JP6954602B2 - Activity inhibitor and skin hyperesthesia inhibitor - Google Patents

Description

The present invention relates to an activity inhibitor and a skin hyperesthesia inhibitor.

Examples of hyperesthesia in the skin include itching and pain in the skin. Of the hyperesthesia, itching in the skin is caused by stimuli such as physical irritation and chemical irritation by a substance that causes itching. As a substance that causes itching in the skin, for example, histamine and the like are known. Therefore, as a substance that suppresses itching in the skin, an external composition containing diphenhydramine having a strong antihistamine action or a salt thereof has been proposed (see, for example, Patent Document 1).

Japanese Unexamined Patent Publication No. 2010-180206

However, the external composition containing diphenhydramine or a salt thereof may be difficult to suppress itching in the skin depending on the type of stimulus that causes itching. In addition, there are various hyperesthesia in the skin other than the itch, which have different causes. Therefore, a new means capable of suppressing hyperesthesia such as itching and pain is desired.

The present invention has been made in view of the above-mentioned prior art, and at least one selected from the group consisting of an activity inhibitor capable of suppressing the activity of TRPA1, TRPV4 and ANO1 and TRPA1, TRPV4 and ANO1 in the skin. It is an object of the present invention to provide a skin hyperesthesia inhibitor capable of suppressing species-related hyperesthesia.

The gist of the present invention is
(1) An activity inhibitor for suppressing the activity of each of TRPA1, TRPV4 and ANO1 as an active ingredient according to the formula (I):

(Wherein, R ¹ represents a hydrogen atom, a hydroxyl group or an alkyl group having 1 to 4 carbon ^{atoms, R 2, R 3, R} 4, R 5, R 6, R 7, R 8, R 9 and R ¹⁰ are hydrogen shows the original child)
An activity inhibitor containing a compound represented by
(2) A cutaneous hypersensitivity inhibitors for at least one selected from the group consisting of TRPAl, TRPV4 and ANO1 in skin can be inhibited hypersensitivity involved, according to (1) as an active ingredient A skin hyperesthesia inhibitor containing an activity inhibitor,
( 3 ) The skin hypersensitivity inhibitor according to (2 ) above, and (4 ) the sensation, wherein the hypersensitivity is an itch involving at least one selected from the group consisting of TRPA1, TRPV4 and ANO1 in the skin. The cutaneous hypersensitivity inhibitor according to (2 ) or ( 3 ) above, wherein the hypersensitivity further comprises pain involving at least one selected from the group consisting of TRPA1, TRPV4 and ANO1 in the skin.

According to the activity inhibitor of the present invention, the activities of TRPA1, TRPV4 and ANO1 can be effectively suppressed. Further, according to the skin hyperesthesia inhibitor of the present invention, skin hyperesthesia involving at least one selected from the group consisting of TRPA1, TRPV4 and ANO1 can be effectively suppressed.

(A) is a change over time in the relative current value of mouse ANO1 when (a) 4-isopropylcyclohexanol, (b) isopropylcyclohexane or (c) 1-isopropyl-4-methylcyclohexane was administered in Test Example 1. (B) is a dose-response curve showing the relationship between the 4-isopropylcyclohexanol concentration and the relative current value of mouse ANO1. In Test Example 2, it is a graph which shows the time-dependent change of the relative current value of human ANO1. (A) is a graph showing the time course of allyl isothiocyanate-induced mouse TRPA1 current before and after administration of 4-isopropylcyclohexanol in Test Example 3, and (B) is the presence or absence of administration of 4-isopropylcyclohexanol and mouse TRPA1 current. It is a graph which shows the relationship with density. (A) is a graph showing the relationship between the presence or absence of 4-isopropylcyclohexanol and the magnitude of the amplitude of the mouse TRPA1 current in Test Example 3, and (B) is the relationship between the presence or absence of 4-isopropylcyclohexanol and the opening time of mouse TRPA1. The graph showing the relationship, (C) is a graph showing the relationship between the presence or absence of 4-isopropylcyclohexanol and the closing time of mouse TRPA1. (A) is a graph showing the time course of allyl isothiocyanate-induced human TRPA1 current before and after administration of 4-isopropylcyclohexanol in Test Example 4, and (B) is the presence or absence of administration of 4-isopropylcyclohexanol and human TRPA1 current. It is a graph which shows the relationship with density. (A) is a graph showing the time course of GSK1016790A-induced mouse TRPV4 current before and after administration of 4-isopropylcyclohexanol in Test Example 5, and (B) is a graph showing the time course of GSK1016790A-induced human TRPV4 current before and after administration of 4-isopropylcyclohexanol. It is a graph which shows the change with time. (A) is a dose-response curve showing the relationship between the 4-isopropylcyclohexanol concentration and the relative current value of mouse TRPA1 in Test Example 8, and (B) is the relationship between the 4-isopropylcyclohexanol concentration and the relative current value of human TRPA1. It is a dose-response curve showing a relationship. (A) is a graph showing the time-dependent change of the relative current value of mouse TRPV4 in Test Example 9, and (B) is a graph showing the time-dependent change of the relative current value of human TRPV4.

1. 1. Activity inhibitor The activity inhibitor of the present invention is an activity inhibitor for suppressing the activity of TRPA1, TRPV4 and ANO1 respectively. The activity inhibitor of the present invention has the formula (I): as an active ingredient.

(In the formula, R ¹ , R ² , R ³ , R ⁴ , R ⁵ , R ⁶ , R ⁷ , R ⁸ , R ⁹ and R ¹⁰ are independently hydrogen atoms, hydroxyl groups or 1 to 4 carbon atoms, respectively. (Indicates the alkyl group of)
One of the major features is that it contains the compound represented by. The compound represented by the formula (I) suppresses the activation of TRPA1, TRPV4 and ANO1 caused by the agonist, respectively. Moreover, since the activity inhibitor of the present invention suppresses the activities of TRPA1, TRPV4 and ANO1, the interaction between TRPA1, TRPV4 and ANO1 can be effectively suppressed.

In formula (I), R ¹ , R ² , R ³ , R ⁴ , R ⁵ , R ⁶ , R ⁷ , R ⁸ , R ⁹ and R ¹⁰ are independently hydrogen atoms, hydroxyl groups or carbon atoms 1, respectively. It is an alkyl group of ~ 4. Examples of the alkyl group having 1 to 4 carbon atoms include a methyl group, an ethyl group, an n-propyl group, an isopropyl group, an n-butyl group, an isobutyl group, a tert-butyl group and the like. It is not limited to illustrations only.

In the formula (I), R ¹ is a hydrogen atom, a hydroxyl group or an alkyl group having 1 to 4 carbon atoms, but from the viewpoint of effectively suppressing the activities of TRPA1, TRPV4 and ANO1, the hydrogen atom and the hydroxyl group are preferable. More preferably, it is a hydroxyl group.

In formula (I), R ² , R ³ , R ⁴ , R ⁵ , R ⁶ , R ⁷ , R ⁸ , R ⁹ and R ¹⁰ are independently hydrogen atoms, hydroxyl groups or 1 to 4 carbon atoms. Although it is an alkyl group, it cannot be unconditionally determined because it differs depending on the use of the activity inhibitor of the present invention, and therefore it is desirable to determine it according to the use of the activity inhibitor of the present invention. If application of the active inhibitor of the present invention, for example, is the use which is required to quickly suppress skin hypersensitivity to TRPAl, TRPV4 and at least one selected from the group consisting of ANO1 are involved, R ^2, R ³ , R ⁴ , R ⁵ , R ⁶ , R ⁷ , R ⁸ , R ⁹ and R ¹⁰ are independent hydrogen atoms or alkyl groups having 1 to 4 carbon atoms from the viewpoint of rapidly exhibiting an inhibitory effect. It is preferable that it is a hydrogen atom, and it is more preferable that it is a hydrogen atom. Further, when the application of the activity inhibitor of the present invention is, for example, an application requiring high affinity for the skin, R ² , R ³ , R ⁴ , R ⁵ , R ⁶ , R ⁷ , R ⁸ , R 8, From the viewpoint of enhancing the affinity for the skin, R ⁹ and R ¹⁰ are preferably hydrogen atoms or alkyl groups having 1 to 4 carbon atoms independently, and more preferably alkyl groups having 1 to 4 carbon atoms. preferable.

When the application of the activity inhibitor of the present invention is an application in which at least one selected from the group consisting of TRPA1, TRPV4 and ANO1 is required to rapidly suppress skin hypersensitivity, the inhibitory effect is rapidly suppressed. From the viewpoint of expression, in the formula (I), R ¹ is a hydrogen atom, a hydroxyl group or an alkyl group having 1 to 4 carbon atoms, and is R ² , R ³ , R ⁴ , R ⁵ , R ⁶ , R ⁷ , R ⁸ , R ⁹ and R ¹⁰ are preferably ^{hydrogen atoms, R 1} is a hydrogen atom or hydroxyl, R ² , R ³ , R ⁴ , R ⁵ , R ⁶ , R ⁷ , R ⁸ , R ⁹ and R ^{Compounds in which 10} is a hydrogen atom are more preferable, R ¹ is a hydroxyl group, and R ² , R ³ , R ⁴ , R ⁵ , R ⁶ , R ⁷ , R ⁸ , R ⁹ and R ¹⁰ are hydrogen atoms. Is even more preferable. Examples of the compound represented by the formula (I) include 4-isopropylcyclohexanol, isopropylcyclohexane, 1-isopropyl-4-methyl-cyclohexane and the like, but the present invention is not limited to these examples. .. Among the compounds represented by these formulas (I), the use of the activity inhibitor of the present invention rapidly suppresses skin hyperesthesia involving at least one selected from the group consisting of TRPA1, TRPV4 and ANO1. From the viewpoint of rapidly exhibiting the inhibitory effect, 4-isopropylcyclohexanol, isopropylcyclohexane and 1-isopropyl-4-methyl-cyclohexane are preferable, and 4-isopropylcyclohexanol is more preferable.

When the cis-trans isomer is present as the compound represented by the formula (I), the compound represented by the formula (I) may be either a trans isomer or a mixture of a trans isomer and a cis isomer.

The content of the compound represented by the formula (I) in the activity inhibitor of the present invention is preferably set as appropriate according to the use of the activity inhibitor of the present invention.

The activity inhibitor of the present invention may contain other components such as an appropriate solvent, a pH adjuster, and a calcium chelating agent within a range that does not interfere with the object of the present invention.

The activity inhibitor of the present invention may be used for in vitro applications or in vivo applications.

TRPA1 and TRPV4 are transient receptor type voltage-gated channels (hereinafter, also referred to as “TRP channels”). In addition, ANO1 is a chloride ion channel activated by intracellular calcium ions.

Examples of TRPA1 include human TRPA1 and mouse TRPA1, but the present invention is not limited to these examples. Human TRPA1 has the amino acid sequence shown in GenBank Accession No .: NM_007332. In addition, mouse TRPA1 has the amino acid sequence shown in GenBank accession number: NM_177781. Further, in the present invention, if TRPA1 exhibits an activity equivalent to the activity of a polypeptide consisting of the amino acid sequence shown in GenBank accession number: NM_007332 or NM_177781 (hereinafter, referred to as "TRPA1 activity"), it is referred to as GenBank access. Session number: It may be a variant of a polypeptide consisting of the amino acid sequence shown in NM_007332 or NM_177781. As a variant of TRPA1, for example, in the amino acid sequence shown in (A1) GenBank accession number: NM_007332 or NM_177781, one or several, preferably 1-3, more preferably 1-2 amino acid residues. Polypeptides that exhibit TRPA1 activity, with substitutions, deletions or additions to
(B1) GenBank Accession No .: A polypeptide having a sequence identity of 95% or more, preferably 98% or more with respect to the amino acid sequence shown in NM_007332 or NM_177781, and exhibiting TRPA1 activity can be mentioned. Is not limited to such examples. In the present specification, "sequence identity" means that the sequence to be evaluated (query sequence) is aligned with the sequence (reference sequence) of a predetermined GenBank accession number using PROTEIN BLAST as a default parameter. It means the value calculated by.

Examples of the TRPA1 activity include the ability to transport calcium ions from the outside of the cell to the inside of the cell, the ability to generate a current caused by TRPA1 in the cell (hereinafter, also referred to as “TRPA1 current”), and the like. It is not limited to such an example. TRPA1 activity is expressed by activating TRPA1 with a TRPA1 agonist. Examples of TRPA1 agonists include, but are not limited to, allyl isothiocyanate, allicin, icylin, and the like. The inhibitory effect of the activity inhibitor of the present invention on TRPA1 is, for example, a TRPA1 current induced by a TRPA1 agonist in a cell expressing TRPA1 (hereinafter referred to as "TRPA1-expressing cell") (hereinafter, also referred to as "agonist-induced TRPA1 current"). ) Etc. can be evaluated. The TRPA1 current can be measured by, for example, the patch clamp method. In this case, the agonist-induced TRPA1 current when the activity inhibitor is administered is smaller than the agonist-induced TRPA1 current when the activity inhibitor is not administered.

Examples of TRPV4 include human TRPV4 and mouse TRPV4, but the present invention is not limited to these examples. Human TRPV4 has the amino acid sequence shown in GenBank Accession No .: NM_021625. In addition, mouse TRPV4 has the amino acid sequence shown in GenBank accession number: NM_022017. Further, in the present invention, if TRPV4 exhibits an activity equivalent to the activity of a polypeptide consisting of the amino acid sequence shown in GenBank accession number: NM_01625 or NM_022017 (hereinafter, referred to as "TRPV4 activity"), it is referred to as GenBank access. Session number: It may be a variant of a polypeptide consisting of the amino acid sequence shown in NM_01625 or NM_022017. As a variant of TRPV4, for example, in the amino acid sequence shown in (A2) GenBank accession number: NM_021625 or NM_022017, one or several amino acid residues, preferably 1 to 3, more preferably 1 to 2 amino acid residues. A polypeptide having TRPV4 activity, with a substitution, deletion or addition of
(B2) GenBank Accession No .: A polypeptide having a sequence identity of 95% or more, preferably 98% or more with respect to the amino acid sequence shown in NM_021625 or NM_022017, and exhibiting TRPV4 activity can be mentioned. Is not limited to such examples.

Examples of the TRPV4 activity include the ability to transport calcium ions from the outside of the cell to the inside of the cell, the ability to generate a current (hereinafter, also referred to as “TRPV4 current”) caused by TRPV4 in the cell, and the like. It is not limited to such an example. TRPV4 activity is expressed by activating TRPV4 with a TRPV4 agonist. Examples of TRPV4 agonists include, for example, GSK1016790A, but the present invention is not limited to these examples. The inhibitory effect on TRPV4 by the activity inhibitor of the present invention is also referred to as, for example, a TRPV4 current induced by a TRPV4 agonist in a cell expressing TRPV4 (hereinafter referred to as “TRPV4 expressing cell”) (hereinafter, “agonist-induced TRPV4 current”). It can be evaluated based on such things as). The TRPV4 current can be measured by, for example, the patch clamp method. In this case, the agonist-induced TRPV4 current when the activity inhibitor is administered is smaller than the agonist-induced TRPV4 current when the activity inhibitor is not administered.

Examples of ANO1 include human ANO1 and mouse ANO1, but the present invention is not limited to these examples. Human ANO1 has the amino acid sequence shown in GenBank Accession No .: NM_018403. In addition, mouse ANO1 has the amino acid sequence shown in GenBank accession number: NM_178642. Further, in the present invention, if ANO1 exhibits an activity equivalent to the activity of a polypeptide consisting of the amino acid sequence shown in GenBank accession number: NM_018403 or NM_178642 (hereinafter referred to as "ANO1 activity"), the ANO1 is GenBank access. Session number: It may be a variant of a polypeptide consisting of the amino acid sequence shown in NM_018403 or NM_178642. As a variant of ANO1, for example, in the amino acid sequence shown in (A3) GenBank accession number: NM_018403 or NM_178642, one or several, preferably 1-3, more preferably 1-2 amino acid residues. Polypeptides that exhibit ANO1 activity, with substitutions, deletions or additions of
(B3) GenBank Accession No .: A polypeptide having a sequence identity of 95% or more, preferably 98% or more with respect to the amino acid sequence shown in NM_018403 or NM_178642, and exhibiting ANO1 activity can be mentioned. Is not limited to such examples.

Examples of the ANO1 activity include the ability to transport chlorine ions in cells and the ability to generate a current (hereinafter, also referred to as “ANO1 current”) caused by ANO1 in cells, but the present invention is limited to such examples. It is not something that is done. ANO1 activity is expressed by activating TRPA1 with an ANO1 agonist. Examples of the ANO1 agonist include calcium ions, but the present invention is not limited to these examples. The inhibitory effect on ANO1 by the activity inhibitor of the present invention is, for example, an ANO1 current induced by an ANO1 agonist (calcium ion) in a cell expressing ANO1 (hereinafter referred to as "ANO1-expressing cell") (hereinafter, "agonist induction"). It can be evaluated based on "ANO1 current" or the like. The ANO1 current can be measured by, for example, the patch clamp method. In this case, the agonist-induced ANO1 current when the activity inhibitor is administered is smaller than the agonist ANO1 current when the activity inhibitor is not administered.

2. Skin hyperesthesia inhibitor The skin hyperesthesia inhibitor of the present invention is a skin hyperesthesia inhibitor for suppressing hyperesthesia involving at least one selected from the group consisting of TRPA1, TRPV4 and ANO1 in the skin. One of the major features is that the activity inhibitor is contained as an active ingredient. Since the skin hyperesthesia inhibitor of the present invention contains the activity inhibitor, at least selected from the group consisting of TRPA1, TRPV4 and ANO1 in the skin by bringing the skin hyperesthesia inhibitor into contact with the skin. Hyperesthesia involving one type can be effectively suppressed. The hyperesthesia includes, for example, hyperesthesia involving TRPA1 and ANO1 in the skin, hyperesthesia involving TRPV4 and ANO1 in the skin, and the like.

Examples of the hyperesthesia include itching involving at least one selected from the group consisting of TRPA1, TRPV4 and ANO1, and pain involving at least one selected from the group consisting of TRPA1, TRPV4 and ANO1. However, the present invention is not limited to such examples. Itching involving at least one selected from the group consisting of TRPA1, TRPV4 and ANO1 includes, for example, histamine-independent itch involving at least one selected from the group consisting of TRPV4 and ANO1, TRPA1, TRPV4. And histamine-dependent itching involving at least one selected from the group consisting of ANO1 and the like, but the present invention is not limited to such examples. Histamine-independent itching involving at least one selected from the group consisting of TRPA1, TRPV4 and ANO1 includes, for example, chloroquine, thymic stromal lymphocyte neoplasia [TSLP], bovine adrenal medulla peptide (8- 22) Itching due to [BAM 8-22], PAR2 agonist peptide [SLIGRL], etc., but the present invention is not limited to these examples. In addition, examples of histamine-dependent itching in which at least one selected from the group consisting of TRPA1, TRPV4 and ANO1 is involved include histamine; itching caused by a substance that causes the release of histamine from mast cells and the like. However, the present invention is not limited to such an example. Pain involving at least one selected from the group consisting of TRPA1, TRPV4 and ANO1 is associated with, for example, the activity of at least one selected from the group consisting of TRPA1, TRPV4 and ANO1 such as allyl isothiocyanate and protease. Pain caused by at least one selected from the group consisting of TRPA1, TRPV4 and ANO1 may be mentioned, but the present invention is not limited to such examples.

Since the skin hyperesthesia inhibitor of the present invention contains the activity inhibitor, it is used for suppressing itching involving at least one selected from the group consisting of TRPA1, TRPV4 and ANO1. Suitable as an antihyperesthesia agent. In addition, the skin hyperesthesia inhibitor of the present invention has a histamine-independent itch-inhibiting action involving at least one selected from the group consisting of TRPA1, TRPV4 and ANO1 and a group consisting of TRPA1, TRPV4 and ANO1. It exhibits both histamine-dependent itching-suppressing effects involving at least one selected species. Therefore, the skin hyperesthesia inhibitor of the present invention is expected to be used in applications that are required to suppress both histamine-independent itch and histamine-dependent itch.

The hyperesthesia to which the skin hyperesthesia inhibitor used for the purpose of suppressing itching is applied further includes pain involving at least one selected from the group consisting of TRPA1, TRPV4 and ANO1 in the skin. May be good. That is, the itching to which the skin hypersensitivity inhibitor of the present invention is applied may be painful involving at least one selected from the group consisting of TRPA1, TRPV4 and ANO1 in the skin.

Since the skin hyperesthesia inhibitor of the present invention contains the activity inhibitor, it is used for suppressing pain involving at least one selected from the group consisting of TRPA1, TRPV4 and ANO1. It may be an agent.

The content of the activity inhibitor in the skin hyperesthesia inhibitor of the present invention may be an amount effective for suppressing skin hyperesthesia. Therefore, it is preferable that the content of the activity inhibitor in the skin hyperesthesia inhibitor of the present invention is appropriately set according to the use of the skin hyperesthesia inhibitor and the like.

The skin hypersensitivity inhibitor of the present invention includes, for example, suitable solvents, surfactants, moisturizers, thickeners, preservatives, antioxidants, chelating agents, as long as the object of the present invention is not impaired. Other ingredients such as a pH adjuster may be blended.

Examples of the dosage form of the skin hypersensitivity inhibitor of the present invention include external solid preparations such as external powders; external liquids such as liniments and lotions; sprays such as external aerosols; ointments; creams; gels. Examples thereof include patches such as tapes and poultices, but the present invention is not limited to these examples.

The application target of the skin hyperesthesia inhibitor of the present invention includes humans, non-human mammals and the like, but the present invention is not limited to such examples.

It is preferable that the dose and the number of times of application of the skin hyperesthesia inhibitor of the present invention are appropriately selected according to the type of hyperesthesia, the type of application target, the age of application target, the body weight of the application target, and the like.

The number of times the skin hyperesthesia inhibitor of the present invention is applied is preferably selected as appropriate according to the type of hyperesthesia, the type of application target, the age of the application target, the weight of the application target, and the like. Further, it is preferable that the usage of the skin hyperesthesia inhibitor of the present invention is appropriately selected according to the dosage form of the skin hyperesthesia inhibitor of the present invention.

Hereinafter, the present invention will be described in more detail with reference to Examples, but the present invention is not limited to such Examples.

<Explanation of abbreviations>
NMDG: N-methyl-D-glucamine NMDG-Cl: N-methyl-D-glucamine hydrochloride EGTA: ethylene glycol tetraacetic acid HEPES: 2- [4- (2-hydroxyethyl) -1-piperazin] ethanesulfonic acid BAPTA : 1,2-bis (o-aminophenoxide) ethane-N, N, N', N'-tetraacetic acid ATP-Mg: adenosine-5'-magnesium triphosphate GTP-Na ₂ : guanosine-5'-3 Ethane phosphate

Manufacturing example 1
In a humidifying chamber maintained at 37 ° C. in an atmosphere containing 5% by volume of carbon dioxide, the medium [manufactured by Wako Pure Chemical Industries, Ltd., trade name: D-MEM high glucose, 044-29765; 584 mg / L. L-glutamine, 15 mg / L phenol red, 10 mass% bovine fetal serum (Life Technologies, 10437-028), 50,000 units / L penicillin / streptomycin mixture (Life Technologies, product) ^{70 5 × 10 5} HEK293T cells in DMEM (high glucose) containing Name: Pen Strep, 15140-122) and a cell culture supplement (manufactured by Life Technologies, trade name: GlutaMax). Cultured until ~ 80% confluency.

Next, 0.5 μg of cDNA carrying mouse ANO1 (GenBank accession number: NM_178642) is transfected into the HEK293T cells using a cationic lipid for gene transfer [manufactured by Invitrogen, trade name: lipofectamine (registered trademark)]. As a result, mouse ANO1-expressing cells were obtained.

Production Examples 2 to 6
In Production Example 1, instead of using the cDNA carrying mouse ANO1 (GenBank accession number: NM_178642), the cDNA carrying human ANO1 (GenBank accession number: NM_018403), mouse TRPA1 (GenBank accession) CDNA holding number: NM_177781) (Production Example 3), cDNA holding human TRPA1 (GenBank accession number: NM_007332) (Production Example 4), cDNA holding mouse TRPV4 (GenBank accession number: NM_022017) (Production) The same operation as in Production Example 1 was performed except that cDNA (Production Example 6) carrying human TRPV4 (GenBank accession number: NM_0216225) was used, and human ANO1-expressing cells (Production Example 2), Mouse TRPA1-expressing cells (Production Example 3), human TRPA1-expressing cells (Production Example 4), mouse TRPV4-expressing cells (Production Example 5), or human TRPV4-expressing cells (Production Example 6) were obtained.

Production example 7
NMDG-Cl, magnesium chloride, EGTA, glucose and HEPES buffer (pH 7.4) are mixed to give the following composition: 140 mM sodium chloride, 1 mM magnesium chloride, 5 mM EGTA, 10 mM glucose and 10 mM HEPES buffer, and standardized. A bath solution was obtained.

Production Example 8
NMDG-Cl, magnesium chloride, BAPTA and HEPES buffer (pH 7.3) were mixed to give the following composition: 140 mM NMDG-Cl, 1 mM magnesium chloride, 5 mM BAPTA and 10 mM HEPES buffer to obtain a pipette solution. .. In addition, the free calcium contained in the pipette solution was maintained at 500 nM.

Example 1
4-Isopropylcyclohexanol, a compound represented by the formula (I), was added to the standard bath solution of Production Example 7 so as to have a concentration of 3 mM to obtain a bath solution of Example 1.

Examples 2 and 3
In Example 1, isopropylcyclohexane (Example 2) or 1-isopropyl-4-methyl-cyclohexane (Example 3) was used instead of 4-isopropylcyclohexanol as the compound represented by the formula (I). Except for this, the same operation as in Example 1 was carried out to obtain bath solutions of Examples 2 and 3.

Comparative Example 1
In Example 1, the same operation as in Example 1 was carried out except that cyclohexanol, which is not the compound represented by the formula (I), was used instead of 4-isopropylcyclohexanol, and the bath solution of Comparative Example 1 was prepared. Obtained.

Examples 4-8
In Example 1, instead of setting the concentration of 4-isopropylcyclohexanol to 3 mM, 0.01 mM (Example 4), 0.03 mM (Example 5), 0.1 mM (Example 6), 0.3 mM (Example 6). The same operation as in Example 1 was carried out except that it was set to Example 7) or 1 mM (Example 8) to obtain bath solutions of Examples 4 to 8.

Test Example 1
(1) Measurement of Mouse ANO1 Current by Fixed Total Cell Potential Recording Method The mouse ANO1-expressing cells of Production Example 1 were placed in a perfusion chamber and washed with the standard bath solution of Production Example 7. Mouse ANO1-expressing cells were aspirated and brought into close contact with the tip opening of a patch electrode containing the pipette solution of Production Example 8. The patch membrane was then punctured by more intense aspiration of mouse ANO1-expressing cells. Next, the standard bath solution in the perfusion chamber is replaced with the bath solution of Example 1, Example 2 or Example 3, and the ANO1-expressing cells are exposed to the bath solution of Example 1, Example 2 or Example 3. At the same time, the measurement of mouse ANO1 current was started according to the whole cell potential fixed recording method. After 5 to 10 minutes from the start of exposure, the bath solution of Example 1, Example 2 or Example 3 in the perfusion chamber was replaced with the standard bath solution of Production Example 7, and ANO1-expressing cells were replaced with the standard bath solution of Production Example 7. Washed with standard bath solution.

The mouse TRPA1 current is measured by the whole cell potential fixed recording method using a patch clamp amplifier [Molecular Devices, trade name: Axopatch 200B amplifier] and a data acquirer (Axon Instruments, trade name: Digidata 1440A). ) And data acquisition / analysis software [Axon Instruments, trade name: pCLAMP 10] to record changes in mouse ANO1 current in mouse ANO1-expressing cells at a holding potential of -60 mV. Therefore, the mouse ANO1 current was measured by the whole cell potential fixed recording method. The relative current value of mouse ANO1 was calculated using the obtained measured value of mouse ANO1 current. Specifically, the relative current value of the mouse ANO1 is divided by the mouse ANO1 current value in the standard bath solution of Production Example 7 by dividing the mouse ANO1 current value in the bath solution of Example 1, Example 2 or Example 3. Was calculated. Using the calculated relative current value of mouse ANO1, the change with time of the relative current value of mouse ANO1 was investigated.

FIG. 1 (A) shows the results of examining the change over time in the relative current value of mouse ANO1 in Test Example 1. In FIG. 1 (A), (a) shows a change over time in the relative current value of mouse ANO1 when the mouse ANO1-expressing cells were exposed to the bath solution of Example 1 (administered with 4-isopropylcyclohexanol). ) Is a change over time in the relative current value of mouse ANO1 when the mouse ANO1 expressing cells are exposed to the bath solution of Example 2 (administered with isopropylcyclohexane), and (c) is the mouse ANO1 expressing cells of Example 3 The time course of the relative current value of mouse ANO1 when exposed to the bath solution (administered with 1-isopropyl-4-methylcyclohexane) is shown. Further, in FIG. 1 (A), “administration” refers to the period during which ANO1-expressing cells are exposed to the bath solution of Example 1, Example 2 or Example 3, and “washout” refers to Example 1 in the perfusion chamber. , The period after replacing the bath solution of Example 2 or Example 3 with the standard bath solution of Production Example 7.

From the results shown in FIG. 1 (A), a bath solution containing 4-isopropylcyclohexanol (Example 1), isopropylcyclohexane (Example 2) or 1-isopropyl-4-methyl-cyclohexane (Example 3) was prepared. It can be seen that the relative current value of mouse ANO1 when used is significantly reduced as compared with the relative current value before exposure of each bath solution. Therefore, it can be seen that the compound represented by the formula (I) such as 4-isopropylcyclohexanol, isopropylcyclohexane, and 1-isopropyl-4-methyl-cyclohexane inhibits the activity of ANO1. When the bath solution of Comparative Example 1 was used instead of the bath solution of Example 1, Example 2 or Example 3, no change in the relative current value before and after the exposure of the bath solution was observed.

The relative current value of mouse ANO1 when the bath solution containing 4-isopropylcyclohexanol of Example 1 was used was the bath solution containing isopropylcyclohexane of Example 2 or 1-isopropyl-4-methyl of Example 3. It can be seen that the relative current value of mouse ANO1 decreases rapidly as compared with the relative current value of mouse ANO1 when a bath solution containing cyclohexane is used [see “Administration” in FIG. 1 (A)]. From these results, it can be seen that the action of 4-isopropylcyclohexanol is expressed more rapidly than the actions of isopropylcyclohexane and 1-isopropyl-4-methyl-cyclohexane, respectively.

Furthermore, from the results shown in FIG. 1 (A), the relative current value of mouse ANO1 when the bath solution containing 4-isopropylcyclohexanol of Example 1 was used was determined by the bath solution of Example 1 in the perfusion chamber. Is replaced with the standard bath solution of Production Example 7, and it can be seen that the amount increases. On the other hand, the relative current value of mouse ANO1 when a bath solution containing isopropylcyclohexane (Example 2) or a bath solution containing 1-isopropyl-4-methyl-cyclohexane (Example 3) was used was in the perfusion chamber. It can be seen that there is no increase after replacing the bath solution of Example 1 with the standard bath solution of Production Example 7.

(2) 4-isopropyl cyclohexanol calculated Test Example IC ₅₀ values for Nord 1 (1), Example 1, each bus of Example 1,4～8 instead of using bath solution of Example 2 or Example 3 The same operation as in Test Example 1 (1) was performed except that the solution was used, and the relative current value of mouse ANO1 was determined.

FIG. 1 (B) shows the results of examining the relationship between the 4-isopropylcyclohexanol concentration and the relative current value of mouse ANO1 in Test Example 1. In FIG. 1 (B), "4-iPr-CyH-OH" represents 4-isopropylcyclohexanol.

From the dose-response curve shown in FIG. 1 (B), the IC ₅₀ value of 4-isopropylcyclohexanol was calculated. As a result, the IC ₅₀ values of 4-isopropyl cyclohexanol, was 1.09 mm.

Test Example 2
In Test Example 1, human ANO1-expressing cells were used instead of mouse ANO1-expressing cells, and the bath solution of Example 1 was used instead of the bath solution of Example 1, Example 2 or Example 3. The same operation as in Test Example 1 was carried out except for the above, and the change over time in the relative current value of human ANO1 was examined.

FIG. 2 shows the results of examining the change over time in the relative current value of human ANO1 in Test Example 2. In FIG. 2, “administration” is the period during which the bath solution of Example 1 is exposed to human ANO1-expressing cells, and “washout” is the standard bath of Production Example 7 in which the bath solution of Example 1 is produced in the perfusion chamber. The period after replacement with the solution is shown. In FIG. 2, "4-iPr-CyH-OH" represents 4-isopropylcyclohexanol.

From the results shown in FIG. 2, the relative current value of human ANO1 when the bath solution containing 4-isopropylcyclohexanol (Example 1) was used was the bath solution containing 4-isopropylcyclohexanol (Example 1). It can be seen that the relative current value of the mouse ANO1 (see FIG. 1) is similar to that of the mouse ANO1 when the above-mentioned is used. Further, when another compound represented by the formula (I) is used instead of 4-isopropylcyclohexanol, the same tendency as when 4-isopropylcyclohexanol is used can be seen. Therefore, it can be seen that the compound represented by the formula (I) suppresses the activity of human ANO1. When the bath solution of Comparative Example 1 was used instead of the bath solution of Example 1, no change in the relative current value before and after the exposure of the bath solution was observed.

Manufacturing example 9
Sodium chloride, magnesium chloride, EGTA, glucose and HEPES buffer (pH 7.4) were mixed to give the following composition: 140 mM sodium chloride, 1 mM magnesium chloride, 5 mM EGTA, 10 mM glucose and HEPES buffer. The obtained mixed solution was used as a standard bath solution.

Production Example 10
NMDG-Cl, magnesium chloride, BAPTA and HEPES buffer (pH 7.3) were mixed to give the following composition: 140 mM NMDG-Cl, 1 mM magnesium chloride, 5 mM BAPTA and 10 mM HEPES buffer to obtain a pipette solution. ..

Example 9
Allyl isothiocyanate and 4-isopropylcyclohexanol were added to the standard bath solution of Production Example 9 so that the concentration of the allyl isothiocyanate was 300 μM and the concentration of the 4-isopropylcyclohexanol was 3 mM. A bath solution was obtained.

Production Example 11
Allyl isothiocyanate was added to the standard bath solution of Production Example 9 so as to have a concentration of 300 μM to obtain a bath solution of Production Example 11.

Production Example 12
Sodium chloride, EGTA, glucose and HEPES buffer (pH 7.4) were mixed to give the following composition: 140 mM sodium chloride, 5 mM EGTA, 10 mM glucose and HEPES buffer. The resulting mixture was used as a standard bath solution and pipette solution.

Example 10
Allyl isothiocyanate and 4-isopropylcyclohexanol were added to the standard bath solution of Production Example 11 so that the concentration of the allyl isothiocyanate was 10 μM and the concentration of the 4-isopropylcyclohexanol was 100 μM. A bath solution was obtained.

Production Example 13
Allyl isothiocyanate was added to the standard bath solution of Production Example 12 so as to have a concentration of 10 μM to obtain the bath solution of Production Example 13.

Test Example 3
(1) Measurement of Mouse TRPA1 Current by Fixed Total Cell Potential Recording Method The mouse TRPA1-expressing cells of Production Example 3 were placed in a perfusion chamber and washed with the standard bath solution of Production Example 9. After washing with the standard bath solution of Production Example 9, the mouse TRPA1-expressing cells were aspirated and brought into close contact with the tip opening of the patch electrode containing the pipette solution of Production Example 10. Then, the patch film was sucked more strongly to make a hole in the patch film. Next, the standard bath solution in the perfusion chamber was replaced with the bath solution of Production Example 11, and the measurement of mouse TRPA1 current was started according to the whole cell potential fixation recording method. After 15 to 30 seconds had elapsed from the start of replacement of the bath solution, the bath solution in the perfusion chamber was replaced with the bath solution of Example 9, and mouse TRPA1-expressing cells were exposed to the bath solution of Example 9. After the mouse TRPA1 current was minimized, the bath solution of Example 9 in the perfusion chamber was replaced with the bath solution of Production Example 11. Next, the mouse TRPA1 current was measured again until the mouse TRPA1 current reached the maximum value, and then the mouse TRPA1 current was replaced with the standard bath solution of Production Example 9.

The mouse TRPA1 current is measured by the whole cell potential fixed recording method using a patch clamp amplifier [Molecular Devices, trade name: Axopatch 200B amplifier] and a data acquirer (Axon Instruments, trade name: Digidata 1440A). ) And data acquisition / analysis software [manufactured by Axon Instruments, trade name: pCLAMP 10], the holding potential is set to -60 mV, and a ramp pulse for 300 milliseconds at -100 to +100 mV is applied to the patch electrode. This was done by giving and recording the change in current over time.

In addition, the maximum value of allyl isothiocyanate-induced mouse TRPA1 current after administration of allyl isothiocyanate was divided by the capacitance of the cell membrane to determine the mouse TRPA1 current density when 4-isopropylcyclohexanol was not administered. .. In addition, after administration of 4-isopropylcyclohexanol, the minimum value of allyl isothiocyanate-induced mouse TRPA1 current was divided by the capacitance of the cell membrane to determine the mouse TRPA1 current density when 4-isopropylcyclohexanol was administered. ..

In Test Example 3 (1), the results of examining the time course of the allyl isothiocyanate-induced mouse TRPA1 current before and after administration of 4-isopropylcyclohexanol are shown in FIG. 3 (A), the presence or absence of administration of 4-isopropylcyclohexanol, and the mice. The result of investigating the relationship with the TRPA1 current density is shown in FIG. 3 (B). In FIGS. 3A and 3B, "4-iPr-CyH-OH" indicates 4-isopropylcyclohexanol and "AITC" indicates allyl isothiocyanate. Further, in FIG. 3B, lane 1 is allyl isothiocyanate-induced mouse TRPA1 current density when 4-isopropylcyclohexanol is not administered, and lane 2 is allyl isothiocyanate induction when 4-isopropylcyclohexanol is administered. The mouse TRPA1 current density is shown.

From the results shown in FIG. 3 (A), it can be seen that the mouse TRPA1 current induced by the administration of allyl isothiocyanate is decreased after the administration of 4-isopropylcyclohexanol. In addition, from the results shown in FIG. 3 (B), the allyl isothiocyanate-induced mouse TRPA1 current density (lane 2) when 4-isopropylcyclohexanol was administered was allyl when 4-isopropylcyclohexanol was not administered. It can be seen that the isothiocyanate-induced mouse TRPA1 current density is lower than that of TRPA1 (lane 1). From these results, it can be seen that 4-isopropylcyclohexanol inhibits the induction of mouse TRPA1 current by allyl isothiocyanate. When another compound represented by the formula (I) is used instead of 4-isopropylcyclohexanol, the same tendency as when 4-isopropylcyclohexanol is used can be seen. Therefore, it can be seen that the compound represented by the formula (I) suppresses the activity of mouse TRPA1.

(2) Measurement of Mouse TRPA1 Current by Single-Channel Potential Fixed Recording Method The mouse TRPA1-expressing cells of Production Example 3 were placed in a perfusion chamber and washed with the standard bath solution of Production Example 12. After washing, the mouse TRPA1-expressing cells were aspirated and brought into close contact with the tip opening of the patch electrode containing the pipette solution of Production Example 12, and the patch membrane was peeled off to form an inside-out patch membrane at the tip opening. Next, the standard bath solution in the perfusion chamber was replaced with the bath solution of Production Example 13. After 1 to 3 minutes from the start of replacement of the bath solution, the bath solution in the perfusion chamber was replaced with the bath solution of Example 10, and the mouse TRPA1-expressing cells were exposed to the bath solution of Example 10. One minute after the start of exposure, the bath solution of Example 10 in the perfusion chamber was replaced with the standard bath solution of Production Example 12.

Patch clamp amplifier [Molecular Devices, product name: Axopatch 200B amplifier], data acquirer (Axon Instruments, product name: Digidata 1440A), and data acquisition / analysis software [Axon Instruments (Axon) The mouse TRPA1 current was measured by the single-channel potential fixed recording method by recording the change over time of the current when the holding potential was −60 mV using a product manufactured by Instruments), trade name: pCLAMP 10]. An amplitude histogram of the mouse TRPA1 current was created based on the obtained measured values of the mouse TRPA current. Using the amplitude histogram, the average value of the amplitude magnitude (pA) of the mouse TRPA1 current, the average value of the opening time of the mouse TRPA1 and the average value of the closing time of the mouse TRPA1 were calculated.

Further, in the above, the same operation as described above was performed except that the bath solution of Production Example 13 was used instead of the bath solution of Example 10, and the average value of the amplitude magnitude (pA) of the mouse TRPA1 current was performed. , The average value of the opening time of the mouse TRPA1 and the average value of the closing time of the mouse TRPA1 were calculated.

In Test Example 3, the results of investigating the relationship between the presence or absence of 4-isopropylcyclohexanol and the magnitude of the amplitude of the mouse TRPA1 current are shown in FIG. 4 (A), and the relationship between the presence or absence of 4-isopropylcyclohexanol and the opening time of mouse TRPA1. The results of examining the above are shown in FIG. 4 (B), and the results of examining the relationship between the presence or absence of 4-isopropylcyclohexanol and the closing time of mouse TRPA1 are shown in FIG. 4 (C). In FIG. 4 (A), lane 1 has a large amplitude of mouse TRPA1 current in the absence of 4-isopropylcyclohexanol, and lane 2 has a large amplitude of mouse TRPA1 current in the presence of 4-isopropylcyclohexanol. Indicates that. In FIG. 4B, lane 1 shows the opening time of mouse TRPA1 in the absence of 4-isopropylcyclohexanol, and lane 2 shows the opening time of mouse TRPA1 in the presence of 4-isopropylcyclohexanol. In FIG. 4C, lane 1 shows the closing time of mouse TRPA1 in the absence of 4-isopropylcyclohexanol, and lane 2 shows the closing time of mouse TRPA1 in the presence of 4-isopropylcyclohexanol.

From the results shown in FIG. 4 (A), the magnitude of the amplitude of the mouse TRPA1 current in the absence of 4-isopropylcyclohexanol (see lane 1) and the mouse TRPA1 in the presence of 4-isopropylcyclohexanol. It can be seen that the magnitude of the amplitude of the current (lane 2) is almost the same. From these results, it can be seen that 4-isopropylcyclohexanol does not affect the conductance of mouse TRPA1.

Student's t-test was performed on the results shown in FIGS. 4 (B) and 4 (C). In the results shown in FIG. 4B, Student's t-test p value was less than 0.01. Therefore, from this result, there is a significant difference between the opening time of mouse TRPA1 in the absence of 4-isopropylcyclohexanol and the opening time of mouse TRPA1 in the presence of 4-isopropylcyclohexanol. Is suggested. On the other hand, in the result shown in FIG. 4 (C), the p value of Student's t-test was 0.068. Therefore, from this result, there is no significant difference between the closing time of mouse TRPA1 in the absence of 4-isopropylcyclohexanol and the closing time of mouse TRPA1 in the presence of 4-isopropylcyclohexanol. Is suggested. From these results, it can be seen that 4-isopropylcyclohexanol affects the gating of mouse TRPA1.

From these results, it can be seen that 4-isopropylcyclohexanol does not affect the conductance in mouse TRPA1, but affects the gating of mouse TRPA1.

Test Example 4
In Test Example 3 (1), the same operation as in Test Example 3 (1) was performed except that the human TRPA1 expressing cells of Production Example 4 were used instead of the mouse TRPA1 expressing cells of Production Example 3, and 4-. The time course of allyl isothiocyanate-induced human TRPA1 current before and after administration of isopropylcyclohexanol and the relationship between the presence or absence of administration of 4-isopropylcyclohexanol and the human TRPA1 current density were investigated.

In Test Example 4, the results of examining the time course of allyl isothiocyanate-induced human TRPA1 current before and after administration of 4-isopropylcyclohexanol are shown in FIG. 5 (A), the presence or absence of administration of 4-isopropylcyclohexanol and the human TRPA1 current density. The result of investigating the relationship with is shown in FIG. 5 (B). In FIG. 5 (A), "4-iPr-CyH-OH" indicates 4-isopropylcyclohexanol, and "AITC" indicates allyl isothiocyanate. Further, in FIG. 5B, lane 1 shows the human TRPA1 current density when 4-isopropylcyclohexanol is not administered, and lane 2 shows the human TRPA1 current density when 4-isopropylcyclohexanol is administered.

From the results shown in FIG. 5 (A), it can be seen that the human TRPA1 current induced by the administration of allyl isothiocyanate is reduced after the administration of 4-isopropylcyclohexanol, similar to the mouse TRPA1 current. In addition, from the results shown in FIG. 5 (B), the allyl isothiocyanate-induced human TRPA1 current density (lane 2) when 4-isopropylcyclohexanol was administered was higher than that when 4-isopropylcyclohexanol was not administered. It can be seen that it is lower than the allyl isothiocyanate-induced human TRPA1 current density (lane 1). From these results, it can be seen that 4-isopropylcyclohexanol inhibits the induction of human TRPA1 current by allyl isothiocyanate. When another compound represented by the formula (I) is used instead of 4-isopropylcyclohexanol, the same tendency as when 4-isopropylcyclohexanol is used can be seen. Therefore, it can be seen that the compound represented by the formula (I) suppresses the activity of human TRPA1.

Example 11
TRPV4 agonist (GSK10167790A) and 4-isopropylcyclohexanol were added to the standard bath solution of Production Example 9 so that the concentration of the TRPV4 agonist (GSK10167790A) was 300 nM and the concentration of the 4-isopropylcyclohexanol was 3 mM. The bath solution of Example 11 was obtained.

Production example 14
A TRPV4 agonist (GSK10167790A) was added to the standard bath solution of Production Example 9 so as to have a concentration of 300 nM to obtain a bath solution of Production Example 14.

Test Example 5
The mouse TRPV4-expressing cells of Production Example 5 were placed in a perfusion chamber and washed with the standard bath solution of Production Example 9. After washing with the standard bath solution of Production Example 9, the mouse TRPV4-expressing cells were aspirated and brought into close contact with the tip opening of the patch electrode containing the pipette solution of Production Example 10. Then, mouse TRPV4-expressing cells were aspirated more strongly to puncture the patch membrane. Next, the standard bath solution in the perfusion chamber was replaced with the bath solution of Production Example 14, and the measurement of mouse TRPV4 current was started according to the whole cell potential fixation recording method. After 30 seconds from the start of replacement of the bath solution, the bath solution in the perfusion chamber was replaced with the bath solution of Example 11, and the mouse TRPV4-expressing cells were exposed to the bath solution of Example 11. After the mouse TRPV4 current was minimized, the bath solution of Example 11 in the perfusion chamber was replaced with the bath solution of Production Example 14. One minute after the start of replacement of the bath solution, the bath solution was replaced with the standard bath solution of Production Example 9.

The mouse TRPV4 current was measured by the whole cell potential fixed recording method using a patch clamp amplifier [Molecular Devices, trade name: Axopatch 200B amplifier] and a data acquirer (Axon Instruments, trade name: Digidata 1440A). ) And data acquisition / analysis software [manufactured by Axon Instruments, trade name: pCLAMP 10], holding potential: -60 mV, -100 to +100 mV, 300 milliseconds ramp pulse to patch electrode This was done by recording the time course of mouse TRPV4 current in mouse TRPV4-expressing cells when fed.

Further, in the above, the same operation as described above was performed except that the mouse TRPV4 expressing cells of Production Example 5 were used instead of the human TRPV4 expressing cells of Production Example 6, and the time course of the human TRPV4 current was measured. ..

In Test Example 5, the results of examining the time course of the GSK1016790A-induced mouse TRPV4 current before and after administration of 4-isopropylcyclohexanol are shown in FIG. 6 (A). The result of examining the target change is shown in FIG. 6 (B). In FIGS. 6 (A) and 6 (B), "4-iPr-CyH-OH" indicates 4-isopropylcyclohexanol, and "GSK101" indicates a TRPV4 agonist (GSK10167990A).

From the results shown in FIGS. 6 (A) and 6 (B), the mouse TRPV4 current and the human TRPV4 current induced by the administration of the TRPV4 agonist (GSK10167990A) were decreased after the administration of 4-isopropylcyclohexanol, respectively. You can see that. Therefore, from these results, it can be seen that 4-isopropylcyclohexanol inhibits the induction of mouse TRPV4 current and human TRPV4 current by the TRPV4 agonist (GSK10167990A). When another compound represented by the formula (I) is used instead of 4-isopropylcyclohexanol, the same tendency as when 4-isopropylcyclohexanol is used can be seen. Therefore, it can be seen that the compound represented by the formula (I) suppresses the activity of mouse TRPV4 and the activity of human TRPV4.

As described above, the compound represented by the formula (I) such as 4-isopropylcyclohexanol has a TRPA1 current induced by a TRPA1 agonist, a TRPV4 current induced by a TRPV4 agonist, and an ANO1 current induced by an ANO1 agonist. From the inhibition, it can be seen that the activity of TRPA1, TRPV4 and ANO1 is suppressed. When the relationship between the activities of TRPA1, TRPV4 and ANO1 and itch was investigated, a correlation was found between the two, suggesting that they are involved in the occurrence of itch. Moreover, when the relationship between the activity of TRPA1, TRPV4 and ANO1 and pain was investigated, a correlation was found between the two, suggesting that they are involved in the occurrence of pain. Therefore, the compound represented by the formula (I) such as 4-isopropylcyclohexanol is selected from the group consisting of itching, TRPA1, TRPV4 and ANO1 in which at least one selected from the group consisting of TRPA1, TRPV4 and ANO1 is involved. It is suggested that at least one of them suppresses hyperesthesia such as pain.

Example 12
4-Isopropylcyclohexanol and a histamine-independent itch-causing itch-causing substance are added to a physiological saline solution containing 0.9% by mass of sodium chloride to obtain a test sample of Example 12.

Comparative Example 2
A histamine-independent itch-causing itch-causing substance is added to a physiological saline solution containing 0.9% by mass sodium chloride to obtain a test sample of Comparative Example 2.

Test Example 6
Using a 50 μL volume Hamilton syringe equipped with a 30 gauge needle, 50 μL of the test sample of Example 12 or the test sample of Comparative Example 2 is subcutaneously injected into the back of a C57 / BL6NCr mouse. Using a digital camera, the scratching behavior time of the mouse for 30 minutes from the end of injection of the test sample is measured. The measurement results are statistically analyzed by the Mann-Whitney U test. When the p value is less than 0.05, it is judged that there is a statistically significant difference.

As a result, the scratching behavior time of the mouse when the test sample containing 4-isopropylcyclohexanol of Example 10 was administered was the scratching behavior time of the mouse when the test sample of Comparative Example 2 containing no 4-isopropylcyclohexanol was administered. It can be confirmed that it is significantly shorter than the action time. From these results, it can be confirmed that 4-isopropylcyclohexanol has an antipruritic effect on histamine-independent itching in which at least one selected from the group consisting of TRPA1, TRPV4 and ANO1 is involved.

Example 13
4-Isopropylcyclohexanol and a pain-causing substance that activates TRPA1, TRPV4 and ANO1 and causes pain are added to a physiological saline solution containing 0.9% by mass sodium chloride to obtain a test sample of Example 13.

Comparative Example 3
A pain-causing substance that activates TRPA1, TRPV4 and ANO1 and causes pain is added to a physiological saline solution containing 0.9% by mass sodium chloride to obtain a test sample of Comparative Example 3.

Test Example 7
Using a 25 μL volume Hamilton syringe equipped with a 30 gauge needle, 10 μL of the test sample of Example 13 or the test sample of Comparative Example 3 is subcutaneously injected into the tip of the hind leg of a C57 / BL6NCr mouse. Using a digital camera, the foot licking time by the mouse is measured for 5 minutes (5 minutes after administration of the pain-causing substance) from the end of injection of the test sample containing the pain-causing substance. The measurement results are statistically analyzed by the Mann-Whitney U test. When the p value is less than 0.05, it is judged that there is a statistically significant difference.

As a result, the foot licking time of the mouse when the test sample containing 4-isopropylcyclohexanol of Example 13 was administered was the foot licking time when the test sample not containing 4-isopropylcyclohexanol of Comparative Example 3 was administered. It can be confirmed that it is remarkably short compared to. Therefore, it can be confirmed that 4-isopropylcyclohexanol has an analgesic effect on pain involving at least one selected from the group consisting of TRPA1, TRPV4 and ANO1.

Examples 14-18
In Example 9, instead of setting the concentration of 4-isopropylcyclohexanol to 3 mM, 0.01 mM (Example 14), 0.03 mM (Example 15), 0.1 mM (Example 16), 0.3 mM (Example 16). The same operation as in Example 9 was carried out except that it was set to Example 17) or 1 mM (Example 18) to obtain bath solutions of Examples 14 to 18.

Test Example 8
The mouse TRPA1-expressing cells of Production Example 3 were placed in a perfusion chamber and washed with the standard bath solution of Production Example 9. The mouse TRPA1-expressing cells were aspirated and brought into close contact with the tip opening of a patch electrode containing the pipette solution of Production Example 10. The patch membrane was then punctured by more intense aspiration of mouse TRPA1-expressing cells. Next, the standard bath solution in the perfusion chamber was replaced with the bath solution of Production Example 11, and the measurement of mouse TRPA1 current was started according to the whole cell potential fixation recording method. After 15 to 30 seconds have passed from the start of replacement of the bath solution, the bath solution in the perfusion chamber was replaced with each bath solution of Examples 9 and 14 to 18, and mouse TRPA1-expressing cells were replaced with Example 9 and Example 14. Each bath solution of ~ 18 was exposed. After the mouse TRPA1 current became the minimum value, the bath solutions of Examples 9 and 14 to 18 in the perfusion chamber were replaced with the bath solutions of Production Example 11. Next, the mouse TRPA1 current was measured again until the mouse TRPA1 current reached the maximum value, and then the mouse TRPA1 current was replaced with the standard bath solution of Production Example 9.

The mouse TRPA1 current is measured by the whole cell potential fixed recording method using a patch clamp amplifier [Molecular Devices, trade name: Axopatch 200B amplifier] and a data acquirer (Axon Instruments, trade name: Digidata 1440A). ) And data acquisition / analysis software [manufactured by Axon Instruments, trade name: pCLAMP 10], holding potential: -60 mV, -100 to +100 mV, 300 milliseconds ramp pulse to patch electrode This was done by giving and recording the change in current over time.

The relative current value of mouse TRPA1 was calculated using the obtained measured value of mouse TRPA1 current. Specifically, the mouse TRPA1 current value when the bath solutions of Examples 9 and 14 to 18 were used was divided by the mouse TRPA1 current value in the standard bath solution of Production Example 11. The relative current value was calculated.

Further, in the above, the same operation as in Test Example 7 was performed except that the human TRPA1 expressing cells of Production Example 4 were used instead of the mouse TRPA1 expressing cells of Production Example 3, and the relative current value of human TRPA1 was calculated. bottom.

In Test Example 8, the result of investigating the relationship between the 4-isopropylcyclohexanol concentration and the relative current value of mouse TRPA1 was examined in FIG. 7 (A), and the relationship between the 4-isopropylcyclohexanol concentration and the relative current value of human TRPA1 was investigated. The results are shown in FIG. 7 (B). In the figure, "4-iPr-CyH-OH" indicates 4-isopropylcyclohexanol.

From the results shown in FIG. 7 (A), it can be seen that the higher the concentration of 4-isopropylcyclohexanol in the bath solution, the higher the relative current value of mouse TRPA1 tends to be. Therefore, it can be seen that 4-isopropylcyclohexanol inhibits mouse TRPA1 in a dose-dependent manner. Further, based on the dose-response curve shown in FIG. 7 (A), IC ₅₀ values were calculated for 4-isopropyl cyclohexanol to mouse TRPAl. As a result, the IC ₅₀ values of 4-isopropyl cyclohexanol to mouse TRPAl, was 0.23 mM.

From the results shown in FIG. 7B, it can be seen that the higher the concentration of 4-isopropylcyclohexanol in the bath solution, the higher the relative current value of human TRPA1 tends to be. Therefore, it can be seen that 4-isopropylcyclohexanol inhibits human TRPA1 in a dose-dependent manner. Further, based on the dose-response curve shown in FIG. 7 (B), IC ₅₀ values were calculated for 4-isopropyl cyclohexanol to human TRPAl. As a result, the IC ₅₀ values of 4-isopropyl cyclohexanol to human TRPAl, was 1.25 mM.

Examples 19-22
The TRPV4 agonist (GSK10167790A) and 4-isopropylcyclohexanol were added to the standard bath solution of Production Example 9, the TRPV4 agonist (GSK10167790A) had a concentration of 300 μM, and the 4-isopropylcyclohexanol concentration was 0.3 mM (Example 19). Additions were made to 1 mM (Example 20), 2 mM (Example 21) or 3 mM (Example 22) to obtain bath solutions of Examples 19 to 22.

Production example 15
A TRPV4 agonist (GSK10167790A) was added to the standard bath solution of Production Example 9 so as to have a concentration of 300 μM to obtain a bath solution of Production Example 15.

Test Example 9
Mouse TRPV4-expressing cells of Production Example 5 were placed in a perfusion chamber and washed with the standard bath solution of Production Example 9. Next, the mouse TRPV4-expressing cells were aspirated and brought into close contact with the tip opening of the patch electrode containing the pipette solution of Production Example 10. The patch membrane was then punctured by more intense aspiration of mouse TRPV4-expressing cells. Next, the standard bath solution in the perfusion chamber was replaced with the bath solution of Production Example 15, and the measurement of mouse TRPV4 current was started according to the whole cell potential fixation recording method. After 30 seconds from the start of replacement of the bath solution, the bath solution in the perfusion chamber was replaced with each bath solution of Examples 19 to 22, and mouse TRPV4-expressing cells were exposed to each of the bath solutions of Examples 19 to 22. After the mouse TRPV4 current was minimized, the bath solution of Examples 19-22 in the perfusion chamber was replaced with the bath solution of Production Example 15. One minute after the start of the replacement, the solution was replaced with the standard bath solution of Production Example 9.

The mouse TRPV4 current was measured by the whole cell potential fixed recording method using a patch clamp amplifier [Molecular Devices, trade name: Axopatch 200B amplifier] and a data acquirer (Axon Instruments, trade name: Digidata 1440A). ) And data acquisition / analysis software [manufactured by Axon Instruments, trade name: pCLAMP 10], holding potential: -60 mV, -100 to +100 mV, 300 milliseconds ramp pulse to patch electrode This was done by recording the time course of mouse TRPV4 current in mouse TRPV4-expressing cells when fed.

The relative current value of mouse TRPV4 was calculated using the obtained measured value of mouse TRPV4 current. Specifically, the relative current value of the mouse TRPV4 is obtained by dividing the mouse TRPV4 current value when each of the bath solutions of Examples 19 to 22 is used by the mouse TRPV4 current value in the standard bath solution of Production Example 9. Calculated.

Further, in the above, instead of using the mouse TRPV4 expressing cells of Production Example 5, the human TRPV4 expressing cells of Production Example 6 were used, and instead of using the bath solutions of Examples 19 to 22, each of Examples 20 to 22 was used. The same operation as described above was performed except that the bath solution was used, and the relative current value of human TRPV4 was calculated.

In Test Example 9, the result of examining the change with time of the relative current value of mouse TRPV4 is shown in FIG. 8 (A), and the result of examining the change with time of the relative current value of human TRPV4 is shown in FIG. 8 (B). In FIG. 8 (A), (a) shows the change over time in the relative current value of mouse TRPV4 when the mouse TRPV4-expressing cells were exposed to the bath solution containing 0.3 mM 4-isopropylcyclohexanol of Example 19. b) is a change over time in the relative current value of mouse TRPV4 when mouse TRPV4-expressing cells are exposed to a bath solution containing 1 mM 4-isopropylcyclohexanol of Example 20, and (c) is 2 mM 4- of Example 21. Changes in relative current values of mouse TRPV4 over time when mouse TRPV4-expressing cells were exposed to a bath solution containing isopropylcyclohexanol, (d) shows mouse TRPV4 in a bath solution containing 3 mM 4-isopropylcyclohexanol of Example 22. The time course of the relative current value of mouse TRPV4 when the expressing cells are exposed is shown. In FIG. 8A, “administration” indicates the duration of exposure of mouse TRPV4-expressing cells to each bath solution of Examples 19-22. Further, in FIG. 8 (B), (a) shows a change over time in the relative current value of mouse TRPV4 when the mouse TRPV4-expressing cells were exposed to the bath solution containing 1 mM 4-isopropylcyclohexanol of Example 20. b) is a change over time in the relative current value of mouse TRPV4 when mouse TRPV4-expressing cells are exposed to a bath solution containing 2 mM 4-isopropylcyclohexanol of Example 21, and (c) is 3 mM 4- of Example 22. The time course of the relative current value of mouse TRPV4 when the mouse TRPV4-expressing cells are exposed to a bath solution containing isopropylcyclohexanol is shown. In FIG. 8B, “administration” indicates the duration of exposure of human TRPV4-expressing cells to each bath solution of Examples 20-22.

From the results shown in FIG. 8 (A), it can be seen that the higher the concentration of 4-isopropylcyclohexanol in the bath solution, the higher the relative current value of mouse TRPV4 tends to be. Therefore, it can be seen that 4-isopropylcyclohexanol suppresses the activity of mouse TRPV4 in a dose-dependent manner. Further, from the results shown in FIG. 8 (B), it can be seen that the higher the concentration of 4-isopropylcyclohexanol in the bath solution, the higher the relative current value of human TRPV4 tends to be.

Example 23
4-Isopropylcyclohexanol and chloroquine were added to a physiological saline solution containing 0.9% by mass sodium chloride so that the concentration of 4-isopropylcyclohexanol was 3 mM and the concentration of chloroquine was 200 μg / 50 μL, and the test of Example 23 was conducted. A sample was obtained.

Comparative Example 4
Chloroquine was added to a physiological saline solution containing 0.9% by mass sodium chloride so that its concentration was 200 μg / 50 μL to obtain a test sample of Comparative Example 4.

Test Example 10
Four 10-week-old male C57 / BL6NCr mice were placed in a gauge and bred for 1-2 hours to acclimatize each C57 / BL6NCr mouse to the environment in the gauge. Using a 500 μL volume syringe [manufactured by Terumo Corporation, trade name: Myjector] equipped with a 29 gauge needle, 50 μL of the test sample of Example 23 was administered intradermally to the back of the neck of each C57 / BL6NCr mouse. The number of scratching motions of each C57 / BL6NCr mouse was measured from the end of administration of the test sample to the lapse of 1 minute. Next, the average value of the number of scratching motions of the four C57 / BL6NCr mice was calculated.

Further, in the above, the same operation as described above was performed except that the test sample of Comparative Example 4 was used instead of the test sample of Example 23, and the average value of the number of scratching operations was obtained.

Table 1 shows the results of examining the number of scratching motions of C57 / BL6NCr mice when each of the test samples of Example 23 and Comparative Example 4 was administered.

From the results shown in Table 1, the number of scratching motions of the mice when the test sample containing 4-isopropylcyclohexanol of Example 23 was administered was the same as that of the test sample containing no 4-isopropylcyclohexanol of Comparative Example 4. It can be seen that the number of scratching motions of the mice at the time of administration is significantly smaller. The measurement results were statistically analyzed by the Mann-Whitney U test. As a result, since the p value was less than 0.05, the number of scratching motions of the mouse when the test sample of Example 23 was administered and the scratching motion of the mouse when the test sample of Comparative Example 4 was administered. It was judged that there was a statistically significant difference from the number of times. Chloroquine-induced itch is considered to be one of histamine-independent itch involving at least one selected from the group consisting of TRPA1, TRPV4 and ANO1. Therefore, it can be seen that 4-isopropylcyclohexanol has an antipruritic effect on itching caused by chloroquine, which is one of histamine-independent itching. When another compound represented by the formula (I) is used instead of 4-isopropylcyclohexanol, the same tendency as when 4-isopropylcyclohexanol is used can be seen.

These results suggest that the compound represented by the formula (I) suppresses itching involving at least one selected from the group consisting of TRPA1, TRPV4 and ANO1.

Example 24
Example 24, 4-isopropylcyclohexanol and histamine were added to a physiological saline solution containing 0.9% by mass sodium chloride so that the concentration of 4-isopropylcyclohexanol was 3 mM and the concentration of histamine was 111.15 ng / 50 μL. Test sample was obtained.

Comparative Example 5
Chloroquine was added to a physiological saline solution containing 0.9% by mass sodium chloride so that the concentration of histamine was 111.15 ng / 50 μL to obtain a test sample of Comparative Example 5.

Test Example 11
Eight 6-week-old male ICR mice were placed in gauges and bred for 1-2 hours to purify each ICR mouse into the environment. Using a 500 μL volume syringe [manufactured by Terumo Corporation, trade name: Myjector] equipped with a 29 gauge needle, 50 μL of the test sample of Example 24 was administered intradermally to the back of the neck of each ICR mouse. Using a digital camera, the number of scratching motions of each ICR mouse was measured from the end of administration of the test sample to the lapse of 5 minutes. Next, the average value of the number of scratching motions of eight ICR mice was calculated.

Further, in the above, the same as above, except that 11 ICR mice were used instead of 8 ICR mice and the test sample of Comparative Example 5 was used instead of the test sample of Example 24. The operation was performed, and the average value of the number of scratching operations was calculated.

Table 2 shows the results of examining the number of scratching motions of the ICR mice when each of the test samples of Example 24 and Comparative Example 5 was administered.

From the results shown in Table 1, the number of scratching motions of the mice when the test sample containing 4-isopropylcyclohexanol of Example 24 was administered was the same as that of the test sample of Comparative Example 5 containing no 4-isopropylcyclohexanol. It can be seen that the number of scratching motions of the mice at the time of administration is significantly smaller. Therefore, it can be seen that 4-isopropylcyclohexanol has an antipruritic effect on histamine-dependent itching. The measurement results were statistically analyzed by the Mann-Whitney U test. As a result, since the p value was less than 0.05, the number of scratching motions of the mouse when the test sample of Example 24 was administered and the scratching motion of the mouse when the test sample of Comparative Example 5 was administered. It was judged that there was a statistically significant difference from the number of times. Itching caused by histamine is considered to be one of the itch involving at least one selected from the group consisting of TRPA1, TRPV4 and ANO1. Therefore, it can be seen that 4-isopropylcyclohexanol has an antipruritic effect on histamine-dependent itching. When another compound represented by the formula (I) is used instead of 4-isopropylcyclohexanol, the same tendency as when 4-isopropylcyclohexanol is used can be seen.

As described above, it is suggested that the compound represented by the formula (I) suppresses itching involving at least one selected from the group consisting of TRPA1, TRPV4 and ANO1.

Claims (4)

An activity inhibitor for suppressing the activities of TRPA1, TRPV4 and ANO1 respectively, and as an active ingredient, the formula (I):

(Wherein, R ¹ represents a hydrogen atom, a hydroxyl group or an alkyl group having 1 to 4 carbon ^{atoms, R 2, R 3, R} 4, R 5, R 6, R 7, R 8, R 9 and R ¹⁰ are hydrogen shows the original child)
An activity inhibitor comprising a compound represented by . A cutaneous hypersensitivity inhibitors for at least one selected from the group consisting of TRPAl, TRPV4 and ANO1 in skin can be inhibited hypersensitivity involved, the activity inhibitor according to claim 1 as an active ingredient Contains a skin hyperesthesia inhibitor. The skin hyperesthesia inhibitor according to claim 2 , wherein the hyperesthesia is an itch involving at least one selected from the group consisting of TRPA1, TRPV4 and ANO1 in the skin. The skin hyperesthesia inhibitor according to claim 2 or 3 , wherein the hyperesthesia further comprises pain involving at least one selected from the group consisting of TRPA1, TRPV4 and ANO1 in the skin.

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