succinate receptor inhibitors and osteoclast formation inhibitors

Abstract

This invention provides succinate receptor inhibitors that are useful for preventing periodontal disease and inhibiting its progression. [Solution] The succinate receptor inhibitor contains at least one selected from the group consisting of β-glycyrrhetinic acid, ursolic acid, resveratrol, shikonin, biochanin A, isoliquitigenin, ethinylestradiol, daidzein, allyl isothiocyanate, riboflavin, cetylpyridinium chloride, dodecylpyridinium chloride, sodium lauroyl sarcosinate, cocamidopropyl betaine, and alkyldiaminoethylglycine hydrochloride.

Description

Technical Field

[0001] The present invention relates to a succinate receptor inhibitor and an osteoclast formation inhibitor.

Background Art

[0002] Periodontal diseases are classified into gingivitis and periodontitis. Gingivitis is caused by bacteria attached to the gingival margin, resulting in mild inflammation, such as redness, bleeding, and swelling of the gingiva, but no loss of supporting tissues such as attachment loss or alveolar bone resorption. When gingivitis progresses, it becomes periodontitis, and the bacterial flora in the periodontal pocket causes inflammatory destruction of the periodontal tissues due to dysbiosis, leading to the absorption of connective tissues and alveolar bone supporting the teeth, and ultimately resulting in tooth loss.

[0003] Non-Patent Document 1 discloses that the concentration of succinic acid increases in the subgingival plaque of periodontal disease patients. It is described that succinic acid stimulates inflammation by activating the succinate receptor (SUCNR1).

[0004] The same document describes that in SUCNR1 knockout mice, suppression of inflammatory cytokine production and suppression of alveolar bone resorption were confirmed. Also, by applying an SUCNR1 antagonist to the gingiva of mice, suppression of inflammatory cytokine production, suppression of osteoclast formation, and suppression of alveolar bone resorption were confirmed. Thus, the relationship between the activation of the succinate receptor and periodontal disease has been reported.

Prior Art Documents

Non-Patent Documents

[0005]

Non-Patent Document 1

Summary of the Invention

[0006] Inhibiting the activation of succinate receptors is thought to lead to the prevention and slowing of the progression of periodontal disease by suppressing inflammation, osteoclast formation, and alveolar bone resorption. Therefore, there is a need for succinate receptor inhibitors that are useful for preventing and slowing the progression of periodontal disease. [Means for solving the problem]

[0007] The following describes various methods for solving the above problems. The succinate receptor inhibitor of Embodiment 1 contains at least one selected from the group consisting of β-glycyrrhetinic acid, ursolic acid, resveratrol, shikonin, biochanin A, isoliquitigenin, ethinylestradiol, daidzein, allyl isothiocyanate, riboflavin, cetylpyridinium chloride, dodecylpyridinium chloride, sodium lauroyl sarcosinate, cocamidopropyl betaine, and alkyldiaminoethylglycine hydrochloride.

[0008] The osteoclast formation inhibitor of embodiment 2 contains at least one selected from the group consisting of β-glycyrrhetinic acid, ursolic acid, resveratrol, shikonin, biochanin A, isoliquitigenin, ethinylestradiol, daidzein, allyl isothiocyanate, and riboflavin. [Effects of the Invention]

[0009] This invention is useful for preventing periodontal disease and inhibiting its progression. [Modes for carrying out the invention]

[0010] Embodiments of succinate receptor inhibitors and osteoclast formation inhibitors will be described. Succinate receptor inhibitors contain specific components, which will be discussed later.

[0011] The succinate receptor inhibitor of this embodiment exerts the effect of inhibiting the activation of succinate receptors, which are associated with inflammation in periodontal disease, as will be described later. As mentioned above, it has been reported that inhibiting the activation of succinate receptors can suppress osteoclast formation. For this reason, the succinate receptor inhibitor of this embodiment can also be used as an osteoclast formation inhibitor.

[0012] <Specific ingredients> This section describes the specific components contained in succinate receptor inhibitors. Succinate receptor inhibitors may contain only one of the following components alone, or two or more components in appropriate combinations. There are no particular restrictions on the amount of each component. The amount of each component can be appropriately set within the range that provides the desired effect as a succinate receptor inhibitor.

[0013] The succinate receptor inhibitor contains at least one selected from the group consisting of β-glycyrrhetinic acid, ursolic acid, resveratrol, shikonin, biochanin A, isoliquitigenin, ethinylestradiol, daidzein, allyl isothiocyanate, riboflavin, cetylpyridinium chloride, dodecylpyridinium chloride, sodium lauroyl sarcosinate, cocamidopropyl betaine, and alkyldiaminoethylglycine hydrochloride.

[0014] The osteoclast formation inhibitor contains, for example, at least one selected from the group consisting of β-glycyrrhetinic acid, ursolic acid, resveratrol, shikonin, biochanin A, isoliquitigenin, ethinylestradiol, daidzein, allyl isothiocyanate, and riboflavin.

[0015] Each component may be chemically synthesized or extracted from natural materials containing that component. Commercially available products can also be used for each component. The extraction method used when obtaining components by extraction is not particularly limited. For example, known extraction methods such as solvent extraction, ultrasonic extraction, compression, and supercritical fluid extraction can be employed.

[0016] β-Glycyrrhetinic acid β-Glycyrrhetinic acid is a component obtained by hydrolyzing glycyrrhizic acid contained in plants of the genus Glycyrrhiza of the legume family. β-Glycyrrhetinic acid is the aglycone of glycyrrhizic acid.

[0017] Ursolic acid Ursolic acid is a component contained in rosemary and apple peels. Resveratrol Resveratrol is a component contained in grapes, peanuts, mulberries, etc.

[0018] Shikonin Shikonin is a component contained in the purple root, which is the root of Lithospermum erythrorhizon. Biochanin A Biochanin A is a component contained in Trifolium pratense. Trifolium pratense is also called Trifolium incarnatum.

[0019] Isoliquiritigenin Isoliquiritigenin is a component contained in licorice. Ethinyl estradiol Ethinyl estradiol is a type of estrogen.

[0020] Daidzein Daidzein is a component contained in leguminous plants such as soybeans, peas, and kudzu. Allyl isothiocyanate Allyl isothiocyanate is a component contained in Japanese horseradish, mustard, etc.

[0021] Riboflavin Riboflavin is a type of vitamin and is also called vitamin B2. Specific surfactant Cetylpyridinium chloride (CPC), dodecylpyridinium chloride (DPC), sodium lauroyl sarcosinate (LSS), cocamidopropyl betaine, and alkyldiaminoethylglycine hydrochloride are surfactants.

[0022] <Other ingredients> Succinate receptor inhibitors and osteoclast formation inhibitors may contain other components besides those mentioned above, depending on the intended use, form, and application. Examples of other components include surfactants, flavoring agents, sweeteners, humectants, thickeners, preservatives, colorants, pH adjusters, chelating agents, pharmaceutically active ingredients, bases, stabilizers, and abrasives. Other components that are known to be incorporated into succinate receptor inhibitors and osteoclast formation inhibitors may be used. Succinate receptor inhibitors and osteoclast formation inhibitors may contain only one of the above-mentioned other components individually, or they may contain two or more in combination.

[0023] Examples of surfactants include nonionic surfactants, anionic surfactants, amphoteric surfactants, and cationic surfactants. Specific examples of nonionic surfactants include sugar fatty acid esters such as sucrose fatty acid esters, maltose fatty acid esters, and lactose fatty acid esters; fatty acid alkanolamides; glycerin fatty acid esters; sorbitan fatty acid esters; fatty acid monoglycerides; polyoxyethylene alkyl ethers with a polyoxyethylene addition coefficient of 8 to 10 and 13 to 15 carbon atoms in the alkyl group; polyoxyethylene alkylphenyl ethers with a polyoxyethylene addition coefficient of 10 to 18 and 9 carbon atoms in the alkyl group; diethyl sebacate; polyoxyethylene hydrogenated castor oil; and fatty acid polyoxyethylene sorbitan (also called polysorbate).

[0024] Specific examples of anionic surfactants include sulfate ester salts such as sodium lauryl sulfate and sodium polyoxyethylene lauryl ether sulfate; sulfosuccinates such as sodium lauryl sulfosuccinate and sodium polyoxyethylene lauryl ether sulfosuccinate; acyl amino acid salts such as sodium lauroyl methylalanine; and sodium cocoyl methyl taurate.

[0025] Specific examples of amphoteric surfactants include, for example, betaine-type surfactants such as lauryldimethylaminoacetic acid betaine and coconut oil fatty acid amidopropyldimethylaminoacetic acid betaine; imidazoline-type surfactants such as N-cocoyl-N-carboxymethyl-N-hydroxyethylethylenediamine sodium; and amino acid-type surfactants such as N-lauryldiaminoethylglycine.

[0026] Specific examples of cationic surfactants include distearyldimethylammonium chloride, stearyldimethylbenzylammonium chloride, and N-coconut oil fatty acid acyl-L-arginine ethyl DL-pyrrolidone carboxylate.

[0027] Specific examples of flavoring agents include menthol, carboxylic acids, anethole, eugenol, methyl salicylate, limonene, ocimene, n-decyl alcohol, citronellol, α-terpineol, methyl acetate, citronenyl acetate, methyl eugenol, cineole, thymol, spearmint oil, peppermint oil, lemon oil, orange oil, sage oil, rosemary oil, perilla oil, wintergreen oil, clove oil, eucalyptus oil, pimento oil, d-camphor, d-borneol, fennel oil, cinnamon oil, anise oil, cinnamaldehyde, peppermint oil, vanillin, and others.

[0028] Specific examples of sweeteners include sodium saccharin, potassium acesulfamethamate, stevioside, neohesperidyl dihydrochalcone, perillartin, thaumatin, aspartylphenylalanyl methyl ester, and p-methoxycinnamic aldehyde.

[0029] Specific examples of wetting agents include, for example, sorbitol, ethylene glycol, propylene glycol, glycerin, 1,3-butylene glycol, polypropylene glycol, xylitol, maltitol, lactitol, and polyoxyethylene glycol.

[0030] Specific examples of thickeners include, for example, cellulose derivatives such as sodium carboxymethylcellulose, carboxymethyl ethylcellulose salt, hydroxyethylcellulose, hydroxypropylcellulose, hydroxypropylmethylcellulose, methylcellulose, ethylcellulose, crystalline cellulose, and crystalline cellulose-carmellose sodium; microbially produced polymers such as xanthan gum; natural polymers or natural rubbers such as tragacanth gum, karaya gum, arabic gum, guar gum, carrageenan, dextrin, agar, pectin, pullulan, gellan gum, locust bean gum, and sodium alginate; synthetic polymers such as polyvinyl alcohol, polyvinylpyrrolidone, carboxyvinyl polymer, polyvinyl methyl ether, and sodium polyacrylate; inorganic thickeners such as thickening silica and bee gum; and cationic thickeners such as O-[2-hydroxy-3-(trimethylammonio)propyl]hydroxyethylcellulose chloride.

[0031] Specific examples of preservatives include parabens such as methylparaben, ethylparaben, propylparaben, and butylparaben, as well as sodium benzoate, phenoxyethanol, and alkyldiaminoethylglycine hydrochloride.

[0032] Specific examples of colorants include legally approved pigments such as Blue No. 1, Yellow No. 4, Red No. 202, and Green No. 3, mineral pigments such as ultramarine, enhanced ultramarine, and Prussian blue, and titanium dioxide. Specific examples of pH adjusters include, for example, citric acid, phosphoric acid, lactic acid, tartaric acid, glycerophosphate, acetic acid, nitric acid, or chemically possible salts thereof, or sodium hydroxide.

[0033] Specific examples of chelating agents include, for example, EDTA, sodium EDTA, potassium EDTA, phytic acid, pyrophosphate, polyphosphate, and malic acid. Specific examples of active ingredients include, for example, vitamin E derivatives such as dl-α-tocopherol acetate, tocopherol succinate, or tocopherol nicotinate; vitamin C derivatives such as ascorbic acid, sodium ascorbate, or magnesium ascorbyl phosphate; vitamin B6 derivatives such as pyridoxine hydrochloride; amphoteric disinfectants such as glycyrrhizinate and its derivatives; nonionic disinfectants such as triclosan, isopropylmethylphenol, and polyoxyethylene lauryl ether; anionic disinfectants such as sodium cocoyl sarcosinate and sorbic acid; chlorhexidine hydrochloride, chlorhexidine gluconate, and benzalkonium chloride. Examples include cationic disinfectants such as ammonium and benzethonium chloride, enzymes such as dextranase, amylase, protease, mutanase, lysozyme, and lytic enzymes (Litec enzyme), alkali metal monofluorophosphates such as sodium monofluorophosphate and potassium monofluorophosphate, fluorides such as sodium fluoride and stannous fluoride, tranexamic acid and epsilon-aminocaproic acid, aluminum chlorohydroxyl allantoin, dihydrocholesterol, hinokitiol, sodium copper chlorophyllin, chlorophyll, sodium chloride, caropeptide, allantoin, carbazochrome, potassium nitrate, and palatinite.

[0034] Specific examples of bases include alcohols, silicones, apatite, white petrolatum, paraffin, liquid paraffin, microcrystalline wax, squalane, and Plastibase.

[0035] Specific examples of alcohols include ethyl alcohol, lauryl alcohol, and myristyl alcohol. Specific examples of stabilizers include sodium thiosulfate, sodium sulfite, calcium lactate, lanolin, triacetin, castor oil, and magnesium sulfate.

[0036] Specific examples of abrasives include calcium carbonate, magnesium carbonate, dicalcium phosphate, tricalcium phosphate, magnesium phosphate, silica, zeolite, sodium metaphosphate, aluminum hydroxide, magnesium hydroxide, calcium pyrophosphate, red iron oxide, calcium sulfate, and anhydrous silicic acid.

[0037] <Application form, dosage form, and use> The applications of succinate receptor inhibitors and osteoclast formation inhibitors are not particularly limited and can be used, for example, as pharmaceuticals, medical devices, quasi-drugs, and cosmetics. Examples of medical devices include dental periodontal protective materials.

[0038] The dosage form of succinate receptor inhibitors and osteoclast formation inhibitors is not particularly limited and can be applied to ointments, pastes, pasta preparations, sprays, gels, liquids, suspensions, gums, sheets, etc., by including a solvent such as water or alcohol.

[0039] The type of water used as a solvent is not particularly limited; for example, distilled water, pure water, ultrapure water, purified water, tap water, etc., can be used. The type of alcohol used as a solvent is not particularly limited; for example, ethanol can be used. A mixture of water and alcohol can also be used.

[0040] The applications of succinate receptor inhibitors and osteoclast formation inhibitors can be those of known use as appropriate. Specifically, examples include masticatory agents, orally dissolving agents, orally disintegrating agents, tongue care agents, mouth fresheners, toothpastes, mouthwashes, rinses, liquid toothpastes, oral ointments, biofilm dispersants, bad breath preventatives, hypersensitivity inhibitors, gum massage agents, oral moisturizing agents, tongue coating removers, oral application agents, oral disinfectants, throat disinfectants, oral and throat agents, periodontal disease treatment agents, denture attachment agents, denture coating agents, denture stabilizers, denture preservatives, denture cleaners, implant care agents, etc.

[0041] <Operation and Effects of This Embodiment> The operation of this embodiment will now be described. Succinate receptor inhibitors inhibit the activation of succinate receptors. Furthermore, by inhibiting succinate receptor activation, the differentiation of osteoclast precursor cells into osteoclasts can be suppressed. This, in turn, inhibits osteoclast formation.

[0042] The effects of this embodiment will now be explained. (1) The succinate receptor inhibitor contains at least one selected from the group consisting of β-glycyrrhetinic acid, ursolic acid, resveratrol, shikonin, biochanin A, isoliquitigenin, ethinylestradiol, daidzein, allyl isothiocyanate, riboflavin, cetylpyridinium chloride, dodecylpyridinium chloride, sodium lauroyl sarcosinate, cocamidopropyl betaine, and alkyldiaminoethylglycine hydrochloride.

[0043] The succinate receptor inhibitor of this embodiment can be expected to suppress the production of inflammatory cytokines, inhibit the formation of osteoclasts, and suppress alveolar bone resorption by inhibiting the activation of succinate receptors. The succinate receptor inhibitor of this embodiment is useful for preventing periodontal disease and inhibiting its progression.

[0044] (2) The osteoclast formation inhibitor contains at least one selected from the group consisting of β-glycyrrhetinic acid, ursolic acid, resveratrol, shikonin, biochanin A, isoliquitigenin, ethinylestradiol, daidzein, allyl isothiocyanate, and riboflavin.

[0045] The osteoclast formation inhibitor of this embodiment can suppress osteoclast formation by inhibiting the differentiation of osteoclast precursor cells into osteoclasts. This is expected to have the effect of suppressing alveolar bone resorption. The osteoclast formation inhibitor of this embodiment is useful for the prevention of periodontal disease and the suppression of its progression.

[0046] <Example of changes> Incidentally, the above embodiment can be implemented with the following modifications. The present embodiment and the following modification examples can be implemented in combination with each other within a technically consistent range.

[0047] · It may be applied to pets other than humans, domestic animals such as livestock, etc.

Example

[0048] The succinate receptor inhibitor and osteoclast formation inhibitor will be further described in more detail based on the following examples. Note that the succinate receptor inhibitor and osteoclast formation inhibitor are not limited to the configurations described in the example section.

[0049] [Succinate Receptor Inhibition Evaluation] <Culture of SUCNR1-Expressing Cells> Ready-to-Assay SUCNR1 / GPR91 Succinate Receptor Frozen Cells (Eurofins) were inoculated into the attached Media Component to obtain a cell suspension adjusted to 5.0×10 4 cells / mL. 100 μL of the cell suspension was transferred to a 96 well black / clear bottom plate and cultured at 37°C under 5% CO2 for 1 day.

[0050] <Preparation of Stock Solutions> Stock solutions of Examples 1 to 15 and Reference Example 1 were prepared respectively. The types of materials contained in each stock solution are as shown in Table 1. Commercially available products were used for each material other than Reference Example 1.

[0051] Antagonist 7a in Reference Example 1 is a compound represented by the following formula. Antagonist 7a is a well-known compound having an effect of inhibiting the succinate receptor (SUCNR1). Antagonist 7a was purchased from MedChemExpress.

[0052]

Chemical Formula

[0053] Each material solution was prepared by using 4.9% DMSO-containing HHBS as the solvent, adjusting the concentration of each material to 30 μM.

[0054] [Table 1]

[0055] <Succinate receptor inhibition evaluation test> To the cultured cells described above, 100 μL of a fluorescent dye solution prepared according to the manual for the Screen Quest Fluo-8 No Wash Calcium Assay Kit (AAT Bioquest) was added, and the cells were cultured at 37°C and 5% CO2 for 30 minutes. 50 μL of each material solution was added to the cells, and the cells were cultured at room temperature for 30 minutes. 50 μL of succinic acid solution, prepared to 3 mM using HHBS, was added to the cells, and the fluorescence intensity at excitation light: 490 nm and emitted light: 520 nm was immediately measured over time. The receptor activation effect of succinic acid was calculated based on the following formula (Equation 1) using the fluorescence intensity immediately after addition of the succinic acid solution (Basal) and the maximum fluorescence intensity during measurement (Peak). As a control sample, the fluorescence intensity was measured in the same manner as above using a solvent without added material instead of the material solution, and the receptor activation effect was calculated. Using the receptor activation effect values ​​of the control sample, the succinic acid receptor inhibitory effect was calculated based on the following formula (Equation 2).

[0056] Receptor activation effect = [(Peak-Basal) / Basal] × 1000 ... (Equation 1) Succinate receptor inhibitory effect (%) = [{(receptor activation effect of control sample) - (receptor activation effect of each example or comparative example)} / (receptor activation effect of control sample)] × 100 ... (Equation 2) The calculated succinate receptor inhibitory effect was evaluated based on the following criteria. The results are shown in Table 1.

[0057] • Criteria for evaluating succinate receptor inhibitory effects A (Excellent): Succinate receptor inhibitory effect is 50% or more. B (Good): Succinate receptor inhibitory effect is between 30% and 50%. D (Not acceptable): Succinate receptor inhibitory effect is less than 30%. <Result> When succinate receptors are activated, intracellular calcium concentration increases. According to this study, the inhibitory effect of succinate receptors can be evaluated using calcium indicators.

[0058] As shown in Table 1, all of Examples 1-15 were confirmed to be able to suppress succinate receptor activation. In particular, excellent succinate receptor inhibitory effects were confirmed in Examples 1-7, 9, and 11-15. The succinate receptor inhibitory effects in Examples 1-7, 9, and 11-15 were equivalent to those of Reference Example 1.

[0059] [Evaluation of Osteoclast Formation Inhibition] <Preparation of the material solution> Material solutions for Examples 16-25 and Reference Example 2 were prepared. The types of materials contained in each material solution are shown in Table 2. Except for Reference Example 2, commercially available materials were used. Antagonist 7a in Reference Example 2 was obtained using the method described above.

[0060] Each material solution was prepared by using αMEM containing 10% FBS and 4.9% DMSO as the solvent, adjusting the concentration of the material to the concentrations shown in Table 2.

[0061] [Table 2]

[0062] <Culture test of osteoclast progenitor cells> Soluble RANK ligand (sRANKL) was dissolved in αMEM containing 10% FBS to prepare a RANKL solution with a concentration of 50 ng / mL.

[0063] RAW264.7 cells were inoculated into 10% FBS-containing αMEM medium in which RANKL solution and a predetermined concentration of material solution were dissolved, and 2.5 × 10⁶ cells were collected. 4Cell suspensions were prepared at a concentration of cells / mL for each material solution. 200 μL of each cell suspension was transferred to a 96-well plate. After incubation at 37°C and 5% CO2 for 3 days, the medium was changed and the cells were incubated for a further 2 days. As a control, a solvent without added material was used instead of the material solution. As a negative control, only the solvent was used instead of the material solution and RANKL solution.

[0064] <Evaluation Method> The TRACP activity of cultured cells was evaluated using the TRACP & ALP Assay Kit (TAKARA) according to the manual. The TRACP activity of the control was set to 100%, and the TRACP activity of the negative control was set to 0%, and the TRACP activity after each material solution treatment was calculated. The TRACP activity after each material solution treatment was evaluated based on the following criteria. The results are shown in Table 2.

[0065] A (Excellent): The decrease in TRACP activity compared to the control is 30% or more. D (Not acceptable): The decrease in TRACP activity compared to the control is less than 30%. <Result> Tartrate-resistant acid phosphatase (TRACP) is used as a marker for osteoclasts. By calculating the decrease in TRACP activity compared to the control TRACP activity during treatment with each material solution, the effect of suppressing the differentiation of osteoclast precursor cells into osteoclasts can be evaluated.

[0066] As shown in Table 2, in all of Examples 16-25, the decrease in TRACP activity compared to the control was 30% or more. This confirms that, similar to Reference Example 2, Examples 16-25 can suppress differentiation into osteoclasts. In other words, it was confirmed that they can suppress osteoclast formation.

Claims

1. A succinate receptor inhibitor containing at least one selected from the group consisting of β-glycyrrhetinic acid, ursolic acid, resveratrol, shikonin, biochanin A, isoliquitigenin, ethinylestradiol, daidzein, allyl isothiocyanate, riboflavin, cetylpyridinium chloride, dodecylpyridinium chloride, sodium lauroyl sarcosinate, cocamidopropyl betaine, and alkyldiaminoethylglycine hydrochloride.

2. An osteoclast inhibitor containing at least one selected from the group consisting of β-glycyrrhetinic acid, ursolic acid, resveratrol, shikonin, biochanin A, isoliquitigenin, ethinylestradiol, daidzein, allyl isothiocyanate, and riboflavin.

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