States, etc., and royal jelly from any country of origin can be used. Also, royal jelly from multiple countries of origin may be appropriately mixed and used.

[0020] Furthermore, royal jelly may be one that has been processed by heating, centrifugation, alcohol extraction, filtration, etc.

[0021] Furthermore, 10-hydroxy-2-decenoic acid may be purified using various chromatographies. Examples of purification methods include gel filtration chromatography, ion exchange chromatography, affinity chromatography, hydrophobic chromatography, reverse phase chromatography, normal phase chromatography, ultrafiltration, etc., and purification can also be performed by using these alone or in combination.

[0022] Gel filtration chromatography has carriers for gel filtration chromatography that can separate proteins of various molecular weights, and carriers for gel filtration chromatography that can separate proteins with a molecular weight of about 10,000 or less are preferred. Examples of ion exchange groups used in ion exchange chromatography include anion exchangers and cation exchangers. Examples of anion exchangers can include diethylaminoethyl group (DEAE group), quaternary aminoethyl group (QAE), etc. Also, examples of cation exchangers can include carboxymethyl (CM) group, sulfopropyl (SP) group. Examples of carriers used in hydrophobic chromatography can include carriers to which a butyl group (Butyl group), ethyl group (Ethyl group), or phenyl group (Phenyl group) is bonded. Examples of carriers used in reverse phase chromatography include carriers to which an octadecyl group (C18), C30, C8, C4, etc. with different alkyl chain lengths are bonded. Examples of carriers used in normal phase chromatography include silica gel, and carriers to which a cyanopropyl group, a functional group having a diol structure, an aminopropyl group, a polyamine, etc. are bonded.

[0023] 10-Hydroxy-2-decenoic acid may be a salt, and examples of such salts include metal salts such as sodium salt, potassium salt, and magnesium salt, and organic amine salts such as monomethylamine and trimethylamine.

[0024] It is also possible to add various components to 10-hydroxy-2-decenoic acid. Examples of the various components include sugars, lipids, emulsifiers, thickeners, seasonings, fragrances, acid regulators, preservatives, fruit juices, flavors, and various nutritional components, etc., and they can be used within the range that does not impair the effects of the present invention. Also, the various components may be used alone or in combination of two or more. For example, examples of sugars include sucrose, isomerized sugar, glucose, fructose, palatinose, trehalose, lactose, xylose, etc. Examples of emulsifiers include sucrose fatty acid esters, glycerin fatty acid esters, lecithin, etc. Examples of thickeners include carrageenan, gum arabic, xanthan gum, guar gum, pectin, locust bean gum, thickening agent starch, gellan gum, etc. Examples of acid regulators include citric acid, lactic acid, malic acid, fumaric acid, gluconic acid, tartaric acid, etc. Examples of preservatives include benzoic acid and its salts, sorbic acid and its salts, parabens, sodium sulfite, pectin degradation products, glycine, etc. Examples of fruit juices include tomato juice, plum juice, apple juice, lemon juice, orange juice, berry-based juices, etc. Examples of flavors include spices such as herbs and spices, fruit-based flavors, flavors such as vanilla, etc. In addition, other preferable nutritional components include vitamins such as vitamin D and minerals such as calcium, magnesium, iron, manganese, zinc, etc.

[0025] The osteoclast differentiation inhibitor or the preventive / ameliorative agent for postmenopausal osteoporosis of the present invention can also be provided as foods and pharmaceuticals. Specific forms of foods include, for example, beverages, confectioneries, candies, gums, breads, meat products, dairy products, retort foods, instant foods, frozen foods, jelly-like foods, beekeeping products, pickles, seasonings, etc. These foods are also useful as so-called health foods, functional foods, foods for specified health uses, nutritional functional foods, dietary supplements, supplements, etc. Also, examples of their shapes as foods include granules, powders, tablets, capsules, chewables, drinks, jellies, pastes, grains, etc.

[0026] Specific forms of pharmaceuticals include powders, tablets, granules, pills, powders, capsules, liquids, syrups, pastes, emulsions, etc.

[0027] Furthermore, the osteoclast differentiation inhibitor or the preventive / ameliorative agent for postmenopausal osteoporosis of the present invention may be taken together with calcium, vitamin D, estrogen, etc.

Examples

[0028] Examples of the present invention are shown below, but the present invention is not limited to the following examples.

[0029] (Method for measuring osteoclast differentiation inhibitory activity) The measurement of the inhibitory activity of the differentiation of osteoclast precursors into osteoclasts was carried out by the method shown below.

[0030] Bone marrow cells were collected from the femurs and tibias of female C57BL / 6J mice, and cultured in aMEM (Minimum essential medium, a modification) medium supplemented with 10 ng / ml of M-CSF (Macrophage-colony stimulating factor) and 10% fetal bovine serum for 2 days to generate bone marrow-derived monocyte / macrophage precursor cells (BMM). These cells were cultured for 3 days in a medium supplemented with RANKL at a concentration of 12.5 - 50 ng / ml to induce osteoclast differentiation (osteoclast differentiation culture). The concentration of RANKL added was appropriately adjusted between 12.5 - 50 ng / ml according to the differentiation state of the bone marrow-derived monocyte / macrophage precursor cells. The test substance was added simultaneously with RANKL. The obtained cells were washed with PBS and fixed with 4% paraformaldehyde at room temperature for 15 minutes. Then, a solution of acetone (50%) and ethanol (50%) was added and left standing for 30 seconds. TRAP (tartrate-resistant acid phosphatase) staining solution (Naphatol AS-MX phosphate 0.1 mg / ml, N,N-Dimethylformamide 10 ml / ml, Fast red violet LB salt 0.6 mg / ml, TRAP Buffer (sodium acetate 5.44 g / l, sodium tartrate 10.5 g / l)) was added and left standing for 5 minutes to perform TRAP staining. Cells with three or more nuclei were regarded as osteoclasts, and the number of osteoclasts with three or more nuclei per field of view was counted to evaluate the osteoclast differentiation inhibitory activity.

[0031] (Method for measuring the osteoclast differentiation inhibitory activity of royal jelly) For raw royal jelly, a test substance was prepared by the method shown below, and the inhibitory activity of osteoclast differentiation was measured. Raw royal jelly was dissolved in DMSO (dimethyl sulfoxide) to obtain a solution with a concentration of 200 mg / ml. Further centrifugation was performed to precipitate the solid components, and the supernatant was collected. The supernatant was sterilized by filtration using an Acrodisc DMSO safe filter (PALL) and used as the test substance to measure the inhibitory activity of osteoclast differentiation. As a result, the inhibitory activity of osteoclast differentiation was confirmed for royal jelly.

[0032] (Animal test) To investigate the effect of royal jelly on bone, experiments were conducted using ovariectomized postmenopausal osteoporosis model mice. Specifically, 9-week-old female C57BL / 6J mice were subjected to ovariectomy (OVX) or Sham surgery. The control group was administered physiological saline, and the test group was administered 1.0 g / kg body weight of raw royal jelly daily via an oral sonde. Four weeks later, the mice were euthanized, and the femurs were subjected to μCT analysis, and the tibias were subjected to bone morphometric analysis. From the results of μCT analysis, images were obtained in which the decrease in bone mass was suppressed in the raw royal jelly-administered OVX group compared to the OVX group. The bone volume (Bone volume / Ttissuevolume (BV / TV)) was found to be suppressed from decreasing in the raw royal jelly-administered OVX group compared to the OVX group (Figure 1). Also, the trabecular number (Tb. N), an index of cancellous bone, decreased due to OVX, but the decrease was suppressed in the raw royal jelly-administered OVX group. Also, the trabecular separation (Tb. Sep) and trabecular spacing (Tb. Spa) increased in the OVX group compared to the Sham group, but the increase was suppressed in the raw royal jelly-administered OVX group. Also, from bone morphometric analysis, the number of osteoclasts (N.Oc / BS) was significantly decreased in the raw royal jelly-administered OVX group compared to the OVX group, and similar trends were observed for the osteoclast surface (Oc.S / BS) and the eroded surface (ES / BS). Also, no significant changes were observed in osteoblast-related parameters such as bone formation rate, bone formation rate (BFR), and osteoblast surface (Ob.S / BS).

[0033] From the above, it was suggested that royal jelly has the effect of suppressing osteoclast differentiation or function and suppressing the decrease in bone mass due to OVX.

[0034] (Separation and identification of the involved components from royal jelly) Since the inhibitory activity of royal jelly on the differentiation of osteoclast precursors into osteoclasts and the effect of suppressing the decrease in bone mass in ovariectomized mice were confirmed, the separation and identification of the components involved in the osteoclast differentiation inhibitory activity were carried out from royal jelly.

[0035] Four times the amount of methanol was added to raw royal jelly, and after stirring, it was left at -20°C overnight. After leaving it overnight, centrifugation was performed at 9,200×g for 10 minutes to obtain a supernatant (A) and a precipitate. The same amount of methanol was further added to the precipitate, and after stirring, it was left at -20°C for 2 hours. After leaving it for 2 hours, centrifugation was performed at 9,200×g for 10 minutes to obtain a supernatant (B) and a precipitate. The supernatant (A) and the supernatant (B) were mixed and filtered through a filter with a pore size of 0.45 μm (50CP045AS Advantec Toyo) to obtain a protein-depleted fraction. After removing the solvent and water from the protein-depleted fraction using a rotary evaporator, the residue was dissolved in ethyl acetate and purified water, and liquid separation was performed using the ethyl acetate and the purified water. Since osteoclast differentiation inhibitory activity was confirmed in the ethyl acetate layer, the ethyl acetate layers were pooled. After removing the solvent from the ethyl acetate layer using a rotary evaporator, the residue was dissolved in chloroform and applied to a silica gel column (ID20×200mm silica gel 60 63 - 200μm Merck) equilibrated with chloroform. After washing with chloroform, fractionation was performed with chloroform containing 10% methanol, and the fractions with confirmed osteoclast differentiation inhibitory activity were pooled to obtain a silica gel fraction. After removing the solvent from the silica gel fraction using a rotary evaporator, the residue was dissolved in methanol, and the methanol concentration was adjusted to 50% with purified water and applied to a Biotage SNAPS 30g Column Ultra C18 (Biotage) equilibrated with 50% methanol. Fractionation was performed with 50% methanol, and the fractions with confirmed osteoclast differentiation inhibitory activity were pooled to obtain a SNAPS fraction. After removing the solvent and water from the SNAPS fraction using a rotary evaporator, the residue was dissolved in methanol, and the methanol concentration was adjusted to 40% with purified water and applied to a COSMOSIL Packed Column 5C18-AR-II (10ID×250mm Nacalai Tesque) equilibrated with 40% methanol. Fractionation was performed with 40% methanol, and the fractions with confirmed osteoclast differentiation inhibitory activity were pooled to obtain an ARII fraction. The ARII fraction evaporated to dryness using an evaporator and a spray-type test tube concentrator was used as the purified fraction.

[0036] Confirmation of the purified fraction by HPLC Analysis by HPLC (High Performance Liquid Chromatography) The purified fraction and 10-hydroxy-2-decenoic acid were each dissolved in methanol and analyzed using a COSMOSIL Packed Column 5C18-AR-II (4.6 ID × 150 mm) controlled by a Chromaster system (Hitachi High-Tech Science). The elution solvent was 50% methanol containing 5.9 mmol / L H 3 PO 4 and 5.0 mmol / L NaH 2 PO 4 Analysis was performed isocratically. The column temperature was maintained at 40 °C. The column effluent was detected at 210 nm. As a result, the retention times of the purified fraction and 10-hydroxy-2-decenoic acid were almost identical at 6.5 minutes, and the purified fraction was presumed to be 10-hydroxy-2-decenoic acid (Figs. 2, 3).

[0037] LC / MS Analysis Since the purified fraction was presumed to be 10-hydroxy-2-decenoic acid, the purified fraction and 10-hydroxy-2-decenoic acid were each dissolved in methanol and compared by LC / MS. LC / MS analysis was performed using an Acquity UPLC system (Waters) and an Xevo QTof MS system (Waters). An Acquity UPLC HSS T3 column (2.1 ID × 100 mm, 1.8 μm, Waters) was controlled by the Acquity UPLC system. The elution solvents were (A) 0.1% acetic acid and (B) methanol. Separation was carried out with a linear concentration gradient from A 99.5%·B 0.5% to A 0%·B 100% for 20 minutes, followed by maintaining B 100% for 5 minutes. The flow rate was controlled at 0.4 mL / min, and the column temperature was maintained at 40 °C.

[0038] Mass spectrometry using the Xevo QTof MS system was performed under the following conditions; Mode: Negative mode Ionization method: ESI, Capillary voltage: 3.0 KV Cone voltage: 15 V Mass range: m / z 50 - 1000

[0039] As a result of LC / MS analysis, a single peak was confirmed at a retention time of 10.7 minutes for both the purified fraction and 10-hydroxy-2-decenoic acid (Figure 4). Furthermore, upon examining the MS spectrum at a retention time of 10.7 minutes, it was confirmed that the mass also matched m / z 185.11 (Figure 5). Additionally, the collision energy for m / z 185.11 was varied from 10 to 25 eV, and the generated fragments were integrated and compared. As a result, the fragmentation pattern was consistent between the purified fraction and 10-hydroxy-2-decenoic acid (Figure 6), and the purified fraction was presumed to be 10-hydroxy-2-decenoic acid.

[0040] (Comparison of the inhibitory activity of royal jelly and 10-hydroxy-2-decenoic acid on osteoclast differentiation) Raw royal jelly was prepared in methanol at concentrations of 0.25 mg / mL and 0.5 mg / mL, and centrifuged to obtain the supernatant. At that time, the 10-hydroxy-2-decenoic acid concentrations in the supernatant were 25 μM and 50 μM respectively. Therefore, 10-hydroxy-2-decenoic acid was dissolved in methanol and prepared at concentrations of 25 μM and 50 μM to compare the inhibitory activity on osteoclast differentiation. As a result, the inhibitory activities of 10-hydroxy-2-decenoic acid and raw royal jelly on osteoclast differentiation were almost the same, and the main component of the involved components contained in raw royal jelly was presumed to be 10-hydroxy-2-decenoic acid (Figure 7).

[0041] (Examination of the receptor for 10-hydroxy-2-decenoic acid) According to the method for measuring the inhibitory activity of osteoclast differentiation shown in this example, bone marrow-derived monocyte macrophage progenitor cells (BMM) and osteoclasts were obtained.

[0042] RT-PCR (real-time PCR) was performed on BMM and osteoclasts by the method shown below. As shown in Figure 8, the mRNA expression of Ffar1, Ffar2, Ffar3, and Gpr84 was decreased compared to BMM, while the mRNA expression of Ffar4 was increased, leading to the possibility that FFAR4 functions as a receptor for 10-hydroxy-2-decenoic acid.

[0043] Note that RT-PCR was measured by the following method. RNA was extracted from BMM and osteoclasts using ISOGEN from NIPPON GENE, and 0.5 μg of total RNA extracted was used to synthesize cDNA using SuperscriptIII reverse transcriptase from Thermo Fisher Scientific. Next, RT-PCR was performed on the cDNA using CFX384 Touch from Bio-Rad as a real-time PCR analysis system and SYBR Green Realtime PCR Master Mix from TOYOBO as a real-time PCR reagent. Note that the primers shown in Table 1 were used and correction was performed using Gapdh.

[0044]

Table 1

[0045] To examine the effect of 10-hydroxy-2-decenoic acid via FFAR4, the affinity between 10-hydroxy-2-decenoic acid and FFAR4 was investigated using the FFAR4 (GPR120) Reporter Assay Kit (Cayman Chemical). As a result, as shown in Figure 9, the affinity with 10-hydroxy-2-decenoic acid was confirmed, and the EC50 was 1.025 mM. Note that the FFAR4 (GPR120) Reporter Assay Kit was operated according to the attached manual.

[0046] Lentiviral vectors were created according to the product protocol and used to infect BMMs for 24 hours. After that, the cells were stimulated with RANKL and 10-hydroxy-2-decenoic acid, and knockdown was performed using an shRNA (short hairpin RNA) lentiviral vector targeting Ffar4 (Kim Y, Hayashi M, Ono T, Yoda T, Takayanagi H, Nakashima T., Mod Rheumatol. 2020 Jan;30(1):85-92.). The knockdown efficiency at that time was about 90%. As shown in Figure 10, in the Control without Ffar4 knockdown, osteoclast differentiation was suppressed by 10-hydroxy-2-decenoic acid, while in shFfar4 with Ffrar4 knockdown, osteoclast differentiation was not suppressed by 10-hydroxy-2-decenoic acid. From these results, the receptor for 10-hydroxy-2-decenoic acid was presumed to be FFAR4.

[0047] Osteoclast differentiation culture was performed in the presence or absence of 10-hydroxy-2-decenoic acid. After serum starvation for 6 hours, RANKL and 10-hydroxy-2-decenoic acid stimulation was carried out. At that time, cells were collected at 0, 5, and 20 minutes and subjected to Western Blot analysis. As a result, as shown in Figure 11, in the Control (in the absence of 10-hydroxy-2-decenoic acid), the band of IκBα gradually became thinner and IκBα was degraded. In 10H2DA (in the presence of 10-hydroxy-2-decenoic acid), there was no change in the band of IκBα, and it was considered that IκBα was not degraded.

[0048] The Western Blot analysis was performed by the following method. The obtained cells were washed with cold PBS and lysed on ice for 10 minutes using lysis buffer containing Roche Bioscience's Complete protease inhibitor cocktail and Roche Bioscience's PhosSTOP. The lysate was centrifuged at 15,000 rpm for 10 minutes for purification. The extracted protein was subjected to SDS-PAGE and transferred to a PVDF membrane (Millipore). The transferred PVDF membrane was blocked with Blocking One P or Bullet Blocking One (Nacalai Tesque) and reacted with the primary antibody at room temperature for 2 hours, followed by reaction with the secondary antibody. The bands were visualized with Chemi-Lumi One Ultra (Nacalai Tesque). For the primary antibody, the antibody against phospho-IκBα (p-IκBα) and IκBα was purchased from Cell Signaling Technology, and the antibody against β-actin was purchased from Sigma-Aldrich. After infecting BMMs with the control (TurboGFP positive control vector) and the knockdown vector (shFfar4) for 24 hours respectively, osteoclast differentiation culture was performed. After serum starvation for 6 hours, RANKL·10-hydroxy-2-decenoic acid stimulation was carried out. At that time, cells were collected at 0, 5, and 20 minutes and Western Blot analysis was performed. As a result, in the control, there was no change in the band of IκBα, and it was considered that IκBα was not degraded. On the other hand, in the knockdown vector (shFfar4), the band of IκBα became thinner and it was considered that IκBα was degraded. The Western Blot analysis was performed in the same manner as the method shown in this paragraph.

[0049] (Consideration of the mechanism of action) The regulation of osteoclast differentiation by RANKL is as follows: when RANKL binds to RANK, the IKK complex is activated. The activated IKK complex phosphorylates and degrades IκBα, causing NF-κB to translocate into the nucleus and inducing osteoclast differentiation. On the other hand, when 10-hydroxy-2-decenoic acid binds to FFAR4, the activation of the IKK complex is suppressed, and the phosphorylation and degradation of IκBα are inhibited. In addition, in the shFfar4 group with knockdown of Ffar4, the inhibition of phosphorylation and degradation of IκBα by 10-hydroxy-2-decenoic acid was attenuated. From this, it is considered that 10-hydroxy-2-decenoic acid inhibits osteoclast differentiation by suppressing the NF-κB pathway.

Industrial Applicability

[0050] According to the present invention, it becomes possible to provide an inhibitor for differentiating osteoclast precursors into osteoclasts and a prophylactic / ameliorating agent for postmenopausal osteoporosis.

Claims

[Claim 1] An agent that contains 10-hydroxy-2-decenoic acid as an active ingredient, which inhibits activation of NF-κB signaling by directly binding to FFAR4 in osteoclasts.

Images