TRPV1 activity inhibitor and use thereof

Abstract

To provide a novel TRPV1 activity inhibitor and use thereof.SOLUTION: A TRPV1 activity inhibitor is used, the inhibitor containing, as an active ingredient, at least one selected from the group consisting of lupeol, guaiazulene, tonghaosu, a derivative of lupeol, a derivative of guaiazulene, a derivative of tonghaosu, and a salt of these derivatives.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to a TRPV1 activity inhibitor and its use. [Background technology]

[0002] TRPV1 (Transient Receptor Potential Vanilloid 1) is known to be an ion channel sensitive to capsaicin and heat, and its activation is also known to be associated with inflammatory pain.

[0003] Therefore, substances that inhibit TRPV1 activity (TRPV1 activity inhibitors) are useful as analgesics for inflammatory pain, etc. Known examples of such substances that inhibit TRPV1 activity include menthol, menthoxypropanediol, menthone glycerin acetal, and menthyl lactate (Patent Documents 1 to 3).

[0004] However, these substances not only inhibit TRPV1 activity but also promote TRPM8 (Transient Receptor Potential Melastatin 8) activity, which can cause a cooling sensation on the skin, making it difficult to use them as ingredients in formulations that do not require a cooling sensation. [Prior art documents] [Patent documents]

[0005] [Patent Document 1] Japanese Patent Application Laid-Open No. 2017-074024 [Patent Document 2] Japanese Patent Application Publication No. 2017-075129 [Patent Document 3] Japanese Patent Publication No. 2022-144971 Summary of the Invention [Problem to be solved by the invention]

[0006] Under the above circumstances, there is a need for the development of novel TRPV1 activity inhibitors.

[0007] An object of one aspect of the present invention is to provide a novel TRPV1 activity inhibitor and use thereof. [Means for solving the problem]

[0008] The present inventors have discovered that lupeol, guaiazulene, tonghaosu, lupeol derivatives, guaiazulene derivatives, tonghaosu derivatives, and salts of these derivatives have the effect of inhibiting TRPV1 activity, and have completed the present invention. The present invention encompasses the following inventions.

[0009] [1] A TRPV1 activity inhibitor containing at least one active ingredient selected from the group consisting of lupeol, guaiazulene, tonghaosu, derivatives of lupeol, derivatives of guaiazulene, derivatives of tonghaosu, and salts of these derivatives.

[0010] [2] The TRPV1 activity inhibitor according to [1], which does not have the effect of promoting TRPM8 activity.

[0011] [3] A skin irritation inhibitor comprising the TRPV1 activity inhibitor according to [1] or [2].

[0012] [4] An external skin preparation comprising the TRPV1 activity inhibitor according to [1] or [2], or the skin irritation inhibitor according to [3]. [Effects of the Invention]

[0013] According to one aspect of the present invention, a novel TRPV1 activity inhibitor and use thereof can be provided. [Brief explanation of the drawings]

[0014] [Figure 1]1 is a graph showing the results of evaluating the TRPV1 activity inhibitory effect of specific compounds using a Ca imaging method in an example of the present invention. [Figure 2] 1 is a graph showing the results of evaluating the TRPV1 activity inhibitory effect of a specific compound using a patch clamp method in an example of the present invention. [Figure 3] 1 is a graph showing the results of evaluating the TRPV1 activity inhibitory effect of specific compounds by measuring the amount of protein produced in an example of the present invention. [Figure 4] 1 is a graph showing the results of evaluating the synergistic effect of a specific compound on the TRPV1 activity inhibitory effect of the specific compound in an example of the present invention. DETAILED DESCRIPTION OF THE INVENTION

[0015] An example of an embodiment of the present invention will be described in detail below, but the present invention is not limited to this. Various modifications of the present invention 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."

[0016] [1. TRPV1 activity inhibitors] A TRPV1 activity inhibitor according to one embodiment of the present invention contains at least one active ingredient (e.g., one, two, three, four, five, six, seven, eight, or nine) selected from the group consisting of lupeol, guaiazulene, tonghaosu, lupeol derivatives, guaiazulene derivatives, tonghaosu derivatives, and salts of these derivatives. Various mixtures (e.g., plant extracts) containing lupeol, lupeol derivatives, guaiazulene, guaiazulene derivatives, tonghaosu, tonghaosu derivatives, and / or salts of these derivatives are known. For example, lupin oil (a product of Laboratoires Expanscience) is known as a mixture containing lupeol, and VEGETOL MATRICARIA MCF 793 HYDRO and VEGETOL MATRICARIA GR 337 HYDRO (manufactured by Gatefosse) are known as mixtures containing guaiazulene and tonghaos. These mixtures, or mixtures obtained by further purifying these mixtures to adjust the amount of active ingredient, can also be used as the active ingredient of the TRPV1 activity inhibitor according to one embodiment of the present invention.

[0017] Tonghuaosu is known to exist as two isomers: (E)-tonghuaosu (i.e., trans-tonghuaosu) and (Z)-tonghuaosu (i.e., cis-tonghuaosu). As shown in the examples below, among these isomers, (E)-tonghuaosu is preferred from the viewpoints of (i) better inhibition of TRPV1 activity by Tonghuaosu alone and (ii) better inhibition of TRPV1 activity by the synergistic effect of Tonghuaosu with other compounds.

[0018] A TRPV1 activity inhibitor according to one embodiment of the present invention preferably contains (i) at least one active ingredient selected from the group consisting of lupeol, guaiazulene, lupeol derivatives, guaiazulene derivatives, and salts of these derivatives, and (ii) at least one active ingredient selected from the group consisting of tonghaosu, tonghaosu derivatives, and salts of these derivatives. A TRPV1 activity inhibitor according to one embodiment of the present invention preferably contains (i') at least one active ingredient selected from the group consisting of lupeol, tonghaosu, lupeol derivatives, tonghaosu derivatives, and salts of these derivatives, and (ii') at least one active ingredient selected from the group consisting of guaiazulene, guaiazulene derivatives, and salts of these derivatives. With this configuration, TRPV1 activity can be more effectively inhibited by using multiple active ingredients that are thought to have different sites of action (different mechanisms of action).

[0019] A more preferred embodiment of the present invention is a TRPV1 activity inhibitor containing (iii) at least one active ingredient selected from the group consisting of guaiazulene, guaiazulene derivatives, and salts of the derivatives, and (iv) at least one active ingredient selected from the group consisting of tonghaosu, tonghaosu derivatives, and salts of the derivatives. This configuration allows for even better inhibition of TRPV1 activity by using multiple active ingredients thought to have different sites of action (different mechanisms of action).

[0020] As shown in the examples below, for example, the synergistic effect of Tonghaosu and other active ingredients can better inhibit TRPV1 activity. In this case, even if the amounts of Tonghaosu and other active ingredients contained in the TRPV1 activity inhibitor according to one embodiment of the present invention are reduced, TRPV1 activity can still be sufficiently inhibited. If Tonghaosu has effect A other than the TRPV1 activity inhibitory effect, and the other active ingredients have effect B other than the TRPV1 activity inhibitory effect, this configuration can fully exhibit the TRPV1 activity inhibitory effect while suppressing the unwanted expression of effect A and effect B.

[0021] A TRPV1 activity inhibitor according to one embodiment of the present invention can effectively inhibit TRPV1 activity.

[0022] TRPV1 is a type of transient receptor potential channel (also referred to as a TRP channel) that functions as a stimulus receptor. When a stimulus (e.g., a skin stimulus) is applied to a living body via TRPV1, an acute stimulus response occurs shortly after TRPV1 activation, and / or a delayed stimulus response occurs after a certain period of time has elapsed after TRPV1 activation. For example, a delayed stimulus response is caused by a response mediated by activation of a TRP channel (e.g., TRPV1) at a nerve ending in the skin, which is transmitted to sensory nerves such as the central nervous system through intercellular crosstalk, resulting in the sensation of pain or irritation. As shown in the Examples below, a TRPV1 activity inhibitor according to one embodiment of the present invention can suppress acute stimulus responses and / or delayed stimulus responses.

[0023] Furthermore, TRPV1 activation is known to be associated with (induce) itching, flushing, rash, pain, eczema, inflammation, and the like. A TRPV1 activity inhibitor according to one embodiment of the present invention can inhibit TRPV1 activity, and thus can suppress the above-mentioned symptoms associated with TRPV1 activation (itching, flushing, rash, pain, eczema, inflammation, and the like). More specifically, a TRPV1 activity inhibitor according to one embodiment of the present invention can be used as a skin irritation inhibitor or a topical skin preparation, as described below, for the purpose of suppressing symptoms associated with TRPV1 activation.

[0024] The lupeol derivatives, guaiazulene derivatives, and tonghaosu derivatives may be derivatives obtained by substituting a portion of lupeol, guaiazulene, or tonghaosu with another functional group or another atom, and the specific structure is not limited.

[0025] Examples of the other functional groups include a sulfo group, a hydroxyl group, a carbonyl group, an acetyl group, an alkyl group, an alkoxy group, an alkylthio group, an aryl group, an aryloxy group, an arylthio group, an arylalkyl group, an arylalkoxy group, an arylalkylthio group, an arylalkenyl group, an arylalkynyl group, an allyl group, an amino group, a substituted amino group, a silyl group, a substituted silyl group, a silyloxy group, a substituted silyloxy group, an arylsulfonyloxy group, an alkylsulfonyloxy group, and a nitro group. Examples of the other atoms include a carbon atom, a hydrogen atom, an oxygen atom, a nitrogen atom, a sulfur atom, a phosphorus atom, and a halogen atom.

[0026] The salt may be any salt that is physiologically acceptable when administered to a subject, and there are no limitations on the specific composition.

[0027] Examples of the salts include alkali metal salts (sodium salts, potassium salts, etc.), alkaline earth metal salts (calcium salts, magnesium salts, etc.), ammonium salts, organic base salts (trimethylamine salts, triethylamine salts, pyridine salts, picoline salts, dicyclohexylamine salts, N,N'-dibenzylethylenediamine salts, etc.), organic acid salts (acetates, maleates, tartrates, methanesulfonates, benzenesulfonates, formates, toluenesulfonates, trifluoroacetates, etc.), and inorganic acid salts (hydrochlorides, hydrobromides, sulfates, phosphates, etc.).

[0028] The derivatives and salts are not limited in their specific configuration as long as they have the effect of inhibiting TRPV1 activity. Whether or not a desired derivative or salt has the effect of inhibiting TRPV1 activity can be determined by Ca imaging, patch clamp analysis, gene expression analysis, and / or measurement of protein production, as described in the Examples below.

[0029] The salt of the guaiazulene derivative is more specifically sodium guaiazulene sulfonate, and may also be sodium guaiazulene sulfonate hydrate.

[0030] The active ingredient can be a mixture containing the active ingredient. Examples of the mixture containing lupeol include lupine oil and dandelion root extract. Examples of the mixture containing guaiazulene include chamomile oil, chamomile flower extract, and eucalyptus oil. Examples of plants containing tonghaos include chamomile, artemisia capillaris, and waldhemia glabra, and these plant extracts can be used as mixtures containing tonghaos.

[0031] A TRPV1 activity inhibitor according to one embodiment of the present invention may not have an effect of promoting TRPM8 activity.

[0032] As shown in the examples below, the compound used as the active ingredient of the TRPV1 activity inhibitor according to one embodiment of the present invention does not promote TRPM8 activity. Therefore, the TRPV1 activity inhibitor according to one embodiment of the present invention can prevent the skin from feeling cool and refreshing. For example, the TRPV1 activity inhibitor according to one embodiment of the present invention can be used as an ingredient in preparations that do not require the skin to feel cool and refreshing (e.g., skin irritation inhibitors and topical skin preparations).

[0033] The amount of the active ingredient contained in the TRPV1 activity inhibitor 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. It may be 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.

[0034] The TRPV1 activity inhibitor according to one embodiment of the present invention may contain ingredients other than the above-mentioned active ingredients.

[0035] 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.

[0036] 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.

[0037] Examples of the pH adjuster include hydrochloric acid, phosphoric acid, citric acid, acetic acid, sodium hydroxide, and potassium hydroxide.

[0038] Examples of the isotonicity agent include ionic isotonicity agents (eg, sodium chloride, potassium chloride, calcium chloride, magnesium chloride) and non-ionic isotonicity agents (eg, glycerin, propylene glycol, sorbitol, mannitol).

[0039] Examples of the preservative include benzalkonium chloride, benzalkonium bromide, benzethonium chloride, sorbic acid, potassium sorbate, methyl parahydroxybenzoate, propyl parahydroxybenzoate, and chlorobutanol.

[0040] Examples of the antioxidant include ascorbic acid, tocopherol, dibutylhydroxytoluene, butylhydroxyanisole, sodium erythorbate, propyl gallate, and sodium sulfite.

[0041] 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.

[0042] Examples of the excipient include lactose, sucrose, D-mannitol, xylitol, sorbitol, erythritol, starch, and crystalline cellulose.

[0043] Examples of the solvent include water, physiological saline, and alcohol.

[0044] Examples of the antibacterial agent 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.

[0045] The amount of ingredients other than the active ingredient contained in the TRPV1 activity inhibitor 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 99.99% by mass. %, may be 10% by mass to 99.9% by mass, may be 20% by mass to 99.9% by mass, may be 30% by mass to 99.9% by mass, may be 40% by mass to 99.9% by mass, may be 50% by mass to 99.9% by mass, may be 60% by mass to 99.9% by mass, may be 70% by mass to 99.9% by mass, may be 80% by mass to 99.9% by mass, or may be 90% by mass to 99.9% by mass.

[0046] The dosage form of the TRPV1 activity inhibitor according to one embodiment of the present invention is not particularly limited, and examples thereof include topical preparations (e.g., liquids, ointments, gels, creams, sticks, sheets, sprays, aerosols), tablets, powders, and granules.

[0047] The TRPV1 activity inhibitor according to one embodiment of the present invention may be in the form of a lotion (e.g., body lotion), deodorant cosmetic, lotion, emulsion, skin care cream, tonic, stick cosmetic, lip, facial cleanser, cleanser, or sheet cosmetic.

[0048] The route of administration of the TRPV1 activity inhibitor according to one embodiment of the present invention is not particularly limited, and examples thereof include subcutaneous administration, intradermal administration, transdermal administration, intraocular administration, oral administration, and transmucosal administration.

[0049] The interval at which the TRPV1 activity inhibitor of 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, and once every 1 to 7 days.

[0050] [2. Skin irritation inhibitors] A skin irritation inhibitor according to one embodiment of the present invention comprises a TRPV1 activity inhibitor according to one embodiment of the present invention.

[0051] Note that the description of the configuration explained in the above section [1. TRPV1 activity inhibitor] will be omitted here.

[0052] More specifically, (1) a TRPV1 activity inhibitor according to one embodiment of the present invention may be used as a skin irritation inhibitor according to one embodiment of the present invention, or (2) a TRPV1 activity inhibitor according to one embodiment of the present invention may be used as an active ingredient contained in a skin irritation inhibitor according to one embodiment of the present invention.

[0053] In the case of (2) above, the skin irritation inhibitor according to one embodiment of the present invention may correspond to an invention in which, for example, the "active ingredient" in the description of [1. TRPV1 activity inhibitor] above is replaced with "TRPV1 activity inhibitor," and the "TRPV1 activity inhibitor" in the description of [1. TRPV1 activity inhibitor] is replaced with "skin irritation inhibitor."

[0054] A skin irritation inhibitor according to one embodiment of the present invention can also be used as a skin irritation inhibitor intended to suppress, for example, symptoms associated with TRPV1 activation (such as itching, flushing, rash, pain, eczema, and inflammation).

[0055] [3. Skin Topical Agents] An external skin preparation according to one embodiment of the present invention comprises a TRPV1 activity inhibitor according to one embodiment of the present invention or a skin irritation inhibitor according to one embodiment of the present invention.

[0056] Note that the description of the configuration explained in the above section [1. TRPV1 activity inhibitor] will be omitted here.

[0057] More specifically, (3) a TRPV1 activity inhibitor according to one embodiment of the present invention may be used as a topical skin preparation according to one embodiment of the present invention, (4) a TRPV1 activity inhibitor according to one embodiment of the present invention may be used as an active ingredient contained in a topical skin preparation according to one embodiment of the present invention, (5) a skin irritation inhibitor according to one embodiment of the present invention may be used as a topical skin preparation according to one embodiment of the present invention, or (6) a skin irritation inhibitor according to one embodiment of the present invention may be used as an active ingredient contained in a topical skin preparation according to one embodiment of the present invention.

[0058] In the case of (4) above, the topical skin preparation according to one embodiment of the present invention may correspond to an invention in which, for example, the "active ingredient" in the description of [1. TRPV1 activity inhibitor] above is replaced with "TRPV1 activity inhibitor," and the "TRPV1 activity inhibitor" in the description of [1. TRPV1 activity inhibitor] is replaced with "topical skin preparation."

[0059] In the case of (6) above, the topical skin preparation according to one embodiment of the present invention may correspond to an invention in which, for example, the "active ingredient" in the description of [1. TRPV1 activity inhibitor] above is replaced with "skin irritation inhibitor," and the "TRPV1 activity inhibitor" in the description of [1. TRPV1 activity inhibitor] is replaced with "topical skin preparation."

[0060] The topical skin preparation according to one embodiment of the present invention can also be used, for example, as a topical skin preparation intended to suppress symptoms associated with TRPV1 activation (such as itching, flushing, rash, pain, eczema, and inflammation).

[0061] [4. Other] An embodiment of the present invention can also be configured as follows.

[0062] <1> A method for inhibiting TRPV1 activity, comprising the step of administering to a subject (e.g., a human) a TRPV1 activity inhibitor containing as an active ingredient at least one selected from the group consisting of lupeol, guaiazulene, tonghaosu, derivatives of lupeol, derivatives of guaiazulene, derivatives of tonghaosu, and salts of these derivatives.

[0063] <2> The TRPV1 activity inhibitor does not have a TRPM8 activity promoting effect. <1> The method for suppressing TRPV1 activity described in .

[0064] <3> A method for suppressing skin irritation, comprising the step of administering to a subject (e.g., a human) a skin irritation inhibitor containing a TRPV1 activity inhibitor containing at least one active ingredient selected from the group consisting of lupeol, guaiazulene, tonghaosu, derivatives of lupeol, derivatives of guaiazulene, derivatives of tonghaosu, and salts of these derivatives.

[0065] <4> A skin treatment method comprising the step of administering to a subject (e.g., a human) (i) a TRPV1 activity inhibitor containing at least one active ingredient selected from the group consisting of lupeol, guaiazulene, tonghaosu, derivatives of lupeol, derivatives of guaiazulene, derivatives of tonghaosu, and salts of these derivatives, or (ii) a skin irritation inhibitor containing a TRPV1 activity inhibitor containing at least one active ingredient selected from the group consisting of lupeol, guaiazulene, tonghaosu, derivatives of lupeol, derivatives of guaiazulene, derivatives of tonghaosu, and salts of these derivatives.

[0066] This invention may also contribute to achieving Goal 3 of the United Nations' Sustainable Development Goals (SDGs), including "Ensure good health and promote well-being for all." [Example]

[0067] The present invention will be described in more detail in the following examples, but the present invention is not limited to these examples.

[0068] <1. Preparation of cell lines used in the test> <1-1. Generation of TRPV1 stable expressing cell lines> A cDNA encoding human TRPV1 (a polynucleotide sequence from positions 276 to 2795 of the nucleotide sequence shown in GenBank accession number MN_080704.3) was inserted into the cloning site of a mammalian cell vector (Invitrogen, product name: pcDNA3.1(+)) to obtain a human TRPV1 expression vector. Five micrograms of the resulting human TRPV1 expression vector was suspended in 10 μL of sterile water to obtain Mixture I.

[0069] Additionally, HEK293 cells were cultured in 10% FBS-containing DMEM medium on a 100 mm diameter Petri dish maintained at 37°C in an atmosphere of 5% carbon dioxide by volume until they reached 70% confluency. The cultured HEK293 cells were suspended in 100 μL of R buffer included in the electroporation gene transfer reagent (Thermo Fisher, product name: Neon Transfection System 100 μL Kit, catalog number: MPK10025) to obtain a cell suspension.

[0070] The cell suspension was mixed with Mixture I, and gene transfer was carried out using the NeonTransfection System (Thermo Fisher, catalog number: MPK5000) under the conditions of Plus Voltage 1100V, Plus Width 20ms, and Plus Number 2.

[0071] The day after gene transfer, the culture medium for HEK293 cells was replaced with 10% FBS-containing DMEM medium supplemented with 1 mg / mL G418 sulfate. The cells were then cultured in a 5% carbon dioxide atmosphere for 3 weeks, with the medium replaced every two days, to obtain a TRPV1-stably expressing cell line in which the human TRPV1 expression vector was introduced into HEK293 cells.

[0072] <1-2. Generation of TRPM8-expressing cell lines> A cDNA encoding human TRPM8 (a polynucleotide sequence consisting of positions 41 to 3355 of the nucleotide sequence shown in GenBank accession number NM_024080.4) was inserted into the cloning site of a mammalian cell vector (Invitrogen, product name: pcDNA3.1(+)) to obtain a human TRPM8 expression vector. Mixture II was obtained by mixing 1 μg of the resulting human TRPM8 expression vector with 6 μL of a gene transfer reagent (Invitrogen, product name: PLUS Reagent, catalog number: 11514-015). Furthermore, 4 μL of a cationic lipid for gene transfer (manufactured by Invitrogen, trade name: Lipofectamine (registered trademark), catalog number: 18324-012) was mixed with 200 μL of a reduced-serum medium (manufactured by Invitrogen, trade name: OPTI-MEM (registered trademark) I Reduced-Serum Medium (catalog number: 11058021)) to obtain mixture III.

[0073] In addition, in a 5% by volume carbon dioxide atmosphere, 5 × 10 cells were cultured in a 10% by mass FBS-containing DMEM medium on a 35 mm diameter dish maintained at 37°C. 5 HEK293 cells were cultured to 70% confluency.

[0074] Mixture II and Mixture III were added to the resulting cell culture, thereby introducing the human TRPM8 expression vector into HEK293 cells and obtaining a TRPM8-expressing cell line.

[0075] 2. Evaluation of TRPV1 activity inhibition using Ca imaging In this study, the inhibitory effects of various compounds on TRPV1 activity were evaluated using the acute stimulus response (ion movement) that occurs shortly after TRPV1 activation as an indicator.

[0076] <2-1. Evaluation of agonists for TRPV1 activity> On the day before the test, 100 μL of medium (Sigma-Aldrich, DMEM) was added per well to a 96-well black clear plate (Corning, BioCoat™ microplate), and 2 × 10 4 were sown individually.

[0077] On the day of the test, the medium in each well was completely removed. Then, 100 μL of the loading reagent Fluo-4NW (Thermo Fisher Scientific) was added to each well and allowed to stand for 1 hour to load the TRPV1 stable expressing cell line with the loading reagent. After the loading process, 50 μL of the loading reagent was removed from each well.

[0078] 25 μL of one test compound solution (test sample X), 25 μL of CaBath solution (composition: 140 mM sodium chloride, 5 mM potassium chloride, 2 mM magnesium chloride, 2 mM calcium chloride, 10 mM glucose, and 10 mM HEPES-HCl buffer (pH 7.4)) (non-treat X), or 25 μL of capsaicin solution with a final concentration of 30 nM (control X) was added to each well, and then the fluorescence value of each well was measured for 3 minutes using an FDSS7000 (Hamamatsu Photonics K.K.) (agonist evaluation).

[0079] 2700 kinds of compounds were used as test compounds in the test compound solution. In addition, the test compound solution was prepared by adding CaBath solution to a DMSO solution (5 μL) containing the test compound so that the final concentration of DMSO was 2% by volume. The concentration of the test compound in the test compound solution was set arbitrarily.

[0080] The TRPV1 agonist activity in the agonist evaluation was the relative intensity of the fluorescence value measured in the well to which the test compound solution was added (test sample X) when the fluorescence value measured in the well to which the capsaicin solution was added (control X) was taken as 100%, and was calculated according to the following formula: TRPV1 agonist activity (%) = {(fluorescence value of test sample X - fluorescence value of non-treat X) / (fluorescence value of control X - fluorescence value of non-treat X)} × 100 ····(formula).

[0081] Test compounds for which no increase in fluorescence value was observed in the agonist evaluation (in other words, test compounds with small TRPV1 agonist activity (%) values ​​described below) were further subjected to antagonist evaluation described below.

[0082] <2-2. Evaluation of antagonists against TRPV1 activity> To wells containing test sample X, where no increase in fluorescence value was observed in the agonist evaluation described above, 25 μL of capsaicin solution (test sample Y) with a final concentration of 30 nM was added, and then the fluorescence value was measured for 2 minutes (antagonist evaluation). Furthermore, to wells containing non-treat X in the same agonist evaluation, 25 μL of CaBath solution (non-treat Y) or 25 μL of capsaicin solution (control Y) with a final concentration of 30 nM was added, and then the fluorescence value was measured for 2 minutes.

[0083] The TRPV1 antagonist activity in the antagonist evaluation was the relative intensity of the fluorescence value measured in the wells to which the test compound solution and the capsaicin solution were added, when the fluorescence value measured in the well to which only the capsaicin solution was added (control Y) was taken as 100%, and was calculated according to the following formula: TRPV1 antagonist activity (%) = {(fluorescence value of test sample Y - non-treat Y) / (fluorescence value of control Y - fluorescence value of non-treat Y)} × 100 ····(formula).

[0084] The test results are shown in 101 to 106 in Figure 1. "V1" indicates the results of the evaluation of each test compound as an antagonist of TRPV1 activity, and "mock" indicates the results of the evaluation of each test compound as an antagonist of TRPV1 activity using mock cells obtained by the method described in <1-1. Preparation of a stable TRPV1-expressing cell line>, except that a mammalian cell vector was used instead of the human TRPV1 expression vector. As shown in Figure 1, 101 to 106, among the test compounds, guaiazulene (Tokyo Chemical Industry Co., Ltd. CAS RN: 489-84-9), sodium guaiazulene sulfonate (Tokyo Chemical Industry Co., Ltd. CAS RN: 6223-35-4), lupeol (Tokyo Chemical Industry Co., Ltd. CAS RN: 545-47-1), menthol (Tokyo Chemical Industry Co., Ltd. CAS RN: 2216-51-5), (Z)-tonghaosu (TargetMol CAS RN: 4575-53-5), and (E)-tonghaosu (Key Organics Limited CAS RN: 50257-98-2) were found to inhibit TRPV1 activity. The horizontal axis of 101 to 106 in the figure shows the logarithmic concentration of each test compound at the end of each evaluation (when measuring fluorescence values).

[0085] <2-3. Evaluation of agonists for TRPM8 activity> On the day before the test, 100 μL of medium (Sigma-Aldrich, DMEM) was added per well to a 96-well black clear plate (Corning, BioCoat™ microplate), and 2 × 10 4 were sown individually.

[0086] On the day of the test, the medium in each well was completely removed. Then, 100 μL of the loading reagent Fluo-4NW (Thermo Fisher Scientific) was added to each well and allowed to stand for 1 hour to load the TRPM8-expressing cell line with the loading reagent. After the loading process, 50 μL of the loading reagent was removed from each well.

[0087] 50 μL of one test compound solution (test sample Z), 50 μL of CaBath solution (non-treat Z), or 50 μL of menthol solution (control Z) with a final concentration of 1 mM was added to each well, and then the fluorescence value of each well was measured for 3 minutes using an FDSS7000 (Hamamatsu Photonics K.K.) (agonist evaluation).

[0088] The TRPM8 agonist activity in the agonist evaluation was the relative intensity of the fluorescence value measured in the well to which the test compound solution was added (test sample Z), when the fluorescence value measured in the well to which menthol was added (control Z) was taken as 100%, and was calculated according to the following formula: TRPM8 agonist activity (%) = {(fluorescence value of test sample Z - fluorescence value of non-treated Z) / (fluorescence value of control Z - fluorescence value of non-treated Z)} × 100 ····(formula).

[0089] The test results are shown in Figure 1. "M8" indicates the results of the agonist evaluation of each test compound on TRPM8 activity. As shown in 101-103 and 105-106 in Figure 1, guaiazulene, sodium guaiazulenesulfonate, lupeol, (Z)-tonghaosu, and (E)-tonghaosu did not promote TRPM8 activity (the test results for M8 and mock are almost identical in Figure 1). On the other hand, as shown in 104 in Figure 1, menthol promoted TRPM8 activity. The horizontal axis of 101-106 in the figure indicates the logarithmic concentration of each test compound at the end of each evaluation (when the fluorescence value was measured).

[0090] 3. Evaluation of TRPV1 activity inhibition using patch clamp method In this study, the inhibitory effects of various compounds on TRPV1 activity were evaluated using the acute stimulus response (ion movement) that occurs shortly after TRPV1 activation as an indicator.

[0091] Capsaicin was dissolved in CaBath solution to a capsaicin concentration of 20 nM or 50 nM to obtain capsaicin-containing samples.

[0092] The TRPV1 stably expressing cell line was washed by incubating the cell line in CaBath solution at 37°C for 2 minutes with shaking.

[0093] After washing, the TRPV1 stable cell line was placed in a constant-temperature chamber containing CaBath solution. The tip of the electrode was placed in contact with the TRPV1 stable cell line in the constant-temperature chamber, and current was measured under a clamped voltage using current recording software (Molecular Devices, product name: pCLAMP10) and a current recording device (Molecular Devices, product name: Axopatch 200B Amplifier).

[0094] After a predetermined time had elapsed since the start of current measurement, the TRPV1 stable-expressing cell line was exposed to capsaicin by circulating a capsaicin-containing sample in a circulating constant-temperature chamber containing the TRPV1 stable-expressing cell line.

[0095] Next, after a predetermined time had elapsed since the start of circulation of the capsaicin-containing sample, the TRPV1 stably expressing cell line was exposed to the desired compound (e.g., guaiazulene, lupeol, etc.) by circulating both the capsaicin-containing sample and a CaBath solution containing the desired compound at a predetermined concentration in the circulating constant temperature chamber into which the TRPV1 stably expressing cell line had been placed.

[0096] Finally, after a predetermined time had elapsed since the start of circulation of the CaBath solution containing the desired compound, the TRPV1 stable-expressing cell line was exposed to capsaicin by circulating a capsaicin-containing sample in a circulating constant-temperature chamber containing the TRPV1 stable-expressing cell line.

[0097] The current was continuously measured during the test. The current measurement results are shown in 201 and 202 of Figure 2. 201 of Figure 2 shows the current measurement results when guaiazulene was used as the desired compound, and 202 of Figure 2 shows the current measurement results when lupeol was used as the desired compound.

[0098] As can be seen from 201 and 202 in Figure 2, when the circulation of the capsaicin-containing sample was started, TRPV1 activity was promoted and the measured current increased (in other words, the amount of ions passing through TRPV1 increased). Next, when the circulation of the CaBath solution containing the desired compound (guaiazulene or lupeol) was started, TRPV1 activity was suppressed and the measured current decreased (in other words, the amount of ions passing through TRPV1 decreased). Finally, when the circulation of the capsaicin-containing sample was resumed, TRPV1 activity was promoted and the measured current increased again (in other words, the amount of ions passing through TRPV1 increased again).

[0099] 4. Evaluation of TRPV1 activity inhibition by measuring protein production In this study, the inhibitory effect of various compounds on TRPV1 activity was evaluated using the delayed stimulus response (change in protein production) that occurs at a certain time after TRPV1 activation as an indicator.

[0100] In a 5% by volume carbon dioxide atmosphere, 5 × 10 cells were cultured in a 35 mm diameter Petri dish in a DMEM medium containing 10% by mass of FBS, maintained at 37°C. 5 The TRPV1 stable expressing cell line was cultured to 70% confluency.

[0101] Next, lupeol was added to the 10% FBS-containing DMEM medium so that the final exposure concentration for the TRPV1 stable-expressing cell line was 5 μM or 50 μM. A control experiment (antagonism (-)) was also conducted in which lupeol was not added to the 10% FBS-containing DMEM medium.

[0102] Next, 3 minutes after adding lupeol, capsaicin, a known TRPV1 agonist, was added to the 10% FBS-containing DMEM medium to a concentration of 30 nM. 10 minutes after adding capsaicin, the TRPV1 stable-expressing cell line was washed with capsaicin-free 10% FBS-containing DMEM medium, and then cultured in the capsaicin-free medium for 24 hours.

[0103] After 24 hours of culture in a capsaicin-free medium, the culture supernatant was collected and the amounts of MCP-1 (Monocye Chemoattractant Protein 1) and MCP-2 (Monocye Chemoattractant Protein 2) proteins expressed in the TRPV1 stable expressing cell line were measured using a Bio-Plex human cytokine assay kit. Note that MCP-1 and MCP-2 are indicators of a delayed stimulus response that occurs a certain time after TRPV1 activation (see, for example, JP 2021-65171 A).

[0104] The test results are shown in 301 and 302 of Figure 3. 301 of Figure 3 shows the test results for MCP-1, and 302 of Figure 3 shows the test results for MCP-2.

[0105] As is clear from 301 and 302 in Figure 3, it was revealed that lupeol suppresses the delayed stimulus response that occurs at a certain time after TRPV1 activation.

[0106] <5. Evaluation of synergistic effects of active ingredients on TRPV1 activity inhibition> The nine types of samples listed in Table 1 below were evaluated for their inhibitory effect on TRPV1 activity according to the above-mentioned <2-1. Evaluation of agonists against TRPV1 activity> and <2-2. Evaluation of antagonists against TRPV1 activity>.

[0107] [Table 1]

[0108] The test results are shown in Figure 4. Comparison of the test results for Samples 1-3, 4-6, and 7-9 revealed that mixing Tonghaos with other compounds resulted in a synergistic effect in inhibiting TRPV1 activity. In Samples 1-3, the concentrations of Tonghaos and other compounds were set low, intentionally creating conditions where Tonghaos alone or other compounds alone would be unlikely to inhibit TRPV1 activity. As a result, it appears that a stronger synergistic effect was observed in Samples 1-3 compared to Samples 4-9. [Industrial Applicability]

[0109] The present invention can be used as a TRPV1 activity inhibitor, a skin irritation inhibitor, or an external skin preparation.

Claims

1. A TRPV1 activity inhibitor containing at least one active ingredient selected from the group consisting of lupeol, guaiazulene, tonghaosu, derivatives of lupeol, derivatives of guaiazulene, derivatives of tonghaosu, and salts of these derivatives.

2. The TRPV1 activity inhibitor according to claim 1, which does not have the effect of promoting TRPM8 activity.

3. A skin irritation inhibitor comprising the TRPV1 activity inhibitor of claim 1.

4. An external skin preparation comprising the TRPV1 activity inhibitor according to claim 1 or claim 2, or the skin irritation inhibitor according to claim 3.

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