WO2019189380A1 - Trp channel activity inhibitor - Google Patents

Abstract

Provided is a TRP channel activity inhibitor capable of effectively inhibiting the activity of TRP channels, wherein the TRP channel activity inhibitor is characterized by containing aluminum ions as an active ingredient for inhibiting the activity of TRP channels.

Description

TRP channel activity inhibitor

The present invention relates to a TRP channel activity inhibitor. More specifically, the present invention relates to a TRP channel activity inhibitor and a TRP channel activity suppression method.

The external preparation for skin is an external preparation that is applied to the skin and has a useful effect on the skin. However, the topical skin preparation may cause an unpleasant sensation or an unpleasant physiological event depending on the skin condition of the user. However, in recent years, due to an increase in safety awareness of users, substances and methods that cause or do not cause unpleasant sensations or unpleasant physiological events to the user and that sufficiently express useful effects are awaited. It is.

Incidentally, the TRP channel is a transient receptor potential channel involved in sensory reception for receiving various stimuli received from the outside world. It has been found by the present inventors that TRPA1, which is one of the TRP channels, is involved in the expression of unpleasant sensations due to, for example, an alkaline agent (see, for example, Patent Document 1).

JP 2012-62304 A

The present invention has been made in view of the above prior art, and an object thereof is to provide a TRP channel activity inhibitor and a TRP channel activity suppression method that effectively suppress the activity of a TRP channel.

The present invention
(1) A TRP channel activity inhibitor for suppressing the activity of a TRP channel, which contains aluminum ions as an active ingredient for suppressing the activity of the TRP channel. Agent,
(2) A TRP channel activity inhibitor for suppressing the activity of the TRP channel, wherein a substance capable of dissociating into aluminum ions is blended as an active ingredient for suppressing the activity of the TRP channel. The present invention relates to a TRP channel activity inhibitor, and (3) an activity suppression method for suppressing the activity of a TRP channel, which comprises contacting an aluminum ion with a TRP channel.

The TRP channel activity inhibitor and the TRP channel activity suppression method of the present invention have an excellent effect of effectively suppressing the activity of the TRP channel.

In Test Example 1, it is a graph showing the results of examining time-dependent changes in current in TRPV1-expressing cells. In Experiment 2, it is a graph which shows the result of having investigated the time-dependent change of the electric current in a TRPV1 expression cell. In Experiment 3, it is a graph which shows the result of having investigated the relationship between aluminum potassium sulfate concentration and TRPV1 activity. In Test Example 4, it is a graph showing the results of examining time-dependent changes in current in TRPA1-expressing cells. In Experiment 5, it is a graph which shows the result of having investigated the relationship between an aluminum chloride density | concentration and a suppression rate. In Experiment 6, it is a graph which shows the result of having investigated the time-dependent change of the electric current in TRPM8 expression cell. In Test Example 7, it is a graph showing the results of examining the change over time of the fluorescence intensity ratio. In Test Example 8, it is a graph showing the results of examining the change over time of the fluorescence intensity ratio. In Experiment 9, it is a graph which shows the result of having investigated the relationship between the kind of sample, and TRPV1 activity. In Experiment 10, it is a graph which shows the result of having investigated the relationship between the kind of sample, and TRPA1 activity. In Experiment 11, it is a graph which shows the result of having investigated the relationship between aluminum potassium sulfate concentration and the inhibition rate. In Experiment 12, it is a graph which shows the result of having investigated the relationship between pH of a test sample, and a suppression rate.

1. TRP channel activity inhibitor In one aspect, the present invention is a TRP channel activity inhibitor for suppressing the activity of a TRP channel, and contains aluminum ions as an active ingredient for suppressing the activity of a TRP channel. The present invention relates to a TRP channel activity inhibitor (hereinafter referred to as “activity inhibitor A”).

Aluminum ions suppress the activity of TRP channels by agonists. Therefore, since the activity inhibitor A of the present invention contains aluminum ions as an active ingredient, the activity of the TRP channel is effectively suppressed by bringing the activity inhibitor A of the present invention into contact with the TRP channel. be able to.

In the activity inhibitor A of the present invention, aluminum ions may be present in a dissociated state in an aqueous solvent, for example. Examples of the aqueous solvent include water, citrate buffer, and phosphate buffer, but the present invention is not limited to such examples. When the aqueous solvent is water, the pH of the activity inhibitor A of the present invention is preferably 7.5 to 14 (hereinafter referred to as “high pH”) from the viewpoint of stably presenting aluminum ions in a dissociated state in water. ) Or 1 to 6.5 (hereinafter referred to as “low pH”). When the pH of the activity inhibitor A of the present invention is high, the pH of the activity inhibitor A of the present invention is preferably 7.5 or more from the viewpoint of stably presenting aluminum ions in a dissociated state in water. More preferably, it is 8 or more, more preferably 8.5 or more, and preferably 14 or less, more preferably 13 or less, from the viewpoint of stably presenting aluminum ions in a dissociated state in water. When the pH of the activity inhibitor A of the present invention is low, it is preferably 1 or more, more preferably 2 or more, and is dissociated in water from the viewpoint of stably presenting aluminum ions in a dissociated state in water. From the viewpoint of stably presenting aluminum ions, it is preferably 6.5 or less, more preferably 6 or less, and even more preferably 5 or less.

Since the aluminum ion concentration in the activity inhibitor A of the present invention varies depending on the type of TRP channel to be applied, the use of the activity inhibitor A of the present invention, and the like, it cannot be determined unconditionally. It is preferable to determine appropriately depending on the type of the agent and the use of the activity inhibitor A of the present invention. The concentration of aluminum ions in the activity inhibitor A of the present invention is usually preferably 10 μM or more, more preferably 100 μM or more from the viewpoint of sufficiently exerting the inhibitory effect on the activity of the TRP channel, and the activity inhibitor A of the present invention. From the viewpoint of improving the storage stability, it is preferably 20 mM or less, more preferably 10 mM or less. In this specification, the aluminum ion concentration is a value measured using a metal indicator (trade name: Cu-PAN, manufactured by Dojindo Laboratories).

The activity inhibitor A of the present invention may contain other components such as a pH adjuster and a surfactant within a range that does not hinder the object of the present invention.

The activity inhibitor A of the present invention can be produced, for example, by mixing a substance that dissociates into aluminum ions and an aqueous solvent. When the activity inhibitor A of the present invention contains other components such as a pH adjuster and a surfactant, the activity inhibitor A is obtained by, for example, mixing a substance that dissociates into aluminum ions and other components. Can be manufactured.

Examples of the substance dissociating into aluminum ions include aluminum halides, inorganic acid aluminum salts, and organic acid aluminum salts, but the present invention is not limited to such examples. Examples of the aluminum halide include aluminum chloride, but the present invention is not limited to such examples. The inorganic acid aluminum salt may be an inorganic acid aluminum single salt or an inorganic acid aluminum double salt. As an inorganic acid aluminum single salt, although aluminum phosphate etc. are mentioned, for example, this invention is not limited only to this illustration. Examples of inorganic acid aluminum double salts include aluminum sulfate double salts, but the present invention is not limited to such examples. Examples of the aluminum sulfate double salt include aluminum sulfate alkali metal double salts such as sodium aluminum sulfate and potassium aluminum sulfate, and aluminum ammonium sulfate, but the present invention is not limited to such examples. Examples of the organic acid aluminum salt include aluminum formate, aluminum acetate, and aluminum citrate, but the present invention is not limited to such examples. Of these substances dissociating into aluminum ions, aluminum halides, inorganic acid aluminum salts and organic acid aluminum salts are preferred from the viewpoint of sufficiently exerting an inhibitory effect on the activity of the TRP channel. More preferred are salts, even more preferred are aluminum chloride, potassium aluminum sulfate and aluminum ammonium sulfate, even more preferred are aluminum chloride and potassium aluminum sulfate, and even more preferred is aluminum chloride.

In another aspect, the present invention is a TRP channel activity inhibitor for suppressing the activity of the TRP channel, and contains a substance that dissociates into aluminum ions as an active ingredient for suppressing the activity of the TRP channel. The present invention relates to a TRP channel activity inhibitor (hereinafter referred to as “activity inhibitor B”).

When the activity inhibitor B of the present invention is brought into contact with the TRP channel, the activity of the TRP channel is effectively inhibited by the contact between the aluminum ion dissociated from the substance dissociating into the aluminum ion and the TRP channel. . Therefore, since the activity inhibitor B of the present invention contains a substance that dissociates into aluminum ions, the activity of the TRP channel can be effectively suppressed.

The substance that dissociates into aluminum ions may be dissociated into aluminum ions when brought into contact with the TRP channel. The substance dissociating into aluminum ions used in the activity inhibitor B of the present invention is the same as the substance dissociating into aluminum ions used in the production of the activity inhibitor A of the present invention. Among the substances dissociating into aluminum ions, aluminum halide, inorganic acid aluminum salt, and organic acid aluminum salt are preferable, and aluminum halide and aluminum sulfate double salt are preferable from the viewpoint of sufficiently exerting an inhibitory effect on the activity of TRP channel. More preferably, aluminum chloride, potassium aluminum sulfate and aluminum ammonium sulfate are more preferable, aluminum chloride and potassium aluminum sulfate are more preferable, and aluminum chloride is still more preferable.

In the activity inhibitor B of the present invention, the substance dissociated into aluminum ions may exist in a dissolved state in an aqueous solvent. The aqueous solvent used for the activity inhibitor B of the present invention is the same as the aqueous solvent used for the activity inhibitor A described above. When the aqueous solvent is water, the pH of the activity inhibitor B of the present invention varies depending on the type of substance that dissociates into aluminum ions and cannot be determined unconditionally. It is preferable to determine appropriately according to the above. The pH of the activity inhibitor B of the present invention when the aqueous solvent is water is preferably the high pH or the low pH from the viewpoint of stably allowing aluminum ions to dissociate in water. When the pH of the activity inhibitor B of the present invention is high, the pH of the activity inhibitor B of the present invention is preferably 7.5 or more from the viewpoint of stably presenting aluminum ions in a dissociated state in water. More preferably, it is 8 or more, more preferably 8.5 or more, and preferably 14 or less, more preferably 13 or less, from the viewpoint of stably presenting aluminum ions in a dissociated state in water. When the pH of the activity inhibitor B of the present invention is low, it is preferably 1 or more, more preferably 2 or more, and is dissociated in water from the viewpoint of stably presenting aluminum ions in a dissociated state in water. From the viewpoint of stably presenting aluminum ions, it is preferably 6.5 or less, more preferably 6 or less, and even more preferably 5 or less.

Since the concentration of the substance dissociating into aluminum ions in the activity inhibitor B of the present invention varies depending on the type of substance dissociating into aluminum ions, the type of TRP channel to be applied, etc., it cannot be determined unconditionally. It is preferable to determine appropriately depending on the type of substance dissociated into ions, the type of TRP channel to be applied, and the like. The amount of the substance dissociated into aluminum ions per 100 parts by mass of the activity inhibitor B of the present invention is preferably 0.01 parts by mass or more, more preferably from the viewpoint of sufficiently exerting the inhibitory effect on the activity of the TRP channel. It is 0.05 parts by mass or more, and is 100 parts by mass or less from the viewpoint of sufficiently exerting an inhibitory effect on the activity of the TRP channel. When the activity inhibitor of the present invention contains components other than the substance that dissociates into aluminum ions, the upper limit of the amount of the substance that dissociates into aluminum ions per 100 parts by mass of the activity inhibitor B of the present invention is the activity suppression of the present invention. From the viewpoint of improving the storage stability of the agent B, it is preferably 10 parts by mass or less, more preferably 1 part by mass or less.

The activity inhibitor B of the present invention may contain other components such as a pH adjuster and a surfactant within a range that does not hinder the purpose of the present invention.

The aluminum ion concentration of the activity inhibitor B of the present invention at the time of use is usually preferably 0.01 mM or more, more preferably 0.1 mM or more, from the viewpoint of sufficiently expressing the TRP channel activity inhibitory action. From the viewpoint of sufficiently expressing the activity suppressing action, it is preferably 10 mM or less, more preferably 5 mM or less.

The activity inhibitor B of the present invention can be produced, for example, by mixing a substance that dissociates into aluminum ions and an aqueous solvent. When the activity inhibitor B of the present invention contains other components such as a pH adjuster and a surfactant, for example, the activity inhibitor B is obtained by mixing a substance that dissociates into aluminum ions and other components. Can be manufactured.

Examples of the TRP channel include TRPA1, TRPM8, TRPV1, TRPV3, and TRPV4, but the present invention is not limited to such examples. Among these TRP channels, TRPA1, TRPM8, TRPV1, TRPV3 and TRPV4 are preferable because the activity can be effectively suppressed.

Examples of TRPA1 include human TRPA1 (for example, GenBank accession number NM_007332) and the like, but the present invention is not limited to such examples. The activity of TRPA1 includes, for example, chemical stimulation with allyl isothiocyanate, high pH stimulation under conditions of pH 10 to 12, cold stimulation at a temperature of around 17 ° C., and stimulation from the outside of cells by stimulation such as mechanical stimulation. The ability to transport calcium ions; the ability to regulate the membrane potential by the stimulus, and the like are exemplified, but the present invention is not limited to such examples. The ability of TRPA1 to transport calcium ions can be examined, for example, by measuring the inflow of calcium ions from the outside into the cell accompanying the binding of a TRPA1 agonist to TRPA1 possessed by a TRPA1 expressing cell. The ability to regulate the membrane potential possessed by TRPA1 can be examined, for example, by measuring the amount of increase in current in the cells accompanying the binding of a TRPA1 agonist to TRPA1 possessed by a TRPA1-expressing cell. Examples of the TRPA1 agonist include allyl isothiocyanate, cinnamaldehyde, and allicin, but the present invention is not limited to such examples. Examples of unpleasant sensations or physiological events resulting from the activation of TRPA1 include pain such as inflammatory pain and neuropathic pain, excessive irritation, excessive cooling, etc. It is not limited only to such illustration.

Examples of TRPM8 include human TRPM8 (for example, GenBank accession number NM_024080), but the present invention is not limited only to such illustration. Examples of the activity of TRPM8 include the ability to transport calcium ions from the outside to the inside of the cell by stimulation such as chemical stimulation by menthol and cold stimulation at a temperature of about 25 to 28 ° C. It is not limited to illustration only. The ability of TRPM8 to transport calcium ions can be examined, for example, by measuring the inflow of calcium ions from the outside to the inside of the cell accompanying the binding of the TRPM8 agonist to TRPM8 possessed by TRPM8-expressing cells. Examples of the TRPM8 agonist include menthol and icilin, but the present invention is not limited to such examples. Examples of unpleasant sensations or physiological events resulting from the activation of TRPM8 include excessive cooling, but the present invention is not limited to such examples.

Examples of TRPV1 include human TRPV1 (eg, GenBank accession number NM — 080704), but the present invention is not limited to such examples. The activity of TRPV1 includes, for example, chemical stimulation with capsaicin, low pH stimulation under conditions of pH 3 to 5.5, thermal stimulation at a temperature around 43 ° C., stimulation from pain, mechanical stimulation, etc. Examples include the ability to transport calcium ions into cells, but the present invention is not limited to such examples. The ability of TRPV1 to transport calcium ions can be examined, for example, by measuring the inflow amount of calcium ions from the outside to the inside of the cell accompanying the binding of a TRPV1 agonist to TRPV1 of a TRPV1-expressing cell. Examples of TRPV1 agonists include capsaicin, camphor, and allicin, but the present invention is not limited to such examples. Examples of unpleasant sensations or physiological events resulting from the activation of TRPV1 include excessive heat sensation, but the present invention is not limited to such examples.

Examples of TRPV3 include human TRPV3 (for example, GenBank accession number: NM_145068), but the present invention is not limited to such examples. The activity of TRPV3 includes, for example, the ability to transport calcium ions from the outside to the inside of the cell by stimulation such as chemical stimulation by camphor, heat stimulation at a temperature of about 33 to 39 ° C. It is not limited only to such illustration. The ability of TRPV3 to transport calcium ions can be examined, for example, by measuring the inflow amount of calcium ions from the outside to the inside of the cell accompanying the binding of a TRPV3 agonist to TRPV3 of a TRPV3-expressing cell. Examples of TRPV3 agonists include camphor, eugenol, carvacrol, and the like, but the present invention is not limited to such examples. Examples of unpleasant sensations or physiological events resulting from the activation of TRPV3 include excessive heat feeling and skin hyperkeratinization, but the present invention is not limited to such examples.

Examples of TRPV4 include human TRPV4 (eg, GenBank accession number: NM — 021625), but the present invention is not limited to such examples. The activity of TRPV4 includes, for example, chemical stimulation with 4α-phorbol-12,13-didecanoate, etc., thermal stimulation at a temperature around 27 to 34 ° C., hypoosmotic stimulation, mechanical stimulation, etc. However, the present invention is not limited to such examples. The ability of TRPV4 to transport calcium ions can be examined, for example, by measuring the inflow of calcium ions from the outside into the cells accompanying the binding of a TRPV4 agonist to TRPV4 possessed by a TRPV4-expressing cell. Examples of the TRPV4 agonist include 4α-phorbol 12,13-didecanoate, but the present invention is not limited to such examples. Examples of unpleasant sensations or physiological events resulting from the activation of TRPV4 include excessive thermal sensation and itchiness, but the present invention is not limited to such examples.

The TRP channel activity inhibiting action of the activity inhibitor A and the activity inhibitor B of the present invention is, for example, the intracellular calcium ion concentration of a TRP channel-expressing cell (hereinafter referred to as “TRP channel-expressing cell”), TRP channel expression. It can be evaluated by using the current in the cell as an index.

The TRP channel-expressing cell may be a cell expressing an endogenous TRP channel or a cell expressing an exogenous TRP channel. The TRP channel-expressing cell may be a cell that expresses one type of TRP channel, or may be a cell that expresses two or more types of TRP channels. Examples of cells that express an exogenous TRP channel include cells obtained by introducing a nucleic acid encoding an exogenous TRP channel into a host cell, but the present invention is limited to such examples only. is not.

Specific examples of the evaluation method for the TRP channel activity inhibitory action include the following evaluation method 1, evaluation method 2, and the like, but the present invention is not limited to such examples.

<Evaluation method 1>
(1A) contacting a TRP channel-expressing cell with the activity inhibitor A or activity inhibitor B of the present invention and an agonist of the TRP channel, and measuring the intracellular calcium ion concentration A of the TRP channel-expressing cell;
(1B) contacting a TRP channel-expressing cell with a TRP channel agonist and measuring the intracellular calcium ion concentration B of the TRP channel-expressing cell; and (1C) intracellular calcium ion concentration A and intracellular calcium ion concentration. A method comprising the step of comparing with B.

<Evaluation method 2>
(2A) contacting a TRP channel-expressing cell with the activity inhibitor A or activity inhibitor B of the present invention and a TRP channel agonist, and measuring the current A in the TRP channel-expressing cell;
(2B) A method comprising the steps of contacting a TRP channel-expressing cell with an agonist of a TRP channel, measuring a current B in the TRP channel-expressing cell, and (2C) comparing the current A and the current B.

Examples of the method for measuring the intracellular calcium ion concentration A and the intracellular calcium ion concentration B in Evaluation Method 1 include a method using a calcium indicator that binds to calcium ions, but the present invention is limited only to such examples. Is not to be done. In the method using a calcium indicator, the intracellular calcium ion concentration is determined by introducing it into a TRP channel-expressing cell, binding the calcium indicator to the calcium ion in the TRP channel-expressing cell, and examining the amount of the calcium indicator bound to the calcium ion. Can be measured. Since the calcium indicator can measure the amount of calcium indicator bound to calcium ions with a simple operation, it must be a reagent that can detect changes before and after binding with calcium ions by changes in optical properties, etc. Is preferred. Examples of changes in optical characteristics include changes in fluorescence intensity and changes in absorbance, but the present invention is not limited to such examples. Examples of the calcium indicator include a fluorescent calcium indicator whose fluorescence intensity changes before and after binding with calcium ions, but the present invention is not limited to such examples. Specific examples of calcium indicators include 1- [6-amino-2- (5-carboxy-2-oxazolyl) -5-benzofuranyloxy] -2- (2-amino-5-methylphenoxy) ethane-N , N, N ′, N′-tetraacetic acid pentaacetoxymethyl ester (Fura 2-AM), 1- [2-amino-5- (2,7-dichloro-6-hydroxy-3-oxo-9-xanthenyl) Phenoxy] -2- (2-amino-5-methylphenoxy) ethane-N, N, N ′, N′-tetraacetic acid tetraacetoxymethyl ester (Fluo 3-AM), 1- [2-amino-5- ( 2,7-difluoro-6-acetoxymethoxy-3-oxo-9-xanthenyl) phenoxy] -2- (2-amino-5-methylphenoxy) ethane-N, N, N ′, N′-tetraacetic acid Although fluorescent calcium indicator, such as tiger acetoxymethyl ester (Fluo 4-AM) and the like, and the present invention is not limited only to those exemplified.

In the evaluation method 1, when the intracellular calcium ion concentration A is lower than the intracellular calcium ion concentration B, it can be evaluated that the activity inhibitor A or the activity inhibitor B of the present invention has a TRP channel activity inhibitory action. it can. In addition, the greater the difference between the intracellular calcium ion concentration A and the intracellular calcium ion concentration B, the higher the activity inhibitor A or activity B of the present invention is evaluated to have a higher TRP channel activity inhibitory effect. Can do.

The measurement method of the current A and the current B in the evaluation method 2 includes, for example, a patch clamp method, but the present invention is not limited to such an example.

In the evaluation method 2, when the absolute value of the current A is smaller than the absolute value of the current B, it can be evaluated that the activity inhibitor A or the activity inhibitor B of the present invention has a TRP channel activity inhibitory action. Moreover, it can be evaluated that the activity inhibitor A or activity inhibitor B of this invention has a high TRP channel activity inhibitory effect, so that the difference between the absolute value of the electric current A and the absolute value of the electric current B is large. .

As described above, the TRP channel activity inhibitor of the present invention can suppress the activity of TRP channels involved in the development of unpleasant sensations or physiological events. Therefore, the TRP channel activity inhibitor of the present invention is used for applications that suppress the expression of unpleasant sensations or physiological events resulting from activation of TRP channels, for example, applications that suppress stimulation caused by activation of TRPA1. Stimulus suppressant, a cooling sensation adjuster used for adjusting the cooling sensation caused by TRPM8 activation, a pain suppressor used for suppressing pain caused by TRPV1 activation, and TRPV3 activation It can be suitably used for a skin cell hyperkeratinization inhibitor used for the purpose of suppressing the resulting hyperkeratinization of skin cells, a stagnation inhibitor used for the purpose of suppressing the itch caused by activation of TRPV4, and the like. .

2. TRP channel activity suppression method The TRP channel activity suppression method of the present invention (hereinafter referred to as "activity suppression method") is an activity suppression method for suppressing the activity of a TRP channel, wherein aluminum ions and a TRP channel are brought into contact with each other. This is a method for inhibiting the activity of a TRP channel. In addition, the medical practice may be excluded from the concept of the activity suppression method of this invention, and does not need to be excluded. In this specification, a medical practice refers to an action in which a doctor and a person who receives instructions from the doctor perform treatment on a human. According to the activity suppressing method of the present invention, since the aluminum ion and the TRP channel are brought into contact with each other, the activity of the TRP channel can be effectively suppressed.

The contact between the aluminum ion and the TRP channel can be performed by supplying the aluminum ion to a portion including the TRP channel. Examples of the site containing the TRP channel include skin epidermis, airway epithelium, peripheral nerve, ocular mucosa, and the like, but the present invention is not limited to such examples.

In the contact between aluminum ions and the TRP channel, aluminum ions and components that stably maintain the aluminum ions can be used in combination. Examples of the component that stably maintains aluminum ions include an aqueous solvent, but the present invention is not limited to such examples. Moreover, you may use the activity inhibitor of this invention in the case of a contact with an aluminum ion and a TRP channel.

Aluminum ions are usually brought into contact with the TRP channel in a state of being contained in the liquid. The amount of aluminum ions contained in the liquid brought into contact with the TRP channel varies depending on the use of the method for suppressing activity of the present invention, the type of TRP channel to be applied, the type of site containing the TRP channel to be applied, etc. Therefore, it is preferable to determine appropriately according to the use of the activity suppression method of the present invention, the type of TRP channel to be applied, the type of site including the TRP channel to be applied, and the like. For example, when the application target of the activity suppression method of the present invention is a TRP channel contained in a peripheral nerve, the amount of aluminum ions in the liquid brought into contact with the TRP channel is usually from the viewpoint of sufficiently exhibiting the TRP channel activity suppression effect. The concentration is preferably 0.01 mM or more, more preferably 0.1 mM or more, and preferably 10 mM or less, more preferably 5 mM or less, from the viewpoint of reducing the action on other tissues.

Since the contact time between the aluminum ion and the TRP channel differs depending on the use of the activity suppression method of the present invention, the type of TRP channel to be applied, etc., it cannot be determined unconditionally, so the use of the activity suppression method of the present invention It is preferable to determine appropriately according to the type of TRP channel to be applied.

The TRP channel activity suppression effect by the activity suppression method of the present invention can be evaluated by the same technique as the evaluation of the TRP channel activity suppression effect by the activity inhibitor.

According to the activity suppression method of the present invention, the activity of the TRP channel that is related to the development of unpleasant sensations such as pain, excessive irritation, excessive cooling, excessive heat, and itching is effectively suppressed. Can be suppressed. Therefore, for example, when using an external preparation containing a component that may give an unpleasant sensation due to activation of the TRP channel, the activity suppression method of the present invention is performed using aluminum ions and the external preparation together. By doing so, it is possible to suppress the activity of the TRP channel and suppress the expression of unpleasant sensations due to the activation of the TRP channel.

As described above, according to the activity suppressing method of the present invention, since the aluminum ion and the TRP channel are brought into contact with each other, the activity of the TRP channel can be effectively suppressed. Therefore, the activity inhibitor of the present invention is used for suppressing an unpleasant sensation caused by activation of a TRP channel or an unpleasant physiological event, for example, an application for suppressing a stimulus caused by activation of TRPA1, TRPM8 Use for adjusting the cooling sensation caused by activation of TRPV1, use for suppressing pain caused by activation of TRPV1, use for suppressing hyperkeratinization of skin cells caused by activation of TRPV3, caused by activation of TRPV4 It is expected to be used for applications that suppress stagnation.

The present invention will be described in more detail with reference to the following examples. However, the present invention is not limited to such examples. In the following, the meaning of each abbreviation is as follows.
<Explanation of abbreviations>
DMEM: Dulbecco's modified Eagle medium FBS: fetal bovine serum NMDG-Cl: N-methyl-D-glucamine chloride BAPTA: 1,2-bis (o-aminophenoxide) ethane-N, N, N ′, N′-tetra Acetic acid HEPES: 2- [4- (2-hydroxyethyl) piperazin-1-yl] ethanesulfonic acid AITC: Allyl isothiocyanate 4αPDD: 4α-phorbol-12,13-didecanoate Fluorescence intensity _{340 nm} : Fluorescence intensity _at excitation wavelength 340 nm Fluorescence Intensity _{380 nm} : fluorescence intensity at excitation wavelength 380 nm

Preparation Example 1
(1) Preparation of TRPV1-expressing cells Human TRPV1 cDNA (cDNA corresponding to positions 276 to 2795 of the base sequence shown in GenBank accession number: NM — 080704) was obtained by using a vector for mammalian cells [trade name: pcDNA3. 1 (+)] to obtain a human TRPV1 expression vector. 1 μg of the obtained human TRPV1 expression vector was mixed with 6 μL of a gene introduction reagent [manufactured by Invitrogen, trade name: PLUS Reagent, catalog number: 11514-015] to obtain a mixture I. In addition, 4 μL of a cationic lipid for gene transfer [manufactured by Invitrogen, trade name: Lipofectamine (registered trademark), catalog number: 18324-012] and a serum-reducing medium [trade name: OPTI-MEM (manufactured by Invitrogen), registered (Trademark) I Reduced-Serum Medium (Cat. No. 11058021)] 200 μL was mixed to obtain a mixture II.

In 10% FBS-containing DMEM on a 35 mm diameter petri dish maintained at 37 ° C. in a 5% by volume carbon dioxide atmosphere, 5 × 10 ⁵ HEK293 cells were cultured to 70% confluency. By adding the mixture I and the mixture II to the obtained cell culture, a human TRPV1 expression vector was introduced into HEK293 cells to obtain TRPV1-expressing cells.

(2) Preparation of TRPA1-expressing cells In Preparation Example 1 (1), instead of using human TRPV1 cDNA, human TRPA1 cDNA [GenBank accession number: cDNA corresponding to positions 63 to 3888 of the nucleotide sequence shown in NM_007332] is used. Except that, TRPA1-expressing cells were obtained in the same manner as in Preparation Example 1 (1).

(3) Preparation of TRPM8-expressing cells In Preparation Example 1 (1), instead of using human TRPV1 cDNA, human TRPM8 cDNA [GenBank accession number: cDNA corresponding to positions 41 to 3355 of the nucleotide sequence shown in NM_024080] is used. Except for the above, the same operation as in Preparation Example 1 (1) was performed to obtain TRPM8-expressing cells.

(4) Preparation of TRPV3-expressing cells In Preparation Example 1 (1), instead of using human TRPV1 cDNA, human TRPV3 cDNA [GenBank accession number: cDNA corresponding to positions 323 to 2695 of the nucleotide sequence shown in NM_145068] is used. Except that, TRPV3-expressing cells were obtained in the same manner as in Preparation Example 1 (1).

(5) Preparation of TRPV4-expressing cells In Preparation Example 1 (1), instead of using human TRPV1 cDNA, human TRPV4 cDNA [GenBank accession number: cDNA corresponding to positions 90 to 2705 of the nucleotide sequence shown in NM — 021625] is used. Except that, TRPV4-expressing cells were obtained in the same manner as in Preparation Example 1 (1).

Example 1
Aluminum chloride was added to solvent A (composition: 140 mM NMDG-Cl, 1 mM magnesium chloride, 5 mM BAPTA and 10 mM HEPES buffer) so that the concentration was 5 mM to obtain a mixture. By adding hydrochloric acid to the obtained mixture, the pH of the mixture was adjusted to 5 to obtain a test sample. In the obtained test sample, aluminum ions were present in a dissociated state (aluminum ion concentration: 5 mM).

Comparative Example 1
By adding hydrochloric acid to solvent A, the pH of solvent A was adjusted to 5 to obtain a sample for low pH stimulation.

Test example 1
The TRPV1-expressing cells obtained in Preparation Example 1 (1) were incubated in solvent A with shaking at 37 ° C. for 2 minutes to wash the TRPV1-expressing cells. Next, the TRPV1-expressing cells after washing were placed in a circulating constant temperature chamber containing solvent A. The tip of the electrode was brought into contact with the TRPV1-expressing cells in the circulation constant temperature chamber, and current recording software [Molecular Devices, product name: pCLAMP10] and current recording device [Molecular Devices, product name: Axopat 200B Amplifier] Was used to measure the current in TRPV1-expressing cells over time when the voltage was fixed at −60 mV. After 30 seconds from the start of measurement, the low pH stimulation sample obtained in Comparative Example 1 was circulated in the circulation constant temperature chamber. The test sample obtained in Example 1 was circulated in the circulation constant temperature chamber after 50 seconds had elapsed since the start of circulation of the low pH stimulation sample. After 30 seconds from the start of circulation of the test sample obtained in Example 1, the sample for low pH stimulation obtained in Comparative Example 1 was circulated in the circulation constant temperature chamber for 30 seconds. Thereafter, measurement of the current change was terminated.

FIG. 1 shows the results of examining time-dependent changes in current in TRPV1-expressing cells in Test Example 1. In the figure, “pH 5” indicates the duration of low pH stimulation, and “test sample” indicates the circulation period of the test sample.

1 shows that the absolute value of the current in the TRPV1-expressing cells increases with low pH stimulation but decreases with the addition of the test sample. From these results, it can be seen that aluminum ions dissociated from aluminum chloride have an action of suppressing the activity of TRPV1 activated by low pH stimulation.

Example 2
Aluminum chloride and capsaicin were added to solvent A so that the concentration of aluminum chloride was 5 mM and the concentration of capsaicin was 100 nM to obtain a mixture. By adding hydrochloric acid to the obtained mixture, the pH of the mixture was adjusted to 5 to obtain a test sample. In the obtained test sample, aluminum ions were present in a dissociated state (aluminum ion concentration: 5 mM).

Comparative Example 2
An agonist-containing sample was obtained by adding capsaicin to solvent A so that its concentration was 100 nM.

Test example 2
In Test Example 1, instead of using the test sample obtained in Example 1, the test sample obtained in Example 2 was used, and in place of using the low pH stimulation sample obtained in Comparative Example 1, Except that the agonist-containing sample obtained in 2 was used, the same operation as in Test Example 1 was performed, and the current in the TRPV1-expressing cells was measured over time.

FIG. 2 shows the results of examining the temporal change of the current in the TRPV1-expressing cells in Test Example 2. In the figure, “CAP” represents the duration of stimulation with capsaicin, and “test sample” represents the circulation period of the test sample.

The results shown in FIG. 2 indicate that the absolute value of the current in the TRPV1-expressing cells increases with stimulation with capsaicin contained in the agonist-containing sample, but decreases with the addition of the test sample. From these results, it can be seen that aluminum ions dissociated from aluminum chloride have an action of suppressing the activity of TRPV1 activated by stimulation with capsaicin.

In Example 2, instead of using aluminum chloride, aluminum chloride was also used when an inorganic acid aluminum salt such as aluminum phosphate or potassium aluminum sulfate; or another substance dissociating into aluminum ions such as aluminum acetate was used. The same trend is seen. From these results, it can be seen that aluminum ions have an action of suppressing the activity of TRPV1.

Test example 3
(1) Preparation of sample Aluminum potassium sulfate and capsaicin with a concentration of 1 μM aluminum potassium sulfate [aluminum ion concentration: 1 μM (experiment number 1)], 10 μM [aluminum ion concentration: 10 μM (experiment number 2)], 100 μM [aluminum ion] Concentration: 100 μM (experiment number 3)], 200 μM [aluminum ion concentration: 200 μM (experiment number 4)], 500 μM [aluminum ion concentration: 500 μM (experiment number 5)], 1000 μM [aluminum ion concentration: 1000 μM (experiment number 6) ] 5000 μM ([aluminum ion concentration: 5000 μM (experiment number 7)] or 10000 μM [aluminum ion concentration: 10000 μM (experiment number 8)] and added to solvent A so that the concentration of capsaicin is 100 nM. To obtain a compound. By addition of hydrochloric acid to the resulting mixture, to obtain a test sample to adjust the pH of the mixture to 5.

(2) Calculation of fluorescence intensity ratio _agonist The TRPV1-expressing cells obtained in Preparation Example 1 (1) contain 10 mass% FBS containing FURA 2-AM (manufactured by Invitrogen), a fluorescent calcium ion indicator, at a final concentration of 5 μM. By incubating in DMEM at room temperature for 60 minutes, FURA 2-AM was introduced into TRPV1-expressing cells to obtain indicator-introduced cells. The indicator-introduced cells were placed in a circulating constant temperature chamber of a fluorescence measuring apparatus with a circulating constant temperature chamber (trade name: ARGUS-50, manufactured by Hamamatsu Photonics Co., Ltd.), and then washed with solvent A. Next, the agonist-containing sample obtained in Comparative Example 2 was placed in a circulation constant temperature chamber. Thereafter, the fluorescence intensity _{340 nm} (fluorescence intensity A) in the presence of the agonist and the fluorescence intensity _{380 nm} (fluorescence intensity B) in the presence of the agonist were measured.

In addition, the same operation as described above was performed except that the control (solvent A) was used instead of the agonist-containing sample obtained in Comparative Example 2, and the fluorescence intensity in the presence of the control was _{340 nm} (fluorescence intensity). C) and fluorescence intensity _{380 nm} (fluorescence intensity D) in the presence of a control were measured.

Using fluorescence intensities A to D, the formula (I):
[Δ fluorescence intensity ratio _agonist ]
= [Fluorescence intensity A / fluorescence intensity B]-[fluorescence intensity C / fluorescence intensity D] (I)
Accordingly, a Δfluorescence intensity ratio _agonist was calculated.

Further, the same operation as that when using the agonist-containing sample obtained in Comparative Example 2 except that each test sample of Experiment Nos. 1 to 8 was used instead of using the agonist-containing sample obtained in Comparative Example 2. The fluorescence intensity _{340 nm} (fluorescence intensity E) in the presence of aluminum ions and agonist and the fluorescence intensity _{380 nm} (fluorescence intensity F) in the presence of aluminum ions and agonist were measured.

Using the fluorescence intensities C to F, the formula (II):
[Δ fluorescence intensity specific _{activity inhibitor} ]
= [Fluorescence intensity E / fluorescence intensity F]-[fluorescence intensity C / fluorescence intensity D] (II)
Accordingly, the Δ fluorescence intensity specific _{activity inhibitor} was calculated.

Using the calculated Δ fluorescence intensity ratio _agonist and Δ fluorescence intensity ratio _{activity inhibitor} , the formula (III):
[TRPV1 activity]
= [Δ fluorescence intensity ratio _{activity inhibitor} / Δ fluorescence intensity ratio _agonist ] (III)

Accordingly, TRPV1 activity was calculated.

FIG. 3 shows the results of examining the relationship between the potassium aluminum sulfate concentration and the TRPV1 activity in Test Example 3.

Using the results shown in FIG. 3, the 50% inhibitory concentration of aluminum potassium sulfate for TRPV1 activity was calculated. As a result, it was found that the 50% inhibitory concentration of aluminum potassium sulfate for TRPV1 activity was 246 μM. From the above results, it was found that the 50% inhibitory concentration of aluminum ions for TRPV1 activity was 246 μM.

Example 3
Aluminum chloride and AITC were added to solvent A so that the concentration of aluminum chloride was 5 mM and the concentration of AITC was 20 μM to obtain a mixture. By adding hydrochloric acid to the obtained mixture, the pH of the mixture was adjusted to 5 to obtain a test sample. In the obtained test sample, aluminum ions were present in a dissociated state (aluminum ion concentration: 5 mM).

Comparative Example 3
An agonist-containing sample was obtained by adding AITC to solvent A so that its concentration was 20 μM.

Test example 4
The TRPA1-expressing cells obtained in Preparation Example 1 (2) were incubated in solvent A with shaking at 37 ° C. for 2 minutes to wash the TRPA1-expressing cells. The washed TRPA1-expressing cells were placed in a circulating constant temperature chamber containing solvent A. The tip of the electrode was brought into contact with the TRPA1-expressing cell in the circulation constant temperature chamber, and the current in the TRPA1-expressing cell when the voltage was fixed at −60 mV was measured over time using current recording software and a current recording device. After 30 seconds from the start of measurement, the agonist-containing sample obtained in Comparative Example 3 was circulated in the circulation constant temperature chamber. The test sample obtained in Example 3 was circulated in the circulation constant temperature chamber after 50 seconds had elapsed since the start of circulation of the agonist-containing sample obtained in Comparative Example 3. After 25 seconds from the start of circulation of the test sample obtained in Example 3, the agonist-containing sample obtained in Comparative Example 3 was circulated in the circulation constant temperature chamber. After 50 seconds from the start of circulation of the agonist-containing sample obtained in Comparative Example 3, the solvent A was circulated in the circulation constant temperature chamber, and the measurement of the current change was completed.

FIG. 4 shows the results of examining time-dependent changes in current in TRPA1-expressing cells in Test Example 4. In the figure, “AITC” indicates the duration of stimulation by AITC, and “Test sample” indicates the circulation period of the test sample.

From the results shown in FIG. 4, it can be seen that the absolute value of the current in the TRPA1-expressing cells increases with stimulation by AITC contained in the agonist-containing sample, but decreases with the addition of the test sample. From these results, it can be seen that aluminum ions dissociated from aluminum chloride have an action of suppressing the activity of TRPA1 activated by stimulation with AITC.

In Example 3, instead of using aluminum chloride, aluminum chloride was used when inorganic acid aluminum salts such as aluminum phosphate and potassium aluminum sulfate; and other substances that dissociate into aluminum ions such as aluminum acetate were used. The same trend is seen. From these results, it can be seen that aluminum ions have an action of suppressing the activity of TRPA1.

Example 4
Aluminum chloride and AITC were added to solvent A so that the concentration of aluminum chloride was 500 μM and the concentration of AITC was 20 μM to obtain a mixture. By adding hydrochloric acid to the obtained mixture, the pH of the mixture was adjusted to 5 to obtain a test sample. In the obtained test sample, aluminum ions were present in a dissociated state (aluminum ion concentration: 500 μM).

Example 5
Aluminum chloride and AITC were added to solvent A so that the concentration of aluminum chloride was 50 μM and the concentration of AITC was 20 μM to obtain a mixture. By adding hydrochloric acid to the obtained mixture, the pH of the mixture was adjusted to 5 to obtain a test sample. In the obtained test sample, aluminum ions were present in a dissociated state (aluminum ion concentration: 50 μM).

Test Example 5
The TRPA1-expressing cells obtained in Preparation Example 1 (2) were incubated in solvent A with shaking at 37 ° C. for 2 minutes to wash the TRPA1-expressing cells. Next, the TRPA1-expressing cells after washing were placed in a circulating constant temperature chamber containing solvent A. The tip of the electrode was brought into contact with the TRPA1-expressing cell in the circulation constant temperature chamber, and the current in the TRPA1-expressing cell when the voltage was fixed at −60 mV was measured over time using current recording software and a current recording device. After 30 seconds from the start of measurement, the agonist-containing sample obtained in Comparative Example 3 was circulated in the circulation constant temperature chamber. After 50 seconds from the start of circulation of the agonist-containing sample obtained in Comparative Example 3, 30 seconds for the test sample obtained in Example 5 and 30 for the test sample obtained in Example 4 in the circulation constant temperature chamber. The test sample obtained in Example 3 was circulated for 30 seconds. Thereafter, the agonist-containing sample obtained in Comparative Example 3 was circulated in a circulation constant temperature chamber. After 30 seconds from the start of circulation of the agonist-containing sample obtained in Comparative Example 3, the solvent A was circulated in the circulation constant temperature chamber, and the measurement of the current change was completed. Formula (IV):
[Inhibition rate]
= [(Change amount of current before and after circulation of agonist-containing sample) − (Change amount of current before and after circulation of test sample)] / [Change amount of current before and after circulation of sample containing agonist] (IV)
Therefore, the inhibition rate was calculated.

Fig. 5 shows the results of examining the relationship between the aluminum chloride concentration and the inhibition rate in Test Example 5.

FIG. 5 shows that aluminum chloride suppresses the activity of TRPA1 in a concentration-dependent manner. From these results, it can be seen that aluminum ions dissociated from aluminum chloride suppress the activity of TRPA1 in a concentration-dependent manner.

Example 6
Aluminum chloride and menthol were added to solvent A so that the concentration of aluminum chloride was 5 mM and the concentration of menthol was 1 mM to obtain a mixture. By adding hydrochloric acid to the obtained mixture, the pH of the mixture was adjusted to 5 to obtain a test sample. In the obtained test sample, aluminum ions were present in a dissociated state (aluminum ion concentration: 5 mM).

Comparative Example 4
Menthol was added to solvent A so that its concentration was 1 mM to obtain an agonist-containing sample.

Test Example 6
The TRPM8-expressing cells obtained in Preparation Example 1 (3) were incubated in solvent A with shaking at 37 ° C. for 2 minutes to wash the TRPM8-expressing cells. Next, the TRPM8-expressing cells after washing were placed in a circulating constant temperature chamber containing solvent A. The tip of the electrode was brought into contact with the TRPM8-expressing cell in the circulating constant temperature chamber, and the current in the TRPM8-expressing cell when the voltage was fixed at −60 mV was measured over time using current recording software and a current recording device. After a lapse of 20 seconds from the start of measurement, the low pH stimulation sample obtained in Comparative Example 1 was circulated in the circulation constant temperature chamber. After 15 seconds from the start of circulation of the low pH stimulation sample, the agonist-containing sample obtained in Comparative Example 4 was circulated in the circulation constant temperature chamber. After 30 seconds from the start of circulation of the agonist-containing sample obtained in Comparative Example 4, the test sample obtained in Example 6 was circulated in the circulation constant temperature chamber. After 30 seconds from the beginning of circulation of the test sample obtained in Example 6, the agonist-containing sample obtained in Comparative Example 4 was circulated in the circulation constant temperature chamber. After a lapse of 50 seconds from the start of circulation of the agonist-containing sample obtained in Comparative Example 4, the low pH stimulation sample obtained in Comparative Example 1 was circulated in the circulation constant temperature chamber. After 60 seconds from the start of circulation of the low pH stimulation sample, the solvent A was circulated in the circulation constant temperature chamber, and the measurement of the current change was completed.

FIG. 6 shows the results of examining time-dependent changes in current in TRPM8-expressing cells in Test Example 6. In the figure, “menthol” indicates the duration of stimulation with menthol, “pH 5” indicates the duration of low pH stimulation, and “test sample” indicates the circulation period of the test sample.

From the results shown in FIG. 6, the absolute value of the current in TRPM8-expressing cells increases by stimulation with menthol contained in the agonist-containing sample, but decreases by low pH stimulation and further decreases by addition of the test sample. I understand. From these results, it can be seen that aluminum ions dissociated from aluminum chloride have an action of suppressing the activity of TRPM8 activated by stimulation with menthol.

In Example 6, instead of using aluminum chloride, aluminum chloride was used when inorganic acid aluminum salts such as aluminum phosphate and potassium aluminum sulfate; and other substances dissociating into aluminum ions such as aluminum acetate were used. The same trend is seen. From these results, it can be seen that aluminum ions have an action of suppressing the activity of TRPM8.

Example 7
Aluminum chloride and camphor were added to solvent A so that the concentration of aluminum chloride was 5 mM and the concentration of camphor was 1 mM to obtain a mixture. By adding hydrochloric acid to the obtained mixture, the pH of the mixture was adjusted to 5 to obtain a test sample. In the obtained test sample, aluminum ions were present in a dissociated state (aluminum ion concentration: 5 mM).

Comparative Example 5
A camphor was added to the solvent A so that the density | concentration might be set to 1 mM, and the agonist containing sample was obtained.

Test Example 7
The TRPV3-expressing cells obtained in Preparation Example 1 (4) are incubated for 60 minutes at room temperature in 10% FBS-containing DMEM containing FURA 2-AM (manufactured by Invitrogen), a fluorescent calcium ion indicator, at a final concentration of 5 μM. Thus, FURA 2-AM was introduced into TRPV3-expressing cells to obtain indicator-introduced cells. The indicator-introduced cells were washed by incubating the indicator-introduced cells in solvent A with shaking at 37 ° C. for 2 minutes. The indicator-introduced cells after washing are placed in a circulating thermostat chamber of a fluorescence measuring apparatus with a circulating thermostat chamber (trade name: ARGUS-50, manufactured by Hamamatsu Photonics Co., Ltd.), and then the control (solvent A) is circulated and the fluorescence intensity is increased. Measurements at _{340 nm} and fluorescence intensity of _{380 nm} were started. After 50 seconds from the start of circulation of the control (solvent A), the agonist-containing sample obtained in Comparative Example 5 was circulated in the circulation constant temperature chamber. After 100 seconds from the start of circulation of the agonist-containing sample obtained in Comparative Example 5, the test sample obtained in Example 7 was circulated in the circulation constant temperature chamber for 100 seconds.

Using a fluorescence intensity of _{340 nm} and a fluorescence intensity of _{380 nm} , the formula (V):
[Fluorescence intensity ratio]
= [Fluorescence intensity _340nm / fluorescence intensity _380nm ] (V)
Accordingly, the fluorescence intensity ratio was calculated.

FIG. 7 shows the results of examining the temporal change in the fluorescence intensity ratio in Test Example 7. In the figure, “camphor” indicates the duration of stimulation by camphor, and “test sample” indicates the circulation period of the test sample.

FIG. 7 shows that the fluorescence intensity ratio increases with the start of stimulation by camphor contained in the agonist-containing sample, but decreases with the addition of the test sample. From these results, it can be seen that aluminum ions dissociated from aluminum chloride have an action of suppressing the activity of TRPV3 activated by stimulation with camphor.

In Example 7, instead of using aluminum chloride, aluminum chloride was used when inorganic acid aluminum salts such as aluminum phosphate and potassium aluminum sulfate; and other substances that dissociate into aluminum ions such as aluminum acetate were used. The same trend is seen. From these results, it can be seen that aluminum ions have an action of suppressing the activity of TRPV3.

Example 8
Aluminum chloride and 4αPDD were added to solvent A so that the concentration of aluminum chloride was 5 mM and the concentration of 4αPDD was 10 μM to obtain a mixture. By adding hydrochloric acid to the obtained mixture, the pH of the mixture was adjusted to 5 to obtain a test sample. In the obtained test sample, aluminum ions were present in a dissociated state (aluminum ion concentration: 5 mM).

Comparative Example 6
An agonist-containing sample was obtained by adding 4αPDD to solvent A so that its concentration was 10 μM.

Test Example 8
In Test Example 7, instead of using the TRPV3-expressing cells obtained in Preparation Example 1 (4), the TRPV4-expressing cells obtained in Preparation Example 1 (5) were used, and the agonist-containing sample obtained in Comparative Example 5 Test example, except that the agonist-containing sample obtained in Comparative Example 6 was used instead of the test sample and that the test sample obtained in Example 8 was used instead of the test sample obtained in Example 7 The same operation as in No. 7 was performed, and the change with time in the fluorescence intensity ratio was examined.

FIG. 8 shows the results of examining the temporal change in the fluorescence intensity ratio in Test Example 8. In the figure, “4αPDD” indicates the duration of stimulation by 4αPDD, and “test sample” indicates the circulation period of the test sample.

FIG. 8 shows that the fluorescence intensity ratio increases with the start of stimulation by 4αPDD contained in the agonist-containing sample, but decreases with the addition of the test sample. From these results, it can be seen that aluminum ions dissociated from aluminum chloride have an action of suppressing the activity of TRPV4 activated by stimulation with 4αPDD.

In Example 8, instead of using aluminum chloride, aluminum chloride was used when inorganic acid aluminum salts such as aluminum phosphate and potassium aluminum sulfate; and other substances dissociating into aluminum ions such as aluminum acetate were used. The same trend is seen. From these results, it can be seen that aluminum ions have an action of suppressing the activity of TRPV4.

Comparative Example 7
Aluminum hydroxide gel (aluminum hydroxide content: 1.3% by mass) and capsaicin were added to solvent A so that the concentration of aluminum hydroxide gel was 5% by mass (diluted 20 times) and the concentration of capsaicin was 1 μM. A test sample was obtained.

Comparative Example 8
Capsaicin was added to solvent A so that its concentration was 1 μM to obtain a test sample.

Test Example 9
The TRPV1-expressing cells obtained in Preparation Example 1 (1) are incubated at room temperature for 60 minutes in 10% by mass FBS-containing DMEM containing FURA 2-AM (manufactured by Invitrogen), a fluorescent calcium ion indicator, at a final concentration of 5 μM. Thus, FURA 2-AM was introduced into TRPV1-expressing cells to obtain indicator-introduced cells. The indicator-introduced cells were washed by incubating the indicator-introduced cells in solvent A with shaking at 37 ° C. for 2 minutes. The indicator-introduced cells after washing were placed in a circulation constant temperature chamber of a fluorescence measurement apparatus with a circulation constant temperature chamber. Thereafter, the solvent A was circulated and measurement of fluorescence intensity _{340 nm} and fluorescence intensity _{380 nm} was started. The fluorescence intensity _{340 nm} (fluorescence intensity G) in the presence of the control and the fluorescence intensity _{380 nm} (fluorescence intensity H) in the presence of the control were obtained when 50 seconds passed from the start of the circulation of the solvent A (at the end of the circulation of the control). In the circulation constant temperature chamber, the test sample obtained in Comparative Example 7 was circulated for 50 seconds. At the end of circulation of the test sample, fluorescence intensity _{340 nm} (fluorescence intensity I) and aluminum hydroxide in the presence of an aluminum hydroxide gel and an agonist A fluorescence intensity of _{380 nm} (fluorescence intensity J) in the presence of gel and agonist was obtained. Next, the control (solvent A) was circulated for 100 seconds in a circulation constant temperature chamber. Thereafter, the test sample obtained in Comparative Example 8 was circulated for 50 seconds in the circulation constant temperature chamber, and the fluorescence intensity _{340 nm} (fluorescence intensity K) in the presence of the agonist and the fluorescence in the presence of the agonist at the end of the circulation of the test sample. An intensity of _{380 nm} (fluorescence intensity L) was obtained.

Using fluorescence intensities G, H, I and J, formula (VI):
[Delta fluorescence intensity ratio _{aluminum hydroxide gel} ]
= [Fluorescence intensity I / fluorescence intensity J]-[fluorescence intensity G / fluorescence intensity H] (VI)
Accordingly, Δ fluorescence intensity ratio _{aluminum hydroxide gel} was calculated.

Using fluorescence intensities G, H, K and J, formula (VII):
[Δ fluorescence intensity ratio _agonist ]
= [Fluorescence intensity K / fluorescence intensity L]-[fluorescence intensity G / fluorescence intensity H] (VII)
Accordingly, a Δfluorescence intensity ratio _agonist was calculated.

Using the calculated Δ fluorescence intensity ratio _{aluminum hydroxide gel} and Δ fluorescence intensity ratio _agonist , the formula (VIII):
[TRPV1 activity or TRPA1 activity]
= [Δ fluorescence intensity ratio _{aluminum hydroxide gel} / Δ fluorescence intensity ratio _agonist ] (VIII)
Accordingly, TRPV1 activity was calculated.

FIG. 9 shows the results of examining the relationship between the type of sample and the TRPV1 activity in Test Example 9. In the figure, lane 1 shows TRPV1 activity when the test sample obtained in Comparative Example 7 is used, and lane 2 shows TRPV1 activity when the test sample obtained in Comparative Example 8 is used.

From the results shown in FIG. 9, the TRPV1 activity when using a test sample (Comparative Example 7) containing an aluminum hydroxide gel and an agonist is TRPV1 when using a test sample containing an agonist (Comparative Example 8). It can be seen that it is higher than the activity. From these results, it can be seen that the aluminum hydroxide gel does not have a TRPV1 activity suppressing action but has a TRPV1 activity promoting action.

Comparative Example 9
Aluminum hydroxide gel (aluminum hydroxide content 1.3 mass%) and AITC were added to solvent A so that the concentration of aluminum hydroxide gel was 5 mass% (diluted 20 times) and the concentration of AITC was 20 μM. A test sample was obtained.

Comparative Example 10
A test sample was obtained by adding AITC to solvent A so that its concentration was 20 μM.

Test Example 10
In Test Example 9, instead of using the TRPV1-expressing cells obtained in Preparation Example 1 (1), the TRPA1-expressing cells obtained in Preparation Example 1 (2) were used, and the test sample obtained in Comparative Example 7 was used. Test Example 9 except that the test sample obtained in Comparative Example 9 was used instead of the test sample and that the test sample obtained in Comparative Example 10 was used instead of the test sample obtained in Comparative Example 8 The same operation was performed, and TRPA1 activity was calculated.

FIG. 10 shows the results of examining the relationship between the type of sample and the TRPA1 activity in Test Example 10. In the figure, lane 1 shows TRPA1 activity when the test sample obtained in Comparative Example 9 is used, and lane 2 shows TRPA1 activity when the test sample obtained in Comparative Example 10 is used.

From the results shown in FIG. 10, the TRPA1 activity when using a test sample (Comparative Example 9) containing an aluminum hydroxide gel and an agonist is TRPA1 when using a test sample containing an agonist (Comparative Example 10). It can be seen that it is higher than the activity. From these results, it can be seen that the aluminum hydroxide gel does not have a TRPA1 activity inhibitory action but has a TRPA1 activity promoting action.

Preparation Example 2
(1) Preparation of test sample A The concentration of potassium aluminum sulfate is 1 μM [aluminum ion concentration: 1 μM (experiment number 9)], 10 μM [aluminum ion concentration: 10 μM (experiment number 10)], 100 μM [aluminum ion concentration: 100 μM]. (Experiment number 11)], 200 μM [aluminum ion concentration: 200 μM (experiment number 12)], 500 μM [aluminum ion concentration: 500 μM (experiment number 13)], 1000 μM [aluminum ion concentration: 1000 μM (experiment number 14)], 5000 μM ([Aluminum ion concentration: 5000 μM (experiment number 15)] or 10000 μM [aluminum ion concentration: 10000 μM (experiment number 16)]) was added to solvent A to obtain a mixture, and hydrochloric acid was added to the resulting mixture. By doing To obtain a test sample A by adjusting the pH of the mixture to 4.

(2) Preparation of test sample B The concentration of potassium aluminum sulfate and AITC is 1 μM [aluminum ion concentration: 1 μM (experiment number 9)], 10 μM [aluminum ion concentration: 10 μM (experiment number 10)], 100 μM [ Aluminum ion concentration: 100 μM (experiment number 11)], 200 μM [aluminum ion concentration: 200 μM (experiment number 12)], 500 μM [aluminum ion concentration: 500 μM (experiment number 13)], 1000 μM [aluminum ion concentration: 1000 μM (experiment number) 14)] 5000 μM ([aluminum ion concentration: 5000 μM (experiment number 15)] or 10000 μM [aluminum ion concentration: 10000 μM (experiment number 16)]) and the solvent A so that the concentration of AITC is 5 μM. To obtain a pressure to the mixture. By the addition of hydrochloric acid to the resulting mixture, to obtain a test sample B to adjust the pH of the mixture to 4.

Preparation Example 3
AITC was added to Solvent A to a concentration of 5 μM to obtain a mixture. By adding hydrochloric acid to the obtained mixture, the pH of the mixture was adjusted to 4 to obtain an agonist-containing sample.

Test Example 11
The TRPA1-expressing cells obtained in Preparation Example 1 (2) were incubated at room temperature for 60 minutes in DMEM containing 10 mass% FBS containing FURA 2-AM (manufactured by Invitrogen) at a final concentration of 5 μM. FURA 2-AM was introduced into the cells to obtain indicator-introduced cells. The indicator-introduced cells were washed by incubating the indicator-introduced cells in solvent A with shaking at 37 ° C. for 2 minutes. The indicator-introduced cells after washing were placed in a circulation constant temperature chamber of a fluorescence measurement apparatus with a circulation constant temperature chamber. Thereafter, the solvent A was circulated in the circulation constant temperature chamber, and the measurement of the fluorescence intensity _{340 nm} and the fluorescence intensity _{380 nm} was started.

The fluorescence intensity _{340 nm} (fluorescence intensity M) in the presence of the control and the fluorescence intensity _{380 nm} (fluorescence intensity N) in the presence of the control were obtained when 50 seconds passed from the start of the circulation of the solvent A (at the end of the circulation of the control). Thereafter, the test sample A of Experiment No. 9 was circulated for 50 seconds in a circulation constant temperature chamber. Next, the test sample B of Experiment No. 9 is circulated for 100 seconds in the circulation constant temperature chamber. At the end of circulation of the test sample B, the fluorescence intensity _{340 nm} (fluorescence intensity O) in the presence of aluminum ions and agonist (AITC) and A fluorescence intensity of _{380 nm} (fluorescence intensity P) in the presence of aluminum ions and agonist (AITC) was obtained. Next, the control (solvent A) was circulated for 100 seconds in a circulation constant temperature chamber. Thereafter, the agonist-containing sample obtained in Preparation Example 3 was circulated for 100 seconds in the circulation constant temperature chamber. At the end of circulation of the agonist-containing sample, the fluorescence intensity _{340 nm} (fluorescence intensity Q) in the presence of the agonist and the presence of the agonist. A fluorescence intensity of _{380 nm} (fluorescence intensity R) was obtained.

Using fluorescence intensities M, N, O and P, the formula (IX):
[Δ fluorescence intensity specific _{activity inhibitor} ]
= [Fluorescence intensity O / fluorescence intensity P]-[fluorescence intensity M / fluorescence intensity N]
(IX)
Accordingly, the Δ fluorescence intensity specific _{activity inhibitor} was calculated.

Using fluorescence intensity M, N, Q and R, the formula (X):
[Δ fluorescence intensity ratio _agonist ]
= [Fluorescence intensity Q / fluorescence intensity R]-[fluorescence intensity M / fluorescence intensity N]
(X)
Accordingly, a Δfluorescence intensity ratio _agonist was calculated.

Using the calculated Δ fluorescence intensity ratio _{activity inhibitor} and Δ fluorescence intensity ratio _agonist , the formula (XI):
[Inhibition rate]
= [Δ fluorescence intensity ratio _agonist- Δ fluorescence intensity ratio _{activity inhibitor} ] / [Δ fluorescence intensity ratio _agonist ]
(XI)
Accordingly, the inhibition rate of TRPA1 activity was calculated.

Further, in the above, test sample A and test sample of experiment number 9 are used except that test sample A and test sample B of experiment number 10 to 16 are used instead of test sample A and test sample B of experiment number 9 The same operation as when B was used was performed, and the inhibition rate of TRPA1 activity was calculated.

FIG. 11 shows the results of examining the relationship between the potassium aluminum sulfate concentration and the inhibition rate of TRPA1 activity in Test Example 11.

Using the results shown in FIG. 11, the 50% inhibitory concentration of potassium aluminum sulfate for TRPA1 activity was calculated. As a result, it was found that the 50% inhibitory concentration of potassium aluminum sulfate for TRPA1 activity was 103 μM. From the above results, it was found that the 50% inhibitory concentration of aluminum ions for TRPA1 activity was 103 μM.

Preparation Example 4
(1) Preparation of test sample C Potassium aluminum sulfate was added to solvent A so that the density | concentration might be set to 1000 micromol [aluminum ion concentration: 1000 micromol], and the mixture was obtained. By adding hydrochloric acid or sodium hydroxide to the obtained mixture, the pH of the mixture was adjusted to 4, 6 (experiment number 17), 6 (experiment number 18) or 7.4 (experiment number 19). Sample C was obtained.

(2) Preparation of test sample D Potassium aluminum sulfate and capsaicin were added to solvent A so that the concentration of potassium aluminum sulfate was 1000 μM [aluminum ion concentration: 1000 μM] and the concentration of capsaicin was 100 nM to obtain a mixture. By adding hydrochloric acid or sodium hydroxide to the obtained mixture, the pH of the mixture was adjusted to 4, 6 (experiment number 17), 6 (experiment number 18) or 7.4 (experiment number 19). Sample C was obtained.

(3) Preparation of test sample E Potassium aluminum sulfate and AITC were added to solvent A so that the concentration of potassium aluminum sulfate was 1000 μM [aluminum ion concentration: 1000 μM] and the concentration of AITC was 5 μM to obtain a mixture. By adding hydrochloric acid or sodium hydroxide to the obtained mixture, the pH of the mixture was adjusted to 4, 6 (experiment number 17), 6 (experiment number 18) or 7.4 (experiment number 19). Sample D was obtained.

Preparation Example 5
Capsaicin was added to solvent A to a concentration of 100 nM to obtain a mixture. By adding hydrochloric acid or sodium hydroxide to the obtained mixture, the pH of the mixture is adjusted to 4, 6 (Experiment No. 17), 6 (Experiment No. 18) or 7.4 (Experiment No. 19) to be agonists. A containing sample was obtained.

Preparation Example 6
AITC was added to Solvent A to a concentration of 5 μM to obtain a mixture. By adding hydrochloric acid or sodium hydroxide to the obtained mixture, the pH of the mixture is adjusted to 4, 6 (Experiment No. 17), 6 (Experiment No. 18) or 7.4 (Experiment No. 19) to be agonists. A containing sample was obtained.

Test Example 12
(1) Calculation of inhibition rate of TRPV1 activity The TRPV1-expressing cells obtained in Preparation Example 1 (1) were mixed with FURA 2-AM (manufactured by Invitrogen) at a final concentration of 5 μM in 10% by mass FBS-containing DMEM at room temperature. By incubating for 60 minutes, FURA 2-AM was introduced into TRPV1-expressing cells to obtain indicator-introduced cells. The indicator-introduced cells were washed by incubating the indicator-introduced cells in solvent A with shaking at 37 ° C. for 2 minutes. The indicator-introduced cells after washing were placed in a circulation constant temperature chamber of a fluorescence measurement apparatus with a circulation constant temperature chamber, and then the solvent A was circulated and measurement of fluorescence intensity _{340 nm} and fluorescence intensity _{380 nm} was started.

The fluorescence intensity _{340 nm} (fluorescence intensity M) in the presence of the control and the fluorescence intensity _{380 nm} (fluorescence intensity N) in the presence of the control were obtained when 50 seconds passed from the start of the circulation of the solvent A (at the end of the circulation of the control). Thereafter, the test sample C of Experiment No. 17 was circulated for 50 seconds in a circulation constant temperature chamber. Next, the test sample D of the experiment number 17 is circulated for 100 seconds in the circulation constant temperature chamber, and at the end of circulation of the test sample D, the fluorescence intensity _{340 nm} (fluorescence intensity O) in the presence of aluminum ions and agonist (capsaicin) and A fluorescence intensity of _{380 nm} (fluorescence intensity P) in the presence of aluminum ions and an agonist (capsaicin) was obtained. Next, the control (solvent A) was circulated for 100 seconds in a circulation constant temperature chamber. Thereafter, the agonist-containing sample of Experiment No. 17 obtained in Preparation Example 5 was circulated for 100 seconds in the circulation constant temperature chamber, and the fluorescence intensity _{340 nm} (fluorescence intensity Q) in the presence of the agonist at the end of circulation of the agonist-containing sample and A fluorescence intensity of _{380 nm} (fluorescence intensity R) in the presence of an agonist was obtained.

Fluorescence intensities M to R were used to calculate the inhibition rate of TRPV1 activity according to formula (IX), formula (X) and formula (XI).

Also, in the above, test sample C and test sample of experiment number 17 are used except that test sample C and test sample D of experiment numbers 18 to 19 are used instead of test sample C and test sample D of experiment number 17 The same operation as when D was used was performed, and the inhibition rate of TRPV1 activity was calculated.

FIG. 12 shows the results of examining the relationship between the pH of the test sample and the inhibition rate of TRPV1 activity in Test Example 12.

(2) Calculation of inhibition rate of TRPA1 activity In Test Example 12 (1), instead of using the TRPV1-expressing cell obtained in Preparation Example 1 (1), the TRPA1-expressing cell obtained in Preparation Example 1 (2) was used. The test sample E was used instead of the test sample D and the agonist-containing sample obtained in Preparation Example 6 was used instead of the agonist-containing sample obtained in Preparation Example 5. The same operation as in Example 12 (1) was performed, and the inhibition rate of TRPA1 activity was calculated.

FIG. 12 shows the results of examining the relationship between the pH of the test sample and the inhibition rate of TRPA1 activity in Test Example 12. In the figure, the black bar shows the inhibition rate of TRPV1 activity, and the white bar shows the inhibition rate of TRPA1 activity.

From the results shown in FIG. 12, it can be seen that the inhibition rate of TRPV1 activity and the inhibition rate of TRPA1 activity are higher in the order of pH 4, 6 and 7.4. From these results, when potassium aluminum sulfate is used as a substance that dissociates into aluminum ions, it is possible to suppress TRPV1 activity and TRPA1 activity by adjusting the pH of the activity inhibitor to 1 to 6.5, preferably 4 to 6. It can be seen that the rate can be improved.

As described above, it can be seen that aluminum ions suppress the activity of TRP channels such as TRPA1, TRPV1, TRPM8, TRPV3, and TRPV4. Therefore, since the activity inhibitor of the present invention contains aluminum ions, it results from the activation of an agent that suppresses or modulates the expression of unpleasant sensations or physiological events resulting from the activation of the TRP channel, for example, TRPA1. Stimulation inhibitor used for the purpose of suppressing stimulation, cooling sensation adjusting agent used for adjusting the sensation of cooling caused by activation of TRPM8, pain suppression used for the purpose of suppressing pain caused by activation of TRPV1 Agents, skin cell hyperkeratinization inhibitor used to suppress skin cell hyperkeratinization caused by TRPV3 activation, itching agent used for curbing caused by TRPV4 activation, etc. It is expected to be used for In addition, since the method for inhibiting activity of the present invention brings aluminum ions into contact with TRP channels, it suppresses or regulates the expression of unpleasant sensations or physiological events caused by activation of TRP channels, for example, activation of TRPA1. Suppression caused by stimulation, adjustment of cooling sensation caused by activation of TRPM8, inhibition of pain caused by activation of TRPV1, suppression of hyperkeratinization of skin cells caused by activation of TRPV3, activation of TRPV4 It is expected to be used for suppressing the itch caused.

Claims (3)

1. A TRP channel activity inhibitor for suppressing the activity of a TRP channel, comprising an aluminum ion as an active ingredient for suppressing the activity of the TRP channel.
2. A TRP channel activity inhibitor for suppressing the activity of a TRP channel, which comprises a substance that dissociates into aluminum ions as an active ingredient for suppressing the activity of the TRP channel. Inhibitor.
3. An activity suppression method for suppressing the activity of a TRP channel, which comprises contacting an aluminum ion with a TRP channel.

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