Method for evaluating test samples

Abstract

To provide a method for evaluating a test sample based on a new index, which can easily evaluate an anti-inflammatory effect of the test sample.SOLUTION: A test sample evaluation method for evaluating an anti-inflammatory effect of a test sample, comprises contacting a test sample with a macrophage, measuring a physiological event caused by TRPV4 activation of the macrophage, and evaluating the anti-inflammatory effect of the test sample based on the physiological event.SELECTED DRAWING: Figure 3

Description

[Technical field]

[0001] The present invention relates to a method for evaluating a test sample for evaluating an anti-inflammatory effect or an anti-cell death effect of the test sample. The present invention also relates to an agent that suppresses the production amount of NLRP3 (NLR family, pyrin domain cantaining 3) and / or Caspase-1, and an anti-inflammatory agent. [Background technology]

[0002] TRP channels are a family of proteins involved in sensory reception. They are known to exist widely from yeast to mammals, and in humans, a total of 29 genes are known to exist, belonging to six subfamilies: TRPC, TRPV, TPPM, TRPA, TRPP, and TRPML.

[0003] TRPV4, one of the TRP channels, was initially identified as an osmotic pressure sensor, but it has since been revealed that it is activated by temperature (25-34°C) and chemicals. TRPV4 is also expressed in various tissues, such as sensory nerves, hypothalamus, skin, kidneys, lungs, and inner ear, and is known to play an important role in transmitting pain caused by osmotic and mechanical stimuli, as well as inflammatory pain.

[0004] The inventors previously conducted research focusing on TRPV4 in monocytes and discovered that activating TRPV4 in monocytes with a TRPV4 activator (GSK1016790A) reduces the production of various cytokines (e.g., inflammatory cytokines, chemokines, etc.) and cell adhesion molecules in response to stimulation with various adjuvants. Based on this discovery, they filed an application for a method for evaluating the anti-inflammatory effect of a test sample targeting monocytes (Patent Document 1). [Prior art documents] [Patent documents]

[0005] [Patent Document 1] JP 2020-51835 A Summary of the Invention [Problem to be solved by the invention]

[0006] The above-mentioned technology is excellent, but since monocytes are present in the blood, it is technically significant to develop an evaluation method based on a new indicator that takes into account their localization in the skin, etc.

[0007] Therefore, one aspect of the present invention aims to provide a method for evaluating a test sample based on a new index, which enables easy evaluation of the anti-inflammatory effect, etc., of a test sample, and a technology for using the same. [Means for solving the problem]

[0008] The present inventors have conducted intensive research to solve the above problems, and have found for the first time that the anti-inflammatory effect of a test sample can be easily evaluated by using as an index a physiological event caused by the activation of TRPV4 in macrophages. Based on these findings, the present invention has been completed. That is, one embodiment of the present invention includes the following configuration. <1> A method for evaluating an anti-inflammatory effect of a test sample, comprising the steps of: A method for evaluating a test sample, comprising contacting the test sample with a macrophage, measuring a physiological event caused by activation of TRPV4 in the macrophage, and evaluating the anti-inflammatory effect of the test sample based on the physiological event. <2> A method for evaluating an anti-cell death effect of a test sample, comprising the steps of: A method for evaluating a test sample, comprising contacting the test sample with a macrophage, measuring a physiological event caused by activation of TRPV4 in the macrophage, and evaluating the anti-cell death effect of the test sample based on the physiological event. <3> The macrophages are GM-CSF macrophages and / or M-CSF macrophages; <1> or <2> A method for evaluating a test sample according to claim 1. <4> The physiological event caused by the activation of TRPV4 is at least one selected from the group consisting of suppression of the production amount of NLRP3, suppression of the production amount of Caspase-1, and suppression of the production amount of cytokines. <1> ~ <3> 2. The method for evaluating a test sample according to any one of the preceding claims. <5> An agent that contains as an active ingredient a substance that activates TRPV4 in macrophages and suppresses the production of NLRP3 and / or Caspase-1. <6> An anti-inflammatory agent that contains as its active ingredient a substance that activates TRPV4 in macrophages. Effect of the Invention

[0009] According to one aspect of the present invention, there is provided a method for evaluating a test sample, which can easily evaluate the anti-inflammatory effect of a test sample. [Brief description of the drawings]

[0010] [Figure 1] FIG. 1 shows the expression of TRPV4 mRNA in monocytes and macrophages. [Diagram 2] 1 is a graph showing TRPV4 mRNA expression in monocytes and macrophages. [Diagram 3] FIG. 1 shows the amount of IL-1β produced after TRPV4 activation in macrophages. [Figure 4] FIG. 1 shows the amount of IL-1β mRNA after TRPV4 activation in macrophages. [Diagram 5] FIG. 1 shows the amount of NLRP3 produced after TRPV4 activation in macrophages. [Figure 6] FIG. 1 shows the amount of Caspase-1 produced after TRPV4 activation in macrophages. [Figure 7]FIG. 1 shows TRPV4 mRNA expression and macrophage staining in skin tissues from patients with atopic dermatitis or healthy individuals. [Figure 8] FIG. 1 shows TRPV4 mRNA expression and macrophage staining in skin tissues (epidermis and dermis) from patients with atopic dermatitis or skin tissues (epidermis and dermis) from healthy individuals. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0011] An embodiment of the present invention will be described below, but the present invention is not limited thereto. The present invention is not limited to each of the configurations described below, and various modifications are possible within the scope of the claims. The technical scope of the present invention also includes embodiments obtained by appropriately combining the technical means disclosed in different embodiments. In this specification, unless otherwise specified, "A to B" representing a numerical range means "A or more, B or less."

[0012] 1. Overview The method for evaluating a test sample according to one embodiment of the present invention is (1) a method for evaluating an anti-inflammatory effect of a test sample (hereinafter also referred to as "the present evaluation method (1)"), or (2) a method for evaluating an anti-cell death (anti-apoptosis) effect of a test sample (hereinafter also referred to as "the present evaluation method (2)"), which is characterized in that the test sample is brought into contact with macrophages, a physiological event caused by activation of TRPV4 in the macrophages is measured, and the anti-inflammatory effect of the test sample is evaluated based on the physiological event. Hereinafter, "the present evaluation method (1)" and "the present evaluation method (2)" may be collectively referred to as "the present evaluation method".

[0013] As mentioned above, the present inventors have previously reported an evaluation method for evaluating the anti-inflammatory effect of a test sample using as an index a physiological event caused by the activation of TRPV4 in monocytes. Although the above technology is excellent, since monocytes exist in blood, it was considered that there is a technical significance in developing an evaluation method based on a new index that takes into account the localization of monocytes in the skin, etc.

[0014] The present inventors have succeeded in obtaining the following findings through extensive investigations into evaluation methods based on new indices. · GM-CSF and M-CSF macrophages, like monocytes, express TRPV4. -The macrophages have higher expression of TRPV4 than monocytes. When TRPV4 in the macrophages was activated by a TRPV4 activator (GSK1016790A), the amount of IL-1β produced and the amount of mRNA were reduced. When TRPV4 in the macrophages was activated by a TRPV4 activator (GSK1016790A), the production of NLRP3 and Caspase-1 was reduced.

[0015] In the conventional evaluation method, monocytes migrate from the blood to skin tissue in vivo and then differentiate into macrophages. Since the differentiated macrophages are localized in the skin tissue, the method of evaluating the anti-inflammatory effect of a test sample using the macrophages as the target is of high technical significance in the development of external skin preparations such as cosmetics, compared with the evaluation method using monocytes that are present in the blood and migrate.

[0016] Furthermore, since macrophages express a higher amount of TRPV4 than monocytes, the present evaluation method targeting macrophages has the advantage of being able to more sensitively evaluate the anti-inflammatory effect of a test sample.

[0017] 2. Evaluation method of test sample In this evaluation method (1), a test sample is contacted with macrophages, a physiological event caused by the activation of TRPV4 in the macrophages is measured, and the anti-inflammatory effect of the test sample is evaluated based on the physiological event. In this evaluation method (1), the anti-inflammatory effect of the test sample can be evaluated by using the physiological event caused by the activation of TRPV4 in macrophages as an index.

[0018] Specific examples of the present evaluation method (1) are not particularly limited, but include, for example, (1A): contacting macrophages with a test sample and measuring physiological events caused by activation of TRPV4; (1B): contacting a macrophage with a test sample under conditions that inhibit the function of TRPV4 in the macrophage, and measuring physiological events caused by activation of TRPV4; (1C): A step of evaluating the activity promoting effect of the test sample on the macrophage TRPV4 based on the physiological events measured in steps (1A) and (1B), and evaluating the anti-inflammatory effect of the test sample based on the evaluation result of the activity promoting effect. The method includes the steps of:

[0019] In addition, in the present evaluation method (2), a test sample is contacted with macrophages, a physiological event caused by the activation of TRPV4 in the macrophages is measured, and the anti-cell death effect of the test sample is evaluated based on the physiological event. In the present evaluation method (2), the anti-cell death effect of the test sample can be evaluated by using the physiological event caused by the activation of TRPV4 in macrophages as an index.

[0020] Specific examples of the present evaluation method (2) are not particularly limited, but include, for example, (2A): contacting macrophages with a test sample and measuring physiological events caused by activation of TRPV4; (2B): contacting a macrophage with a test sample under conditions that inhibit the function of TRPV4 in the macrophage, and measuring physiological events caused by activation of TRPV4; and (2C): A step of evaluating the activity promoting effect of the test sample on macrophage TRPV4 based on the physiological events measured in steps (2A) and (2B), and evaluating the anti-cell death effect of the test sample based on the evaluation result of the activity promoting effect. The method includes the steps of:

[0021] In one embodiment of the present invention, the above steps (2A) and (2B) may be the same as steps (1A) and (1B) in the present evaluation method (1).

[0022] Hereinafter, "Step (1A)" and "Step (2A)" may be collectively referred to as "Step (A)", "Step (1B)" and "Step (2B)" may be collectively referred to as "Step (B)", and "Step (1C)" and "Step (2C)" may be collectively referred to as "Step (C)".

[0023] (Step (A)) In step (A), macrophages are contacted with a test sample, and physiological events caused by activation of TRPV4 are measured.

[0024] The macrophages may be granulocyte macrophage-colony stimulating factor (GM-CSF) macrophages (also referred to as "M1 type macrophages"), which are inflammatory macrophages, or may be macrophage-colony stimulating factor (M-CSF) macrophages (also referred to as "M2 type macrophages"), which are anti-inflammatory macrophages. Hereinafter, "GM-CSF macrophages" and "M-CSF macrophages" are collectively referred to as "macrophages."

[0025] The macrophages may be isolated, unestablished macrophages (hereinafter referred to as "isolated macrophages"), may be established macrophage cells, or may be macrophages differentiated in vitro from monocytes. The source of the isolated macrophages is not particularly limited, and examples thereof include human peripheral blood, human bone marrow, mouse peripheral blood, mouse bone marrow, etc. Examples of the isolated macrophages include human isolated macrophages, mouse isolated macrophages, etc. Human isolated GM-CSF macrophages are CD14 negative, CD11b positive, CD80 positive, TNFalpha positive, and CD68 positive (CD14 - CD11b + CD68 + Human isolated M-CSF macrophages are CD68 positive, CD163 positive, Arginase 1 positive, IL-10 positive, CD206 positive, TNFalpha negative, and CD80 negative (CD68 + CD163 + Arginase 1 + IL-10 + CD206 + TNFalpha - CD80 - ) are shown. Mouse isolated GM-CSF macrophages are CD80 positive, CD86 positive, CD36 positive, CD68 positive, CD32 positive, CD16 positive, IFNgammaR positive, COX2 positive, iNOS positive, IRF5 positive and CD163 negative (CD80 + CD86 + CD36 + CD68 + CD32 + CD16 + IFNgammaR + COX2 + iNOS + IRF5 + CD163 - ) The mouse isolated M-CSF macrophages were positive for CD163, CD200, CD301, CXCR1, DC209, Dectin-1, Arginase 1, IRF4, PPARgamma, and IL-10 (CD163 + CD200+ CD301 + CXCR1 + DC209 + Dectin-1 + Arginase 1 + IRF4 + PPARgamma + IL-10 + ) is shown.

[0026] Examples of macrophage cell lines include, but are not limited to, RAW-264.7, J774, MV-4-11, and KG-1.

[0027] The method for differentiating monocytes into macrophages in vitro is not particularly limited, but can be, for example, by culturing monocytes in a monocyte medium containing GM-CSF and / or M-CSF under culture conditions suitable for maintaining the physiological functions of monocytes.

[0028] The amount of GM-CSF and / or M-CSF in the medium for monocytes varies depending on the number of monocytes, etc., and therefore cannot be determined in general terms. It is preferable to determine the amount appropriately according to the number of monocytes, etc. The amount of GM-CSF and / or M-CSF in the medium for monocytes is not particularly limited, but is, for example, 5 ng / mL or more, preferably 10 ng / mL or more. The upper limit of the amount of GM-CSF and / or M-CSF in the medium for monocytes is not particularly limited, but is, for example, 200 ng / mL or less, preferably 100 ng / mL or less. The medium for monocytes is not particularly limited, but is, for example, the same as the medium for macrophages used as a solvent for the test sample described later. The culture conditions are not particularly limited, but are, for example, the same as the culture conditions used when the macrophages are contacted with the test sample described later.

[0029] The contact time between monocytes and GM-CSF and / or M-CSF is not particularly limited as long as it is a time sufficient for differentiation of monocytes into macrophages, but is, for example, 3 days or more, preferably 5 days or more.

[0030] In this evaluation method, monocytes differentiated into macrophages by the above-mentioned method may be used as they are, or purified macrophages may be used.

[0031] The method for purifying macrophages is not particularly limited, but may be, for example, flow cytometry, magnetic cell separation, etc. When purifying GM-CSF macrophages by flow cytometry or magnetic cell separation, the markers of GM-CSF macrophages are not particularly limited, but may be, for example, CD14, CD11b, CD68, CD80, CD163, etc. Among these markers of GM-CSF macrophages, CD14, CD11b, and CD68 are preferred from the viewpoint of easily distinguishing monocytes from GM-CSF macrophages. GM-CSF macrophages can be detected and separated using the indicator of "CD14 negative, CD11b positive, and CD68 positive." When purifying M-CSF macrophages by flow cytometry, the markers of M-CSF macrophages are not particularly limited, but may be, for example, CD68, CD163, Arginase-1, IL-10, etc. Among these M-CSF macrophage markers, Arginase-1, IL-10, and CD163 are preferred from the viewpoint of easily distinguishing M-CSF macrophages from monocytes. M-CSF macrophages can be detected and separated using the indicators "CD68 positivity, CD163 positivity, Arginase 1 positivity, IL-10 positivity, CD206 positivity, TNFalpha negativity, and CD80 negativity."

[0032] TRPV4 is one of the nonselective cation channels that is activated, for example, by a decrease in the osmotic pressure of the extracellular fluid, and has the amino acid sequence shown in GenBank Accession No. NM_021625.

[0033] The test sample is not particularly limited, but examples thereof include inorganic compounds, organic compounds, plant extracts, cell culture supernatants, cell extracts, etc. When the test sample is liquid, it may be used as is, or may be diluted with a solvent as necessary. When the test sample is solid, it may be dissolved in a solvent before use.

[0034] The solvent varies depending on the type of test sample, the type of physiological event to be measured, etc., and therefore cannot be generally determined, and it is preferable to appropriately determine the solvent depending on the type of test sample, the type of physiological event to be measured, etc. The solvent is not particularly limited, but examples thereof include physiological saline, phosphate buffered physiological saline, purified water, ethanol, ethanol aqueous solution, calcium-containing solution [composition: 140 mM sodium chloride, 5 mM potassium chloride, 2 mM magnesium chloride, 2 mM calcium chloride, 10 mM glucose, and 10 mM 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid (HEPES) hydrochloric acid buffer (pH 7.4)], calcium-free solution [composition: 140 mM sodium chloride, 5 mM potassium chloride, 2 mM magnesium chloride, 5 mM glycol ether diamine tetraacetic acid, 10 mM glucose, and 10 mM HEPES hydrochloric acid buffer (pH 7.4)], dimethyl sulfoxide (DMSO), and the like.

[0035] Contact between macrophages and a test sample can be achieved, for example, by culturing macrophages in a macrophage medium containing the test sample under culture conditions suitable for maintaining the physiological functions of macrophages.

[0036] The macrophage medium can be prepared, for example, by adding a medium additive to a basal medium. The medium additive is not particularly limited, but examples thereof include serum, antibiotics, etc. The basal medium is not particularly limited, but examples thereof include RPMI1640 medium, MEM medium, IMDM medium, Ham's F12 medium, etc. The macrophage medium may be a serum-starved medium or a non-serum-starved medium. The serum-starved medium usually has a serum concentration of preferably 0 to 1% (v / v), more preferably 0 to 0.5% (v / v). The non-serum-starved medium usually has a serum concentration of preferably 2 to 10% (v / v), more preferably 5 to 10% (v / v). Among these macrophage media, the serum-starved medium is preferred because it contains serum containing various physiologically active substances, and therefore the effect of the test sample can be evaluated more specifically.

[0037] The amount of the test sample in the macrophage medium varies depending on the type of test sample, the number of macrophages, etc., and therefore cannot be determined in general, and is preferably determined appropriately according to the type of test sample, the number of macrophages, etc. The macrophage medium used for contacting the macrophage with the test sample is the same as the macrophage medium used as a solvent for the test sample. The contact time between the macrophage and the test sample varies depending on the type of test sample, the culture temperature, etc., and therefore cannot be determined in general, and is preferably determined appropriately according to the type of test sample, the culture temperature, etc. The contact time between the macrophage and the test sample is preferably 0.15 hours or more, more preferably 0.5 hours or more, even more preferably 1 hour or more, and particularly preferably 3 hours or more, from the viewpoint of accurately evaluating the anti-inflammatory effect or anti-cell death effect, preferably the anti-inflammatory effect or anti-cell death effect mediated by the activation of TRPV4 in the macrophage. Furthermore, from the viewpoint of accurately evaluating the anti-inflammatory effect or anti-cell death effect, preferably the anti-inflammatory effect or anti-cell death effect mediated by the activation of TRPV4 in macrophages, as described above, the upper limit of the contact time between the macrophages and the test sample is preferably 72 hours or less, more preferably 12 hours or less, and even more preferably 8 hours or less.

[0038] The culture conditions include the culture temperature, the carbon dioxide concentration in the culture atmosphere, etc. The culture temperature is preferably 35°C or higher, more preferably 36.5°C or higher, from the viewpoint of accurately evaluating the anti-inflammatory or anti-cell death effect, preferably the anti-inflammatory or anti-cell death effect mediated by the activation of TRPV4 in macrophages. The upper limit of the culture temperature is preferably 38°C or lower, more preferably 37.5°C or lower, from the viewpoint of accurately evaluating the anti-inflammatory or anti-cell death effect, preferably the anti-inflammatory or anti-cell death effect mediated by the activation of TRPV4 in macrophages, as described above. The carbon dioxide concentration in the culture atmosphere is preferably 4% (v / v) or higher, more preferably 5% (v / v) or higher, from the viewpoint of accurately evaluating the anti-inflammatory or anti-cell death effect, preferably the anti-inflammatory or anti-cell death effect mediated by the activation of TRPV4 in macrophages. The upper limit of the carbon dioxide concentration in the culture atmosphere is preferably 10% (v / v) or less, and more preferably 7% (v / v) or less, from the viewpoint of accurately evaluating the anti-inflammatory effect or anti-cell death effect, preferably the anti-inflammatory effect or anti-cell death effect mediated by the activation of TRPV4 in macrophages.

[0039] Physiological events caused via activation of TRPV4 include, but are not limited to, (i) a decrease in the expression level of an inflammation-related factor or its mRNA due to activation of TRPV4 in macrophages, (ii) a decrease in the expression level of NLRP3 and / or Caspase-1, or its mRNA, which are necessary for inflammation-related factors to mature from their precursors, (iii) an increase in intracellular calcium ion concentration in macrophages due to activation of TRPV4 in macrophages, and (iv) an increase in membrane potential in macrophages due to activation of TRPV4 in macrophages.

[0040] Methods for measuring physiological events caused via the activation of TRPV4 are not particularly limited, but include, for example, the following Measurement Methods 1 to 4.

[0041] <Measurement method 1> In the measurement method 1, the physiological event is the above-mentioned "(i) a decrease in the expression level of an inflammation-related factor or its mRNA due to activation of TRPV4 in macrophages." (1a): Contacting macrophages with an inflammation-inducing substance; and (1b): Measuring the expression level of an inflammation-related factor or its mRNA Includes.

[0042] Contact of macrophages with an inflammation-inducing substance can be achieved, for example, by culturing macrophages in a macrophage medium containing the inflammation-inducing substance under culture conditions suitable for maintaining the physiological functions of macrophages.

[0043] Examples of inflammation-inducing substances include, but are not limited to, lipopolysaccharides; CpG oligonucleotides such as K3CpG; lipopeptides such as Pam3CSK4; Poly I:C; and the like.

[0044] The amount of the inflammation-inducing substance in the macrophage medium cannot be determined in general because it varies depending on the type of inflammation-inducing substance, the number of macrophages, etc., and is therefore preferably determined appropriately depending on the type of inflammation-inducing substance, the number of macrophages, etc. The culture conditions are similar to those used when the macrophages are brought into contact with the test sample.

[0045] When measurement method 1 is used in step (A), in step (1a) of measurement method 1, the test sample and the inflammation-inducing substance may be contacted with the macrophages simultaneously or separately.

[0046] When the test sample and the inflammation-inducing substance are simultaneously contacted with the macrophages, for example, a macrophage medium containing the test sample and the inflammation-inducing substance can be used. The culture temperature when the test sample and the inflammation-inducing substance are simultaneously contacted with the macrophages is the same as the culture temperature in the above culture conditions. The contact time between the macrophages, the test sample, and the inflammation-inducing substance varies depending on the type of the test sample, the type of the inflammation-inducing substance, the culture temperature, etc., and therefore cannot be determined in general, so it is preferable to appropriately determine the contact time depending on the type of the test sample, the type of the inflammation-inducing substance, the culture temperature, etc.

[0047] When the test sample and the inflammation-inducing substance are contacted with the macrophage separately, the test sample may be contacted with the macrophage and then the inflammation-inducing substance may be contacted with the macrophage, or the inflammation-inducing substance may be contacted with the macrophage and then the test sample may be contacted with the macrophage. The culture temperature when the inflammation-inducing substance is contacted with the macrophage is the same as the culture temperature in the culture conditions. The contact time between the macrophage and the inflammation-inducing substance varies depending on the type of inflammation-inducing substance, the culture temperature, etc., and cannot be determined in general, so it is preferable to appropriately determine it according to the type of inflammation-inducing substance, the culture temperature, etc. The contact time between the macrophage and the inflammation-inducing substance is preferably 0.15 hours or more, more preferably 0.5 hours or more, even more preferably 1 hour or more, particularly preferably 3 hours or more, and particularly preferably 6 hours or more, from the viewpoint of accurately evaluating the anti-inflammatory effect or anti-cell death effect, preferably the anti-inflammatory effect or anti-cell death effect mediated by the activation of TRPV4 in the macrophage. The upper limit of the contact time between the macrophages and the inflammation-inducing substance is, from the viewpoint of accurately evaluating the anti-inflammatory effect or anti-cell death effect, preferably the anti-inflammatory effect or anti-cell death effect mediated by the activation of TRPV4 in macrophages, as described above, preferably 72 hours or less, more preferably 12 hours or less, and even more preferably 8 hours or less.

[0048] The inflammation-related factor is a factor present at the site of inflammation. Examples of the inflammation-related factor include, but are not limited to, inflammatory cytokines such as IFN-α, IFN-γ, IL-1a, IL-1β, IL-12p70, IL-13, IL-17α, IL-4, IL-6, TNF-α, and GM-CSF; chemokines such as IP-10, MCP-1, MIP-1α, and MIP-1β; and cell adhesion factors such as E-selectin, P-selectin, and sICAM-1. Among these inflammation-related factors, from the viewpoint of accurately evaluating the anti-inflammatory effect or anti-cell death effect, preferably the anti-inflammatory effect or anti-cell death effect mediated by the activation of TRPV4 in macrophages, IFN-α, IFN-γ, IL-1a, IL-1β, IL-12p70, IL-13, IL-17α, IL-4, IL-6, TNF-α, GM-CSF, IP-10, MCP-1, MIP-1α, MIP-1β, E-selectin, P-selectin, and sICAM-1 are preferred.

[0049] Methods for measuring the expression level of inflammation-associated factors include, but are not limited to, enzyme-labeled immunosorbent assay (hereinafter referred to as "ELISA"), Western blotting, immunofluorescence staining, fluorescence-activated cell sorting (hereinafter referred to as "FACS"), multiplex assay using fluorescently labeled beads, etc. Methods for measuring the expression level of inflammation-associated factor mRNA include, but are not limited to, real-time RT-PCR, Northern blotting, etc.

[0050] The antibody used in ELISA and Western blotting is not particularly limited, but may be, for example, an antibody against an inflammation-related substance or an antibody fragment thereof. The antibody against an inflammation-related substance may be a monoclonal antibody or a polyclonal antibody. As the antibody against an inflammation-related substance or an antibody fragment thereof, a commercially available antibody or an antibody fragment thereof may be used.

[0051] The primer pair used in the real-time RT-PCR method is not particularly limited, and may be, for example, a primer consisting of a part of the base sequence of a nucleic acid encoding an inflammation-related factor and a primer consisting of a part of the base sequence of the antisense strand of the nucleic acid, etc. As the primer pair, a commercially available primer pair can be used.

[0052] The probe used in the Northern blotting method is not particularly limited, and examples thereof include a nucleic acid consisting of all or a part of a nucleic acid encoding an inflammation-associated factor, a nucleic acid consisting of all or a part of an antisense strand of a nucleic acid encoding an inflammation-associated factor, etc. When the probe is a nucleic acid consisting of a part of a nucleic acid encoding an inflammation-associated factor or a nucleic acid consisting of a part of an antisense strand of the nucleic acid, the length of the probe varies depending on the type of inflammation-associated factor, etc., and therefore cannot be determined in general, and is preferably determined appropriately depending on the type of inflammation-associated factor, etc. The length of the probe is usually preferably 20 to 500 nucleotides long, from the viewpoint of improving the accuracy of measuring the expression level of an inflammation-associated factor.

[0053] <Measurement method 2> In the measurement method 2, the physiological event is the aforementioned "decrease in the expression level of NLRP3 and / or Caspase-1, or their mRNA, which are necessary for the maturation of inflammation-related factors from their precursors." (2a): Steps similar to step (1a) in <Measurement Method 1> (2b): Measuring the amount of NLRP3 and / or Caspase-1 produced or the amount of mRNA expression thereof Includes.

[0054] When macrophages receive inflammatory stimuli (e.g., stimulation by LPS), they form a protein complex called the NLRP3 inflammasome via NLRP3, which reacts to irritating metabolites (urate crystals, free fatty acids, etc.) that have accumulated in the body due to overnutrition. The NLRP3 inflammasome activates caspase-1, cleaves the precursor of inflammation-related factors, and matures them. For example, when the inflammation-related factor is IL-1β, the NLRP3 inflammasome cleaves pro-IL-1β, the precursor of IL-1β, and activates it. The activated IL-1β released outside the cell promotes the subsequent inflammatory response. Excessive activation of the NLRP3 inflammasome is closely related to the onset of inflammatory diseases such as gouty arthritis or diabetes. In addition, activation of caspase-1, which constitutes the NLRP3 inflammasome, induces pyroptosis, a type of cell death. Thus, since NLRP3 and Caspase-1 are associated with the maturation of inflammation-related factors and cell death, the anti-inflammatory and / or anti-cell death effects of a test sample can be evaluated by using the production amount of NLRP3 and / or Caspase-1, or the expression amount of their mRNA, as an indicator.

[0055] As mentioned above, inflammation-related factors including IL-1β are controlled at least at two stages: mRNA expression and subsequent protein maturation. In other words, activation of TRPV4 in macrophages not only directly reduces the production of inflammation-related factors, but also suppresses the production and activation of NLRP3 inflammasome, which is involved in the maturation mechanism and promotes inflammatory responses. IL-1β, NLRP3, and Caspase-1 are genes represented by the following GenBank accession numbers. IL-1β: NC_000002.12 (human) NLRP3: NC_000001.11 (human) Caspase-1: NC_000011.10 (human) Step (2a) is the same as step (1a) in <Measurement method 1>, and the contents described in <Measurement method 1> are incorporated herein by reference.

[0056] Methods for measuring the expression levels of NLRP3 and Caspase-1 include, but are not limited to, ELISA, Western blotting, immunofluorescence staining, FACS, multiplex assay using fluorescently labeled beads, etc. Methods for measuring the expression levels of NLRP3 and Caspase-1 mRNA include, but are not limited to, real-time RT-PCR, Northern blotting, etc.

[0057] The antibody used in ELISA and Western blotting is not particularly limited, but may be, for example, an antibody against NLRP3 and Caspase-1 or an antibody fragment thereof. The antibody against NLRP3 and Caspase-1 may be a monoclonal antibody or a polyclonal antibody. As the antibody against inflammation-related substances and an antibody fragment thereof, commercially available antibodies and antibody fragments thereof may be used.

[0058] The primer pair used in the real-time RT-PCR method is not particularly limited, but may be, for example, a primer consisting of a part of the base sequence of a nucleic acid encoding NLRP3 and Caspase-1 and a primer consisting of a part of the base sequence of the antisense strand of the nucleic acid. Commercially available primer pairs can be used as the primer pair.

[0059] The probe used in the Northern blotting method is not particularly limited, and examples thereof include a nucleic acid consisting of all or a part of a nucleic acid encoding NLRP3 and Caspase-1, a nucleic acid consisting of all or a part of an antisense strand of a nucleic acid encoding NLRP3 and Caspase-1, etc. When the probe is a nucleic acid consisting of a part of a nucleic acid encoding NLRP3 and Caspase-1 or a nucleic acid consisting of a part of an antisense strand of the nucleic acid, it is preferable to appropriately determine the length of the probe. From the viewpoint of improving the accuracy of measuring the expression levels of NLRP3 and Caspase-1, the length of the probe is usually preferably 20 to 500 nucleotides.

[0060] <Measurement method 3> In measurement method 3, the physiological event used is the above-mentioned "(iii) increase in intracellular calcium ion concentration in macrophages caused by activation of TRPV4 in macrophages." The intracellular calcium ion concentration in macrophages can be measured by introducing a calcium indicator into the macrophages and using the amount of calcium indicator bound to calcium ions in the macrophages as an index.

[0061] Since the amount of calcium indicator bound to calcium ions can be measured by a simple operation, the calcium indicator is preferably a reagent that can detect changes before and after binding with calcium ions by changes in optical properties, etc. Examples of changes in optical properties include, but are not limited to, changes in fluorescence intensity and changes in absorbance, etc. Examples of calcium indicators include, but are not limited to, fluorescent calcium indicators whose fluorescence intensity changes before and after binding with calcium ions, etc. Specific examples of calcium indicators include, but are not limited to, 1-[6-amino-2-(5-carboxy-2-oxazolyl)-5-benzofuranyloxy]-2-(2-amino-5-methylphenoxy)ethane-N,N,N',N'-tetraacetic acid pentaacetoxymethyl ester (hereinafter referred to as "Fura 2-AM"), 1-[2-amino-5-(2,7-dichloro-6-hydroxy-3-oxo-9-xanthenyl)phenoxy]-2-(2-amino-5-methylphenoxy)ethane-N,N,N',N'-tetraacetic acid tetraacetoxymethyl ester (hereinafter referred to as "Fluo Examples of the fluorescent calcium indicator include Fura 2-AM, 1-[2-amino-5-(2,7-difluoro-6-acetoxymethoxy-3-oxo-9-xanthenyl)phenoxy]-2-(2-amino-5-methylphenoxy)ethane-N,N,N',N'-tetraacetic acid tetraacetoxymethyl ester (hereinafter referred to as "Fluo 4-AM"). Among the calcium indicators, from the viewpoint of distinguishing the dynamics of calcium ions from the dynamics of impurities and accurately evaluating the anti-inflammatory effect or anti-cell death effect, preferably the anti-inflammatory effect or anti-cell death effect mediated by the activation of TRPV4 in macrophages, fluorescent calcium indicators whose fluorescence intensity changes before and after binding with calcium ions are preferred, and Fura 2-AM, Fluo 3-AM and Fluo 4-AM are more preferred.

[0062] The fluorescent calcium indicator may have one kind of excitation wavelength, or may have two or more kinds of excitation wavelengths. When the calcium indicator is a fluorescent calcium indicator, the fluorescent calcium indicator is preferred because the fluorescent intensity is easy to measure and the detection intensity is high. When a fluorescent calcium indicator having one kind of excitation wavelength is used in measuring the change in intracellular calcium concentration, the change in intracellular calcium concentration can be measured based on the fluorescence intensity at the excitation wavelength. When a fluorescent calcium indicator having two or more kinds of excitation wavelengths is used in measuring the change in intracellular calcium concentration, two kinds of excitation wavelengths (a first excitation wavelength and a second excitation wavelength) are selected from the two or more kinds of excitation wavelengths from the viewpoint of accurately evaluating the anti-inflammatory effect or anti-cell death effect, preferably the anti-inflammatory effect or anti-cell death effect mediated by the activation of TRPV4 in macrophages, and the change in intracellular calcium concentration can be measured based on the fluorescence intensity ratio calculated from the fluorescence intensity at each of the first excitation wavelength and the second excitation wavelength. When measuring intracellular calcium concentration, for example, when using FURA 2-AM, a fluorescent calcium indicator having two excitation wavelengths, the fluorescence intensity at the first excitation wavelength (hereinafter also referred to as "first fluorescence intensity") can be the fluorescence intensity at an excitation wavelength of 340 nm, and the fluorescence intensity at the second excitation wavelength (hereinafter also referred to as "second fluorescence intensity") can be the fluorescence intensity at an excitation wavelength of 380 nm. The fluorescence intensity ratio can be calculated, for example, by the formula (I): [Fluorescence intensity ratio] = [First fluorescence intensity] / [Second fluorescence intensity] (I) It can be determined based on the following.

[0063] The method of introducing a fluorescent calcium indicator into a macrophage is not particularly limited, but for example, a method of circulating a buffer solution containing a fluorescent calcium indicator in a reflux chamber containing a macrophage. The method of contacting a single macrophage with a test sample after the indicator introduction is not particularly limited, but for example, a method of circulating a buffer solution containing a fluorescent calcium indicator in a reflux chamber containing a macrophage with a fluorescent calcium indicator introduced therein. The method of contacting a cell with calcium ions after the test sample contact is not particularly limited, but for example, a method of circulating a buffer solution containing calcium ions and not containing a test sample. The buffer solution is not particularly limited, but for example, HEPES buffer solution. The temperature when introducing a fluorescent calcium indicator into a macrophage, the contact temperature between the macrophage and the test sample, and the contact temperature between the macrophage and calcium ions are preferably 35°C or higher, more preferably 36.5°C or higher, from the viewpoint of accurately evaluating the anti-inflammatory effect or anti-cell death effect, preferably the anti-inflammatory effect or anti-cell death effect via the activation of TRPV4 in macrophages. The upper limits of the temperature when the fluorescent calcium indicator is introduced into the macrophage, the contact temperature between the macrophage and the test sample, and the contact temperature between the macrophage and calcium ions are preferably 38° C. or lower, more preferably 37.5° C. or lower, from the viewpoint of accurately evaluating the anti-inflammatory or anti-cell death effect, preferably the anti-inflammatory or anti-cell death effect mediated by the activation of TRPV4 in the macrophage, as described above. The contact time between the macrophage and the test sample is preferably 0.5 minutes or more, more preferably 1 minute or more, from the viewpoint of accurately evaluating the anti-inflammatory or anti-cell death effect, preferably the anti-inflammatory or anti-cell death effect mediated by the activation of TRPV4 in the macrophage, and is preferably 1 hour or less, more preferably 0.5 hours or less, from the viewpoint of accurately evaluating the change in intracellular calcium.The contact time between macrophages and calcium ions is preferably 1 minute or more, more preferably 5 minutes or more, from the viewpoint of accurately evaluating the anti-inflammatory or anti-cell death effect, preferably the anti-inflammatory or anti-cell death effect mediated by the activation of TRPV4 in macrophages. The upper limit of the contact time between macrophages and calcium ions is preferably 2 hours or less, more preferably 1 hour or less, from the viewpoint of accurately evaluating the anti-inflammatory or anti-cell death effect, preferably the anti-inflammatory or anti-cell death effect mediated by the activation of TRPV4 in macrophages, as described above.

[0064] <Measurement method 4> In the measurement method 4, the physiological event is the above-mentioned "(iv) increase in membrane potential of macrophages caused by activation of TRPV4 in macrophages." The method for measuring the membrane potential of macrophages is not particularly limited, but examples thereof include patch clamp methods such as the whole cell method and the cell attachment method.

[0065] (Step (B)) In step (B), the macrophage is contacted with a test sample under conditions in which the function of TRPV4 in the macrophage is inhibited, and physiological events caused by activation of TRPV4 are measured. The measurement of the physiological events in step (B) can be performed under the same conditions and by the same method as in step (A), except that the measurement is performed under conditions in which the function of TRPV4 in the macrophage is inhibited. Furthermore, the physiological events measured in step (B) are the same type of physiological events as those measured in step (A).

[0066] Methods for suppressing the function of TRPV4 in macrophages include, but are not limited to, a method of contacting a TRPV4 activity inhibitor with TRPV4 in macrophages, a method of knocking down or knocking out TRPV4 in macrophages, and the like.

[0067] Examples of the TRPV4 activity inhibitor include, but are not limited to, 2-methyl-1-[3-(4-morpholinyl)propyl]-5-phenyl-N-[3-(trifluoromethyl)phenyl]-1H-pyrrole-3-carboxamide (hereinafter referred to as "HC-067047"), 3-([1,4'-bipiperidine]-1'-ylmethyl)-7-bromo-N-(1-phenylcyclopropyl)-2-[3-(trifluoromethyl)phenyl]-4-quinolinecarboxamide (hereinafter referred to as " and TRPV4 antagonists such as N-[4-[[4-(1-methylethyl)-1-piperazinyl]sulfonyl]phenyl]-2-nitro-4-(trifluoromethyl)benzamide hydrochloride (hereinafter referred to as "RN9893 hydrochloride"), 2,4-dichloro-N-(1-methylethyl)-N-{2-[(1-methylethyl)amino]ethyl}benzenesulfonamide (hereinafter referred to as "RN-1734"), and ruthenium red. Among these TRPV4 activity inhibitors, from the viewpoint of accurately measuring physiological events caused by activation of TRPV4 in macrophages, TRPV4 antagonists are preferred, with HC-067047, GSK2193874, RN9893 hydrochloride, RN-1734 and ruthenium red being more preferred, and HC-067047 and GSK2193874 being even more preferred.

[0068] The amount of TRPV4 activity inhibitor to be contacted with macrophage TRPV4 cannot be determined in general terms because it varies depending on the type of TRPV4 activity inhibitor, the number of macrophages, etc., so it is preferable to determine the amount appropriately depending on the type of TRPV4 activity inhibitor, the number of macrophages, etc.

[0069] From the viewpoint of accurately evaluating the action of a test sample under conditions that inhibit the function of TRPV4 in macrophages, it is preferable to contact a TRPV4 activity inhibitor with TRPV4 in macrophages before or simultaneously with the contact of the macrophages with the test sample.

[0070] When the contact between the TRPV4 activity inhibitor and the TRPV4 of macrophage is performed before the contact between the macrophage and the test sample, the contact time between the TRPV4 activity inhibitor and the TRPV4 of macrophage is preferably 5 minutes or more, more preferably 15 minutes or more, from the viewpoint of accurately evaluating the anti-inflammatory effect or anti-cell death effect, preferably the anti-inflammatory effect or anti-cell death effect via the activation of the TRPV4 of macrophage.When the contact between the TRPV4 activity inhibitor and the TRPV4 of macrophage is performed before the contact between the macrophage and the test sample, the upper limit of the contact time between the TRPV4 activity inhibitor and the TRPV4 of macrophage is preferably 24 hours or less, more preferably 1 hour or less, from the viewpoint of accurately evaluating the anti-inflammatory effect or anti-cell death effect, preferably the anti-inflammatory effect or anti-cell death effect via the activation of the TRPV4 of macrophage, as described above.

[0071] When the contact of the TRPV4 activity inhibitor with the TRPV4 of the macrophages is carried out simultaneously with the contact of the macrophages with the test sample, the contact time of the TRPV4 activity inhibitor with the TRPV4 of the macrophages is the same as the contact time of the TRPV4 of the macrophages with the test substance.

[0072] Methods for knocking down TRPV4 in macrophages include, but are not limited to, a method for inhibiting the expression of the TRPV4 gene by RNA silencing, a method for inhibiting normal TRPV4 function by expressing a dominant-negative mutant, etc. Methods for knocking out TRPV4 in macrophages include, but are not limited to, a method for disrupting the TRPV4 gene by homologous recombination, a method for disrupting the TRPV4 gene by genome editing technology, etc.

[0073] (Step (C)) In step (C), the activity of the test sample in promoting the activity of macrophages against TRPV4 is evaluated based on the physiological events measured in steps (A) and (B), and the anti-inflammatory and / or anti-cell death activity of the test sample is evaluated based on the evaluation result of the activity promoting activity.

[0074] When the physiological event measured in steps (A) and (B) is "(i) a decrease in the expression level of an inflammation-related factor or its mRNA due to activation of TRPV4 in macrophages," the test sample is evaluated as having an activity-promoting effect on TRPV4 in macrophages based on at least one of the following indicators (1a) and (1b).

[0075] <Indicator (1a)> The expression level of the inflammation-related factor measured in step (A) is lower than the expression level of the inflammation-related factor measured in step (B).

[0076] <Indicator (1b)> The expression level of the mRNA of the inflammation-related factor measured in step (A) is lower than the expression level of the mRNA of the inflammation-related factor measured in step (B).

[0077] When the physiological event measured in steps (A) and (B) is "(ii) a decrease in the expression level of NLRP3 and / or Caspase-1, or their mRNA, which are necessary for inflammation-related factors to mature from their precursors," the test sample is evaluated as having an activity-promoting effect on TRPV4 in macrophages based on at least one of the following indicators (2a) and (2b).

[0078] <Indicator (2a)> The amount of NLRP3 and / or Caspase-1 produced measured in step (A) is lower than the amount of NLRP3 and / or Caspase-1 produced measured in step (B).

[0079] <Indicator (2b)> The expression level of NLRP3 and / or Caspase-1 mRNA measured in step (A) is lower than the expression level of NLRP3 and / or Caspase-1 mRNA measured in step (B).

[0080] When the physiological event measured in steps (A) and (B) is "(iii) an increase in intracellular calcium ion concentration in macrophages due to activation of TRPV4 in macrophages," the test sample is evaluated as having an activity-promoting effect on TRPV4 in macrophages based on the following indicator (3a).

[0081] <Indicator (3a)> The intracellular calcium ion concentration of the macrophage measured in step (A) is higher than the intracellular calcium ion concentration of the macrophage measured in step (B).

[0082] When the physiological event measured in steps (A) and (B) is "(iv) an increase in the membrane potential of macrophages due to activation of TRPV4 in macrophages," the test sample is evaluated as having an activity-promoting effect on TRPV4 in macrophages based on the following indicator (4a).

[0083] <Indicator (4a)> The membrane potential of the macrophage measured in step (A) is greater than the membrane potential of the macrophage measured in step (B).

[0084] If the test sample has an activity-promoting effect on TRPV4 in macrophages, it can be evaluated as having an anti-inflammatory effect and / or an anti-cell death effect, preferably an anti-inflammatory effect and / or an anti-cell death effect mediated by the activation of TRPV4.If the test sample does not have an activity-promoting effect on TRPV4 in macrophages, it can be evaluated as not having an anti-inflammatory effect and / or an anti-cell death effect, preferably an anti-inflammatory effect and / or an anti-cell death effect mediated by the activation of TRPV4.

[0085] As described above, according to the method for evaluating a test sample of the present invention, the anti-inflammatory effect and / or anti-cell death effect of the test sample, preferably the anti-inflammatory effect and / or anti-cell death effect mediated by the activation of TRPV4, can be easily evaluated.

[0086] (others) In one embodiment of the present invention, the present evaluation method may be performed using a combination of macrophages and monocytes. By using a plurality of cells, evaluation can be performed using a plurality of indicators, enabling more precise and diverse screening.

[0087] The combination of macrophages and monocytes may be, for example, a combination of GM-CSF macrophages and monocytes, a combination of M-CSF macrophages and monocytes, or a combination of GM-CSF macrophages, M-CSF macrophages and monocytes. In this case, the evaluation method using monocytes can be performed, for example, by replacing "macrophages" in the above steps (1A) to (1C) or steps (2A) to (2C) with "monocytes."

[0088] 3. Agents that suppress the production of NLRP3 and / or Caspase-1, and anti-inflammatory agents In one embodiment of the present invention, there is provided an agent (hereinafter referred to as "the present inhibitor") that suppresses the production amount of NLRP3 and / or Caspase-1, comprising as an active ingredient a substance that activates TRPV4 in macrophages.

[0089] As used herein, the term "agent that suppresses the production amount of NLRP3 and / or Caspase-1" refers to a substance that reduces (suppresses) the production amount of NLRP3 and / or Caspase-1 in the presence of the substance compared to the absence of the substance. The inhibitor can be used, for example, as a drug exhibiting an anti-cell death effect.

[0090] Moreover, in one embodiment of the present invention, there is provided an anti-inflammatory agent (hereinafter referred to as "the present anti-inflammatory agent") comprising, as an active ingredient, a substance that activates TRPV4 in macrophages.

[0091] As used herein, the term "anti-inflammatory agent" refers to a substance that reduces (suppresses) inflammation in the presence of the substance compared to its absence. The anti-inflammatory agent can reduce inflammation caused by, for example, chemical stimuli such as chemical substances or drugs, thermal stimuli (e.g., stimuli of around 43°C), pain stimuli, mechanical stimuli, etc.

[0092] (Active ingredient) The present inhibitor and anti-inflammatory agent contain, as an active ingredient, a substance that activates TRPV4 in macrophages.

[0093] In this specification, the term "substance that activates macrophage TRPV4" is not particularly limited as long as it is a substance that can positively regulate the activity of macrophage TRPV4. Examples of substances that activate macrophage TRPV4 include, but are not limited to, GSK1016790A, 4alpha-PDD, 5',6'-Epoxyeicosatrienoic acid, arachidonic acid, etc.

[0094] Furthermore, in one embodiment of the present invention, a substance that activates TRPV4 in macrophages may be a test substance that is evaluated using the present evaluation method as having an anti-inflammatory effect and / or an anti-cell death effect, preferably an anti-inflammatory effect and / or an anti-cell death effect mediated by the activation of TRPV4.

[0095] The present inhibitor and the present anti-inflammatory agent may contain, as an active ingredient, only one substance that activates TRPV4 in macrophages, or may contain two or more substances.

[0096] (Other Ingredients) The present inhibitor and the present anti-inflammatory agent may contain other components (other components) other than the above-mentioned active ingredient (substance that activates TRPV4 in macrophages). The other components may be any pharma- ceutically acceptable components, such as buffers, pH adjusters, isotonicity agents, preservatives, antioxidants, high molecular weight polymers, excipients, solvents, transdermal absorption enhancers, etc.

[0097] Examples of the buffer include phosphoric acid or a phosphate, boric acid or a borate, citric acid or a citrate, acetic acid or a acetate, carbonic acid or a carbonate, tartaric acid or a tartrate, ε-aminocaproic acid, trometamol, etc. Examples of the phosphate include sodium phosphate, sodium dihydrogen phosphate, disodium hydrogen phosphate, potassium phosphate, potassium dihydrogen phosphate, dipotassium hydrogen phosphate, etc. Examples of the borate include borax, sodium borate, potassium borate, etc. Examples of the citrate include sodium citrate, disodium citrate, trisodium citrate, etc. Examples of the acetate include sodium acetate, potassium acetate, etc. Examples of the carbonate include sodium carbonate, sodium bicarbonate, etc. Examples of the tartrate include sodium tartrate, potassium tartrate, etc.

[0098] Examples of the pH adjuster include hydrochloric acid, phosphoric acid, citric acid, acetic acid, sodium hydroxide, and potassium hydroxide.

[0099] Examples of the isotonicity agent include ionic isotonicity agents (sodium chloride, potassium chloride, calcium chloride, magnesium chloride, etc.) and non-ionic isotonicity agents (glycerin, propylene glycol, sorbitol, mannitol, etc.).

[0100] Examples of the preservatives include benzalkonium chloride, benzalkonium bromide, benzethonium chloride, sorbic acid, potassium sorbate, methyl parahydroxybenzoate, propyl parahydroxybenzoate, and chlorobutanol.

[0101] Examples of the antioxidant include ascorbic acid, tocopherol, dibutylhydroxytoluene, butylhydroxyanisole, sodium erythorbate, propyl gallate, and sodium sulfite.

[0102] Examples of the high molecular weight polymer include methyl cellulose, ethyl cellulose, hydroxymethyl cellulose, hydroxyethyl cellulose, hydroxypropyl cellulose, hydroxyethyl methyl cellulose, hydroxypropyl methyl cellulose, carboxymethyl cellulose, sodium carboxymethyl cellulose, hydroxypropyl methyl cellulose acetate succinate, hydroxypropyl methyl cellulose phthalate, carboxymethyl ethyl cellulose, cellulose acetate phthalate, polyvinylpyrrolidone, polyvinyl alcohol, carboxyvinyl polymer, polyethylene glycol, and atelocollagen.

[0103] Examples of the excipient include lactose, sucrose, D-mannitol, xylitol, sorbitol, erythritol, starch, and crystalline cellulose.

[0104] Examples of the solvent include water, physiological saline, alcohol, and the like.

[0105] The present inhibitor and the present anti-inflammatory agent may contain, as the other ingredient described above, a substance having the effect of improving the effect of the active ingredient (hereinafter, sometimes referred to as an "enhancing agent"). The enhancing agent is a substance that has the effect of improving the effect of the active ingredient when used in combination with the active ingredient. The enhancing agents may be used alone or in combination of two or more kinds.

[0106] Examples of the enhancer include inorganic compounds, organic compounds, plant extracts, microbial cultures, microbial extracts, surfactants such as nonionic surfactants, etc. Among these substances, surfactants are preferred because they can further enhance the effects of the active ingredient, and nonionic surfactants are more preferred.

[0107] Examples of nonionic surfactants include nonionic surfactants having a polyoxyalkylene chain, such as polyoxyalkylene fatty acid ester, polyoxyalkylene glycerin fatty acid ester, polyoxyalkylene sorbitan fatty acid ester, polyoxyalkylene alkyl ether, polyoxyalkylene alkylphenyl ether, polyoxyethylene (hardened) castor oil, etc.; nonionic surfactants not having a polyoxyalkylene chain, such as glycerin fatty acid ester, polyglycerin fatty acid ester, propylene glycol fatty acid ester, sorbitan fatty acid ester, sucrose fatty acid ester, fatty acid alkylolamide, etc. Among these nonionic surfactants, nonionic surfactants having a polyoxyalkylene chain are preferred from the viewpoint of further improving the effect of the active ingredient.

[0108] (Content of active ingredients and other ingredients) The amount of the active ingredient of the present inhibitor and the present anti-inflammatory agent is not particularly limited as long as the active ingredient exerts the desired effect. For example, the amount of the active ingredient may be 0.001% by weight to 100% by weight, 0.01% by weight to 100% by weight, 0.1% by weight to 100% by weight, 0.1% by weight to 95% by weight, 0.1% by weight to 90% by weight, 0.1% by weight to 80% by weight, 0.1% by weight to 70% by weight, 0.1% by weight to 60% by weight, 0.1% by weight to 50% by weight, 0.1% by weight to 40% by weight, 0.1% by weight to 30% by weight, 0.1% by weight to 20% by weight, or 0.1% by weight to 10% by weight, based on the total weight of the drug.

[0109] The amount of the components other than the active ingredient contained in the present inhibitor and the present anti-inflammatory agent is not particularly limited. For example, the amount of the components other than the active ingredient may be 0% by weight to 99.999% by weight, 0% by weight to 99.99% by weight, 0% by weight to 99.9% by weight, 5% by weight to 99.9% by weight, 10% by weight to 99.9% by weight, 20% by weight to 99.9% by weight, 30% by weight to 99.9% by weight, 40% by weight to 99.9% by weight, 50% by weight to 99.9% by weight, 60% by weight to 99.9% by weight, 70% by weight to 99.9% by weight, 80% by weight to 99.9% by weight, or 90% by weight to 99.9% by weight.

[0110] (Subject and route of administration) The subjects to which the present inhibitor and the present anti-inflammatory agent are administered are not particularly limited, and may be humans or non-human animals (e.g., livestock, pets, laboratory animals, etc.). Examples of non-human animals include monkeys, chimpanzees, cows, pigs, sheep, goats, horses, dogs, cats, rabbits, mice, and rats.

[0111] The present inhibitor and the present anti-inflammatory agent can be administered to a subject by any administration route. Examples of administration routes include subcutaneous administration, intradermal administration, transdermal administration, intraocular administration, oral administration, transmucosal administration, etc. Parenteral administration is preferred because it is easy to maintain the function of the active ingredient, and among parenteral administration, transdermal administration or transmucosal administration via the eyeball, oral cavity, or other mucous membranes is preferred.

[0112] (Formulations and formulations) The active ingredient and other ingredients can be used as raw materials and mixed by a known method, for example, using a homomixer or a universal mixer, to prepare the present inhibitor and anti-inflammatory agent.

[0113] When administering the present inhibitor and the present anti-inflammatory agent, there is no limit to the dosage, so long as the desired effect is obtained. When administering the present inhibitor and the present anti-inflammatory agent, there is no limit to the administration interval, so long as the desired effect is obtained. The administration interval can be, for example, once every hour to 6 months, once every hour, once every 2 hours, once every 3 hours, once every 6 hours, once every 12 hours, once every day, once every 2 days, once every 3 days, once every 4 days, once every 5 days, once every 6 days, once every week, once every 2 weeks, once every 3 weeks, once every month, once every 2 months, once every 3 months, once every 4 months, once every 5 months, once every 6 months, etc.

[0114] (Methods of treatment, improvement or prevention) The present invention also includes a method for treating, improving, or preventing skin itch, flush, rash, pain, eczema, and / or inflammation, which comprises administering the present inhibitor or the present anti-inflammatory agent to a subject. The subject may be a subject in need of treatment, improvement, or prevention of a disease accompanied by skin itch, flush, rash, pain, eczema, and / or inflammation, or a subject in need of treatment, improvement, or prevention of skin itch, flush, rash, pain, eczema, and / or inflammation associated with irritation by a chemical substance or a drug, etc. For example, the subject may be a subject suffering from the disease, or a subject in contact with the chemical substance, etc. The subject may be a human or a non-human animal. Examples of diseases associated with skin itch, flush, rash, pain, eczema, and / or inflammation include irritant contact dermatitis, allergic contact dermatitis, psoriasis, etc. In addition, the present invention also includes a method for suppressing the activity of TRPV4, which comprises administering the present inhibitor or the present anti-inflammatory agent to a subject. The inhibitor or anti-inflammatory agent can, for example, be applied to the skin of a subject.

[0115] (others) In one embodiment of the present invention, the inhibitor or anti-inflammatory agent can be formulated as a skin topical preparation. When the inhibitor or anti-inflammatory agent is formulated as a skin topical preparation, the inhibitor or anti-inflammatory agent may contain other optional ingredients other than the active ingredient. Examples of other optional ingredients include additives such as lower alcohols, ultraviolet absorbers, powders, antioxidants, preservatives (methylparaben, etc.), fragrances, colorants, chelating agents, cooling agents, thickeners, vitamins, neutralizing agents, amino acids, pH adjusters, whitening agents, anti-inflammatory agents, deodorants, animal and plant extracts, and metal sequestering agents (sodium salt of ethylenediaminetetraacetic acid, etc.). These other optional ingredients may be used alone or in combination of two or more.

[0116] When the present inhibitor or anti-inflammatory agent is formulated as a skin topical preparation, the dosage form of the present inhibitor or anti-inflammatory agent is not particularly limited, but is preferably an ointment, cream, gel, gel-like cream, liniment, lotion, spray, aerosol, emulsion, lotion, serum, gel, etc.

[0117] Furthermore, when the present inhibitor or anti-inflammatory agent is formulated as a skin topical preparation, the application site of the present inhibitor or anti-inflammatory agent is not particularly limited, and examples include the face (forehead, eyes, corners of the eyes, cheeks, mouth, etc.), arms, elbows, backs of the hands, fingertips, feet, knees, heels, neck, armpits, back, etc.

[0118] The present invention is not limited to the above-described embodiments, and various modifications are possible within the scope of the claims. Embodiments obtained by appropriately combining the technical means disclosed in different embodiments are also included in the technical scope of the present invention. EXAMPLES

[0119] The present invention will be described in more detail below with reference to examples, but the present invention is not limited to these examples.

[0120] Isolation of Monocytes Peripheral blood mononuclear cells (PBMCs) were isolated from peripheral blood of healthy volunteers by density gradient centrifugation using human lymphocyte separation medium (GE Healthcare, product name: Ficoll-Paque PLUS). ACK buffer (150 mM NH4Cl, 10 mM KHCO3, 0.1 mM EDTA) was added to the isolated PBMCs and left at room temperature for 15 minutes to hemolyze contaminating red blood cells. Next, dead cells were magnetically labeled using a Dead Cell Removal Kit (Miltenyi Biotec, product name: LS column, product number: 130-042-401) and removed by passing through a MACS column (Miltenyi Biotec, product name: LS column, product number: 130-042-401). CD14 positive cells contained in PBMC were magnetically labeled using CD14 MicroBeads (Miltenyi Biotec, model number: 130-050-201). A CD14 positive cell population was obtained by magnetic separation using a MACS column. Hereinafter, this cell population was defined as monocytes.

[0121] [Cell culture method] Monocytes obtained by the method described above in "Isolation of monocytes" were cultured in RPMI1640 medium (Gibco, model number: 11875-093) containing 20% ​​FBS (fetal bovine serum) (SIGMA, model number: 172012).

[0122] [Method of Differentiating Macrophages] Monocytes obtained by the method described above in [Isolation of monocytes] were cultured for 7 days in RPMI1640 medium containing 20% ​​FBS and antibiotics (Gibco, antibiotic-antimycotic). To differentiate the monocytes into GM-CSF macrophages, 50ng / ml GM-CSF (SHENANDOAH, model number: 100-08) was added and cultured. To differentiate the monocytes into M-CSF macrophages, 50ng / ml M-CSF (SHENANDOAH, model number: 100-03) was added and cultured. On the second or fifth day after the start of culture, RPMI1640 medium containing the same reagents as at the start of culture was added. Adherent cells after 7 days of culture were defined as GM-CSF macrophages or M-CSF macrophages.

[0123] [1. Expression of TRPV4 in macrophages] To confirm the expression of TRPV4 mRNA, monocytes (day 0 monocytes) obtained by the method described in [Isolation of monocytes] above, monocytes cultured for 7 days as a control (day 7 monocytes), and macrophages obtained by the method described in [Differentiation of macrophages] above were prepared. The cells were washed with PBS, and total RNA was extracted using Trireagent (Molecular Research, model number: TR118). The extracted total RNA was stored at -80°C until the experiment.

[0124] After returning to room temperature, chloroform was added, and the aqueous layer containing RNA was collected, to which isopropanol was added to precipitate the RNA. The RNA sample was obtained by washing with 70% ethanol and dissolving in RNAse-free water. cDNA was prepared from this RNA sample using reverse transcriptase (QIAGEN, product name: QuantiTect reverse transcription kit, model number: 205315). This cDNA was detected using a real-time PCR device with SYBR Green mix (TOYOBO, product name: THUNDERBIRD SYBR qPCR Mix, model number: QPS-201). The amount of mRNA was calculated from the obtained data. 18s RNA was used as an endogenous control. The following primers were used. The results are shown in Figures 1 and 2 (n=10 in Figure 2).

[0125] [Table 1]

[0126] As shown in Figure 1, TRPV4 was expressed in monocytes, GM-CSF macrophages, and M-CSF macrophages. As shown in Figure 2, the median expression level of TRPV4 in macrophages was approximately 6.6 times higher in GM-CSF macrophages and approximately 6.9 times higher in M-CSF macrophages than in monocytes.

[0127] 2. TRPV4 activation suppresses IL-1β production Macrophages prepared by the method described in the above [Method for Differentiating Macrophages] were added to a dish containing the macrophages, and the dish was then left at 37°C for 15 minutes and then pipetted to recover the macrophages. The macrophages were cultured for 30 minutes in RPMI1640 medium (Gibco, model number: 11875-093) containing 0.5% FBS (fetal bovine serum) and 30 μM HC067047, and then stimulated with 10 ng / ml LPS and / or 10 μM GSK1016790A for 6 hours.

[0128] After stimulation, polyethylene glycol tert-octylphenyl ether (TritonX-100) was added to the culture supernatant containing the macrophages to a concentration of 0.1 v / v%, and the mixture was allowed to stand on ice to prepare a cell lysate. The prepared cell lysate was stored at 4°C until use in the assay. The mixture was centrifuged at 14000 rpm at 4°C for 5 minutes, and the supernatant was used as a sample. IL-1β was detected using the sample according to the protocol of the kit (manufactured by R&D, product name: Human IL-1β / IL-1F2 Duo Set ELISA). In addition, absorbance was measured using a plate reader (TECAN, product name: Infinite F200 PRO). The change in the amount of cytokine production relative to the amount of cytokine produced when stimulated with LPS alone was compared using a Steel test to see if there was a significant difference. The results are shown in Figure 3 (n=10 in Figure 3).

[0129] As shown in Figure 3, in both GM-CSF and M-CSF macrophages, the simultaneous addition of GSK1016790A, a TRPV4 activator, significantly reduced the amount of IL-1β produced compared to LPS stimulation alone. In addition, it was confirmed that the effect was counteracted in both macrophages by pretreatment with HC067047, a TRPV4 inhibitor.

[0130] [3. TRPV4 activation suppresses IL-1β mRNA levels] To confirm the expression of IL-1β mRNA, macrophages obtained by the method described above in [Macrophage Differentiation Method] were cultured for 6 hours in RPMI1640 medium (gibco, model number: 11875-093) containing 0.5% FBS (fetal bovine serum). After treatment with HC067047 (TCORIS, model number: 4100), a TRPV4 inhibitor, at a concentration of 30 μM for 30 minutes, the cells were stimulated with 10 ng / ml LPS (SIGMA, model number: L2630) to induce IL-1β. Simultaneously with the LPS stimulation, 10 μM of GSK1016790A (wako, model number: 073-06491), a TRPV4 activator, was added. After 6 hours of culture, the cells were washed with PBS and total RNA was extracted with Trireagent (Molecular Research, model number: TR118). The extracted total RNA was stored at −80° C. until the experiment.

[0131] cDNA was prepared in the same manner as described above in [1. Expression of TRPV4 in macrophages], and the cDNA was detected using a real-time PCR device. The amount of mRNA was calculated from the obtained data. GAPDH was used as an endogenous control. The following primers were used. The results are shown in Figure 4 (n=10 in Figure 4).

[0132] [Table 2]

[0133] As shown in Figure 4, in both GM-CSF and M-CSF macrophages, the amount of IL-1β mRNA was significantly reduced by simultaneous addition of GSK1016790A, a TRPV4 activator, compared to LPS stimulation alone. It was also confirmed that the effect was counteracted in both macrophages by pretreatment with HC067047, a TRPV4 inhibitor.

[0134] [4. TRPV4 activation suppresses NLRP3 and caspase-1 production] To confirm the expression of TRPV4, monocytes (day 0 monocytes) obtained by the method described in the above section "Isolation of monocytes" and macrophages obtained by the method described in the above section "Differentiation of macrophages" were prepared. In addition, pcDNA3.1-TRPV4 was expressed in HEK293T cells using polyethleneimine (Polysciences) as a positive control.

[0135] To confirm the protein expression levels of NLRP3 and Caspase-1, macrophages prepared by the method described above in "Macrophage differentiation method" were cultured for 30 minutes in RPMI1640 medium (Gibco, model number: 11875-093) containing 0.5% FBS (fetal bovine serum) and 30 μM HC067047, and then stimulated with 10 ng / ml LPS and / or 10 μM GSK1016790A for 6 hours.

[0136] After washing the cells with PBS, they were dissolved in 2x Laemmli Sample Buffer (BIO RAD, model number: #1610737) containing protease inhibitors and phosphatase inhibitors. DNA contained in the cell lysate was disrupted by ultrasonication and further heated at 95°C for 10 minutes to prepare samples. To detect each protein contained in the obtained samples, anti-TRPV4 antibody (abcam, model number: ab39260), anti-NLRP3 antibody (abcam, model number: ab263899), and anti-Caspase-1 antibody (Cell Singaling TECHNOLOGY, model number: #3866) were used as primary antibodies. To detect β-actin, which is a standard for quantifying proteins, anti-β-actin antibody (Santa Cruz Biotechnology, model number: sc-1616) was used. The results are shown in Figures 5 and 6.

[0137] As shown in Figures 5 and 6, in both GM-CSF and M-CSF macrophages, the simultaneous addition of GSK1016790A, a TRPV4 activator, significantly reduced the production of NLRP3 and Caspase-1 compared to LPS stimulation alone. In addition, it was confirmed that the effect was counteracted in both macrophages by pretreatment with HC067047, a TRPV4 inhibitor.

[0138] 5. Detection of TRPV4 in diseased skin Skin tissues from patients with atopic dermatitis or healthy subjects were labeled by hybridization with a probe that labels TRPV4 mRNA, and immunostained with an antibody that recognizes CD11b, a macrophage marker.

[0139] Hybridization was performed according to the protocol of RNAscope Multiplex Fluorescent Reagent Kit v2 (ACD, product number: 323110). The TRPV4-recognizing probe (ACD, product name: RNAscope Probe Hs-TRPV4, product number: 452221) was stained with 1500-fold diluted Opa1520 (ACD, product name: Opa1520 Reagent Pack, product number: FP1487001KT). Nuclei were stained with DAPI included in RNAscope Multiplex Fluorescent Reagent Kit v2. After hybridization, the skin tissue was reacted with PBS containing 10 v / v% FBS and 0.1 v / v% TritonX-100 at room temperature for 30 minutes and blocked. Then, anti-CD11b antibody (abcam, product name: Anti-CD11b antibody, product number: ab52478) was reacted overnight at 4°C. The skin tissue after the reaction was washed with a surfactant-containing PBS solution. The washed skin tissue was stained by reacting it with an anti-rabbit IgG antibody labeled with a fluorescent dye (manufactured by Thermo Fisher Scientific, product name: Donkey Anti-Rabbit IgG H&L (fluorescent dye: Alexa Fluor (registered trademark) 594, catalog number: A-21207)) at room temperature (25°C) for 2 hours. The stained skin tissue was washed with a surfactant-containing PBS solution. The washed skin tissue was then observed using a confocal microscope (manufactured by Olympus Corporation, product number: FV1200). The results are shown in Figures 7 and 8.

[0140] 7 and 8, it was confirmed that there was a difference in the expression level of TRPV4 in macrophages between skin tissues from patients with atopic dermatitis and those from healthy individuals. This result suggests that the evaluation of anti-inflammatory substances using TRPV4 in macrophages as an indicator is useful. [Industrial Applicability]

[0141] The present invention can be suitably used for the development of anti-inflammatory cosmetics, anti-inflammatory agents, and the like.

Claims

1. A method for evaluating an anti-inflammatory effect of a test sample, comprising the steps of: A method for evaluating a test sample, comprising contacting the test sample with a macrophage, measuring a physiological event caused by activation of TRPV4 in the macrophage, and evaluating the anti-inflammatory effect of the test sample based on the physiological event.

2. A method for evaluating an anti-cell death effect of a test sample, comprising the steps of: A method for evaluating a test sample, comprising contacting the test sample with a macrophage, measuring a physiological event caused by activation of TRPV4 in the macrophage, and evaluating the anti-cell death effect of the test sample based on the physiological event.

3. The method for evaluating a test sample according to claim 1 or 2, wherein the macrophages are GM-CSF macrophages and / or M-CSF macrophages.

4. The method for evaluating a test sample according to any one of claims 1 to 3, wherein the physiological event caused by activation of TRPV4 is at least one selected from the group consisting of suppression of the amount of NLRP3 produced upon inflammatory stimulation, suppression of the amount of Caspase-1 produced upon inflammatory stimulation, and suppression of the amount of cytokine produced upon inflammatory stimulation.

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