Method for analyzing distribution of component of skin external preparation having penetrated skin, and method for predicting penetration path of component of skin external preparation having penetrated skin

Abstract

To provide an analysis method for analyzing the distribution of a component of a skin external preparation having penetrated the skin.SOLUTION: A method for analyzing the distribution of a component of a skin external preparation having penetrated the skin includes: a first mapping step including preparing a tissue piece of a region containing a horny layer of the skin, after bringing a skin external preparation containing a labeled target component into contact with the skin, preparing a section exposing a cross section in a depth direction of the horny layer in the tissue piece, and mapping the distribution of ion detected from the section by using NanoSIMS; a second mapping step including collecting a sample using a tape stripping method from the skin, after bringing the skin external preparation containing the labeled target component into contact with the skin, and mapping the distribution of ion detected from the sample by using the NanoSIMS; and a determination step for determining the distribution of the labeled target component from the ion distribution obtained in the first mapping step and from the ion distribution obtained in the second mapping step.SELECTED DRAWING: Figure 3A

Description

[Technical field]

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

[0002] Skin topical preparations have been developed for various applications, such as those that act from the skin to the whole body, those that act locally on the skin, and functional cosmetics. In order to evaluate the efficacy and safety of such skin topical preparations, it is important to analyze the permeability of the active ingredient, such as the permeation rate, permeation amount, and permeation route. In particular, in the case of skin topical preparations that act locally on the skin, for example, 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 effects, and the like, so that a simple method for evaluating the permeability of the active ingredient is strongly desired.

[0003] Conventionally, the permeability of active ingredients through the skin has been measured mainly by the tape stripping method, in which the 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 surface of the skin, and it is difficult to analyze the permeability into the interior of the skin or the permeation route.

[0004] In response to this, a method has been proposed for analyzing the distribution of components of a topical skin preparation that has penetrated 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 the ions generated from the adhesive tape to which the keratin has adhered, and the distribution of the components of the topical skin preparation, the keratinocytes, and the ions of the tape generated from the adhesive tape to which the keratin has adhered are 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 the problem that it is difficult to quantify the components of the topical skin preparation that have penetrated into the skin. [Prior art documents] [Patent documents]

[0006] [Patent Document 1] Patent No. 6530165 Summary of the Invention [Problem to be solved by the invention]

[0007] In view of the above, an object of one aspect of the present invention is to provide an analysis method capable of analyzing the distribution of components of an external preparation for skin that has penetrated into the skin.Another object of the present invention is to provide a prediction method capable of predicting the penetration path of the components of an external preparation for skin that has penetrated into the skin. [Means for solving the problem]

[0008] In order to solve the above problems, one embodiment of the analytical method of the present invention is 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 topical skin preparation containing a labeled target component with the skin, preparing a tissue slice of a region including the stratum corneum of the skin, 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 topical skin preparation containing the labeled target component with the skin, 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 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. Effect of the Invention

[0009] According to one aspect of the present invention, there is provided an analysis method capable of analyzing the distribution of components of an external preparation for skin that has penetrated into the skin. Also, according to another aspect of the present invention, there is provided a prediction method capable of predicting the penetration path of components of an external preparation for skin that has penetrated into the skin. [Brief description of the drawings]

[0010] [Figure 1] 1 is a CCD camera image of a frozen section sample in Test Example 1. The scale bar indicates 200 μm. [Figure 2A] 2A is a diagram showing the distribution of 12C14N- in the region boxed in FIG. 1 of the frozen section sample in Test Example 1, imaged by Nano-SIMS. 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. [Figure 2B] 2B is a diagram showing the distribution of the ratio [13C15N- / 12C14N-] of the Nano-SIMS imaging in the region shown in the box in FIG. 1 of the frozen section sample in Test Example 1. The distribution of the ratio [13C15N- / 12C14N-] shows the distribution of tranexamic acid (TA). The scale bar indicates 10 μm. [Figure 2C] 2C is a diagram showing the distribution of the ratio [2D- / 1H-] of the Nano-SIMS imaging of the frozen section sample in the region shown in the box in FIG. 1 of the frozen section sample in Test Example 1. The distribution of the ratio [2D- / 1H-] shows the distribution of 4-methoxysalicylic acid potassium salt (4MSK). The scale bar indicates 10 μm. [Figure 2D] FIG. 2D is a diagram showing the distribution of 32S- in Nano-SIMS imaging of a frozen section sample in the region shown in the box in FIG. 1 of the frozen section sample in Test Example 1. The distribution of 32S- mainly shows the distribution of the keratinized outer membrane. The scale bar indicates 10 μm. [Figure 2E]FIG. 2E is a diagram showing the distribution of 31P- in Nano-SIMS imaging of the frozen section sample in the region shown in the box in FIG. 1 of the frozen section sample in Test Example 1. The distribution of 31P- mainly shows the distribution of cells in the granular layer and spinous layer. The scale bar indicates 10 μm. [Figure 3A] Figure 3A is a diagram showing the distribution of tranexamic acid (TA) by superimposing the distribution of the ratio [13C15N- / 12C14N-] shown in Figure 2B, which shows the distribution of TA, and the distribution of 32S- shown in Figure 2D, which shows the distribution mainly in the outer keratinized membrane. The scale bar indicates 10 μm. [Figure 3B] Figure 3B is a diagram showing the distribution of 4-methoxysalicylic acid potassium salt (4MSK) by superimposing the distribution of the ratio [2D- / 1H-] shown in Figure 2C, which shows the distribution of 4MSK, and the distribution of 32S- shown in Figure 2D, which shows the distribution mainly in the outer keratinized membrane. The scale bar indicates 10 μm. [Figure 3C] Figure 3C is a diagram showing the distribution of the ratio [13C15N- / 12C14N-] shown in Figure 2B, which shows the distribution of TA, and the distribution of the ratio [2D- / 1H-] shown in Figure 2C, which shows the distribution of 4MSK. The scale bar indicates 10 μm. [Figure 4A] 4A is a CCD camera image of a peeled stratum corneum sample in Test Example 1. The scale bar indicates 200 μm. [Figure 4B] 4B is a CCD camera image of another exfoliated stratum corneum sample in Test Example 1. The scale bar indicates 200 μm. [Figure 5A] Fig. 5A is a diagram showing the distribution of 1H- in Nano-SIMS imaging of the region boxed in Fig. 4A of the exfoliated stratum corneum sample in Test Example 1. The scale bar indicates 10 µm. [Figure 5B] Fig. 5B is a diagram showing the distribution of 12C14N- in the region boxed in Fig. 4A of the exfoliated stratum corneum sample in Test Example 1, as imaged by Nano-SIMS. The scale bar indicates 10 µm. [Figure 5C]Fig. 5C is a diagram showing the distribution of the ratio [13C15N- / 12C14N-] in Nano-SIMS imaging in the region boxed in Fig. 4A of the exfoliated stratum corneum sample in Test Example 1. The scale bar indicates 10 µm. [Figure 5D] Fig. 5D is a diagram showing the distribution of the ratio [2D- / 1H-] of Nano-SIMS imaging in the boxed region of Fig. 4A of the exfoliated stratum corneum sample in Test Example 1. The scale bar indicates 10 µm. [Figure 6A] Fig. 6A is a diagram showing the distribution of 1H- in Nano-SIMS imaging of the region shown in the box in Fig. 4B of another exfoliated stratum corneum sample in Test Example 1. The scale bar indicates 10 µm. [Figure 6B] Fig. 6B is a diagram showing the distribution of 12C14N- in Nano-SIMS imaging of the region boxed in Fig. 4B of another exfoliated stratum corneum sample in Test Example 1. The scale bar indicates 10 µm. [Figure 6C] Fig. 6C is a diagram showing the distribution of the ratio [13C15N- / 12C14N-] in Nano-SIMS imaging in the boxed region of Fig. 4B of another exfoliated stratum corneum sample in Test Example 1. The scale bar indicates 10 µm. [Figure 6D] Fig. 6D is a diagram showing the distribution of the ratio [2D- / 1H-] in Nano-SIMS imaging in the boxed region in Fig. 4B of another exfoliated stratum corneum sample in Test Example 1. The scale bar indicates 10 µm. [Figure 7A] FIG. 7A is an enlarged view of the boxed area in FIG. 6C of another exfoliated stratum corneum sample in Test Example 1, and shows the distribution of the ratio [13C15N- / 12C14N-] imaged by Nano-SIMS. [Figure 7B] FIG. 7B is an enlarged view of the boxed area in FIG. 6D of another exfoliated stratum corneum sample in Test Example 1, and shows the distribution of the ratio [2D- / 1H-] imaged by Nano-SIMS. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0011] Hereinafter, the embodiments of the present invention will be described in detail. Note that the embodiments are not limited by the following description, and can be appropriately modified without departing from the gist of the present invention. In addition, in this specification, "to" indicating a numerical range means that the numerical values ​​before and after it are included as the lower and upper limits, unless otherwise specified.

[0012] (Method for analyzing the distribution of ingredients in topical skin preparations that have penetrated into the skin) In one embodiment, a method for analyzing the distribution of a component of a topical skin preparation that has penetrated into the skin (hereinafter, sometimes abbreviated as "analysis method") includes a first mapping step of contacting the topical skin preparation containing a labeled target component with the skin, 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 topical skin preparation containing the labeled target component with the 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 further including other steps as necessary.

[0013] <First mapping step> The first mapping step is a 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.

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

[0015] The tissue slice may be a commercially available product or may be directly collected from an organism, but it is preferable to use a commercially available product. The commercially available tissue slice 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, but it is preferable that the tissue slice further contains a granular layer from the viewpoint of analyzing the distribution of the components of the skin topical agent that has penetrated into the skin in the depth direction of the skin (the same direction as the depth direction of the stratum corneum).

[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, cows, sheep, goats, dogs, cats, mice, rats, and birds. Among these, the origin of the tissue slice is preferably humans. In addition, 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, for example, a method in which the tissue slice is frozen and prepared using a frozen section preparation device (e.g., a cryostat), a method in which the tissue slice is embedded in paraffin and prepared using a microtome, etc. Among these, the method in which the tissue slice is frozen and prepared 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 a method of freezing with liquid nitrogen is preferred because it causes less damage to the tissue slice.

[0019] In one embodiment of the analysis method, the "depth direction of the stratum corneum" means the direction from the stratum corneum toward the granular layer.

[0020] Nano-SIMS is a two-dimensional high-resolution secondary ion mass spectrometer. In Nano-SIMS, the surface of a solid sample is irradiated with primary ions, which causes the surface of the irradiated sample to be sputtered, releasing particles such as electrons, neutrons, and ions. Among these, only ions (secondary ions) are selected and passed through a magnetic field, where their trajectory is bent according to the ratio of mass to charge of the secondary ions, and then they reach a detector, where the relative amount of each emitted secondary ion can be detected. Visualization of isotope and trace element distribution is performed 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 a known device can be appropriately used. In the analysis method according to one embodiment, the Nano-SIMS can be used to analyze the distribution of the components of the topical agent for skin that has penetrated into the skin in the depth direction of the stratum corneum with high sensitivity.

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

[0023] The primary ions used in Nano-SIMS are not particularly limited and can be appropriately selected depending on the purpose. Examples of the primary ions 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. However, it is preferable that the secondary ions are 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] CN ions are derived from the coexistence of carbon and nitrogen, which are generally contained in organic matter, so 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 especially in the epidermis (including the stratum corneum). Here, for example, if the target component is 13 C and 15 In the case where the nuclei are labeled with N, the nuclei are analyzed by Nano-SIMS in the discrimination process described below. 12 C 14 N - The imaging results are shown 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 and 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 that contain large amounts of these amino acids. In the stratum corneum, S ions are abundant 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 granular layer and spinous layer. Note that stratum corneum cells do not contain DNA, and phospholipids are decomposed in the granular layer and are therefore almost nonexistent in the stratum corneum. Therefore, the distribution of P ions can be used to distinguish between the stratum corneum and the granular layer.

[0029] <<External skin preparations>> The topical skin preparation contains a labeled target component and, if necessary, further contains other components.

[0030] The type of the topical skin preparation is not particularly limited and can be appropriately selected depending on the purpose. Examples of the topical skin preparation include basic cosmetics such as creams, milky lotions, lotions, and the like; medicated cosmetics for the purpose of whitening, etc.; makeup cosmetics such as foundations; sunscreen cosmetics; cleansing cosmetics such as facial cleansers; and topical skin medicines for the treatment and improvement of age spots, freckles, melasma, chloasma, and the like.

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

[0032] -Target ingredients- The target component is a component that is to be analyzed for distribution after penetration into the skin.Therefore, the target component is not particularly limited, and can be appropriately selected from components that are generally used in skin external preparations according to the purpose.Examples of components that are generally used in skin external preparations include tranexamic acid (hereinafter sometimes referred to as "TA"), 4-methoxysalicylic acid potassium salt (hereinafter sometimes referred to as "4MSK"), nicotinamide, retinol, sodium pyrrolidone carboxylate, glycerin, glycolic acid, trimethylglycine, cholesterol, etc.

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

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

[0035] The labeled target component may be a commercially available product, or may be appropriately synthesized by a known method, as necessary. For example, the commercially available labeled target component may be tranexamic acid (TA) in which carbon and nitrogen are substituted with stable isotopes ( 13 C and 15 N) substituted TA- 13 C2, 15 N (TRONTO Research Chemicals), nicotinamide 13 C6 (manufactured by Cambridge Isotope Laboratories), retinol-D6 (manufactured by Cambridge Isotope Laboratories), sodium pyrrolidone carboxylate-D5 (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.), glycerin-D5 (manufactured by Cambridge Isotope Laboratories), glycolic acid-D4 (manufactured by Cambridge Isotope Laboratories), trimethylglycine- 15 N (manufactured by TRONTO Research Chemicals), cholesterol-D7 (manufactured by TRONTO Research Chemicals), etc.

[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 and the type of label, for example, a chemical modification method such as deuterium substitution method, etc. Alternatively, the target component can be substituted or modified with a desired label by entrusting the synthesis to an institution that synthesizes labeled compounds, such as NARD Laboratory Co., Ltd.

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

[0038] -Other ingredients- The other components in the skin external preparation are not particularly limited, and can be appropriately selected from components usually contained in skin external preparations depending on the type of skin external preparation, and examples thereof 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, whitening active ingredients, anti-inflammatory ingredients, antibacterial ingredients, cell activating ingredients, astringent ingredients, antioxidant ingredients, acne improving ingredients, anti-aging ingredients, biocomponent synthesis promoting ingredients 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 external 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 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 the Nano-SIMS.

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

[0042] The method for collecting a sample from the skin by tape stripping is not particularly limited, and any known tape stripping method can be used, but it is preferable that the method be capable of peeling off the stratum corneum of the skin. For example, 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] The tape used in the tape stripping method is not particularly limited, 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 to collect the sample from the skin is not particularly limited and can be appropriately selected depending on the purpose, and may be one time or two or more times. In addition, the sample may be collected from the same site of the skin two or more times, or may be collected from a different site from the second time onwards.

[0046] The sample taken from the skin by the tape stripping method for use in the Nano-SIMS measurement preferably contains at least the stratum corneum, and is preferably an area where the boundaries of stratum corneum cells can be distinguished, from the viewpoint of facilitating the analysis of the distribution of the components of the skin topical agent that has penetrated into the skin. Therefore, it is preferable that the sample taken from the skin by the tape stripping method for use in the Nano-SIMS measurement is subjected to a microscope examination of the stratum corneum cells and the measurement target is selected before the sample is subjected to the Nano-SIMS measurement.

[0047] The measurement conditions for Nano-SIMS and the secondary ions detected by Nano-SIMS are not particularly limited and can be appropriately selected depending on the purpose, and examples thereof include the same as those in the first mapping step.

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

[0049] <Discrimination process> 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] When only the first mapping step is performed, the Nano-SIMS measurement result may not be able to confirm the influence of the change in molecular distribution of the skin caused by freezing and thawing.In contrast, in the analysis method according to one embodiment, in addition to the first mapping step, the second mapping step performs Nano-SIMS measurement using a biological sample that is not pretreated such as frozen.Even if the first mapping step has an influence on the skin due to the pretreatment, the second mapping step can eliminate the influence.Furthermore, the first mapping step can evaluate the distribution of the components of the skin topical agent in the depth direction of the skin, and the second mapping step can evaluate the distribution of the components of the skin topical agent in the horizontal direction of the stratum corneum (the direction approximately perpendicular to the depth direction of the stratum corneum), so that the distribution of the components of the skin topical agent that has penetrated into the skin can be analyzed widely.

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

[0052] The discrimination step is preferably carried out 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 analysis method, the "relative amount" refers to the signal intensity expressed relatively by calculating the signal intensities obtained from the intracellular region and the intercellular lipid region, respectively, when the signal intensity obtained from the entire region measured by Nano-SIMS 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. For example, 2 D - / 1 H - ]of, 32 S - When the distribution of the target component is 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 cornified outer 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 addition, 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, so that the effect of the other components on the permeability of the target component can also be analyzed.

[0057] (Method for predicting the permeation pathway of ingredients of topical skin preparations that have penetrated into the skin) In one embodiment, a method for predicting the permeation pathway of a component of a topical skin preparation that has permeated into the skin (hereinafter, sometimes abbreviated as "prediction method") includes a first mapping step of contacting the topical skin preparation containing a labeled target component with the skin, 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 topical skin preparation containing the labeled target component with the skin, 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, and further including other steps as necessary.

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

[0059] <Prediction process> 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 discriminating the signals of each ion in a manner similar to the discrimination step of the analysis method according to one embodiment.

[0061] In the prediction step, furthermore, by performing Nano-SIMS measurements over time from the contact of the topical skin preparation containing a labeled target component with the skin in the first mapping step and the second mapping step, it is possible to observe how the target component of the topical skin preparation penetrates over time, and thereby it is also possible to predict the penetration rate and penetration route.

[0062] In addition, in the prediction method according to one embodiment, 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, so that the effect of the other components on the permeability of the target component can also be predicted. This makes it possible to carry out a formulation design of a topical skin preparation having a desired permeability. EXAMPLES

[0063] The embodiments will be described in more detail below with reference to test examples, but the embodiments are not limited to these test examples.

[0064] (Test Example 1) <Preparation of medication> The carbon and nitrogen of tranexamic acid (TA) represented by the following structural formula (1) are substituted with stable isotopes ( 13 C and 15 N) substituted TA- 13 C2, 15 N (manufactured by TRONTO Research Chemicals) and 4MSK-D3 (synthesis outsourced to NARD Laboratory Co., Ltd.), in which the hydrogen of 4-methoxysalicylic acid potassium salt (4MSK) represented by the following structural formula (2) was replaced with a stable isotope (D3), were used to evaluate the permeability.

[0065] [ka]

[0066] [ka]

[0067] <Tracer penetration into skin samples> The surface of a fresh human skin sample (fresh abdominal skin of a Caucasian female, a disk of 2-5 mm thickness and 8-10 mm diameter, manufactured by BIOPREDIC International) was washed with pure water, and then excess water was absorbed with a cotton swab. 13 C2, 15An aqueous solution containing N and 2 mass% 4MSK-D3 was applied at 2 μL / cm 2 to the surface of the skin sample and allowed 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 sample was frozen in liquid nitrogen, and using a cryostat (CryoStar NX70, manufactured by Thermo SCIENTIFIC), sections with a thickness of 5 μm were thinly cut in the depth direction of the stratum corneum. The prepared thin sections were transferred onto indium tin oxide (ITO)-coated glass (manufactured by Bruker Daltonics).

[0069] <Collection of exfoliated stratum corneum samples> After standing for 2 hours, the stratum corneum was continuously peeled from the surface of the skin sample using an adhesive tape (D-Squame (registered trademark), manufactured by Clinical & Derm). A professional evaluator confirmed the exfoliated stratum corneum cells under a microscope, and tapes with almost no exfoliated stratum corneum cells and the first and second tapes close to the surface were avoided. Areas where the boundaries between stratum corneum cells could be distinguished were selected as the measurement targets.

[0070] <Evaluation of permeability by Nano-SIMS> Using an evaporation device (Ion Sputter MC1000, manufactured by Hitachi High-Technologies Fielding Co., Ltd.), platinum palladium was deposited on the surfaces of the frozen section samples and the exfoliated stratum corneum samples under the condition of an average thickness of 12.5 nm. Then, after photographing with a CCD camera, measurements were performed using Nano-SIMS under the following conditions to evaluate the permeability of the drug.

[0071] [Nano-SIMS measurement conditions] · Instrument: NanoSIMS 50L (manufactured by CAMECA) · Primary ion source: Cs + · Primary ion beam diameter: An appropriate beam diameter was selected according to the measurement area between 40 nm and 200 nm. · Number of pixels for imaging: 256 pixels × 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 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. 2 D - were measured simultaneously. 1 H - The ratio of ion intensities [ 2 D - / 1 H - ] was analyzed as the distribution of 4MSK-D3. 13 C2, 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 C2, 15 The analysis was carried out as a distribution of N.

[0073] <<Evaluation results of frozen section samples>> An image of a frozen section sample taken with a CCD camera is shown in FIG. 1. In FIG. 1, the scale bar indicates 200 μm. Nano-SIMS imaging of the area enclosed in a square in FIG. 1 is shown in FIG. 2A to FIG. 2E. In FIG. 2A to FIG. 2E, the scale bar indicates 10 μm. Furthermore, superimposed images of FIG. 2B and FIG. 2D, FIG. 2C and FIG. 2D, and FIG. 2B and FIG. 2C are shown in FIG. 3A to FIG. 3C. In FIG. 3A to FIG. 3C, the scale bar indicates 10 μm.

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

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

[0076] Figure 2C shows the ratio [ 2 D - / 1 H - As a result, 4MSK was not found to have a layered distribution like TA in the stratum corneum, but was distributed relatively uniformly.

[0077] FIG. 2D shows 32 S - The distribution of is shown. 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] Figure 2E shows 31 P - The distribution of is shown. 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. However, 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 granular layer can be distinguished based on the distribution of

[0079] From Figure 3A, the ratio showing the distribution of TA [ 13 C 15 N - / 12 C 14 N - ] and the distribution of the keratinized epithelium. 32 S - The distribution of TA in the stratum corneum was complementary to that in the control, suggesting that TA penetrated into the interior of the stratum corneum cells.

[0080] From Figure 3B, the ratio showing the distribution of 4MSK [ 2 D - / 1 H - ] and the distribution of the keratinized epithelium. 32 S - The distribution of the two was relatively consistent. However, 4MSK had a higher degree of similarity than TA and 32 S - The lamellar distribution shown by the distribution of 4MSK was not observed, suggesting that 4MSK penetrated mainly into the outer keratinized membrane of the stratum corneum, but also partially into the interior of the stratum corneum cells.

[0081] From Figure 3C, the ratio showing the distribution of TA [ 13 C 15 N - / 12 C 14 N - ] and the ratio showing the distribution of 4MSK [ 2 D - / 1 H - ] also showed complementary distributions for TA and 4MSK, as in Figure 3A, suggesting that the penetration pathways of the two components are different.

[0082] <<Evaluation results of peeled stratum corneum samples>> Images of the peeled stratum corneum sample taken by a CCD camera are shown in Figures 4A and 4B. Figures 4A and 4B are images of different human skin samples (fresh abdominal skin of a Caucasian woman, disks with a thickness of 2 mm to 5 mm and a diameter of 8 mm to 10 mm, manufactured by BIOPREDIC International). In Figures 4A and 4B, the scale bar indicates 200 μm. Furthermore, Nano-SIMS images of the area enclosed in a square in Figure 4A are shown in Figures 5A to 5D. In Figures 5A to 5D, the scale bar indicates 10 μm. Furthermore, Nano-SIMS images of the area enclosed in a square in Figure 4B are shown in Figures 6A to 6D. In Figures 5A to 5D, the scale bar indicates 10 μm. Furthermore, an enlarged image of the area enclosed in a square in Figure 6C is shown in Figure 7A, and an enlarged image of the area enclosed in a square in Figure 6D is shown in Figure 7B. In Figures 7A and 7B, the scale bars indicate 2 µm.

[0083] From Fig. 5A to Fig. 5D, the ratio showing the distribution of TA [ 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 was relatively uniform 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- ] was observed to have complementary distributions. This suggests that the main penetration routes of TA and 4MSK are intracellular and extracellular, respectively, and the results of the Nano-SIMS analysis of the exfoliated stratum corneum samples were consistent with the results of the Nano-SIMS analysis of the frozen section samples.

[0084] Examples of aspects of the present invention include the following. <1> A method for analyzing the distribution of components of an external 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 the Nano-SIMS; a discrimination step of discriminating a 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; The analytical method is characterized by comprising: <2> the label of the component of interest is a trace isotope; <1> This is the analysis method described in <3> 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. <1> or <2> This is the analysis method described in <4> the discrimination step is carried out 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; <3> This is the analysis method described in <5> The determining step further determines the relative amount of the target component of the topical skin preparation that has penetrated into the skin. <1> from <4> The present invention relates to an analytical method according to any one of the above. <6> A method for predicting a permeation pathway of a component of an external skin preparation that has permeated 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 the Nano-SIMS; a prediction step of predicting a permeation path 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; The prediction method is characterized by comprising: <7> the label of the component of interest is a trace isotope; <6> This is a prediction method described in <8> 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. <6> or <7> This is a prediction method described in <9> the prediction step is carried out 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; <8> This is a prediction method described in <10> The prediction step further includes determining a relative amount of the target component of the topical skin preparation that has penetrated the skin. <6> from <9> The prediction method according to any one of the preceding claims.

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

Claims

1. A method for analyzing the distribution of components of an external 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 the Nano-SIMS; a discrimination step of discriminating a 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; An analytical method comprising the steps of:

2. The method of claim 1 , wherein the label of the component of interest is a trace isotope.

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

4. 4. The analytical method according to claim 3, wherein the discrimination step is performed by comparing a 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 a distribution of ions derived from the label of the target component.

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

6. A method for predicting a permeation pathway of a component of an external skin preparation that has permeated 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 the Nano-SIMS; a prediction step of predicting a permeation path 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; A prediction method comprising:

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

8. 7. 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. 9. The prediction method according to claim 8, wherein the prediction step is performed by comparing a 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 a distribution of ions derived from the label of the target component.

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

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