Anti-inflammatory agent comprising lysophosphatidic acid, lysophosphatidic acid precursor, or lysophosphatidic acid receptor agonist as active ingredient

Abstract

The present invention addresses the problem of providing a novel anti-inflammatory agent. The present invention is an anti-inflammatory agent comprising lysophosphatidic acid (LPA) or a precursor thereof, an LPAR6 agonist, or a pharmaceutically acceptable salt of these.

Description

Anti-inflammatory agents containing lysophosphatidic acid, its precursor, or its receptor agonist as active ingredients

[0001] The present invention relates to an anti-inflammatory agent comprising lysophosphatidic acid, its precursor, or its receptor agonist as an active ingredient.

[0002] Various treatments have been developed for inflammatory skin diseases. Corticosteroids have traditionally been widely used. However, corticosteroids are known to have side effects such as skin thinning, telangiectasia, rosacea-like dermatitis, and exacerbation of skin infections. Many patients avoid steroids due to these side effects. While non-steroidal anti-inflammatory drugs (NSAIDs) have been developed in recent years, their anti-inflammatory effects are often weak, and side effects such as irritation are problematic. Therefore, the development of new anti-inflammatory agents is desired.

[0003] Lysophosphatidic acid (LPA) is a lysophospholipid and is known as one of the lipid mediators. There are several LPA analogs depending on the binding mode. LPA is mostly found in plasma, but it is also known to be present in skin blisters, hair follicle fluid, saliva, and synovial fluid. LPAR1 to LPAR6 are LPA receptors, all of which are G protein-coupled receptors (GPCRs). The expression patterns of the receptors and downstream signaling differ depending on the tissue, and they perform a variety of functions. Among the main functions, they are widely known to regulate cell proliferation, platelet aggregation, wound healing, and angiogenesis. In the skin, although there are a few reports, such as the association of LPAR1 with systemic sclerosis (SSc) (Non-patent Literature 1), little was known about the function of LPA and LPA receptors in skin immune cells. In particular, the function of LPAR6 (lysophosphatidic acid receptor 6), among LPA receptors, in skin immune cells, and especially its role in regulating immune cell migration in inflammatory diseases, has remained largely unknown until now.

[0004] Signal Transduct Target Ther., 2021;6:45J Immunol., 2014;192(9):4361-4369J Invest Dermatol., 2019;139(5):1010-1022Sci Immunol., 2017;2(18):eaao1135J Lipid Res., 2021;62:100029Anal Chem., 2021;93(8):3867-3875J Immunol., 2009;182(9):5836-5845Sci Immunol., 2022;7(70):eabl9165

[0005] The object of this invention is to provide a novel anti-inflammatory agent.

[0006] The inventors focused on lysophosphatidic acid (LPA) and its receptor as novel targets. Using an imiquimod-induced psoriasis-like dermatitis mouse model, a representative inflammatory skin disease, they confirmed an increase in LPA in psoriatic lesions, finding that LPA contributes to skin inflammation. Furthermore, they found that dermatitis was attenuated by both subcutaneous injection and topical application of LPA. In addition, in psoriatic skin, TCRγδ cells, which produce high levels of the cytokine (interleukin 17: IL-17) important in the pathogenesis, were found to contribute to LPA. ｌｏｗ We discovered that the number of cells decreases with LPA administration, that these cells highly express LPAR6, the receptor for LPA, and that the LPA-LPAR6 axis suppresses the migration of immune cells. Based on these findings, we clarified that the LPA-LPAR6 axis contributes to the suppression of skin inflammation. Based on these findings, we completed the present invention.

[0007] In other words, the present invention is as follows: [1] An anti-inflammatory agent comprising lysophosphatidic acid (LPA) or a precursor thereof, an LPAR6 agonist, or a pharmaceutically acceptable salt thereof. [2] The anti-inflammatory agent according to [1], wherein the LPA has an acyl group having 16 to 22 carbon atoms. [3] The anti-inflammatory agent according to [1] or [2], wherein the LPA has an acyl group having 0 to 6 double bonds. [4] The anti-inflammatory agent according to any one of [1] to [3], wherein the LPA has an acyl group having 18 carbon atoms and 1 double bond. [5] The anti-inflammatory agent according to any one of [1] to [4], wherein the LPA is 1-oleoyl-2-hydroxy-sn-glycero-3-phosphate. [6] The anti-inflammatory agent according to any one of [1] to [5], wherein the precursor is a precursor selected from the group consisting of lysophosphatidylcholine (LPC) and phosphatidic acid (PA). [7] The anti-inflammatory agent according to [1], wherein the LPAR6 agonist is an agonist selected from the group consisting of 9Z-Octadecenicyl phosphorate (ODP) and D-sn-1-O-oleyl-2-methyl-glyceryl-3-phosphothionate (Alkyl-OMPT). [8] The anti-inflammatory agent according to any one of [1] to [7] for suppressing inflammation of the skin or other organs involved in the pathology by immune cells expressing LPAR6. [9] The anti-inflammatory agent according to any one of [1] to [8], which is a topical preparation or an injectable preparation.

[10] A pharmaceutical composition comprising the anti-inflammatory agent according to any one of [1] to [9].

[11] The pharmaceutical composition according to

[10] for treating and / or preventing inflammatory diseases of the skin or other organs involved in the pathology by immune cells expressing LPAR6.

[12] The pharmaceutical composition according to

[11] , wherein the inflammatory disease is a disease related to the migration of cells expressing LPAR6.

[13] The pharmaceutical composition according to

[11] or

[12] , wherein the inflammatory disease is a disease selected from the group consisting of diseases related to IL-17 and skin diseases related to immune cells expressing LPAR6.

[14] The pharmaceutical composition according to any one of

[11] to

[13] , wherein the inflammatory disease is a disease selected from the group consisting of psoriasis vulgaris, psoriatic arthritis, pustular psoriasis, lichen planus, hidradenitis suppurativa, multiple sclerosis, ulcerative colitis, Crohn's disease, rheumatoid arthritis, and atopic dermatitis.

[15] A method for screening an anti-inflammatory agent, comprising the steps of: administering a test substance to immune cells expressing LPAR6; measuring the migratory ability of the immune cells administered the test substance; comparing the migratory ability of the immune cells administered the test substance with the migratory ability of immune cells not administered the test substance; and screening the test substance as a candidate anti-inflammatory agent because the migratory ability of the immune cells administered the test substance is lower than that of immune cells not administered the test substance.

[0008] The present invention may also be in the following embodiments:

[16] A method for suppressing inflammation, comprising administering lysophosphatidic acid (LPA) or its precursor, LPAR6 agonist, or a pharmaceutically acceptable salt thereof to a subject.

[17] Use of lysophosphatidic acid (LPA) or its precursor, LPAR6 agonist, or a pharmaceutically acceptable salt thereof for the suppression of inflammation.

[18] Use of lysophosphatidic acid (LPA) or its precursor, LPAR6 agonist, or a pharmaceutically acceptable salt thereof for use in the suppression of inflammation.

[19] Use of lysophosphatidic acid (LPA) or its precursor, LPAR6 agonist, or a pharmaceutically acceptable salt thereof in the manufacture of an anti-inflammatory agent.

[20] A method for treating and / or prophylactic a disease of the skin or other organs in which LPAR6-expressing immune cells are involved in the pathogenesis, comprising administering lysophosphatidic acid (LPA) or its precursor, an LPAR6 agonist, or a pharmaceutically acceptable salt thereof to a subject.

[21] Use of lysophosphatidic acid (LPA) or its precursor, an LPAR6 agonist, or a pharmaceutically acceptable salt thereof for the treatment and / or prophylactic a disease of the skin or other organs in which LPAR6-expressing immune cells are involved in the pathogenesis.

[22] Use of lysophosphatidic acid (LPA) or its precursor, an LPAR6 agonist, or a pharmaceutically acceptable salt thereof for use in the treatment and / or prophylactic a disease of the skin or other organs in which LPAR6-expressing immune cells are involved in the pathogenesis.

[23] Use of lysophosphatidic acid (LPA) or its precursor, LPAR6 agonist, or pharmaceutically acceptable salt thereof in the manufacture of a pharmaceutical composition for treating and / or preventing inflammatory diseases of the skin or other organs in which immune cells expressing LPAR6 are involved in the pathogenesis.

[0009] This invention provides a novel anti-inflammatory agent targeting the LPA-LPAR6 axis. For example, inflammation can be suppressed by topical application or subcutaneous injection of LPA to the inflamed lesion. Furthermore, it has been suggested that an agonist of LPAR6, an LPA receptor, can exert a similar effect. Since LPA is not a compound but a lysophospholipid that is naturally present in the body, side effects from administration to the body are expected to be limited, and because it is administered locally to the skin, it is expected to be highly safe in terms of side effects.

[0010] Figure 1 shows the results of LPA measurement by matrix-assisted laser desorption / ionization mass spectrometry imaging (MALDI-MSI) using psoriasis skin samples obtained by skin biopsy from human psoriasis patients. Figure 2 shows the results of LPA measurement by liquid chromatography-tandem mass spectrometry (LC-MS / MS) using mouse psoriasis-like skin. A is a diagram showing the administration schedule of imiquimod (IMQ) for creating a mouse model of psoriasis-like dermatitis. B is a graph showing the results of LPA mass spectrometry using ear skin. Error bars indicate standard error (SEM). C is a graph showing the results of LPA mass spectrometry using dorsal skin. Error bars indicate standard error (SEM). Figures 3A-C show the effects of LPA subcutaneous injection on mouse psoriasis-like skin. Figure 3A shows the administration schedule when LPA subcutaneous injection was added to a psoriasis-like dermatitis model in which imiquimod (IMQ) was applied daily to the dorsal surface. Figure 3B is a graph showing various scores that evaluate the degree of psoriasis-like dermatitis on the back skin when IMQ was applied and LPA was injected subcutaneously. As a control, mice administered only IMQ were used (IMQ + Vehicle in the figure). In the figure, ** indicates P < 0.01 and **** indicates P < 0.0001. Error bars indicate the standard error (SEM). Figure 3C is a micrograph used to evaluate the effect of LPA subcutaneous injection on Day 4 when IMQ was applied and LPA subcutaneously was injected. The scale bar indicates 100 μm. Figures 4A to 4E show the effects of topical LPA application on mouse psoriasis-like skin. Figure 4A shows the administration schedule when topical LPA application was added to a psoriasis-like dermatitis model in which imiquimod (IMQ) was applied daily to the back or ear. Figure 4B shows the permeability of LPA into the dermis of the skin 30 minutes and 3 hours after application of LPA cream (Cr) in psoriasis-like dermatitis (Day 3). Figure 4C is a photograph comparing dermatitis on Day 5 when IMQ was applied to the back and when LPA topical application was performed. Figure 4D is a graph showing various scores that evaluate the degree of psoriasis-like dermatitis on the skin of the back when IMQ was applied to the back and when LPA topical application was performed.As a control, mice administered only IMQ were used (IMQ + Vehicle in the figure). In the figure, * indicates P < 0.05, ** indicates P < 0.01, *** indicates P < 0.001, and **** indicates P < 0.0001. Error bars indicate standard error (SEM). Figure 4E is a graph showing the results of comparing the degree of psoriasis-like dermatitis of the ear skin when IMQ was applied to the ear and when LPA was applied topically, based on skin thickening. In the figure, * indicates P < 0.05, ** indicates P < 0.01. Error bars indicate standard error (SEM). Figure 5 is a figure showing the effect of topical application of LPA cream on immune cells in mouse psoriasis-like skin and the results of LPAR6 expression analysis. A is a graph showing the flow cytometry analysis results using ear skin (Day 4) of IMQ-induced psoriasis-like dermatitis. In the figure, * indicates P < 0.05 and ** indicates P < 0.01. Error bars indicate standard error (SEM). B is a graph showing the results of LPAR6 mRNA expression analysis by quantitative PCR in each cell isolated from ear skin (Day 4) of IMQ-induced psoriasis-like dermatitis. Error bars indicate standard error (SEM). Figure 6 shows the results of a migration experiment using Jurkat cells (human acute T-cell leukemia cell line). A shows the results of flow cytometry expression analysis of LPAR6 in Jurkat cells. B shows the results of a migration experiment using Jurkat cells. It shows the migration inhibitory effect of LPA. C shows the results of a migration experiment using Jurkat cells. It shows the migration inhibitory effect of 9Z-Octadecenylphosphonate (ODP), which is known as an agonist of LPAR4, 5, and 6. Figure D shows the results of a migration experiment using Jurkat cells. It shows the migration inhibitory effect of D-sn-1-O-oleyl-2-methyl-glyceryl-3-phosphothionate (Alkyl-OMPT), which is known as an agonist of LPAR1, 3, (4), and 6. Figure E shows the results of a migration experiment using Jurkat cells. It shows the migration inhibitory effect of T7, which is known as an agonist of LPAR4. Figures 7A-C show wild-type mice (in the figures, WT or Lpar6). ＋／＋ ) and LPA6-deficient mice (in the figure, Lpar6KO or Lpar6)－／－ This figure shows the comparative results of psoriasis-like dermatitis in LPA6-deficient mice (Lpar6 in the figure). Figure 7A is a graph showing various scores that evaluate the degree of psoriasis-like dermatitis on the back. In the figure, * indicates P < 0.05, ** indicates P < 0.01, and *** indicates P < 0.001. Error bars indicate standard error (SEM). Figure 7B is a graph showing the results of comparing the degree of skin thickening in psoriasis-like dermatitis on the ear by skin thickening. In the figure, ** indicates P < 0.01, and **** indicates P < 0.0001. Error bars indicate standard error (SEM). Figure 7C is a graph showing the flow cytometry analysis results using the ear (Day 4). In the figure, ** indicates P < 0.01. Error bars indicate standard error (SEM). Figures 8A-B show LPA6-deficient mice (Lpar6 in the figure). －／－ This figure shows the effect of topical application of LPA on psoriasis-like dermatitis in the following cases. Figure 8A is a graph showing the results of evaluating the degree of psoriasis-like dermatitis on the back using a score. Error bars indicate the standard error (SEM). Figure 8B is a graph showing the results of comparison of psoriasis-like dermatitis on the ear by skin thickening. Error bars indicate the standard error (SEM). Figures 9A to 9C show the results of reanalysis of single-cell RNA sequencing data of human skin CD45-positive cells published in Non-Patent Literature 8. Figure 9A is a graph showing all skin CD45-positive cells from healthy skin, atopic dermatitis, and psoriasis in a UMAP (Uniform Manifold Application and Projection) plot. Figure 9B is a graph showing only cells that highly express IL-17A in a UMAP plot for healthy skin, atopic dermatitis, and psoriasis, and showing the mRNA expression level of LPAR6 by the intensity of the color. Figure 9C is a graph showing the mRNA expression levels of LPAR6 in the cells shown in Figure 9B.

[0011] <Anti-inflammatory agent> One embodiment of the present invention is an anti-inflammatory agent comprising lysophosphatidic acid (LPA) or its precursor, an LPAR6 agonist, or a pharmaceutically acceptable salt thereof.

[0012] LPA is a lysophospholipid with a structure in which one fatty acid and a phosphate group are bonded to a glycerol backbone. LPA is a lysophospholipid that is also present in the body, and in the body, it is synthesized from its precursor, lysophosphatidylcholine (LPC), by the action of autotaxin (ATX), a phospholipid metabolic enzyme. In addition, LPA is also synthesized in the body from its precursor, phosphatidic acid (PA), by the action of PA-PLA1α. Therefore, even when these precursors are administered, they are expected to be converted to LPA in the body, thereby exerting the anti-inflammatory effect of the present invention.

[0013] In the present invention, the type of LPA is not particularly limited as long as the anti-inflammatory agent of the present invention exhibits an anti-inflammatory effect. For example, structures of 1-acyl-sn-glycero-3-phosphate or 2-acyl-sn-glycero-3-phosphate can be preferably used.

[0014] The acyl groups constituting LPA may be linear or branched.

[0015] The number of carbon atoms in the acyl group constituting LPA is arbitrary; for example, the number of carbon atoms may be 16 to 22, 17 to 20, or 18.

[0016] Furthermore, the number of double bonds in the acyl group constituting LPA is arbitrary; for example, the number of double bonds may be 0 to 6, 1 to 4, or 1.

[0017] The carbon-to-double-bond ratio of the acyl group constituting the LPA may be, for example, 16:0, 16:1, 17:0, 18:0, 18:1, 18:2, 18:3, 20:3, 20:4, 20:5, or 22:6. Hereinafter, LPAs with a specified carbon-to-double-bond ratio of the acyl group may simply be referred to as 16:0 LPA, 16:1 LPA, 17:0 LPA, 18:0 LPA, 18:1 LPA, 18:2 LPA, 18:3 LPA, 20:3 LPA, 20:4 LPA, 20:5 LPA, or 22:6 LPA. Preferably, LPA has an acyl group with 18 carbon atoms and 1 double bond.

[0018] Examples of LPA having an acyl group with 16 carbon atoms and 0 double bonds include 1-palmitoyl-2-hydroxy-sn-glycero-3-phosphate. Examples of LPA having an acyl group with 16 carbon atoms and 1 double bond include 1-palmitoreyl-2-hydroxy-sn-glycero-3-phosphate. Examples of LPA having an acyl group with 17 carbon atoms and 0 double bonds include 1-heptadecanoyl-2-hydroxy-sn-glycero-3-phosphate. Examples of LPA having an acyl group with 18 carbon atoms and 0 double bonds include 1-stearoyl-2-hydroxy-sn-glycero-3-phosphate. Examples of LPA having an acyl group with 18 carbon atoms and 1 double bond include 1-oleoyl-2-hydroxy-sn-glycero-3-phosphate. Examples of LPA having an acyl group with 18 carbon atoms and 2 double bonds include 1-linoleoyl-2-hydroxy-sn-glycero-3-phosphate. Examples of LPA having an acyl group with 18 carbon atoms and 3 double bonds include 1-linolenoyl-2-hydroxy-sn-glycero-3-phosphate. Examples of LPA having an acyl group with 20 carbon atoms and 3 double bonds include 1-eicosatrienoyl-2-hydroxy-sn-glycero-3-phosphate. Examples of LPA having an acyl group with 20 carbon atoms and 4 double bonds include 1-arachidonoyl-2-hydroxy-sn-glycero-3-phosphate. Examples of LPA having an acyl group with 20 carbon atoms and 5 double bonds include 1-eicosapentaenoyl-2-hydroxy-sn-glycero-3-phosphate. Examples of LPAs having an acyl group with 22 carbon atoms and 6 double bonds include 1-docosahexaenoyl-2-hydroxy-sn-glycero-3-phosphate.

[0019] Precursors of LPA include, for example, lysophosphatidylcholine (LPC) and phosphatidic acid (PA). LPC has a structure in which choline is bonded to the phosphate group of LPA, and PA has a structure in which a fatty acid is bonded to the hydroxyl group of LPA. The description of the acyl group above can be applied to LPA.

[0020] The LPAR6 agonist is not particularly limited as long as it exerts its anti-inflammatory effect as an anti-inflammatory agent in this embodiment by binding to LPAR6, one of the LPA receptors. For example, by binding to LPAR6, it can exert its anti-inflammatory effect on immune cells that highly express LPAR6 (especially TCRγδ ｌｏｗ It can be a substance that suppresses the migration of cells to the site of inflammation. The LPAR6 agonist may bind specifically to LPAR6 only, or it may bind to LPA receptors other than LPAR6. For example, an agonist selected from the group consisting of 9Z-octadeceneyl phosphorate (ODP) and D-sn-1-O-oleyl-2-methyl-glyceryl-3-phosphothionate (Alkyl-OMPT) can be used.

[0021] The components of the anti-inflammatory agent in this embodiment, namely LPA, LPA precursors, and LPAR6 agonists, may be included in the anti-inflammatory agent as pharmaceutically acceptable salts. These may include, but are not particularly limited, salts of alkali metals such as potassium and sodium; salts of alkaline earth metals such as calcium and magnesium; ammonium salts, etc.

[0022] LPA, LPA precursors, and LPAR6 agonists may be prepared by methods known to those skilled in the art, or they may be commercially available products.

[0023] The anti-inflammatory agent of this embodiment can be used to suppress inflammation at any site of administration. For example, it can be preferably used to suppress inflammation in the skin or other organs involved in the pathological condition by immune cells expressing LPAR6. While not particularly limited, other organs include, for example, joints, brain, heart, lungs, liver, kidneys, stomach, small intestine, large intestine, pancreas, spleen, gallbladder, bladder, esophagus, trachea, thyroid gland, adrenal gland, ovary, testis, uterus, lymphoid tissue, and joints, with joints being preferred.

[0024] Suppression of inflammation may mean, for example, that by administering the anti-inflammatory agent of this embodiment, inflammation has disappeared in the affected area, or the degree of inflammation has been reduced, or it may also mean that the occurrence of inflammation has been prevented in areas where inflammation is likely to occur. The degree of inflammation can be evaluated using known indicators, for example, evaluation may be performed based on an indicator that scores the degree of erythema, infiltration, desquamation, etc., or evaluation may be performed based on the body surface area of ​​the inflamed area, or these evaluation methods may be combined with other evaluation methods.

[0025] LPA, LPA precursors, and LPAR6 agonists may be used individually or in combination of multiple types.

[0026] The target animals for administration are not particularly limited and may include mammals in general, such as humans, mice, rats, guinea pigs, hamsters, monkeys, cattle, pigs, goats, sheep, dogs, and cats.

[0027] The anti-inflammatory agent of this embodiment contains lysophosphatidic acid (LPA) or its precursor, an LPAR6 agonist, or a pharmaceutically acceptable salt thereof as an active ingredient. However, the anti-inflammatory agent may consist only of these active ingredients, or may contain any other ingredients as long as the anti-inflammatory agent exerts an anti-inflammatory effect. For example, for formulation purposes, it may contain carriers, excipients, disintegrants, buffers, emulsifiers, suspending agents, analgesics, stabilizers, preservatives, antiseptics, surfactants, lubricants, diluents, coatings, sugar coatings, flavoring and deodorizing agents, pH adjusters, isotonic agents, solubilizers, fragrances, colorants, solubilizers, physiological saline, etc.

[0028] The method of administering the anti-inflammatory agent in this embodiment is not particularly limited as long as it can exert an anti-inflammatory effect, and may be administered parenterally or orally, but parenteral administration is preferred, and it is preferable to administer it locally to the site of inflammation.

[0029] The dosage forms of the anti-inflammatory agent of the present embodiment include, for example, external preparations, injections, tablets, powders, fine granules, granules, capsules, syrups, solutions, suspensions, emulsions, etc. However, in order to directly act on the inflammation site, an external preparation or an injection is preferred.

[0030] The contents of LPA, the precursor of LPA, and the LPAR6 agonist in the anti-inflammatory agent are not particularly limited as long as they can exert an anti-inflammatory effect in the administered subject, and generally vary depending on the dosage form. However, in order to achieve the desired dosage, for example, it may be set within the range of about 0.01% by mass to about 99.9% by mass. Also, the contents of LPA, the precursor of LPA, and the LPAR6 agonist with respect to the total amount of the anti-inflammatory agent may be 1 ng / mL to 1 mg / mL, and also, for example, 1 ng / mg to 1 mg / mg.

[0031] The dosage of the anti-inflammatory agent administered to the subject can be appropriately adjusted according to the age, sex, weight, degree of inflammation, etc. of the administered subject. For example, the amounts of LPA, the precursor of LPA, and the LPAR6 agonist contained in the anti-inflammatory agent may be appropriately set within the range of 0.001 μg to 10000 mg per day. The anti-inflammatory agent with the above dosage may be administered once a day, or may be administered in multiple divided doses per day.

[0032] <Pharmaceutical composition> Another embodiment of the present invention is a pharmaceutical composition containing the above anti-inflammatory agent. Note that all the descriptions in the section <Anti-inflammatory agent> above can be incorporated into this embodiment.

[0033] The pharmaceutical composition of the present embodiment can be used for treating and / or preventing inflammatory diseases. The treatment of a disease may mean that the disease completely disappears, or may mean improving the state of the disease or suppressing the progression of the disease. The prevention of a disease may mean completely preventing the occurrence of the disease, or may mean preventing its aggravation even if the disease has occurred.

[0034] As long as the pharmaceutical composition according to the present embodiment can exert an anti-inflammatory effect, the site of onset of the inflammatory disease is not particularly limited. For example, the skin or other organs in which immune cells expressing LPAR6 are involved in the disease state can be mentioned. Although not particularly limited, examples of other organs include joints, brain, heart, lungs, liver, kidneys, stomach, small intestine, large intestine, pancreas, spleen, gallbladder, bladder, esophagus, trachea, thyroid gland, adrenal glands, ovaries, testes, uterus, lymphoid tissue, etc., and joints are preferred.

[0035] In the examples described later, LPA reduces the number of TCRγδ ｌｏｗ cells in the living body at the inflammatory site, that is, reduces the migration of TCRγδ ｌｏｗ cells to the inflammatory site. In this specification, "TCRγδ ｌｏｗ cells" means γδ T cells that express TCRγδ at a low level, and the expression of TCRγδ is confirmed by flow cytometry, but the expression level refers to a cell population that is at a lower level compared to normal γδ T cells (for example, dendritic epidermal T cells). TCRγδ ｌｏｗ cells are immune cells that express LPAR6, and typically are immune cells that highly express LPAR6. Here, high expression of LPAR6 means that the expression level is higher compared to control cells (for example, TCRβ cells, keratinocytes, fibroblasts, etc.), and when it can be represented by a numerical value such as gene expression level, for example, the expression level is 1.5 times or more, 2 times or more, 3 times or more, 4 times or more, 5 times or more, 10 times or more, 15 times or more higher compared to control cells. TCRγδ ｌｏｗ cells are cells that contribute to IL-17 production, so it was suggested that the amount of IL-17 at the inflammatory site decreases. Also, TCRγδ ｌｏｗ cells were shown to have high gene expression of LPAR6, which is one of the LPA receptors, and it was shown that the inhibition of the migration of TCRγδ ｌｏｗ cells to the inflammatory site by LPA is LPAR6-dependent.

[0036] In other words, the pharmaceutical composition of this embodiment can treat and / or prevent inflammatory diseases associated with the migration of cells expressing LPAR6, preferably cells that highly express LPAR6, through the active ingredients LPA, a precursor of LPA, and an LPAR6 agonist, for example, it can treat and / or prevent inflammatory diseases associated with IL-17. Specifically, it can treat and / or prevent inflammatory diseases selected from the group consisting of psoriasis vulgaris, psoriatic arthritis, pustular psoriasis, lichen planus, hidradenitis suppurativa, multiple sclerosis, ulcerative colitis, Crohn's disease, rheumatoid arthritis, and atopic dermatitis. The mechanism of action of the present invention is thought to be as follows: LPA or its agonist, via LPAR6, affects immune cells that highly express LPAR6 (especially TCRγδ ｌｏｗ It suppresses the migration of IL-17 cells to the site of inflammation. As a result, the number of IL-17-producing cells at the site of inflammation decreases, and inflammation is suppressed. However, the present invention is not limited to the above mechanism of action.

[0037] <Screening Method for Anti-inflammatory Agents> Another embodiment of the present invention is a screening method for anti-inflammatory agents, comprising the steps of: administering a test substance to immune cells that express LPAR6, preferably those that highly express LPAR6; measuring the migratory ability of the immune cells administered the test substance; comparing the migratory ability of the immune cells administered the test substance with the migratory ability of immune cells that have not been administered the test substance; and screening the test substance as a candidate anti-inflammatory agent because the migratory ability of the immune cells administered the test substance is lower than that of immune cells that have not been administered the test substance.

[0038] The immune cells expressing LPAR6 are not particularly limited as long as the screening method of this embodiment can be used, but examples include lymphocyte cell lines such as Jurkat cells (human acute T cell leukemia cell line) and TCRγδ. ｌｏｗ Cells can be used.

[0039] The test substance is not particularly limited as long as it can be used with the screening method of this embodiment, but examples include compounds, antibodies, antigen-binding fragments, peptides, nucleic acids, etc.

[0040] The method for measuring swiftness can be carried out by methods known to those skilled in the art.

[0041] The method of this embodiment may optionally include additional steps.

[0042] The examples are provided for disclosure purposes only and are not intended to limit the scope of the invention.

[0043] <Human Samples> Skin samples were collected from psoriasis patients via skin biopsy. In accordance with the principles of the Declaration of Helsinki, informed consent was obtained from all patients.

[0044] <Mice> All mice used were of the C57BL / 6 strain. The original C57BL / 6 (B6, CD45.2) wild-type (WT) mice were obtained from Nippon SLC Co., Ltd. (Hamamatsu, Japan). All mice were raised in a pathogen-free environment or a standard facility environment, provided with a 12-hour light-dark cycle, ad libitum access to water and standard feed, and mice aged 6 to 10 weeks were used in the experiments.

[0045] <Cell line and cell culture> Jurkat cells (human acute T-cell leukemia cell line) were obtained from the Cell Materials Development Laboratory at the RIKEN BioResource Research Center. The cells were cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS) and various additives (penicillin G sodium, streptomycin sulfate, amphotericin B) at 37°C and 5% CO2. ２ The culture was performed at the specified concentration.

[0046] <RNA Extraction and qRT-PCR> Total RNA was extracted from isolated cells using the RNeasy Fibrous Tissue Mini Kit (Qiagen, California, USA). Complementary DNA was synthesized from the obtained RNA using the RiverTra Ace qPCR RT Master Mix (Toyobo, Osaka, Japan). qRT-PCR was performed on the obtained complementary DNA using the THUNDERBIRD SYBR qPCR Mix (Toyobo) and the ABI Prism 7000 sequence detector (Applied Biosystems, California, USA). mRNA levels were normalized to the glyceraldehyde-3-phosphate dehydrogenase (Gapdh) gene. Data were obtained from 2 －ΔΔＣｔ The analysis was performed using a standard method. The primers are listed in Table 1.

[0047] <Imiquimod (IMQ)-induced psoriasis-like dermatitis mouse model and LPA treatment protocol> For six days, mice aged 6-12 weeks were anesthetized with isoflurane, and 10 mg of 5% IMQ cream (Mochida Pharmaceutical, Tokyo, Japan) was applied to the ear skin and / or 62.5 mg to the back skin. The thickness of the back skin and ear was measured, and various scores were recorded (Non-Patent Literature 2). The thickness of the ear skin was measured using an electronic caliper (4-3086-03; Azwan Corporation). Skin inflammation was evaluated daily before and during IMQ application. To confirm the response of LPA to psoriasis-like dermatitis, 200 μL of 10 μML LPA dissolved in 50% ethanol was subcutaneously injected, or 25 mg of 1% LPA cream was applied, simultaneously with the start of IMQ application. In particular, for topical application of LPA, the cream composition followed previous reports (Non-Patent Literature 3). Specifically, 1-oleoyl-2-hydroxy-sn-glycero-3-sodium phosphate (LPA, Aavnti) was added to a cream consisting of the following substances: 56% water, 10% propylene glycol (#2921745; Nacalai Tesque, Kyoto, Japan), 10% paraffin solution (#2611475; Nacalai Tesque), 5% petrolatum (#3620205; Nacalai Tesque), 10% glycerol monostearate (#43883; Alfa Aesar, Massachusetts, USA), 6% 1-hexadecanol (#0782272; Nacalai Tesque), and 2% polyoxyethylene hexadecyl ether (#0540184; Nacalai Tesque). Each component was mixed and homogenized to prepare the product according to Non-Patent Literature 3. As a control, solvent cream (25 mg) or 1% LPA cream (25 mg) was applied topically to both sides of the right ear for four consecutive days. Four days after the start of the study, skin tissue was collected from the back of the mice and subjected to hematoxylin-eosin staining.

[0048] <Cell Isolation and Preparation> To examine skin cells from mice that developed IMQ-induced psoriasis-like dermatitis, lymphocytes and epidermal keratinocytes were isolated from the entire skin layer. First, the entire mouse skin layer was placed in actase solution (AT104, Innovative Cell Technologies, California, USA) and shaken at 37°C for 10 minutes. Then, liberase™ (05401119001; Roche Diagnostics, Indiana, USA) and DNase I (D4263; Sigma-Aldrich, Massachusetts, USA) were incubated at 37°C for 30 minutes in two sets with shaking. The solution that passed through a 40 μm nylon mesh filter was used as the cell suspension.

[0049] <Antibodies and Flow Cytometry> Skin infiltrating cells and Jurkat cells were used for flow cytometry analysis. The cells were washed twice with FACS buffer (PBS containing 2% FBS, 1 mMEDTA, and 0.1% sodium azide) and stained with fluorescently labeled antibodies at 4°C for 30 minutes. The following monoclonal or polyclonal antibodies were used for flow cytometry: Alexa Fluor 700 anti-mouse CD45.2 antibody (clone 104; 109822, Biolegend), PE / Cyanine 5 anti-mouse CD3ε antibody (clone 145-2C11; 100310, Biolegend), PE / Cyanine 7 anti-mouse TCRβ antibody (clone H57-597; 109222, Biolegend), PE anti-mouse TCRγ / δ antibody (clone GL3; 118108, Biolegend), PerCP / Cyanine 5 anti-mouse CD4 antibody (clone GK1.5; 100434, Biolegend), and Fixed Viability Dye. eFluor780 (65-0865-14, eBiosscience, California, USA). Anti-human / mouse LPAR6 / P2RY5 antibody (rabbit-derived, A05725-1, Boster Bio, California, USA) was used as the primary antibody, and Alexa Fluor488 anti-rabbit IgG (goat-derived, ab150157, Abcam, Cambridge, UK) was used as the secondary antibody. Flow cytometry of stained samples was performed using CytoFLEX S (Beckman Coulter, California, USA) and analyzed with FlowJo software (v10.10.0, TreeStar, Oregon, USA).

[0050] <Cell sorting for QPCR> Dermal TCRγδ ｌｏｗFor QPCR analysis using T cells, TCRβ+ T cells, dermal fibroblasts, and keratinocytes, cells infiltrating mouse skin were sorted using a cell sorter MA900 (Sony, Tokyo, Japan) and separated as TCRγδ+, TCRβ+, EpCAM+, and CD140a (PDGFRa)+, respectively. The following monoclonal antibodies were used for cell isolation: Pacific Blue anti-mouse CD45.2 antibody (clone 104; 109820, Biolegend), PE anti-mouse CD3 antibody (clone 17A2; 100206, Biolegend), APC anti-mouse CD326 (EpCAM) antibody (clone G8.8; 118213, Biolegend), PE / Cyanine7 anti-mouse CD140a antibody (clone APA5; 135911, Biolegend), and Fixable Viability Dye eFluor780 (65-0865-14, eBiosscience, California, USA).

[0051] <Migration Assay> A migration assay to evaluate the motility of cells was performed using a 6.5 mm Transwell (3422; Corning, New York, USA) with a polycarbonate membrane insert with a pore size of 8.0 μm, according to the method described in Non-Patent Literature 4. For Jurkat cells, recombinant human CXC chemokine ligand 12 (CXCL12; final concentration 100 ng / ml) or a solvent control was diluted in migration medium (RPMI1640 containing 0.1% fatty acid-free bovine serum albumin (BSA) and penicillin / streptomycin) and placed in the lower chamber. Furthermore, LPA (1-oleoyl lysophosphatidic acid; 10010093; Michigan, USA) was added to the bottom of the lower chamber to final concentrations of 0, 0.1, 1, and 10 μM, and the effect of LPA on cell migration was investigated. To evaluate the effects of LPA receptor modulators on Jurkat cell migration, ODP (0, 10 μM), Alkyl-OMPT (0, 10 μM), or T7 (0, 10 μM) were used in place of LPA. Jurkat cells (5 × 10) after being subjected to serum starvation for 30 minutes were then examined. ６A suspension containing cells (100 μL) was placed in an upper chamber with a filter having a pore size of 8.0 μm. After incubation at 37°C for 3 hours, the number of cells that moved to the lower chamber was measured using a hemocytometer or flow cytometer.

[0052] <Liquid Chromatography-Tandem Mass Spectrometry (LC-MS / MS) Analysis> Skin samples from mice that developed IMQ-induced psoriasis-like dermatitis were placed in 1.5 mL sample tubes containing 1 mL of methanol and internal standards (17:0 LPA and 17:0 LPC, Alabama, USA), and finely chopped with scissors. Zirconium beads were inserted into the mixture in the tubes, and homogenization was performed using Micro Smash MS-100R (Tommy Seikou Co., Ltd.). The resulting supernatant was collected in tubes and centrifuged. These samples were subjected to LC-MS / MS analysis by the method described in Non-Patent Literature 5. Specifically, LC-ESI-MS / MS analysis was performed using a combination of a UHPLC system (Vanquish, Thermo Fisher Scientific, Tokyo, Japan) and a triple quadrupole mass spectrometer (TSQ Altis, Thermo Fisher Scientific, Tokyo, Japan). A methanol extract (10 μL) was analyzed using an INERT C18 ACR column (2.0 mm × 100 mm, Osaka Soda Co., Ltd., Osaka, Japan) with a gradient program using mobile phase A (5 mM ammonium formate, 95% (v / v) water, pH 4.0) and mobile phase B (5 mM ammonium formate, 95% (v / v) acetonitrile, pH 4.0) (B: ​​60%, then linearly gradient to B: 100% over 6 minutes, maintaining B: 100% for 5 minutes, returning to initial conditions and maintaining for 1.5 minutes). LPA and LPC were monitored by MRM (Multiple Reaction Monitoring) in negative and positive ion modes, respectively. The optimal product ions were m / z 153.0 (LPA) and m / z 184.1 (LPC), and various types of lysophospholipids (LPLs) with different acyl chains (carbon number:double bond number = 16:0, 16:1, 17:0, 18:0, 18:1, 18:2, 18:3, 20:3, 20:4, 22:6) were monitored. Analyte quantification was performed based on the analyte and the corresponding IS peak area ratio (100 nM for 17:0 LPA species and 1 μM for 17:0 LPC species), and this peak area ratio was corrected for the weight ratio of each sample.

[0053] <Matrix-Assisted Laser Desorption / Ionization Mass Spectrometry Imaging (MALDI-MSI)> Human skin from patients or mouse skin from IMQ-induced psoriasis-like dermatitis was immediately frozen in liquid nitrogen and sectioned to a thickness of 10 μm in a cryostat (CM 1860 UV) at -35°C. The sections were mounted on indium tin oxide-coated glass slides (Bruker Daltonics, Massachusetts, USA). These samples were subjected to matrix-assisted laser desorption / ionization mass spectrometry imaging (MALDI-MSI) by the method described in Non-Patent Literature 6. Specifically, the prepared sections were immersed in 50 mM ammonium formate for 5 seconds and then completely dried. To derivatize LPA on the tissue sections, Phos-tag MS-101H ( ６８ A solution (1 mM) of Zn) dissolved in ultrapure water was sprayed using a SunCollect automatic sprayer (Sunchrom, Germany). Next, the MALDI matrix, paranitroaniline, was sprayed using SunCollect. MALDI-MSI analysis was performed using a Fourier transform orbital trap MS (QExactive, Thermo Fisher Scientific, California, USA) connected to a MALDI laser system (AP-SMALDI5, TransMIT, Giessen, Germany) in full scan mode with a mass resolution of 140,000 and a mass range of 700–1400. The laser scan pitch was set to 40 μm. Ion images were reconstructed using an IMAGEREVEAL™ MS (Shimadzu Corporation, Kyoto, Japan) with a mass tolerance of ±0.01 Da.

[0054] <Example 1: Increased LPA in lesions of human psoriasis skin> LPA was measured by imaging mass spectrometry using skin samples taken from human psoriasis patients. LPA with different acyl chains (number of carbon atoms: number of double bonds = 16:0, 18:0, 18:1) was measured. The results showed that various LPAs were increased in lesions compared to non-lesion areas (Figure 1).

[0055] <Example 2: Creation and Evaluation of an Imiquimod (IMQ)-Induced Psoriasis-like Dermatitis Mouse Model> A psoriasis-like dermatitis mouse model was created by applying imiquimod (IMQ) daily for six days (Figure 2A). IMQ was applied to the skin of the back and ears, respectively. LPA was measured daily from Day 0 to Day 6, with the first day of IMQ application designated as Day 0. Various types of LPA with different acyl chains (carbon number:double bond number = 16:0, 16:1, 17:0, 18:0, 18:1, 18:2, 18:3, 20:3, 20:4, 22:6) were measured. The vertical axis of each graph shows the relative value with respect to LPA with an acyl chain having a carbon number:double bond ratio of 17:0. The ear and back skin of the psoriasis-like dermatitis mouse model showed elevated levels of various LPAs, similar to human psoriasis skin (Figures 2B-C). Therefore, the following analyses were performed using the IMQ-induced psoriasis-like dermatitis mouse model.

[0056] <Example 3: Effects of LPA Subcutaneous Injection on Mouse Psoriasis-like Skin> To confirm the effects of LPA subcutaneous injection on mouse psoriasis-like skin, a psoriasis-like dermatitis model in which imiquimod (IMQ) was applied daily to the back was further enhanced with LPA subcutaneous injection (Figure 3A). In this example, LPA with an acyl chain having a carbon-to-double-bond ratio of 18:1 was used. Day 0 was designated as the first day of IMQ application to the back of the mouse and LPA subcutaneous injection, and the degree of psoriasis-like dermatitis was evaluated daily from Day 0 to Day 6 using various inflammation scores (erythema score, desquamation score, infiltration score, and total score). As a result, mice that received LPA subcutaneous injection showed a decrease in all scores compared to mice that did not receive LPA subcutaneous injection, and a particularly significant decrease in all scores was shown on Day 4 (Figure 3B). Furthermore, tissue evaluation on Day 4 showed that mice that received LPA subcutaneously exhibited suppressed skin thickening (Figure 3C). Therefore, it was demonstrated that local administration of LPA via subcutaneous injection reduces inflammation.

[0057] <Example 4: Effects of topical application of LPA on mouse psoriasis-like skin> To confirm the effects of topical application of LPA on mouse psoriasis-like skin, a psoriasis-like dermatitis model in which imiquimod (IMQ) was applied daily to the back or ear was further enhanced by the topical application of a cream containing LPA (Figure 4A). In this example, LPA with an acyl chain having a carbon-to-double-bond ratio of 18:1 was used. First, imaging mass spectrometry was used to confirm whether LPA penetrated into the subcutaneous tissue when the LPA cream was applied topically. As a result, it was confirmed that a large amount of LPA remained near the epidermis 30 minutes after application of the LPA cream, but significant penetration occurred after 3 hours (Figure 4B). When comparing the dermatitis on the backs of mice on Day 5, it was confirmed that the dermatitis was reduced in the mice to which LPA was applied topically (Figure 4C). Furthermore, the degree of psoriasis-like dermatitis was evaluated daily from Day 0 to Day 6, with the first day of IMQ application and LPA topical application to the back of the mice designated as Day 0. Various inflammation scores (erythema score, desquamation score, infiltration score, and sum of scores) were used to assess the condition. The results showed that mice treated with LPA topical application showed a decrease in all scores compared to mice not treated with LPA topical application (Figure 4D). Additionally, the degree of psoriasis-like dermatitis was evaluated daily from Day 0 to Day 6, with the first day of IMQ application and LPA topical application to the ears designated as Day 0, based on skin thickening associated with ear swelling. The results showed that mice treated with LPA topical application showed reduced ear thickening compared to mice not treated with LPA topical application (Figure 4E). Therefore, it was demonstrated that topical application of LPA reduces inflammation.

[0058] <Example 5: Effect of topical application of LPA cream on immune cells in mouse psoriasis-like skin and analysis of LPAR6 expression> To analyze the reduction of inflammation by topical application of LPA cream, the number of immune cells was analyzed by flow cytometry (Figure 5A). In this example, LPA with an acyl chain having a carbon-to-double-bond ratio of 18:1 was used. As a result, topical application of LPA resulted in an increase in TCRγδ, an important cell that produces interleukin-17. ｌｏｗA decrease in the number of cells was observed. Here, interleukin 17 is known to be important in psoriatic dermatitis (Non-Patent Literature 7). Furthermore, when the gene expression of various cells collected from ear skin in imiquimod-induced psoriasis was examined, TCRγδ ｌｏｗ In cells, high gene expression of LPAR6, one of the LPA receptors, was shown (Figure 5B).

[0059] <Example 6: Migration Experiment Using Jurkat Cells> A migration experiment was conducted to investigate the function of LPAR6 in immune cells. Since it is difficult to isolate a sufficient number of immune cells from the skin for in vitro experiments, Jurkat cells (human acute T-cell leukemia cell line), a representative lymphocyte cell line, were used. In Jurkat cells, endogenous expression of LPAR6 was confirmed by flow cytometry (Figure 6A). Furthermore, when a migration experiment was performed on Jurkat cells, a concentration-dependent inhibitory effect on migration was confirmed (Figure 6B). In this example, LPA with an acyl chain having a carbon-to-double-bond ratio of 18:1 was used as the LPA. Next, various existing LPA receptor agonists were used to identify the responsible receptor. Specifically, migration experiments were conducted on Jurkat cells using 9Z-Octadecenyl phosphorate (ODP), known as an agonist for LPAR4, 5, and 6; D-sn-1-O-oleyl-2-methyl-glyceryl-3-phosphothionate (Alkyl-OMPT), known as an agonist for LPAR1, 3, (4), and 6; and T7, known as an agonist for LPAR4 (Figures 6C-E, respectively). As a result, a migration inhibitory effect on Jurkat cells was confirmed when using ODP or Alkyl-OMPT, while no migration inhibitory effect was confirmed when using T7. Therefore, it was shown that the inhibition of Jurkat cell migration by LPA is LPAR6-dependent.

[0060] <Example 7: Comparison of the contribution of LPAR6 to mouse psoriasis-like skin in wild-type mice and LPAR6-deficient mice> Based on the results of Examples 5 and 6, LPAR6 and TCRγδ ｌｏｗBecause cells were expected to be important for skin inflammation, wild-type and LPAR6-deficient (Lpar6 －／－ A comparative study was conducted using mice. LPAR6-deficient mice were created by crossing conditional LPAR6 fl / fl mice (accession number: CDB0977K; RIKEN) with CAGcre mice that systemically express Cre recombinase. As a result, dermatitis was exacerbated in LPAR6-deficient mice in both the back (Figure 7A) and ears (Figure 7B), and TCRγδ in the skin of the ear was also increased. ｌｏｗ The number of cells was increased in LPAR6-deficient mice (Figure 7C). This suggests that LPAR6 is involved in TCRγδ ｌｏｗ This suggests that it negatively controls the number of cells and has an anti-inflammatory effect.

[0061] <Example 8: Comparison of the effect of topical LPA application on mouse psoriasis-like skin in wild-type mice and LPAR6-deficient mice> Based on the results of Examples 5, 6, and 7, in order to confirm whether the anti-inflammatory effect of topical LPA application observed in Example 4 is LPAR6-dependent, the effect of topical LPA containing an 18:1 acyl chain on mouse psoriasis-like skin was compared between wild-type mice and LPAR6-deficient mice. As a result, the anti-inflammatory effect of topical LPA disappeared in both the back (Figure 8A) and the ear (Figure 8B). This suggests that the therapeutic effect of topical LPA is LPAR6-dependent.

[0062] <Example 9: High Expression of LPAR6 in IL-17A High-Expression Cells in Human Psoriasis Lesions> Re-analysis of single-cell RNA sequencing data of human skin CD45-positive cells reported in Non-Patent Literature 8 (Figure 9A) revealed that IL-17A-expressing cells were more numerous in psoriasis skin than in healthy skin or atopic dermatitis skin (Figures 9B and 9C). This suggests that the present invention can be applied at least to human psoriasis.

[0063] Based on these results, it became clear that the reason why topical application of LPA suppressed inflammation and reduced immune cells is that the administered LPA exerted a migration-inhibiting effect via LPAR6, one of the LPA receptors on immune cells.

Claims

1. An anti-inflammatory agent comprising lysophosphatidic acid (LPA) or its precursor, an LPAR6 agonist, or a pharmaceutically acceptable salt thereof.

2. The anti-inflammatory agent according to claim 1, wherein the LPA has an acyl group having 16 to 22 carbon atoms.

3. The anti-inflammatory agent according to claim 1, wherein the LPA has acyl groups with 0 to 6 double bonds.

4. The anti-inflammatory agent according to claim 1, wherein the LPA has an acyl group having 18 carbon atoms and 1 double bond.

5. The anti-inflammatory agent according to claim 1, wherein the LPA is 1-oleoyl-2-hydroxy-sn-glycero-3-phosphate.

6. The anti-inflammatory agent according to claim 1, wherein the precursor is a precursor selected from the group consisting of lysophosphatidylcholine (LPC) and phosphatidic acid (PA).

7. The anti-inflammatory agent according to claim 1, wherein the LPAR6 agonist is an agonist selected from the group consisting of 9Z-Octadecenyl phosphorate (ODP) and D-sn-1-O-oleyl-2-methyl-glyceryl-3-phosphothionate (Alkyl-OMPT).

8. The anti-inflammatory agent according to claim 1, for suppressing inflammation of the skin or other organs involved in a pathological condition by immune cells expressing LPAR6.

9. The anti-inflammatory agent according to claim 1, which is a topical preparation or an injectable preparation.

10. A pharmaceutical composition comprising the anti-inflammatory agent described in any one of claims 1 to 9.

11. The pharmaceutical composition according to claim 10 for treating and / or preventing inflammatory diseases of the skin or other organs in which immune cells expressing LPAR6 are involved in the pathogenesis.

12. The pharmaceutical composition according to claim 11, wherein the inflammatory disease is a disease related to the migration of cells expressing LPAR6.

13. The pharmaceutical composition according to claim 11, wherein the inflammatory disease is a disease selected from the group consisting of diseases related to IL-17 and skin diseases related to immune cells expressing LPAR6.

14. The pharmaceutical composition according to claim 11, wherein the inflammatory disease is a disease selected from the group consisting of psoriasis vulgaris, psoriatic arthritis, pustular psoriasis, lichen planus, hidradenitis suppurativa, multiple sclerosis, ulcerative colitis, Crohn's disease, rheumatoid arthritis, and atopic dermatitis.

15. A method for screening anti-inflammatory agents, comprising the steps of: administering a test substance to immune cells expressing LPAR6; measuring the migratory ability of the immune cells administered the test substance; comparing the migratory ability of the immune cells administered the test substance with the migratory ability of immune cells not administered the test substance; and screening the test substance as a candidate anti-inflammatory agent based on the fact that the migratory ability of the immune cells administered the test substance is lower than that of immune cells not administered the test substance.

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