Inhibitors of primary cilia in immune-related cells and their use

Abstract

This invention provides an inhibitor of primary cilia in immune-related cells and its use. [Solution] An inhibitor of primary cilia of immune-related cells containing an ERK1 / 2 inhibitor as an active ingredient is used.

Description

Technical Field

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

Background Art

[0002] Inflammation is one of the biological reactions caused by chemical stimuli and / or physical stimuli. Since inflammation may damage living tissues, anti-inflammatory agents may be applied to inflammation.

[0003] As research progresses, it is becoming clear that the pathogenesis of inflammation is diverse. This indicates that there are appropriate anti-inflammatory agents depending on the type of inflammation, and therefore, the development of new anti-inflammatory agents is being vigorously promoted.

[0004] The present inventors have hitherto found that there is an organelle called primary cilia in immune-related cells, and that the expression of primary cilia is enhanced in skin diseases accompanied by inflammation (see, for example, Patent Documents 1 to 3). This indicates that an inhibitor of primary cilia of immune-related cells can be used as an anti-inflammatory agent.

Prior Art Documents

Patent Documents

[0005]

Patent Document 1

Patent Document 2

Patent Document 3

Summary of the Invention

Problems to be Solved by the Invention

[0006] As mentioned above, inflammation has various mechanisms, and in dealing with it, it is necessary to select the appropriate anti-inflammatory agent according to the type of inflammation. Therefore, the development of new compounds that can be used as anti-inflammatory agents is still needed. In particular, the primary cilia of immune-related cells have recently been found to be related to inflammation, and there is much room for research into inhibitors and their use.

[0007] In the circumstances described above, one aspect of the present invention aims to provide an inhibitor of primary cilia of immune-related cells and its use. [Means for solving the problem]

[0008] As a result of diligent research to solve the aforementioned problems, the present inventors discovered that ERK1 / 2 inhibitors have the effect of suppressing the expression of primary cilia in immune-related cells, and thus completed the present invention. That is, the present invention includes the following configuration.

[0009] [1] An inhibitor of primary cilia of immune-related cells, containing an ERK1 / 2 inhibitor as an active ingredient.

[0010] [2] The inhibitor according to [1], wherein the ERK1 / 2 inhibitor is at least one selected from the group consisting of cucurbitacin IIa and SCH772984.

[0011] [3] An anti-inflammatory topical preparation containing the inhibitor described in [1] or [2] as an active ingredient. [Effects of the Invention]

[0012] According to one aspect of the present invention, an inhibitor of primary cilia of immune-related cells and its use can be provided. [Brief explanation of the drawing]

[0013] [Figure 1] This graph shows the primary ciliary inhibitory effect of cucurbitacin IIa in RPE1 cells. [Figure 2]This graph shows the primary ciliary inhibitory effect of SCH772984 in RPE1 cells. [Figure 3] This graph shows the primary ciliary inhibitory effect of cucurbitacin IIa in NHEK cells. [Figure 4] This graph shows the primary cilia inhibitory effect of cucurbitacin IIa in HaCaT cells. [Figure 5] This graph shows the primary ciliary suppression effect of SCH772984 in HaCaT cells. [Modes for carrying out the invention]

[0014] One embodiment of the present invention is described below, but the present invention is not limited thereto. The present invention is not limited to the 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. Moreover, new technical features can be formed by combining the technical means disclosed in each embodiment. All academic and patent documents mentioned herein are incorporated herein by reference. Unless otherwise specified herein, "X~Y" representing a numerical range is intended to mean "X or more and Y or less".

[0015] [1. Inhibitors of primary cilia in immune-related cells] An inhibitor of primary cilia of immune-related cells according to one embodiment of the present invention (hereinafter sometimes referred to as "this inhibitor") contains an ERK1 / 2 inhibitor as an active ingredient.

[0016] This inhibitor may suppress the expression of primary cilia in immune-related cells. More specifically, this inhibitor may (i) reduce the number and / or proportion of immune-related cells having primary cilia, or (ii) suppress the increase in the number and / or proportion of immune-related cells having primary cilia.

[0017] The immune-related cells related to this inhibitor include immune cells mainly responsible for immune reactions and immune function-bearing cells indirectly involved in immune cells. That is, this inhibitor may be an inhibitor of the primary cilia of immune cells or an inhibitor of the primary cilia of immune function-bearing cells.

[0018] Immune function-bearing cells have, for example, a function of activating immune cells. Examples of immune cells include skin dendritic cells (e.g., Langerhans cells, dermal dendritic cells), lymphocyte-based immune cells (e.g., T cells, NK cells, B cells), and monocyte-based immune cells (e.g., conventional dendritic cells, monocyte-based dendritic cells (e.g., plasmacytoid dendritic cells)). On the other hand, examples of immune function-bearing cells include keratinocytes (keratinocytes, e.g., EK cells), fibroblasts, and epithelial cells (e.g., retinal pigment epithelial cells). In addition, the immune-related cells related to this inhibitor may be immune-related cells collected from a living body or immortalized immune-related cells (e.g., HaCaT cells).

[0019] The ERK1 / 2 inhibitor, which is the active ingredient of this inhibitor, is not particularly limited as long as it is a substance capable of inhibiting the activity or expression of ERK1 / 2. The ERK1 / 2 inhibitor in this specification may inhibit the activity of ERK1, inhibit the activity of ERK2, inhibit the activities of ERK1 and ERK2, inhibit the expression of ERK1, inhibit the expression of ERK2, or inhibit the expressions of ERK1 and ERK2, and may inhibit the activity or expression of ERK1 / 2 by any of these methods.

[0020] The active ingredients of this inhibitor, which are ERK1 / 2 inhibitors, include, specifically, cucurbitacin IIa, SCH772984, trametinib (CAS No. 871700-17-3), U0126 (CAS No. 109511-58-2), selumetinib (CAS No. 606143-52-6), cobimetinib (CAS No. 934660-93-2), PD0325901 (CAS No. 391210-10-9), prullipotin (CAS No. 839707-37-8), kaempaulon (CAS No. 142273-20-9), evodiamine (CAS No. 518-17-2), FR180204 (CAS No. 865362-74-9), and p38 MAP Kinase Inhibitor IV. Examples include (CAS No. 1638-41-1), 3-(2-Aminoethyl)-5-((4-ethoxyphenyl)methylene)-2,4-thiazolidinedione (CAS No. 1049738-54-6), Pyrazolylpyrrole ERK Inhibitor (CAS No. 933786-58-4), CR8,(S)-Isomer (CAS No. 1084893-56-0), and 5-Iodotubercidin (CAS No. 24386-93-4). The active ingredient of this inhibitor may be one of the above substances, or two or more in combination.

[0021] Cucurbitacin IIa is a compound represented by the following chemical formula and is known to be found in plants of the Cucurbitaceae family (e.g., cucumbers). Cucurbitacin IIa is also known as CAS No. 58546-34-2.

[0022] [ka]

[0023] When using cucurbitacin IIa as the active ingredient in this inhibitor, commercially available cucurbitacin IIa may be used, or it may be obtained from an extract of a cucurbitaceous plant.

[0024] SCH772984 is a compound known as CAS No. 942183-80-4. When using SCH772984 as the active ingredient in this inhibitor, commercially available products can be preferably used.

[0025] The amount of the ERK1 / 2 inhibitor, which is the active ingredient in this inhibitor, is not particularly limited. For example, if this inhibitor is considered as 100% by mass, the amount may be 0.00001% to 100% by mass, 0.0001% to 100% by mass, 0.001% to 100% by mass, 0.01% to 100% by mass, 0.1% to 100% by mass, 0.1% to 100% by mass, 0.1% 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.

[0026] When the active ingredient is cucurbitacin IIa, it is preferable that the inhibitor contains an amount of cucurbitacin IIa that allows cucurbitacin IIa at a concentration of 100 nM or higher, 250 nM or higher, 500 nM or higher, or 1 μM or higher (the upper limit of the concentration is not limited and may be, for example, 10 μM or lower, or 100 μM or lower) to come into contact with immune-related cells when the inhibitor is administered to the target. Of course, the inhibitor (for example, in the form of a liquid, gel, or cream) may contain cucurbitacin IIa at the above concentration. With this configuration, the expression of primary cilia on immune-related cells can be suppressed more effectively.

[0027] When the active ingredient is SCH772984, it is preferable that the inhibitor contains an amount of SCH772984 that allows SCH772984 at a concentration of 1 nM or higher, 2.5 nM or higher, 5 nM or higher, or 10 nM or higher (the upper limit of the concentration is not limited and may be, for example, 50 nM or less, 100 nM or less, or 1000 nM or less) to come into contact with immune-related cells when the inhibitor is administered to the target. Of course, the inhibitor (for example, in the form of a liquid, gel, or cream) may contain SCH772984 at the above concentration. With this configuration, the expression of primary cilia on immune-related cells can be suppressed more effectively.

[0028] This inhibitor may contain ingredients other than the active ingredient described above (other ingredients).

[0029] Other components that this inhibitor may contain are not particularly limited and may include, for example, buffering agents, pH adjusters, isotonic agents, preservatives, antioxidants, high molecular weight polymers, excipients, solvents, and antibacterial agents.

[0030] Examples of the buffering agent include phosphoric acid or phosphate, boric acid or borate, citric acid or citrate, acetic acid or acetate, carbonate or carbonate, tartaric acid or tartrate, ε-aminocaproic acid, and trometamol. Examples of the phosphate include sodium phosphate, sodium dihydrogen phosphate, disodium hydrogen phosphate, potassium phosphate, potassium dihydrogen phosphate, and dipotassium hydrogen phosphate. Examples of the borate include borax, sodium borate, and potassium borate. Examples of the citrate include sodium citrate, disodium citrate, and trisodium citrate. Examples of the acetate include sodium acetate and potassium acetate. Examples of the carbonate include sodium carbonate and sodium bicarbonate. Examples of the tartrate include sodium tartrate and potassium tartrate.

[0031] Examples of the pH adjusting agents include hydrochloric acid, phosphoric acid, citric acid, acetic acid, sodium hydroxide, and potassium hydroxide.

[0032] Examples of the isotonic agents include ionic isotonic agents (e.g., sodium chloride, potassium chloride, calcium chloride, magnesium chloride) and nonionic isotonic agents (e.g., glycerin, propylene glycol, sorbitol, mannitol).

[0033] Examples of the aforementioned preservatives include benzalkonium chloride, benzalkonium bromide, benzethonium chloride, sorbic acid, potassium sorbate, methyl parahydroxybenzoate, propyl parahydroxybenzoate, and chlorobutanol.

[0034] Examples of the aforementioned antioxidants include ascorbic acid, tocopherol, dibutylhydroxytoluene, butylhydroxyanisole, sodium erythorbate, propyl gallate, and sodium sulfite.

[0035] Examples of the high molecular weight polymers include methylcellulose, ethylcellulose, hydroxymethylcellulose, hydroxyethylcellulose, hydroxypropylcellulose, hydroxyethylmethylcellulose, hydroxypropylmethylcellulose, carboxymethylcellulose, sodium carboxymethylcellulose, hydroxypropylmethylcellulose acetate succinate, hydroxypropylmethylcellulose phthalate, carboxymethylethylcellulose, cellulose phthalate acetate, polyvinylpyrrolidone, polyvinyl alcohol, carboxyvinyl polymer, polyethylene glycol, and atelocollagen.

[0036] Examples of the excipients include lactose, sucrose, D-mannitol, xylitol, sorbitol, erythritol, starch, and crystalline cellulose.

[0037] Examples of the aforementioned solvents include water, physiological saline solution, and alcohol.

[0038] Examples of the aforementioned antibacterial agents include β-lactam, aminoglycoside, tetracycline, lincomycin, chloramphenicol, macrolide, ketolide, polypeptide, and glycopeptide antibiotics; and synthetic antibacterial agents such as pyridonecarboxylic acid (quinolone), fluoroquinolone, oxazolidinone, and sulfonamide drugs.

[0039] The amount of other components contained in this inhibitor is not particularly limited, and may be 0% to 99.99999% by mass, equivalent to 100% by mass of this inhibitor, or 0% to 99.9999% by mass, or 0% to 99.999% by mass, or 0% to 99.99% by mass, or 0% to 99.9% by mass, or 5% to 99.9% by mass, or 10% to 99.9% by mass, or 20% to 99.9% by mass, or 30% to 99.9% by mass, or 40% to 99.9% by mass, or 50% to 99.9% by mass, or 60% to 99.9% by mass, or 70% to 99.9% by mass, or 80% to 99.9% by mass, or 90% to 99.9% by mass.

[0040] The dosage form of this inhibitor is not particularly limited and includes, for example, topical preparations (e.g., liquids, gels, creams, sticks, sheets), tablets, powders, granules, etc.

[0041] The target population for administration of this inhibitor is not particularly limited and includes, for example, humans, non-human animals (e.g., livestock, pets, and laboratory animals), tissues collected from them, cells collected from them, and cell lines. The aforementioned non-human animals are not particularly limited and include, for example, monkeys, chimpanzees, cattle, pigs, sheep, goats, horses, dogs, cats, rabbits, mice, and rats.

[0042] The route of administration of this inhibitor is not particularly limited and includes, for example, parenteral administration (e.g., transdermal administration) and oral administration.

[0043] The administration interval for this inhibitor is not particularly limited and may include, for example, 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 every 1 month, once every 2 months, once every 3 months, once every 4 months, once every 5 months, once every 6 months, or once every year.

[0044] [2. Topical anti-inflammatory agents] An anti-inflammatory topical agent (this topical agent) according to one embodiment of the present invention contains the inhibitor as an active ingredient. Because this topical agent contains the inhibitor which has a primary ciliary inhibitory effect, it can suppress the expression of primary cilia in immune-related cells, and as a result, it can suppress inflammation involving immune-related cells.

[0045] This topical preparation may be a topical preparation for the prevention of inflammation, a topical preparation for the treatment of inflammation, or may have both effects. Furthermore, this topical preparation may consist of this inhibitor.

[0046] The inhibitor contained in this topical preparation has already been explained, so we will omit that explanation here.

[0047] The inflammation to which this topical agent is applied is not particularly limited, as long as it involves immune-related cells, and includes, for example, inflammation caused by chemical and / or physical stimuli, and inflammation associated with inflammatory diseases. Examples of such inflammatory diseases include atopic dermatitis, psoriasis, eczema, acne, and contact dermatitis.

[0048] More specifically, this topical preparation may be an anti-inflammatory agent, an anti-inflammatory agent, an anti-inflammatory agent, an anti-inflammatory cosmetic, an anti-inflammatory cosmetic, or an anti-inflammatory cosmetic.

[0049] Specific examples of cosmetic preparations that constitute one form of this external preparation include, for example, body lotion, deodorant cosmetic, toner, emulsion, skincare cream, tonic, stick cosmetic, lip balm, facial cleanser, cleanser, sheet cosmetic, shaving cosmetic, and hair growth stimulant.

[0050] The dosage form of this external preparation is not particularly limited and includes, for example, liquids, gels, creams, sticks, and sheets. The degree of gelation of the gel preparation may vary depending on the application and is therefore not particularly limited.

[0051] The skin to which this topical agent can be applied is not particularly limited, but includes, for example, the head, face, neck, arms, hands, elbows, shins, back, torso, and feet.

[0052] The amount of the inhibitor contained in this external preparation is not particularly limited. For example, if the external preparation is considered to be 100% by mass, the amount may be 0.00001% to 100% by mass, 0.0001% to 100% by mass, 0.001% to 100% by mass, 0.01% to 100% by mass, 0.1% to 100% by mass, 0.1% to 100% by mass, 0.1% 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.

[0053] This topical preparation may contain ingredients other than the inhibitor. Such ingredients other than the inhibitor are not particularly limited and include, for example, the "other ingredients" described above in [1. Inhibitors of primary cilia of immune-related cells].

[0054] The amount of components other than the inhibitor contained in this topical preparation is not particularly limited. For example, if the topical preparation is considered to be 100% by mass, the amount may be 0% to 99.99999% by mass, 0% to 99.9999% by mass, 0% to 99.999% by mass, 0% to 99.99% by mass, 0% to 99.9% by mass, 5% to 99.9% by mass, and 10% by mass. It may be % to 99.9 mass%, 20 mass% to 99.9 mass%, 30 mass% to 99.9 mass%, 40 mass% to 99.9 mass%, 50 mass% to 99.9 mass%, 60 mass% to 99.9 mass%, 70 mass% to 99.9 mass%, 80 mass% to 99.9 mass%, or 90 mass% to 99.9 mass%.

[0055] The target population, route of administration, and administration interval of this topical agent are not particularly limited and may be the same as those for the inhibitor described above.

[0056] [3. Other] One embodiment of this invention can also be configured as follows:

[0057] <1> An inhibitor of primary cilia in immune-related cells, containing an ERK1 / 2 inhibitor as its active ingredient.

[0058] <2> <1> An anti-inflammatory topical agent containing the inhibitor described above as an active ingredient.

[0059] <3> A method for inhibiting primary cilia of immune-related cells, comprising the step of administering an inhibitor of primary cilia of immune-related cells containing an ERK1 / 2 inhibitor as an active ingredient to a subject (e.g., a human or a non-human animal (e.g., monkeys, chimpanzees, cattle, pigs, sheep, goats, horses, dogs, cats, rabbits, mice, and rats)).

[0060] <4> A method for treating inflammation (e.g., a method for preventing inflammation, or a method for treating inflammation), comprising the step of administering a topical drug containing an ERK1 / 2 inhibitor as an active ingredient to a subject (e.g., a human, or a non-human animal (e.g., monkeys, chimpanzees, cattle, pigs, sheep, goats, horses, dogs, cats, rabbits, mice, and rats)).

[0061] <5> Use of ERK1 / 2 inhibitors to produce inhibitors of primary cilia in immune-related cells.

[0062] <6> Use of ERK1 / 2 inhibitors or primary cilia inhibitors of immune-associated cells for the manufacture of topical anti-inflammatory agents. [Examples]

[0063] Examples of the present invention are described below.

[0064] <1. Detection of primary cilia in RPE1 cells> Retinal pigment epithelial cells (RPE1) were seeded at a rate of 5000 cells per well in 24-well plates and cultured for 24 hours. Then, culture media containing ERK1 / 2 inhibitors of the types and concentrations shown below were supplied, and the cells were pre-treated for 1 hour. Subsequently, IL-31 (10 ng / ml), which has primary cilia-inducing activity, was added to the medium, and the cells were cultured for 24 hours (inhibitor + IL-31 group). As a control, RPE1 cells were also cultured for 24 hours in media without inhibitors or IL-31 (control group), or in media without inhibitors but containing IL-31 (IL-31 group).

[0065] The types and final concentrations of the ERK1 / 2 inhibitors in the culture medium containing the aforementioned ERK1 / 2 inhibitors were as follows: • Cucurbitacin IIa [Manufactured by Selleck, trade name: Cucurbitacin IIA, catalog number: S5463] Final concentration: 100nM, 250nM, 500nM • SCH772984 (Manufactured by Selleck, Product name: SCH772984, Catalog number: S7101) Final concentrations: 1nM, 2.5nM, 5nM, 10nM.

[0066] After culturing, RPE1 cells were collected from the inhibitor + IL-31 group, the control group, and the IL-31 group. These RPE1 cells were then placed in 4% paraformaldehyde-phosphate buffer (4% PFA) [Fujifilm Corporation, product name: 4% paraformaldehyde-phosphate buffer, catalog number: 163-20145] for tissue fixation and fixed at 4°C for 1 hour. After fixation, the RPE1 cells were washed three times with phosphate-buffered saline (PBS), and then blocked by incubation in 10% fetal bovine serum (FBS) / PBS / 0.25% Triton-X100 [Sigma Corporation, product name: Triton® X-100, catalog number: T8787] at 4°C for 1 hour.

[0067] Furthermore, RPE1 that had been blocked was reacted overnight at 4°C with anti-ARL13b antibody (Proteintech, trade name: ARL13B Polyclonal antibody, catalog number: 17711-1-AP) and anti-FGFR10P antibody (abnova, trade name: FGFR1OP monoclonal antibody (M01), clone 2B1, catalog number: H00011116-M01) dissolved in PBS at 0.1% by volume. ARL13b and FGFR10P are known as markers for identifying primary cilia.

[0068] RPE1, reacted with anti-ARL13b antibody and anti-FGFR10P antibody, was washed twice with PBS. Each RPE1 was then mixed with 0.1% by volume of PBS, a fluorescently labeled anti-rabbit IgG antibody (manufactured by Thermo Fisher Scientific, trade name: Goat Anti-Rabbit IgG H&L (fluorescent dye: Goat anti-Rabbit IgG (H+L) Cross-Adsorbed Secondary Antibody, Alexa Fluor 488, catalog number: A11008)), a fluorescently labeled anti-mouse IgG antibody (manufactured by Thermo Fisher Scientific, trade name: Donkey anti-Mouse IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor® 594, catalog number: A-21203)), and the nuclear stain Hoechst. RPE1 was stained by reacting it with 33342 [manufactured by Thermo Fisher Scientific Co., Ltd., product name: Hoechst 33342, Trihydrochloride, Trihydrate - 10 mg / mL Solution in Water, catalog number: H3570] at room temperature (25°C) for 1 to 2 hours.

[0069] After staining, each RPE1 sample was washed twice with surfactant-containing PBS. Then, the RPE1 samples were mounted in a fade-preventing mounting medium (manufactured by Thermo Fisher Scientific, Inc., product name: ProLong® Gold fade-preventing mounting medium, catalog number: P36934) to prepare mounted samples for observation.

[0070] Using a confocal microscope (Olympus Corporation, model number: FV3000) and a system microscope (Olympus Corporation, model number: VS200), we observed cells containing primary cilia in each sample by detecting cell nuclei and primary ciliary markers contained in the mounted samples.

[0071] For each sample, more than 300 cell nuclei were observed, and then the primary cilia formation rate was calculated according to formula (Ia); [Primary cilia formation rate] = [Number of primary cilia in the image] / [Total number of cell nuclei in the image] × 100 ...(Ia).

[0072] The primary cilia formation rates for each sample are shown in Figures 1 and 2. Figure 1 shows the primary cilia formation rates for samples supplied with cucurbitacin IIa (CuIIa) as an ERK1 / 2 inhibitor and controls (control group and IL-31 group), while Figure 2 shows the primary cilia formation rates for samples supplied with SCH772984 (SCH) as an ERK1 / 2 inhibitor and controls (control group and IL-31 group).

[0073] In Figure 1, the values ​​on the vertical axis represent the relative primary cilia formation rate of each sample, with the primary cilia formation rate in the control group set to 1. The horizontal axis represents the type of sample; a + in the IL-31 column indicates the IL-31 administration group, and a - indicates the IL-31 non-administration group. A - in the CuIIa column indicates that cucurbitacin IIa was not supplied (control), and the numerical value represents the final concentration (nM) of supplied cucurbitacin IIa.

[0074] In Figure 2, the values ​​on the vertical axis and the meaning of the IL-31 term on the horizontal axis are the same as in Figure 1. A - in the SCH term on the horizontal axis indicates that SCH772984 was not supplied (control), and the numerical value represents the final concentration (nM) of supplied SCH772984.

[0075] As is clear from Figures 1 and 2, it was shown that administering an ERK1 / 2 inhibitor to RPE1 cells, which are immune-related cells, can suppress the formation of primary cilia during inflammatory stimuli (IL-31 administration group).

[0076] <2. Detection of primary cilia in NHEK cells> Normal human epidermal keratinocytes (NHEKs) were placed on an 8-well chamber slide, with 1 × 10⁶ cells per well. 5 Cells were seeded individually and cultured for 24 hours. Next, a culture medium containing cucurbitacin IIa at the following concentrations was supplied to a chamber slide, and pre-treatment was performed for 1 hour. Subsequently, the cells were stimulated with IL-31 (10 ng / ml), which has activity to induce primary cilia, and cultured for 48 hours (CuIIa + IL-31 group). In addition, as a control, NHEK cells were cultured for 48 hours in a medium without CuIIa and IL-31 (control group), or in a medium without CuIIa but containing IL-31 (IL-31 group).

[0077] The final concentration of cucurbitacin IIa in the culture medium containing cucurbitacin IIa was as follows:

[0078] Cucurbitacin IIa [Manufactured by Selleck, product name: CucurbitacinIIA, catalog number: S5463] Final concentration: 1 μM.

[0079] After culturing, NHEK was collected from the CuIIa+IL-31 group, the control group, and the IL-31 group. This NHEK was placed in 4% PFA and fixed at 4°C for 1 hour. After fixation, the NHEK was washed three times with Phosphate Buffered Saline (PBS), and then blocked by incubation overnight at 4°C in 10% FBS / milliQ / 0.1% triton-X100 / 0.1% SDS (Fujifilm Corporation, trade name: sodium dodecyl sulfate, catalog number: 191-07145).

[0080] Furthermore, blocked NHEK was reacted overnight at 4°C with anti-acetylated tubulin antibody (Sigma, trade name: Monoclonal Anti-Tubulin Acetylated Mouse Host Antibody, catalog number: T6793) and anti-pericentrin antibody (Thermo Fisher Scientific, trade name: Pericentrin / Kendrin Polyclonal Antibody, catalog number: IHC-00264), dissolved in PBS at 0.1% by volume. Acetylated tubulin and pericentrin are known as markers for identifying primary cilia.

[0081] NHEK reacted with anti-acetylated tubulin antibody and anti-pericentrin antibody, washed twice with PBS, and then each NHEK was dissolved in PBS at a volume of 0.1% with fluorescently labeled anti-mouse IgG antibody [Thermo Fisher Scientific Co., Ltd., trade name: Goat Anti-Mouse IgG H&L (fluorescent dye: Alexa Fluor® 488, catalog number: ab150113)], fluorescently labeled anti-rabbit IgG antibody [Thermo Fisher Scientific Co., Ltd., trade name: Donkey anti-Rabbit IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor® 594, catalog number: A-21207)], and nuclear stain Hoechst 33342 [Thermo Fisher Scientific Co., Ltd., trade name: Hoechst NHEK was stained by reacting it with [33342, Trihydrochloride, Trihydrate - 10 mg / mL Solution in Water, Catalog Number: H3570] at room temperature (25°C) for 1 to 2 hours.

[0082] After staining, each NHEK was washed three times for 5 minutes with surfactant-containing PBS. Then, the NHEK was mounted in a fade-preventing mounting medium [Thermo Fisher Scientific Co., Ltd., product name: ProLong® Gold fade-preventing mounting medium, product number: P36934] to prepare mounted samples for observation.

[0083] Using a confocal microscope (Olympus Corporation, model number: FV3000), we observed cells containing primary cilia in each sample by detecting cell nuclei and primary ciliary markers contained in the mounted samples.

[0084] For each sample, more than 300 cell nuclei were observed, and then the primary cilia formation rate was calculated according to formula (Ib); [Primary cilia formation rate] = [Number of primary cilia in the image] / [Total number of cell nuclei in the image] × 100 ...(Ib).

[0085] Figure 3 shows the primary cilia formation rates for each sample. Figure 3 shows the primary cilia formation rates for samples supplied with IL-31 and cucurbitacin IIa (CuIIa) (CuIIa + IL-31 group), and for controls (control group and IL-31 group).

[0086] In Figure 3, the values ​​on the vertical axis represent the relative primary cilia formation rate of each sample, with the primary cilia formation rate in the control group (NT) set to 1. The horizontal axis represents the type of sample.

[0087] As is clear from Figure 3, it was shown that administering cucurbitacin IIa, an ERK1 / 2 inhibitor, to NHEK cells, which are immune-related cells, can suppress the formation of primary cilia during inflammatory stimuli (IL-31 administration group).

[0088] <3. Detection of primary cilia in HaCaT cells> HaCaT cells (human epidermal keratinocyte line) are placed on an 8-well chamber slide, 1 × 10⁶ cells per well.5 Cells were seeded individually and cultured for 24 hours. Next, a culture medium containing the following types and concentrations of ERK1 / 2 inhibitors was supplied to a chamber slide, and pre-treated for 1 hour. Subsequently, HaCaT cells were stimulated with IL-31 (10 ng / ml), which has primary cilia-inducing activity, and cultured for 48 hours (inhibitor + IL-31 group). As a control, HaCaT cells were also cultured for 48 hours in a medium without inhibitors or IL-31 (control group), or in a medium without inhibitors but containing IL-31 (IL-31 group).

[0089] The final concentrations of the ERK1 / 2 inhibitor in the culture medium containing the aforementioned ERK1 / 2 inhibitor were as follows:

[0090] • Cucurbitacin IIa [Manufactured by Selleck, product name: CucurbitacinIIA, catalog number: S5463] Final concentration: 100nM, 500nM • SCH772984 (Manufactured by Selleck, Product name: SCH772984, Catalog number: S7101) Final concentration: 1nM, 5nM.

[0091] After culturing, HaCaT was collected from the inhibitor + IL-31 group, the control group, and the IL-31 group. The HaCaT was placed in 4% PFA and fixed at 4°C for 1 hour. After fixation, the HaCaT was washed three times with Phosphate Buffered Saline (PBS), and then blocked by incubation overnight at 4°C in 10% FBS / milliQ / 0.1% triton-X100 / 0.1% SDS [Fujifilm Corporation, trade name: sodium dodecyl sulfate, catalog number: 191-07145].

[0092] Furthermore, blocked HaCaT was reacted overnight at 4°C with anti-acetylated tubulin antibody (Sigma, trade name: Monoclonal Anti-Tubulin Acetylated Mouse Host Antibody, catalog number: T6793) and anti-pericentrin antibody (Thermo Fisher Scientific, trade name: Pericentrin / Kendrin Polyclonal Antibody, catalog number: IHC-00264), dissolved in PBS at 0.1% by volume. Acetylated tubulin and pericentrin are known as markers for identifying primary cilia.

[0093] HaCaT samples reacted with anti-acetylated tubulin antibody and anti-pericentrin antibody were washed twice with PBS. Each HaCaT sample was then mixed with 0.1% by volume of PBS containing fluorescently labeled anti-mouse IgG antibody [Thermo Fisher Scientific Co., Ltd., trade name: Goat Anti-Mouse IgG H&L (fluorescent dye: Alexa Fluor® 488, catalog number: ab150113)], fluorescently labeled anti-rabbit IgG antibody [Thermo Fisher Scientific Co., Ltd., trade name: Donkey anti-Rabbit IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor® 594, catalog number: A-21207)], and the nuclear stain Hoechst 33342 [Thermo Fisher Scientific Co., Ltd., trade name: Hoechst HaCaT was stained by reacting it with [33342, Trihydrochloride, Trihydrate - 10 mg / mL Solution in Water, Catalog Number: H3570] at room temperature (25°C) for 1 to 2 hours.

[0094] After staining, each HaCaT sample was washed three times for 5 minutes with surfactant-containing PBS. Then, the HaCaT samples were mounted in a fade-preventing mounting medium (manufactured by Thermo Fisher Scientific Co., Ltd., product name: ProLong® Gold fade-preventing mounting medium, product number: P36934) to prepare mounted samples for observation.

[0095] Using a confocal microscope (Olympus Corporation, model number: FV3000), we observed cells containing primary cilia in each sample by detecting cell nuclei and primary ciliary markers contained in the mounted samples.

[0096] For each sample, more than 300 cell nuclei were observed, and then the primary cilia formation rate was calculated according to formula (Ic); [Primary cilia formation rate] = [Number of primary cilia in the image] / [Total number of cell nuclei in the image] × 100 ·····(I C).

[0097] The primary cilia formation rates for each calculated sample are shown in Figures 4 and 5. Figure 4 shows the primary cilia formation rates for samples supplied with cucurbitacin IIa (CuIIa) as an ERK1 / 2 inhibitor and controls (control group and IL-31 group), while Figure 5 shows the primary cilia formation rates for samples supplied with SCH772984 (SCH) as an ERK1 / 2 inhibitor and controls (control group and IL-31 group).

[0098] In Figure 4, the values ​​on the vertical axis represent the relative primary cilia formation rate of each sample, with the primary cilia formation rate in the control group set to 1. The horizontal axis represents the type of sample; a + in the IL-31 column indicates the IL-31 administration group, and a - indicates the IL-31 non-administration group. A - in the CuIIa column indicates that cucurbitacin IIa was not supplied (control), and the numerical value represents the final concentration (nM) of supplied cucurbitacin IIa.

[0099] In Figure 5, the values ​​on the vertical axis and the meaning of the IL-31 term on the horizontal axis are the same as in Figure 1. A negative value for the SCH term on the horizontal axis indicates that SCH772984 was not supplied (control), and the numerical value represents the final concentration (nM) of supplied SCH772984.

[0100] As is evident from Figures 4 and 5, it was shown that administering an ERK1 / 2 inhibitor to RPE1 cells, which are immune-related cells, can suppress the formation of primary cilia during inflammatory stimuli (IL-31 administration group).

[0101] Given that immune-related cells possess organelles called primary cilia, and that primary cilia expression is enhanced in inflammatory skin diseases, it was suggested that this inhibitor, which includes an ERK1 / 2 inhibitor that suppresses primary cilia expression, also has an anti-inflammatory effect, as demonstrated above. [Industrial applicability]

[0102] The present invention can be used as an inhibitor of primary cilia of immune-related cells, and more specifically, as an anti-inflammatory topical agent (e.g., anti-inflammatory agent, anti-inflammatory agent, anti-inflammatory agent, anti-inflammatory cosmetic, anti-inflammatory cosmetic, anti-inflammatory cosmetic).

Claims

1. An inhibitor of primary cilia in immune-related cells, containing an ERK1 / 2 inhibitor as its active ingredient.

2. The inhibitor according to claim 1, wherein the ERK1 / 2 inhibitor is at least one selected from the group consisting of cucurbitacin IIa and SCH772984.

3. An anti-inflammatory topical agent containing the inhibitor described in claim 1 or 2 as an active ingredient.

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