Method for screening stratum corneum adhesive structure decomposition accelerator

Abstract

To provide a method for screening a stratum corneum peeling accelerator that allows a degrading enzyme to effectively permeate an adhesion region between stratum corneum cells through which the degrading enzyme hardly permeates.SOLUTION: The problem has been solved by finding a method for screening a stratum corneum adhesive structure decomposition accelerator through evaluation of permeability of a stratum corneum adhesive structure decomposition enzyme relative to an adhesive region between stratum corneum cells.EFFECT: The present invention enables provision of a stratum corneum adhesive structure decomposition accelerator, and in addition, enables acquisition of an effect accompanying stratum corneum adhesive structure decomposition, and thereby, enables provision of a stratum corneum peeling accelerator, a skin transparency improving agent, and a stratum corneum thickness improving agent.SELECTED DRAWING: Figure 1

Description

[Technical field]

[0001] The present invention relates to a method for screening for a stratum corneum adhesive structure decomposition promoter, which comprises evaluating the permeability of a stratum corneum adhesive structure decomposition enzyme into the adhesion region between stratum corneum cells. [Background technology]

[0002] The human skin is made up of three layers, the epidermis, dermis, and subcutaneous tissue, in that order from the surface layer that comes into contact with the outside air. The epidermis is made up of cells called keratinocytes, which are further divided into the basal layer, spinous layer, granular layer, and stratum corneum (horny layer) from the deepest part close to the dermis. In the epidermis, keratinocytes divide and differentiate, pushing up from the basal layer toward the stratum corneum, becoming keratinocytes, and at the outermost layer of the stratum corneum, they are exfoliated as so-called dirt.

[0003] The stratum corneum, located at the outermost surface of the skin, acts as a barrier for the skin and has important functions such as retaining moisture in the skin and protecting it from external stimuli. To maintain this function, the stratum corneum forms a structure called the brick-and-mortar model, in which stratum corneum cells are piled up like bricks, and the spaces between them are filled with intercellular lipids like mortar. The stratum corneum cells are connected by corneodesmosomes, which are the adhesive structure of the stratum corneum. When these corneodesmosomes are decomposed by adhesive structure-degrading enzymes, the bonds between the stratum corneum cells collapse, and the stratum corneum eventually becomes dandruff and falls off.

[0004] Corneodesmosomes are composed of desmoglein 1, desmocollin 1, corneodesmosin, etc., and are degraded by serine proteases such as kallikrein (KLK) 1, 5, 6, 7, and 14 (Non-Patent Document 1). Therefore, conventional methods for selecting components that promote stratum corneum peeling or degradation of stratum corneum adhesive structure include a method that uses a decrease in constituent factors of corneodesmosomes as an indicator by immunostaining (Patent Document 1) and a method that uses promoted expression of the KLK family as an indicator (Patent Document 2).

[0005] The effect of promoting the decomposition of corneodesmosomes is, first, that the adhesion between keratinocytes is eliminated, promoting the peeling of the stratum corneum. In addition, the number of keratinocytes decreases as a result of the promotion of peeling of the stratum corneum, which reduces the thickness of the stratum corneum, i.e., leads to the improvement of stratum corneum thickening. Furthermore, since it is known that the thicker the stratum corneum, the lower the transparency of the skin, the improvement of stratum corneum thickening results in the improvement of skin transparency. [Prior art documents] [Patent documents]

[0006] [Patent Document 1] Patent Publication No. 2022-82369 [Patent Document 2] Patent Publication No. 2022-97632 [Non-patent literature]

[0007] [Non-Patent Document 1] Akemi Yamamoto, Tetsuko Igawa. Adhesion structure of stratum corneum cells, corneodesmosomes and skin diseases. Nishinippon Dermatology, 2015;77:5-9. Summary of the Invention [Problem to be solved by the invention]

[0008] The present inventors have found that, since the corneal adhesive structure between stratum corneum cells is decomposed by an adhesive structure decomposing enzyme, the bond between the stratum corneum cells is broken down and stratum corneum peeling occurs, but the stratum corneum adhesive structure decomposing enzyme is difficult to penetrate into the entire adhesion area because the stratum corneum cells are in close contact with each other by the corneodesmosome, which is the stratum corneum adhesive structure.Even if the activity of the decomposing enzyme is increased by a conventional stratum corneum peeling promotion technique, the effect is limited if the decomposing enzyme itself cannot reach the site where the corneodesmosome is present.Therefore, the present invention aims to provide a method for screening a stratum corneum peeling promoter that effectively allows the decomposing enzyme to penetrate into the inside of the adhesion area where the decomposing enzyme is difficult to penetrate. [Means for solving the problem]

[0009] The above-mentioned problem was solved by finding a method for screening for stratum corneum adhesive structure decomposition promoters by evaluating the permeability of a stratum corneum adhesive structure decomposition enzyme to the adhesion region between stratum corneum cells. Effect of the Invention

[0010] The present invention makes it possible to provide an agent for promoting the decomposition of a stratum corneum adhesive structure, and in addition, since the effects associated with the decomposition of the stratum corneum adhesive structure are also obtained, it is possible to provide an agent for promoting stratum corneum peeling, an agent for improving skin transparency, and an agent for improving stratum corneum thickening. [Brief description of the drawings]

[0011] [Figure 1] Schematic diagram of adhesion areas between stratum corneum cells. [Diagram 2] Detection of desmoglein 1 in the stratum corneum with and without permeation enhancement treatment using Triton X-100. [Diagram 3] Detectability of desmoglein 1 in the stratum corneum after trypsin treatment with and without permeation enhancement treatment using Triton X-100. [Figure 4] Percentage of fluorescent area for each test substance. [Diagram 5] Fluorescent area ratio after trypsin treatment with TritonX-100. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0012] In the present invention, as shown in the examples, it was shown that the stratum corneum adhesive structure-degrading enzyme has difficulty penetrating the adhesion region between stratum corneum cells, and while the stratum corneum adhesive structure around the adhesion region is relatively easily degraded, the stratum corneum adhesive structure inside the adhesion region is difficult to degrade. Based on this result, we conducted extensive research and found that a component that increases the permeability of the stratum corneum adhesive structure-degrading enzyme to the adhesion region promotes the decomposition of the stratum corneum adhesive structure.

[0013] In the present invention, the stratum corneum adhesive structure is corneodesmosome as mentioned above, but as long as it is a cell adhesion device that attracts cells in the stratum corneum, stratum corneum adhesive structures other than corneodesmosomes do not contradict the gist of the present invention.For example, in the stratum corneum where some of the adhesive structures between epidermal keratinocytes such as tight junctions remain, these adhesive structures can also be considered as stratum corneum adhesive structures.In addition, stratum corneum adhesive structure degrading enzymes refer to enzymes that can degrade molecules that constitute stratum corneum adhesive structures such as corneodesmosomes, and for example, the KLK family can be considered as stratum corneum adhesive structure degrading enzymes.

[0014] When one stratum corneum adhesive structure is taken into consideration, the adhesion that the structure enables is adhesion at a very small point on the surface of the stratum corneum cells, which is the scale of the molecules that constitute the stratum corneum adhesive structure, compared to the size of the stratum corneum cells. However, if multiple stratum corneum adhesive structures are present on the surface of the stratum corneum cells, the adhesion between the stratum corneum cells will be adhesion across the area where the stratum corneum adhesive structures are present. In the present invention, the adhesion area between stratum corneum cells refers to the area in the stratum corneum cells where two stratum corneum cells are adhered to each other via the stratum corneum adhesive structure and are thus adhered to each other. More specifically, as shown in FIG. 1, it is the localized area of ​​the stratum corneum adhesive structure between two overlapping stratum corneum cells. In many cases, the adhesion area between stratum corneum cells is mainly present at the periphery of the stratum corneum cells that overlap with the adjacent stratum corneum cells. In addition, it is synonymous even if it is simply described as the adhesion area.

[0015] The step of obtaining a stratum corneum in which the adhesion areas between stratum corneum cells are maintained is a step of obtaining a stratum corneum to be used in screening. The stratum corneum in which the adhesion areas between stratum corneum cells are maintained refers to a stratum corneum that maintains the adhesion areas due to the stratum corneum adhesive structure, and the stratum corneum adhesive structure may be somewhat damaged as long as it is maintained to an extent that the permeability of the stratum corneum adhesive structure-degrading enzyme to the adhesion areas can be evaluated. Examples of stratum corneum in which the adhesion areas between stratum corneum cells are maintained include stratum corneum peeled from the skin by tape stripping or abrasion, excised skin tissue, and the skin surface of a living organism. On the other hand, a stratum corneum that has been treated with trypsin, an enzyme that has the effect of degrading the stratum corneum adhesive structure, and in which the stratum corneum adhesive structure has been degraded to an extent that the permeability of the stratum corneum adhesive structure-degrading enzyme to the adhesion areas cannot be evaluated is not suitable for this screening.

[0016] The method for the step of allowing the test substance to coexist with the stratum corneum in which the adhesion area between stratum corneum cells is maintained is not particularly limited as long as the test substance can act on the adhesion area between stratum corneum cells, and the type and properties of the test substance are not particularly limited as long as the test substance can coexist with the stratum corneum in which the adhesion area between stratum corneum cells is maintained. Note that, when allowing the test substance to coexist with the stratum corneum, it is preferable that the test substance and the stratum corneum are in physical contact with each other, but there is no problem even if they are not in contact as long as the conditions for the test substance to have some kind of action are met. For example, a vaporizing test substance may be allowed to coexist without contact, and the generated gas may act.

[0017] The step of evaluating the permeability of the stratum corneum adhesive structure decomposing enzyme to the adhesion region between stratum corneum cells is a step of evaluating how easily the stratum corneum adhesive structure decomposing enzyme is permeable between the stratum corneum cells in the adhesion region between stratum corneum cells, and the method is not limited as long as the permeability can be evaluated, and the method may be directly or indirectly evaluated. A method for directly evaluating the permeability of the stratum corneum adhesive structure decomposing enzyme to the adhesion region between stratum corneum cells may be, for example, a method for evaluating the location of the stratum corneum adhesive structure decomposing enzyme labeled with fluorescence or the like. In addition, a method for indirectly evaluating the permeability of the stratum corneum adhesive structure decomposing enzyme to the adhesion region between stratum corneum cells may be, for example, a method for evaluating the permeability of a model component instead of the stratum corneum adhesive structure decomposing enzyme itself as an indicator substance, and a model component having at least some properties of the stratum corneum adhesive structure decomposing enzyme may be used as the indicator substance. For example, the permeability of the stratum corneum adhesive structure decomposing enzyme can be indirectly evaluated by using a component with the same hydrophilicity as the stratum corneum adhesive structure decomposing enzyme as an indicator substance and evaluating the permeability of the indicator substance to the adhesion region between stratum corneum cells. The indicator substance must be a substance whose permeability can be evaluated by evaluating its location by fluorescence or the like. In addition, if the indicator substance has properties similar to those of the stratum corneum adhesive structure-degrading enzyme, the accuracy will be higher, and it is preferable that the indicator substance is, for example, a polymer. For example, the permeability of the stratum corneum adhesive structure-degrading enzyme can be indirectly evaluated by using an antibody against a protein constituting the stratum corneum adhesive structure as an indicator substance, immunostaining the protein constituting the stratum corneum adhesive structure, and measuring the degree of staining in the adhesion area between stratum corneum cells.

[0018] The step of selecting a test substance that increases the permeability of the stratum corneum adhesive structure decomposition enzyme to the adhesion area between stratum corneum cells compared to when the test substance is not present is a step of determining whether the test substance is a stratum corneum adhesive structure decomposition promoter, and if the permeability of the stratum corneum adhesive structure decomposition enzyme to the adhesion area between stratum corneum cells is increased by the step of allowing the test substance to coexist with the stratum corneum in which the adhesion area between stratum corneum cells is maintained, the test substance is determined to be a stratum corneum adhesive structure decomposition promoter. Regarding the determination of the increase in permeability, it is sufficient that the permeability is increased compared to the control (when treated with water instead of the treatment with the test substance or when untreated), and the higher the permeability, the better. When quantified, the permeability is preferably 1.2 times or more than the control, more preferably 1.5 times or more, and even more preferably 2.0 times or more. In addition, it is even more preferable that a significant difference is detected from the control, taking into account the variation.

[0019] The effect of promoting the decomposition of the stratum corneum adhesive structure is, first, the dissolution of adhesion between stratum corneum cells, which promotes stratum corneum peeling. In addition, the number of stratum corneum cells is reduced by promoting stratum corneum peeling, which reduces the thickness of the stratum corneum, i.e., leads to improvement of stratum corneum thickening. Furthermore, the thicker the stratum corneum, the lower the transparency of the skin, and therefore the improvement of stratum corneum thickening results in improvement of skin transparency, so that the screening method for the stratum corneum adhesive structure decomposition promoter can also be used as a screening method for stratum corneum peeling promoters, stratum corneum thickening improving agents, and / or skin transparency improving agents.

[0020] The stratum corneum adhesive structure decomposition promoter selected by the screening method of the present invention can be suitably incorporated as an active ingredient in a composition for promoting the decomposition of stratum corneum adhesive structure. That is, the present specification also provides a method for designing a composition, which includes a step of carrying out the screening method of the present invention and incorporating a substance (stratum corneum adhesive structure decomposition promoter) selected thereby. Such a composition can be in the form of a composition administered percutaneously (for example, a cosmetic, a quasi-drug, a skin topical agent such as a pharmaceutical). In addition, the formulation of the composition is not particularly limited. For example, the formulation when designed as a cosmetic composition can be a commonly known lotion formulation, emulsion formulation, essence formulation, cream formulation, powder-containing formulation, etc. Furthermore, the composition may be designed to incorporate necessary ingredients as appropriate. For example, when designed as a cosmetic composition, it is possible to incorporate a wide range of ingredients that are commonly used in cosmetics.

[0021] An example of the step of evaluating the permeability of the stratum corneum adhesive structure degrading enzyme to the adhesion region between stratum corneum cells in the present invention is a step of immunostaining the proteins constituting the stratum corneum adhesive structure and measuring the staining intensity in the adhesion region between stratum corneum cells. In this case, if the permeability increases, i.e., the staining intensity increases, the test substance is considered to be a stratum corneum adhesive structure decomposition promoter. A method for selecting a stratum corneum adhesive structure reducer using the amount of stratum corneum adhesive structure as an index by immunostaining the proteins constituting the stratum corneum adhesive structure has been known in the past, but in the past, the lower the staining intensity, the more the stratum corneum adhesive structure was considered to be reduced, and a component that reduces the staining intensity was selected, whereas in the present invention, the higher the staining intensity, the more the antibody permeability increases, and the component is considered to be one that promotes the degradation of the stratum corneum adhesive structure, so the judgment criteria are completely different. EXAMPLES

[0022] The present invention will be described in more detail below with reference to examples. However, the present invention is not limited to the following examples as long as it does not depart from the gist of the present invention.

[0023] <Examination of desmoglein 1 (DSG1) degradation by DSG1 immunostaining> A stratum corneum sample was taken from the inside of the forearm using a skin checker (Promo Tool), and an area of ​​approximately 7 mm square was circled on the skin checker with a pen, and the following treatment was performed within this area. 90 μL of 0.25% trypsin was dropped, left to stand at room temperature for 10 minutes, and then washed with PBS(-) to perform DSG1 degradation treatment. 90 μL of 20% TritonX-100 (Promega) / PBS(-) solution was dropped, left to stand at room temperature for 90 minutes, and then washed with PBS(-) to perform antibody permeation promotion treatment. 90 μL of 3% BSA / PBS(-) solution was dropped, left to stand at room temperature for 60 minutes, and then washed with PBS(-) to perform blocking treatment. Anti-desmoglein 1 (DSG1) primary antibody (anti-Desmoglein 1 mouse monoclonal, Dsg1-P124, supernatant (PROGEN)) was diluted 10-fold with 1% BSA / PBS(-) solution, 90 μL was dropped, and the sample was left to stand at room temperature for 60 minutes and then washed with PBS(-) for primary antibody treatment. The secondary antibody (Goat Anti-Mouse IgG H&L (Alexa Fluor568) preadsorbed (Abcam)) was diluted 200-fold with 1% BSA / PBS(-) solution, 90 μL of which was dropped onto the plate and left to stand in the dark at room temperature for 60 minutes. The plate was then washed with PBS(-) for secondary antibody treatment. After air drying, the specimens were observed under a fluorescence microscope (BZ-X700, KEYENCE) to obtain fluorescent images, and the fluorescent area was quantified using ImageJ. The fluorescent area of ​​the sample that had been subjected to DSG1 degradation treatment relative to the fluorescent area of ​​the sample that had not been subjected to DSG1 degradation treatment was defined as the fluorescent area residual rate.

[0024]

number

[0025] The treatment using Triton X-100 is generally aimed at promoting cell membrane permeation by antibodies. However, when treated at a high concentration, as shown in Fig. 2, antibody permeation into the adhesion region between stratum corneum cells, where adjacent stratum corneum cells overlap and adhere closely to each other, is also promoted, and it becomes possible to stain DSG1 inside the adhesion region. Therefore, it was used as a treatment for promoting antibody permeation into the adhesion region between stratum corneum cells.

[0026] As shown in Fig. 3, when the antibody permeation promotion treatment was not performed, the fluorescence of DSG1 almost disappeared due to the degradation treatment of DSG1 by trypsin, a serine protease. However, when the antibody permeation promotion treatment was performed, a large amount of DSG1 fluorescence remained. From these results, it was found that while DSG1 around the adhesion region between stratum corneum cells was degraded by the degradation treatment of DSG1 by trypsin, trypsin hardly reached the inside of the adhesion region between stratum corneum cells and DSG1 was hardly degraded. When the fluorescence region residual rate around the adhesion region and the fluorescence region residual rate inside the adhesion region after trypsin treatment were actually calculated, the fluorescence region residual rate around the adhesion region was 14%, while the fluorescence region residual rate inside the adhesion region was 38%, and it was confirmed that a large amount of DSG1 inside the adhesion region remained undegraded. That is, it was found that DSG1 is localized in the adhesion region of stratum corneum cells, but it is difficult for the degrading enzyme to reach the inside of the adhesion region, and it is important to deliver the degrading enzyme to the inside of the adhesion region in promoting the decomposition of the stratum corneum adhesion structure and promoting stratum corneum exfoliation.

[0027] <Screening by DSG1 immunostaining> A stratum corneum sample was collected from the inner part of the forearm using a skin checker (promo tool). A range of about 7 mm square was outlined with a pen on the skin checker, and the following treatment was performed on this range. As mentioned above, the test substances selected were 10% TritonX-100 aqueous solution, which is known to promote the penetration of antibodies into the adhesion area between stratum corneum cells, as well as fennel extract, amacha extract, oyster mushroom culture solution, and soybean seed extract. 90μL of each test substance was dropped onto the sample, left to stand at room temperature for 120 minutes, and then washed with PBS(-). 90μL of 3%BSA / PBS(-) solution was dropped onto the sample, left to stand at room temperature for 60 minutes, and then washed with PBS(-) to perform blocking treatment. Anti-desmoglein 1 (DSG1) primary antibody (anti-Desmoglein 1 mouse monoclonal, Dsg1-P124, supernatant (PROGEN)) was diluted 10-fold with 1% BSA / PBS(-) solution, 90 μL was dropped, and the sample was left to stand at room temperature for 60 minutes and then washed with PBS(-) for primary antibody treatment. The secondary antibody (Goat Anti-Mouse IgG H&L (Alexa Fluor568) preadsorbed (Abcam)) was diluted 200-fold with 1% BSA / PBS(-) solution, 90 μL of which was dropped onto the plate and left to stand in the dark at room temperature for 60 minutes. The plate was then washed with PBS(-) for secondary antibody treatment. After air drying, the samples were observed under a fluorescence microscope (BZ-X700, KEYENCE), fluorescent images were taken, and the fluorescent area was quantified using ImageJ. Visible light images were also taken of the same samples, and the area of ​​the stratum corneum region in the images was calculated as the visible light area. When the fluorescent area area was divided by the visible light area to obtain the fluorescent area area ratio, the higher the fluorescent area area ratio, the larger the fluorescent area relative to the stratum corneum region, which means that water-soluble components such as antibodies can easily penetrate into the adhesive area. Therefore, it can be determined that the test substance with a higher fluorescent area area ratio compared to the control (water) has a greater effect of promoting the penetration of decomposition enzymes.

[0028] As shown in Fig. 4, with 10% Triton X-100, the fluorescence area ratio was about five times that of the control, indicating that the DSG1 antibody, which is a hydrophilic component, could be delivered to the adhesion region between stratum corneum cells. It could be considered that the permeability of the stratum corneum adhesion-degrading enzyme to the adhesion region between stratum corneum cells was high. Thus, by immunostaining DSG1 and measuring the staining intensity in the adhesion region between stratum corneum cells, in other words, using the permeability of the antibody to the adhesion region between stratum corneum cells as an index, a substance with high permeability of the stratum corneum adhesion-degrading enzyme to the adhesion region between stratum corneum cells, such as Triton X-100, can be found.

[0029] <Promoting Effect of Triton X-100 on Degradation of Stratum Corneum Adhesion Structure> A stratum corneum sample was collected from the inner part of the forearm using a skin checker (promotion tool). A square area of about 7 mm on each side was outlined with a pen on the skin checker, and the following treatments were performed on this area. 90 μL of 10% Triton X-100 or control (water) was dropped, left standing at room temperature for 120 minutes, and then washed with PBS(-). 90 μL of 0.25% trypsin was dropped, left standing at room temperature for 10 minutes, and then washed with PBS(-) for DSG1 degradation treatment. 90 μL of 20% Triton X-100 / PBS(-) solution was dropped, left standing at room temperature for 90 minutes, and then washed with PBS(-) for antibody permeability promotion treatment. 90 μL of 3% BSA / PBS(-) solution was dropped, left standing at room temperature for 60 minutes, and then washed with PBS(-) for blocking treatment. The anti-desmoglein 1 (DSG1) primary antibody (anti-Desmoglein 1 mouse monoclonal, Dsg1-P124, supernatant (PROGEN)) was diluted 10-fold with 1% BSA / PBS(-) solution, 90 μL was dropped, left standing at room temperature for 60 minutes, and then washed with PBS(-) for primary antibody treatment. The secondary antibody (Goat Anti-Mouse IgG H&L (Alexa Fluor568) preadsorbed (Abcam)) was diluted 200-fold with 1% BSA / PBS(-) solution, 90 μL was dropped, left standing in the dark at room temperature for 60 minutes, and then washed with PBS(-) for secondary antibody treatment. After air drying, the samples were observed under a fluorescence microscope (BZ-X700, KEYENCE), fluorescent images were taken, and the fluorescent area was quantified using ImageJ. Visible light images were also taken of the same samples, and the area of ​​the stratum corneum region in the images was calculated as the visible light area. The fluorescent area was divided by the visible light area to obtain the fluorescent area area ratio. The lower the fluorescent area ratio, the less DSG1 there was, which means that the decomposition of DSG1 by trypsin was promoted.

[0030] As a result, as shown in Figure 5, by applying TritonX-100, which has high permeability to the adhesion area for enzymes that degrade stratum corneum adhesive structures, and by carrying out a treatment to promote trypsin permeation to the adhesion area between stratum corneum cells, more DSG1 was actually degraded compared to the control (water). From this, it was confirmed that it is possible to screen for ingredients that promote the degradation of stratum corneum adhesive structures by promoting the permeability of stratum corneum adhesive structure-degrading enzymes to the adhesion regions between stratum corneum cells using a screening method that uses the permeability of stratum corneum adhesive structure-degrading enzymes to the adhesion regions between stratum corneum cells as an index. Furthermore, since it is known that degradation of stratum corneum adhesive structures leads to the promotion of stratum corneum peeling, improvement of stratum corneum thickening, and improvement of skin transparency, it is also possible to screen for agents that improve these.

Claims

1. A method for screening for a stratum corneum adhesive structure decomposition promoter, comprising the following steps [1] to [4]: [1] A process for obtaining a stratum corneum in which adhesion areas between stratum corneum cells are maintained [2] A step of allowing a test substance to coexist with a stratum corneum in which adhesion areas between stratum corneum cells are maintained [3] A step of evaluating the permeability of a stratum corneum adhesive structure-degrading enzyme to the adhesion area between stratum corneum cells [4] A step of selecting, as a stratum corneum adhesive structure decomposition promoter, a test substance that increases the permeability of a stratum corneum adhesive structure decomposition enzyme to the adhesion region between stratum corneum cells compared to when the test substance is not coexistent.

2. a step of evaluating the permeability of a stratum corneum adhesive structure degrading enzyme to the adhesion region between the stratum corneum cells; This is a step for evaluating the permeability of an indicator substance to the adhesion area between stratum corneum cells. The screening method of claim 1.

3. a step of evaluating the permeability of a stratum corneum adhesive structure degrading enzyme to the adhesion region between the stratum corneum cells; This is a process in which proteins that make up the stratum corneum adhesive structure are immunostained and the degree of staining in the adhesive areas between stratum corneum cells is measured. The screening method of claim 1.

4. The screening method according to any one of claims 1 to 3, wherein the stratum corneum adhesive structure decomposition promoter is a stratum corneum peeling promoter, a stratum corneum thickening improving agent, and / or a skin transparency improving agent.

5. A step of incorporating a substance selected by the method according to any one of claims 1 to 3; A method for designing a composition comprising:

6. The design method according to claim 5, wherein the composition of claim 5 is a cosmetic for promoting decomposition of the adhesive structure of the stratum corneum, a cosmetic for promoting peeling of the stratum corneum, a cosmetic for improving stratum corneum thickening, or a cosmetic for improving skin transparency.

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