Method for analyzing distribution of component in skin topical agent having permeated through skin and method for predicting permeation route of component of skin topical agent permeating into skin

Abstract

A method for analyzing the distribution of a component of a skin topical agent having permeated through the skin according to the present disclosure comprises: a first mapping step for bringing a skin topical agent containing a labeled component of interest into contact with the skin, and thereafter, preparing a tissue piece in a region including the stratum corneum of the skin, preparing slices that expose cross sections along the depth direction of the stratum corneum in the tissue piece, and mapping the distribution of ions detected from the slices using Nano-SIMS; a second mapping step for bringing the skin topical agent containing the labeled component of interest into contact with the skin, and thereafter, taking a sample from the skin by a tape-stripping method, and mapping the distribution of ions detected from the sample using the Nano-SIMS; and a determination step for determining the distribution of the labeled component of interest from the ion distribution obtained in the first mapping step and the ion distribution obtained in the second mapping step.

Description

Method for analyzing distribution of components of topical skin preparations that have penetrated into the skin and method for predicting the penetration route of components of topical skin preparations that have penetrated into the skin

[0001] The present invention relates to a method for analyzing the distribution of components of an external skin preparation that has penetrated into the skin, and a method for predicting the penetration pathway of components of an external skin preparation that has penetrated into the skin.

[0002] Various topical skin preparations have been developed for various applications, including those that act systemically through the skin, those that act locally on the skin, and functional cosmetics. To evaluate the efficacy and safety of such topical skin preparations, it is important to analyze the permeability of the active ingredient, such as the permeation rate, permeation amount, and permeation route. In particular, for topical skin preparations that act locally on the skin, the concentration of the active ingredient on the skin surface is important for sunscreens and skin protectants, while the concentration in the epidermis or dermis is important for functional cosmetics with antibacterial, antipruritic, and whitening properties. Since the site of action on the skin varies depending on the application, a simple method for evaluating the permeability of active ingredients is highly desirable.

[0003] Traditionally, the permeability of active ingredients through the skin has been measured mainly by tape stripping, in which skin to which a topical skin preparation has been applied is stripped with tape and analyzed. However, this method is limited to evaluating the stratum corneum on the skin surface, and it is difficult to analyze the permeability and permeation pathways into the interior of the skin.

[0004] In response to this, a method has been proposed for analyzing the distribution of components of a topical skin preparation that has penetrated into the skin, in which the topical skin preparation is applied to the skin, multiple layers of keratin are peeled off from the area of ​​the skin where the topical skin preparation has been applied using adhesive tape, and a TOF-SIMS is used to perform mass analysis of ions generated from the adhesive tape to which the keratin has adhered, and the distribution of ions from the components of the topical skin preparation, keratinocytes, and tape generated from the adhesive tape to which the keratin has adhered is mapped on a plane (see Patent Document 1).

[0005] However, even with the analytical method of Patent Document 1, it is difficult to clearly visualize the penetration pathway or analyze the penetration into the inside of the skin, and further, there is a problem in that it is difficult to quantify the components of the topical skin preparation that have penetrated into the skin.

[0006] Patent No. 6530165

[0007] In view of the above, one aspect of the present invention aims to provide an analytical method capable of analyzing the distribution of components of a topical skin preparation that has penetrated into the skin. Another aspect of the present invention aims to provide a prediction method capable of predicting the penetration pathway of components of a topical skin preparation that has penetrated into the skin.

[0008] In order to solve the above-mentioned problems, one aspect of the analytical method of the present invention is a method for analyzing the distribution of components of a topical skin preparation that has penetrated into skin, comprising: a first mapping step of contacting the skin with the topical skin preparation containing a labeled target component, preparing a tissue slice of a region of the skin that includes the stratum corneum, preparing a slice of the tissue slice that exposes a cross section of the stratum corneum in the depth direction, and mapping the distribution of ions detected from the slice using Nano-SIMS; a second mapping step of contacting the skin with the topical skin preparation containing the labeled target component, collecting a sample from the skin using a tape stripping method, and mapping the distribution of ions detected from the sample using Nano-SIMS; and a discrimination step of discriminating the distribution of the labeled target component from the distribution of ions obtained in the first mapping step and the distribution of ions obtained in the second mapping step.

[0009] According to one aspect of the present invention, an analytical method can be provided that can analyze the distribution of components of an external skin preparation that has penetrated into the skin. Also, according to another aspect of the present invention, a prediction method can be provided that can predict the penetration pathway of components of an external skin preparation that has penetrated into the skin.

[0010] Fig. 1 is a CCD camera image of a frozen section sample in Test Example 1. The scale bar indicates 200 μm. Fig. 2A is a Nano-SIMS image of the frozen section sample in Test Example 1 in the boxed area in Fig. 1. １２ C １４ N －2B is a diagram showing the distribution of SC and Epi in the frozen section sample of Test Example 1. SC indicates the stratum corneum, Epi indicates the epidermis, and the dotted line indicates the boundary between SC and Epi. The scale bar indicates 10 μm. FIG. 2B is a diagram showing the distribution of SC and Epi in the frozen section sample of Test Example 1. The ratio [ １３ C １５ N － / １２ C １４ N － ] is a diagram showing the distribution of the ratio [ １３ C １５ N － / １２ C １４ N － ] indicates the distribution of tranexamic acid (TA). The scale bar indicates 10 μm. Figure 2C shows the ratio [ ２ D － / １ H － ] is a diagram showing the distribution of the ratio [ ２ D － / １ H － ] indicates the distribution of 4-methoxysalicylic acid potassium salt (4MSK). The scale bar indicates 10 μm. FIG. 2D shows the results of imaging of the frozen section sample in the boxed region of FIG. 1 by Nano-SIMS in Test Example 1. ３２ S － FIG. ３２ S － The distribution of the keratinocytes mainly indicates the distribution of the outer keratinocytes. The scale bar indicates 10 μm. FIG. 2E shows the results of Nano-SIMS imaging of the frozen section sample in the boxed region of FIG. 1 in Test Example 1. ３１ P － FIG. ３１ P － The distribution of tranexamic acid (TA) is mainly in the granular and spinous layers. The scale bar indicates 10 μm. Figure 3A shows the distribution of tranexamic acid (TA) at the ratio [ １３ C １５ N － / １２ C １４ N－ ] and mainly the distribution of the keratinized outer membrane, as shown in Figure 2D. ３２ S － The scale bar indicates 10 μm. Figure 3B shows the distribution of 4-methoxysalicylic acid potassium salt (4MSK) at the ratio [ ２ D － / １ H － ] and mainly the distribution of the keratinized outer membrane, as shown in Figure 2D. ３２ S － The scale bar indicates 10 μm. Figure 3C shows the distribution of TA, with the ratio [ １３ C １５ N － / １２ C １４ N － ] and the ratio [ ２ D － / １ H － 4A is a CCD camera image of a peeled stratum corneum sample in Test Example 1. The scale bar indicates 200 μm. FIG. 4B is a CCD camera image of another peeled stratum corneum sample in Test Example 1. The scale bar indicates 200 μm. FIG. 5A is a Nano-SIMS image of the boxed region of FIG. 4A of the peeled stratum corneum sample in Test Example 1. １ H － 5B is a graph showing the distribution of the area indicated by the box in FIG. 4A of the peeled stratum corneum sample in Test Example 1, obtained by Nano-SIMS imaging. The scale bar indicates 10 μm. １２ C １４ N － The scale bar indicates 10 μm. Fig. 5C shows the ratio [ １３ C １５ N － / １２ C １４ N －5D shows the distribution of the ratio [ ] of the area of ​​the peeled stratum corneum sample in Test Example 1, which is shown in the box in FIG. 4A, obtained by Nano-SIMS imaging. ２ D － / １ H － 6A is a graph showing the distribution of the stratum corneum extract in the region indicated by the box in FIG. 4B of another exfoliated stratum corneum sample in Test Example 1, as imaged by Nano-SIMS. １ H － 6B is a graph showing the distribution of the stratum corneum in the region indicated by the box in FIG. 4B of another exfoliated stratum corneum sample in Test Example 1, as imaged by Nano-SIMS. The scale bar indicates 10 μm. １２ C １４ N － The scale bar indicates 10 μm. Fig. 6C shows the ratio [ １３ C １５ N － / １２ C １４ N － 6D shows the distribution of the ratio [ ] of the area shown in the box in FIG. 4B of another exfoliated stratum corneum sample in Test Example 1, as imaged by Nano-SIMS. ２ D － / １ H － 7A is an enlarged view of the boxed area in FIG. 6C of another exfoliated stratum corneum sample in Test Example 1, showing the distribution of [ １３ C １５ N － / １２ C １４ N － 7B is an enlarged view of the boxed area in FIG. 6D of another exfoliated stratum corneum sample in Test Example 1, showing the distribution of the ratio [ ２ D － / １ H －] is a diagram showing the distribution of

[0011] Hereinafter, embodiments of the present invention will be described in detail. Note that the embodiments are not limited by the following description and can be modified as appropriate within the scope of the present invention. Furthermore, in this specification, unless otherwise specified, the term "to" indicating a range of values ​​means that the values ​​before and after it are included as the lower and upper limits.

[0012] (Method for Analyzing the Distribution of Components of a Topical Skin Preparation that Has Penetrated into Skin) In one embodiment, the method for analyzing the distribution of components of a topical skin preparation that has penetrated into skin (hereinafter, may be abbreviated as "analysis method") comprises: a first mapping step of contacting the topical skin preparation containing a labeled target component with skin, preparing a tissue slice of a region of the skin that includes the stratum corneum, preparing a slice that exposes a cross section of the stratum corneum in the depth direction of the tissue slice, and mapping the distribution of ions detected from the slice using Nano-SIMS; a second mapping step of contacting the topical skin preparation containing the labeled target component with skin, collecting a sample from the skin by tape stripping, and mapping the distribution of ions detected from the sample using Nano-SIMS; and a discrimination step of discriminating the distribution of the labeled target component from the distribution of ions obtained in the first mapping step and the distribution of ions obtained in the second mapping step, and may further comprise other steps as necessary.

[0013] <First Mapping Step> The first mapping step is a step of bringing the topical skin preparation containing a labeled target component into contact with skin, preparing a tissue slice from a region of the skin that includes the stratum corneum, preparing a slice that exposes a cross section of the stratum corneum in the depth direction of the tissue slice, and mapping the distribution of ions detected from the slice using Nano-SIMS.

[0014] The method for contacting the topical skin preparation containing the labeled target component with the skin is not particularly limited and can be appropriately selected depending on the method of use of the topical skin preparation, and examples thereof include a method of applying the topical skin preparation to the surface of the skin, a method of immersing a skin sample consisting of a fragment of skin in the topical skin preparation, etc. Furthermore, when contacting the topical skin preparation containing the labeled target component with the skin, the amount of the topical skin preparation applied, the application area, the contact time, the number of applications, etc. are also not particularly limited and can be appropriately selected depending on the purpose, such as the method of use of the topical skin preparation.

[0015] The tissue slice may be a commercially available product or may be one directly collected from an organism, but it is preferable to use a commercially available product. Commercially available tissue slices can be obtained, for example, from BIOPREDIC International. The shape and size of the tissue slice are not particularly limited as long as it contains the stratum corneum of the skin. However, from the viewpoint of analyzing the distribution of components of the topical skin preparation that have penetrated the skin in the depth direction of the skin (the same direction as the depth direction of the stratum corneum), it is preferable that the tissue slice further contains a granular layer.

[0016] The origin of the tissue slice is not particularly limited and can be appropriately selected depending on the purpose, and examples thereof include humans, monkeys, pigs, cattle, sheep, goats, dogs, cats, mice, rats, and birds. Among these, the origin of the tissue slice is preferably humans. Furthermore, the age, sex, condition, etc. of the organism from which the tissue slice of the region containing the stratum corneum of the skin is derived are not particularly limited.

[0017] The method for preparing a section exposing a cross section of the stratum corneum in the depth direction of the tissue slice is not particularly limited and can be appropriately selected from known methods, such as a method of freezing the tissue slice and preparing it using a frozen section preparation device (e.g., a cryostat), a method of embedding the tissue slice in paraffin and preparing it using a microtome, etc. Among these, the method of freezing the tissue slice and preparing it using a frozen section preparation device is preferred.

[0018] The method for freezing the tissue slice is not particularly limited and can be appropriately selected from known methods depending on the purpose, but the method of freezing with liquid nitrogen is preferred because it causes less damage to the tissue slice.

[0019] In an analysis method according to one embodiment, the term "depth direction of the stratum corneum" refers to the direction from the stratum corneum toward the granular layer.

[0020] Nano-SIMS is a two-dimensional high-resolution secondary ion mass spectrometer. Nano-SIMS involves irradiating the surface of a solid sample with primary ions, sputtering the surface and releasing particles such as electrons, neutrons, and ions. This instrument selects only the ions (secondary ions) and passes them through a magnetic field, bending their trajectories according to the mass-to-charge ratio of the secondary ions. The ions then reach a detector, where the relative amounts of each emitted secondary ion can be detected. Visualization of isotope and trace element distributions is achieved by repeating this process for each measurement point, matching the measurement positions with the mass analysis results on a computer, and reconstructing the image.

[0021] The Nano-SIMS is not particularly limited, and any known device can be used as appropriate. In an analysis method according to one embodiment, the use of Nano-SIMS enables highly sensitive analysis of the distribution of components of a topical skin preparation that has penetrated the skin in the depth direction of the stratum corneum.

[0022] The conditions for the Nano-SIMS measurement are not particularly limited and can be appropriately selected depending on the purpose. For example, the measurement can be performed under the conditions described in the examples below.

[0023] The primary ions used in Nano-SIMS are not particularly limited and can be appropriately selected depending on the purpose, and examples thereof include Cs ions and O ions.

[0024] The secondary ions detected by Nano-SIMS are not particularly limited and can be appropriately selected depending on the purpose, but are preferably at least one selected from the group consisting of CN ions, C ions, O ions, H ions, S ions, and P ions.１２ C １４ N － , １３ C １５ N － , １ H － , ２ D － , ３２ S － , and ３１ P － It is more preferable that the material is at least one selected from the group consisting of:

[0025] Since CN ions originate from the coexistence of carbon and nitrogen, which are generally contained in organic matter, the area where CN ions are detected in Nano-SIMS measurements mainly represents proteins. Since the main component of skin is protein, the distribution of CN ions represents the entire skin, and they are detected with high intensity in particular in the epidermis (including the stratum corneum). Here, for example, if the target component is １３ C and １５ When the compound is labeled with N, it is detected by Nano-SIMS in the determination step described later. １２ C １４ N － Imaging results by Nano-SIMS １３ C １５ N － By correcting the imaging results, the intradermal distribution of the target component can be analyzed.

[0026] H ions originate from hydrogen contained not only in the skin but also in the glass or preparation on which the slice is placed during Nano-SIMS measurement. Therefore, H ions represent the background of the skin, glass, preparation, etc.

[0027] S ions are derived from sulfur contained in amino acids such as cysteine ​​and methionine, and therefore can detect the distribution of proteins containing large amounts of these amino acids. In the stratum corneum, S ions are present in large amounts in loricrin, cornifin, and elafin, which are the main cell envelope proteins of keratinocytes, and therefore can detect the distribution of the cornified outer membrane (also called the "cornified envelope").

[0028] P ions are derived from phosphorus, which is abundant in DNA and phospholipids, and can therefore be used to detect the distribution of cells in the stratum granulosum and stratum spinosum. Corneocytes do not contain DNA, and phospholipids are decomposed in the stratum granulosum, meaning they are almost absent in the stratum corneum. Therefore, the distribution of P ions can be used to distinguish between the stratum granulosum and stratum spinosum.

[0029] <<External Skin Preparation>> The external skin preparation contains the labeled target component, and further contains other components as necessary.

[0030] The type of the topical skin preparation is not particularly limited and can be appropriately selected depending on the purpose. Examples include basic cosmetics such as creams, milky lotions, and lotions; medicated cosmetics for whitening or the like; makeup cosmetics such as foundation; sunscreen cosmetics; cleansing cosmetics such as face washes; and topical skin pharmaceuticals for treating and improving age spots, freckles, melasma, chloasma, and the like.

[0031] The dosage form of the topical skin preparation is not particularly limited and can be appropriately selected depending on the purpose. Examples include creams, lotions, emulsions, gels, ointments, pastes, sprays, and patches.

[0032] -Target Component- The target component is a component whose distribution after penetration into the skin is to be analyzed. Therefore, the target component is not particularly limited and can be appropriately selected from components commonly used in topical skin preparations depending on the purpose. Examples of components commonly used in topical skin preparations include tranexamic acid (hereinafter sometimes referred to as "TA"), 4-methoxysalicylic acid potassium salt (hereinafter sometimes referred to as "4MSK"), nicotinamide, retinol, sodium pyrrolidonecarboxylate, glycerin, glycolic acid, trimethylglycine, and cholesterol.

[0033] --Labeling-- The label of the target component is attached in order to distinguish the distribution of the target component from the distribution of components inherent in the skin. The label is not particularly limited and can be appropriately selected depending on the purpose, but stable isotopes are preferred, and trace isotopes are more preferred. In the analysis method according to one embodiment, the term "trace isotope" refers to an isotope with a low abundance ratio. Specific examples of trace isotopes include carbon-13 ( １３ C), nitrogen 15 ( １５ N), oxygen 18 ( １８ O), deuterium ( ２ D). These may be used alone or in combination of two or more. Therefore, the labeled target component may be, for example, a component substituted or modified with one or more stable isotopes.

[0034] Note that many of the components present in the skin are abundant isotopes. In the analysis method according to one embodiment, the term "abundant isotope" refers to an isotope with a high abundance ratio. Specific examples of abundant isotopes include carbon-12 ( １２ C), nitrogen 14 ( １４ N), oxygen 16 ( １６ O), hydrogen 1 ( １ H), sulfur 32 ( ３２ S), phosphorus 31 ( ３１ P) and the like.

[0035] The labeled target component may be a commercially available product, or may be appropriately synthesized by a known method, as necessary. Examples of commercially available labeled target components include tranexamic acid (TA) in which carbon and nitrogen are substituted with stable isotopes ( １３ C and １５ N) substituted TA- １３ C ２ , １５ N (manufactured by TRONTO Research Chemicals), nicotinamide １３ C ６ (Cambridge Isotope Laboratories), Retinol-D ６ (Cambridge Isotope Laboratories), Sodium pyrrolidone carboxylate-D ５(Fujifilm Wako Pure Chemical Industries, Ltd.), Glycerin-D ５ (Cambridge Isotope Laboratories), Glycolic Acid-D ４ (Cambridge Isotope Laboratories), trimethylglycine- １５ N (manufactured by TRONTO Research Chemicals), Cholesterol-D ７ (manufactured by TRONTO Research Chemicals).

[0036] The method for substituting or modifying the target component with a desired label is not particularly limited and can be appropriately selected from known methods depending on the structure of the component, the type of label, etc. Examples include chemical modification methods such as deuterium substitution. Alternatively, the target component can be substituted or modified with a desired label by outsourcing the synthesis to an institution that synthesizes labeled compounds, such as NARD Research Institute, Inc.

[0037] The content of the labeled target component in the topical skin preparation is not particularly limited and can be appropriately selected depending on the purpose. The topical skin preparation may consist solely of the labeled target component.

[0038] Other Components The other components in the topical skin preparation are not particularly limited and can be appropriately selected from components typically contained in topical skin preparations depending on the type of topical skin preparation, and examples include solvents (e.g., water, alcohol, etc.), liquid oils, water-soluble polymers (e.g., thickeners, etc.), oil-soluble polymers, solid oils, moisturizers, surfactants, powder components, preservatives, antioxidants, pH adjusters, active whitening ingredients, anti-inflammatory ingredients, antibacterial ingredients, cell-activating ingredients, astringent ingredients, antioxidant ingredients, acne-reducing ingredients, anti-aging ingredients, ingredients that promote the synthesis of biological components such as collagen, blood circulation-promoting ingredients, plant extracts, etc. These may be used alone or in combination of two or more.

[0039] The content of the other ingredients in the topical skin preparation is not particularly limited and can be appropriately selected depending on the purpose.

[0040] <Second Mapping Step> The second mapping step is a step of bringing the topical skin preparation containing the labeled target component into contact with skin, collecting a sample from the skin by tape stripping, and mapping the distribution of ions detected from the sample using the Nano-SIMS.

[0041] The method for contacting the topical skin preparation containing the labeled target component with the skin is not particularly limited and can be appropriately selected depending on the method of use of the topical skin preparation, and examples thereof include a method of applying the topical skin preparation to the surface of the skin, a method of immersing a skin sample consisting of a fragment of skin in the topical skin preparation, etc. Furthermore, when contacting the topical skin preparation containing the labeled target component with the skin, the amount of the topical skin preparation applied, the application area, the contact time, the number of applications, etc. are also not particularly limited and can be appropriately selected depending on the purpose, such as the method of use of the topical skin preparation.

[0042] There are no particular limitations on the method for collecting a sample from the skin by tape stripping, and any known tape stripping method can be used, but it is preferable that the method be one that can peel off the stratum corneum of the skin.For example, there is a method in which a desired tape is attached to the area from which the skin is to be collected, a certain amount of pressure is applied, and then the tape is peeled off.

[0043] There are no particular restrictions on the tape used in the tape stripping method, and any known tape can be used, but it is preferable that the tape be capable of peeling off the stratum corneum of the skin, and examples thereof include mending tape, cellophane tape, vinyl tape, craft tape, surgical tape, cloth adhesive tape, aluminum tape, and carbon tape.

[0044] The shape, structure, size, etc. of the tape are not particularly limited and can be appropriately selected depending on the purpose.

[0045] The number of times that the sample is collected from the skin is not particularly limited and can be appropriately selected depending on the purpose, and may be one or more times. Furthermore, the sample may be collected from the same site of the skin two or more times, or the second and subsequent samples may be collected from different sites.

[0046] The sample collected from the skin by tape stripping and used for the Nano-SIMS measurement preferably contains at least the stratum corneum, and it is more preferable that the sample be an area where the boundaries of stratum corneum cells can be distinguished, as this facilitates analysis of the distribution of components of the topical skin preparation that has penetrated the skin. Therefore, it is preferable that the sample collected from the skin by tape stripping and used for the Nano-SIMS measurement be subjected to microscopic examination of stratum corneum cells and selection of measurement targets before subjecting the sample to Nano-SIMS measurement.

[0047] There are no particular limitations on the measurement conditions for Nano-SIMS or the secondary ions detected by Nano-SIMS, and they can be appropriately selected depending on the purpose, and examples thereof include the same as those in the first mapping step.

[0048] The topical skin preparation in the second mapping step is the same as that in the first mapping step.

[0049] <Discrimination Step> The discrimination step is a step of discriminating the distribution of the labeled target component from the distribution of the ions obtained in the first mapping step and the distribution of the ions obtained in the second mapping step.

[0050] If only the first mapping step is performed, it may be impossible to confirm the influence of changes in the molecular distribution of the skin caused by freezing and thawing on the Nano-SIMS measurement results. In contrast, in one embodiment, in addition to the first mapping step, a second mapping step is performed using Nano-SIMS measurements on a biological sample without pretreatment such as freezing. This is advantageous in that even if the pretreatment influence on the skin is present in the first mapping step, the second mapping step can eliminate that influence. Furthermore, the first mapping step can evaluate the distribution of the components of the topical skin preparation in the depth direction of the skin, and the second mapping step can evaluate the distribution of the components of the topical skin preparation in the horizontal direction of the stratum corneum (a direction approximately perpendicular to the depth direction of the stratum corneum), which is advantageous in that it allows for a wide range of analysis of the distribution of the components of the topical skin preparation that has penetrated the skin.

[0051] The method for determining the distribution of the labeled target component from the ion distribution obtained in the first mapping step and the ion distribution obtained in the second mapping step is not particularly limited, and examples thereof include a method of comparing the ion distribution image obtained in the first mapping step with the ion distribution image obtained in the second mapping step, and further preferably a comparison with a microscope image. These comparisons may be performed visually by a professional evaluator or using image processing software (e.g., OpenMIMS in ImageJ, etc.).

[0052] The discrimination step is preferably performed by comparing a distribution of at least one ion selected from the group consisting of CN ions, C ions, O ions, H ions, S ions, and P ions with a distribution of ions derived from the label of the target component; １２ C １４ N － , １３ C １５ N － , １ H － , ２ D － , ３２ S － , and ３１ P － It is more preferable to compare the distribution of at least one ion selected from the group consisting of: with the distribution of ions derived from the label of the target component.

[0053] In the discrimination step, when discriminating the distribution of the labeled target component, the ion distribution obtained in the first mapping step and the ion distribution obtained in the second mapping step may be further corrected for the ion intensity of the trace isotope derived from the label of the labeled target component with the ion intensity of the major isotope derived from the component present in the skin, and the ratio of these ion intensities may be calculated, and the ratio of the ion intensities in the first mapping step and the ratio of the ion intensities in the second mapping step may be compared. ２ D － When labeled with １ H －The ratio of ion intensities [ ２ D － / １ H － ] can be analyzed as the distribution of the target component, thereby making it possible to determine the relative amount of the target component in the topical skin preparation that has penetrated the skin.

[0054] In one embodiment of the analytical method, the term "relative amount" refers to the signal intensity calculated from the intracellular region and the intercellular lipid region, relative to the signal intensity obtained from the entire region measured by Nano-SIMS, which is set to 100.

[0055] In the discrimination step, in addition to the ratio of the ion intensities, a comparison may be made with ion intensities having different distributions. ２ D － / １ H － ]of, ３２ S － When the distribution of the target component is overlaid and compared with that of the target component, it is possible to analyze whether the target component is distributed on the outside or inside of the outer cornified membrane. ２ D － / １ H － ]of, ３１ P － When compared with the distribution of the target component, it is possible to analyze whether the target component is distributed in the stratum corneum or the granular layer.

[0056] In one embodiment of the analytical method, when the topical skin preparation contains the other components, the labeled target component and its content are kept constant, and the type or content of the other components is changed, thereby enabling the effect of the other components on the permeability of the target component to be analyzed.

[0057] (Method for predicting the permeation pathway of a component of a topical skin preparation that has penetrated into the skin) According to one embodiment, the method for predicting the permeation pathway of a component of a topical skin preparation that has penetrated into the skin (hereinafter, sometimes abbreviated as "prediction method") comprises: a first mapping step of contacting the skin with the topical skin preparation containing a labeled target component, preparing a tissue slice of a region of the skin that includes the stratum corneum, preparing a slice that exposes a cross section of the stratum corneum in the depth direction of the tissue slice, and mapping the distribution of ions detected from the slice using Nano-SIMS; a second mapping step of contacting the skin with the topical skin preparation containing the labeled target component, collecting a sample from the skin by tape stripping, and mapping the distribution of ions detected from the sample using Nano-SIMS; and a prediction step of predicting the permeation pathway of the labeled target component from the distribution of ions obtained in the first mapping step and the distribution of ions obtained in the second mapping step, and further comprising other steps as necessary.

[0058] In the prediction method according to one embodiment, the first mapping step and the second mapping step are the same as those in the analysis method according to one embodiment, and therefore a description thereof will be omitted.

[0059] <Prediction Step> The prediction step is a step of predicting the permeation pathway of the labeled target component from the distribution of the ions obtained in the first mapping step and the distribution of the ions obtained in the second mapping step.

[0060] The prediction step includes distinguishing the signals of each ion in the same manner as the distinguishing step of the analysis method according to one embodiment.

[0061] In the prediction step, Nano-SIMS measurements are further performed over time from the contact of the topical skin preparation containing the labeled target component with the skin in the first mapping step and the second mapping step, thereby making it possible to observe the permeation of the target component of the topical skin preparation over time, and thereby making it possible to predict the permeation rate and permeation route.

[0062] In one embodiment, when the topical skin preparation contains other components, the effects of the other components on the permeability of the target component can be predicted by keeping the labeled target component and its content constant and varying the type or content of the other components, thereby enabling formulation design of a topical skin preparation with desired permeability.

[0063] The following test examples will be used to further explain the embodiments, but the embodiments are not limited to these test examples.

[0064] (Test Example 1) <Preparation of Drug> Carbon and nitrogen of tranexamic acid (TA) represented by the following structural formula (1) were substituted with stable isotopes ( １３ C and １５ N) substituted TA- １３ C ２ , １５ N (manufactured by TRONTO Research Chemicals), and 4-methoxysalicylic acid potassium salt (4MSK) represented by the following structural formula (2) were derivatized with a stable isotope (D ３ ) substituted 4MSK-D ３ (Synthesis was outsourced to NARD Laboratory Co., Ltd.) was used to evaluate the permeability.

[0065]

[0066]

[0067] <Penetration of tracer into skin sample> The surface of a fresh human skin sample (fresh abdominal skin of a Caucasian woman, a disk with a thickness of 2 mm to 5 mm and a diameter of 8 mm to 10 mm, manufactured by BIOPREDIC International) was washed with pure water, and excess water was absorbed with a cotton swab. １３ C ２ , １５ N, and 2 mass% 4MSK-D ３ An aqueous solution containing ２ The solution was applied to the surface of the skin sample and left to stand at room temperature (25°C ± 5°C) for 2 hours.

[0068] <Preparation of frozen section samples> After standing for 2 hours, the skin samples were frozen with liquid nitrogen and sliced ​​into 5 μm-thick sections in the depth direction of the stratum corneum using a frozen section preparation device (Cryostar NX70, manufactured by Thermo SCIENTIFIC). The prepared thin sections were transferred to indium tin oxide (ITO)-coated glass (manufactured by Bruker Daltonics).

[0069] <Collection of exfoliated stratum corneum samples> After leaving the samples to stand for 2 hours, the stratum corneum was successively peeled from the surface of the skin samples using adhesive tape (D-Squame (registered trademark), manufactured by Clinical & Derm). A specialist evaluator examined the exfoliated stratum corneum cells under a microscope, avoiding tapes with few exfoliated stratum corneum cells and the first and second tapes close to the surface, and selected areas for measurement where the boundaries between stratum corneum cells could be distinguished.

[0070] <Evaluation of Penetration by Nano-SIMS> Using a vapor deposition device (Ion Sputter MC1000, manufactured by Hitachi High-Tech Fielding Corp.), platinum-palladium was vapor-deposited onto the surface of the frozen section sample and the exfoliated stratum corneum sample to an average thickness of 12.5 nm. Images were then taken with a CCD camera, and measurements were taken using Nano-SIMS under the following conditions to evaluate the drug permeability.

[0071] [Nano-SIMS measurement conditions] - Device: NanoSIMS 50L (manufactured by CAMECA) - Primary ion source: Cs ＋ Primary ion beam diameter: An appropriate beam diameter was selected between 40 nm and 200 nm depending on the measurement area. Number of imaging pixels: 256 pixels x 256 pixels. Dwell time: 10 msec / pixel. Measurement ions: １ H － , ２ D － , １２ C １４ N － , １３ C １５ N － , ３１ P － , and ３２ S －The required ions were selected and measured using a multi-collector by switching between two magnetic fields, low and high.

[0072] [Analysis] The images obtained by Nano-SIMS were analyzed using ImageJ's OpenMIMS. ３ It is thought to be the origin ２ D － were measured simultaneously. １ H － The ratio of ion intensities [ ２ D － / １ H － ], 4MSK-D ３ The distribution of TA- １３ C ２ , １５ It is thought to be derived from N １３ C １５ N － were measured simultaneously. １２ C １４ N － The ratio of ion intensities [ １３ C １５ N － / １２ C １４ N － ], TA- １３ C ２ , １５ The analysis was carried out as a distribution of N.

[0073] <<Evaluation Results of Frozen Section Samples>> Images of frozen section samples taken with a CCD camera are shown in Figure 1. In Figure 1, the scale bar represents 200 μm. Nano-SIMS images of the area enclosed in a square in Figure 1 are shown in Figures 2A to 2E. In Figures 2A to 2E, the scale bar represents 10 μm. Furthermore, superimposed images of Figures 2B and 2D, Figures 2C and 2D, and Figures 2B and 2C are shown in Figures 3A to 3C. In Figures 3A to 3C, the scale bar represents 10 μm.

[0074] FIG. 2A is a １２ C １４ N － The distribution of １２ C １４ N－ indicates nitrogen-containing organic matter, and therefore can detect the distribution of mainly proteins.

[0075] FIG. 2B shows the ratio [ １３ C １５ N － / １２ C １４ N － From this, it was confirmed that TA was distributed in layers within the stratum corneum.

[0076] FIG. 2C shows the ratio [ ２ D － / １ H － As can be seen, 4MSK was distributed relatively uniformly in the stratum corneum, without any layered distribution as in TA.

[0077] FIG. 2D shows ３２ S － The distribution of ３２ S － is found in large amounts in amino acids such as cysteine ​​and methionine, and can be detected mainly in the outer cornified membrane (also called the "cornified envelope").

[0078] FIG. 2E shows ３１ P － The distribution of ３１ P － is found in large amounts in DNA and phospholipids, and can be detected mainly in the distribution of cells in the granular layer and spinous layer. Note that DNA is not present in stratum corneum cells, and phospholipids are decomposed in the granular layer and are hardly present in the stratum corneum. ３１ P － The stratum corneum and stratum granulosum can be distinguished by the distribution of

[0079] From Fig. 3A, the ratio [ １３ C １５ N － / １２ C １４ N － ] and the distribution of the outer keratinized membrane. ３２ S － The distributions of TA and TA were complementary to each other, suggesting that TA penetrated into the interior of the keratinocytes.

[0080] From FIG. 3B, the ratio [ ２ D － / １ H － ] and the distribution of the outer keratinized membrane. ３２ S － However, 4MSK has a relatively large number of coincident distributions. ３２ S － The layered distribution shown in the distribution of 4MSK was not observed, suggesting that 4MSK penetrated mainly into the outer cornified membrane of the stratum corneum, but also partially into the interior of the cornified cells.

[0081] From Fig. 3C, the ratio [ １３ C １５ N － / １２ C １４ N － ] and the ratio showing the distribution of 4MSK [ ２ D － / １ H － ], similar to FIG. 3A, TA and 4MSK showed complementary distributions, suggesting that the penetration pathways of the two components were different.

[0082] <<Evaluation Results of Exfoliated Stratum Corneum Samples>> Images of the exfoliated stratum corneum samples taken with a CCD camera are shown in Figures 4A and 4B. Figures 4A and 4B are images of different human skin samples (fresh abdominal skin from a Caucasian female, 2-5 mm thick, 8-10 mm diameter disks, manufactured by BIOPREDIC International). In Figures 4A and 4B, the scale bars represent 200 μm. Nano-SIMS images of the boxed region in Figure 4A are shown in Figures 5A to 5D. The scale bars represent 10 μm in Figures 5A to 5D. Nano-SIMS images of the boxed region in Figure 4B are shown in Figures 6A to 6D. The scale bars represent 10 μm in Figures 5A to 5D. Furthermore, an enlarged image of the boxed region in Fig. 6C is shown in Fig. 7A, and an enlarged image of the boxed region in Fig. 6D is shown in Fig. 7B. In Fig. 7A and Fig. 7B, the scale bars indicate 2 μm.

[0083] 5A to 5D, the ratio [１３ C １５ N － / １２ C １４ N － ] and the ratio showing the distribution of 4MSK [ ２ D － / １ H － The distribution of TA was clearly different around the stratum corneum cells. １ H － and １３ C １５ N － The distribution of TA indicated a relatively uniform distribution at the location of each individual keratinocyte. On the other hand, 4MSK showed a relatively strong signal at the periphery of the keratinocyte. Furthermore, in the keratinocytes shown in Figures 6A to 6D, as shown in Figures 7A and 7B, the ratio [ １３ C １５ N － / １２ C １４ N － ] and the ratio showing the distribution of 4MSK [ ２ D － / １ H － ] were observed. This suggests that the main penetration routes for TA and 4MSK are intracellular and extracellular, respectively. The results of the Nano-SIMS analysis using the exfoliated stratum corneum samples were consistent with the results of the Nano-SIMS analysis using the frozen section samples.

[0084] Examples of aspects of the present invention include the following: <1> A method for analyzing the distribution of a component of a topical skin preparation that has penetrated into skin, comprising: a first mapping step of contacting the skin with the topical skin preparation containing a labeled target component, preparing a tissue slice from a region of the skin containing the stratum corneum, preparing a section of the tissue slice that exposes a cross section of the stratum corneum in the depth direction, and mapping the distribution of ions detected from the section using Nano-SIMS; a second mapping step of contacting the skin with the topical skin preparation containing the labeled target component, collecting a sample from the skin by tape stripping, and mapping the distribution of ions detected from the sample using Nano-SIMS; and a discrimination step of discriminating the distribution of the labeled target component from the distribution of ions obtained in the first mapping step and the distribution of ions obtained in the second mapping step. <2> The analysis method according to <1>, wherein the label of the target component is a trace isotope. <3> The analytical method according to <1> or <2>, wherein the ions detected in the first mapping step and the second mapping step are at least one selected from the group consisting of CN ions, C ions, O ions, H ions, S ions, and P ions. <4> The analytical method according to <3>, wherein the discrimination step is performed by comparing the distribution of at least one ion selected from the group consisting of CN ions, C ions, O ions, H ions, S ions, and P ions with the distribution of ions derived from the label of the target component. <5> The analytical method according to any one of <1> to <4>, wherein the discrimination step further discriminates the relative amount of the target component in the topical skin preparation that has penetrated the skin.<6> A method for predicting the penetration pathway of a component of a topical skin preparation that has penetrated into skin, comprising: a first mapping step of contacting skin with the topical skin preparation containing a labeled target component, preparing a tissue slice from a region of the skin including the stratum corneum, preparing a section of the tissue slice exposing a cross section of the stratum corneum in the depth direction, and mapping the distribution of ions detected from the section using Nano-SIMS; a second mapping step of contacting skin with the topical skin preparation containing the labeled target component, collecting a sample from the skin by tape stripping, and mapping the distribution of ions detected from the sample using Nano-SIMS; and a prediction step of predicting the penetration pathway of the labeled target component from the distribution of ions obtained in the first mapping step and the distribution of ions obtained in the second mapping step. <7> The prediction method according to <6>, wherein the label of the target component is a trace isotope. <8> The prediction method according to <6> or <7>, wherein the ions detected in the first mapping step and the second mapping step are at least one selected from the group consisting of CN ions, C ions, O ions, H ions, S ions, and P ions. <9> The prediction method according to <8>, wherein the prediction step is performed by comparing the distribution of at least one ion selected from the group consisting of CN ions, C ions, O ions, H ions, S ions, and P ions with the distribution of ions derived from the label of the target component. <10> The prediction method according to any one of <6> to <9>, wherein the prediction step further determines the relative amount of the target component of the topical skin preparation that has penetrated the skin.

[0085] As described above, the present invention has been described based on specific embodiments and examples, but these embodiments and examples are presented merely as examples, and the present invention is not limited to the above embodiments and examples. The above embodiments can be embodied in various other forms, and various combinations, omissions, substitutions, additions, modifications, etc. can be made without departing from the spirit of the invention. These embodiments and their modifications are included in the scope and spirit of the invention, and are also included in the inventions described in the claims and their equivalents.

[0086] This international application claims priority based on Japanese Patent Application No. 2023-142220, filed on September 1, 2023, the entire contents of which are incorporated herein by reference.

Claims

1. A method for analyzing the distribution of a component of a topical skin preparation that has penetrated into the skin, comprising: a first mapping step of contacting the skin with the topical skin preparation containing a labeled target component, preparing a tissue slice of a region of the skin including the stratum corneum, preparing a slice exposing a cross section of the stratum corneum in the depth direction of the tissue slice, and mapping the distribution of ions detected from the slice using Nano-SIMS; a second mapping step of contacting the skin with the topical skin preparation containing the labeled target component, collecting a sample from the skin by tape stripping, and mapping the distribution of ions detected from the sample using Nano-SIMS; and a discrimination step of discriminating the distribution of the labeled target component from the distribution of ions obtained in the first mapping step and the distribution of ions obtained in the second mapping step.

2. The analytical method according to claim 1, wherein the label of the target component is a trace isotope.

3. The analytical method according to claim 1, wherein the ions detected in the first mapping step and the second mapping step are at least one selected from the group consisting of CN ions, C ions, O ions, H ions, S ions, and P ions.

4. The analytical method according to claim 3, wherein the discrimination step is performed by comparing the distribution of at least any one of the ions selected from the group consisting of the CN ions, the C ions, the O ions, the H ions, the S ions, and the P ions with the distribution of ions derived from the label of the target component.

5. The analytical method according to claim 1, wherein the determining step further determines the relative amount of the target component of the topical skin preparation that has penetrated the skin.

6. A method for predicting the permeation pathway of a component of a topical skin preparation that has penetrated into the skin, comprising: a first mapping step of contacting the skin with the topical skin preparation containing a labeled target component, preparing a tissue slice of a region of the skin including the stratum corneum, preparing a slice exposing a cross section of the stratum corneum in the depth direction of the tissue slice, and mapping the distribution of ions detected from the slice using Nano-SIMS; a second mapping step of contacting the skin with the topical skin preparation containing the labeled target component, collecting a sample from the skin by a tape stripping method, and mapping the distribution of ions detected from the sample using Nano-SIMS; and a prediction step of predicting the permeation pathway of the labeled target component from the distribution of ions obtained in the first mapping step and the distribution of ions obtained in the second mapping step.

7. The prediction method according to claim 6, wherein the label of the target component is a trace isotope.

8. The prediction method according to claim 6, wherein the ions detected in the first mapping step and the second mapping step are at least one selected from the group consisting of CN ions, C ions, O ions, H ions, S ions, and P ions.

9. The prediction method according to claim 8, wherein the prediction step is performed by comparing a distribution of at least one of the ions selected from the group consisting of the CN ions, the C ions, the O ions, the H ions, the S ions, and the P ions with a distribution of ions derived from the label of the target component.

10. The prediction method of claim 6, wherein the prediction step further determines a relative amount of the target component of the topical skin preparation that has penetrated the skin.

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