Inhibitor of primary cilia of immune-related cells and use thereof

Abstract

The present invention addresses the problem of providing a regulator for primary cilia of immune-related cells and a technique for the use thereof. Use is made of a regulator for primary cilia of immune-related cells that contains at least one selected from the group consisting of silymarin, silybin, dehydrosilybin, annonacin and quercetin.

Description

[Technical Field]

[0001] The present invention relates to an inhibitor of primary cilia of immune-related cells and use thereof. [Background technology]

[0002] Inflammation is a biological response caused by chemical and / or physical stimuli. Because inflammation can damage biological tissue, anti-inflammatory drugs are sometimes used to treat inflammation.

[0003] As research progresses, it is becoming clear that there are various mechanisms by which inflammation develops. This indicates that there are anti-inflammatory drugs that are appropriate for each type of inflammation, and therefore, the development of new anti-inflammatory drugs is being actively pursued.

[0004] The present inventors have previously found that immune-related cells contain organelles called primary cilia, and that the expression of primary cilia is increased in inflammatory skin diseases (see, for example, Patent Documents 1 to 3). This suggests that inhibitors of primary cilia in immune-related cells can be used as anti-inflammatory agents. [Prior art documents] [Patent documents]

[0005] [Patent Document 1] International Publication No. WO2019 / 172419 [Patent Document 2] Patent Publication No. 2021-73928 [Patent Document 3] Patent Publication No. 2021-75516 Summary of the Invention [Problem to be solved by the invention]

[0006] However, no inhibitors of primary cilia of immune-related cells have been found, and the development of such inhibitors has been desired.

[0007] An object of one aspect of the present invention is to provide an agent for inhibiting primary cilia of immune-related cells and a technique for using the same. [Means for solving the problem]

[0008] As a result of extensive research to solve the above problems, the present inventors have found that (i) silymarin, silybin, and dehydrosilybin, which are components of an extract of milk thistle (Silybum marianum), and (ii) annonacin and quercetin, which are components of an extract of graviola (Annona muricata), have the effect of suppressing the expression of primary cilia in immune-related cells, and have completed the present invention. That is, the present invention comprises the following features.

[0009] <1> An inhibitor of primary cilia of immune-related cells, comprising at least one selected from the group consisting of A to E below: A: Silymarin, B: Silybin, C: Dehydrosilybin, D: Annonacin, E: Quercetin. [Effects of the Invention]

[0010] According to one aspect of the present invention, an agent for inhibiting primary cilia of immune-related cells and a technique for using the same can be provided. [Brief explanation of the drawings]

[0011] [Figure 1] Graph 101 shows the inhibitory effect of silymarin on primary cilia in immune-related cells, and graph 102 shows the inhibitory effect of silybin on primary cilia in immune-related cells. [Figure 2] Graphs 201 and 202 show the inhibitory effects of 2,3-dehydrosilybin A and 2,3-dehydrosilybin B on primary cilia in immune-related cells, respectively. [Figure 3]Graphs 301, 302 and 303 respectively show the inhibitory effect of cosmetic raw materials containing materials derived from Milk Thistle on primary cilia in immune-related cells. [Figure 4] 401 and 402 are graphs showing the inhibitory effects of annonacin and quercetin hydrate on primary cilia in immune-related cells, respectively. DETAILED DESCRIPTION OF THE INVENTION

[0012] The present invention is described below, but is not limited thereto. The present invention is not limited to the respective configurations described below, and various modifications are possible within the scope of the claims. Furthermore, embodiments or examples obtained by appropriately combining the technical means disclosed in different embodiments or examples are also included in the technical scope of the present invention. Furthermore, new technical features can be formed by combining the technical means disclosed in each embodiment. All academic literature and patent documents described in this specification are incorporated herein by reference. Unless otherwise specified in this specification, the term "X to Y" representing a numerical range means "greater than or equal to X and less than or equal to Y."

[0013] [1. Inhibitors of primary cilia in immune-related cells] An inhibitor of primary cilia of immune-related cells in one embodiment of the present invention contains at least one selected from the group consisting of A to E below: A: silymarin, B: silybin, C: dehydrosilybin, D: annonacin, E: quercetin.

[0014] The immune-related cells include immune cells that are primarily responsible for immune responses and immune function-retaining cells that are indirectly involved in immune cells. Immune function-retaining cells have, for example, the function of activating immune cells. Examples of immune cells include skin dendritic cells (e.g., Langerhans cells, dermal dendritic cells), lymphocytic immune cells (e.g., T cells, NK cells, B cells), and monocytic immune cells (e.g., conventional dendritic cells, monocytic dendritic cells (e.g., plasmacytoid dendritic cells)). On the other hand, examples of immune function-retaining cells include keratinocytes, fibroblasts, and epithelial cells. Immune-related cells may be immune-related cells collected from a living body or may be established immune-related cell lines (e.g., HaCaT cells).

[0015] Silymarin (CAS No. 65666-07-1) is a mixture of flavonolignans obtained from milk thistle (Silybum marianum). Silymarin contains silybin and dehydrosilybin from the compounds B to E mentioned above.

[0016] As the silymarin, commercially available silymarin can be used, or silymarin obtained from an extract of milk thistle can also be used. The method for obtaining silymarin from an extract of milk thistle is not limited. For example, first, milk thistle seeds are extracted with an organic solvent (e.g., ethanol, methanol, acetone, or ethyl acetate) to obtain an extract. Next, lipids and highly polar impurities are removed from the extract, and the extract is dried by a method such as spray drying to obtain silymarin.

[0017] As described above, silymarin can be obtained from an extract of Milk thistle. Therefore, an inhibitor of primary cilia of immune-related cells according to one embodiment of the present invention may be an inhibitor of primary cilia of immune-related cells containing a material derived from Milk thistle (e.g., an extract or pulverized product of Milk thistle (e.g., seeds, leaves, stems, buds, flowers, or roots of Milk thistle)).

[0018] Silybin (CAS No. 22888-70-6) is one of the compounds contained in the above-mentioned silymarin.

[0019] As silybin, commercially available silybin can be used, or silybin obtained from an extract of milk thistle can be used. The method for obtaining silybin from a milk thistle extract is not limited. For example, milk thistle seeds are first extracted with an organic solvent (e.g., ethanol, methanol, acetone, or ethyl acetate) to obtain an extract. Silybin can then be obtained by further purifying the extract using known purification methods (e.g., column chromatography, high-performance liquid chromatography).

[0020] As described above, silybin can be obtained from an extract of Milk thistle. Therefore, an agent for inhibiting primary cilia of immune-related cells according to one embodiment of the present invention may be an agent for inhibiting primary cilia of immune-related cells that contains a material derived from Milk thistle (e.g., an extract or pulverized product of Milk thistle (e.g., seeds, leaves, stems, buds, flowers, or roots of Milk thistle)).

[0021] Dehydrosilybin (for example, 2,3-dehydrosilybin A (CAS No. 25166-14-7) and 2,3-dehydrosilybin B (CAS No. 142796-24-5)) is one of the compounds contained in the above-mentioned silymarin.

[0022] As dehydrosilybin, commercially available dehydrosilybin can be used, or dehydrosilybin obtained from an extract of milk thistle can also be used. The method for obtaining dehydrosilybin from a milk thistle extract is not limited. For example, milk thistle seeds are first extracted with an organic solvent (e.g., ethanol, methanol, acetone, or ethyl acetate) to obtain an extract. The extract can then be further purified by known purification methods (e.g., column chromatography, high-performance liquid chromatography) to obtain dehydrosilybin.

[0023] As described above, dehydrosilybin can be obtained from an extract of Milk thistle. Therefore, an agent for inhibiting primary cilia of immune-related cells according to one embodiment of the present invention may be an agent for inhibiting primary cilia of immune-related cells that contains a material derived from Milk thistle (e.g., an extract or pulverized product of Milk thistle (e.g., seeds, leaves, stems, buds, flowers, or roots of Milk thistle)).

[0024] Annonacin (CAS No. 111035-65-5) is a compound obtained from graviola (Annona muricata).

[0025] As annonacin, commercially available annonacin can be used, or annonacin obtained from an extract of graviola can also be used. The method for obtaining annonacin from an extract of graviola is not limited. For example, first, graviola seeds or leaves are extracted with an organic solvent (e.g., ethanol, methanol, acetone, or ethyl acetate) to obtain an extract. Next, the extract can be further purified by a known purification method (e.g., column chromatography, high-performance liquid chromatography) to obtain annonacin.

[0026] As described above, annonacin can be obtained from an extract of graviola. Therefore, an inhibitor of primary cilia of immune-related cells according to one embodiment of the present invention may be an inhibitor of primary cilia of immune-related cells containing a material derived from graviola (e.g., an extract or pulverized product of graviola (e.g., seeds, leaves, stems, buds, flowers, or roots of graviola)).

[0027] Quercetin (CAS No. 117-39-5) is a compound obtained from graviola (Annona muricata). The quercetin may be in the form of a hydrate (quercetin hydrate).

[0028] As the quercetin, commercially available quercetin can be used, or quercetin obtained from a graviola extract can be used. The method for obtaining quercetin from a graviola extract is not limited. For example, first, graviola seeds or leaves are extracted with an organic solvent (e.g., ethanol, methanol, acetone, or ethyl acetate) to obtain an extract. Then, the extract can be further purified by a known purification method (e.g., column chromatography, high-performance liquid chromatography) to obtain quercetin.

[0029] As described above, quercetin can be obtained from an extract of graviola. Therefore, an inhibitor of primary cilia of immune-related cells according to one embodiment of the present invention may be an inhibitor of primary cilia of immune-related cells containing a material derived from graviola (e.g., an extract or pulverized product of graviola (e.g., seeds, leaves, stems, buds, flowers, or roots of graviola)).

[0030] The amount of the active ingredient (in other words, at least one selected from the group consisting of silymarin, silybin, dehydrosilybin, annonacin, and quercetin) contained in the inhibitor of primary cilia of immune-related cells according to one embodiment of the present invention is not particularly limited, and may be, for example, 0.00001% by mass to 100% by mass, 0.0001% by mass to 100% by mass, 0.001% by mass to 100% by mass, or 0.01% by mass to 100% by mass, assuming the inhibitor to be 100% by mass. It may be 0.1% by mass to 100% by mass, 0.1% by mass to 95% by mass, 0.1% by mass to 90% by mass, 0.1% by mass to 80% by mass, 0.1% by mass to 70% by mass, 0.1% by mass to 60% by mass, 0.1% by mass to 50% by mass, 0.1% by mass to 40% by mass, 0.1% by mass to 30% by mass, 0.1% by mass to 20% by mass, or 0.1% by mass to 10% by mass.

[0031] As can be seen from the examples described below, silymarin, silybin, dehydrosilybin, annonacin, and quercetin can better suppress the expression of primary cilia in immune-related cells when contacted with the cells at concentrations of 48 μg / mL or more, 100 μM or more, 10 μM or more, 0.5 μM or more, and 10 μM or more, respectively.

[0032] When the active ingredient is silymarin, an inhibitor of primary cilia of immune-related cells according to one embodiment of the present invention preferably contains the active ingredient in an amount that allows the active ingredient to come into contact with immune-related cells at a concentration of 30 μg / mL or more, 48 μg / mL or more, 75 μg / mL or more, 1 mg / mL or more, or 5 mg / mL or more (the upper limit of the concentration is not limited, for example, 100 mg / mL or 10 mg / mL) when administered to a subject. Of course, an inhibitor of primary cilia of immune-related cells according to one embodiment of the present invention (e.g., a liquid, gel, or cream) may contain silymarin or silybin at the above concentrations. This configuration allows for better inhibition of primary cilia expression in immune-related cells.

[0033] When the active ingredient is silybin, an inhibitor of primary cilia in immune-related cells according to one embodiment of the present invention preferably contains the active ingredient in an amount that allows the active ingredient to come into contact with immune-related cells at a concentration of 75 μM or more, 100 μM or more, 500 μM or more, 1 mM or more, or 5 mM or more (the upper limit of the concentration is not limited, and is, for example, 1 mM, 100 mM, or 10 mM) when administered to a subject. Of course, an inhibitor of primary cilia in immune-related cells according to one embodiment of the present invention (e.g., a liquid, gel, or cream) may also contain silymarin or silybin at the above concentrations. This configuration allows for better inhibition of the expression of primary cilia in immune-related cells.

[0034] When the active ingredient is dehydrosilybin, an inhibitor of primary cilia in immune-related cells according to one embodiment of the present invention preferably contains the active ingredient in an amount that allows the active ingredient to come into contact with immune-related cells at a concentration of 8 μM or more, 10 μM or more, 25 μM or more, 50 μM or more, 100 μM or more, or 500 μM or more (the upper limit of the concentration is not limited, and is, for example, 1 μM, 100 mM, or 10 mM) when administered to a subject. Of course, an inhibitor of primary cilia in immune-related cells according to one embodiment of the present invention (e.g., a liquid, gel, or cream) may contain dehydrosilybin at this concentration. This configuration allows for better inhibition of the expression of primary cilia in immune-related cells.

[0035] When the active ingredient is annonacin, an inhibitor of primary cilia of immune-related cells according to one embodiment of the present invention preferably contains the active ingredient in an amount that allows the active ingredient to come into contact with immune-related cells at a concentration of 0.2 μM or more, 0.5 μM or more, 1 μM or more, 5 μM or more, or 10 μM or more (the upper limit of the concentration is not limited, and is, for example, 1 μM, 100 mM, or 10 mM) when the inhibitor is administered to a subject. Of course, an inhibitor of primary cilia of immune-related cells according to one embodiment of the present invention (e.g., a liquid, gel, or cream) may contain annonacin at such a concentration. With this configuration, the expression of primary cilia in immune-related cells can be more effectively inhibited.

[0036] When the active ingredient is quercetin, an inhibitor of primary cilia of immune-related cells according to one embodiment of the present invention preferably contains the active ingredient in an amount that allows the active ingredient to come into contact with immune-related cells at a concentration of 8 μM or more, 10 μM or more, 50 μM or more, 100 μM or more, or 500 μM or more (the upper limit of the concentration is not limited, and is, for example, 1 μM, 100 mM, or 10 mM) when administered to a subject. An inhibitor of primary cilia of immune-related cells according to one embodiment of the present invention (e.g., a liquid, gel, or cream) may contain quercetin at this concentration. With this configuration, the expression of primary cilia in immune-related cells can be more effectively suppressed.

[0037] Dehydrosilybin and annonacin can more efficiently suppress the expression of primary cilia in immune-related cells at lower concentrations than silymarin, silybin, and quercetin. Therefore, it is thought that inhibitors containing dehydrosilybin and annonacin have a stronger inhibitory effect on the expression of primary cilia in immune-related cells than inhibitors containing silymarin, silybin, and quercetin.

[0038] The agent for inhibiting primary cilia of immune-related cells according to one embodiment of the present invention may contain ingredients other than the above-mentioned active ingredients.

[0039] The ingredients other than the active ingredient are not particularly limited and may be, for example, a buffering agent, a pH adjusting agent, an isotonicity agent, a preservative, an antioxidant, a high molecular weight polymer, an excipient, a solvent, an antibacterial agent, or the like.

[0040] Examples of the buffering agent include phosphoric acid or phosphate salts, boric acid or borates, citric acid or citrate salts, acetic acid or acetate salts, carbonic acid or carbonate salts, tartaric acid or tartrate salts, ε-aminocaproic acid, and trometamol. Examples of the phosphate salts include sodium phosphate, sodium dihydrogen phosphate, disodium hydrogen phosphate, potassium phosphate, potassium dihydrogen phosphate, and dipotassium hydrogen phosphate. Examples of the borates include borax, sodium borate, and potassium borate. Examples of the citrate salts include sodium citrate, disodium citrate, and trisodium citrate. Examples of the acetate salts include sodium acetate and potassium acetate. Examples of the carbonate salts include sodium carbonate and sodium bicarbonate. Examples of the tartrate salts include sodium tartrate and potassium tartrate.

[0041] Examples of the pH adjuster include hydrochloric acid, phosphoric acid, citric acid, acetic acid, sodium hydroxide, and potassium hydroxide.

[0042] Examples of the isotonic agent include ionic isotonic agents (eg, sodium chloride, potassium chloride, calcium chloride, magnesium chloride) and non-ionic isotonic agents (eg, glycerin, propylene glycol, sorbitol, mannitol).

[0043] Examples of the preservative include benzalkonium chloride, benzalkonium bromide, benzethonium chloride, sorbic acid, potassium sorbate, methyl parahydroxybenzoate, propyl parahydroxybenzoate, and chlorobutanol.

[0044] Examples of the antioxidant include ascorbic acid, tocopherol, dibutylhydroxytoluene, butylhydroxyanisole, sodium erythorbate, propyl gallate, and sodium sulfite.

[0045] Examples of the high molecular weight polymer include methyl cellulose, ethyl cellulose, hydroxymethyl cellulose, hydroxyethyl cellulose, hydroxypropyl cellulose, hydroxyethyl methyl cellulose, hydroxypropyl methyl cellulose, carboxymethyl cellulose, sodium carboxymethyl cellulose, hydroxypropyl methyl cellulose acetate succinate, hydroxypropyl methyl cellulose phthalate, carboxymethyl ethyl cellulose, cellulose acetate phthalate, polyvinylpyrrolidone, polyvinyl alcohol, carboxyvinyl polymer, polyethylene glycol, and atelocollagen.

[0046] Examples of the excipient include lactose, sucrose, D-mannitol, xylitol, sorbitol, erythritol, starch, and crystalline cellulose.

[0047] Examples of the solvent include water, physiological saline, and alcohol.

[0048] Examples of the antibacterial agents include β-lactam, aminoglycoside, tetracycline, lincomycin, chloramphenicol, macrolide, ketolide, polypeptide, and glycopeptide antibiotics; and pyridonecarboxylic acid (quinolone), new quinolone, oxazolidinone, and sulfonamide synthetic antibacterial agents.

[0049] The amount of ingredients other than the active ingredient contained in the inhibitor of primary cilia of immune-related cells according to one embodiment of the present invention is not particularly limited, and may be, for example, 0% by mass to 99.99999% by mass, 0% by mass to 99.9999% by mass, 0% by mass to 99.9999% by mass, 0% by mass to 99.9999% by mass, 0% by mass to 99.999% by mass, 0% by mass to 99.99% by mass, or 5% by mass to 99.9% by mass. It may be 10% by mass to 99.9% by mass, 20% by mass to 99.9% by mass, 30% by mass to 99.9% by mass, 40% by mass to 99.9% by mass, 50% by mass to 99.9% by mass, 60% by mass to 99.9% by mass, 70% by mass to 99.9% by mass, 80% by mass to 99.9% by mass, or 90% by mass to 99.9% by mass.

[0050] The dosage form of the inhibitor of primary cilia of immune-related cells in one embodiment of the present invention is not particularly limited, and examples include topical preparations (e.g., liquids, gels, creams, sticks, sheets), tablets, powders, and granules.

[0051] The subjects to which the inhibitor of primary cilia of immune-related cells according to one embodiment of the present invention is administered are not particularly limited, and examples thereof include humans, non-human animals (e.g., livestock, pets, and laboratory animals), tissues collected therefrom, cells collected therefrom, and established cell lines. The non-human animals are not particularly limited, and examples thereof include monkeys, chimpanzees, cows, pigs, sheep, goats, horses, dogs, cats, rabbits, mice, and rats.

[0052] The route of administration of the inhibitor of primary cilia of immune-related cells according to one embodiment of the present invention is not particularly limited, and examples include parenteral administration (e.g., transdermal administration) and oral administration.

[0053] The interval at which an inhibitor of primary cilia of immune-related cells according to one embodiment of the present invention is administered to a subject is not particularly limited, and examples include once every hour, once every 1 to 6 hours, once every 6 to 12 hours, once every 12 hours to 1 day, once every 1 to 3 days, once every 1 to 5 days, once every 1 to 7 days, once every 7 to 14 days, once every 14 to 21 days, once per month, once per 2 months, once per 3 months, once per 4 months, once per 5 months, once per 6 months, or once per year.

[0054] 2. Anti-inflammatory topical agents An anti-inflammatory (e.g., inflammation prevention, inflammation treatment) topical preparation according to one embodiment of the present invention contains an inhibitor of primary cilia of immune-related cells according to one embodiment of the present invention. An anti-inflammatory (e.g., inflammation prevention, inflammation treatment) topical preparation according to one embodiment of the present invention may consist of an inhibitor of primary cilia of immune-related cells according to one embodiment of the present invention.

[0055] The inhibitor of primary cilia of immune-related cells has already been explained, so its explanation will be omitted here.

[0056] The inflammation to which the topical preparation is applied is not particularly limited, and examples thereof include inflammation caused by chemical and / or physical stimuli, and inflammation associated with inflammatory diseases, such as atopic dermatitis, psoriasis, eczema, comedones, and contact dermatitis.

[0057] More specifically, the anti-inflammatory topical agent may be an anti-inflammatory agent, an inflammation preventive agent, an inflammation treatment agent, an anti-inflammatory cosmetic, an inflammation preventive cosmetic, or an inflammation treatment cosmetic.

[0058] Specific examples of the cosmetics include body lotions, deodorant cosmetics, lotions, emulsions, skin care creams, tonics, stick cosmetics, lip products, facial cleansers, cleansers, sheet cosmetics, shaving cosmetics, and hair growth agents.

[0059] The dosage form of the topical preparation is not particularly limited, and examples thereof include liquids, gels, creams, sticks, and sheets. The degree of gelation of the gels is not particularly limited, as it may vary depending on the application.

[0060] The skin to which the topical agent can be applied is not particularly limited, and examples include the head, face, neck, arms, hands, elbows, shins, back, trunk, and feet.

[0061] The amount of the inhibitor of primary cilia of immune-related cells contained in the anti-inflammatory topical preparation according to one embodiment of the present invention is not particularly limited, and may be, for example, 0.00001% by mass to 100% by mass, 0.0001% by mass to 100% by mass, 0.0001% by mass to 100% by mass, 0.01% by mass to 100% by mass, 0.1% by mass to 100% by mass, or 0.1% by mass to 100% by mass, for example. % to 95% by mass, 0.1% to 90% by mass, 0.1% to 80% by mass, 0.1% to 70% by mass, 0.1% to 60% by mass, 0.1% to 50% by mass, 0.1% to 40% by mass, 0.1% to 30% by mass, 0.1% to 20% by mass, or 0.1% to 10% by mass.

[0062] The anti-inflammatory topical preparation according to one embodiment of the present invention may contain ingredients other than the inhibitor of primary cilia of immune-related cells described above. The "ingredients other than inhibitors of primary cilia of immune-related cells" are not particularly limited, and examples include the "ingredients other than the active ingredient" described above in [1. Inhibitors of primary cilia of immune-related cells].

[0063] The amount of components other than the inhibitor of primary cilia of immune-related cells contained in the anti-inflammatory topical preparation according to one embodiment of the present invention is not particularly limited, and may be, for example, 0% by mass to 99.99999% by mass, 0% by mass to 99.9999% by mass, 0% by mass to 99.9999% by mass, 0% by mass to 99.9999% by mass, 0% by mass to 99.999% by mass, 0% by mass to 99.99% by mass, 5% by mass to 99.9 It may be % by mass, 10% by mass to 99.9% by mass, 20% by mass to 99.9% by mass, 30% by mass to 99.9% by mass, 40% by mass to 99.9% by mass, 50% by mass to 99.9% by mass, 60% by mass to 99.9% by mass, 70% by mass to 99.9% by mass, 80% by mass to 99.9% by mass, or 90% by mass to 99.9% by mass.

[0064] The administration target, administration route, and administration interval of the anti-inflammatory topical preparation according to one embodiment of the present invention are not particularly limited, and may be the same as the administration target, administration route, and administration interval of the inhibitor of primary cilia of immune-related cells according to one embodiment of the present invention described above.

[0065] [3. Other] An embodiment of the present invention can also be configured as follows.

[0066] <1> An inhibitor of primary cilia of immune-related cells, comprising at least one selected from the group consisting of A to E below: A: silymarin, B: silybin, C: dehydrosilybin, D: annonacin, E: quercetin.

[0067] <2> <1> 1. An anti-inflammatory topical preparation containing the inhibitor described in .

[0068] <3> A method for inhibiting primary cilia of immune-related cells, comprising the step of administering to a subject (e.g., a human or a non-human animal (e.g., a monkey, chimpanzee, cow, pig, sheep, goat, horse, dog, cat, rabbit, mouse, and rat)) an inhibitor of primary cilia of immune-related cells, which contains at least one selected from the group consisting of A to E below: A: silymarin, B: silybin, C: dehydrosilybin, D: annonacin, E: quercetin.

[0069] <4> A method for treating inflammation (e.g., a method for preventing or treating inflammation), comprising the step of administering to a subject (e.g., a human or a non-human animal (e.g., a monkey, chimpanzee, cow, pig, sheep, goat, horse, dog, cat, rabbit, mouse, and rat)) an external agent containing an inhibitor of primary cilia of immune-related cells, which contains at least one selected from the group consisting of A to E below: A: silymarin, B: silybin, C: dehydrosilybin, D: annonacin, E: quercetin.

[0070] <5> Use of at least one selected from the group consisting of A to E below for producing an inhibitor of primary cilia of immune-related cells: A: silymarin, B: silybin, C: dehydrosilybin, D: annonacin, E: quercetin.

[0071] <6> Use of at least one selected from the group consisting of A to E below for producing an anti-inflammatory topical agent: A: silymarin, B: silybin, C: dehydrosilybin, D: annonacin, E: quercetin. [Example]

[0072] An embodiment of the present invention will be described below.

[0073] (1. Cell culture method) Neonatal normal human keratinocytes (NHEK, Lonza), a type of immune-related cell, were cultured in KGM-Gold medium (Lonza).

[0074] (2. Method for inducing primary cilia) KGM-Gold medium was added to each well of an 8-well chamber slide (Thermo Fisher Scientific, model number: 154534).

[0075] NHEK cells were subcultured and then collected. 1.0 × 10 cells were added to each well of the 8-well chamber slide. 5 The cells were seeded at 1000 cells / well and cultured overnight at 37°C in 5% CO2.

[0076] IL-13 (PeproTech, model number AF-200-13) was added to each well to a final concentration of 100 ng / mL, and the cells were cultured at 37°C and 5% CO for 48 hours to induce the expression of primary cilia in the NHEK cells.

[0077] (3. Cell immunostaining method) The NHEK cells on the slide were fixed using 4% paraformaldehyde phosphate buffer (Fujifilm Corporation, model number: 163-20145) at 4°C for 20 minutes.

[0078] After the fixation, the NHEK cells were washed three times with phosphate-buffered saline (PBS), and then blocked by incubating them in 10% FBS (fetal bovine serum) / 0.1% Triton X-100 (Sigma, model number T8787) at room temperature for 30 minutes.

[0079] After blocking, NHEK cells were incubated with primary antibodies overnight at 4°C. In the following experiments, ARL13B, a marker protein for primary cilia, was stained to detect primary cilia, and CEP164, a marker protein for centrosomes, was stained to detect centrosomes. Information about the primary antibodies used and their dilution ratios are listed below. [Table 1] After incubation with the primary antibody, the NHEK cells were washed with phosphate-buffered saline (PBST) containing 0.1% Tween-20 (Sigma, model number P9416) and then incubated with the secondary antibody for 1 hour at room temperature in the dark. During incubation with the secondary antibody, the nuclei of the NHEK cells were simultaneously stained with Hoechst 33342 (1:1000) (Thermo Fisher Scientific, model number H3570).

[0080] The secondary antibodies used were anti-mouse IgG donkey polyclonal antibody (1:1000) (Thermo Fisher Scientific, model number: A-21202) and anti-rabbit IgG donkey polyclonal antibody (1:1000) (Thermo Fisher Scientific, model number: A-21207).

[0081] After reacting with the secondary antibody, the NHEK cells were washed in PBST, and then ProLong Gold (registered trademark) (Thermo Fisher Scientific, model number: P36980) was dropped onto the NHEK cells on the slide.The NHEK cells were then covered with a cover glass, and the NHEK cells were sealed between the slide and the cover glass.

[0082] The NHEK cells sealed between the slide and the cover glass were observed using a confocal laser scanning microscope (manufactured by OLYMPUS, trade name: FV1200 IX83 or FV3000 IX83).

[0083] The percentage of cells with primary cilia was calculated according to the following formula (I): [Cells with primary cilia (%)] = [Number of cells with primary cilia in the image observed by microscope] / [Total number of cells in the image observed by microscope] × 100 ··· Equation (I).

[0084] <Test 1> NHEK cells were induced to express primary cilia in KGM-Gold medium containing (i) various concentrations (0 μM, 5 μM, 10 μM, 50 μM, or 100 μM) of silybin (Tokyo Chemical Industry Co., Ltd.), 2,3-dehydrosilybin A (Phytolab), or 2,3-dehydrosilybin B (Phytolab), or various concentrations (0 μg / mL, 2.4 μg / mL, 4.8 μg / mL, 24 μg / mL, or 48 μg / mL) of silymarin (Sigma-Aldrich), and (ii) IL-13 (100 ng / mL).

[0085] After inducing the expression of primary cilia, NHEK cells were immunostained, and the presence or absence of ARL13B and CEP164 staining was used as an indicator to calculate the percentage of NHEK cells with primary cilia among all NHEK cells observed from images observed with a confocal laser scanning microscope. The test results are shown in Figures 1 and 2.

[0086] 101 and 102 are graphs showing the inhibitory effect of silymarin on primary cilia in immune-related cells.

[0087] As is clear from Figure 1, 101, silymarin inhibited the expression of primary cilia in HEK cells in a concentration-dependent manner. More specifically, silymarin inhibited the expression of primary cilia in HEK cells more effectively at concentrations of 48 μg / mL or higher.

[0088] As is clear from Figure 1, 102, silybin inhibited the expression of primary cilia in HEK cells in a concentration-dependent manner. More specifically, silybin inhibited the expression of primary cilia in HEK cells more effectively at concentrations of 100 μM or higher.

[0089] 201 and 202 in FIG. 2 are graphs showing the inhibitory effects of 2,3-dehydrosilybin A and 2,3-dehydrosilybin B on primary cilia in immune-related cells, respectively.

[0090] As is clear from Figure 2, 201, 2,3-dehydrosilybin A was found to inhibit the expression of primary cilia in NHEK cells in a concentration-dependent manner. More specifically, 2,3-dehydrosilybin A was found to inhibit the expression of primary cilia in HEK cells more effectively at concentrations of 10 μM or higher.

[0091] As is clear from 202 in Figure 2, 2,3-dehydrosilybin B was found to inhibit the expression of primary cilia in NHEK cells in a concentration-dependent manner. More specifically, 2,3-dehydrosilybin B was found to more effectively inhibit the expression of primary cilia in HEK cells at concentrations of 10 μM or higher.

[0092] <Test 2> Known cosmetic ingredients containing milk thistle extract include Organic Milk Thistle Extract (manufactured by Koei Kogyo Co., Ltd.; ingredient name: Organic Milk Thistle Extract BG-50; label name: Milk Thistle Seed Extract), Ameliox (manufactured by H. Holstein; ingredient name: Ameliox; label name: lecithin, carnosine, tocopherol, milk thistle fruit extract, glycerin, ethanol, water), and SilstemU (manufactured by H. Holstein; ingredient name: Silstem-U; label name: glycerin, water, propanediol, milk thistle extract, uva-ursi leaf extract).

[0093] NHEK cells were induced to express primary cilia in KGM-Gold medium containing (i) various concentrations (0%, 0.1%, or 1%) of cosmetic ingredients and (ii) IL-13 (100 ng / mL).

[0094] After inducing the expression of primary cilia, NHEK cells were immunostained, and the presence or absence of ARL13B and CEP164 staining was used as an indicator to calculate the percentage of NHEK cells with primary cilia among all NHEK cells observed from images observed with a confocal laser scanning microscope. The test results are shown in Figure 3.

[0095] Graphs 301, 302 and 303 in FIG. 3 are graphs showing the inhibitory effects of cosmetic ingredients containing organic milk thistle extract, Ameliox and SilstemU on primary cilia in immune-related cells, respectively.

[0096] As is clear from 301 to 303 in Figure 3, it was revealed that cosmetic ingredients containing organic milk thistle extract, Ameliox, and SilstemU inhibit the expression of primary cilia in NHEK cells in a concentration-dependent manner. In other words, it was revealed that milk thistle extract contains compounds that inhibit the expression of primary cilia.

[0097] <Test 3> NHEK cells were induced to express primary cilia in KGM-Gold medium containing (i) various concentrations (0 μM, 0.5 μM, 1 μM, 10 μM, or 100 μM) of annonacin (Cayman Chemical) or quercetin hydrate (Tokyo Chemical Industry Co., Ltd.) and (ii) IL-13 (100 ng / mL).

[0098] After inducing the expression of primary cilia, NHEK cells were immunostained, and the presence or absence of ARL13B and CEP164 staining was used as an indicator to calculate the percentage of NHEK cells with primary cilia among all NHEK cells observed from images observed with a confocal laser scanning microscope. The test results are shown in Figure 4.

[0099] 401 and 402 in FIG. 4 are graphs showing the inhibitory effects of annonacin and quercetin (quercetin hydrate) on primary cilia in immune-related cells, respectively.

[0100] As is clear from 401 in Figure 4, annonacin was found to inhibit the expression of primary cilia in NHEK cells in a concentration-dependent manner. More specifically, annonacin was found to inhibit the expression of primary cilia in HEK cells more effectively at concentrations of 0.5 μM or higher.

[0101] As is clear from 402 in Figure 4, quercetin (quercetin hydrate) was found to inhibit the expression of primary cilia in HEK cells in a concentration-dependent manner. More specifically, quercetin (quercetin hydrate) was found to more effectively inhibit the expression of primary cilia in HEK cells at concentrations of 10 μM or higher.

[0102] It is known that immune-related cells have organelles called primary cilia, and that the expression of primary cilia is increased in skin diseases accompanied by inflammation. As described above, the inhibitor of primary cilia in immune-related cells of the present invention has the effect of inhibiting the expression of primary cilia in immune-related cells. This suggests that the inhibitor of primary cilia in immune-related cells of the present invention also has the effect of suppressing inflammation. [Industrial Applicability]

[0103] The present invention can be used as an inhibitor of primary cilia of immune-related cells, and more specifically, can be used as an anti-inflammatory topical agent (e.g., an anti-inflammatory agent, an inflammation preventive agent, an inflammation treatment agent, an anti-inflammatory cosmetic, an inflammation preventive cosmetic, an inflammation treatment cosmetic).

Claims

1. An inhibitor of primary cilia of immune-related cells, comprising at least one selected from the group consisting of A to E below, wherein the immune-related cells are keratinocytes: A: Silymarin, B: Silybin, C: Dehydrosilybin, D: Annonacin, E: Quercetin.

2. An anti-inflammatory topical preparation containing an inhibitor of primary cilia of immune-related cells containing annonacin, wherein the immune-related cells are keratinocytes.

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