JP6508969B2 - Evaluation method of test substance - Google Patents

Description

  The present invention relates to a method of evaluating a test substance. More specifically, the present invention relates to a method of evaluating a test substance useful for the development of an external preparation such as a cosmetic.

  When the external preparation such as a cosmetic contains a refreshing agent, parabens, an alkaline agent, etc., some users may feel unpleasant irritation on the skin when using the external preparation. However, in recent years, due to an increase in safety awareness of users, external preparations that do not give such unpleasant stimuli or have few unpleasant stimuli tend to be preferred.

  The evaluation of the irritant properties of substances to the skin is performed by methods such as human patch test and human stinging test. However, in the above-mentioned method, an error may occur in the evaluation result because the subject is likely to cause a variation or the like. In addition, in the above method, the sensitivity to sensory stimulation varies depending on the subject, so that the difference in the degree of sensory stimulation on the skin may not be evaluated with sufficient sensitivity. Therefore, the method has the disadvantage that it is difficult to evaluate sensory stimuli in the skin with high reproducibility and high sensitivity.

  By the way, for example, a refreshing agent, parabens, an alkaline agent, etc. activate TRPA1, which is one of the transient receptor potential channels, to cause unpleasant stimulation via the activated TRPA1. They are found by the inventors (see, for example, Patent Documents 1 to 4).

JP, 2008-079528, A JP, 2009-082053, A JP, 2009-225733, A JP 2012-062304 A

  However, at present, the present inventors have described the literature specifically describing the relationship between TRPA1 and hypotonicity, and a method for evaluating the TRPA1 activating activity of a test substance using the aforementioned relationship. I have not found it.

  The present invention has been made in view of the above-mentioned prior art, and an object of the present invention is to provide a method for evaluating a test substance which can evaluate TRPAl activating activity of a test substance with high reproducibility and high sensitivity.

This onset Ming,
(1) A method of evaluating a test substance for evaluating the activity of activating TRPA1 possessed by the test substance, which is a hypotonic fluid having an osmotic pressure lower than the osmotic pressure of the intracellular fluid of TRPA1-expressing cells, the test substance, and the test substance TRPA1 expressing cells and contacting the said when the hypotonic liquid was measured physiological events triggered through TRPA1, based on the physiological events, assessing the TRPA1 activation activity with the test substance, the Changes in calcium ion concentration in TRPAl -expressing cells caused by TRPA1 by the hypotonic fluid before and after contact with the test substance as physiological events, TRPA1 by the hypotonic fluid before and after contact with the test substance be characterized by using a change or a combination of these currents of TRPA1 expressing cells caused Te Evaluation method of the test substance,
(2) The method according to (1), wherein the hypotonic fluid has an osmotic pressure 40 to 200 mOsm lower than the osmotic pressure of the intracellular fluid, and (3) the physiological event is that of the test substance The method according to (1) or (2), which is a change in calcium ion concentration in the TRPAl -expressing cell caused by TRPRP1 by the hypotonic fluid before and after contact.

  The evaluation method of the test substance of the present invention exhibits an excellent effect that the TRPA1 activating activity of the test substance can be evaluated with high reproducibility and high sensitivity.

In Experiment 1, it is a graph which shows the result of having investigated the relationship between the difference with the osmotic pressure of the intracellular fluid, and the cell area increase rate. In Experiment 1, it is a graph which shows the result of having investigated the relationship between the difference with the osmotic pressure of intracellular fluid, and the degree of TRPA1 activation. (A) is a graph showing the results of examining the relationship between the cell area increase rate when contacted with a hypotonic fluid and the TRPA1 activation degree when contacted with a hypotonic fluid in Test Example 1 ( B) is a graph showing the results of examining the relationship between the cell area increase rate in the presence of TRPAl antagonist and the degree of TRPAl activation in the presence of TRPAl antagonist. In Example 1, it is a graph which shows the result of having investigated the relationship between the difference with the osmotic pressure of the intracellular fluid, and a fluorescence-intensity ratio.

1. Evaluation method of test substance The evaluation method of a test substance of the present invention is an evaluation method of a test substance for evaluating TRPAl activating activity of a test substance, and the osmotic pressure is lower than the osmotic pressure of intracellular fluid of TRPA1 expressing cells The test substance and the TRPAl -expressing cell are brought into contact with each other (ie, the test substance is brought into contact with the TRPAl -expressing cell in the hypotonic liquid), and the TRPAl is treated with the hypotonic liquid. It is characterized by measuring the physiological event triggered via, and evaluating the TRPA1 activation effect which the said to-be-tested substance has based on the said physiological event.

The method for evaluating a test substance of the present invention comprises bringing the hypotonic fluid, the test substance and the TRPAl -expressing cell into contact, and measuring the physiological event caused by the hypotonic fluid via TRPAl, the physiology It has one big feature in the point that operation of evaluating the TRPA1 activation effect which the above-mentioned test substance has based on specific event is performed. According to the hypotonic solution, TRPA1 is activated. Therefore, according to the method for evaluating a test substance of the present invention, since the above operation is performed, physiology caused via TRPA1 as compared with the case where an isotonic liquid having the same osmotic pressure as the intracellular fluid is used. Events can be measured with high sensitivity. Therefore, according to the method for evaluating a test substance of the present invention, since the above operation is carried out, even if the test substance is in a small amount, it is possible to evaluate with high sensitivity the TRPA1 activating action of the test substance. . Further, according to the method for evaluating a test substance of the present invention, when evaluating the TRPAl activating effect of the test substance, physiological events triggered via TRPAl are measured using the TRPAl -expressing cells. Therefore, evaluation can be performed simultaneously in parallel many times under the same conditions. Therefore, according to the evaluation method of the test substance of the present invention can be evaluated with good reproducibility for TRPA1 activation operation possessed by the test substance.

  Examples of the test substance include, for example, inorganic compounds, organic compounds, plant extracts, microorganism cultures, microorganism extracts and the like, but the present invention is not limited to such examples. The test substance may be used as it is, or may be used by being dissolved in a solvent, if necessary. The solvent is preferably a solvent that does not affect the growth of TRPAl -expressing cells and the expression of physiological functions of TRPAl. Examples of the solvent in which the test substance is dissolved include ethanol, physiological saline, water and the like, but the present invention is not limited to such examples.

  In the present specification, the osmotic pressure [mOsm (ion amount conversion)] is represented by the formula (I) unless otherwise specified.

[Osmotic pressure (mOsm)]
= [Amount of electrolyte ion] + [amount of osmotic pressure regulator] (I)

(In the formula, “the amount of electrolyte ion” is the sum of the number of moles of each of the electrolyte anion and electrolyte cation that constitute the electrolyte that affects the osmotic pressure, and “the amount of osmotic pressure regulator” indicates the number of moles of osmotic pressure regulator
Calculated based on

  Examples of the electrolyte include alkali metal halides such as sodium chloride and potassium chloride, and alkaline earth metal halides such as calcium chloride and magnesium chloride, but the present invention is limited to only such examples. is not. Examples of the osmotic pressure adjusting agent include sugars such as mannitol, glucose, and sucrose, but the present invention is not limited to such examples.

  Examples of the osmotic pressure adjusting agent include sugars such as mannitol, glucose, and sucrose, but the present invention is not limited to such examples.

  In the present specification, the difference between the osmotic pressure of the intracellular fluid (hereinafter also referred to as “osmotic pressure A”) and the difference between the osmotic pressure of the low osmotic pressure liquid (hereinafter also referred to as “osmotic pressure B”) (osmotic pressure A − The osmotic pressure B) is preferably 40 mOsm (908 hPa) from the viewpoint of more reliably generating a physiological event triggered by TRPA1 by a hypotonic fluid and evaluating TRPA1 activation activity easily and accurately. The above, more preferably 80 mOsm (1816 hPa) or more, and from the viewpoint of suppressing the response of cells independent of TRPA1, preferably 200 mOsm (4540 hPa) or less, more preferably 160 mOsm (3632 hPa) or less. The osmotic pressure B varies depending on the osmotic pressure of the intracellular fluid of the TRPA1-expressing cells, and can not be determined indiscriminately. Therefore, the osmotic pressure B may be appropriately set according to the osmotic pressure of the intracellular fluid of the TRPA1-expressing cells. Is preferred. For example, the osmotic pressure of the intracellular fluid of human cells is usually 280 to 320 mOsm (osmotic pressure at 25 ° C: 6356 to 7264 hPa), preferably 312 mOsm (osmotic pressure at 25 ° C: 7082 hPa). Therefore, when TRPAl -expressing cells are derived from human cells, the osmotic pressure of the hypotonic fluid is preferably 100 mOsm (osmotic pressure at 25 ° C: 2270 hPa) from the viewpoint of suppressing cell responses not dependent on TRPAl. Or more, more preferably 140 (osmotic pressure at 25 ° C .: 3178 hPa) mOsm or more, which more reliably generates a physiological event triggered by TRPA1 by the hypotonic liquid, and facilitates evaluation of TRPA1 activation action In order to perform accurately and precisely, it is preferably 260 mOsm (osmotic pressure at 25 ° C: 5902 hPa) or less, more preferably 240 mOsm (osmotic pressure at 25 ° C: 5448 hPa) or less.

  The TRPAl-expressing cells are cells that express the physiological function of TRPAl, which is one of transient receptor potential channels (TRP channels). Physiological functions of TRPA1 include, for example, the ability to transport cations such as calcium ions and sodium ions from outside to inside of cells by stimulation such as chemical stimulation by cold TRP1 agonist, cold stimulation, mechanical stimulation by TRPAl agonist; Although the ability to control the membrane potential in cells and the like can be mentioned, the present invention is not limited to such examples. Examples of the TRPA1 agonist include menthol, ethanol, 1,3-butylene glycol, propylene glycol, alkaline agents (eg, ammonia, monoethanolamine, potassium hydroxide etc.), allyl isothiocyanate, methyl paraben, allicin, icillin, Although hydrogen peroxide, bradykinin, acrolein, a perfume oil component (eg, citral, eugenol, cinnamaldehyde etc.) and the like can be mentioned, the present invention is not limited to such examples.

  The TRPAl-expressing cell may be an endogenous TRPAl-expressing cell expressing endogenous TRPAl, or an exogenous TRPAl-expressing cell in which a nucleic acid encoding TRPAl has been introduced into a host cell in an expressible manner. Good. Among the TRPAl-expressing cells, exogenous TRPAl-expressing cells are preferable, from the viewpoint of being able to accurately measure physiological events caused via TRPAl by a simple operation.

  Examples of the endogenous TRPAl -expressing cells include sensory neurons and inner ear cells, but the present invention is not limited to such examples. The above-mentioned endogenous TRPAl -expressing cell is preferably a human-derived cell from the viewpoint of appropriately evaluating the TRPAl activating action of the test substance in human.

  The exogenous TRPA1-expressing cell may be a cell in which the nucleic acid introduced into the host cell is present as an extrachromosomal element, and the nucleic acid introduced into the host cell is integrated into the chromosome by integration. It may be a cell.

  The exogenous TRPA1-expressing cells are transformed with a host cell by a recombinant vector carrying a nucleic acid encoding TRPA1, for example, by a transformation method such as electroporation, lipofection, transfection, particle gun method, etc. It is obtained by

  The nucleic acid encoding TRPAl may be a nucleic acid encoding human TRPA1 or a nucleic acid encoding TRPAl of another animal. From the viewpoint of appropriately evaluating the TRPAl activating activity of the test substance in human, the nucleic acid encoding TRPAl is preferably a nucleic acid encoding human TRPAl. Examples of the nucleic acid encoding TRPAl include a nucleic acid consisting of the base sequence shown in SEQ ID NO: 1, and the like. The nucleotide sequence shown in SEQ ID NO: 1 is the nucleotide sequence of a nucleic acid encoding human TRPA1 registered in GenBank under Accession No. NM — 007332. The nucleic acid encoding TRPAl is one or several nucleotide residues inside or at the end of the nucleotide sequence of the structural gene of TRPAl if the polypeptide encoded by the nucleic acid expresses the above-mentioned physiological function. Or a mutant nucleic acid having a substitution, deletion or insertion of The nucleic acid encoding the TRPAl is, for example, a hybridization method using a probe prepared based on the nucleotide sequence shown in SEQ ID NO: 1, and two types designed and synthesized based on the nucleotide sequence shown in SEQ ID NO: 1 And a nucleic acid amplification method using a primer pair consisting of oligonucleotide primers of

As a variant type nucleic acid, for example, conditions of Cost to open gap 11, Cost to extend gap 1, expect value 10 and wordsize 11 (default) according to the BLAST algorithm for the base sequence shown in SEQ ID NO: 1 Value) The sequence homology value calculated by aligning the sequence to be evaluated is preferably 80% or more, more preferably 90% or more from the viewpoint of sufficiently exerting the physiological function of TRPA1. A nucleic acid which is a polypeptide consisting of a base sequence and encoded and which at least expresses a function of permeating cations;
(B) In SEQ ID NO: 2, encoding an amino acid sequence having a substitution, deletion or addition of one or several amino acid residues and the encoded polypeptide at least expresses a function of permeating cations A nucleic acid which is a polypeptide,
(C) a polypeptide which hybridizes under stringent conditions with a complementary strand nucleic acid to a nucleic acid consisting of the base sequence shown in SEQ ID NO: 1 under stringent conditions and the encoded polypeptide at least expresses a function of permeating cations Although a certain nucleic acid etc. are mentioned, this invention is not limited only to this illustration.

  In the present specification, "stringent conditions" refers to a polypeptide having a physiological function that is equivalent to or greater than the physiological function of a polypeptide encoded by a nucleic acid consisting of the base sequence shown in SEQ ID NO: 1 From the viewpoint of obtaining a nucleic acid encoding SEQ ID NO: 1, preferably the nucleic acid consisting of the base sequence shown in SEQ ID NO: 1 and the nucleic acid consisting of the base sequence having at least 80% sequence homology to SEQ ID NO: 1 are specifically high. Conditions for hybridization. As the stringent conditions, for example, a nucleic acid consisting of the base sequence shown in SEQ ID NO: 1 and a nucleic acid to be hybridized, a hybridization solution [composition: 6 × SSC (composition: 0.9 M sodium chloride, In 0.09 M sodium citrate, pH 7.0), 0.5 wt% sodium dodecyl sulfate, 5 × Denhardt's solution, 100 μg / ml denatured salmon sperm DNA, 50% by volume formamide], as a more stringent condition at room temperature or higher Incubate for 10 hours at a temperature of 60 ° C. or higher as 42 ° C. or higher, more stringent condition, and then, for example, 2 × SSC, 0.1 × SSC ionic strength condition as more stringent condition, and room temperature Above, as more stringent conditions, 42 ° C or more, Although conditions under which washing is performed at a temperature of 60 ° C. or higher, and the like may be mentioned as the essential conditions, the present invention is not limited to such examples.

  The recombinant vector can be obtained by ligating the above-mentioned TRPA1-encoding nucleic acid with a conventional vector. The vector may be any vector that is easy to prepare, can be efficiently introduced into host cells, and can efficiently express TRPA1 in the host cells. The vector is preferably a vector having a selectable marker gene because selection of exogenous TRPA1 expressing cells from among transformed cells is easy. In addition, the vector may optionally have an expression promoter suitable for expressing TRPA1 in a host cell, upstream of the site for linking TRPA1. Examples of the vector include plasmid vectors such as E. coli plasmid vectors, yeast plasmid vectors, and viral vectors such as retroviral vectors, but the present invention is not limited to such examples.

  As the host cell, any nucleic acid encoding the TRPA1 can be efficiently expressed and culture can be easily performed, and examples thereof include animal cells, bacterial cells, plant cells, insect cells and the like, but the present invention Is not limited to such an example. Among these host cells, preferred are animal cells from the viewpoint of appropriately evaluating the TRPAl activating action of the test substance in human. Examples of the animal cells include human cells such as HEK 293 cells and Hela cells; monkey cells such as COS-7 cells; mouse cells such as CHO cells and NIH 3T3 cells; It is not limited. Among these animal cells, HEK293 cells, CHO cells, COS-7 cells and NIH3T3 cells are preferable, and HEK293 cells are more preferable, from the viewpoint of appropriately evaluating the TRPAl activating action of the test substance in human.

  Selection of exogenous TRPAl -expressing cells from cells after transformation can be carried out, for example, by culturing in a selection medium corresponding to the selection marker gene, etc. if the recombinant vector used has the selection marker gene . In addition, confirmation that the obtained cell is an exogenous TRPA1-expressing cell is carried out, for example, by bringing the cell into contact with 1 to 10 mM parahydroxybenzoic acid methyl ester, and after the contact according to the method for measuring intracellular calcium ion concentration described later. It can be carried out by measuring the intracellular calcium ion concentration of the cells of When the cell is an exogenous TRPAl -expressing cell, the intracellular calcium ion concentration of the cell after contact is higher than the intracellular calcium ion concentration of the cell not in contact with parahydroxybenzoic acid methyl ester.

  The hypotonic solution can be obtained, for example, by mixing purified water, an electrolyte, and an osmotic pressure regulator which is not related to ionic strength.

  Since the amount of the test substance varies depending on the type of the test substance, the number of TRP1-expressing cells, etc. upon contact of the test substance with the TRP1-expressing cells, the type of the test substance can not be determined. It is desirable to determine appropriately according to the number of expressing cells and the like.

The number of TRPAl -expressing cells per 1 cm ² when contacting the test substance with TRPAl -expressing cells is 1 × 10 ³ cells or more, more preferably 1 × 10 ⁴ cells or more from the viewpoint of the reliability of test data. From the viewpoint of sufficiently exerting the physiological function of TRPA1 in TRPAl -expressing cells, it is 1 × 10 ⁶ cells or less, more preferably 5 × 10 ⁵ cells or less.

  As the physiological event, for example, a change in calcium ion concentration in TRPAl -expressing cells caused by the hypotonic fluid via TRPA1 before and after the contact with the test substance, and the hypotonic pressure before and after the contact with the test substance The changes in the current in TRPAl -expressing cells caused by TRP1 by the fluid, combinations thereof, and the like can be mentioned, but the present invention is not limited to such examples.

  In the present invention, from the viewpoint of being able to measure with a simple operation and with high sensitivity, the physiological event is a cell that expresses TRPA1 caused by the hypotonic fluid via TRPA1 before and after contact with the test substance. It is preferable that it is a change of the calcium ion concentration inside.

  The TRPAl activating activity of the test substance is, for example, when the calcium ion concentration in TRPAl -expressing cells when the hypotonic liquid and the test substance are brought into contact, and when the hypotonic liquid is brought into contact Difference with calcium ion concentration in TRPA1-expressing cells; TRPA1 mediated potential in TRPAl-expressing cells when contacting with the hypotonic fluid and the test substance, and TRPA1 when contacting with the hypotonic fluid It is measured by examining differences in potentials mediated by TRPAl in expressing cells, combinations thereof and the like.

When using a change in calcium ion concentration in TRPAl -expressing cells caused by TRPPA by the hypotonic fluid before and after contact with the test substance as the physiological event, TRPAl expression in contact with the hypotonic fluid Intracellularly, the influx of calcium ion from TRPA1-expressing extracellular cells to TRPA1-expressing cells via TRPA1 compared to TRPA1-expressing cells in contact with iso-osmotic solution having the same osmotic pressure as the intracellular fluid osmotic pressure. Increases. Therefore, in the TRPAl -expressing cells contacted with the hypotonic fluid and the test substance, the TRPAl -expressing cells via TRPAl with the test substance as compared to the TRPAl -expressing cells contacted with the iso-osmotic fluid and the test substance The influx of calcium ions from the outside into TRPAl -expressing cells relatively increases, and the range of increase in intracellular calcium ion concentration increases. Therefore, the intracellular calcium ion concentration can be measured with higher sensitivity by the contact between the hypotonic fluid, the test substance, and the TRPAl -expressing cells. In this case, for example, as a criterion for the test substance to have TRPAl activating activity,
(I) The calcium ion concentration in the TRPA1-expressing cell when contacted with the hypotonic fluid and the test substance is based on the calcium ion concentration in the TRPA1-expressing cell when contacted with the hypotonic fluid Too big,
(II) When the iso-osmotic fluid having the same osmotic pressure as the intracellular fluid and the test substance are brought into contact with the calcium ion concentration in the TRPAl -expressing cell when the hypotonic fluid and the test substance are contacted The difference between the calcium ion concentration in the TRPA1-expressing cells and the calcium ion concentration in the TRPA1-expressing cells when the hypotonic solution and the known agonist of the TRPA1 are in contact with each other, and It may be equal to or greater than the difference from the intracellular calcium ion concentration of TRPAl -expressing cells when contacted with a known agonist.

  The calcium ion concentration is, for example, introducing a calcium indicator that specifically binds to calcium ion into TRPAl-expressing cells, causing the calcium indicator to bind to calcium ion in the TRPAl-expressing cell, and combining the calcium indicator with calcium ion It can measure by the method of quantifying etc.

  The calcium indicator is a reagent capable of detecting a change in optical properties before and after binding to a calcium ion, since the calcium indicator bound to a calcium ion can be quantified by a simple operation. preferable. Examples of the change in the optical properties include a change in fluorescence intensity, a change in absorbance, and the like, but the present invention is not limited to such examples. Examples of the calcium indicator include, for example, a fluorescent calcium indicator whose fluorescence intensity changes before and after binding to a calcium ion, but the present invention is not limited to such an example. Among the calcium indicators, fluorescent calcium whose fluorescence intensity changes before and after binding to calcium ions, from the viewpoint of easy measurement and easy differentiation from the dynamics of contaminating substances present in TRPAl -expressing cells. Indicators are preferred. Examples of the fluorescent calcium indicator include FURA 2, FURA 2-AM, Fluo-3 and the like, but the present invention is not limited to such examples. The fluorescent calcium indicator may have one excitation wavelength or may have two or more excitation wavelengths. When the calcium indicator is the fluorescent calcium indicator, the fluorescent magnesium indicator is easy to measure the fluorescence intensity, and can further enhance the accuracy of the evaluation of the TRPA1 activating activity of the test substance, so two types can be used. Preferred are fluorescent magnesium indicators having an excitation wavelength of

  In the case of using a fluorescent magnesium indicator having one type of excitation wavelength when measuring the calcium ion concentration, the TRPA1 activating action can be evaluated based on the fluorescence intensity at the excitation wavelength. In addition, in the case of using a fluorescent magnesium indicator having two or more types of excitation wavelengths in the measurement of calcium ion concentration, from the viewpoint of enhancing the accuracy of evaluation of the TRPA1 activation effect of the test substance, from the two or more types of excitation wavelengths The selected two excitation wavelengths (the first excitation wavelength and the second excitation wavelength) are selected, and the TRPA1 activating action is based on the fluorescence intensity ratio calculated from the fluorescence intensity at each of the first excitation wavelength and the second excitation wavelength. Can be evaluated.

  In the case of using, for example, FURA 2-AM, which is a fluorescent calcium indicator having two types of excitation wavelengths, in the measurement of calcium ion concentration, it is 340 nm as the fluorescence intensity at the first excitation wavelength (hereinafter also referred to as "first fluorescence intensity") The fluorescence intensity at 380 nm can be used as the fluorescence intensity at and the fluorescence intensity at the second excitation wavelength (hereinafter also referred to as “second fluorescence intensity”). In this case, the fluorescence intensity ratio is, for example, the formula (II):

Fluorescence intensity ratio = 1st fluorescence intensity / 2nd fluorescence intensity (II)

It can be determined according to In this case, for example, as a criterion for the test substance to have TRPAl activating activity,
(I) The fluorescence intensity ratio (hereinafter referred to as "fluorescence intensity (A)") when the hypotonic solution and the test substance are brought into contact with each other is the fluorescence intensity ratio when the hypotonic solution is brought into contact Hereinafter referred to as "fluorescence intensity (B)"
(Ii) The fluorescence intensity ratio (A) when the hypoosmotic liquid and the test substance are brought into contact, and the fluorescence intensity ratio when the iso-osmotic liquid and the test substance are brought into contact [hereinafter referred to as "fluorescence intensity (C) ”[fluorescence intensity ratio (A) −fluorescence intensity ratio (C)] is the fluorescence intensity ratio when the hypotonic solution and TRPA1 known agonist are in contact with each other [hereinafter, Difference between fluorescence intensity ratio [referred to as “fluorescence intensity (D)”] and fluorescence intensity ratio [hereinafter referred to as “fluorescence intensity (E)”] when said iso-osmotic solution and a known agonist of TRPA1 are brought into contact [fluorescence intensity The same as or larger than the ratio (D) −fluorescence intensity ratio (E)], and the like.

  When a change in current in TRPAl-expressing cells caused via TRPAP1 is used as the physiological event, for example, the hypoosmotic fluid may be used as a criterion for TRP1 activating activity of the test substance. The current under constant potential in TRPAl -expressing cells when contacted with a test substance [hereinafter referred to as "current (A)"] is constant in TRPAl -expressing cells when contacted with said hypotonic liquid It may be larger than the current under potential [hereinafter referred to as “current (B)”]. Examples of the method of measuring the change in the current include a patch clamp method and the like, but the present invention is not limited to such an example.

  In addition, prior to bringing the hypoosmotic fluid, the test substance, and the TRPAl -expressing cell into contact, in order to maintain the TRPAl -expressing cell in a state suitable for expressing a physiological function, as necessary, TRPAl The expressing cells may be cultured for any time. The method for culturing TRPAl -expressing cells may be any method according to the type of cells used, and examples include methods such as monolayer static culture, suspension culture, rotation culture, and three-dimensional carrier culture. . In addition, culture conditions such as culture temperature, culture time, pH of culture solution, carbon dioxide concentration and the like are appropriately set according to the host cell to be used. When the host cell used for TRPAl-expressing cells is HEK293 cells, TRPAl-expressing cells are usually 36 ° C to 38 ° C, preferably 36.5 ° C, in 5% by volume of carbon dioxide, from the viewpoint of well growing the cells. Incubate at ~ 37.5 ° C. As a medium used for culturing TRPAl-expressing cells, components suitable for growing TRPAl-expressing cells, such as glucose, amino acids, peptones, vitamins, cell growth promoting factors (eg, cell growth factors, hormones, binding proteins, etc. Any medium may be used as long as it contains, for example, cell adhesion factor, lipids, serum, such as fetal calf serum, calcium chloride, magnesium chloride, etc. The medium may be a conventional basic medium supplemented with the components, or may be a commercially available medium. The basic medium is not particularly limited, and examples thereof include MEM medium, DMEM medium, RPMI 1640 medium and the like. The culture medium used to culture TRPAl -expressing cells can not be determined indiscriminately because the culture conditions differ depending on the type of TRPAl -expressing cells. For example, when the TRPAl -expressing cell to be used is a cell obtained from HEK 293 cells, a 10 mass% fetal calf serum-containing DMEM medium or the like is used as a medium to be used for culturing TRPAl -expressing cells.

  It is considered that the test substance found to be an activator of TRPA1 by being subjected to the method for evaluating a test substance of the present invention is a substance that causes sensory irritation to skin etc. via TRPA1. Therefore, according to the method of evaluating a test substance of the present invention, whether or not the compound or the like causes sensory stimulation by using, for example, a compound used as a component of an external preparation such as a cosmetic agent as a test substance It can be examined. This also makes it possible to exclude in advance substances that cause sensory stimulation. In addition, according to the method for evaluating a test substance of the present invention, screening for substances that cause sensory stimulation, pharmacological evaluation and the like can be performed. Therefore, the evaluation method of the test substance of the present invention is expected to be used for the development of external preparations such as cosmetics suitable for human beings who easily cause skin problems.

  The present invention will be described in more detail by the following examples, but the present invention is not limited to these examples. In addition, the osmotic pressure (ion amount conversion) of a bath solution is a value calculated according to Formula (I) below.

(Production Example 1)
A cDNA encoding human TRPA1 [polynucleotide of position 63 to 3888 of the base sequence shown in SEQ ID NO: 1 (GenBank accession number: NM — 007332)] is used as a mammalian cell vector [Invitrogen, trade name: pcDNA3]. 1 (+)] was inserted to obtain a human TRPA1 expression vector. A mixture I was obtained by mixing 1 μg of the obtained human TRPA1 expression vector with 6 μL of a gene transfer reagent (manufactured by Invitrogen, trade name: PLUS Reagent (plus reagent), catalog number: 11514-015). 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 medium for reducing the amount of serum used (manufactured by Invitrogen, trade name: OPTI-MEM (registered trademark) It mixed with 200 microliters of (trademark) I Reduced-Serum Medium (catalog number: 11058021)], and obtained the mixture II.

In addition, 5 × 10 ⁵ HEK293 cells are cultured to 70% confluency in DMEM medium containing 10 mass% FBS on a 35 mm diameter petri dish maintained at 37 ° C. in a 5 vol% carbon dioxide atmosphere. did.

  The human TRPA1 expression vector was introduced into HEK293 cells by adding the mixture I and the mixture II to the obtained cell culture, to obtain TRPA1 expressing cells.

(Production Example 2)
Sodium chloride, mannitol, potassium chloride, magnesium chloride, calcium chloride, glucose and 2- [4- (2-hydroxyethyl) piperazin-1-yl] ethanesulfonic acid buffer (hereinafter also referred to as “HEPES buffer”) pH 7.4) is mixed to the following composition: 60 mM sodium chloride, 160 mM mannitol, 5 mM potassium chloride, 2 mM magnesium chloride, 2 mM calcium chloride, 10 mM glucose and 10 mM HEPES buffer, and the same as the osmotic pressure of the intracellular fluid A bath solution having an osmotic pressure [osmotic pressure (ion amount conversion): 312 mOsm (osmotic pressure at 25 ° C .: 7082 hPa)] was obtained.

(Production Example 3)
In Production Example 2, the same procedure as in Production Example 2 is carried out except that the concentration of mannitol is set to 80 mM instead of setting the concentration of mannitol to 160 mM, and the osmotic pressure of the intracellular fluid is 80 mOsm ( A bath solution [osmotic pressure (ion amount conversion): 232 mOsm (osmotic pressure at 25 ° C .: 5266 hPa)] which is 1816 hPa) was obtained.

(Production Example 4)
In Production Example 2, the same procedure as in Production Example 2 is carried out except that the concentration of mannitol is set to 40 mM instead of setting the concentration of mannitol to 160 mM, and the difference with the osmotic pressure of the intracellular fluid is 120 mOsm ( A bath solution [osmotic pressure (ion amount conversion): 192 mOsm (osmotic pressure at 25 ° C .: 4358 hPa)] which is 2724 hPa) was obtained.

(Production Example 5)
In Production Example 2, the same procedure as in Production Example 2 is performed except that the concentration of mannitol is set to 0 mM instead of setting the concentration of mannitol to 160 mM, and the difference with the osmotic pressure of the intracellular fluid is 160 mOsm ( A bath solution [osmotic pressure (ion amount conversion): 152 mOsm (osmotic pressure at 25 ° C .: 3450 hPa)] having 3632 hPa) was obtained.

(Test Example 1)
(1) Evaluation of relationship between hypotonic stimulation and cell area increase rate TRPA1-expressing cells (5 × 10 ⁴ cells) obtained in Production Example 1 were added to a circulating chamber [Warner Instruments, trade name: RC-26 I put it in]. Thereafter, a standard bath solution having an osmotic pressure (in terms of ion amount): 312 mOsm (osmotic pressure at 25 ° C .: 7082 hPa) in the chamber [composition: 140 mM sodium chloride, 5 mM potassium chloride, 2 mM magnesium chloride, 2 mM Washed with calcium chloride, 10 mM glucose and 10 mM HEPES buffer (pH 7.4). The washed TRPA1 expressing cells were photographed with a digital camera. The obtained image and the image processing software Image J [US National Institutes of Health web page http: // imagej. nih. cell surface area (hereinafter also referred to as “cell area A ₀ ”) when contacted with an iso-osmotic fluid having the same osmotic pressure as the intracellular fluid.

Next, the bath solution obtained in Production Examples 2 to 5 was added to the chamber containing the washed TRPAl -expressing cells. The cells after addition of the bath solution were photographed with a digital camera. The area of the cells (hereinafter also referred to as “cell area A ₁ ”) when they are brought into contact with a low osmotic pressure fluid having an osmotic pressure lower than that of the intracellular fluid using the obtained image and the image processing software Image J I asked for.

Next, using cell area A ₀ and cell area A ₁ , formula (III):
Cell area increase rate = (cell area A ₁ -cell area A ₀ ) / cell area A ₀ (III)

The cell area increase rate was determined based on.

  The results of examining the relationship between the difference between the osmotic pressure of the intracellular fluid and the cell area increase rate in Test Example 1 are shown in FIG.

  From the results shown in FIG. 1, it can be seen that the cell area increase rate is higher as the difference with the osmotic pressure of the intracellular fluid is larger.

(2) Evaluation of relationship between hypotonic stimulation and degree of activation of TRPA1 in TRPA1-expressing cells Standard bath solution [composition: 140 mM sodium chloride, 5 mM potassium chloride, 2 mM magnesium chloride, TRPA1-expressing cells obtained in Production Example 1 The cells were washed with 2 mM calcium chloride, 10 mM glucose and 10 mM HEPES buffer (pH 7.4). Next, TRPAl -expressing cells were exposed to the bath solution obtained in Production Example 2, Production Example 3, Production Example 4 or Production Example 5. One minute after exposure, the tip of an electrode containing solution B (composition: 140 mM potassium chloride, 5 mM glycol ether diamine tetraacetic acid and 10 mM HEPES buffer (pH 7.4)) is brought into contact with the TRPAl -expressing cells, and current recording software The change in current when the voltage was fixed at 60 mV was measured using [Molecular Device, Inc., trade name: pCLAMP10] and a current recording device [Molecular Device, Inc., trade name: Axopatch 200B Amplifier].

  Using the calculated open probability (Po) and the number of TRPAl in TRPAl expressing cells, Formula (IV):

TRPA1 activation degree (NPo)
= Number of TRPA1 in TRPA1-expressing cells (N) × opening probability (Po) (IV)

The degree of TRPA1 activation was determined according to

  The results of examining the relationship between the difference between the osmotic pressure of the intracellular fluid and the degree of activation of TRPA1 in Test Example 1 are shown in FIG.

  From the results shown in FIG. 2, it can be seen that the degree of TRPA1 activation (NPo) increases as the difference with the osmotic pressure of the intracellular fluid increases. From these results, it is understood that a hypotonic fluid having an osmotic pressure lower than that of the intracellular fluid more activates TRPA1 in the cells expressing TRPA1. Therefore, these results suggest that, in humans, hypotensive fluid having an osmotic pressure lower than that of the intracellular fluid enhances the activity of TRPA1 to cause pain. In addition, from these results, for example, when a hypotonic solution comes into contact with human eyes, the eye irritation perceived by humans is TRPA1 with a hypotonic solution having an osmotic pressure lower than the osmotic pressure of the intracellular fluid. It is suggested that this is caused by the enhanced activity of

(3) Evaluation of relationship between cell area increase rate and change in activation degree of TRPA1 In Test Example 1 (1), various methods are used instead of using the bus solution obtained in Production Example 2, Production Example 3 or Production Example 5. The cell area increase rate was determined in the same manner as in Test Example 1 (1) except that a bath solution having an osmotic pressure of was used. In addition, in Test Example 1 (2), the same as Test Example 1 (2) except that the bus solutions having various osmotic pressures are used instead of using the bus solutions obtained in Production Examples 2 to 5. The following procedure was performed to determine the degree of TRPA1 activation (NPo). Next, cell area increase rate and TRPA1 activation degree (NPo) when each bath solution is used, x axis is cell area increase rate, y axis is TRPA1 activation degree (NPo) 2 Plotted on dimensional coordinates.

  Also, in the above, instead of using the bath solution having various osmotic pressure, the same operation as the above except using the 1 μM TRPAl agonist (allyl isothiocyanate) and using the bath solution having various osmotic pressure To determine the cell area increase rate and the degree of TRPA1 activation (NPo). Next, cell area increase rate and TRPA1 activation degree (NPo) when each bath solution is used, x axis is cell area increase rate, y axis is TRPA1 activation degree (NPo) 2 Plotted on dimensional coordinates.

  The results of examining the relationship between the cell area increase rate when the hypotonic fluid was contacted and the TRPA1 activation degree (NPo) when the hypotonic fluid was contacted in Test Example 1 are shown in FIG. 3 (A). FIG. 3 (B) shows the results of examining the relationship between the cell area increase rate in the presence of TRPAl agonist and the degree of TRPAl activation (NPo) in the presence of TRPAl agonist. In the figure, R shows a correlation coefficient.

  From the results shown in FIGS. 3 (A) and 3 (B), it can be seen that the cell area increase rate when the hypotonic fluid is in contact and the degree of TRPA1 activation (NPo) when the hypotonic fluid is in contact The correlation coefficient R is found to be 0.51 both between the increase in cell area between and in the presence of the TRPAl agonist and the degree of TRPAl activation (NPo) in the presence of the TRPAl agonist. Therefore, based on these results, it is understood that in the presence of a TRPA1 agonist, the cell area increase rate when the hypotonic solution is in contact and the TRPA1 activation degree (NPo) when the hypotonic solution is in contact. It can be seen that there is a positive correlation as in the case between the cell area elevation rate in and the degree of TRPAl activation (NPo) in the presence of TRPAl agonist. From these results, it can be seen that cell expansion by hypotonic fluid is associated with an increase in intracellular calcium concentration. Therefore, these results suggest that expansion of cells by hypotonic pressure increases the activity of TRPA1.

(Production Example 6)
Allyl isothiocyanate, sodium chloride, mannitol, potassium chloride, magnesium chloride, calcium chloride, glucose and HEPES buffer (pH 7.4), the following composition: 1 μM allyl isothiocyanate, 60 mM sodium chloride, 160 mM mannitol, 5 mM potassium chloride, 2 mM Mix so that it becomes magnesium chloride, 2mM calcium chloride, 10mM glucose and 10mM HEPES buffer solution, and bath solution with the same osmotic pressure as intracellular liquid [Osmotic pressure (ion amount conversion): 312mOsm (at 25 ° C] Osmotic pressure: 7082 hPa)] was obtained.

(Production Example 7)
In Production Example 6, the same procedure as in Production Example 6 is carried out except that the concentration of mannitol is set to 80 mM instead of setting the concentration of mannitol to 160 mM, and the difference between the osmotic pressure of the intracellular fluid and the osmotic pressure is 80 mOsm ( A bath solution [osmotic pressure (ion amount conversion): 232 mOsm (osmotic pressure at 25 ° C .: 5266 hPa)] which is 1816 hPa) was obtained.

Production Example 8
In Production Example 6, the same procedure as in Production Example 6 is carried out except that the concentration of mannitol is set to 40 mM instead of setting the concentration of mannitol to 160 mM, and the difference with the osmotic pressure of the intracellular fluid is 120 mOsm ( A bath solution [osmotic pressure (ion amount conversion): 192 mOsm (osmotic pressure at 25 ° C .: 4358 hPa)] which is 2724 hPa) was obtained.

Production Example 9
In Production Example 6, the same procedure as in Production Example 6 is performed except that the concentration of mannitol is set to 0 mM instead of setting the concentration of mannitol to 160 mM, and the difference with the osmotic pressure of the intracellular fluid is 160 mOsm ( A bath solution [osmotic pressure (ion amount conversion): 152 mOsm (osmotic pressure at 25 ° C .: 3450 hPa)] which is 3632 hPa) was obtained.

Example 1
The TRPA1-expressing cells obtained in Production Example 1 were treated at 60 ° C. in DMEM medium containing 10% by weight of fetal calf serum containing FURA 2-AM (manufactured by Invitrogen) as a reagent for measuring intracellular calcium ions at a final concentration of 5 μM. By incubation for a minute, FURA 2-AM was introduced into the TRPA1-expressing cells to obtain FURA 2-AM-introduced TRPA1-expressing cells.

  The obtained FURA 2-AM-introduced TRPA1-expressing cells were placed in the chambers of a fluorescence measuring apparatus with a circulating constant temperature chamber (manufactured by Hamamatsu Photonics K.K., trade name: ARGUS-50). Then, FURA 2-AM introduced TRPA1 expressing cells in the chamber were treated with solution A [composition: 140 mM sodium chloride, 5 mM potassium chloride, 2 mM magnesium chloride, 2 mM calcium chloride, 10 mM glucose and 10 mM HEPES buffer (pH 7.4)]. It was washed.

  Next, the bath solution obtained in Production Examples 2 to 9 was added to a chamber containing FURA 2-AM-introduced TRPA1-expressing cells after washing. The osmotic pressure (ion amount conversion) of the mixture containing the bath solution obtained in Production Example 2 and TRPA1 expressing cells is 312 mOsm (osmotic pressure at 25 ° C .: 7082 hPa), and the bath solution obtained in Production Example 3 The osmotic pressure (ion amount conversion) of the mixture containing TRPA1-expressing cells is 232 mOsm (osmotic pressure at 25 ° C .: 5266 hPa), and the osmotic pressure of the mixture containing the bath solution obtained in Production Example 4 and TRPA1-expressing cells (ion Amount conversion) is 192 mOsm (osmotic pressure at 25 ° C: 4358 hPa), and the osmotic pressure (ion amount conversion) of the mixture containing the bath solution obtained in Production Example 5 and TRPA1 expressing cells is 152 mOsm (osmotic pressure at 25 ° C.) : 3450 hPa), a mixture containing the bath solution obtained in Production Example 6 and TRPA1 expressing cells The osmotic pressure (ion amount conversion) of the mixture is 312 mOsm (osmotic pressure at 25.degree. C .: 7082 hPa), and the osmotic pressure (ion amount conversion) of the mixture containing the bath solution obtained in Production Example 7 and TRPA1 expressing cells is Osmotic pressure at 5 ° C .: 5266 hPa), Osmotic pressure (in terms of ion amount) of the mixture containing the bath solution obtained in Production Example 8 and TRPAl-expressing cells is 192 mOsm (osmotic pressure at 25 ° C .: 4358 hPa), Production Example 9 The osmotic pressure (ion amount conversion) of the mixture containing the bath solution obtained in and the cells containing TRPA1 was 152 mOsm (osmotic pressure at 25 ° C .: 3450 hPa).

Thereafter, in the chamber, the intensity of fluorescence based on FURA 2-AM introduced into TRPAl-expressing cells at an excitation wavelength of 340 nm and bound to intracellular calcium ions (hereinafter referred to as "fluorescence intensity _{340 nm} ") and TRPAl at an excitation wavelength of 380 nm The intensity of the fluorescence based on FURA 2-AM introduced into the expressing cells (hereinafter referred to as "fluorescence intensity _{380 nm} ") was measured.

Using the measured fluorescence intensity _{340 nm} and fluorescence intensity _{380 nm} , the formula (V):
Fluorescence intensity ratio = fluorescence intensity _{340 nm} / fluorescence intensity _{380 nm} (V)
The fluorescence intensity ratio was determined based on.

  FIG. 4 shows the result of examining the relationship between the difference between the osmotic pressure of the intracellular fluid and the fluorescence intensity ratio in Example 1. In the figure, the black bars indicate the fluorescence intensity ratio when using a bath solution (bath solutions of Preparation Examples 6 to 9) containing allyl isothiocyanate which is a known TRPA1 agonist as a test substance, and the white bars contain allyl isothiocyanate. The fluorescence intensity ratio at the time of using the bus solution (the bus solution of manufacture examples 1-5) is shown.

  From the results shown in FIG. 4, it is found that the lower the osmotic pressure of the bath solution, the bath solution having the same osmotic pressure as the intracellular fluid [osmotic pressure (ion amount conversion): 312 mOsm (osmotic pressure at 25 ° C .: 7082 hPa)]. It can be seen that the fluorescence intensity ratio is larger than the fluorescence intensity ratio when using. In addition, it can be seen that the fluorescence intensity ratio when using a bath solution containing allyl isothiocyanate which is a TRPA1 agonist is significantly higher than the fluorescence intensity ratio when using a bath solution not containing allyl isothiocyanate. From these results, it is understood that the degree of calcium ion influx, which is a physiological event when a test substance is brought into contact with a test substance and a cell with TRPA1 expression, which has an osmotic pressure lower than that of the intracellular fluid, Compared with the degree of calcium influx, which is a physiological event when the test substance is brought into contact with the TRPA1-expressing cells under an isotonic pressure solution (bath solution of Production Example 2) having the same osmotic pressure as that of the solution. Know that

Therefore, based on these results, the hypotensive fluid having an osmotic pressure lower than the osmotic pressure of the intracellular fluid is brought into contact with the test substance and the TRPAl -expressing cells, and the physiology caused by the hypotonic fluid via TRPAl Chemical substances are measured, and based on the physiological events, by evaluating the TRPAl activating effect of the test substance, the TRP1 activating effect of the test substance can be highly sensitive even if the test substance is in a small amount It can be seen that it can be evaluated. In addition, when evaluating the TRPA1 activation activity of the test substance, when measuring a physiological event triggered via TRPA1 using the TRPAl-expressing cells, evaluation is performed simultaneously under the same conditions many times simultaneously from it can be performed, by performing the operation, it is understood that it is possible to evaluate good reproducibility for TRPA1 activation operation possessed by the test substance.

  As described above, it is considered that the test substance found to be an activator of TRPA1 by being subjected to the method for evaluating a test substance of the present invention is a substance that causes sensory irritation to skin etc. via TRPA1. Be Therefore, according to the method of evaluating a test substance of the present invention, whether or not the compound or the like causes sensory stimulation by using, for example, a compound used as a component of an external preparation such as a cosmetic agent as a test substance It can be examined. In addition, according to the method for evaluating a test substance of the present invention, screening for substances that cause sensory stimulation, pharmacological evaluation and the like can be performed. Therefore, the evaluation method of the test substance of the present invention is expected to be used for the development of external preparations such as cosmetics suitable for human beings who easily cause skin problems.

Claims (3)

A method of evaluating a test substance for evaluating the activity of activating TRPA1 possessed by a test substance, comprising: a hypotonic fluid having an osmotic pressure lower than the osmotic pressure of the intracellular fluid of TRPA1-expressing cells, the test substance, and the TRPA1-expressing cells The physiological events caused by the hypoosmotic fluid are measured by the hypotonic fluid, and when evaluating the TRPA1 activating action of the test substance based on the physiological events, the physiological events. The change in calcium ion concentration in TRPAl -expressing cells caused by TRPAP1 by the hypotonic fluid before and after contact with the test substance, as triggered by TRPRP1 by the hypotonic fluid before and after contact with the test substance change in current TRPA1-expressing cell or subject, which comprises using a combination of these Evaluation method of quality.   The method according to claim 1, wherein the hypotonic fluid has an osmotic pressure 40 to 200 mOsm lower than that of the intracellular fluid.   The method according to claim 1 or 2, wherein the physiological event is a change in calcium ion concentration in the TRPAl -expressing cells triggered by TRPAl by the hypotonic fluid before and after contact with the test substance.