Inflammatory cytokine inhibitor

Abstract

To provide an inflammatory cytokine inhibitor which allows inhibition of interleukin-6 and interleukin-1β production while sufficiently ensuring safety.SOLUTION: Provided is a cytokine inhibitor for suppressing production of interleukin-6 and interleukin-1β, comprising as an active ingredient a triglyceride represented by formula (I) (wherein R1, R2, and R3 each represent a saturated fatty acid residue, and at least one of them is a pentadecanoic acid residue).SELECTED DRAWING: Figure 3

Description

[Technical Field]

[0001] The present invention relates to an inflammatory cytokine suppressor, and more particularly to a cytokine suppressor capable of suppressing the production of interleukin-6 and interleukin-1β. [Background technology]

[0002] Cytokines are proteins primarily produced by immune cells. They are broadly classified into pro-inflammatory cytokines, which induce inflammatory symptoms in the body, and anti-inflammatory cytokines, which suppress inflammatory responses. Among pro-inflammatory cytokines, interleukin-6 (IL-6), interleukin-1β (IL-1β), and tumor necrosis factor-α (TNF-α) are representative inflammatory factors. While IL-6 is involved in homeostasis, such as inducing cell proliferation, it is also known to abnormally activate the immune response and cause excessive IL-6 production. IL-6 overproduction can trigger a cytokine storm, in which cytokines become uncontrollable. IL-1β also enhances inflammation by increasing the production of prostaglandin E2 (PGE2), a physiologically active substance that promotes leukocyte extravasation and chemotaxis. In particular, IL-1β is converted from its precursor to its mature form by caspase-1, an enzyme involved in cell death, leading to its overproduction. Excessive production of IL-1β is a factor that causes diseases such as CAPS (cryopyrin-associated periodic syndrome).

[0003] Under these circumstances, various studies have been conducted to suppress the production of inflammatory cytokines, and for example, Patent Document 1 proposes an inflammatory cytokine production inhibitor containing a tomato extract as an active ingredient, while Patent Document 2 proposes a cytokine production inhibitor containing an aluminum compound as an active ingredient.

[0004] However, there is still room for improvement in terms of efficacy, safety, and the like, of inflammatory cytokine inhibitors. [Prior art documents] [Patent documents]

[0005] [Patent Document 1] International Publication No. 2020 / 017489 [Patent Document 2] Japanese Patent Publication No. 2022-68272 Summary of the Invention [Problem to be solved by the invention]

[0006] A main object of the present invention is to provide an inflammatory cytokine inhibitor that can suppress the production of interleukin-6 and interleukin-1β while fully ensuring safety. [Means for solving the problem]

[0007] An inflammatory cytokine suppressor according to one embodiment of the present invention is an inflammatory cytokine suppressor for suppressing the production of interleukin-6 and interleukin-1β, and comprises a triglyceride represented by the following formula (I) as an active ingredient.

[0008] [ka]

[0009] In the formula, R 1 , R 2 and R 3 are each saturated fatty acid residues, at least one of which is a pentadecanoic acid residue.

[0010] In one embodiment of the above-mentioned inflammatory cytokine suppressor, it is preferable that the active ingredient is contained in an amount of 1 mg or more and 100 mg or less.

[0011] In one embodiment of the above-mentioned inflammatory cytokine suppressor, R 1 , R 2 and R 3 and a triglyceride in which all of R in formula (I) are pentadecanoic acid residues. 1 , R 2 and R 3 Preferably, the triglyceride contains a triglyceride in which any two of the triglycerides are pentadecanoic acid residues.

[0012] In one embodiment of the above-mentioned inflammatory cytokine suppressor, R 1 , R 2 and R 3 and a triglyceride in which all of R in formula (I) are pentadecanoic acid residues. 1 , R 2 and R 3 It is preferable that any two of the above triglycerides contain pentadecanoic acid residues, and the total mass of the triglycerides is 30% by mass or more of the total mass of the active ingredient.

[0013] In one embodiment of the above-mentioned inflammatory cytokine suppressor, R 1 , R 2 and R 3 It is preferable that the mass of the triglyceride in which all of the residues are pentadecanoic acid is 10% by mass or more of the total mass of the active ingredient. [Effects of the Invention]

[0014] The proinflammatory cytokine inhibitor of the present invention can suppress the production of interleukin-6 and interleukin-1β while fully ensuring safety. [Brief explanation of the drawings]

[0015] [Figure 1] FIG. 1 shows the results of evaluation by the MTT method. [Figure 2] FIG. 1 shows the results of evaluation by the LIVE / DEAD method, where (A) shows a fluorescent microscope image, (B) shows the cell death rate, and (C) shows the total cell count. [Figure 3] This figure shows the mRNA levels of inflammatory cytokines determined by RT-PCR. (A) shows the mRNA level of interleukin 6 (IL-6), (B) shows the mRNA level of interleukin 1β (IL-1β), and (C) shows the mRNA level of tumor necrosis factor α (TNF-α). [Figure 4] 1 shows the mRNA levels of anti-inflammatory cytokines determined by RT-PCR. (A) shows the mRNA level of transforming growth factor beta (TGF-β), and (B) shows the mRNA level of interleukin-10 (IL-10). DETAILED DESCRIPTION OF THE INVENTION

[0016] The proinflammatory cytokine suppressor of the present invention will be specifically described below. Note that the embodiments described below do not limit the invention according to the claims, and not all of the elements and combinations thereof described in the embodiments are necessarily essential to the solution of the present invention.

[0017] <<Inflammatory cytokine inhibitor>> An inflammatory cytokine suppressor according to an embodiment of the present disclosure (hereinafter referred to as an inflammatory cytokine suppressor of one embodiment) is an inflammatory cytokine suppressor used to suppress the production of interleukin-6 and interleukin-1β. The inflammatory cytokine suppressor of one embodiment contains a triglyceride represented by the following formula (I) as an active ingredient. Hereinafter, interleukin-6 may be abbreviated as IL-6, and interleukin-1β may be abbreviated as IL-1β. Hereinafter, the terms "active ingredient," "pentadecanoic acid triglyceride," or "PdATG" refer to the triglyceride represented by the following formula (I).

[0018] [ka]

[0019] (In the formula, R 1 , R 2and R 3 are each saturated fatty acid residues, at least one of which is a pentadecanoic acid residue.

[0020] In the formula, R 1 , R 2 and R 3 When any one or two of the residues are pentadecanoic acid residues, the other residue may be a saturated fatty acid residue other than pentadecanoic acid residue. "Saturated fatty acid" is a general term for fatty acids that do not have double or triple bonds in the molecule, and C n H 2n+1 The saturated fatty acids are represented by the chemical formula COOH. These saturated fatty acids are linear or branched, and include linear saturated fatty acids such as capric acid (C10), lauric acid (C12), tridecylic acid (C13), myristic acid (C14), pentadecanoic acid (C15), palmitic acid (C16), margaric acid (C17), stearic acid (C18), arachidic acid (C20), behenic acid (C22), lignoceric acid (C24), and cerotic acid (C26), as well as branched saturated fatty acids such as 2-hexyldecanoic acid (C16), 13-methylpentadecanoic acid (C16), and 16-methylheptadecanoic acid (C18). Examples of saturated fatty acids include, but are not limited to, R 1 , R 2 and R 3 In the case where one or two of the above are saturated fatty acid residues other than pentadecanoic acid (C15) residues, representative examples of the saturated fatty acid residues other than pentadecanoic acid (C15) residues include saturated fatty acid residues in the range of C10 to C20, particularly C13 to C17. 1 , R 2 and R 3 The total number of carbon atoms in the three saturated fatty acid residues is preferably in the range of C40 to C50, particularly C42 to C48.

[0021] In a preferred embodiment, the inflammatory cytokine suppressor is represented by the formula (I) 1 , R 2 and R 3 and a triglyceride in which all of R in formula (I) are pentadecanoic acid residues.1 , R 2 and R 3 It contains triglycerides in which any two of the residues are pentadecanoic acid.

[0022] In a preferred embodiment, the inflammatory cytokine suppressor is represented by the formula (I) 1 , R 2 and R 3 and a triglyceride in which all of R in formula (I) are pentadecanoic acid residues. 1 , R 2 and R 3 The triglyceride contains triglycerides in which any two of the above are pentadecanoic acid residues, and the total mass thereof is 30% by mass or more of the total mass of the active ingredient. An example upper limit is 100% by mass.

[0023] In a preferred embodiment, the inflammatory cytokine suppressor is represented by the formula (I) 1 , R 2 and R 3 In a more preferred embodiment, the proinflammatory cytokine suppressor is a triglyceride represented by the formula (I), wherein R is a pentadecanoic acid residue, and the mass of the triglyceride is 10% by mass or more of the total mass of the active ingredient. In one example, the upper limit is 100% by mass. 1 , R 2 and R 3 wherein the mass of the triglyceride in which all of the residues are pentadecanoic acid residues is 10% by mass or more and 80% by mass or less of the total mass of the active ingredient, and 1 , R 2 and R 3 The mass of the triglyceride in which any two of the above are pentadecanoic acid residues is 20% by mass or more and 90% by mass or less of the total mass of the active ingredient.

[0024] A preferred embodiment of the inflammatory cytokine inhibitor includes a triglyceride represented by formula (I) composed of saturated fatty acids mainly containing pentadecanoic acid (C15) (hereinafter sometimes referred to as "DM-PdATG").

[0025] In a more preferred embodiment, PdATG is represented by the following formula (II) or (III):

[0026] [ka]

[0027] In the formulas (II) and (III), R is a C14 to C16 saturated fatty acid.

[0028] The total mass of DM-PdATG or triglycerides represented by formula (II) and / or formula (III) is preferably 30% by mass or more, more preferably 50% by mass or more and 100% by mass or less, of the total mass of the active ingredient. When the content of triglycerides containing two or more pentadecanoic acid residues relative to the total mass of the active ingredient is less than 30% by mass, the objective can be achieved by increasing the intake amount. In the present invention, the active ingredient may be present in the form of a mixture of triglycerides containing two or more pentadecanoic acid residues. The mass of the mixture of triglycerides containing two or more pentadecanoic acid residues is at least 1% by mass, preferably 30% by mass or more, more preferably 50% by mass or more, and particularly preferably 90% by mass or more, relative to the total mass of the active ingredient, so long as the mixture itself is contained at a purity of at least 1% by mass, preferably 30% by mass or more, more preferably 50% by mass or more, and particularly preferably 90% by mass or more, of the total mass of the active ingredient, so that the mixture itself can fully function as the active ingredient.

[0029] In the present invention, the active ingredient may be present in the form of a mixture with a triglyceride other than the triglyceride represented by formula (I) (a triglyceride other than the active ingredient). In this case, the mass of the active ingredient is at least 1% by mass, preferably 50% by mass or more, and more preferably 90% by mass or more, based on the total mass of the mixture (a mixture of the active ingredient and triglycerides other than the active ingredient), so long as the mass of the active ingredient is at a purity of at least 1% by mass, preferably 50% by mass or more, and more preferably 90% by mass or more, so that the mixture itself can fully exhibit its function as an active ingredient.

[0030] The daily intake of the active ingredient is preferably adjusted to about 1 to 100 mg per adult. In a preferred embodiment, the proinflammatory cytokine suppressor contains the active ingredient in an amount of 1 mg to 100 mg.

[0031] (Action and effect) The active ingredient of the present invention inhibits the production of IL-6 and IL-1β at the transcriptional level, thereby improving diseases caused by excessive production of IL-6 and IL-1β and the poor physical condition that precedes the onset of the disease. In particular, the active ingredient of the present invention can inhibit the production of IL-6 and IL-1β induced by lipopolysaccharide (hereinafter referred to as LPS), improving diseases caused by excessive production of IL-6 and IL-1β and the poor physical condition that precedes the onset of the disease. The active ingredient of the present invention has no or very little cytotoxicity, making it safe for oral administration.

[0032] (Production of inflammatory cytokines) Inflammatory cytokines are produced by various cells, including T cells, B cells, fibroblasts, monocytes, endothelial cells, and mesangial cells. Macrophages produce inflammatory cytokines such as IL-6 and IL-1β upon stimulation with LPS via Toll-like receptors on their cell surface. For example, exposure of BV-2 cells to LPS activates the cells, enhancing their production of inflammatory cytokines such as IL-6 and IL-1β. BV-2 cells are a cell line derived from immortalized microglia from C57BL / 6 mice and are widely used as an immortalized model cell line to replace primary cultured microglia. As used herein, the term "production of inflammatory cytokines" includes the expression and associated secretion of inflammatory cytokines.

[0033] (Production method of active ingredient) The active ingredient, pentadecanoic acid triglyceride (including pentadecanoic acid triglyceride mixture), may be chemically synthesized or naturally occurring. If it is natural, its source is not particularly limited. Examples include lipids produced by living organisms, such as livestock and poultry fats, seafood oils and vegetable oils, and lipid-producing microorganisms. From the viewpoint of industrial productivity, microorganisms such as algae, bacteria, fungi (including yeast), and / or protists are preferred. Preferred microorganisms include those selected from the group consisting of golden algae (microorganisms of the kingdom Stramenopile), green algae, diatoms, dinoflagellates, yeast, and fungi of the genera Mucor and Mortierella. Members of the microbial group Stramenopile include microalgae. Microalgae refer to organisms that perform oxygenic photosynthesis, excluding bryophytes, ferns, and spermatophytes, and have a cell size of 1 μm to 100 μm in diameter. This also includes Labyrinthula, a group of protists closely related to microalgae. Labyrinthula are heterotrophic, non-photosynthetic marine eukaryotic microorganisms that are widely distributed, mainly in the subtropics and tropics. Generally, Labyrinthula are broadly divided into the Labyrinthulidae and Thraustochytriidae families, and include the genera Labyrinthula, Aurantiochytrium, Schizochytrium, Thraustochytrium, Aplanochytrium, Oblongichytrium, Botryochytrium, and Japonochytrium.

[0034] As the Labyrinthula to be cultured, the genus Aurantiochytrium, Schizochytrium, or Thraustochytrium is more preferred. These species have a relatively high ability to produce lipids and other substances, and are capable of producing hydrocarbons such as squalene, and are therefore suitable for use as food or as a raw material for biofuels.

[0035] Labyrinthulea may be cultured by any culture method, such as batch culture, continuous culture, or fed-batch culture. Labyrinthulea can be cultured by any appropriate culture method, such as shaking culture, aeration culture, aeration-agitation culture, airlift culture, or static culture. Among these culture methods, aeration-agitation culture or airlift culture is more preferred. Examples of culture devices that can be used for culturing Labyrinthulea include mechanically agitated reactors, airlift reactors, packed-bed reactors, and fluidized-bed reactors. Various types of containers, such as tanks, jar fermenters, flasks, dishes, culture bags, tubes, and test tubes, can be used as culture vessels depending on the purpose of the culture, the culture volume, and the like. Culture vessels may be made of any appropriate material, such as inorganic materials, such as stainless steel and glass, or organic materials, such as polystyrene, polyethylene terephthalate copolymer, and polypropylene.

[0036] Labyrinthulea can be cultured under appropriate temperature, pH, aeration, etc. The culture temperature is preferably 5°C to 40°C, more preferably 10°C to 35°C, and even more preferably 10°C to 30°C. The pH is preferably 2 to 11, more preferably 4 to 9, and even more preferably 6 to 8.

[0037] Labyrinthules can be cultured by subculturing at appropriate intervals depending on the genus and species of Labyrinthules, medium composition, culture conditions, etc. For example, after the start of culture, Labyrinthules complete their logarithmic growth phase about two days later and enter their death phase about seven days later. Therefore, Labyrinthules are preferably subcultured at intervals of 1 to 10 days, more preferably at intervals of 2 to 7 days, and even more preferably at intervals of 2 to 5 days. Labyrinthules can be cultured for an appropriate period depending on the genus and species of Labyrinthules, medium composition, culture conditions, and the purpose of the culture. Labyrinthules of the genus Aurantiochytrium are particularly preferred because they are heterotrophic algae that live in brackish waters and are characterized by assimilating nutrients in water to produce lipids, which accumulate intracellularly.

[0038] It is preferable to use Aurantiochytrium algae strains with excellent ability to produce the desired triglycerides. Such algae strains may be naturally collected and isolated, cloned through mutagenesis and screening, or established using genetic engineering. For example, Aurantiochytrium sp. SA-96, NIES-3737, Aurantiochytrium NB6-3, and Aurantiochytrium mh1959 are particularly preferred microorganisms for use in producing the active ingredient of the present invention because they have the ability to intracellularly accumulate large amounts of triglycerides containing the odd-chain fatty acid pentadecanoic acid (PDA) and triglycerides containing the highly unsaturated fatty acids docosahexaenoic acid (DHA) and docosapentaenoic acid (DPA).

[0039] The cultivation of the Aurantiochytrium algae is carried out by methods established in the art. That is, typical maintenance cultivation is carried out by seeding the algae in a medium with appropriately prepared ingredients, according to a standard method. The medium for culturing Aurantiochytrium algae essentially contains salt, a carbon source, and a nitrogen source. Generally, the so-called GTY medium (artificial seawater 10-40 g / L, D(+) glucose 20-100 g / L, tryptone 10-60 g / L, yeast extract 5-40 g / L) is used for culturing microalgae.

[0040] Carbon sources include sugars such as glucose, fructose, sucrose, etc. These carbon sources are added at a concentration of, for example, 20 to 120 g per liter of medium.

[0041] Aurantiochytrium algae are marine algae, and an appropriate amount of artificial seawater is added to the culture medium. Preferably, the artificial seawater is added so that the final salinity of the culture medium is about 10% (v / v) to about 100% (v / v) of seawater (salinity 3.4% (w / v)), for example, about 1.0 to 3.0% (w / v).

[0042] Generally, various nitrogen sources can be added to microalgae culture media, such as organic nitrogen sources (e.g., sodium glutamate, urea, etc.), inorganic nitrogen sources (e.g., ammonium acetate, ammonium sulfate, ammonium chloride, sodium nitrate, ammonium nitrate, etc.), or biological digests (e.g., yeast extract, corn steep liquor, polypeptone, peptone, tryptone, etc.). Cell extracts obtained by extracting liquid components from various animal cells are particularly preferred as nitrogen sources for the culture media used to culture Aurantiochytrium algae. When cells must be mass-cultured on an industrial scale to obtain cultured cell products, the use of cell extracts, which are rich in cell-derived nutrients (e.g., amino acids, nucleic acids, vitamins, minerals, etc.) and available at low cost, is extremely advantageous.

[0043] However, as described above, when a medium prepared based on a cell extract is used, the proportion of odd-numbered fatty acids in the triglycerides produced by the cultured algae is significantly reduced, making it impossible to use a cell extract as a nitrogen source for the medium when efficiently producing the target product of the present invention. Therefore, the present inventors cultured Aurantiochytrium algae in an algal culture medium prepared by adding a cell extract that had been treated with a strong acid, and found that the amount of odd-numbered fatty acids produced was dramatically increased compared to when a cell extract that had not been treated with the strong acid was added. They have already reported a method for producing triglycerides containing odd-numbered fatty acids as the main component (Japanese Patent Laid-Open Publication No. 2017-063633).

[0044] Furthermore, in a preferred embodiment of the present invention, a basal medium for culturing Aurantiochytrium algae is prepared by adding 10-50 mM valine and 10-50 mM sodium propionate to a medium containing 2% or more glucose, 0.5-4% sodium glutamate, 0.1-2% yeast extract, 1-3.3% sea salt, and 2-20% whey (animal or vegetable). The animal or vegetable whey is preferably tofu whey (soybean whey). To this basal medium, 2% or more of an Aurantiochytrium culture solution pre-cultured in 2% or more glucose, 0.5-4% sodium glutamate, 0.1-2% yeast extract, 1-3.3% sea salt, and 2-20% whey (animal or vegetable) at 20-30°C for 72 hours is added. The Aurantiochytrium-added culture solution is aerated and gently stirred. The culture is carried out for 48 to 200 hours at 20 to 30°C and a pH maintained at 5.0 to 8.5 (pH is adjusted using 1.0 M NaOH solution). After the culture, Aurantiochytrium cells that produce pentadecanoic acid triglyceride can be collected by centrifugation (see WO2020 / 054804).

[0045] The pellet recovered from the culture medium obtained by the above-mentioned method, for example, by centrifugation or filtration, is then dried by freeze-drying or heating. Alternatively, the culture medium in which the cultured algal cells are suspended may be used directly for the triglyceride extraction step. Extraction may be performed multiple times using different organic solvents. Examples of organic solvents that can be used include mixtures of polar and weakly polar solvents, such as n-hexane / ethanol mixtures, chloroform / methanol mixtures, or ethanol / diethyl ether mixtures. The resulting extract is purified using methods known to those skilled in the art.

[0046] The triglyceride separation method employs a fractionation method known to those skilled in the art. Separation and purification may be carried out by utilizing various physicochemical properties of the triglyceride molecules to be fractionated, such as polarity, solubility in a solvent, melting point, specific gravity, and molecular weight, and preferably employs column chromatography. The conditions for the triglyceride separation method can be determined by those skilled in the art through routine condition studies, depending on the composition of the triglyceride mixture and the type of triglyceride to be fractionated.

[0047] The algae of the genus Schizochytrium and Aurantiochytrium can synthesize and accumulate both odd-chain fatty acid triglycerides and highly unsaturated fatty acid triglycerides within their cells. Therefore, ethanol, hexane, or ethyl acetate is added to the obtained algal cells to extract lipids, and the solvent is then distilled off to obtain algal lipids. The active ingredient, pentadecanoic acid triglyceride, can be precipitated by leaving the lipids at 5°C. The composition of the purified pentadecanoic acid triglyceride can be analyzed using HPLC-MS, HPLC, gas chromatography, etc.

[0048] Aurantiochytrium algae can synthesize and accumulate both odd-chain fatty acid triglycerides and highly unsaturated fatty acid triglycerides intracellularly. Therefore, hexane or ethyl acetate is added to the resulting Aurantiochytrium cells to extract lipids. The lipid solution is then oxidized by adding hydrogen peroxide or by aerating ozone. After the reaction is complete, the oxidized products are removed using sodium bicarbonate and sodium carbonate or an ion exchange resin to obtain the active ingredient, pentadecanoic acid triglyceride. The composition of the purified pentadecanoic acid triglyceride can be analyzed by HPLC-MS, HPLC, gas chromatography, etc. The pentadecanoic acid triglyceride obtained in this method for producing the active ingredient may also be referred to as "DM-PdATG."

[0049] (Pharmaceuticals) A pharmaceutical according to an embodiment of the present disclosure (hereinafter referred to as a pharmaceutical according to an embodiment) contains a triglyceride represented by formula (I) as an active ingredient. A pharmaceutical composition according to an embodiment of the present disclosure (hereinafter referred to as a pharmaceutical composition according to an embodiment) contains a triglyceride represented by formula (I) (pentadecanoic acid triglyceride) as an active ingredient. The triglyceride represented by formula (I) can be appropriately selected from the various forms described for the proinflammatory cytokine suppressor according to the embodiment above. In a preferred embodiment of a pharmaceutical composition or pharmaceutical according to the pharmaceutical composition, the triglyceride represented by formula (I) is "DM-PdATG." The term "pharmaceutical" refers to a therapeutic agent for preventing, inhibiting the progression of, and / or ameliorating symptoms in patients with disorders manifested by the production of IL-6 and IL-1β and related disorders, as described above, by suppressing the production of IL-6 and IL-1β.

[0050] The pharmaceutical composition of one embodiment or a drug made from the same is effective not only for humans but also for mammals including livestock such as cows, horses, pigs, and goats, as well as pets such as dogs and cats.

[0051] The pharmaceutical composition or drug product of one embodiment may contain only the pentadecanoic acid triglyceride represented by formula (I) as the active ingredient, or may contain other ingredients as long as they do not inhibit the inhibitory effect on IL-6 and IL-1β production. The other ingredients may be, for example, conventionally used therapeutic or prophylactic drugs. Therefore, in a further aspect, the proinflammatory cytokine inhibitor of this embodiment provides a pharmaceutical composition for inhibiting IL-6 and IL-1β production.

[0052] In addition, the pharmaceutical composition of one embodiment or a pharmaceutical product thereof can be administered orally and can be prepared in dosage forms suitable for oral administration, such as granules, powders, tablets (including sugar-coated tablets), pills, capsules, syrups, emulsions, and suspensions. These preparations can be formulated using pharmaceutically acceptable carriers by methods commonly used in the art. Pharmaceutically acceptable carriers include excipients, binders, diluents, additives, flavorings, buffers, thickeners, colorants, stabilizers, emulsifiers, dispersants, suspending agents, and preservatives.

[0053] More specifically, for example, when a pharmaceutical product is produced by blending pentadecanoic acid triglyceride represented by formula (I), any auxiliary agent can be added, for example, sugars such as dextrin and starch; proteins such as gelatin, soybean protein and corn protein; amino acids such as alanine, glutamine and isoleucine; polysaccharides such as cellulose and gum arabic; and fats and oils such as soybean oil and medium-chain fatty acid triglyceride, and the like, to formulate the product into any dosage form.

[0054] The amount of pentadecanoic acid triglyceride represented by formula (I) in the pharmaceutical product of one embodiment is not particularly limited, but is preferably adjusted so that the effective concentration of pentadecanoic acid triglyceride is approximately 5 to 1000 mg per day for an adult. The pharmaceutical product of a preferred embodiment contains 5 mg to 1000 mg of the active ingredient.

[0055] Furthermore, the pharmaceutical composition of one embodiment and the pharmaceutical product thereof are not limited to oral administration forms, but can also be parenterally administered, for example, in the form of an injection, an infusion, etc. In this case, too, they can be formulated using pharmaceutically acceptable adjuvants, carriers, etc., by methods commonly used in the art.

[0056] (food) A food product according to an embodiment of the present disclosure (hereinafter referred to as a food product of one embodiment) contains a pentadecanoic acid triglyceride represented by formula (I). The triglyceride represented by formula (I) can be appropriately selected from the various forms described for the proinflammatory cytokine inhibitor of one embodiment above. A preferred food product contains "DM-PdATG." The food product of one embodiment can be taken over a long period of time as a preventive food or drink, starting before the onset of IL-6 and IL-1β production, and is useful as a health food. Pentadecanoic acid, which constitutes the active ingredient, has been reported to be found in small amounts in edible parts of meat from beef, pork, chicken, and lamb, fish found in rivers and seas, and mushrooms. Furthermore, pentadecanoic acid triglyceride compounds are also found in extremely small amounts, and their safety is inferred from years of dietary experience.

[0057] Therefore, the food of this embodiment is useful as a health food to be taken for health promotion. Here, "health food" means food and drink intended for use in promoting health in daily life and for preventing, preventing the progression of, alleviating, or improving symptoms such as swelling that is prone to occur with age, swelling of the fingers, face, shins, and insteps of the feet, changes in urine, difficulty urinating, frequent urination, abnormally foamy urine, urine that turns bloody and brown, heavy body, lethargy, fatigue, and cystitis, and refers to "health foods" in a broad sense, including functional foods, foods with nutrient claims, or foods for specified health uses under the "Food with Health Claims System" that meet national standards for safety and efficacy.

[0058] When producing a food by blending the pentadecanoic acid triglyceride represented by formula (I), any auxiliary agent can be added, for example, sugars such as dextrin and starch; proteins such as gelatin, soybean protein and corn protein; amino acids such as alanine, glutamine and isoleucine; polysaccharides such as cellulose and gum arabic; oils and fats such as soybean oil and medium-chain fatty acid triglycerides, and the like, to formulate the food into any dosage form.

[0059] The amount of active ingredient in the food product of one embodiment is not particularly limited, but is preferably adjusted so that the daily intake of the active ingredient per adult is about 1 to 100 mg, taking into consideration the general intake of the food product to be added. The food product of a preferred embodiment contains the active ingredient in an amount of 1 mg to 100 mg.

[0060] Specific examples of the above foods include beverages such as soft drinks, carbonated drinks, energy drinks, fruit drinks, and lactic acid drinks (including concentrates and powders for adjusting these beverages); frozen desserts such as ice cream, ice sherbet, and shaved ice; noodles such as soba, udon, vermicelli, gyoza wrappers, shumai wrappers, Chinese noodles, and instant noodles; sweets such as candy, candy, gum, chocolate, snacks, biscuits, jelly, jam, cream, and baked goods; processed seafood and livestock foods such as kamaboko, ham, and sausage; dairy products such as processed milk and fermented milk; oils and fats and oil-based foods such as salad oil, tempura oil, margarine, mayonnaise, shortening, whipped cream, and dressings; condiments such as sauces and dressings; health and nutritional supplements in various forms such as tablets and granules; and other foods such as soups, stews, salads, side dishes, and pickles.

[0061] In one embodiment, the food product may contain various food additives, such as antioxidants, flavorings, various esters, organic acids, organic acid salts, inorganic acids, inorganic acid salts, inorganic salts, colorants, emulsifiers, preservatives, seasonings, sweeteners, acidulants, fruit juice extracts, vegetable extracts, nectar extracts, pH adjusters, and quality stabilizers, either alone or in combination.

[0062] The content concentration of pentadecanoic acid triglyceride in the food product of one embodiment is about 0.00001 to 100% by mass (hereinafter expressed in %), preferably about 0.0005 to 50%, as solid content, to obtain ease of use and good effects.

[0063] The present invention will be described in more detail below with reference to examples, but the present invention is not limited to these examples. In the following examples, the unit % used to indicate the amount of each component added means % by mass. All data are expressed as mean ± standard error (SE). Significance tests were performed using one-way ANOVA, with Tukey's test used post hoc. [Example]

[0064] (Cytotoxicity study) BV-2 cells were placed in a 96-well microplate for the MTT assay and in an 8-well glass chamber slide for the LIVE / DEAD assay at a density of 1.2 × 10 4 cells / cm 2 The cells were seeded at a density of 100 μg / well and cultured for 24 hours in a humidified 10% CO2 incubator at 37°C. After culture, PdATG and LPS (lipopolysaccharide) were added to each well in the amounts shown in Figure 1, and the cells were cultured for 24 hours in a 10% CO2 incubator.

[0065] The "PdATG" used in the examples is a compound having a ratio of R in the above formula (I) to the total mass of "PdATG". 1 , R 2 and R 3 30.6% by mass of triglyceride in which all of R in the formula (I) are pentadecanoic acid residues, 1 , R 2 and R 3 and 18.8% by mass of a triglyceride in which two of the residues are pentadecanoic acid residues and the other is a myristic acid residue, 1 , R 2 and R 3 The composition contains 22.3% by mass of triglycerides in which two of the residues are pentadecanoic acid residues and the other is a palmitic acid residue. The remainder is other triglycerides represented by the above formula (I).

[0066] (Assessed by MTT method) After exposure, all medium was removed, and 100 μL of 0.25 mg / mL 3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (Dojindo Laboratories) was added. The cells were incubated at 37°C for 3 hours. The reaction was stopped using a solution containing 50% dimethylformamide and 20% sodium dodecyl sulfate. The absorbance at 570 nm was measured to assess the number of viable cells. The viability of each treatment group was calculated based on the absorbance of untreated cells cultured without solvent (no medium treatment). Figure 1 shows the results of cytotoxicity evaluation using the MTT assay. Sample No. A1 in Figure 1 is cells untreated with LPS and "PdATG," Sample Nos. A2 to A4 are cells treated with "PdATG" alone, Sample No. B1 is cells treated with LPS alone, and Sample Nos. B2 to B4 are cells co-treated with LPS and "PdATG."

[0067] As is clear from the results in Figure 1, cells treated with "PdATG" alone (samples No.: A2-A4) had no effect on MTT reduction ability (viability) compared to control cells treated with medium (sample No.: A1). Cells treated with LPS alone (sample No.: B1) showed a slight decrease in MTT reduction ability to 87.2±1.3% compared to control cells, indicating a gradual decrease in viability. It was confirmed that "PdATG" did not affect the decrease in MTT reduction ability caused by LPS, regardless of the concentration (samples No.: B2-B4).

[0068] (Evaluation by LIVE / DEAD method) LIVE / DEAD TMViable and dead cells were stained for each of the samples shown in Figure 1 (A1, A4, B1, and B4) using the Viability / Cytotoxicity Kit for Mammalian Cells (Thermo Fisher Scientific). Two μM calcein AM and 4 μM ethidium homodimer-1 (EthD-1) were added to each chamber, and the cells were incubated at 37°C in a humidified, 10% CO2 incubator for 40 minutes. Fluorescence microscopy images were then acquired using a confocal laser scanning microscope (LSM-710; Zeiss). The cell death rate was calculated by counting live calcein AM-positive cells (green) and dead EthD-1-positive cells (red), and then expressing the percentage of dead EthD-1-positive cells relative to the total number of live calcein AM-positive cells and dead EthD-1-positive cells. Figure 2 shows the results of the LIVE / DEAD assay (n = 4, scale bar = 100 μm, ns: no significance). FIG. 2(A) shows a fluorescence microscope image, FIG. 2(B) shows the cell death rate, and FIG. 2(C) shows the total cell number.

[0069] As can be seen from the fluorescence microscopy images in Figure 2(A), cells treated with LPS alone (sample No. B1) did not show any significant increase in floating, degenerated, or atrophic cells. Furthermore, cells treated with PdATG alone (sample No. A4) or cells co-treated with LPS and PdATG (sample No. B4) showed no significant morphological changes. Furthermore, as shown in Figures 2(B) and 2(C), cells treated with PdATG alone, LPS alone, or co-treated with LPS and PdATG showed no changes in the cell death rate (Figure 2(B)) or total cell number (Figure 2(C)). These results suggest that the decrease in MTT reduction activity induced by LPS is not due to cell death in BV-2 cells, but is instead related to the inhibition of cell division. It was also suggested that "PdATG" alone is not cytotoxic and has no protective effect against LPS-induced inhibition of BV-2 cell division.

[0070] (Effects on cytokine production) To evaluate the effect of "PdATG" on the production of various cytokines, BV-2 cells were cultured in a 60 mm dish at 1.2 × 10 4 cells / cm 2 The cells were seeded at a density of 10 μg / mL and cultured for 24 hours in an incubator at 37°C with 10% CO2 and humidified atmosphere. After culture, PdATG and / or LPS were added to each dish, and the cells were cultured for 4 hours in an incubator at 37°C with 10% CO2 and humidified atmosphere. The amounts of PdATG and LPS added (μg / mL) are shown in Figures 3 and 4. Negative controls were cells treated with solvent alone, and positive controls were cells treated with LPS alone. To measure the mRNA levels of various cytokines, total RNA was extracted from the cells using a High Pure RNA Isolation Kit (Roche). RNA was reverse transcribed into cDNA using a Transcriptor First Strand cDNA Synthesis Kit (Roche). After cDNA synthesis, TB Green® Premix Ex Taq TM Real-time PCR was performed using a PCR-PCR kit (TaKaRa). The mRNA expression of various cytokines was calculated by the ΔΔC method using the expression level of GAPDH as an internal standard (n=4). The calculation results are shown in Figures 3 and 4. Figure 3 is a graph showing the mRNA levels of inflammatory cytokines induced by LPS by RT-PCR. Figure 3(A) shows the mRNA levels of inflammatory cytokine (IL-6), Figure 3(B) shows the mRNA levels of inflammatory cytokine (IL-1β), and Figure 3(C) shows the mRNA levels of inflammatory cytokine (TNF-α).

[0071] As is clear from the results in Figure 3, no increase or decrease in inflammatory cytokines (IL-6, IL-1β, TNF-α) was observed in cells treated with "PdATG" alone. On the other hand, it was confirmed that "PdATG" can selectively suppress the increased production of IL-6 and IL-1β mRNA induced by LPS.

[0072] As is clear from the results in Figure 4, exposure of BV-2 cells to LPS did not induce the production of anti-inflammatory cytokines TGF-β and IL-10. Furthermore, no induction of TGF-β or IL-10 expression was observed in cells treated with "PdATG" alone. Similarly, no increase in the production of anti-inflammatory cytokines TGF-β or IL-10 was observed in cells co-treated with "PdATG" and LPS.

[0073] From the above results, it was confirmed that "PdATG" has extremely low cytotoxicity and selectively suppresses the production of inflammatory cytokines without inhibiting the proliferation of microglia.

[0074] (Industrial Applicability) The PdATG of the present invention is effective in treating disorders manifested by the overproduction of IL-6 and IL-1β, which are representative inflammatory factors of inflammatory cytokines, such as Neurological disorders such as Alzheimer's disease, Parkinson's disease, multiple sclerosis, dementia with Lewy bodies, neuromyelitis optica, spinocerebellar degeneration, Huntington's disease, spinal cord injury, neuropathic pain, and amyotrophic lateral sclerosis. Mental illnesses such as schizophrenia, depression, bipolar disorder, autism, and sleep disorders Arthritis, muscle pain, muscle fatigue Blood vessel diseases such as cerebral infarction, myocardial infarction, vasculitis, and sepsis Allergic diseases such as hay fever, allergic rhinitis, and atopic dermatitis Lifestyle-related diseases such as obesity, hyperglycemia, hypertension, cancer, liver disease, and lung disease Complications or aftereffects of pneumonia, encephalitis, upper respiratory tract infections, rhinitis, etc. associated with viral or bacterial infections such as COVID-19, influenza, and pneumococcal infections, The present invention provides an inflammatory cytokine suppressor that can be used to prevent, inhibit the progression of, and / or ameliorate inflammatory disease, has no or little cytotoxicity, and is capable of suppressing the production of IL-6 and IL-1β.

Claims

1. The following formula (I) 【Chemical 1】 (In the formula, R １ , R ２ and R ３ are saturated fatty acid residues, at least one of which is a pentadecanoic acid residue. To suppress the production of interleukin-6 and interleukin-1β, Inflammatory cytokine inhibitor.

2. Contains 1 mg or more and 100 mg or less of the active ingredient, The inflammatory cytokine suppressor according to claim 1.

3. R in formula (I) １ , R ２ and R ３ wherein all of R in formula (I) are pentadecanoic acid residues, １ , R ２ and R ３ any two of which are pentadecanoic acid residues, The inflammatory cytokine suppressor according to claim 1 or 2.

4. R in formula (I) １ , R ２ and R ３ wherein all of R in formula (I) are pentadecanoic acid residues, １ , R ２ and R ３ Any two of the above triglycerides are pentadecanoic acid residues, and the total mass of the triglycerides is 30% by mass or more of the total mass of the active ingredient. The inflammatory cytokine suppressor according to claim 1 or 2.

5. R in formula (I) １ , R ２ and R ３ wherein the mass of the triglyceride in which all of the residues are pentadecanoic acid is 10% by mass or more of the total mass of the active ingredient; The inflammatory cytokine suppressor according to claim 4.

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