JP6917040B2 - Osteoclast formation or activation inhibitor - Google Patents

Description

The present invention relates to an agent that inhibits the formation or activation of osteoclasts. Specifically, it relates to the field of prevention or treatment of periodontal disease.

Periodontal disease is an inflammatory disease caused by periodontopathic bacteria, and is a disease in which the bone tissue (alveolar bone) that supports the teeth is destroyed as the inflammation increases and becomes chronic. The number of patients with periodontal disease in Japan is estimated to exceed 20 million. In recent years, the importance of oral care for improving quality of life has been attracting attention, but it is expected that it will become even more important with the advent of a super-aging society.

Conventionally, bactericidal agents and antibacterial agents for the purpose of sterilizing and removing periodontal pathogenic bacteria, which are the main causes of periodontal disease, have been used for prevention and improvement of periodontal disease, and prevention of inflammation of periodontal tissue. , Plaque suppression with anti-plaque agents and anti-inflammatory agents are used for treatment. It is known that by sterilizing and removing periodontal pathogenic bacteria and maintaining an oral environment with plaque removed, inflammation is improved in the case of mild periodontal disease and the periodontal tissue returns to a healthy state. Has been done. Surgical treatment methods for periodontal disease; drug treatment methods with antibiotics such as perioclin and fungizone; various bone diseases such as osteoporosis, fractures, and periodontal disease consisting of thiazole compounds or pharmaceutically acceptable salts thereof. -Therapeutic agent for disorders (Patent Document 1); Periodontal disease therapeutic agent containing an anti-CD14 antibody as an active ingredient (Patent Document 2); Includes an antibody against vascular endothelial cell proliferation factor / vascular permeability factor (VEGF / VPF). A therapeutic agent for periodontal disease (Patent Document 3) and the like, which are characterized by being composed of a preparation, are known.

On the other hand, regarding bone resorption, research is being carried out from the problem of osteoporosis, and agents having an effect of promoting bone formation and agents having an effect of suppressing bone resorption have been found and used in pharmaceuticals and food ingredients. Various physiological actions have been reported for amino acids, and for example, a method using acyl amino acids for bone growth, bone mass increase, and prevention of osteoporosis is known (Patent Document 4). It is known that oleoylserine, which is an endogenous N-long-chain acyl amino acid, inhibits bone resorption and has bone remodeling and bone mass regulating effects (Non-Patent Document 1). Acyl amino acids are also known to be used for the prevention and amelioration of periodontal disease. For example, dentifrices containing N-long-chain acylamino acids, Lactobacillus bacteria in which N-long-chain acylamino acids are the main cause of dental caries, and Staphylococcus, which is the main cause of periodontal disease. It is known to have a remarkable antibacterial action against Staphylococcus spp. (Patent Document 5). A dentifrice containing an N-long-chain acyl amino acid salt and a water-soluble secondary phosphate, which is an amino acid salt having a saturated or unsaturated acyl group having 8 to 22 carbon atoms and a water-soluble secondary phosphorus. It is known that the effect of preventing the adhesion of dentin when used in combination with a phosphate is known (Patent Document 6).

Japanese Unexamined Patent Publication No. 11-209284 Japanese Unexamined Patent Publication No. 10-114679 Japanese Unexamined Patent Publication No. 2002-097157 International Publication No. 2009/125409 Tokukousho No. 45-24480 Japanese Unexamined Patent Publication No. 50-53545

Proc. Natl. Acad. Sci. USA, Vol.107, No.41, 17710-17715 (2010)

However, as periodontal disease progresses to a level that causes alveolar bone resorption, the chances of improvement diminish, much less if teeth are lost as a result of alveolar bone resorption. Periodontal tissue and tooth recovery cannot be expected. Resorption of alveolar bone by periodontal disease progresses locally and at a faster rate than osteoporosis because it is promoted by periodontopathic bacteria. Therefore, there is a demand for an alveolar bone resorption inhibitor that is effective for alveolar bone resorption due to periodontal disease, not an agent that is effective for osteoporosis.
An object of the present invention is to provide an osteoclast formation or activation inhibitor useful as an alveolar bone resorption inhibitor, and an oral composition effective for preventing or treating periodontal disease. be.

Few studies have been conducted on amino acids related to bone resorption, and their effects on oral inflammation are unknown. Therefore, the present inventors focused on amino acids for which various physiological actions have been reported, and earnestly focused on the effects of amino acids and related factors (amino acids) on the regulation of bone metabolism as the main cause of the maintenance action of the oral environment by amino acids. As a result of the examination, it was found that a specific N-acyl branched chain amino acid suppresses the formation or activation of osteoclasts, and the present invention has been completed.

That is, the present invention is as follows.
[1] An osteoclast formation or activation inhibitor containing an N-acyl branched chain amino acid or a salt thereof.
[2] The inhibitor according to [1], wherein the acyl group has 8 to 16 carbon atoms.
[3] The inhibitor according to [1] or [2], wherein the N-acyl branched chain amino acid is N-acylvaline.
[4] The inhibitor according to [3], wherein the N-acylvaline is N-decanoylvaline or N-lauroylvaline.
[5] The inhibitor according to any one of [1] to [4], which is an alveolar bone resorption inhibitor.
[6] An oral composition for preventing or treating periodontal disease, which comprises an N-acyl branched chain amino acid or a salt thereof.
[7] The composition according to [6], wherein the acyl group has 8 to 16 carbon atoms.
[8] The composition according to [6] or [7], wherein the N-acyl branched chain amino acid is N-acylvaline.
[9] The composition according to [8], wherein the N-acylvaline is N-decanoylvaline or N-lauroylvaline.
[10] The composition according to any one of [6] to [9], which is a dentifrice, a mouthwash, an oral ointment, an oral gel, an oral refreshing agent, an oral pasta or a mouthwash. ..

The osteoclast formation or activation inhibitor of the present invention is a bone in alveolar bone, in particular, because the active ingredient N-acyl branched chain amino acid or a salt thereof suppresses the differentiation of progenitor cells into osteoclasts. It has the effect of significantly reducing absorption activity.
Therefore, the osteoclast formation or activation inhibitor of the present invention is particularly useful as an alveolar bone resorption inhibitor.
Further, in the oral composition for prevention or treatment of periodontal disease of the present invention, the active ingredient N-acyl branched chain amino acid or a salt thereof significantly reduces the bone resorption activity in the alveolar bone and the surface active action. By combining with, it is excellent as an oral care product for the purpose of prevention or treatment of periodontal disease.

In Example 1, it is a graph which shows the influence of valine and N-acylvaline on the differentiation of osteoclasts in the co-culture system of mouse bone marrow cells and osteoblasts. The upper part of the graph shows the result of TRAP staining. In Example 2, it is a graph which shows the influence of valine and N-acylvaline on the bone resorption activity in a mouse parietal bone organ culture system. In Example 3, it is a graph which shows the influence of valine and N-acylvaline on the bone resorption activity in a mouse alveolar bone organ culture system. In Example 4, the result of TRAP staining in the co-culture system of mouse bone marrow cells and osteoblasts is shown. In the figure, C12 indicates C12-Val, and the number in parentheses indicates the concentration (μM). In Example 4, it is a graph which shows the inhibitory effect of N-acylamino acid on the differentiation of osteoclast in the co-culture system of mouse bone marrow cell and osteoblast.

(Inhibitor of osteoclast formation or activation)
The osteoclast formation or activation inhibitor of the present invention (hereinafter, also referred to as “the agent of the present invention” in the present specification) contains an N-acyl branched chain amino acid or a salt thereof as an active ingredient.

In the present invention, osteoclasts are cells that play a role in destroying bone (bone resorption) in the process of bone remodeling, and are several (for example, five) to several tens (for example, 20) or more. It is a multinucleated giant cell with a nucleus of. The cytoplasm of osteoclasts is acidophilic and has tartrate-resistant acid phosphatase activity. Whether or not the cells are osteoclasts can be confirmed by measuring tartrate-resistant acid phosphatase (TRAP) activity. Methods for measuring TRAP activity are described in the Examples.

In the present invention, suppression of osteoclast formation or activation means suppressing an increase in the number of differentiated osteoclasts, suppressing differentiation of progenitor cells into osteoclasts, or differentiating osteoclasts. It means to suppress the activation (increase in bone resorption activity). Here, examples of progenitor cells include bone marrow-derived monocytes, macrophages, and the like.

The N-acyl branched chain amino acid contained as an active ingredient in the agent of the present invention is one in which the amino group of the branched chain amino acid is replaced with an acyl group. Here, examples of the branched chain amino acid include valine, leucine and isoleucine, and valine is preferable. The branched chain amino acid may be any of L-form, D-form and DL-form, but is preferably L-form and DL-form, and more preferably L-form.

The acyl group is preferably a saturated or unsaturated acyl group having 8 to 16 carbon atoms. Specific examples thereof include an octanoyl group, a nonanoyl group, a decanoyyl group, a lauroyl group, a myristoyl group, a palmitoyl group, a myristolail group, and a palmitrail group. More preferably, it is a saturated or unsaturated acyl group having 10 to 14 carbon atoms, and specific examples thereof include a decanoyyl group, a lauroyl group, a myristyl group, and a myristolail group. More preferably, it is a saturated acyl group having 10 to 12 carbon atoms, and specific examples thereof include a decanoyl group and a lauroyl group.

As the N-acyl branched chain amino acid, only one type may be used, or two or more types may be used in combination. From the viewpoint of suppressing the formation or activation of osteoclasts, N-acylvaline is preferable, and N-decanoylvaline or N-lauroylvaline is more preferable.

Further, the N-acyl branched chain amino acid can be used not only in the free form but also in the form of a salt. Examples of the salt form include alkali metal salts, alkaline earth metal salts, ammonium salts, lower alcohol amine salts and the like, and it is preferable to select a pharmacologically acceptable salt.
Examples of the pharmacologically acceptable salt include alkali metal salts such as sodium salt and potassium salt, alkaline earth metal salts such as calcium salt, and lower alcohol amine salts such as ammonium salt and triethanolamine salt.

The N-acyl branched chain amino acid or a salt thereof can be produced by a known method. Known methods include, for example, the Schotten-Baumann reaction of amino acids with fatty acid halides. Further, as the N-acyl branched chain amino acid or a salt thereof, a commercially available product can also be used.

The agent of the present invention may suppress the formation or activation of osteoclasts in any bone, but it is the alveolar bone that suppresses the formation or activation of osteoclasts in the alveolar bone. It is preferable because it is useful as an absorption inhibitor of.

The content of the N-acyl branched chain amino acid or a salt thereof contained in the agent of the present invention is not particularly limited as long as it exerts an effect of suppressing the formation or activation of osteoclasts, but is usually 0.001 weight by weight. % To 100% by weight, preferably 0.01% by weight to 99% by weight.

The agent of the present invention may be in any form of liquid, semi-solid or solid, and may be a liquid agent, a suspension agent, an emulsion, a cream agent, a gel agent, an ointment agent, a plaster (pasta) agent, or a plaster agent. , Powders, granules, tablets and the like, and can be provided in various dosage forms.

The agent of the present invention may contain any carrier or additive in addition to the N-acyl branched chain amino acid or a salt thereof. The carrier or additive is not particularly limited as long as it does not inhibit the action of the N-acyl branched chain amino acid or a salt thereof that suppresses the formation or activation of osteoclasts, and the agent of the agent of the present invention is not particularly limited. Depending on the form, it can be selected without limitation from any carrier or additive used in the oral composition for preventing or treating periodontal disease of the present invention described later.

The agents of the present invention in various dosage forms can be prepared according to general formulation means, for example, the production method described in each article of the 17th revised Japanese Pharmacopoeia General Regulations for Formulation [3] Formulation.

The application target of the agent of the present invention includes mammals that may suffer from periodontal disease, for example, humans, monkeys, mice, rats, hamsters, rabbits, cats, dogs, cows, pigs, sheep, horses and the like. Can be mentioned.
The ingestion or dose of the agent of the present invention is appropriately determined depending on the application target, age, symptoms and the like. The daily ingestion or dose of the agent of the present invention may be ingested or administered at one time, or may be ingested or administered in several divided doses.

The agent of the present invention can suppress the differentiation of progenitor cells into osteoclasts, and in particular, can satisfactorily reduce bone resorption activity in alveolar bone.
Therefore, the agent of the present invention is useful as an alveolar bone resorption inhibitor.

(Oral composition for prevention or treatment of periodontal disease)
The present invention also refers to an oral composition for the prevention or treatment of periodontal disease (hereinafter, also referred to as "the composition of the present invention" in the present specification) containing an N-acyl branched chain amino acid or a salt thereof as an active ingredient. offer.

In the composition of the present invention, the N-acyl branched chain amino acid or a salt thereof contained as an active ingredient is the same as that contained in the above-mentioned agent of the present invention.

The composition of the present invention is applied orally for the purpose of preventing or treating periodontal disease. Here, the treatment of periodontal disease is a concept that includes not only treatment but also improvement of periodontal disease and at least suppression of progression. For this purpose, the compositions of the present invention include, for example, oral solutions, oral gels, oral ointments, oral pasta, oral creams, oral tablets (chewing tablets, troches, sublingual). Tablets, buccal tablets, adhesive tablets, gums), periodontal pocket inserts, oral patches, mouthwashes, oral sprays, various dentifrices, mouthwashes, mouth refreshers (mouthwash), artificial teeth stability It can be prepared and applied in various dosage forms such as liquid, milky, creamy, gelled, pasty and solid. Preferred dosage forms of the compositions of the present invention are dentifrices, mouthwashes, oral ointments, oral gels, oral refreshers, oral pastas or mouthwashes.

The content of the N-acyl branched chain amino acid or a salt thereof in the composition of the present invention may be appropriately set according to the pathophysiology of periodontal disease, the degree of achievement target of prevention or treatment, the dosage form of the composition, and the like. can. It is usually 0.01% by weight to 50% by weight, preferably 0.05% by weight to 20% by weight, and more preferably 0.1% by weight to 10% by weight.

The composition of the present invention may contain various medicinal ingredients and any carrier or additive in addition to the active ingredient N-acyl branched chain amino acid or a salt thereof. The medicinal ingredient, carrier or additive is not particularly limited as long as it does not inhibit the action of the N-acyl branched chain amino acid or a salt thereof, and can be appropriately selected depending on the dosage form of the composition.

Examples of various medicinal ingredients include antibacterial agents such as chlorhexidine, benzethonium chloride, triclosan, cetylpyridinium chloride and hinokithiol; fluorine compounds such as sodium fluoride, stannous fluoride and sodium monofluorophosphate; dextranase and mutanase. , Anti-plaque formation inhibitors such as proteases; Hemostatic agents such as tranexamic acid and ε-aminocaproic acid; Anti-inflammatory agents such as azulene, allantin, lysozyme chloride, glycyrrhizic acids and glycyrrhizic acids; Toothstone preventive agents such as polyphosphates; Sodium chloride And other gingival astringents; enzymes such as dextranase and amylase; plant extracts such as plaque, plaque, chow, ginger, and benibana; various vitamins such as tocopherol acetate; amino acids such as glycine, proline, glutamic acid, and arginine. Be done.

Carriers or additives include, for example, solvents, bases, foaming agents, abrasives, wetting agents, surfactants, sweeteners, acidulants, fragrances, stabilizers, preservatives, pH regulators, thickeners, etc. Examples thereof include ingredients that can be incorporated into oral products of foods or pharmaceuticals, such as colorants, pigments, organic powders, and excipients.

Examples of the solvent include lower alcohols such as ethanol, propanol, isoptanol, butanol and isobutanol; purified water; physiological saline; Ringer's solution and the like.

As a base, vegetable fats and oils such as olive oil, cacao butter, and castor oil; hydrocarbons such as white vaseline, liquid paraffin, and microcrystalin wax; waxes such as carnauba wax and salasimitsu wax; myristyl alcohol, cetanol, stearyl alcohol, and cetostearyl. Fatty alcohols such as alcohols; fatty acids such as myristic acid and stearic acid; polysaccharides such as carrageenan and xanthan gum; cellulose derivatives such as sodium carboxymethyl cellulose and hydroxypropyl cellulose; polyoxyethylene glycol 1500, polyoxyethylene glycol 4000, polyoxy Examples thereof include polyoxyethylene glycol such as ethylene glycol 6000.

Examples of the foaming agent include sodium lauryl sulfate, sodium lauroyl sarcosine and the like. In addition, N-acyl branched chain amino acid or a salt thereof, which is an active ingredient of the composition of the present invention, and a surfactant described later can also act as a foaming agent.

Examples of the polishing agent include dihydrate calcium dihydrate and anhydride, calcium carbonate, calcium pyrophosphate, insoluble sodium metaphosphate, silicic anhydride, hydrous silicic acid, aluminosilicate, alumina, aluminum hydroxide and the like.

Wetting agents include polyhydric alcohols such as ethylene glycol, diethylene glycol, propylene glycol, dipropylene glycol, 1,3-butylene glycol, glycerin, 1,5-pentanediol, sorbit, polyethylene glycol 300; di-lauroyl glutamate (cholesteryl /). Amino acid esters such as behenyl / octyldodecyl) and di lauroyl glutamate (cholesteryl / octyldodecyl); amino acid polymers such as sodium polyaspartate; pyrrolidonecarboxylic acids and salts thereof and the like.

Examples of the surfactant include sorbitan fatty acid ester, glycerin fatty acid ester, polyglycerin fatty acid ester (decaglycerin fatty acid ester, etc.), propylene glycol / pentaeristol fatty acid ester, polyoxyethylene sorbitan fatty acid ester, polyoxyethylene glycerin fatty acid ester, and poly. Oxyethylene sorbit fatty acid ester, polyethylene glycol fatty acid ester, polyoxyethylene alkyl ether, polyoxyethylene polyoxypropylene alkyl ether, polyoxyethylene alkyl phenyl ether, polyoxyethylene castor oil / hardened castor oil, polyoxyethylene lanolin / lanolin alcohol -Nonionic surfactants such as beeswax derivatives, polyoxyethylene alkylamine fatty acid amides, polyoxyethylene alkylphenylformaldehyde condensates, single chain length polyoxyethylene alkyl ethers; alkyl sulfates, polyoxyethylene alkyl sulfates , N-acylamino acid and its salts, N-acylmethyl taurine salt, polyoxyethylene alkyl ether acetate, alkyl sulfocarboxylate, α-olefin sulfonate, alkyl phosphate, polyoxyethylene alkyl ether phosphate Anionic surfactants such as: alkylammonium salts, alkylbenzylammonium salts, cationic surfactants such as fatty acid acylarginine esters; amphoteric surfactants such as betaine acetate, imidazolinium betaine, and lecithin.

Sweeteners include sodium saccharin, stebioside, neohesperidyldihydrochalcone, glycyrrhizin, periraltin, p-methoxycinnamic aldehyde, aspartame, acesulfame potassium, sucralose, erythritol, xylitol, sorbitol, maltitol, thaumatin, glucose, fructose, sucrose. , Lactose, maltitol, etc.

Acidulants include adipic acid, ascorbic acid, aspartic acid, glutamate, citric acid, gluconodeltalactone, gluconic acid, succinic acid, acetic acid, tartaric acid, lactic acid, butyric acid, glacial acetic acid, fumaric acid, maleic acid, malic acid, etc. Examples thereof include organic acids such as citrate, malate, lactate, succinate, tartrate, and ascorbate.

Fragrances include essential oils such as peppermint oil and spearmint oil, l-menthol, carboxylic, eugenol, anetol, citrus flavors such as orange, lemon and grapefruit that can be added as food additives, apples, bananas, grapes, etc. Fruit flavors such as peach, strawberry and pineapple, spice flavors such as pepper, cinnamon, nutmeg and clove, nut flavors such as vanilla, coffee, cocoa and hazelnut, tea flavors such as tea and green tea, linarol, citronellol, etc. Various floral fragrances can be mentioned.

Stabilizers include chelating agents such as ethylenediamine tetraacetate disodium and ethylenediamine tetraacetate disodium; vitamin C, vitamin E, sodium sulfite, sodium pyrosulfite, sodium hydrogen sulfite, butylhydroxytoluene, propyl gallate, butylhydroxy. Antioxidants such as anisole; dispersants such as sodium alginate and polyvinylpyrrolidone; inclusion agents such as β-cyclodextrin and the like can be mentioned.

Examples of the preservative include paraoxybenzoic acid esters such as butyl parapen and ethyl parapen, sodium benzoate and the like.

Examples of the pH adjuster include hydrochloric acid, sodium hydroxide, sodium carbonate, citrate, sodium citrate, glycine, gluconic acid, phosphoric acid, sodium hydrogen phosphate, sodium dihydrogen phosphate, succinic acid, acetic acid, sodium acetate and the like. Can be mentioned.

Examples of the thickener include thickeners such as glycerin, sorbitol, propylene glycol and polyethylene glycol; and binders such as carboxymethyl cellulose, carrageenan, sodium alginate, gum arabic, bee gum, hydroxyethyl cellulose and polyvinyl alcohol.

Examples of the colorant include tar pigments such as edible blue No. 1, edible yellow No. 4, and edible red No. 2, sodium copper chlorophyllin, and the like.

Examples of the pigment include zinc oxide and titanium oxide.

Examples of the organic powder include lauroyl lysine and undecinoyl glycine.

Examples of the excipient include hydroxyapatite, crystalline cellulose, corn starch, talc, lactose and the like.

The blending amount of the above-mentioned medicinal ingredient, carrier or additive can be an amount usually used in oral compositions of various dosage forms as long as the effect of the present invention is not impaired.

The compositions of the present invention in various dosage forms can be prepared according to ordinary formulation means, for example, the production method described in each article of the 17th revised Japanese Pharmacopoeia General Regulations for Preparation [3] Preparation.
For example, in a paste-like composition such as a dentifrice, an N-acyl branched chain amino acid or a salt thereof, which is an active ingredient, is mixed with a carrier or an additive such as an excipient or a base, and water is added and kneaded. Can be prepared.
Further, in the liquid composition such as a mouthwash, a mouthwash, a mouthwash, etc., the active ingredient N-acyl branched chain amino acid or a salt thereof is appropriately added together with various additives such as water and ethanol. It can be prepared by dissolving it in a solvent, emulsifying or suspending it, and filtering it if necessary.
In the oral gel, the active ingredient N-acyl branched chain amino acid or a salt thereof is added to a solvent such as water together with an additive such as a thickener, mixed, heated to dissolve, and then cooled. Can be prepared.
Oral ointments and oral pasta are prepared by heating and melting an oily base, adding the active ingredient N-acyl branched chain amino acid or a salt thereof, mixing and dissolving or dispersing until homogenous. It can be prepared by kneading or by heating and melting a water-soluble base, adding the active ingredient N-acyl branched chain amino acid or a salt thereof, and kneading until homogeneous.

Applicable objects of the composition of the present invention include mammals (for example, humans, monkeys, mice, rats, hamsters, rabbits, cats, dogs, cows, pigs, sheep, horses, etc.).

The amount and number of applications of the composition of the present invention may be appropriately set according to the type, age, severity of symptoms, degree of achievement target of prevention or treatment, dosage form of the composition, and the like. can.
In the case of a human adult, the composition of the present invention can be applied orally about 1 to 10 times a day, preferably 1 to 5 times a day.

The composition of the present invention is excellent in the effect of preventing or treating periodontal disease by the above-mentioned action of reducing the bone resorption activity in alveolar bone of the N-acyl branched chain amino acid or a salt thereof contained as an active ingredient.
It is also excellent as an oral care product due to the surface-active action inherent in N-acyl branched chain amino acids or salts thereof.

Hereinafter, the present invention will be described in more detail with reference to Examples, but the scope of implementation of the present invention is not limited to these Examples.

Val, N-octanoyl valine (C8-Val), N-decanoyl valine (C10-Val) to clarify the effect of acyl group addition on valine (Val) on osteoclast formation, differentiation, etc. And N-lauroylvalin (C12-Val) were used to compare the effects of osteoclast differentiation in bone marrow cell and osteoblast coexistence culture system and bone resorption activity in parietal bone organ culture system. Furthermore, alveolar bone organ culture, which is an ex vivo evaluation system for periodontal disease, was carried out, and the effects of Vals on inflammatory bone resorption activity assuming periodontal disease were compared and examined.
Moreover, using N-oleoylserine (O-Ser) as a comparative example, the action on osteoclast formation and differentiation in the bone marrow cell and osteoblast coexistence culture system was compared with the action of N-acylvarin.

(reagent)
Amino acids (Val and Ser) used were manufactured by Tokyo Chemical Industry Co., Ltd. As the N-acyl amino acid, one synthesized as shown in the reference example below was used.
The reagents and media used were purchased from Funakoshi Co., Ltd., Sigma-Aldrich Co., Ltd. and Cosmo Bio Co., Ltd.
The mouse was purchased from Nippon SLC Co., Ltd. and used.

[Reference Example 1] Synthesis of C10-V Valine (29.29 g, 250.02 mmol), 78.6 g of water, and 36.3 g of acetone were added to a 50 mL beaker and dissolved. Under ice cooling, 27 wt% sodium hydroxide was added to adjust the pH to 12. Caprinoyl chloride (46.42 g, 243.42 mmol) and 25 wt% sodium hydroxide were simultaneously added dropwise to this solution over 70 minutes so as to maintain pH 12, and the mixture was stirred at room temperature for 1.5 hours. 75% by weight sulfuric acid was added to the reaction mixture to lower the pH to 1.3 to precipitate crystals, which were collected by filtration, washed with water and hexane, and dried under reduced pressure overnight.

[Reference Example 2] Synthesis of C12-V Valine (29.30 g, 250.11 mmol), 78.6 g of water, and 36.3 g of acetone were added to a 50 mL beaker and dissolved. Under ice cooling, 25 wt% sodium hydroxide was added to adjust the pH to 12. Lauroyl chloride (53.25 g, 241.42 mmol) and 25 wt% sodium hydroxide were simultaneously added dropwise to this solution over 45 minutes so as to maintain pH 12, and the mixture was stirred at room temperature for 1.5 hours. 75% by weight sulfuric acid was added to the reaction mixture to lower the pH to 2.01 to precipitate crystals, which were collected by filtration, washed with water and hexane, and dried under reduced pressure overnight.
Further, N-acyl valine having an acyl group having a different chain length can be easily synthesized by carrying out the same reaction using an acid chloride having a corresponding carbon chain length.

[Reference Example 3] Synthesis of O-Ser Serine (2.13 g, 20.27 mmol), 12.9 g of water, and 3.7 g of isopropanol were added to a 50 mL beaker and dissolved. Under ice cooling, 25 wt% sodium hydroxide was added to adjust the pH to 10. Oleoil chloride (5.54 g, 18.41 mmol) and 25 wt% sodium hydroxide were simultaneously added dropwise to this solution over 30 minutes so as to maintain pH 10, and the mixture was stirred at room temperature for 2.5 hours. 75% by weight sulfuric acid was added to the reaction mixture to lower the pH to 1.48 to precipitate crystals, which were collected by filtration and dried under reduced pressure overnight at room temperature. The obtained crystal was recrystallized from ethyl acetate to obtain the desired product.

(Statistical analysis)
The obtained data was expressed as mean ± standard error, and a significant difference test was performed using Student's t-test.

[Example 1] Osteoclast formation experiment 1
(1) Bone marrow cells were collected from dddy mice (male, 6 weeks old) by a conventional method, and bone marrow cells were seeded at a cell concentration of ^{2 × 10 6} cells / well and primary osteoblasts at ^{a cell concentration of 1 × 10 4 cells / well.} Then, 10 (v / v)% bovine fetal serum (FBS) / phenol red (PR) (-) α-modified Eagle's minimum essential medium (αMEM) containing interleukin-1 (IL-1) at a final concentration of 2 ng / mL. _using, under 5% CO 2 -95% air vapor were co-cultured for 7 days at 37 ° C.. To this culture system, 150 μM each of valine and N-acylvaline were added together with IL-1.
The case of culturing without adding both IL-1 and valines was used as a control.
(2) Osteoclast assay staining (TRAP staining)
After culturing, the cells were stained with a TRAP staining kit (manufactured by Cosmo Bio Co., Ltd.) and dried at room temperature to detect tartrate-resistant acid phosphatase (TRAP), which is a marker enzyme for osteoclasts. TRAP-positive multinucleated cells with 3 or more nuclei were measured as osteoclasts and evaluated as the number of osteoclasts formed.

(3) Results Microscopic observation of stained cells (“inverted microscope CKX41”, manufactured by Olympus Corporation, magnification = 40 times) and evaluation results of the number of osteoclasts formed are shown in FIG.
As shown in FIG. 1, addition of IL-1 induces osteoclast differentiation (significant at p <0.001). The addition of 150 μM Val tended to suppress the differentiation of IL-1-induced osteoclasts. Significant (p <0.01) suppression was observed in the 150 μM C8-Val addition group, and osteoclast differentiation was suppressed to the control level in each of the C10-Val and C12-Val addition groups (p <0.01). 0.001).

[Example 2] Bone resorption activity experiment using a mouse parietal bone organ culture system (1) A parietal bone was collected from a dddy pup (5 days old), and BGJb medium (penicillin (50 mg / L) and streptomycin (penicillin (50 mg / L)) and streptomycin ( (Containing 100 mg / L)) was cultured at 37 ° C. for 24 hours. Then, the culture medium was replaced with a medium supplemented with IL-1 (2 ng / mL) and a medium supplemented with IL-1 and valine and N-acylvaline, respectively, at 0.2 mM to 1 mM, and the culture was continued. After culturing for 6 days, the calcium concentration in the culture supernatant was measured by the o-Cresolphthalein Complexone (OCPC) method. From the measurement results, the increase in calcium concentration (ΔCa) obtained by subtracting the calcium concentration when cultured only in the culture solution (control) was determined to be calcium ejected from the parietal bone, and was used as an index of bone resorption activity.

(2) Results The evaluation results of the bone resorption activity using the calcium released in the culture supernatant as an index are shown in FIG.
As shown in FIG. 2, IL-1 significantly increased the bone resorption activity (p <0.001), but no inhibitory effect on the bone resorption activity was observed in the Val-added group, and C8-Val was not observed. A mild inhibitory effect was observed in the addition group (p <0.05). In addition, in the C10-Val and C12-Val-added groups, the bone resorption activity induced by IL-1 was suppressed in a concentration-dependent manner. In the C10-Val-added group, it was significantly suppressed at a concentration of 0.75 mM or more (p <0.001), and in the C12-Val-added group, it was significantly suppressed at a concentration of 0.2 mM (p <0.05). , 0.3 mM or higher was significantly suppressed (P <0.001).

[Example 3] Bone resorption activity experiment using a mouse alveolar bone organ culture system (1) According to a known method described in Japanese Patent No. 4662043, etc., lipopolysaccharide (LPS) -induced bone resorption in alveolar bone organ culture The effects of valine and N-acylvaline on activity were investigated.
Addition of LPS to the alveolar bone causes inflammatory alveolar bone destruction. Specifically, alveolar bone is collected from a mouse according to the method described in Japanese Patent No. 4662043, organ-cultured, and alveolar bone destruction (alveolar bone) caused by LPS, which is a Toll-like receptor 4 (TLR4) ligand. The inhibitory effects of valine and N-acylvaline on (inflammatory bone resorption) were analyzed by adding valins together with LPS and comparing the effects on bone resorption activity. The LPS concentration was 1 μg / mL, and the valine concentration was 0.5 mM. As for the bone resorption activity, as in Example 2, the increase in calcium concentration (ΔCa) calculated from the measurement result of the calcium concentration in the culture supernatant was evaluated as calcium ejected from the alveolar bone.

(2) Results The evaluation results are shown in FIG.
As shown in FIG. 3, the Val-added group tended to suppress the inflammatory bone resorption of the alveolar bone induced by LPS, and the C8-Val-added group showed a mild inhibitory effect (p <. 0.01). In the C10-Val and C12-Val-added groups, more remarkable inhibitory effects were observed (p <0.001).

(Discussion on Examples 1 to 3)
As a result of examining the effects of valine and N-acylvarin on IL-1-induced bone resorption activation, a strong inhibitory effect was observed on C10-Val and C12-Val, and N-acylvaline may have an inhibitory effect on bone resorption. Sex was suggested.
In addition, in alveolar bone organ culture, which is an Exvivo evaluation system for periodontal disease using LPS, which is an endotoxin of periodontopathic bacteria, C10-Val and C12-Val inhibit inflammatory bone resorption by LPS. Was clarified, suggesting an improving effect of N-acylvarin on periodontal disease.
This test system showed that C10-Val and C12-Val may have an effective therapeutic effect on bone destruction caused by bone resorption factors.

[Example 4] Osteoclast formation experiment 2
(1) In the same procedure as in Example 1, N-acylvarin and oleoylserine as a comparative example were added to the medium at a concentration of 5 μM to 300 μM, respectively, in a co-culture system of bone marrow cells and mouse osteoblasts. After culturing, osteoclasts were detected and measured.
C12-Val was used as N-acylvarin, and the inhibitory effect on osteoclast differentiation in the co-culture system was compared and examined.

(2) Results The microscopic observation image of the TRAP-stained cells is shown in FIG.
As shown in FIG. 4, IL-1 stimulation induced TRAP-positive polynuclear osteoclast formation. A decrease in the number of polynuclear osteoclasts was observed in both the C12-Val and O-Ser-added groups in a concentration-dependent manner. It was found that cell differentiation was completely suppressed. The suppression of osteoclast differentiation was accompanied by morphological shrinkage of osteoclasts.
The measurement results of the number of osteoclasts formed are shown in FIG.
Similarly, from the results shown in FIG. 5, osteoclast differentiation was completely suppressed at a concentration of 150 μM in the C12-Val-added group (p <0.001), and fractured at a concentration of 300 μM in the O-Ser-added group. Osteoclast differentiation was completely suppressed (p <0.001).
From the above results, it was suggested that C12-Val has a stronger inhibitory effect on osteoclast differentiation than oleoylserine described in Non-Patent Document 1.

(Discussion on Example 4)
As a result of examining the action of N-acylamino acids on IL-1-induced bone resorption activity, strong suppression of osteoclast differentiation was observed in C12-Val, and it is possible that it has an effective bone resorption inhibitory action. It was suggested. This study suggests that C12-Val may have the effect of improving bone destruction caused by bone resorption factors.

INDUSTRIAL APPLICABILITY According to the present invention, it is possible to provide an agent for suppressing the formation or activation of osteoclasts, which can effectively suppress the formation or differentiation of osteoclasts. The osteoclast formation or activation inhibitor of the present invention has an effect of significantly reducing bone resorption activity in alveolar bone, in particular.
Further, according to the present invention, it is possible to provide an oral composition for prevention or treatment of periodontal disease, which is excellent as an oral care product for the purpose of prevention or improvement of periodontal disease.

Claims (4)

An osteoclast formation or activation inhibitor containing an N-acyl branched chain amino acid or a salt thereof having a saturated or unsaturated acyl group having 8 to 16 carbon atoms. Claim 1 is that the N-acyl branched chain amino acid having a saturated or unsaturated acyl group having 8 to 16 carbon atoms is an N-acyl valine having a saturated or unsaturated acyl group having 8 to 16 carbon atoms. The inhibitor described. The inhibitor according to claim 2, wherein the N-acylvaline having a saturated or unsaturated acyl group having 8 to 16 carbon atoms is N-decanoylvaline or N-lauroylvaline. The inhibitor according to any one of claims 1 to 3, which is an alveolar bone resorption inhibitor.

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