Method for detecting sensitization of test substance, method for screening sensitization-suppressing substance, and method for producing composition for administration to living organisms

Abstract

To provide a method capable of confirming safety properties with more excellent reliability in vitro.SOLUTION: A detection method of sensitization by a mutual action of a test substance and a target cell comprises: a co-culture step for co-culturing a target cell which coexists with a test substance and a maturation marker expression cell; a detection step for detecting expression of a maturation marker in the maturation marker expression cell; and an evaluation step for, on the basis of an expression amount of the maturation marker detected in the detection step, evaluating presence / absence of sensitization generated by the mutual action of the test substance and the target cell, in which the maturation marker expression cell is a cell expressing a maturation marker which functions in antigen presentation in an immunity sensitization step.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] The present invention relates to a method for detecting sensitization, a method for screening for a sensitization-suppressing substance, and a method for producing a composition for administration to a living body. [Background technology]

[0002] Rhododenol (RD) is an inhibitor of tyrosinase, which is involved in melanin synthesis, and has been used as a so-called skin-whitening ingredient. However, it has been found that vitiligo develops as a result of the induction of autoimmune disease. This is thought to be due to the following mechanism: RD is not only an inhibitor of tyrosinase, but also serves as a substrate for tyrosinase, producing RD quinones in melanin-producing cells (melanocytes). During the metabolism of RD, reactive oxygen species are generated, which damage melanin-producing cells. Furthermore, it is thought that the RD quinones form a complex with tyrosinase (a hapten), which acts as a new antigen and induces melanin-specific cytotoxic lymphocytes (CTLs), inducing autoimmune disease as a side effect.

[0003] Safety verification is being conducted for various drugs to prevent the induction of side effects. However, in the case of cosmetics and other products, the current international trend is to require verification using methods that do not use laboratory animals, and so-called in vitro safety verification is being conducted. However, as with RD, even if safety is confirmed in vitro, side effects are only confirmed after actual use in humans. For this reason, more reliable in vitro safety verification is required. Therefore, the human cell line activation test (h-CLAT) is currently commonly used to evaluate drug sensitization, using human monocyte-based cultured cells THP-1, which, like dendritic cells, express maturation markers such as CD86 upon activation (Non-Patent Document 1). However, further reliability is required. Summary of the Invention [Problem to be solved by the invention]

[0004] Therefore, an object of the present invention is to provide a new method for confirming safety in vitro with greater reliability. [Means for solving the problem]

[0005] In order to achieve the above-mentioned object, the method of the present invention for detecting sensitization due to the interaction between a test substance and target cells comprises a co-culture step of co-culturing target cells in which the test substance has been co-presented with maturation marker-expressing cells, a detection step of detecting the expression of a maturation marker in the maturation marker-expressing cells, and an evaluation step of evaluating the presence or absence of sensitization caused by the interaction between the test substance and target cells based on the expression level of the maturation marker detected in the detection step, wherein the maturation marker-expressing cells are cells that express a maturation marker that functions during antigen presentation in the immune sensitization process.

[0006] The screening method for sensitization-inhibiting substances of the present invention is characterized in that a sensitization-induction-related substance involved in the induction of sensitization is used as the first test substance, a sensitization-inhibiting test substance is used as the second test substance, the sensitization-inhibiting test substance and the sensitization-inhibiting test substance are allowed to coexist, the sensitization detection method of the present invention is performed, and the sensitization-inhibiting ability of the sensitization-inhibiting test substance is evaluated by comparing the expression level of the maturation marker in the absence of the sensitization-inhibiting test substance with the expression level of the maturation marker in the presence of the sensitization-inhibiting test substance.

[0007] The method for producing a composition for administration to a living body of the present invention is characterized in that a test substance is confirmed to be capable of inducing sensitization due to the interaction between the test substance and target cells using the sensitization detection method of the present invention, and a test substance that does not induce sensitization is used as a component of the composition for administration to a living body in the production of the composition for administration to a living body. [Effects of the Invention]

[0008] According to the present invention, by co-culturing target cells in the presence of a test substance with maturation marker-expressing cells and detecting the expression of the maturation marker in the maturation marker-expressing cells, it is possible to detect with greater reliability whether sensitization is induced by treating the target cells of interest with the test substance.

[0009] The aforementioned h-CLAT can evaluate sensitization by culturing THP-1 cells in the presence of a test substance and confirming the expression of maturation markers such as CD86 in the THP-1 cells. However, when the aforementioned RD is evaluated using this method, CD86 expression is not confirmed in the THP-1 cells. On the other hand, according to the method of the present invention, when THP-1 cells are used as the maturation marker-expressing cells, melanocytes are used as the target cells, and RD is used as the test substance, CD86 expression can be confirmed in the THP-1 cells. Thus, according to the present invention, even when sensitization is not determined to be induced by the h-CLAT, the induction of sensitization can be detected. In particular, the induction of sensitization can be detected not only when the test substance itself induces sensitization, but also when sensitization is induced as a result of the interaction between the test substance and target cells. Therefore, the present invention can detect the induction of sensitization with higher reliability. [Brief explanation of the drawings]

[0010] [Figure 1] FIG. 1(A) is a model diagram of co-culture (h-CLAT / wM) with the addition of rhododenol, FIG. 1(B) is a graph showing changes in CD86 expression levels in an example of the co-culture and a comparative example using h-CLAT, and FIG. 1(C) is a graph showing changes in cell viability in the example and comparative example. [Figure 2] FIG. 2 shows an example of a histogram of CD86 expression levels in a co-culture (h-CLAT / wM) supplemented with rhododenol and a comparative example using h-CLAT. [Figure 3]Figure 3 is a graph showing the change in CD86 expression level (A) and the change in cell viability (B) in the example of co-culture (h-CLAT / wM) with the addition of raspberry ketone, a substance that induces vitiligo, and the comparative example using h-CLAT. [Figure 4] Figure 4 is a graph showing the change in CD86 expression level (A) and the change in cell viability (B) in the example of co-culture (h-CLAT / wM) with the addition of ascorbic acid, a non-vitiligo-inducing substance, and the comparative example using h-CLAT. [Figure 5] Figure 5 is a graph showing the change in CD86 expression level (A) and the change in cell viability (B) in the example of co-culture (h-CLAT / wM) with the addition of tranexamic acid, a non-vitiligo-inducing substance, and the comparative example using h-CLAT. [Figure 6] FIG. 6(A) is a graph showing the amount of ROS generated in an example of co-culture (h-CLAT / wM) supplemented with rhododenol and a comparative example using h-CLAT. FIG. 6(B) is a graph showing the change in CD86 expression level in an example of co-culture (h-CLAT / wM) supplemented with rhododenol and NAC (N-acetylcysteine), an antioxidant that scavenges reactive oxygen species, and a comparative example using h-CLAT. [Figure 7] FIG. 7(A) is a graph showing changes in extracellular ATP concentration in the culture supernatant in an example of co-culture (h-CLAT / wM) with the addition of rhododenol and a comparative example of h-CLAT. FIG. 7(B) is a graph showing changes in CD86 expression levels in an example of co-culture (h-CLAT / wM) with the addition of rhododenol and BBG and a comparative example using h-CLAT. DETAILED DESCRIPTION OF THE INVENTION

[0011] The detection method of the present invention includes, for example, a culture step of culturing the target cells prior to the co-culture step, and after the culture step, the test substance is allowed to coexist with the target cells, and co-culture with the maturation marker-expressing cells is performed.

[0012] In the detection method of the present invention, for example, the maturation marker-expressing cells are at least one selected from the group consisting of dendritic cells, dendritic cell-like cells, and monocyte-derived cells.

[0013] In the detection method of the present invention, for example, the monocyte-derived cells are THP-1 cells.

[0014] In the detection method of the present invention, for example, the maturation marker is at least one selected from the group consisting of CD86, CD54, CD80, OX40L, and HLA-DR.

[0015] In the detection method of the present invention, for example, in the evaluation step, the presence or absence of sensitization is evaluated by comparing the expression level of the maturation marker detected in the detection step with a standard, and the standard is the expression level of the maturation marker in maturation marker-expressing cells in the absence of the test substance, and if the expression level is significantly higher than the standard, it is evaluated as exhibiting sensitization.

[0016] In the detection method of the present invention, for example, the target cells are at least one selected from the group consisting of epithelial cells, keratinocytes, basal cells, spinous cells, granular cells, melanocytes, Langerhans cells, Merkel cells, and hepatocytes.

[0017] In the detection method of the present invention, for example, the tissue from which the target cells are derived is at least one selected from the group consisting of skin, respiratory organs, and liver.

[0018] In the detection method of the present invention, for example, the target cells are tissue-mimicking cells.

[0019] In the detection method of the present invention, for example, in the detection step, the maturation marker is separated from the target cells and the expression of the maturation marker is detected.

[0020] The detection method of the present invention uses, for example, a co-culture set consisting of an outer container and an inner container, where the outer container is an incubator for the maturation marker-expressing cells and the inner container is an incubator for the target cells, and the co-culture set is used with an inner container that can be placed inside the outer container and has a bottom that allows components in the culture medium other than the cells to pass through to the outer container, and the culture step involves placing the target cells and culture medium in the inner container and culturing the target cells, and the co-culture step involves placing the maturation marker-expressing cells and culture medium in the outer container, further placing the inner container inside the outer container, adding the test substance to the inner container, and co-culturing the outer container and the inner container.

[0021] In the method of the present invention for producing a composition for administration to a living body, for example, the composition for administration to a living body is at least one selected from the group consisting of pharmaceuticals, quasi-drugs, and cosmetics.

[0022] <Method for detecting sensitization> As described above, the present invention comprises a co-culture step of co-culturing target cells in which a test substance has been co-cultured with maturation marker-expressing cells, a detection step of detecting the expression of a maturation marker in the maturation marker-expressing cells, and an evaluation step of evaluating the presence or absence of sensitization caused by the interaction between the test substance and the target cells based on the expression level of the maturation marker detected in the detection step, wherein the maturation marker-expressing cells are cells that express a maturation marker that functions during antigen presentation in the immune sensitization process.

[0023] In the present invention, sensitization due to the interaction between the test substance and the target cells means that maturation marker-expressing cells are activated by treating the target cells with the test substance, specifically, the activation means that maturation markers that function during antigen presentation in the immune sensitization process are expressed. The degree of sensitization is, for example, the degree of activation, and can be analyzed and evaluated based on the expression level of the maturation marker. The analysis may be, for example, qualitative or quantitative.

[0024] For example, when a metabolite produced by the metabolism of the test substance in the target cells activates the maturation marker-expressing cells and induces sensitization, this could not be detected by the h-CLAT, but the present invention can detect the induction of sensitization. In such cases, the metabolite is also referred to as, for example, a prehapten (a general term for substances that require autoxidation to acquire sensitization ability) or a prohapten (a general term for substances that require metabolism to acquire sensitization ability). However, the present invention is not limited thereto. For example, the induction of sensitization can also be detected when the test substance itself acts as an antigen and activates the maturation marker-expressing cells to induce sensitization. Therefore, for example, by detecting sensitization using the present invention and the h-CLAT for the same test substance, it is possible to determine whether sensitization is induced directly by the test substance, by the metabolite, or by both.

[0025] As described above, the maturation marker-expressing cells are cells that express a maturation marker that functions during antigen presentation in the immune sensitization process. An example of such cells is dendritic cells. Initially, dendritic cells are immature dendritic cells that have the ability to uptake antigens. Upon uptake of antigens, they migrate to lymphoid organs, acquire the ability to activate T cells, lose their antigen uptake ability, and express maturation markers such as CD86 on their surfaces, becoming mature dendritic cells.

[0026] Other examples of the maturation marker-expressing cells include dendritic cell-like cells and monocyte-derived cells, which express the maturation marker upon antigen presentation, similar to dendritic cells. Thus, various cells, such as the aforementioned THP-1, are known that express the maturation marker upon activation by antigen uptake, similar to dendritic cells, and can be applied to the present invention. Examples of the dendritic cell-like cells include THP-1, MUTZ-3, KG-1, CD14-ML-DC, ELD-1, and CAL-1. Examples of the monocyte-derived cells include THP-1, U937, and CD14-ML. The maturation marker-expressing cells may be, for example, cells isolated from a living organism (e.g., primary cells), cell lines isolated as a culture system, or recombinant cells constructed by genetic engineering.

[0027] The maturation marker is not particularly limited and examples thereof include markers expressed by the dendritic cells etc. upon antigen presentation, specific examples of which include costimulatory molecules such as CD86 and CD80, CD54, HLA-DR, OX40L etc. As the maturation marker-expressing cells, for example, cells that express a maturation marker upon activation can be used depending on the type of maturation marker to be detected in the detection step described below.

[0028] The target cells are not particularly limited, and any cells can be selected, for example. Examples of the target cells include epithelial cells and hepatic cells. The origin of these tissues is not particularly limited, and examples include skin, epithelial cells of various organs, and interstitial cells. The organs are not particularly limited, and examples include the respiratory system, digestive system, and liver. Specific examples of the cells include basal cells (e.g., melanoma cells), keratinocytes (e.g., keratinocyte cells), spinous cells, granular cells, Langerhans cells, and Merkel cells. The tissue from which the target cells are derived is not particularly limited, and examples include the skin, respiratory system, and liver. The target cells may be, for example, tissue-mimicking cells, specifically, target tissue-mimicking cells. The mimicking cells are, for example, mimicking cells of the target tissue of a human or non-human animal, as described below, and specific examples include human tissue-mimicking cells.

[0029] The organism from which the cells are derived is not particularly limited and may be, for example, an organism to which the test substance is intended to be applied, specifically, a human or a non-human animal. Examples of the non-human animal include a mouse, rat, rabbit, dog, cat, monkey, cow, pig, sheep, and horse.

[0030] The target cells used in the present invention may be, for example, one type or a combination of two or more types. In the former case, for example, a monolayer composed of one type of cell may be used in the present invention. In the latter case, for example, a laminate of layers of cells may be used in the present invention. As a specific example, when confirming the effect on skin, the target cells may be used in the co-culture as a monolayer composed of one type of cell, such as basal cells or keratinocytes. Furthermore, as a combination of two or more types, for example, a laminate composed of, from bottom to top, the basal layer, the spinous layer, the granular layer, and the stratum corneum may be used in the co-culture, simulating skin tissue, or a laminate combining two or three of these layers may be used in the co-culture.

[0031] The test substance is not limited in any way, and various substances, such as low-molecular-weight compounds, high-molecular-weight compounds, nucleic acids, proteins, and peptides, can be applied to the present invention. According to the present invention, for example, not only test substances as active ingredients for a desired application, but also additives such as excipients used in preparing a composition can be used to detect sensitization due to their interaction with the target cells. In this way, the safety of various components constituting various compositions administered to the human body, such as cosmetics, pharmaceuticals, and foods and beverages, can be evaluated, making it possible to provide compositions with superior safety.

[0032] As described above, the detection method of the present invention involves co-culturing the target cells and the maturation marker-expressing cells, and further detecting the expression of the maturation marker in the maturation marker-expressing cells after the co-culturing step. Therefore, it is preferable that the target cells and the maturation marker-expressing cells are co-cultured in a state that allows them to be easily separated after co-culture. For example, a culture tool for culturing two types of cells in a separable manner can be used in such co-culture. For example, a co-culture set combining an outer container and an inner container can be used as the culture tool.

[0033] The outer container is, for example, a culture vessel (also called a culture tank) for the maturation marker-expressing cells, and is preferably a container capable of two-dimensional suspension culture, such as a 6-well plate or a 12-well plate.

[0034] On the other hand, the inner container is, for example, a culture vessel for maturation marker-expressing cells, and can be placed inside the outer container. The inner container is preferably a container that can deliver components of the culture medium of target cells cultured in the presence of the test substance into the culture environment of the maturation marker cells, without intermixing the target cells with the maturation marker cells, during co-culture. That is, for example, an inner container having a partition (e.g., a bottom) that allows components of the culture medium other than the cells seeded in the inner container to pass through (or be permeable to) into the outer container is preferred. A specific example is a container with a scaffold for three-dimensional culture, the bottom of which is made of, for example, a cell scaffold. The bottom of the container may be, for example, a filter of a so-called transwell insert. By using such an inner container, for example, the scaffold at the bottom can capture target cells, and during co-culture, components in the culture medium can be delivered to the outside through the voids in the scaffold, i.e., into the medium inside the outer container. Specifically, for example, contact between the medium inside the outer container and the bottom of the inner container allows components in the culture medium inside the inner container to be delivered to the medium inside the outer container. The type of scaffold material is not particularly limited, and examples include so-called porous bodies, such as those made of resins such as polystyrene. For example, a 3D culture substrate such as Alvetex (registered trademark) Scaffold can be used as the inner container.

[0035] The method for detecting sensitization of the present invention first involves a co-culture step in which target cells co-cultured with a test substance are co-cultured with maturation marker-expressing cells.

[0036] As described above, for the detection step after the co-culture step, it is preferable to co-culture, for example, the target cells and the maturation marker-expressing cells in a state that allows easy separation after co-culture, and as described above, it is preferable to use the co-culture set.

[0037] The target cells are seeded in a medium placed in the inner container. The medium is not particularly limited and can be selected appropriately depending on the type of the target cells, for example.

[0038] Furthermore, the target cells may be pre-cultured, for example, prior to this co-culture step. In this case, the culture temperature and culture time are not particularly limited and can be set appropriately depending on, for example, the type of the target cells. Specific examples include a culture temperature of, for example, 36 to 37°C, and a culture time of, for example, 24 to 48 hours. Furthermore, the target cells may be treated with the test substance prior to co-culture, for example; specifically, the target cells may be pre-cultured and then treated by adding the test substance. The treatment time with the test substance is, for example, 15 to 60 minutes.

[0039] On the other hand, the maturation marker-expressing cells are seeded in a medium placed in the outer container. The medium is not particularly limited and can be selected appropriately depending on, for example, the type of the maturation marker-expressing cells. The medium may be the same as or different from the medium used for the target cells.

[0040] Then, for example, the inner container seeded with the target cells is set inside the outer container, the test substance is added to the target cells in the inner container, and the target cells and the maturation marker-expressing cells are co-cultured. The treatment of the target cells with the test substance may be performed prior to the co-culture, as described above, or the treatment and co-culture may be performed in parallel by adding the test substance to the target cells under co-culture conditions.

[0041] The culture conditions for the co-culture are not particularly limited, and the culture temperature is, for example, 36 to 37° C. The total culture time until the maturation marker is detected is, for example, 48 to 72 hours for target cells and 24 to 48 hours for maturation marker-expressing cells. The amount of the test substance to be added is not particularly limited.

[0042] In the co-culture, the ratio of the target cells to the maturation marker-expressing cells is not particularly limited. 6 3 × 10 cells expressing the maturation marker 5 ~5×10 5 There are individuals.

[0043] When using the co-culture set, it is preferable to set the inner container inside the outer container so that the bottom of the inner container comes into contact with the medium in the outer container, thereby allowing, for example, components in the culture medium in the inner container, specifically, the test substance and metabolic products produced by treating the target cells with the test substance, to be delivered to the medium in the outer container through the bottom of the inner container.

[0044] Next, a detection step is carried out in which the expression of the maturation marker in the maturation marker-expressing cells is detected.

[0045] The detection of the expression marker is preferably carried out, for example, by separating the co-cultured target cells and the maturation marker-expressing cells, and then detecting the maturation marker-expressing cells. The method of separation is not particularly limited, and, for example, when using the co-culture set described above, the inner container may be removed from the outer container, and only the maturation marker-expressing cells in the outer container may be subjected to expression detection.

[0046] The method for detecting the expression of the maturation marker in the maturation marker-expressing cells is not particularly limited and can be appropriately determined depending on, for example, the type of the maturation marker. Examples of methods for detecting the maturation marker expressed on the surface of the maturation marker-expressing cells include flow cytometry such as fluorescence-activated cell sorting (FACS) and RT-qPCR.

[0047] Next, an evaluation step is carried out to evaluate the presence or absence of sensitization caused by the interaction between the test substance and the target cells based on the expression level of the maturation marker detected in the detection step.

[0048] The presence or absence of sensitization, i.e., whether sensitization has been induced, can be determined, for example, by comparing the expression level of the maturation marker detected in the detection step with a predetermined standard. The standard can be set arbitrarily, for example. Specific examples include, for example, when the expression level of the maturation marker when the maturation marker-expressing cells are cultured under conditions in which sensitization is not induced is used as the standard, if the expression level is significantly higher than the standard, it can be evaluated that sensitization has been induced, and if the expression level is lower than the standard or equivalent to the standard (no significant difference), it can be evaluated that sensitization has not been induced.

[0049] Furthermore, the sensitization detection method of the present invention can also be used, for example, in combination with the h-CLAT method, to determine whether the sensitization is induced by the test substance or another culture medium component by treating the maturation marker-expressing cells with the test substance in the absence of target cells, determining the expression level of the maturation marker, and comparing the expression level detected by the method of the present invention. That is, if the test substance, not the metabolite of the test substance, induces sensitization, the expression is not detected by the h-CLAT method, but the expression of the maturation marker is detected in the present invention. Furthermore, if the test substance induces sensitization, the expression of the maturation marker is also detected in the present invention, and the expression is also detected by the h-CLAT method, and at a comparable expression level. Furthermore, if both the test substance and the metabolite induce sensitization, the expression is also detected by the h-CLAT method, but a greater expression level is detected in the present invention. In this way, by performing detection in the absence of target cells using the method of the present invention and detection using the h-CLAT method and comparing the results of both methods, it is possible to determine, for example, whether it is the test substance itself or a metabolic product that induces sensitization.

[0050] Furthermore, in the detection method of the present invention, for example, the test substance (hereinafter also referred to as the first test substance) may be co-cultured with a sensitization-suppressing test substance (hereinafter also referred to as the second test substance). When the first test substance is involved in inducing sensitization, the presence or absence of sensitization suppression by the second test substance (sensitization-suppressing test substance) can be detected by further co-culture with the first test substance (sensitization-suppressing test substance). In this case, the detection method of the present invention can also be referred to as a method for screening for sensitization-suppressing substances from sensitization-suppressing test substances, or a method for evaluating sensitization-suppressing substances, as described below.

[0051] <Screening method for sensitization inhibitors> As described above, the screening method for sensitization-suppressing substances of the present invention is characterized in that it uses a sensitization-related substance involved in the induction of sensitization as a first test substance, a sensitization-suppressing test substance as a second test substance, performs the sensitization detection method of the present invention in the presence of the sensitization-related substance and the sensitization-suppressing test substance, and evaluates the sensitization-suppressing ability of the sensitization-suppressing test substance by comparing the expression level of the maturation marker in the absence of the sensitization-suppressing test substance with the expression level of the maturation marker in the presence of the sensitization-suppressing test substance. As described above, the screening method for sensitization-suppressing substances of the present invention can also be referred to as a method for evaluating sensitization-suppressing substances.

[0052] The screening method of the present invention can be carried out in the same manner as the detection method of the present invention, for example, except that co-culture is carried out in the further presence of the sensitization-suppressing test substance, and the description of the detection method of the present invention can be cited.

[0053] The screening method of the present invention includes, for example, a co-culture step of co-culturing the target cells, which have been made to coexist with the sensitization-related substance and the sensitization-suppressing test substance, with the maturation marker-expressing cells, and a detection step of detecting the expression of the maturation marker in the maturation marker-expressing cells.The screening method of the present invention further includes an evaluation step of comparing the expression level of the maturation marker in the maturation marker-expressing cells co-cultured in the absence of the sensitization-suppressing test substance but in the presence of the sensitization-related substance with the expression level of the maturation marker in the maturation marker-expressing cells co-cultured in the presence of the sensitization-suppressing test substance but in the presence of the sensitization-related substance. In the evaluation step, for example, if the expression level in the presence of the sensitization-inhibitory test substance is not significantly different from the expression level in the absence of the sensitization-inhibitory test substance, the sensitization-inhibitory test substance can be evaluated as not having sensitization-inhibitory ability, and if the expression level in the presence of the sensitization-inhibitory test substance is significantly reduced compared to the expression level in the absence of the sensitization-inhibitory test substance, the sensitization-inhibitory test substance can be evaluated as a sensitization-inhibitory substance having sensitization-inhibitory ability.

[0054] <Method for detecting sensitization> As described above, the method for producing a composition for administration to a living body of the present invention is characterized in that a test substance is confirmed to be capable of inducing sensitization due to the interaction between the test substance and target cells using the sensitization detection method of the present invention, and a test substance that does not induce sensitization is used as a component of the composition for administration to a living body in the production of the composition for administration to a living body.

[0055] The present invention is characterized in that, in the production of a composition for administration to a living body, the safety of the components constituting the composition is confirmed by the detection method of the present invention, and other conditions and steps are not limited in any way.

[0056] In the present invention, the test substance may be, for example, a candidate active ingredient that exhibits the desired effect of the composition for administration to a living body, or may be any other additive ingredient.

[0057] The composition for administration to a living body is not limited in any way, and examples thereof include pharmaceuticals, quasi-drugs, foods and beverages, and cosmetics. [Example]

[0058] Next, examples of the present invention will be described. However, the present invention is not limited to the following examples. Commercially available reagents were used according to their protocols unless otherwise specified. [Example]

[0059] The detection method of the present invention was used to confirm the sensitization of immune cell lines when melanin-producing cell lines were treated with drugs. An immune cell line (THP-1) mimicking human skin structure was used as the maturation marker-expressing cells, a melanin-producing cell line (melanoma cells: SK-MEL-37) was used as the target cells, and rhododenol (RD) was used as the drug. Cell observation under a microscope confirmed that rhododenol is selectively toxic to the melanoma cells.

[0060] (1) Detection of sensitization in co-culture The example was carried out as follows, based on Figure 1(A). Figure 1(A) is a model diagram of co-culture (h-CLAT / wM) with the addition of rhododenol. A scaffold container 21 (trade name Alvetex (registered trademark) Scaffold 12-well insert, ReproCell) was used as the inner container, which is a 3D culture substrate. The scaffold container 21 was placed in well 10 (trade name 6-well plate, Greiner), and then 4 x 10 melanoma cells were added to the scaffold container 21. 6 The cells were seeded, 4 mL of DMEM (containing 10% FBS and 1% PS) medium was added, and the cells were cultured at 37°C for 24 hours.

[0061] Next, a well plate (trade name: 12-well plate, Greiner) capable of setting the scaffold container 21 inside was used as the outer container. First, 0.5 mL of RPMI (containing 10% FBS and 1% PS) medium was introduced into each well 22 of the well plate, and 3 × 10 THP-1 cells were cultured. 5The scaffold container 21 seeded with the melanoma cells was transferred onto the scaffold container 21. The melanoma cells in the scaffold container 21 were treated with the drug by adding the drug and culturing for a certain period of time (30 minutes). Then, 1.5 mL of a mixed medium, a 1:1 (volume ratio) mixture of DMEM (containing 10% FBS and 1% PS) and RPMI (containing 10% FBS and 1% PS), was introduced. This brought the total volume of the medium in the well 22 to 2 mL. The melanoma cells in the scaffold container 21 were immersed in the medium in the well 22. The wells were then incubated at 37°C for 24 hours to perform co-culture. After incubation, the scaffold container 21 was removed, and the THP-1 cells in the well 22 were subjected to FACS analysis to measure the expression level of CD86.

[0062] In the comparative example, so-called h-CLAT was performed as follows. 2.0 mL of RPMI (containing 10% FBS and 1% PS) medium and the drug were introduced into well 22 of the same well plate as in the above example, and 3 × 10 5 The cells were then incubated at 37°C for 24 hours, and the THP-1 cells in well 22 were subjected to FACS analysis to measure the expression level of CD86. The drug concentration (relative to THP-1 cells) during co-culture of the two types of cells in the Example was set to the same concentration as the drug concentration (relative to THP-1 cells) during culture of THP-1 cells alone in the Comparative Example.

[0063] First, the results of an example and a comparative example in which rhododenol was used as the drug are shown in Figures 1 and 2.

[0064] Figure 1(B) is a graph showing the change in CD86 expression level in an example of coculture with rhododenol (h-CLAT / wM) and a comparative example using h-CLAT. The vertical axis represents the relative expression level (RFI) (%), and the horizontal axis represents the rhododenol concentration (mM, mmol / L) during culture. Figure 1(C) is a graph showing the change in cell viability over time. The vertical axis represents viability (%), and the horizontal axis represents the rhododenol concentration added during culture. The dotted line in Figure 1(C) represents the 75% viability border. In the h-CLAT method, viabilities below 75% are not used due to the significant effect of cell death. As shown in Figure 1(B), the h-CLAT did not demonstrate a significant concentration-dependent increase in CD86 expression, even with increasing amounts of rhododenol added. In contrast, coculture (h-CLAT / wM) demonstrated a significant, concentration-dependent increase in CD86 expression with increasing amounts of rhododenol. This indicates the induction of autoimmune disease, a result consistent with the development of vitiligo by rhododenol treatment. Furthermore, as shown in Figure 1(C), coculture (h-CLAT / wM) demonstrated higher THP-1 cell viability than h-CLAT, allowing for the effects of higher concentrations of the test substance to be observed.

[0065] FIG. 2 shows an example of histograms of CD86 expression levels in an example of co-culture (h-CLAT / wM) with the addition of rhododenol (top row) and a comparative example using h-CLAT (bottom row). Specifically, the top row shows the results of the co-culture, the bottom row shows the results of the h-CLAT, and the histograms on the right side of each row show the results for a reference culture in which only solvent was added. FIG. 2 also shows graphs depicting statistical data obtained from FACS histograms. In FIG. 2, the rhododenol concentration in the co-culture was 5 mmol / L (mM) in the example, and the rhododenol concentration in the h-CLAT in the comparative example. In FIG. 2, the vertical axis represents the cell count, and the horizontal axis represents the expression intensity of the CD86 antigen. The solid line represents data obtained using an anti-CD86 antibody for detection, and the gray scale represents data obtained using a control antibody for detection. As shown in Figure 2, the h-CLAT comparative example (bottom left) was almost the same as the solvent-only reference example (bottom right). In contrast, the co-culture example (top left) showed a peak shifted to the right compared to the solvent-only reference example (bottom right) and the h-CLAT comparative example (bottom left), confirming an increase in the expression level of CD86.

[0066] These results demonstrate that the method of the present invention can accurately detect sensitization, which cannot be fully confirmed by conventional in vitro tests. In particular, it can detect not only the sensitization of the active ingredient itself that exhibits the desired effect, but also the sensitization caused by the indirect influence of the active ingredient, specifically, for example, the sensitization caused by the interaction with a metabolite produced by the action of the active ingredient on target cells.

[0067] (2) Co-culture time In the above examples, for a system in which the rhododenol concentration during co-culture was 5 mmol / L, the co-culture time was set to 24 hours and 48 hours, and the CD86 expression level was compared. The results showed no significant difference between the two. Therefore, it is understood that there is no effect when the co-culture time is 24 to 48 hours. [Example]

[0068] The sensitization of immune cell lines to drugs when melanin-producing cell lines were treated with drugs was confirmed using the detection method of the present invention in the same manner as in Example 1, except that drugs other than rhododenol were used.

[0069] (1) Raspberry Ketone Raspberry ketone is known as a vitiligo substance. Therefore, the same method as in Example 1 was performed using raspberry ketone. The results are shown in Figure 3. Figure 3(A) is a graph showing the change in CD86 expression level in the example of co-culture (h-CLAT / wM) with raspberry ketone added and the comparative example using h-CLAT. The vertical axis represents the RFI (%), which indicates the relative expression level, and the horizontal axis represents the concentration of raspberry ketone added during culture. Figure 3(B) is a graph showing the change in cell viability over time. The vertical axis represents the viability (%) and the horizontal axis represents the concentration of raspberry ketone added during culture. As shown in Figure 3(A), when raspberry ketone was used, a slight concentration-dependent increase in CD86 was observed in the comparative example using h-CLAT. Furthermore, a slightly stronger concentration-dependent increase in CD86 was observed with increasing amounts of raspberry ketone added in the example of co-culture (h-CLATw / M). This result is consistent with the fact that raspberry ketone is known to be a substance responsible for vitiligo. Furthermore, these results demonstrate that the detection method of the present invention can be used to confirm not only the sensitization of metabolites, as in the case of rhododenol in Example 1, but also the sensitization of the drug itself.

[0070] (2) Ascorbic Acid Ascorbic acid is known not to induce leukoplakia. Therefore, the same method as in Example 1 was performed using ascorbic acid. The results are shown in Figure 4. Figure 4(A) is a graph showing the change in CD86 expression level in the example of co-culture (h-CLAT / wM) with ascorbic acid and the comparative example using h-CLAT. The vertical axis represents the RFI (%), which indicates the relative expression level, and the horizontal axis represents the concentration of ascorbic acid added during culture. Figure 4(B) is a graph showing the change in cell viability over time. The vertical axis represents viability (%) and the horizontal axis represents the concentration of ascorbic acid added during culture. As shown in Figure 4, when ascorbic acid was used, no significant concentration-dependent increase in CD86 expression was observed with increasing amounts of ascorbic acid, either in the co-culture (h-CLATw / M) or in the h-CLAT. This result is consistent with the fact that ascorbic acid treatment does not cause leukoplakia.

[0071] (3) Tranexamic acid Tranexamic acid is known not to induce vitiligo. Therefore, the results of a similar experiment using tranexamic acid to the above-described example are shown in Figure 5. Figure 5(A) is a graph showing the change in CD86 expression level in the example of co-culture (h-CLAT / wM) with tranexamic acid and the comparative example using h-CLAT. The vertical axis represents the RFI (%), which indicates the relative expression level, and the horizontal axis represents the concentration of tranexamic acid added during culture. Figure 5(B) is a graph showing the change in cell viability over time. The vertical axis represents viability (%) and the horizontal axis represents the concentration of tranexamic acid added during culture. As shown in Figure 5, when tranexamic acid was used, no significant concentration-dependent increase in CD86 expression was observed with increasing amounts of tranexamic acid, either in the co-culture (h-CLATw / M) or in the h-CLAT. This result is consistent with the fact that treatment with tranexamic acid does not cause vitiligo. [Example]

[0072] (1) Involvement of reactive oxygen species As mentioned above, reactive oxygen species generated by the metabolism of rhododenol in melanin-producing cells may be involved in the induction of sensitization caused by rhododenol treatment. Therefore, the detection method of the present invention was performed in the presence of rhododenol in the presence of N-acetylcysteine ​​(NAC), an antioxidant that scavenges reactive oxygen species.

[0073] FACS was performed in the same manner as in Example 1, except that NAC was added together with rhododenol during co-culture. The rhododenol concentration during co-culture was 0 or 4 mmol / L, and the NAC concentration was set to the specified concentrations (0, 5, 10, 20, or 30 nmol / L).

[0074] The results are shown in Figures 6(A) and 6(B). Figure 6(A) is a graph showing the change in ROS (reactive oxygen species) generation upon addition of 4 mmol / L rhododenol to the example coculture (h-CLAT / wM) and the comparative h-CLAT coculture. The vertical axis represents the amount of ROS generated. Figure 6(B) is a graph showing the change in CD86 expression in the example coculture (h-CLAT / wM) in which 4 mmol / L rhododenol and NAC were cocultured, and the comparative h-CLAT coculture. The vertical axis represents the RFI (%), which indicates the relative expression level. As shown in Figure 6(A), a dramatic increase in ROS was observed only when 4 mmol / L rhododenol was added to the coculture (h-CLAT / wM). At the same time, as shown in Figure 6(B), the addition of NAC resulted in a concentration-dependent decrease in CD86 in the coculture (h-CLAT / wM) compared to the coculture without NAC (0 nmol / L). This result is consistent with NAC scavenging reactive oxygen species and suppressing immune cell sensitization.

[0075] (2) Involvement of ATP ATP is thought to be involved as a causative factor inducing sensitization, other than reactive oxygen species generated by rhododenol treatment. Therefore, the ATP receptor inhibitor BBG (Brilliant Blue G) was cocultured with THP-1 cells, and the detection method of the present invention was performed. Specifically, FACS was performed as in Example 1, except that BBG was added together with THP-1 cells during coculture. The rhododenol concentration during coculture was 5 mmol / L, and the BBG concentration was the specified concentration (200 nmol / L).

[0076] The results are shown in Figure 7. Figure 7(A) is a graph showing the change in extracellular ATP concentration in the culture supernatant in an example of co-culture (h-CLAT / wM) in which 5 mmol / L rhododenol and BBG were co-cultured, and a comparative example of h-CLAT. Figure 7(B) is a graph showing the change in CD86 expression level in the example and the comparative example. The vertical axis of Figure 7(A) is ATP concentration, and the vertical axis of Figure 7(B) is RFI (%), which indicates the relative expression level. The horizontal axes of Figures 7(A) and (B) indicate the culture conditions. As shown in Figure 7, the co-culture of RD and melanoma increased the ATP concentration in the culture supernatant, whereas the co-culture of THP-1 with BBG confirmed a decrease in CD86. This result is consistent with BBG inhibiting the ATP receptor of THP-1 and suppressing immune cell sensitization. Thus, it was found that the present invention can confirm not only the induction of sensitization but also the inhibitory ability of test substances to suppress it.

[0077] These results demonstrate that the present invention makes it possible to confirm not only the induction of sensitization but also the inhibitory ability of test substances to inhibit it.

[0078] Although the present invention has been described above with reference to the embodiments and examples, the present invention is not limited to the above-described embodiments and examples. Various modifications that can be understood by those skilled in the art can be made to the configuration and details of the present invention within the scope of the present invention.

[0079] This application claims priority based on Japanese Patent Application No. 2020-179860, filed on October 27, 2020, the disclosure of which is incorporated herein in its entirety. [Industrial Applicability]

[0080] According to the present invention, by co-culturing target cells in the presence of a test substance with maturation marker-expressing cells and detecting the expression of the maturation marker in the maturation marker-expressing cells, it is possible to detect with greater reliability whether sensitization is induced by treating the target cells of interest with the test substance.

Claims

1. a co-culturing step of co-culturing the target cells in the presence of the test substance with maturation marker-expressing cells; a detecting step of detecting expression of a maturation marker in the maturation marker-expressing cells; and an evaluation step of evaluating the presence or absence of sensitization caused by the interaction between the test substance and the target cells as a possibility of vitiligo induction, based on the expression level of the maturation marker detected in the detection step; the target cells are melanocytes, A method for detecting sensitization due to the interaction between a test substance and target cells, characterized in that the maturation marker-expressing cells are cells that express a maturation marker that functions during antigen presentation in the immune sensitization process.

2. a culture step of culturing the target cells prior to the co-culture step, The method for detecting sensitization according to claim 1 , wherein after the culturing step, the test substance is allowed to coexist with the target cells, and co-culture with the maturation marker-expressing cells is carried out.

3. 3. The method for detecting sensitization according to claim 1, wherein the maturation marker-expressing cells are at least one selected from the group consisting of dendritic cells, dendritic cell-like cells, and monocyte-derived cells.

4. The method for detecting sensitization according to claim 3, wherein the monocyte-derived cells are THP-1 cells.

5. The method for detecting sensitization according to any one of claims 1 to 4, wherein the maturation marker is at least one selected from the group consisting of CD86, CD54, CD80, OX40L, and HLA-DR.

6. In the evaluation step, the presence or absence of sensitization is evaluated by comparing the expression level of the maturation marker detected in the detection step with a standard; A method for detecting sensitization described in any one of claims 1 to 5, wherein the standard is the expression level of a maturation marker in maturation marker-expressing cells in the absence of the test substance, and if the expression level is significantly higher than the standard, the cell is evaluated as exhibiting sensitization.

7. The method for detecting sensitization according to claim 1 , wherein in the detection step, the maturation marker-expressing cells are separated from the target cells, and the expression of the maturation marker is detected.

8. The method includes a culture step of culturing the target cells prior to the co-culture step, After the culturing step, the test substance is allowed to coexist with the target cells, and co-culture is performed with the maturation marker-expressing cells; Using a co-culture set consisting of an outer container and an inner container, the outer container is an incubator for the maturation marker-expressing cells, the inner container is an incubator for the target cells, can be placed inside the outer container, and has a bottom that allows components in the culture medium other than the cells to pass through to the outer container; the culturing step includes placing the target cells and a medium in the inner container and culturing the target cells; The method for detecting sensitization according to any one of claims 1 to 7, wherein the co-culture step comprises placing the maturation marker-expressing cells and a culture medium in the outer container, placing the inner container inside the outer container, adding the test substance to the inner container, and co-culturing the outer container and the inner container.

9. The method includes a culture step of culturing the target cells prior to the co-culture step, After the culturing step, the test substance is allowed to coexist with the target cells, and co-culture is performed with the maturation marker-expressing cells; The method for detecting sensitization according to claim 1 , wherein a sensitization-suppressing test substance is allowed to coexist with the test substance in the co-culture step.

10. As the first test substance, a sensitization-related substance involved in the induction of sensitization is used, As the second test substance, a sensitization-inhibiting test substance is used, The method for detecting sensitization according to any one of claims 1 to 9 is carried out in the presence of the sensitization-related substance and the sensitization-suppressing test substance, A method for screening a sensitization-inhibiting substance, characterized by evaluating the sensitization-inhibiting ability of the sensitization-inhibiting test substance by comparing the expression level of the maturation marker in the absence of the sensitization-inhibiting test substance with the expression level of the maturation marker in the presence of the sensitization-inhibiting test substance.

11. a method for detecting sensitization according to any one of claims 1 to 9, wherein the test substance is confirmed to induce sensitization due to an interaction between the test substance and a target cell; A method for producing a composition for administration to a living body, comprising using a test substance that does not induce sensitization as a component of the composition for administration to a living body.

12. The method according to claim 11, wherein the composition for administration to a living body is at least one selected from the group consisting of pharmaceuticals, quasi-drugs, and cosmetics.

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