Method for evaluating elicitation of foreign body response reaction in skin of test sample, method for searching substance having ability to inhibit foreign body response reaction in skin, and agent for inhibiting foreign body response reaction in skin

Abstract

To provide a method for simply and efficiently evaluating the inducibility of a foreign matter response reaction in the skin of a test sample without using an animal, and to provide a substance having the inhibitory properties of the foreign matter response reaction in the skin and a method for searching the same.SOLUTION: The method includes a step of bringing keratinocytes transformed with a vector having an enhancer sequence containing a foreign body-responsive sequence and a sequence encoding a reporter protein downstream of the enhancer sequence into contact with a solution containing a test sample, a step of measuring the expression level of the reporter protein in the keratinocytes, and a step of evaluating the inducibility of a foreign body response reaction of the test sample in the skin based on the expression level of the reporter protein. The problem is solved by providing an agent for suppressing foreign matter response reaction in the skin containing fregula 1 found by the search method.SELECTED DRAWING: Figure 3

Description

[Technical Field]

[0001] The present invention relates to a foreign body response reaction in the skin, and more specifically, to a method for evaluating the ability of a test sample to induce a foreign body response reaction in the skin, a method for searching for substances that have the ability to inhibit a foreign body response reaction in the skin, and an agent that inhibits a foreign body response reaction in the skin discovered using the searching method. [Background technology]

[0002] The skin is an organ that covers the entire body and protects the body from external influences. Various chemicals exist in the environment, and the skin is constantly exposed to these chemicals. For example, many chemicals are contained in essential daily necessities such as cosmetics, detergents, toiletries, food, and medicines. Furthermore, as the problem of environmental pollution has worsened in recent years, the environment, including the atmosphere, oceans, and soil, also contains a large number of chemicals. Furthermore, the number of chemicals surrounding us continues to increase. For example, the number of chemicals registered in the Chemical Abstracts Service (CAS), the world's largest chemical substance database, reached 100 million in June 2015 and has already exceeded 177 million as of February 2021. Given this situation, it is extremely important to accurately assess the effects of environmental chemicals on the skin.

[0003] Known methods for evaluating the effects of chemicals on the skin include methods using mice or guinea pigs, such as the Guinea Pig Maximization Test (GPMT) (Non-Patent Document 1) and the Local Lymph Node Assay (LLNA) (Non-Patent Document 2). However, these methods, which involve sacrificing the lives of animals, are not only unethical but also inefficient because they require a relatively long time to perform. Various in vitro testing methods have been developed to replace the above-mentioned animal-based methods (Non-Patent Document 3), but all of these test methods focus on the activation of immune responses in the skin by exogenous chemicals and model this activation step, and therefore only evaluate a small portion of the effects of exogenous chemicals on the skin.

[0004] Meanwhile, in recent years, it has gradually become clear that the aryl hydrocarbon receptor (AhR) and xenobiotic response element (XRE) are involved in the foreign body response in skin to chemicals present in the environment. For example, Non-Patent Document 4 suggests that environmental pollutants in the air induce a foreign body response in skin involving AhR and XRE, and that the induction of such a foreign body response enhances the expression of the neurotrophic factor artemin, leading to abnormal nerve outgrowth into the epidermis. This abnormal nerve outgrowth causes pruritic hypersensitivity (alloneosis), which in turn leads to loss of skin barrier function and atopic dermatitis. Meanwhile, Non-Patent Document 5 suggests that activation of AhR in skin induces the expression of matrix metalloproteinases (MMPs), and that collagen degradation due to MMP expression contributes to skin aging and wrinkle formation. Thus, the foreign body response in the skin involving AhR and XRE causes various skin problems such as atopic dermatitis and wrinkles.

[0005] The aromatic hydrocarbon receptor (AhR) is a receptor-type transcription factor discovered as a receptor for dioxins. It is known to bind to ligand molecules such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and induce the expression of xenobiotic-metabolizing enzymes. When activated by a ligand molecule, AhR translocates into the cell nucleus, recognizes a specific base sequence in the enhancer element of the transcriptional regulatory region, and activates the expression of downstream genes. This specific base sequence is called the xenobiotic response element (XRE) and is known to have the sequence 5'-CACGC-3'. Because it was discovered as a receptor for dioxins, the ligands that activate the aromatic hydrocarbon receptor (AhR) have been thought to be primarily polycyclic aromatic hydrocarbons (PAHs) and halogenated aromatic hydrocarbons (HAHs). However, in recent years, it has become clear that the aromatic hydrocarbon receptor (AhR) is activated by binding with not only polycyclic aromatic hydrocarbons and halogenated aromatic hydrocarbons but also chemicals with a wide variety of structures, and that this activation plays an important role in maintaining homeostasis in the body (Non-Patent Document 6). [Prior art documents] [Non-patent literature]

[0006] [Non-Patent Document 1] B. Magnusson, and AM Kligman, Journal of Investigative Dermatology 1969,52:268-276. [Non-patent document 2] DA Basketter et al., Food and Chemical Toxicology 2002,40:593-598. [Non-patent document 3] Sebastian Hoffmann et al., Critical Reviews in Toxicology 2018,48:344-358. [Non-patent document 4] Takanori Hidaka et al., Nature Immunology 2017,18:64-73. [Non-Patent Document 5] E. Dupont, J. Gomez, and D. Bilodeau, International Journal of Cosmetic Science 2013,35:224-232. [Non-patent document 6] Michael S. Denison and Scott R. Nagy, Annual Review of Pharmacology and Toxicology 2003,43:309-334. Summary of the Invention [Problem to be solved by the invention]

[0007] As described above, when a foreign body response is induced in the skin, it can cause various skin problems such as the onset of atopic dermatitis, skin aging, and wrinkle formation. It has become clear that activation of the aromatic hydrocarbon receptor (AhR), which can cause a foreign body response in the skin, can be induced by a wide variety of chemicals. However, to the inventors' knowledge, the foreign body response in the skin, which involves AhR and XRE, has not been taken into consideration at all when evaluating the safety and effects of various chemical-containing products, such as cosmetics, quasi-drugs, and food products, on the skin. Furthermore, no substance is known that can be safely applied to the human body and that inhibits such a foreign body response.

[0008] The present invention has been made to solve the problems of the prior art as described above, and aims to provide a method for easily and efficiently evaluating the ability of various test samples to induce a foreign body response in the skin without using animals, from the viewpoint of animal welfare. In another aspect, the present invention aims to provide a substance that has the ability to inhibit a foreign body response in the skin, and a method for searching for such a substance. [Means for solving the problem]

[0009] In the course of intensive research efforts to solve the above-mentioned problems, the present inventors have noticed that genes whose expression is enhanced by a foreign body response reaction involving the aromatic hydrocarbon receptor (AhR) and a foreign body response element (XRE) have an enhancer sequence containing XRE, an element that controls the expression level of downstream genes in response to a foreign body response reaction, in their transcriptional regulatory region. They have then discovered that by transforming keratinocytes with a reporter vector that has an XRE-containing enhancer sequence in its transcriptional regulatory region and a sequence encoding an easily detectable reporter protein downstream of the enhancer sequence, and then contacting the transformed keratinocytes with a test sample, it is possible to measure the expression level of the reporter protein, which reflects the level of induction of a foreign body response reaction in the skin by the test sample, and have completed the present invention.

[0010] That is, the present invention provides a method for evaluating the ability of a test sample to induce a foreign body response in the skin, comprising: (1) contacting a keratinocyte transformed with a reporter vector having an enhancer sequence containing a foreign body response element (XRE) and a sequence encoding a reporter protein downstream of the enhancer sequence with a solution containing the test sample; (2) measuring the expression level of the reporter protein in the keratinocytes that have been contacted with a solution containing the test sample; and (3) evaluating the ability of the test sample to induce a foreign body response in the skin based on the measured expression level of the reporter protein; The present invention solves the above problems by providing an evaluation method including the steps of:

[0011] In another aspect, the present invention provides a method for searching for a substance that has an inhibitory effect on a foreign body response in skin, the method comprising: (1) contacting a keratinocyte transformed with a reporter vector having an enhancer sequence containing a foreign substance response element (XRE) and a sequence encoding a reporter protein downstream of the enhancer sequence with a test solution containing a candidate substance and a reference solution not containing the candidate substance; (2) measuring the expression level of the reporter protein in the keratinocytes contacted with the test solution and in the keratinocytes contacted with the reference solution; and (3) evaluating the candidate substance as having an inhibitory effect on a foreign body response in skin when the expression level of the reporter protein in the keratinocytes contacted with the test solution is lower than the expression level of the reporter protein in the keratinocytes contacted with the reference solution; The present invention solves the above-mentioned problems by providing a searching method comprising the steps of: In a preferred embodiment, the test solution and the reference solution contain a substance that induces a foreign body response in the skin.

[0012] Incidentally, as mentioned above, activation of the foreign body response in the skin is known to enhance the production of the neurotrophic factor artemin and matrix metalloproteinase 9, leading to the formation of atopic dermatitis and wrinkles. That is, in a preferred embodiment, the above-mentioned method for searching for substances that have the ability to inhibit the foreign body response in the skin is advantageously used to search for substances for preventing and / or treating atopic dermatitis and substances for preventing and / or ameliorating wrinkle formation.

[0013] As a result of further research efforts using the above-mentioned search method, the present inventors discovered that 5-hydroxy-4-phenyl-2(5H)butenolide (hereinafter sometimes referred to as "Fregulide 1"), a butenolide contained in fragrant vinegar, has the ability to inhibit the foreign body response in the skin. Further detailed investigation of the ability of 5-hydroxy-4-phenyl-2(5H)butenolide to inhibit the foreign body response in the skin revealed that 5-hydroxy-4-phenyl-2(5H)butenolide significantly inhibits the production of genes transcribed in conjunction with the xenobiotic response element (XRE), specifically the neurotrophic factor artemin and matrix metalloproteinase 9.

[0014] That is, in yet another aspect, the present invention solves the above-mentioned problems by providing an agent for suppressing the production of matrix metalloproteinase 9, which contains 5-hydroxy-4-phenyl-2(5H)butenolide and / or a salt thereof as an active ingredient. Matrix metalloproteinase 9 is known to degrade type IV collagen and laminin, which are basement membrane components constituting the extracellular matrix, and elastin, which is a dermal matrix component, and the production of matrix metalloproteinase 9 is thought to cause wrinkles. That is, the agent for suppressing the production of matrix metalloproteinase 9, which contains 5-hydroxy-4-phenyl-2(5H)butenolide and / or a salt thereof as an active ingredient, can be suitably used for preventing and / or improving wrinkles.

[0015] Meanwhile, in yet another aspect, the present invention solves the above-mentioned problems by providing an agent for inhibiting the production of artemin, which contains 5-hydroxy-4-phenyl-2(5H)butenolide and / or a salt thereof as an active ingredient. As described above, the production of artemin in the skin is thought to induce abnormal proliferation of nerve cells, leading to atopic dermatitis. That is, the agent for inhibiting the production of artemin, which contains 5-hydroxy-4-phenyl-2(5H)butenolide and / or a salt thereof as an active ingredient, can be suitably used for the prevention and / or treatment of atopic dermatitis.

[0016] Currently, steroids (e.g., prednisolone, hydrocortisone, etc.) and immunosuppressants (e.g., tacrolimus, pimecrolimus, etc.) are known as drugs commonly used to treat allergic dermatitis, including atopic dermatitis. However, these drugs all aim to suppress allergic reactions in the skin and alleviate the symptoms of allergic dermatitis, but do not fundamentally treat the cause of allergic dermatitis. The agent that suppresses the production of artemin according to one aspect of the present invention is distinct from the above-mentioned conventional agents for treating atopic dermatitis, and has the advantage of being able to completely cure atopic dermatitis because it can suppress the production of artemin, which is thought to be the cause of atopic dermatitis. [Effects of the Invention]

[0017] According to the evaluation method of the present invention, the ability of a test sample to induce a foreign body response in the skin can be easily and quantitatively evaluated. Furthermore, according to the screening method of the present invention, a substance capable of inhibiting the foreign body response in the skin can be easily and quickly screened. Furthermore, according to the agent of the present invention, the expression of a gene transcribed in association with a foreign body response element (XRE) can be suppressed, and the production of matrix metalloproteinase 9, which causes wrinkles, and / or artemin, which causes atopic dermatitis, can be suppressed. [Brief explanation of the drawings]

[0018] [Figure 1] FIG. 1 shows the structure of a reporter vector encoding a foreign substance response element (XRE) and, downstream of the element, a minimal promoter and a reporter protein. [Figure 2] FIG. 1 shows the appearance of keratinocytes (XRE-NLuc::HaCaT cells) transformed with a reporter vector encoding a foreign body response element (XRE) and a minimal promoter and reporter protein downstream of the element. [Figure 3] FIG. 1 shows the expression level of a reporter protein in XRE-NLuc::HaCaT cells contacted with a given concentration of FICZ. [Figure 4] FIG. 1 shows the results of quantifying, by PCR, the expression level of artemin mRNA in HaCaT cells that had been contacted with a given concentration of FICZ. [Figure 5] FIG. 1 shows the results of quantifying, by PCR, the expression level of matrix metalloproteinase 9 mRNA in HaCaT cells that had been contacted with a given concentration of FICZ. [Figure 6] FIG. 1 shows the expression level of a reporter protein in XRE-NLuc::HaCaT cells contacted with a medium containing a predetermined concentration of FICZ as well as a predetermined concentration of flegulin 1 (FG1) or cinnamaldehyde (CA). [Figure 7] FIG. 1 shows the expression level of artemin mRNA in HaCaT cells contacted with a medium containing a predetermined concentration of FICZ and a predetermined concentration of flegulin 1 (FG1). [Figure 8] FIG. 1 shows the expression level of artemin mRNA in HaCaT cells contacted with a medium containing a predetermined concentration of FICZ and a predetermined concentration of flegulin 1 (FG1). [Figure 9] FIG. 1 shows the results of quantifying, by PCR, the expression level of matrix metalloproteinase 9 (MMP9) mRNA in HaCaT cells that had been contacted with a given concentration of Fleglide 1. [Figure 10]FIG. 1 shows the results of quantifying, by PCR, the expression level of matrix metalloproteinase 2 (MMP2) mRNA in HaCaT cells that had been contacted with a given concentration of fleglide 1. [Figure 11] FIG. 1 shows the results of evaluating, using gelatin zymography, the protein level expression level of matrix metalloproteinase 9 (MMP9) in HaCaT cells contacted with a given concentration of fleglide 1. [Figure 12] FIG. 1 shows the results of quantifying, by PCR, the expression level of matrix metalloproteinase 9 (MMP9) mRNA in HaCaT cells that have been contacted with a given concentration of retinoic acid or nicotinamide. [Figure 13] FIG. 1 shows the results of quantifying, by PCR, the expression level of matrix metalloproteinase 2 (MMP2) mRNA in HaCaT cells that have been contacted with a given concentration of retinoic acid or nicotinamide. [Figure 14] FIG. 1 shows the results of quantifying, by PCR, the expression level of TIMP-1 (Tissue Inhibitor of Matrix Metalloprotease-1) mRNA in HaCaT cells contacted with a given concentration of Fleguride 1. [Figure 15] FIG. 1 shows the results of quantifying, by PCR, the expression level of TIMP-1 (Tissue Inhibitor of Matrix Metalloprotease-1) mRNA in HaCaT cells that had been contacted with a given concentration of retinoic acid or nicotinamide. [Figure 16] FIG. 1 shows the results of evaluating the cell viability of HaCaT cells treated with a given concentration of Fregulide 1 using the WST-1 method. DETAILED DESCRIPTION OF THE INVENTION

[0019] The present invention will be described in more detail below.

[0020] <1. Method for evaluating the induction of foreign body responses in the skin> First, a method for evaluating the ability of a test sample to induce a foreign body response in the skin according to the present invention will be described.

[0021] As used herein, the term "foreign body response in skin" refers to a foreign body response in skin involving a foreign body response element (XRE). When a foreign body response in skin involving a foreign body response element (XRE) is induced, the expression of a gene having the foreign body response element (XRE) as an enhancer in its transcriptional regulatory region is activated. This is presumably due primarily to the aryl hydrocarbon receptor (AhR) recognizing the foreign body response element (XRE) and specifically activating the expression of genes located downstream of it. The foreign body response in skin can also be referred to as a foreign body response in skin involving the aryl hydrocarbon receptor (AhR) and the foreign body response element (XRE).

[0022] On the other hand, the ability to induce a foreign body response refers to the property of inducing such a foreign body response, and is a concept that encompasses its strength. A high ability to induce a foreign body response means that the foreign body response is strongly activated, and a low ability to induce a foreign body response means that the property of activating the foreign body response is weak. Inducing a foreign body response, more specifically, means expressing and / or enhancing the expression of a gene transcribed with the involvement of a foreign body response element (XRE). Therefore, when a substance capable of inducing a foreign body response comes into contact with skin cells, it expresses or enhances the expression of a gene transcribed with the involvement of a foreign body response element (XRE) in the cells. Incidentally, gene expression means the production of a protein encoded by that gene, and enhancing gene expression means increasing the amount of protein produced by that gene.

[0023] To reiterate, the xenobiotic response element (XRE) is an enhancer sequence found to be specifically recognized by the activated aryl hydrocarbon receptor (AhR), and is known to have the sequence 5'-CACGC-3'. The cytoplasmic aryl hydrocarbon receptor (AhR) is known to translocate to the cell nucleus upon binding with a self-activating ligand molecule and becoming activated, where it specifically enhances the expression of genes that have enhancer sequences containing the xenobiotic response element (XRE) in their transcriptional regulatory regions.

[0024] Meanwhile, the skin is an organ that protects the outer surface of the living body and is composed of skin cells such as keratinocytes, fibroblasts, and corneocytes. The skin is composed of three layers: the epidermis, dermis, and subcutaneous tissue. The foreign body response in the skin is thought to be primarily triggered by the binding of exogenous foreign substances to the aromatic hydrocarbon receptor (AhR) expressed in keratinocytes, which account for approximately 95% of all epidermal cells. Therefore, keratinocytes are particularly suitable for use in the evaluation method of the present invention.

[0025] The keratinocytes used in the evaluation method of the present invention are not particularly limited, and keratinocytes derived from animals such as humans, mice, rats, hamsters, rabbits, dogs, cats, pigs, sheep, horses, and cows can be used, but human-derived keratinocytes are preferred. Keratinocytes may be collected from animals, or may be commercially available established cell lines. For example, established cell lines of human-derived keratinocytes include HaCaT cells and NHEK cells.

[0026] The evaluation method of the present invention uses transformed keratinocytes such as those described above. The reporter vector used for transformation need only contain at least an enhancer sequence containing a foreign-substance response element (XRE) and a sequence encoding a reporter protein downstream of the enhancer sequence. Here, the enhancer sequence is a nucleotide sequence present in the transcriptional regulatory region of a gene that has the function of activating the expression of that gene in response to a specific stimulus, and is sometimes simply referred to as an enhancer. Expression of a reporter protein containing an enhancer sequence containing a foreign-substance response element (XRE) in its upstream transcriptional regulatory region is activated by the induction of a foreign-substance response in the skin, and therefore the expression level of the reporter protein in the transformed keratinocytes reflects the level of the foreign-substance response. Therefore, the ability of a test sample to induce a foreign-substance response in the skin can be evaluated by using keratinocytes transformed with such a reporter vector.

[0027] As described above, a xenobiotic response element (XRE) is a base sequence found in the transcriptional regulatory region of a gene whose expression is increased by activation of the aryl hydrocarbon receptor (AhR). It is an enhancer sequence having the basic sequence 5'-CACGC-3'. The reporter vector used in the method of the present invention has at least one xenobiotic response element (XRE) in the transcriptional regulatory region upstream of the sequence encoding the reporter protein. However, the xenobiotic response element (XRE) may be repeated. From the viewpoint of increasing detection sensitivity, it is preferable to contain two or more xenobiotic response elements (XRE), and more preferably three or more xenobiotic response elements (XRE). Furthermore, enhancer sequences containing xenobiotic response elements (XRE) may be those found upstream of genes such as the P4501A1 (CYP1A1) gene, the P4501B1 (CYP1B1) gene, the glutathione S-transferase gene, the quinone reductase gene, and the UDP-glucuronosyltransferase gene. The nucleotide sequences of these genes can be obtained from public databases, such as the database of the National Center for Biotechnology Information (NCBI).

[0028] On the other hand, the reporter vector used for transformation contains a sequence encoding a reporter protein downstream of an enhancer sequence containing a xenobiotic response element (XRE). Any reporter protein can be used as long as a signal reflecting its expression level can be measured directly or indirectly. The reporter protein can be selected appropriately depending on the measurement equipment available to the tester. For example, if a luminescence spectrometer is available, luciferase proteins such as firefly luciferase, Renilla luciferase, marine copepod luciferase, and P. punctatus luciferase can be used. If a fluorometer or fluorescence microscope is available, fluorescent proteins such as blue fluorescent protein (BFP), green fluorescent protein (GFP), yellow fluorescent protein (YFP), and red fluorescent protein (RFP) can be used. If an absorption spectrometer is available, a chromogenic enzyme protein such as β-galactosidase can be used. The nucleotide sequence encoding the reporter protein can be obtained from a public database, such as the database of the National Center for Biotechnology Information (NCBI).

[0029] In addition to the sequences described above, the reporter vector used for transformation may contain various sequences necessary for expressing the reporter protein in keratinocytes into which the reporter vector has been introduced. For example, the reporter vector used for transformation may contain a promoter sequence, a sequence that provides a ribosome binding site to mRNA, a terminator sequence for transcription termination, a replication origin sequence, a sequence encoding a resistance gene to a selective drug, etc.

[0030] The reporter vector used in the evaluation method of the present invention may be a commercially available product or one that has been prepared by the user. For example, a reporter vector containing a foreign substance response element (XRE) in its enhancer sequence can be purchased from Promega Corporation (e.g., product name "pNL[NLucP / XRE / Hygro]Vector", catalog number: CS186808). Alternatively, a reporter vector used in the evaluation method of the present invention can be prepared by purchasing a reporter vector equipped with a multicloning site and incorporating an enhancer sequence containing a foreign substance response element (XRE) into it according to standard methods.

[0031] Furthermore, there are no particular limitations on the type of reporter vector used for transformation, and for example, a plasmid, a cosmid, a phagemid, a viral vector, etc. In any case, it is sufficient to appropriately select a reporter vector that can be expressed in the target keratinocytes.

[0032] To prepare transformed keratinocytes, the reporter vector described above may be introduced into keratinocytes according to conventional methods in the art. The method for introducing the reporter vector into keratinocytes is not particularly limited, and any appropriate method, whether physical or chemical, may be used. Examples of physical introduction methods include electroporation, sonoporation, and microinjection. Examples of chemical introduction methods include calcium phosphate and lipofection using liposomes, as well as transfection methods using appropriate delivery means such as cationic lipids, lipidoids, cationic polymers, membrane-permeable peptides, antibodies, antibody fragments, proteins, nanoparticles, microparticles, and emulsions.

[0033] On the other hand, from the viewpoint of obtaining stable measurement results, the transformed keratinocytes used in the evaluation method of the present invention are preferably transformants into which DNA introduced via the reporter vector has been stably introduced, i.e., stably transformed keratinocytes. Stably transformed keratinocytes can be established according to conventional methods in the art. For example, a reporter vector further comprising a resistance gene to a selective drug may be introduced into keratinocytes, and then the reporter vector-introduced keratinocytes may be cultured for approximately two weeks in a cell culture medium containing a predetermined amount of the selective drug. Cells stably introduced with the reporter vector express the resistance gene to the selective drug, while cells only transiently introduced with the reporter vector gradually lose expression of the resistance gene to the selective drug and therefore die in the cell culture medium containing the selective drug. Therefore, stably transformed keratinocytes can be obtained by culturing keratinocytes for a predetermined period of time in a cell culture medium containing the selective drug. Examples of selective drugs that can be used include hygromycin B, dioscin, geneticin, puromycin, and blasticidin.

[0034] On the other hand, there are no particular limitations on the type of substance used as the test sample in the evaluation method of the present invention, and it may be a naturally occurring substance or an artificially synthesized substance, and may also be an organic compound or an inorganic compound. Examples include low-molecular-weight organic compounds, amino acids, lipids, peptides, proteins, nucleotides, nucleic acids, carbohydrates, natural polymers, synthetic polymers, or combinations thereof. Furthermore, the test sample does not necessarily have to be an isolated substance, but may be a mixture of multiple substances. Examples of such mixtures include, but are not limited to, plant extracts, cosmetics such as lotions, makeup bases, serums, moisturizing creams, emulsions, blushers, lipsticks, hand creams, lip balms, mascaras, and eye shadows, daily necessities such as laundry detergents, dishwashing detergents, facial soaps, bath additives, hand soaps, body soaps, shampoos, and hand sanitizers, ointments, insect repellent sprays, medicated cosmetics, and quasi-drugs such as sunscreen creams. When the test sample is a product as described above, the evaluation method according to the present invention is suitably used for evaluating the safety and skin compatibility of the product. The test sample may be water from a river, lake, well, spring, tap water, or seawater.

[0035] It is known that when a foreign body response reaction in the skin, i.e., a foreign body response reaction involving a foreign body response element (XRE) in the skin, is induced, the production of matrix metalloproteinase 9, which causes wrinkles, and artemin, which causes atopic dermatitis, is activated (e.g., Non-Patent Documents 4 and 5). Therefore, the evaluation method of the present invention can be suitably used as a method for evaluating the wrinkle-inducing ability and atopic dermatitis-inducing ability of a test sample.

[0036] Each step of the evaluation method of the present invention will be described below.

[0037] (1) contacting transformed keratinocytes with a solution containing a test sample This step involves contacting transformed keratinocytes with a solution containing a test sample to be evaluated. The test sample-containing solution can be prepared by mixing and dissolving the test sample in an appropriate solvent, such as purified water, physiological saline, or cell culture medium. If the test sample is water-insoluble, it may be used in a water-suspended or dispersed state. Alternatively, it may be dissolved in a water-miscible organic solvent, such as DMSO, that has low cytotoxicity, and is then diluted with cell culture medium or physiological saline. A predetermined amount of the test sample-containing solution / suspension / dispersion (hereinafter, unless otherwise specified) can be added to a cell culture plate or dish containing transformed keratinocytes, thereby contacting the transformed keratinocytes with the test sample-containing solution. It is preferable that components other than the test sample contained in the test sample solution do not substantially induce a foreign body response in the skin; for example, cell culture medium or physiological saline can be used.

[0038] From the viewpoint of sufficiently contacting the test sample with the keratinocytes and expressing an amount of reporter protein sufficient to reflect the foreign body response reaction elicited by the test sample, it is preferable to contact the keratinocytes with a solution containing the test sample, then incubate for a predetermined period of time, and then use the keratinocytes in step (2) described below. For example, after contacting the keratinocytes with the solution containing the test sample, the keratinocytes are preferably incubated for at least 6 hours, more preferably 12 hours, even more preferably 18 hours, and even more preferably 24 hours. Incubation can be carried out according to standard methods under conditions of 37°C and 5% CO2.

[0039] On the other hand, by using a negative control solution that is substantially the same as the solution containing the test sample except that it does not contain the test sample (hereinafter, also referred to as a "solution not containing the test sample") as a comparative control, the ability of the test sample to induce a foreign body response in the skin can be more accurately evaluated. The solution not containing the test sample can be, for example, the cell culture medium used to prepare the solution containing the test sample. When using a negative control, keratinocytes can be contacted with a solution not containing the test sample instead of the solution containing the test sample, and the resulting solution can be used in step (2) described below.

[0040] (2) Measuring the expression level of the reporter protein This step is a step of measuring the expression level of the reporter protein in the keratinocytes that have been contacted with a solution containing the test sample in step (1) above, and, when a negative control is used, the expression level of the reporter protein in the keratinocytes that have been contacted with a solution that does not contain the test sample.

[0041] The expression level of a reporter protein can be determined by quantitatively measuring the intensity of a signal derived from the reporter protein, which reflects the expression level of the reporter protein. The intensity of the signal derived from the reporter protein can be measured by an appropriate means depending on the type of reporter protein. For example, when a luciferase protein such as firefly luciferase, Renilla luciferase, marine copepod luciferase, or Pterocephalus luciferase is used as the reporter protein, the intensity of the signal derived from the reporter protein can be measured using a substrate that emits light upon enzymatic reaction with the luciferase protein and a luminescence spectrometer that measures the luminescence intensity. On the other hand, when a fluorescent protein such as blue fluorescent protein (BFP), green fluorescent protein (GFP), yellow fluorescent protein (YFP), or red fluorescent protein (RFP) is used as the reporter protein, the fluorescence intensity emitted by the fluorescent protein can be measured using a fluorometer. Furthermore, when a chromogenic enzyme protein such as β-galactosidase is used as the reporter protein, the intensity of the signal derived from the reporter protein can be measured using a substrate that absorbs light of a specific wavelength or emits fluorescence upon enzymatic reaction by the chromogenic enzyme protein (for example, when the chromogenic enzyme protein is β-galactosidase, 5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside, 2-nitrophenyl-β-D-galactopyranoside, fluorescein-β-D-galactopyranoside, etc.) and an absorptiometer that measures absorbance or a fluorometer that measures fluorescence intensity.

[0042] (3) A step of evaluating the ability to induce a foreign body response The expression level of the reporter protein produced by the transformed keratinocytes used in the present invention reflects the level of a foreign-body response involving the foreign-body response element (XRE). Therefore, the ability to induce a foreign-body response can be evaluated based on the expression level of the reporter protein. In other words, the greater the expression level of the reporter protein, the stronger the ability to induce a foreign-body response. The expression level of the reporter protein can typically be evaluated by comparing the expression level of the reporter protein in keratinocytes contacted with a solution containing a test sample with the expression level of the reporter protein in keratinocytes contacted with a solution not containing the test sample (negative control). That is, if the expression level of the reporter protein in contact with a solution containing the test sample is greater than the expression level in contact with a solution not containing the test sample (negative control), the test sample can be evaluated as having the ability to induce a foreign-body response in skin. Needless to say, a greater increase in the expression level of the reporter protein compared to the negative control indicates a stronger ability to induce a foreign-body response.

[0043] In a preferred embodiment, the evaluation method of the present invention may further include, after or before step (2), a step of measuring the viable cell count of keratinocytes contacted with a solution containing the test sample, or, when a negative control is used, keratinocytes contacted with a solution not containing the test sample. When measuring the viable cell count, the reporter protein expression level measured in step (2) can be divided by the viable cell count measured in this step to obtain the reporter protein expression level per unit viable cell count. This allows for correction of the influence of variations in cell count between samples, enabling a more accurate evaluation of the induction of a foreign body response. The viable cell count can be measured using a method commonly used in the art, such as, but not limited to, the MTT method, which uses a reductive color-developing reagent that develops color depending on the activity of mitochondrial dehydrogenase in viable cells, or the XTT method, MTS method, or WST method, which are colorimetric methods similar to the MTT method.

[0044] <2. Method for searching for substances that inhibit foreign body responses in the skin> On the other hand, a substance that has the effect of reducing the expression level of the reporter protein in the transformed keratinocytes can be evaluated as a substance that has the ability to suppress the foreign body response in the skin. Therefore, in another aspect, the present invention provides a method for searching for a substance that has the ability to suppress the foreign body response in the skin. This searching method is described below.

[0045] The suppression of a foreign body response reaction refers to the property of suppressing a foreign body response reaction, and more specifically, suppressing a foreign body response reaction means reducing the expression of a gene transcribed with the involvement of a foreign body response element (XRE). As mentioned above, gene expression means the production of the protein encoded by that gene, and suppressing gene expression means reducing the amount of production of the protein encoded by that gene.

[0046] There are no particular limitations on the type of candidate substance to be searched for, and it may be a naturally occurring substance or an artificially synthesized substance. It may also be an organic or inorganic compound. Examples include low-molecular-weight organic compounds, amino acids, lipids, peptides, proteins, nucleotides, nucleic acids, carbohydrates, natural polymers, synthetic polymers, or combinations thereof. Furthermore, the candidate substance does not necessarily have to be an isolated substance, but may be a mixture of multiple substances. Examples of such mixtures include, but are not limited to, plant extracts.

[0047] Genes transcribed with the involvement of foreign body response elements (XREs) include, for example, genes encoding matrix metalloproteinase 9, which causes wrinkles, and artemin, which causes atopic dermatitis. Therefore, it can be said that substances that have the ability to inhibit foreign body response reactions in the skin have the property of inhibiting the expression of the above-mentioned genes. In other words, the screening method of the present invention can be suitably used as a method for screening substances that prevent and / or improve wrinkles or substances that have the effect of preventing and / or treating atopic dermatitis.

[0048] In the screening method of the present invention, the same transformed keratinocytes as those already explained in the evaluation method can be used. Therefore, for the transformed keratinocytes and the method for establishing them, please refer to the explanation for the evaluation method above.

[0049] Each step of the search method of the present invention will now be described.

[0050] (1) contacting transformed keratinocytes with a test solution containing a candidate substance or a reference solution not containing the candidate substance; This step involves contacting transformed keratinocytes with a test solution containing a candidate substance and a standard solution that does not contain the candidate substance as a control. The methods for preparing the test solution containing the candidate substance and the standard solution that does not contain the candidate substance, and the methods for contacting these solutions with keratinocytes, are the same as those described for the solution containing the test sample in the explanation of the evaluation method.

[0051] Preferably, the test solution containing the candidate substance and the reference solution containing the candidate substance contain a substance that induces a foreign body response in the skin. Detection sensitivity can be improved by evaluating the suppression of the foreign body response based on the expression level of the reporter protein induced by the reference solution containing the substance that induces the foreign body response. The substance that induces a foreign body response in the skin may be any substance that increases the expression of genes transcribed in the skin with the involvement of the foreign body response element (XRE). Examples of substances that can be used include, but are not limited to, 6-formylindolo[3,2-b]carbazole (FICZ, CAS number: 17922-91-7), indole-3-carbinol (I3C, CAS number: 700-06-1), 2-(1'H-indole-3'-carbonyl)-thiazole-4-carboxylic acid methyl ester (ITE, CAS number: 448906-42-1), and indirubin (CAS number: 479-41-4). Whether or not a substance induces a foreign body response in the skin can be evaluated using, for example, an evaluation method according to another aspect of the present invention.

[0052] Furthermore, from the viewpoint of sufficiently contacting the candidate substance with the keratinocytes and obtaining test results that adequately reflect the inhibitory effect of the candidate substance on the foreign body response reaction, it is preferable to contact the keratinocytes with a test solution containing the candidate substance and a reference solution not containing the candidate substance, and then incubate for a predetermined period of time before using the keratinocytes in step (2) described below. There are no particular limitations on the incubation time, but, for example, after contacting the keratinocytes with the test solution and the keratinocytes with the reference solution, it is preferable to incubate the keratinocytes for at least 6 hours before using the keratinocytes in the subsequent step (2), more preferably for 12 hours or more, even more preferably for 18 hours or more, and even more preferably for 24 hours or more.

[0053] (2) Measuring the expression level of the reporter protein This step involves measuring the expression level of the reporter protein in keratinocytes contacted with a test solution containing the candidate substance in step (1) above, and the expression level of the reporter protein in keratinocytes contacted with a reference solution not containing the candidate substance. Specific methods for measuring the expression level of the reporter protein are as described above in the section "1. Method for evaluating the ability to induce a foreign body response in the skin."

[0054] (3) Comparing the expression levels of the reporter proteins This step involves comparing the reporter protein expression level in keratinocytes contacted with a test solution containing a candidate substance measured in step (2) with the reporter protein expression level in keratinocytes contacted with a standard solution not containing the candidate substance, and evaluating whether the candidate substance has the ability to inhibit a foreign body response in skin based on the comparison results. Specifically, if the reporter protein expression level in keratinocytes contacted with a test solution containing the candidate substance is lower than the reporter protein expression level in keratinocytes contacted with a standard solution not containing the candidate substance, the candidate substance can be evaluated as having the ability to inhibit a foreign body response in skin. Note that a greater decrease in the reporter protein expression level in keratinocytes contacted with the test solution compared to the reporter protein expression level in keratinocytes contacted with the standard solution indicates a stronger inhibitory effect on a foreign body response in skin. Therefore, the screening method of the present invention can evaluate not only whether a candidate substance has the ability to inhibit a foreign body response, but also the degree of inhibitory effect of the candidate substance on a foreign body response.

[0055] In a preferred embodiment, the screening method of the present invention may further include a step of measuring the number of viable keratinocytes contacted with the test solution and the number of viable keratinocytes contacted with the reference solution, either before or after step (2). When measuring the number of viable cells, the expression level of the reporter protein measured in step (2) can be divided by the number of viable cells measured in this step to obtain the expression level of the reporter protein per unit number of viable cells. This allows the influence of variations in cell number between samples to be corrected, enabling a more accurate evaluation of the inhibitory effect on foreign body response. As mentioned above, the number of viable cells can be measured according to standard methods in the art.

[0056] <3. Agents that suppress foreign body response in the skin> Next, an agent for suppressing a foreign body response in the skin according to another aspect of the present invention will be described.

[0057] As mentioned above, suppressing a foreign body response reaction means reducing the expression of genes transcribed with the involvement of foreign body response elements (XRE). In other words, an agent that suppresses a foreign body response reaction in the skin can also be said to be an agent that suppresses the expression of genes transcribed with the involvement of foreign body response elements (XRE).

[0058] The agent for suppressing a foreign body response in skin of the present invention contains 5-hydroxy-4-phenyl-2(5H)butenolide and / or a salt thereof as an active ingredient. 5-Hydroxy-4-phenyl-2(5H)butenolide is a butenolide compound discovered in fragrant vinegar, also known as fregulide 1 or 5-hydroxy-4-phenyl-2(5H)furanone, and its structural formula is as shown in chemical formula (I) (Aydan H. Yatmaz et al., Journal of Oleo Science, 2017, 66:1381-1386).

[0059] [ka]

[0060] 5-Hydroxy-4-phenyl-2(5H)butenolide is known to exhibit tautomerism, and known tautomers are represented by chemical formula (II) and chemical formula (III). In this specification, unless otherwise specified, 5-hydroxy-4-phenyl-2(5H)butenolide also includes its tautomers.

[0061] [ka]

[0062] [ka]

[0063] 5-Hydroxy-4-phenyl-2(5H)butenolide can be obtained by extraction from fragrant vinegar. A method for obtaining 5-hydroxy-4-phenyl-2(5H)butenolide from fragrant vinegar is described, for example, in International Publication No. 2016 / 006548. On the other hand, 5-hydroxy-4-phenyl-2(5H)butenolide can also be artificially synthesized. 5-Hydroxy-4-phenyl-2(5H)butenolide can be synthesized, for example, by the methods described in JP-A-6-211825 and JP-A-11-152280.

[0064] On the other hand, the salt of 5-hydroxy-4-phenyl-2(5H)butenolide is not particularly limited as long as it is a pharmaceutically acceptable salt. 5-Hydroxy-4-phenyl-2(5H)butenolide and its salts may be hydrates, solvates with organic solvents such as alcohols, or anhydrides. Furthermore, prodrugs in which functional groups have been modified so that 5-hydroxy-4-phenyl-2(5H)butenolide and its salts can be produced in vivo may also be used.

[0065] According to the findings of the present inventors, 5-hydroxy-4-phenyl-2(5H)butenolide and / or its salts have the effect of suppressing the foreign body response reaction in the skin, i.e., suppressing the expression of genes transcribed in the skin with the involvement of the foreign body response element (XRE). Genes transcribed in the skin with the involvement of the foreign body response element (XRE) have an enhancer sequence containing the foreign body response element (XRE) in their transcriptional regulatory region. As mentioned above, it is known that the expression of such genes is enhanced in the presence of an activated aryl hydrocarbon receptor (AhR).

[0066] An example of a gene transcribed in conjunction with a foreign body response element (XRE) is the neurotrophic factor artemin, which is believed to be a cause of atopic dermatitis. As shown in the experimental examples described below, 5-hydroxy-4-phenyl-2(5H)butenolide has the effect of suppressing the expression of artemin in keratinocytes. In other words, an agent containing 5-hydroxy-4-phenyl-2(5H)butenolide and / or a salt thereof as an active ingredient can be used as an agent for suppressing the production of artemin.

[0067] Artemin is a member of the glial cell line-derived neurotrophic factor (GDNF) family and induces neuronal proliferation and outgrowth through binding to the GDNF family receptor α3 (Gfrα3). Artemin is expressed in the skin of mice with constitutively activated AhR, which causes abnormal outgrowth of nerves into the epidermis, resulting in hypersensitive itching, which is thought to lead to the loss of skin barrier function and atopic dermatitis.

[0068] Therefore, an agent that suppresses the production of artemin and contains 5-hydroxy-4-phenyl-2(5H)butenolide and / or a salt thereof as an active ingredient can be suitably used for the prevention and / or treatment of atopic dermatitis. Here, the prevention and / or treatment of atopic dermatitis means suppressing one or more symptoms of atopic dermatitis or symptoms associated therewith (e.g., itching, eczema, lichenification, etc.), or preventing the onset of such symptoms or reducing the incidence.

[0069] On the other hand, another gene transcribed with the involvement of the foreign body response element (XRE) is known to be matrix metalloproteinase 9, which is believed to cause wrinkles, and as shown in the experimental examples described below, 5-hydroxy-4-phenyl-2(5H)butenolide has the effect of suppressing the expression of matrix metalloproteinase 9 in keratinocytes. In other words, an agent containing 5-hydroxy-4-phenyl-2(5H)butenolide and / or a salt thereof as an active ingredient can be used as an agent for suppressing the production of matrix metalloproteinase 9.

[0070] Matrix metalloproteinases (MMPs), including matrix metalloproteinase 9, are a collective term for metalloproteinases (proteases with a metal ion in the catalytic site of the active center) that degrade the extracellular matrix. At least 11 types of MMPs are known in humans, each with different substrate proteins and expression sites. Matrix metalloproteinase 9 (MMP9) is an MMP with gelatinase activity. It has been reported that its production is increased in rats with severe wrinkles caused by UV irradiation. Therefore, inhibition of MMP9 expression is thought to be effective in preventing skin aging and wrinkle formation (Japanese Patent Publication No. 2011-178747; Shinji Inomata et al., Journal of Investigative Dermatology 2003, 120:128-134).

[0071] Therefore, an agent for suppressing the production of matrix metalloproteinase 9, which contains 5-hydroxy-4-phenyl-2(5H)butenolide and / or a salt thereof as an active ingredient, can be suitably used for preventing and / or ameliorating wrinkles. Here, preventing and / or ameliorating wrinkles means preventing wrinkle formation or ameliorating wrinkles that have already formed.

[0072] Incidentally, matrix metalloproteinase 9 is a factor involved not only in wrinkle formation but also in numerous inflammatory skin diseases, such as vitiligo, psoriasis, atopic dermatitis, scleroderma, hypomelanosis, and leukoderma. It is known, for example, to be involved in UV-induced skin inflammation and blister formation in pemphigoid (Takashi Kobayashi, Inflammation and Regeneration 2004, 24: 578-583). It is also known to be involved in depigmentation disorders associated with the above-mentioned inflammatory skin diseases (Patent Publication No. 2020-504719). Therefore, the agent according to the present invention, particularly an agent that suppresses the production of matrix metalloproteinase 9, can be effectively used for the prevention and / or treatment of various inflammatory skin diseases caused by increased expression of matrix metalloproteinase 9 and depigmentation disorders associated with such inflammatory skin diseases. As mentioned above, prevention and / or treatment herein means suppressing one or more of these various diseases or symptoms associated therewith, or preventing the onset of such symptoms or reducing the incidence thereof.

[0073] Furthermore, matrix metalloproteinase 9, which has gelatinase activity and functions to degrade extracellular matrices such as collagen, gelatin, and elastin that constitute elastic tissues in arteries, tendons, and skin, has been shown to be involved not only in inflammatory diseases of the skin but also in various diseases in many organs. For example, it is known to play an important role in the invasion and metastasis of cancer cells (Takashi Kobayashi, Inflammation and Regeneration 2004, 24:578-583), and to be involved in rheumatoid arthritis, osteoporosis, periodontitis, and the like (Japanese Patent Laid-Open Publication No. 2015-17048). Therefore, the agent according to the present invention, particularly an agent that suppresses the production of matrix metalloproteinase 9, can be suitably used for the prevention and / or treatment of various diseases caused by increased expression of matrix metalloproteinase 9, such as cancer, rheumatoid arthritis, osteoporosis, and periodontitis.

[0074] On the other hand, as shown in the experimental examples described below, 5-hydroxy-4-phenyl-2(5H)butenolide and / or a salt thereof not only suppresses the production of matrix metalloproteinase 9, but also promotes the production of TIMP-1 (Tissue Inhibitor of Matrix Metalloprotease-1), an inhibitor of matrix metalloproteinases. That is, in yet another aspect, the present invention provides an agent for promoting TIMP-1 production, which contains 5-hydroxy-4-phenyl-2(5H)butenolide and / or a salt thereof as an active ingredient. As described above, since TIMP-1 acts as an inhibitor of matrix metalloproteinases, an agent for promoting TIMP-1 production, which contains 5-hydroxy-4-phenyl-2(5H)butenolide and / or a salt thereof as an active ingredient, can be suitably used for preventing and / or improving wrinkles. It goes without saying that the above-mentioned agents that promote the production of TIMP-1 can be effectively used not only to prevent and / or improve wrinkles, but also to prevent and / or treat various diseases caused by increased expression of matrix metalloproteinase 9.

[0075] There are no particular limitations on the content of 5-hydroxy-4-phenyl-2(5H)butenolide and / or a salt thereof contained in the agent of the present invention, and it may be, for example, 0.0001% by weight to 10% by weight, 0.0005% by weight to 5% by weight, 0.001% by weight to 2% by weight, or 0.005% by weight to 1% by weight.

[0076] The agent of the present invention can be particularly suitably used for skin cells, more suitably for epidermal cells of the skin, and even more suitably for keratinocytes. However, it goes without saying that the agent of the present invention may also be applied to other types of cells other than skin. For example, the agent of the present invention may be applied to cancer cells.

[0077] The agent according to the present invention may be provided as a pharmaceutical product, but may also be provided as a quasi-drug, cosmetic, daily necessities, food, etc. Food products include health foods (functional foods, nutritional supplements, health supplements, nutritionally fortified foods, nutritionally balanced foods, supplements), special dietary foods, and functional health foods (foods for specified health uses, foods with nutritional functions), and cosmetics include skin care cosmetics (lotion, emulsion, cream, serum, pack, cleanser, face wash, soap, hand soap, etc.), makeup cosmetics (makeup base, foundation, concealer, face powder, lipstick, blush, eye shadow, eyeliner, eyebrow, mascara, etc.), body care cosmetics (body soap, body milk, body lotion, body cream, body oil, body powder, soap, etc.), and the like. Examples of cosmetics include, but are not limited to, hair care products (shampoo, rinse, rinse-in shampoo, dry shampoo, hair treatment, conditioner, hair milk, hair cream, hair oil, hair mist, tonic, hair foam, hair gel, wax, pomade, hair liquid, waving agent, hair color, bleach, color rinse, hair growth agent, hair growth agent), toothpaste, mouthwash, gargle, bath cosmetics (bath milk, bath salts, bath oil, etc.), and fragrance cosmetics (perfume, eau de cologne, etc.). Examples of daily necessities include, but are not limited to, laundry detergent, kitchen detergent, household cleaner, bathroom cleaner, disinfectant, insect repellent, insect repellent, wet wipes, etc. These quasi-drugs, cosmetics, daily necessities, or food products containing Fregulide 1 can be suitably used for the prevention and / or treatment of inflammatory skin diseases such as atopic dermatitis, or for the prevention and / or improvement of wrinkles.

[0078] Furthermore, there are no particular limitations on the method of administration of the agent according to the present invention, and it may be administered orally or parenterally, where parenteral administration includes dermal administration, transdermal administration, transmucosal administration, ocular administration, nasal administration, tube administration, inhalation administration, spray administration, subcutaneous injection, intradermal injection, intravenous injection, intramuscular injection, etc.

[0079] There are no particular limitations on the formulation form of the agent according to the present invention, and an appropriate dosage form may be selected depending on the route of administration. For oral administration, examples include tablets, capsules, powders, powders, granules, and syrups, and for parenteral administration, examples include injections, liquids, ointments, poultices, creams, lotions, gels, topical skin preparations, sprays, and poultices, but are not limited to these.

[0080] The agent of the present invention may further contain other ingredients depending on its formulation form and / or application, and may be provided as a composition containing, for example, one or more ingredients such as excipients, base materials, buffering agents, preservatives, chelating agents, antioxidants, surfactants, pearlescent agents, pH adjusters, thickeners, and / or other ingredients that are commonly incorporated into pharmaceuticals, quasi-drugs, cosmetics, daily necessities, food products, etc.

[0081] Examples of excipients include, but are not limited to, monosaccharides such as glucose, galactose, and fructose; disaccharides such as maltose, trehalose, and lactose; oligosaccharides such as maltotriose and maltotetraose; sugar alcohols such as sorbitol, mannitol, and maltitol; and polysaccharides such as cellulose (including crystalline cellulose), starch, dextrin, dextran, and cyclic sugars (cyclodextrin, isomaltodextrin, etc.).

[0082] Examples of base materials include, but are not limited to, water; seawater, deep seawater; lower alcohols such as ethanol and isopropanol; higher alcohols such as cetanol and stearyl alcohol; polyhydric alcohols such as glycerin, butylene glycol, propylene glycol, and polyethylene glycol; hydrocarbons such as petrolatum and liquid paraffin; silicone oils such as dimethiconol and modified silicone; oils and fats such as coconut oil, olive oil, and rice bran oil; and waxes such as jojoba oil and beeswax.

[0083] Buffers may include, for example, but are not limited to, phosphate buffers, borate buffers, citrate buffers, tartrate buffers, acetate buffers, amino acids, and the like.

[0084] Preservatives may include, but are not limited to, for example, benzoic acid, sodium benzoate, parahydroxybenzoic acid esters such as methyl parahydroxybenzoate, quaternary ammonium salts such as benzalkonium chloride, benzyl alcohol, sorbic acid and its salts, thimerosal, methylparaben, ethylparaben, propylparaben, butylparaben, cetylpyridinium chloride, catechin, phenoxyethanol, and the like.

[0085] Chelating agents may include, for example, but are not limited to, sodium edetate, disodium edetate, citric acid, phytic acid, gluconic acid, sodium gluconate, etidronic acid, tetrasodium etidronate, pentasodium pentetate, and the like.

[0086] Antioxidants may include, but are not limited to, for example, sodium bisulfite, sodium sulfite, sodium metabisulfite, ascorbic acid, ergothioneine, reduced 3,4-dihydroxyphenylethanol elenolic acid, and the like.

[0087] The surfactant may be any of nonionic surfactants, cationic surfactants, anionic surfactants, amphoteric surfactants, and may also be silicone surfactants. Examples of nonionic surfactants include, but are not limited to, polyoxyethylene alkyl ethers such as ceteareth-13 and laureth-7; polyoxyethylene fatty acid esters; polyglycerin fatty acid esters such as polyglyceryl-2 oleate, polyglyceryl-10 oleate, and polyglyceryl-10 decaisostearate; polyoxyethylene sorbitan fatty acid esters such as polysorbate-20; polyoxyethylene hydrogenated castor oil; sorbitan fatty acid esters such as sorbitan sesquioleate; glycerin fatty acid esters such as glyceryl stearate; fatty acid alkanolamides such as cocamide DEA, cocamide MEA, and cocamide methyl MEA; alkyl glycosides. Examples of cationic surfactants include, but are not limited to, cationized cellulose such as polyquaternium-10; fatty acid amide amine salts; monoalkyl quaternary ammonium salts such as behentrimonium methosulfate; dialkyl quaternary ammonium salts. Examples of anionic surfactants include, but are not limited to, fatty acid salts such as laurate, stearate, myristate, and palmitate; alkyl sulfate ester salts such as sodium lauryl sulfate; alkyl ether sulfate ester salts such as sodium laureth sulfate; cocoyl alanine salts such as sodium cocoyl alanine and triethanolamine cocoyl; cocoyl methyl alanine salts such as sodium cocoyl methyl alanine; lauroyl methyl alanine salts such as sodium lauroyl; lauroyl methyl alanine salts such as triethanolamine lauroyl; cocoyl glutamine salts. Examples of amphoteric surfactants include, but are not limited to, betaine surfactants such as lauryl betaine. Silicone surfactants may include, but are not limited to, dimethicone (methylpolysiloxane), amodimethicone (aminoethylaminopropylmethylsiloxane-dimethylsiloxane copolymer), PEG-10 dimethicone (polyoxyethylene-methylpolysiloxane copolymer), and the like.

[0088] Pearlizing agents may include, for example, but are not limited to, ethylene glycol di-stearate, ethylene glycol mono-stearate, and the like.

[0089] Examples of pH adjusters include, but are not limited to, inorganic acids and salts thereof such as hydrochloric acid and sulfuric acid, organic acids and salts thereof such as lactic acid and succinic acid, inorganic bases such as potassium hydroxide and sodium hydroxide, and organic bases such as triethanolamine.

[0090] Examples of thickeners include, but are not limited to, vinyl thickeners such as polyvinyl alcohol, polyvinylpyrrolidone, and carbomer (carboxyvinyl polymer); cellulose thickeners such as methyl cellulose, ethyl cellulose, hydroxymethyl cellulose, hydroxyethyl cellulose, and hydroxypropyl methyl cellulose; and polysaccharides such as guar gum, pectin, pullulan, gelatin, carrageenan, and alginic acid.

[0091] The disclosures of all documents cited herein are incorporated by reference in their entirety. Also, when this specification is translated into English, the words "a," "an," and "the" in the singular form shall include the plural as well as the singular, unless the context clearly indicates otherwise.

[0092] The present invention will be explained in more detail below by way of experimental examples. However, these experimental examples are merely illustrative of embodiments of the present invention and do not limit the scope of the present invention in any way.

[0093] <Experiment 1-1: Transformation of human epidermal keratinocytes, HaCaT> <XRE-NLucベクター> Keratinocytes transformed with a reporter vector containing an enhancer sequence containing a foreign body response element (XRE) and a sequence encoding the reporter protein NLuc (NanoLuc®) downstream of the enhancer sequence were prepared using the following procedure. First, the structure of the reporter vector used for transformation (hereinafter sometimes referred to as the "XRE-NLuc vector") is shown in Figure 1. As shown in Figure 1, the XRE-NLuc vector used for transformation is a circular plasmid DNA consisting of 4930 base pairs. It contains an enhancer sequence (designated "XRE1," "XRE2," and "XRE3" in Figure 1) consisting of three XREs linked in tandem. Downstream of this sequence are the Minimal Promoter, a sequence encoding the reporter protein NLuc (designated "NLucP reporter" in Figure 1), and a polyadenylation signal derived from Simian Virus 40 (SV40) (designated "SV40 Late poly(A)" in Figure 1). Further downstream are the SV40 promoter (designated "SV40 Promoter" in Figure 1) and a sequence encoding the resistance gene to the selective drug hygromycin B (designated "Hyg R" in Figure 1). Furthermore, a polyadenylation signal (designated "Poly(A) signal" in Figure 1) is located upstream of the enhancer sequence to reduce background. In this experiment, we used a reporter vector with this structure purchased from Promega Corporation (product name: "pNL[NLucP / XRE / Hygro]Vector", catalog number: CS186808). However, it goes without saying that we can also use a reporter vector we have created ourselves by inserting an XRE sequence or a minimal promoter into a reporter vector with a multicloning site.

[0094] <Transformation> Next, the XRE-NLuc vector was introduced into HaCaT cells using the following procedure, followed by treatment with the selective drug hygromycin B to establish a transformed cell line (hereinafter sometimes referred to as "XRE-NLuc::HaCaT") that stably expresses a luciferase reporter transcriptionally controlled by the xenobiotic response element (XRE). First, HaCaT cells (CLI, catalog number: 300493) were cultured at a cell count of 2 × 10 6 The cells were seeded onto cell culture plates (product name: "True Line Dish," Nippon Genetics, catalog number TR4002) so that each cell was 1000 μg / mL. The culture medium used was DMEM (Dulbecco's Modified Eagle's Medium) supplemented with fetal bovine serum (FBS) at a final concentration of 10%. Seven hours after seeding, the XRE-NLuc vector was introduced into HaCaT cells using a gene transfer reagent (product name: "PEImax," Polysciences, catalog number 24765-1). Specifically, 20 μL of a 0.1% aqueous solution of PEImax was mixed with 10 μL of a 500 μg / mL XRE-NLuc vector, and the entire mixture was added to the cell culture medium. After 48 hours of incubation at 37°C under 5% CO2, the DMEM medium was removed and replaced with DMEM medium containing 0.08 wt% hygromycin B, a selection drug. Thereafter, by continuing to culture the cells in DMEM medium containing 0.08 wt% hygromycin B for a total of 14 days, with the medium changed approximately every 4 days, we obtained the cell line XRE-NLuc::HaCaT, which stably expresses XRE-NLuc. Figure 2 shows an optical microscope photograph of the resulting XRE-NLuc::HaCaT cells. As can be seen in Figure 2, the morphology of the XRE-NLuc::HaCaT cells was unchanged from that of the HaCaT cells before transformation.

[0095] <Experiment 1-2: Evaluation of the ability of XRE-NLuc::HaCaT cells to induce a foreign body response> Using the XRE-NLuc::HaCaT cells obtained in Experiment 1-1, the ability of the test sample to induce a foreign body response in the skin was evaluated. The specific procedure is as follows.

[0096] <Contact with test sample> First, 1 × 10 XRE-NLuc::HaCaT cells were cultured in DMEM medium. 5 The cells were suspended at a concentration of 10,000 cells / mL. 100 μL of the resulting cell suspension was added to each well of a white 96-well plate (ThermoFisher Scientific, catalog number: 136102) so that the cell count per well was 10,000 cells. After 24 hours of incubation at 5% CO2 and 37°C, the DMEM medium was removed. 100 μL of DMEM medium containing the specified concentration of FICZ (6-Formylindolo[3,2-b]carbazole, CAS number: 172922-91-7), a substance known to activate the XRE-mediated foreign body response, was added as a test sample and incubated for another 24 hours at 5% CO2 and 37°C. A negative control was also prepared by adding 100 μL of DMEM medium without FICZ instead of the FICZ-containing DMEM medium and incubating for 24 hours at 5% CO2 and 37°C.

[0097] <Measurement of viable cell count> After 24 hours of incubation, cell numbers in each well were determined by a colorimetric assay based on the decomposition of tetrazolium salt (WST-1) by mitochondrial dehydrogenase in viable cells. Specifically, 3 μL of Premix WST-1 (product name: "Premix WST-1 Cell Proliferation Assay System," Takara Bio Inc., catalog number: MK400) was added to each well of a 96-well plate and incubated at 37°C and 5% CO2 for 1 hour. Then, 93 μL of supernatant was removed from each well to measure the absorbance of the supernatant. The removed supernatant was transferred to a clear 96-well plate and the absorbance at 450 nm (A450) and at 670 nm (A670) was measured using an iMark microplate reader (Bio-Rad, catalog number: 168-1130JA). The value (A450-A670) was calculated by subtracting the absorbance at 670 nm (A670) from the absorbance at 450 nm (A450), and the value obtained for the negative control (A450-A670) was set to 100%, which was then expressed as a relative value to define the relative viable cell count (%).

[0098] <Measurement of reporter protein expression level> After removing the supernatant for measuring viable cell count, the remaining sample in the white 96-well plate was used to measure reporter protein expression. Specifically, 7 μL of Nano-Glo Luciferase Assay System (Promega, catalog number: N110) was added to each well of the white 96-well plate. After 3 minutes, the luminescence intensity from the reporter protein produced by the XRE-NLuc::HaCaT cells in each well was measured using a GloMax Navigator Microplate Luminometer (Promega, catalog number: GM2000). The luminescence intensity was divided by the relative viable cell count obtained by the colorimetric assay described above to obtain the reporter protein expression level (luminescence intensity) per unit viable cell count.

[0099] The results are shown in Figure 3. In Figure 3, the luminescence intensity per unit viable cell number obtained from XRE-NLuc::HaCaT cells incubated in DMEM medium containing each concentration of FICZ is shown as a relative value, with the luminescence intensity per unit viable cell number obtained for the negative control set at 1. The results shown in Figure 3 are the average values ​​obtained from four independent experiments, and the error bars represent the standard error of the mean (SEM). In the figure, "*" and "**" indicate statistically significant differences (p<0.05 and p<0.01), respectively, from the negative control ("FICZ Concentration 0 nM" in Figure 3).

[0100] As shown in Figure 3, XRE-NLuc::HaCaT cells exhibited a concentration-dependent increase in luminescence intensity, which is mediated by FICZ, a compound that activates the XRE. These results indicate that the reporter protein expression level in XRE-NLuc::HaCaT cells reflects the level of activation of the XRE-mediated XRE-mediated XRE-mediated XRE-mediated XRE response. Specifically, the ability of XRE-NLuc::HaCaT cells to induce a XRE response in the skin of a test sample can be assessed by monitoring the reporter protein expression level (i.e., luminescence intensity in this experiment) produced by XRE-NLuc::HaCaT cells, which are keratinocytes transformed with the XRE-NLuc vector, a reporter vector containing an enhancer sequence containing the XRE and a reporter protein coding sequence downstream of the enhancer sequence. In particular, XRE-NLuc::HaCaT cells exhibited approximately three times higher luminescence intensity than the negative control without FICZ, even when cultured in a medium containing only 4 nM FICZ. This result indicates that the evaluation method according to the present invention makes it possible to evaluate the induction of a foreign body response in the skin with extremely high detection sensitivity.

[0101] <Experiment 1-3: Evaluation of the induction of foreign body responses in the skin by PCR method> Next, in order to compare the sensitivity of the evaluation method according to the present invention using XRE-NLuc::HaCaT cells with that of the conventional evaluation method, the change in the expression level of mRNA induced by the foreign body response reaction in HaCaT cells was measured by the PCR method to evaluate the inducibility of the foreign body response reaction. In the following experiments, the target genes were artemin and matrix metalloprotease 9. The expression of these genes is known to be activated by the foreign body response reaction in which the foreign body response sequence XRE is involved in the skin. The following shows the specific procedures.

[0102] <Contact with the test sample> First, HaCaT cells (CLI, catalog number: 300493) were suspended in DMEM medium at a cell concentration of 1×10 5 cells / mL. The obtained cell suspension was added to a white 96-well plate (ThermoFisher Scientific, catalog number: 136102), 100 μL per well so that the number of cells in each well was 10,000. According to a conventional method, after incubating at 37°C for 24 hours in the presence of 5% CO2, the DMEM medium was removed, and 100 μL of DMEM medium containing FICZ at a predetermined concentration (5 nM, 50 nM, 500 nM, or 5,000 nM) was added, and further incubated for 24 hours under the conditions of 5% CO2 and 37°C. Also, a negative control was prepared by adding 100 μL of DMEM medium not containing FICZ instead of the DMEM medium containing FICZ, and incubated for 24 hours under the conditions of 5% CO2 and 37°C.

[0103] <RNA extraction and cDNA preparation> After the 24-hour incubation, total RNA was extracted and cDNA was prepared using the SuperPrep II Cell Lysis & RT Kit for qPCR (catalog number: SCQ-401) manufactured by Toyobo Co., Ltd., according to the instructions included with the kit. Specifically, after the 24-hour incubation, the medium was removed from each well, and the cells were washed with phosphate-buffered saline (PBS). Next, 10 μL of lysis reagent (prepared by mixing 58.7 μL of lysis solution provided with the PCR kit with 0.3 μL of gDNA Remover and 1 μL of RNase Inhibitor) was added to each well. The mixture was shaken for approximately 30 seconds and allowed to stand at room temperature for 5 minutes to obtain a cell lysate containing total RNA that could be used as a template for reverse transcription. The lysis solution is a cell lysing agent that lyses cells, the gDNA Remover is a reagent containing DNase I that degrades genomic DNA, and the RNase Inhibitor is a reagent that inhibits RNA degradation by cellular components such as RNase.

[0104] Next, cDNA was synthesized from the extracted RNA by reverse transcription. First, a master mix for reverse transcription was prepared by mixing 8 μL of 5x RT Master Mix (included in the SuperPrep II Cell Lysis & RT Kit for qPCR, manufactured by Toyobo Co., Ltd.) with 24 μL of nuclease-free water. The master mix was prepared on ice. 32 μL of the resulting master mix was dispensed into PCR tubes, and 8 μL of the cell lysate in each well was added. This total volume was 40 μL and used for the reverse transcription reaction. The reverse transcription reaction conditions were 37°C for 15 min, 50°C for 5 min, and 98°C for 5 min. Eighty μL of sterile water was added to the resulting cDNA solution, which was then frozen and stored at -20°C.

[0105] <Quantitative PCR> Quantitative PCR was performed using THUNDERBIRD Next SYBR qPCR Mix (Toyobo Co., Ltd., catalog number QPX-201) and a StepOne Real-Time PCR System (ThermoFisher Scientific). Specifically, THUNDERBIRD Next SYBR qPCR Mix (provided as a 2x premixed reagent) was diluted to 1x with sterile water, and 5 μL of this solution was mixed with 6 pmoles of forward and reverse primers for each target gene, with the nucleotide sequences shown in Table 1 below. Separately, a frozen cDNA solution was thawed and then diluted 5x with sterile water to obtain a cDNA dilution. 5 μL of the resulting cDNA dilution was mixed with 5 μL of the above mixture to a total volume of 10 μL, which was used as a PCR reaction solution and subjected to quantitative PCR. The amount of target gene mRNA was determined from the Ct value obtained by quantitative PCR. The amount of mRNA of the endogenous gene GAPDHA was used as a reference, and the amount of mRNA of each target gene was corrected by dividing it by the amount of GAPDH mRNA.

[0106] [Table 1]

[0107] The quantitative PCR conditions are as shown in Table 2. The PCR reaction solution was held at 95°C for 20 seconds, followed by a denaturation step at 95°C for 3 seconds and an annealing / extension step at 60°C for 30 seconds, for a total of 40 cycles. After the 40 cycles, the solution was held at 95°C for 15 seconds, then at 60°C for 60 seconds, and then at 95°C for another 15 seconds.

[0108] [Table 2]

[0109] The results are shown in Figures 4 and 5. In Figures 4 and 5, the mRNA expression levels in HaCaT cells incubated in DMEM medium containing FICZ at various concentrations are shown as relative values, with the mRNA expression level in HaCaT cells incubated for 24 hours in DMEM medium without FICZ (as a negative control) set to 1. The results shown in Figures 4 and 5 are the average values ​​obtained from four or three independent experiments, respectively, and the error bars represent the standard deviation (SD).

[0110] As shown in Figure 4, the expression level of artemin mRNA in HaCaT cells increased depending on the concentration of FICZ added to the cell culture medium. This result indicates that the activation of the foreign body response in HaCaT cells can be detected by quantitating mRNA by PCR. However, no change in the expression level of artemin mRNA was observed at FICZ concentrations below 50 nM. To observe a significant increase in the expression level of artemin mRNA, a high concentration of FICZ of at least 500 nM, and more preferably 5,000 nM, was required.

[0111] On the other hand, as shown in Figure 5, an increase in MMP9 mRNA expression in HaCaT cells was confirmed depending on the concentration of FICZ added to the cell culture medium, similar to that observed for artemin. However, the degree of increase in expression was also extremely small, and a high concentration of FICZ of approximately 5,000 nM was required to observe a significant increase in MMP9 mRNA expression.

[0112] In contrast, as shown in Experiment 1-2, when an evaluation method according to one embodiment of the present invention using XRE-NLuc::HaCaT cells was used, even when an extremely low concentration of FICZ, as low as 4 nM, was used, a luminescence intensity approximately three times stronger than that of the negative control without FICZ was observed. This result demonstrates that the evaluation method of the present invention allows for the evaluation of the ability of each test sample to induce a foreign body response in the skin with far greater sensitivity and speed than conventional evaluation methods that quantify mRNA expression levels by PCR.

[0113] <Experiment 1-4: Evaluation of the ability of XRE-NLuc::HaCaT cells to induce a foreign body response - Part 2> The foreign body response induction ability of each test sample was evaluated using XRE-NLuc::HaCaT cells in the same manner as in Experiment 1-2, except that I3C (indole-3-carbinol, CAS No.: 700-06-1), ITE (2-(1'H-indole-3'-carbonyl)-thiazole-4-carboxylic acid methyl ester, CAS No.: 448906-42-1), and indirubin (CAS No.: 479-41-4) were used instead of FICZ. The amount of each test sample added to DMEM medium was adjusted appropriately. From the evaluation results, the maximum effective concentration, EC50 (median effective concentration), and LC50 (median lethal concentration), which are the concentrations of the test samples that maximized reporter protein expression, were calculated using standard methods. The obtained values ​​are shown in Table 3.

[0114] [Table 3]

[0115] As shown in Table 3, XRE-NLuc::HaCaT cells exhibited responsiveness to a variety of chemicals. In other words, the evaluation method of the present invention makes it possible to evaluate the ability of test samples containing chemicals with a wide variety of structures to induce a foreign body response in the skin. Furthermore, since the expression level of the reporter protein by XRE-NLuc::HaCaT cells reflects the level of a foreign body response involving XRE, the evaluation method of the present invention not only determines whether or not an individual test sample has the property of inducing a foreign body response in the skin, but also quantitatively evaluates the level of its ability to induce a foreign body response in the skin, as shown in Table 3.

[0116] Table 3 also shows the literature values ​​for the maximum effective concentration of each test sample, i.e., the concentration of the test sample at which the foreign body response reached a plateau. These literature values ​​are the maximum effective concentrations obtained in experiments using human hepatoma-derived cell line HepG2 cells, not keratinocytes. However, when compared with the maximum effective concentration in keratinocytes obtained in this experiment, it is clear that the degree of foreign body response elicited in human hepatoma-derived cells and keratinocytes is significantly different. For example, the maximum effective concentration of indirubin in human hepatoma-derived cells was 5-10 μM, whereas the maximum effective concentration in keratinocytes was 0.25 μM, less than one-twentieth of that in human hepatoma-derived cells. This result suggests that indirubin can elicit a foreign body response in keratinocytes at a lower dose than in human hepatoma-derived cells. Thus, the evaluation method of the present invention, which uses keratinocytes, allows accurate evaluation of the ability of a test sample to elicit a foreign body response in the skin, and is therefore extremely advantageous for evaluating the compatibility and safety of a test sample with skin.

[0117] <Experiment 2: Searching for substances that inhibit the foreign body response in the skin> As shown in Experiment 1-2, XRE-NLuc::HaCaT cells expressed reporter protein in amounts corresponding to the degree of activation of the foreign body response reaction involving the XRE foreign body response element. Therefore, the inventors believed that by using XRE-NLuc::HaCaT cells, it would be possible to search for substances that suppress the foreign body response reaction involving the XRE foreign body response element, in contrast to Experiment 1-2, and conducted an investigation. The experimental procedure is described below.

[0118] First, XRE-NLuc::HaCaT cells were cultured in DMEM medium at 1 × 10 5 The cells were suspended at a concentration of 10,000 cells / mL. 100 μL of the resulting cell suspension was added to each well of a white 96-well plate (ThermoFisher Scientific, catalog number 136102) so that each well contained 10,000 cells. The resulting suspension was then incubated for 24 hours at 37°C under 5% CO2 conditions. After removing the DMEM medium, 100 μL of DMEM medium containing 5 nM FICZ, which was confirmed to activate reporter protein expression in Experiment 1-2, and the candidate substances at the indicated concentrations was added. A control sample was also prepared by adding 100 μL of DMEM containing 5,000 nM FICZ but no candidate substances. In this experiment, 5-hydroxy-4-phenyl-2(5H)butenolide (hereinafter sometimes referred to as "Fregulide 1" or "FG1") was used as the candidate substance, and cinnamaldehyde, known to inhibit the XRE-mediated foreign body response, was used as a positive control. After 24 hours of incubation under conditions of 5% CO2 and 37°C, the expression level (luminescence intensity) of the reporter protein and the number of viable cells were measured according to the method described in Experiment 1-2, the expression level of the reporter protein per unit number of viable cells was calculated, and the inhibition rate (Inhibition (%)) of reporter protein expression exerted by each candidate substance was calculated according to the following formula.

[0119]

number

[0120] In the above formula, (Reporter Expression) FICZ indicates the reporter protein expression level per unit viable cell number obtained from the cells incubated in the medium containing 5,000 nM of FICZ but not the candidate substance. FICZ+TEST SAMPLE indicates the reporter protein expression level per unit viable cell count obtained from cells incubated in a medium containing 5,000 nM FICZ and a candidate substance. The results are shown in Figure 6. The results shown in Figure 6 are the average values ​​obtained from three independent experiments, and the error bars represent the standard error (SE).

[0121] As shown in Figure 6, in XRE-NLuc::HaCaT cells treated with the positive control cinnamaldehyde ("CA" in Figure 6) at 12.5 μM, 25 μM, and 50 μM, the expression of the reporter protein induced by FICZ (5,000 nM) was suppressed by 8%, 14%, and 16%, respectively. This result indicates that the addition of cinnamaldehyde suppresses the foreign body response induced by FICZ. Furthermore, by adding a candidate substance to be screened together with a substance that induces a foreign body response ("FICZ" in this experiment) to XRE-NLuc::HaCaT cells and selecting a candidate substance that reduces reporter protein expression compared to a control sample that does not contain the candidate substance, it is possible to obtain a substance that inhibits the foreign body response.

[0122] Surprisingly, when FG1 (Figure 6) was added to XRE-NLuc::HaCaT cells at concentrations of 12.5 μM, 25 μM, and 50 μM, the foreign body response induced by FICZ (5,000 nM) was suppressed by 58%, 68%, and 83%, respectively. These results indicate that FG1 suppresses the foreign body response mediated by the XRE foreign body response element, and that this suppression is far greater than that of cinnamaldehyde.

[0123] <Experiment 3-1: Inhibition of Artemin Production by Phleglide 1> These experiments demonstrated that Fregulide 1 has the property of significantly suppressing the foreign body response. Therefore, to further investigate the foreign body response-inhibitory effect of Fregulide 1, we examined the effect of Fregulide 1 on the expression level of artemin, a gene known to be transcriptionally regulated by the xenobiotic response element, in HaCaT keratinocytes. Specifically, HaCaT cells were exposed to FCTZ, a substance that induces the foreign body response, and Fregulide 1, and then quantitative PCR was used to observe the change in the expression level of artemin mRNA due to Fregulide 1. The procedure is as follows:

[0124] First, HaCaT cells (CLI, catalog number: 300493) were cultured in DMEM medium at a concentration of 1 × 10 5 The cells were suspended to a cell concentration of 10,000 cells / mL. 100 μL of the resulting cell suspension was added to each well of a white 96-well plate (ThermoFisher Scientific, catalog number: 136102) so that the number of cells per well was 10,000. After 24 hours of incubation at 5% CO and 37°C, the DMEM medium was removed, and 100 μL of DMEM medium containing the designated concentrations of FICZ (500 nM or 5,000 nM) and flegulin 1 (0 nM or 250 μM) was added. After 24 hours of incubation at 5% CO and 37°C, RNA was extracted and cDNA was prepared as described in Experiment 2. Quantitative PCR was performed using the obtained cDNA. The results are shown in Figure 7. In Figure 7, the results obtained for each sample are shown as relative values, with the expression level of Artemin mRNA in HaCaT cells incubated for 24 hours in DMEM medium containing neither FICZ nor fregulide 1 set to 1.

[0125] As shown in Figure 7, in HaCaT cells incubated in medium containing 500 nM or 5,000 nM FICZ ("FG1 -" in Figure 7), the expression level of artemin mRNA was increased by approximately 3.0-fold and approximately 3.7-fold compared to HaCaT cells incubated in medium without FICZ. This is thought to be due to FICZ-mediated activation of the XRE-mediated foreign body response. In contrast, in HaCaT cells incubated in medium containing 500 nM or 5,000 nM FICZ and 250 μM Flegulin 1 ("FG1 +" in Figure 7), no increase in artemin mRNA expression was observed compared to HaCaT cells incubated in medium without FICZ. This result indicates that Flegulin 1 suppresses the expression of artemin induced by the foreign body response, which is thought to be due to Flegulin 1's inhibition of FICZ-mediated activation of the foreign body response. Furthermore, when Fregulide 1 was applied, the level of artemin mRNA expression was at a similar level to that in HaCaT cells incubated in a medium without FICZ, and there was no excessive decrease in artemin expression. In other words, Fregulide 1 is thought to have the effect of suppressing abnormal artemin expression induced by suppressing the foreign body response in the skin, in other words, to normalize artemin expression.

[0126] <Experiment 3-2: Inhibition of Artemin Production by Phlegmide 1 - Part 2> As described above, Flegulide 1 inhibited FICZ-induced artemin production. Therefore, to quantitatively evaluate the inhibitory effect of Flegulide 1 on artemin production, the following experiment was performed. Specifically, HaCaT cells were treated with FICZ and Flegulide 1 in the same manner as in Experiment 3-1, except that the FICZ concentration was 5,000 nM and the Flegulide 1 concentrations were 0 μM, 0.01 μM, 0.1 μM, 1 μM, 10 μM, and 100 μM. RNA was extracted, cDNA was prepared, and quantitative PCR was performed. Based on the results, the inhibition rate (%) of FICZ-induced artemin mRNA expression obtained when 5,000 nM FICZ and a predetermined concentration of Flegulide 1 were added to the cell culture medium was calculated according to the following formula:

[0127]

number

[0128] In the above formula, (mRNA Expression) CONTROL is the expression level of Artemin mRNA in HaCaT cells incubated in a medium containing neither FICZ nor fregulide 1 (i.e., untreated HaCaT cells), (mRNA Expression) FICZ is the amount of Artemin mRNA expression in HaCaT cells incubated in a medium containing 5,000 nM FICZ. FICZ+TEST SAMPLE shows the expression level of artemin mRNA in HaCaT cells incubated in a medium containing 5,000 nM FICZ and a given concentration of flegulin 1.

[0129] As shown in Figure 8, Fregulide 1 concentration-dependently suppressed artemin expression induced by FICZ. Surprisingly, Fregulide 1 at a low concentration of only 100 nM (0.1 μM) suppressed artemin expression induced by FICZ at 5,000 nM (5 μM) by approximately 50%. These results indicate that Fregulide 1 highly effectively suppresses the XRE-mediated foreign body response and normalizes artemin expression. As mentioned above, induction of artemin expression is thought to be a cause of atopic dermatitis, and it has been reported that artemin expression is also induced by chemicals contained in air pollutants (e.g., Non-Patent Document 4). Fregulide 1, which highly effectively suppresses artemin expression in the skin, is considered to be extremely useful for the prevention and / or treatment of atopic dermatitis.

[0130] <Experiment 4-1: Inhibition of matrix metalloproteinase 9 production by fregulide 1> Next, we examined the effect of fregulide 1 on the expression of matrix metalloproteinase 9 (MMP9), another protein known to be transcriptionally regulated by the xenobiotic response element in HaCaT cells.

[0131] In this experiment, we first examined the effect of Fregulide 1 on the expression level of MMP9 mRNA. Specifically, HaCaT cells were treated with Fregulide 1 as described in Experiment 3-1, except that instead of incubating them for 24 hours in medium containing both FICZ and Fregulide 1, HaCaT cells were incubated for 24 hours in medium containing Fregulide 1 at 0 μM, 1 μM, 10 μM, or 100 μM. RNA was extracted, cDNA was prepared, and quantitative PCR was performed using the resulting cDNA. The quantitative PCR procedure was the same as that described in Experiment 3-1, except that the primers listed in Table 4 below were used. In addition to MMP9 mRNA, this experiment also quantified the mRNA of MMP2, an MMP not subject to transcriptional control by the xenobiotic response element (XRE).

[0132] [Table 4]

[0133] The results are shown in Figures 9 and 10. In Figures 9 and 10, the results obtained for each sample are shown as relative values, with the expression level of MMP9 or MMP2 mRNA in HaCaT cells incubated for 24 hours in DMEM medium not containing flegulin 1 (i.e., untreated HaCaT cells) set at 1. The results shown in Figures 9 and 10 are the average values ​​obtained from three independent experiments, and the error bars represent the standard deviation (SD).

[0134] As shown in Figure 9, HaCaT cells incubated in medium containing 1 μM, 10 μM, and 100 μM of Flegulide 1 showed a 42%, 52%, and 80% reduction in MMP9 mRNA expression compared to HaCaT cells incubated in medium without Flegulide 1. These results indicate that Flegulide 1 has the effect of reducing the expression of MMP9, an MMP whose transcription is regulated by the xenobiotic response element (XRE). In contrast, as shown in Figure 10, HaCaT cells incubated in medium containing 1 μM, 10 μM, and 100 μM of Flegulide 1 did not show a reduction in the expression of MMP2 mRNA, an MMP whose transcription is not regulated by the XRE. Thus, Flegulide 1 was shown to selectively reduce the expression of MMP9, whose transcription is regulated by the XRE-mediated xenobiotic response.

[0135] Next, we examined the effect of fregulide 1 on the expression level of MMP9 at the protein level. Specifically, HaCaT cells were cultured in DMEM medium at a concentration of 1 × 10 6 The cells were suspended at a cell concentration of 1 × 10 cells / mL, and the resulting cell suspension was placed in a 96-well plate at a cell density of 1 × 10 cells per well. 5100 μL of the culture supernatant was seeded into each well so that cells / well were 100 μL. After overnight incubation at 5% CO2 and 37°C, the DMEM culture medium was removed, and 100 μL of serum-free medium containing 0 μM and 100 μM fregulide 1 was added. The cells were then incubated for an additional 24 hours at 5% CO2 and 37°C. 10 μL of the culture supernatant was subjected to gelatin zymography.

[0136] Gelatin zymography was performed according to standard methods (https: / / www.abcam.com / protocols / gelatin-zymography-protocol). Specifically, proteins in the culture supernatant were separated by SDS-PAGE using a 7.5% polyacrylamide gel containing 1 mg / mL gelatin. After electrophoresis, the gel was washed with 5 mM Ca. 2+ , 1 μM Zn 2+ The gel was then immersed in a washing buffer containing 2.5% Triton X-100 and 5 mM CaCl. 2+ , 1 μM Zn 2+ The gel was then immersed overnight at 37°C in an incubation buffer containing 1% Triton X-100 and the enzyme reaction was carried out. The gelatin in the resulting gel was visualized by Coomassie blue staining. In areas where active MMP9 was present, the gelatin in the gel was degraded by its gelatinase activity. In contrast, in areas where active MMP9 was not present, the gelatin was not degraded and was stained blue by Coomassie blue staining. Therefore, active MMP9 was detected as a transparent band in the gel stained blue by Coomassie blue staining. The results are shown in Figure 11.

[0137] As shown in Figure 11, in HaCaT cells incubated in medium containing 100 μM Fleglide 1, the gelatinase activity of MMP9 was reduced by approximately 54% or 24%, compared to HaCaT cells incubated in medium without Fleglide 1. This result indicates that Fleglide 1 reduced the amount of MMP9 protein, which is consistent with the experimental result shown in Figure 9 that Fleglide 1 significantly reduced the expression level of MMP9 mRNA.

[0138] <Experiment 4-2 Comparison with retinoic acid and nicotinamide> Next, we conducted an experiment to inhibit MMP9 and MMP2 in HaCaT cells using retinoic acid (CAS number: 302-79-4) and nicotinamide (CAS number: 98-92-0), which are known to have anti-wrinkle effects. Specifically, we contacted HaCaT cells with retinoic acid and nicotinamide in the same manner as in Experiment 4-1, except that retinoic acid and nicotinamide were used instead of fleguride 1. RNA was extracted, cDNA was prepared, and quantitative PCR was performed using the obtained cDNA.

[0139] The results are shown in Figures 12 and 13. In Figures 12 and 13, the results obtained for each sample are shown as relative values, with the expression level of MMP9 or MMP2 mRNA in HaCaT cells incubated for 24 hours in DMEM medium not containing retinoic acid or nicotinamide (i.e., untreated HaCaT cells) set to 1. The results shown in Figures 12 and 13 are the average values ​​obtained from three independent experiments, and the error bars represent the standard deviation (SD).

[0140] As shown in Figure 12, a decrease in MMP9 expression was also confirmed in HaCaT cells incubated in medium containing retinoic acid ("RA" in Figure 12, same in Figure 13) or nicotinamide ("NA" in Figure 12, same in Figure 13). However, as shown in Figure 13, in HaCaT cells incubated in medium containing retinoic acid or nicotinamide, a decrease in the expression of not only MMP9, which is transcriptionally regulated by the XRE, but also MMP2, which is not transcriptionally regulated by the XRE and therefore should be unrelated to the XRE response in the skin, was also confirmed. These results indicate that retinoic acid and nicotinamide, which have traditionally been used as active ingredients in anti-wrinkle agents, nonspecifically suppress the expression of many genes, including MMP2, a matrix metalloproteinase other than MMP9. Such nonspecific suppression of MMP expression is undesirable because it may inhibit the extracellular matrix degradation required in normal tissues.

[0141] In contrast, as shown in Experiment 4-1, Fregulide 1 selectively inhibits the expression of only MMP9, which is transcriptionally controlled by the Xenobiotic Response Element (XRE), but not MMP2, and therefore has the advantage of being less likely to degrade the extracellular matrix required in normal tissues. In other words, Fregulide 1 selectively inhibits only the expression of MMP9, which is caused by a foreign body response reaction in the skin induced by various chemicals such as environmental pollutants, and is therefore less likely to cause side effects, making it particularly suitable for use as an active ingredient in anti-wrinkle agents. Needless to say, Fregulide 1, which selectively inhibits only MMP9 expression, is extremely useful not only as an active ingredient in anti-wrinkle agents, but also as an active ingredient in agents for treating and / or preventing various diseases known to be associated with increased MMP9 expression.

[0142] <Experiment 5: Effect of Fregulide 1 on TIMP-1 expression> These results demonstrate that fregulide 1 selectively suppresses the expression of matrix metalloproteinase 9, a substance that causes wrinkles. To investigate the effects of fregulide 1 on other factors related to wrinkles, we investigated its effect on the expression of TIMP-1 (tissue inhibitor of metalloproteinase-1), a protein that inhibits MMPs. It is known that collagen homeostasis is maintained in vivo through antagonistic interactions between matrix metalloproteinases (MMPs), which are collagen-degrading enzymes, and TIMPs (tissue inhibitors of metalloproteinases), which inhibit MMPs. When the balance between the two is disrupted and MMP expression becomes dominant, excessive collagen degradation occurs, which is thought to be the cause of wrinkles.

[0143] Specifically, quantitative PCR was performed using the cDNA obtained in Experiment 4-1 to evaluate the expression level of TIMP-1 mRNA in HaCaT cells treated with Fregulide 1. The quantitative PCR procedure was the same as that described in Experiment 4-1, except that the primers shown in Table 5 below were used.

[0144] [Table 5]

[0145] The results are shown in Figure 14. Note that the results for each sample in Figure 14 are shown as relative values, with the expression level of TIMP-1 in HaCaT cells incubated for 24 hours in DMEM medium not containing flegulin 1 (i.e., untreated HaCaT cells) set at 1. The results shown in Figure 14 are the average values ​​obtained from three independent experiments, and the error bars represent the standard deviation (SD).

[0146] As shown in Figure 14, in HaCaT cells incubated in medium containing 1 μM, 10 μM, and 100 μM Flegulide 1, TIMP-1 expression increased in a concentration-dependent manner depending on the Flegulide 1 concentration. In particular, when incubated in medium containing 100 μM Flegulide 1, TIMP-1 expression increased approximately fourfold compared to untreated HaCaT cells. This result indicates that Flegulide 1 not only reduces MMP-9 expression but also increases the expression of TIMP-1, which acts as an MMP9 inhibitor, thereby inhibiting the activity of MMP9. In other words, Flegulide 1 can function not only as an active ingredient in agents that suppress MMP-9 production but also as an active ingredient in agents that promote TIMP-1 production. Flegulide 1, which exerts such multifaceted anti-wrinkle effects, is considered to be extremely useful for improving and / or preventing wrinkles.

[0147] For comparison, retinoic acid and nicotinamide, which are known to be active ingredients in anti-wrinkle agents, were also evaluated in the same manner using the cDNA obtained in Experiment 4-2. The quantitative PCR procedure was the same as that described in Experiment 3-1, except that the primers shown in Table 5 were used.

[0148] The results are shown in Figure 15. In Figure 15, the results obtained for each sample are shown as relative values, with the expression level of TIMP-1 in HaCaT cells incubated for 24 hours in DMEM medium containing no retinoic acid or nicotinamide (untreated HaCaT cells) set at 1. The results shown in Figure 15 are the average values ​​obtained from three independent experiments, and the error bars represent the standard deviation (SD).

[0149] As shown in Figure 15, no increase in TIMP-1 expression was observed in HaCaT cells incubated in medium containing either retinoic acid or nicotinamide. This result indicates that Fregulide 1, which not only reduces the expression level of MMP9 but also increases the expression level of TIMP-1, is extremely useful as an active ingredient in anti-wrinkle agents compared to retinoic acid and nicotinamide, which are the active ingredients in existing anti-wrinkle agents.

[0150] <Experiment 6: Safety evaluation of Phleglide 1> The safety of Fregulide 1 was examined by measuring the number of viable cells using a water-soluble tetrazolium salt (WST-1). Specifically, HaCaT cells were cultured in DMEM medium at a concentration of 1 × 10 5 The cells were suspended at a concentration of 10,000 cells / mL. The resulting cell suspension was added to a white 96-well plate (ThermoFisher Scientific, catalog number: 136102) at 100 μL per well to achieve a cell density of 10,000 cells per well. Following standard incubation procedures at 37°C under 5% CO2 for 24 hours, the DMEM medium was removed, and 100 μL of DMEM medium containing a predetermined concentration of flegulin 1 and 100 μL of DMEM medium without flegulin 1 as a control were added. The cells were then incubated for another 24 hours under 5% CO2 and 37°C conditions. After the 24-hour incubation, cell counts were measured using the "Premix WST-1 Cell Proliferation Assay System" (Takara Bio Inc., catalog number: MK400), a premixed reagent containing WST-1, according to the procedure described in Experiment 1-2. The results are shown in Figure 16. The results shown in FIG. 16 are the average values ​​obtained from three independent experiments, and the error bars represent the standard error (SE).

[0151] As shown in Figure 16, even when HaCaT cells were incubated for 24 hours in DMEM medium containing 500 μM of Flegulide 1, the cell viability exceeded 50%, indicating that the LC50 (median lethal concentration) of Flegulide 1 for HaCaT cells was at least 500 μM. These results indicate that Flegulide 1 has low cytotoxicity and is a highly safe substance. Therefore, Flegulide 1 can be advantageously used as an ingredient in a wide range of products, including pharmaceuticals, cosmetics, health foods, functional foods, and daily necessities.

[0152] The present invention also encompasses various modifications within the scope of the claims that are easily conceivable by a person skilled in the art. The contents of papers, published patent applications, patent publications, and other documents explicitly stated in this specification are hereby incorporated by reference in their entirety.

[0153] Examples of formulations of the agent according to the present invention are shown below, but the present invention is not limited to these in any way.

[0154] <Prescription example 1: Shampoo> Water 87.20% by weight Lauroylmethylalanine TEA 6.00% by weight Lauryl betaine 3.00% by weight Polyquaternium-10 0.30% by weight Sodium bicarbonate 0.02% by weight Sodium lactate 0.20% by weight Cocamidomethyl MEA 1.00% by weight Cocamide DEA 1.00% by weight Lactic acid 0.28% by weight Etidronic acid 0.06% by weight Pentasodium pentetate 0.04% by weight Sodium benzoate 0.40% by weight Fragrance 0.30% by weight Phleglide 1 0.20% by weight

[0155] <Formulation example 2: Hair treatment> Water 86.73% by weight Glycerin 5.00% by weight Cetyl alcohol 3.00% by weight Stearyl alcohol 1.50% by weight Behentrimonium methosulfate 1.50% by weight Amodimethicone 0.50% by weight Dimethicone 1.00% by weight Hydroxyethyl cellulose 0.05% by weight Phenoxyethanol 0.32% by weight Fragrance 0.30% by weight Phleglide 1 0.10% by weight

[0156] <Formulation example 3: Hair milk (leave-on treatment)> Water 93.85% by weight Dimethiconol 2.50% by weight Polyacrylamide 1.20% by weight (C13,14) isoparaffin 0.72% by weight Sodium sulfate 0.02% by weight Hydroxyethyl cellulose 0.01% by weight Sodium lauryl sulfate 0.08% by weight Sodium laureth sulfate 0.08% by weight Polysorbate 20 0.50% by weight Laureth-7 0.18% by weight Phenoxyethanol 0.33% by weight Methylparaben 0.01% by weight Ethylparaben 0.30% by weight Propylparaben 0.05% by weight Butylparaben 0.02% by weight Fragrance 0.10% by weight Phleglide 1 0.05% by weight

[0157] <Formulation Example 4: Skin Milk (Emulsion)> Water 82.89% by weight EDTA-2Na 0.01% by weight Carbomer 0.20% by weight Glycerin 3.00% by weight 1,3-butylene glycol 7.00% by weight Phenoxyethanol 0.30% by weight Methylparaben 0.20% by weight Potassium hydroxide 0.06% by weight Ceteareth-13 1.00% by weight Sorbitan sesquioleate 0.30% by weight Mineral oil 5.00% by weight Phleglide 1 0.04% by weight

[0158] <Formulation example 5: Facial cleanser> Water 37.16% by weight Myristic acid 20.00% by weight Palmitic acid 4.00% by weight Lauric acid 2.00% by weight Stearic acid 1.00% by weight Potassium hydroxide 5.80% by weight Polyglyceryl lauryl ether 3.00% by weight Glyceryl stearate 1.00% by weight Glycol distearate 2.00% by weight Glycerin 20.00% by weight 1,3-butylene glycol 1.00% by weight Lauryl betaine 3.00% by weight Phleglide 1 0.04% by weight

[0159] <Prescription example 6: Body soap> Water 65.50% by weight Lauric acid 5.50% by weight Myristic acid 10.00% by weight Stearic acid 1.00% by weight Lauryl betaine 3.50% by weight Polyglyceryl lauryl ether 2.00% by weight Propylene glycol 5.00% by weight Glycol distearate 2.00% by weight (Acrylates / alkyl acrylate) crosspolymer 0.10% by weight Sodium hydroxide 4.00% by weight Sodium chloride 1.00% by weight Fragrance 0.30% by weight Phleglide 1 0.10% by weight

[0160] <Formulation example 7: Body milk> Water 85.60% by weight EDTA-2Na 0.01% by weight Carbomer 0.20% by weight Hydroxyethyl cellulose 0.10% by weight 1,3-butylene glycol 8.00% by weight Phenoxyethanol 0.30% by weight Methylparaben 0.20% by weight Potassium hydroxide 0.17% by weight Polyglyceryl decaisostearate 3.00% by weight Olive oil 2.00% by weight (Acrylates / alkyl acrylate) crosspolymer 0.20% by weight Fragrance 0.20% by weight Phleglide 1 0.02% by weight

[0161] <Formulation Example 8: Bath Milk> Water 24.37% by weight Olive oil 45.00% by weight 1,3-butylene glycol 8.00% by weight Polyglyceryl oleate 5.00% by weight Polyglyceryl decaisostearate 2.00% by weight Bentonite 15.00% by weight Methylparaben 0.20% by weight Citric acid / sodium citrate 0.40% by weight Phleglide 1 0.03% by weight [Industrial Applicability]

[0162] In modern society, where a variety of chemicals are used in various products around us, it is extremely important to understand the effects of chemicals on the skin. The evaluation method of the present invention, which can easily and quickly evaluate the ability of various test samples to induce a foreign body response in the skin, is expected to be applied in a wide range of industrial fields, including cosmetics, pharmaceuticals, and food products. Furthermore, the agent of the present invention for suppressing a foreign body response in the skin can be incorporated into various products to maintain healthy skin, such as for the prevention and / or treatment of atopic dermatitis and the prevention and / or improvement of wrinkles. Furthermore, the discovery method of the present invention makes it possible to easily and quickly screen for substances that have the ability to suppress a foreign body response in the skin and that can be used as active ingredients in such agents. As described above, the present invention has great industrial applicability.

Claims

1. An agent for suppressing the expression of a gene transcribed with the involvement of a xenobiotic response element (XRE), comprising 5-hydroxy-4-phenyl-2(5H)butenolide and / or a salt thereof.

2. The agent according to claim 1, wherein the gene transcribed in association with a foreign body response element (XRE) is artemin.

3. The agent according to claim 2, which is used for preventing and / or treating atopic dermatitis.

4. The agent according to claim 1, wherein the gene transcribed in association with a foreign body response element (XRE) is matrix metalloproteinase 9.

5. The agent according to claim 4, which is used to prevent and / or improve wrinkles.

6. A pharmaceutical product, quasi-drug, cosmetic, daily necessities, or food product containing the agent according to any one of claims 1 to 5.

7. 7. The cosmetic product according to claim 6, which is a lotion, emulsion, cream, serum, cleanser, pack, facial cleanser, soap, makeup base, foundation, concealer, face powder, lipstick, blush, eye shadow, eyeliner, eyebrow makeup, mascara, body soap, body milk, body lotion, body cream, body oil, body powder, sunscreen, sun oil, deodorant spray, bleaching cream, depilatory cream, hand cream, shampoo, rinse, rinse-in shampoo, dry shampoo, hair treatment, conditioner, hair milk, hair cream, hair oil, hair mist, tonic, hair foam, hair gel, wax, pomade, hair liquid, waving agent, hair color, bleach, color rinse, hair growth agent, hair regrowth agent, toothpaste, mouthwash, gargle, bath milk, bath salts, bath oil, perfume, or cologne.

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