JP2024084133A - Osteoclast differentiation inhibitor, bone resorption inhibitor, and use thereof - Google Patents

Abstract

To provide a novel material superior in osteoclast differentiation inhibitory action.SOLUTION: An osteoclast differentiation inhibitor includes as an active principle at least one selected from the group consisting of fatty acids and fatty acid esters including the fatty acids, where the fatty acids are at least one selected from the group consisting of fatty acids having 13 to 15 carbon atoms. A bone resorption inhibitor includes as an active principle at least one selected from the group consisting of fatty acids and fatty acid esters including the fatty acids, where the fatty acids are at least one kind selected from the group consisting of fatty acids having 13 to 15 carbon atoms.SELECTED DRAWING: None

Description

The present invention relates to an osteoclast differentiation inhibitor, a bone resorption inhibitor, and uses thereof.

Maintaining bone function is important for improving quality of life and extending healthy lifespan. Bones are constantly being regenerated while maintaining homeostasis through bone metabolism, in which old bone is broken down by osteoclasts (bone resorption) and new bone is created by osteoblasts (bone formation). However, if the balance between bone resorption and bone formation is disrupted, bone tissue becomes abnormal, and it is known that various bone diseases such as osteoporosis can occur.

In order to prevent or improve such abnormalities in bone metabolism, various methods for inhibiting osteoclast differentiation and bone resorption have been investigated. For example, Patent Document 1 describes an osteoclast differentiation inhibitor that contains watercress extract as an active ingredient. Patent Document 2 describes an osteoclast differentiation inhibitor and bone resorption inhibitor that contain a lignan compound derived from a plant of the Piperaceae family as an active ingredient.

JP 2022-143322 A JP 2020-142993 A

In this situation, there is still a need for new materials that have excellent inhibitory effects on osteoclast differentiation. Therefore, the object of the present invention is to provide new materials that have excellent inhibitory effects on osteoclast differentiation.

The present inventors have conducted intensive research to achieve the above object and have completed the present invention. That is, according to the present invention, there are provided the following osteoclast differentiation inhibitors, bone resorption inhibitors, food and drink compositions, oil and fat compositions, supplements, and pharmaceutical compositions.
[1] An osteoclast differentiation inhibitor comprising at least one selected from the group consisting of fatty acids and fatty acid esters containing the fatty acids as an active ingredient,
The osteoclast differentiation inhibitor, wherein the fatty acid has at least one carbon atom selected from the group consisting of 13 and 15 carbon atoms.
[2] The osteoclast differentiation inhibitor according to the above-mentioned [1], wherein the fatty acid is a saturated fatty acid.
[3] The osteoclast differentiation inhibitor according to the above [1] or [2], wherein the fatty acid ester is a glycerol fatty acid ester.
[4] A bone resorption inhibitor comprising at least one selected from the group consisting of fatty acids and fatty acid esters containing the fatty acids as an active ingredient,
The bone resorption inhibitor as described above, wherein the fatty acid has at least one carbon atom selected from the group consisting of 13 and 15 carbon atoms.
[5] A food or drink composition comprising the agent according to any one of [1] to [4] above.
[6] An oil composition comprising the agent according to any one of [1] to [4] above.
[7] A supplement comprising the agent according to any one of [1] to [4] above.
[8] A pharmaceutical composition comprising the agent according to any one of the above [1] to [4].

The present invention provides novel osteoclast differentiation inhibitors and bone resorption inhibitors.

The following describes an embodiment of the present invention. Note that unless otherwise specified, "to" in a numerical range indicates from above to below, and both ends of the range are included. When an upper limit and a lower limit of a numerical range are indicated, the upper limit and the lower limit can be appropriately combined, and the numerical range obtained by combining them is also considered to be disclosed.

(Osteoclast differentiation inhibitor, bone resorption inhibitor)
The osteoclast differentiation inhibitor of the present invention contains as an active ingredient at least one selected from the group consisting of fatty acids having 13 and 15 carbon atoms and fatty acid esters containing such fatty acids.
The bone resorption inhibitor of the present invention contains as an active ingredient at least one selected from the group consisting of fatty acids having 13 and 15 carbon atoms and fatty acid esters containing said fatty acids.

The osteoclast differentiation inhibitor has the effect of inhibiting the differentiation of precursor cells into osteoclasts.

The bone resorption inhibitor inhibits the differentiation of precursor cells into osteoclasts, thereby inhibiting bone resorption (the destruction of old bone by osteoclasts).

The osteoclast differentiation inhibitor and bone resorption inhibitor have the effect of inhibiting differentiation into osteoclasts and inhibiting bone resorption as described above, and therefore can be suitably used for preventing or improving bone resorption diseases (meaning diseases that develop when bone resorption exceeds bone formation) or for preventing or treating bone resorption diseases. Examples of bone resorption diseases include osteoporosis, osteopenia, fractures, decreased bone density, periodontal disease, rheumatoid arthritis, osteoarthritis, arthritis, Paget's disease, myeloma, and bone metastasis of cancer.

(fatty acid)
The fatty acid, which is an active ingredient of the present invention, may be at least one fatty acid selected from the group consisting of 13 and 15 carbon atoms, and there is no particular restriction on its origin. For example, it may be derived from a natural product or may be synthetic. In addition, it may be decomposed in the living body and used as at least one fatty acid selected from the group consisting of 13 and 15 carbon atoms, for example, a fatty acid ester containing the above fatty acid. In order to provide it in the form of a food and beverage composition or a pharmaceutical composition, it is preferable that it is prepared from a natural product. In the following, for convenience of explanation, at least one fatty acid selected from the group consisting of 13 and 15 carbon atoms used in the present invention is referred to as "fatty acid (C13, C15)", a fatty acid ester containing at least one fatty acid selected from the group consisting of 13 and 15 carbon atoms is referred to as "fatty acid (C13, C15) ester", and at least one selected from the group consisting of at least one fatty acid selected from the group consisting of 13 and 15 carbon atoms and fatty acid esters containing the fatty acid is referred to as "fatty acid (C13, C15) etc.".

The above fatty acids (C13, C15) include, for example, tridecanoic acid (C13:0) and pentadecanoic acid (C15:0). In addition, they are not limited to saturated fatty acids, and may be unsaturated fatty acids having a double bond in part of the fatty acid alkyl chain, or part of the fatty acid alkyl chain may be branched. The above fatty acids (C13, C15) are preferably saturated fatty acids.

Examples of the fatty acid (C13, C15) esters include glycerol fatty acid esters, sucrose fatty acid esters, organic acid fatty acid esters, fatty acid alcohol esters, sphingolipids, sorbitan fatty acid esters, polypropylene fatty acid esters, sterol esters, and the like, all of which contain the above fatty acids (C13, C15). Of these, glycerol fatty acid esters are preferred. Glycerol fatty acid esters are esters that have glycerol as a constituent component, and specific examples include triacylglycerol, diacylglycerol, monoacylglycerol, glycerophospholipids, and glyceroglycolipids. Of these, triacylglycerol is preferred.

The above fatty acids (C13, C15) etc. may be used alone or in combination of two or more kinds. In addition, when enantiomers or diastereomers exist, one kind may be used alone as a single molecular species, or two or more kinds of molecular species of these isomers may be used in combination.

The method of obtaining the above fatty acids (C13, C15) from natural products is not limited to this, but for example, JP 2016-89025 A reveals that Aurantiochytrium algae, known as a high-oil-producing algae, produce a large amount of triglycerides containing fatty acids (C13) and (C15) as constituent fatty acids. Therefore, by using such edible oils and fats or high-oil-producing algae as a raw material, the above fatty acids (C13, C15) can be efficiently obtained. Typically, for example, organic solvents such as n-hexane, chloroform, diethyl ether, methanol, ethanol, or mixed organic solvents thereof are added to a dried product of a natural product such as algae, and extraction is performed to obtain a triglyceride-containing lipid containing the above fatty acids (C13) and fatty acids (C15) as constituent fatty acids. The obtained triglyceride-containing lipid may be adsorbed with silica gel, acid clay, activated clay, ion exchange resin, or the like to remove polar lipids, thereby obtaining triglycerides. In addition, the triglyceride-containing lipid may be subjected to a general refining process for vegetable oils and fats, i.e., a degumming process, a deacidification process, a bleaching process, or a deodorizing process to remove impurities, thereby obtaining a triglyceride-containing purified lipid. Furthermore, the obtained triglyceride, triglyceride-containing lipid, or triglyceride-containing purified lipid may be subjected to a hydrolysis process using sodium hydroxide or lipase, etc., to liberate fatty acids from the ester structure of glycerol. The liberated fatty acids may be further separated and purified by a method such as molecular distillation, urea addition, or column chromatography. Furthermore, the obtained fatty acids, triglycerides, triglyceride-containing lipids, or triglyceride-containing purified lipids may be used as raw materials for further transesterification or ester synthesis of triglycerides.

When preparing and using a material containing the above fatty acids (C13, C15) etc. using natural products as raw materials, it is preferable to use a material in which the content (purity) of the above fatty acids (C13, C15) etc. is increased to 1% by mass or more, more preferably to use a material in which the content (purity) is increased to 2% by mass or more, even more preferably to use a material in which the content is increased to 5% by mass or more, and particularly preferably to use a material in which the content is increased to 10% by mass or more. Alternatively, as the total content of fatty acids other than fatty acids (C13, C15), it is preferable to use a material with a total content of 99% by mass or less, more preferably to use a material with a total content of 95% by mass or less, even more preferably to use a material with a total content of 90% by mass or less, and particularly preferably to use a material with a total content of 80% by mass or less.

The above fatty acids (C13, C15) etc. can be quantified by methods well known to those skilled in the art. For example, they can be quantified by analyzing them by gas chromatography, liquid chromatography, etc., as specified in the Standard Methods for Analysis of Fats, Oils, and Related Materials established by the Japan Oil Chemists' Society, and applying the concentration obtained when an arbitrarily determined standard sample is analyzed. In this specification, the quantitative determination of the above fatty acids (C13, C15) etc. can be treated as the total amount of one or more types of detectable fatty acids (C13, C15) etc.

The osteoclast differentiation inhibitor and bone resorption inhibitor of the present invention may consist only of the above-mentioned active ingredient, or may contain other ingredients acceptable for use in foods, beverages, and pharmaceuticals in addition to the above-mentioned active ingredient, depending on the specific form of the osteoclast differentiation inhibitor and bone resorption inhibitor.

The osteoclast differentiation inhibitor and bone resorption inhibitor of the present invention may be in any form, such as a solid, liquid (including a solution and a suspension), or a paste.

The osteoclast differentiation inhibitor and bone resorption inhibitor of the present invention may be used to administer the above-mentioned fatty acids (C13, C15) to a subject, and there are no particular limitations on the form of use. For example, fatty acids (C13, C15) may be incorporated into a specific dosage form, which may be administered orally, applied to the skin, administered by inhalation, or injected intramuscularly, or in any other suitable form. The osteoclast differentiation inhibitor and bone resorption inhibitor may be administered once a day, or in multiple divided doses per day.

The osteoclast differentiation inhibitor and bone resorption inhibitor of the present invention can be applied not only to humans but also to non-human animals such as livestock such as cows, pigs, chickens, sheep, and horses, and pets such as dogs and cats. They are preferably used in humans, and more preferably, can be used in healthy middle-aged and elderly people (specifically, healthy people aged 40 or older).

The content of fatty acids (C13, C15) in the osteoclast differentiation inhibitor and bone resorption inhibitor is not particularly limited and may be appropriately adjusted from the viewpoint of ensuring the desired dosage. For example, the content may be 0.1% by mass or more, 0.2% by mass or more, 0.5% by mass or more, 1% by mass or more, 2% by mass or more, 5% by mass or more, 10% by mass or more, or 30% by mass or more. The content may be 100% by mass or less, 90% by mass or less, 80% by mass or less, 60% by mass or less, 50% by mass or less, or 30% by mass or less. In addition, the material that provides the fatty acids (C13, C15) may itself constitute the osteoclast differentiation inhibitor and bone resorption inhibitor.

In addition, it is generally known that there are enzymes in the body that release free fatty acids from esters, such as lingual lipase present in the oral cavity, gastric lipase present in the stomach, and pancreatic lipase secreted into pancreatic juice. Furthermore, esters such as triglycerides contained in the blood are decomposed into free fatty acids and glycerol by lipoprotein lipase present on the cell surface of muscles, etc. In addition, some phospholipases release free fatty acids from glycerophospholipids, which are esters, by hydrolysis. It is also known that there are various free fatty acid uptake receptors on the cell surface, such as GPR40, GPR41, GPR43, and GPR120, which are G protein-coupled receptors (GPR). Therefore, the fatty acids (C13, C15) are released from the fatty acid esters of the above fatty acids (C13, C15) administered to the subject in the subject's body at an appropriate time, and the effects of the fatty acids can be exerted in the subject's body.

When the osteoclast differentiation inhibitor and bone resorption inhibitor of the present invention are orally ingested by humans, the dosage of the fatty acids (C13, C15) and the like can be appropriately determined depending on the age and health condition of the recipient, the duration of administration, and the frequency of administration. Examples of typical dosage amounts include a daily dosage of 5 mg to 5,000 mg for a 60 kg adult, a dosage of 10 mg to 3,000 mg, a dosage of 50 mg to 2,000 mg, or a dosage of 100 mg to 1,000 mg.

(Food and drink composition)
The food and drink composition of the present invention contains at least one of the osteoclast differentiation inhibitor and bone resorption inhibitor described above, and may further contain additives that are usually used in foods and drinks, as necessary.

Examples of the form of food and beverage compositions include oil and fat compositions, confectioneries (e.g., snacks such as potato chips, baked goods such as cookies or cakes, Japanese sweets, chocolate, candy, pudding, jelly, ice cream, gummy candy, gum, etc.), breads (e.g., sweet breads, savory breads, croissants, Danish bread, etc.), noodles (e.g., ramen, udon, pasta, etc.), rice (e.g., rice balls, porridge, fried rice, etc.), cereal foods (e.g., corn flakes, oatmeal, etc.), dairy products (e.g., cheese, yogurt, etc.), processed meat products (e.g., ham, sausage, etc.), processed seafood products (e.g., kamaboko, fish sausage, etc.), These include seasonings (e.g., mayonnaise, sauces, dressings, etc.), soups (e.g., miso soup, vegetable soup, etc.), processed foods and beverages (e.g., simmered foods, fried foods, grilled foods, curries, etc.), premixed flours (e.g., okonomiyaki flour, fried chicken flour, confectionery mix flour, etc.), solid rouxes (e.g., curry roux, etc.), beverages (e.g., alcoholic beverages such as beer, soft drinks such as sports drinks, lactic acid drinks, or vegetable juice, teas such as black tea, coffee, etc.), foods and beverages for nursing care (e.g., liquid diets, etc.), health foods, health functional foods, foods for specified health uses, foods with functional claims, foods with nutritional functions, nutritional supplements, supplements, proteins, animal feed, etc.

The food and drink composition of the present invention contains at least one of the osteoclast differentiation inhibitor and bone resorption inhibitor, and can therefore be used as a food and drink composition for inhibiting osteoclast differentiation or bone resorption. It can also be suitably used as a food and drink composition for preventing or improving the above-mentioned bone resorption diseases.

(Oil and fat composition)
Specifically, the above-mentioned oil and fat composition can be prepared by, for example, blending edible oil and fat with optional excipients, auxiliary agents, emulsifiers, pH adjusters, etc., as necessary, and by a known method, into an oil and fat composition in any form, such as liquid, solid, powder, or paste. Examples of the form of the above-mentioned oil and fat composition include liquid oil and fat, margarine, fat spread, shortening, powdered oil and fat, etc., which are mainly composed of oil and fat components. Alternatively, it may be prepared into a solution, powder, gel, granule, etc., in which the amount of oil and fat components is small. In addition, for example, when powdering, an auxiliary agent such as corn syrup can be used, and further, an emulsifier may be added to prepare an emulsion raw material, which may then be powdered. Examples of the powdering method include spray drying, freeze drying, etc.

Examples of the edible oils and fats include vegetable oils and fats such as rapeseed oil (including high oleic acid type), soybean oil (including high oleic acid type), palm oil, palm kernel oil, corn oil, olive oil, sesame oil, safflower oil (including high oleic acid type), sunflower oil (including high oleic acid type), cottonseed oil, rice oil, peanut oil, coconut oil, grapeseed oil, and cacao butter; algae oil; animal oils and fats such as beef tallow, lard, chicken fat, milk fat, and fish oil; and medium-chain fatty acid triglycerides. In addition, examples of the edible oils and fats include fractionated oils (mid-melting point fractionated oil of palm oil, soft fractionated oil of palm oil, hard fractionated oil of palm oil, etc.), transesterified oil, hydrogenated oil, and other processed oils and fats. The edible oils and fats may be used alone or in a mixture of two or more types.

The above-mentioned oil and fat composition may contain an auxiliary agent that is usually added to edible oil and fat compositions. Examples of the auxiliary agent include antioxidants, antifoaming agents, emulsifiers, fragrances, flavoring agents, colorants, and biologically active substances. Specific examples include lignans, coenzyme Q, γ-oryzanol, tocopherol, and silicones.

There are no particular limitations on the content of the fatty acids (C13, C15) contained in the oil composition. For example, the content may be 0.05% by mass or more, 0.1% by mass or more, 0.2% by mass or more, 0.5% by mass or more, 1% by mass or more, 2% by mass or more, 5% by mass or more, 8% by mass or more, 10% by mass or more, or 12% by mass or more. The content may be 100% by mass or less, 90% by mass or less, 80% by mass or less, 60% by mass or less, 50% by mass or less, 30% by mass or less, 20% by mass or less, 15% by mass or less, or 10% by mass or less.

(supplement)
Examples of the form of the supplement include tablets (including plain tablets, sugar-coated tablets, effervescent tablets, film-coated tablets, chewable tablets, troches, etc.), pills, capsules (hard capsules, soft capsules, seamless capsules, etc.), granules, powders, liquids, syrups, jellies, candies, gummies, etc.

There are no particular limitations on the content of the fatty acids (C13, C15) contained in the supplement. For example, the content may be 0.05% by mass or more, 0.1% by mass or more, 0.2% by mass or more, 0.5% by mass or more, 1% by mass or more, 2% by mass or more, 5% by mass or more, 8% by mass or more, 10% by mass or more, or 12% by mass or more. The content may be 100% by mass or less, 90% by mass or less, 80% by mass or less, 60% by mass or less, 50% by mass or less, 30% by mass or less, 20% by mass or less, 15% by mass or less, or 10% by mass or less.

Pharmaceutical Compositions
The pharmaceutical composition of the present invention contains at least one of the osteoclast differentiation inhibitor and the bone resorption inhibitor, and may further contain additives that are usually used in pharmaceutical compositions, if necessary. The pharmaceutical composition may be a drug or a quasi-drug.

The above pharmaceutical compositions may be in the form of, for example, tablets (including plain tablets, sugar-coated tablets, effervescent tablets, film-coated tablets, chewable tablets, troches, etc.), pills, capsules (hard capsules, soft capsules, seamless capsules, etc.), granules, powders, liquids, syrups, jellies, candies, gummies, and other oral preparations; parenteral preparations such as injections, drips, and suppositories; and topical preparations such as ointments and plasters.

The pharmaceutical composition of the present invention contains at least one of the osteoclast differentiation inhibitor and bone resorption inhibitor, and can therefore be used as a pharmaceutical composition for inhibiting osteoclast differentiation or bone resorption. It can also be suitably used as a pharmaceutical composition for preventing or treating the above-mentioned bone resorption diseases.

The present invention will be explained in more detail below with reference to test examples, but these test examples are not intended to limit the scope of the present invention in any way.

The materials and methods used in this test example are as follows.
[1. cell〕
Mouse macrophage-like cells Raw264.7 (ATCC, Catalog No. TIB-71)

2. Culture medium
Growth medium: DMEM (low glucose) supplemented with 10% FBS, 1% PS
Differentiation medium: MEMα supplemented with 10% charcoal stripped FBS, 1% PS, and 50 ng/mL RANKL
Details of the materials used in the medium are as follows:
FBS: fetal bovine serum (Thermo Fisher Scientific, Catalog No.: 10437-028)
PS: Penicillin/streptomycin mixed solution (Nacalai Tesque, Catalog No. 09367-34)
DMEM (low glucose): Dulbecco's modified Eagle's medium (low glucose) (Nacalai Tesque, catalogue no. 08456-65)
Charcoal stripped FBS: charcoal stripped fetal bovine serum (Thermo Fisher Scientific, Catalog No. A33821-01)
RANKL: Receptor activator of NF-κB ligand (R&D Systems, Catalog No. 462-TEC)

3. Preparation of test substances
Each of the test substances (1) to (2) below was dissolved in dimethyl sulfoxide (DMSO) (manufactured by Nacalai Tesque) to prepare a solution with a concentration of 150 mM. These solutions were added in an amount of 1/1000 to the differentiation medium to prepare a medium with a target final concentration of the test substance of 150 μM (Examples 1 to 2).
As a positive control, the test substance shown in (3) below, which is known to have an inhibitory effect on osteoclast differentiation, was dissolved in the differentiation medium to a target final concentration of 100 μg/mL (Control Example 3).
(Test substance)
(1) Pentadecanoic acid (C15:0) (purity 98.8%, manufactured by Sigma-Aldrich)
(2) Tridecanoic acid (C13:0) (purity 99.2%, manufactured by Sigma-Aldrich)
(3) Heparin (Sigma-Aldrich, model number: H3149)

4. Cell culture and test substance treatment
Raw264.7 cells were put to sleep in a T75 flask using a growth medium, and cultured in a _CO2 incubator (Astec Corporation "SCA-165DRS", conditions: 37°C, 5% _CO2 , humidified, the same below). When the required number of cells was reached, the cells were detached using a cell scraper, and then seeded on a 96-well plate using fresh growth medium. The next day, the medium was replaced with each differentiation medium (differentiation medium without test substance and without RANKL (Control Example 1), differentiation medium with test substance and without RANKL (Control Example 2), differentiation medium prepared in [3. Preparation of test substance] (Examples 1-2, Control Example 3)), and cultured for 2-3 days in a _CO2 incubator.

[5. TRAP activity measurement, cell viability measurement]
After the treatment, tartrate-resistant acid phosphatase (TRAP) activity, which is a differentiation marker for osteoclasts, was measured using a TRAP activity measurement kit (TAKARA BIO INC., "TRACP & ALP Assay Kit") according to the method described in the kit. Specifically, the cells were washed and solubilized to extract TRAP, and then reacted with pNPP (p-nitro-phenyl phosphate), a substrate for TRAP, in the presence of a buffer and tartaric acid. The TRAP activity was determined by measuring absorbance, and the cell viability was measured by the WST-8 method.

[6. result〕
Measured TRAP activity, cell viability, and corrected TRAP activity (values calculated by correcting TRAP activity with cell viability in consideration of the possibility of cell death. Calculated using the following formula. Formula: corrected TRAP activity The mean cell viability (TRAP activity) and standard error (standard error) are shown in Table 1. The mean cell viability and standard error are expressed as relative values (%) when the mean value of Control Example 2 is set to 100%. In addition, the significant difference in the corrected TRAP activity between Control Example 2 and the test substance treatment groups (Control Example 3 and each Example) was examined by Dunnett's test. The results are also shown in Table 1.

As shown in the corrected TRAP activity in Table 1, a comparison of control example 1 and control example 2 confirmed that TRAP activity was increased by differentiation treatment, and a comparison of control example 2 and control example 3 (positive control) confirmed that the increased TRAP activity was suppressed by heparin.

As shown in the corrected TRAP activity in Table 1, the TRAP activity in each Example was significantly decreased compared to Control Example 2. This indicates that pentadecanoic acid and tridecanoic acid have the effect of suppressing osteoclast differentiation.

Claims (8)

An osteoclast differentiation inhibitor comprising at least one active ingredient selected from the group consisting of fatty acids and fatty acid esters containing the fatty acids,
The osteoclast differentiation inhibitor, wherein the fatty acid has at least one carbon atom selected from the group consisting of 13 and 15 carbon atoms. The osteoclast differentiation inhibitor according to claim 1, wherein the fatty acid is a saturated fatty acid. The osteoclast differentiation inhibitor according to claim 1 or 2, wherein the fatty acid ester is a glycerol fatty acid ester. A bone resorption inhibitor comprising at least one active ingredient selected from the group consisting of fatty acids and fatty acid esters containing the fatty acids,
The bone resorption inhibitor, wherein the fatty acid has at least one carbon atom selected from the group consisting of 13 and 15 carbon atoms. A food or beverage composition comprising the agent according to claim 1 or 4. An oil composition comprising the agent according to claim 1 or 4. A supplement comprising the agent according to claim 1 or 4. A pharmaceutical composition comprising the agent according to claim 1 or 4.