JP6105252B2 - Skin behavior evaluation method - Google Patents

Description

  The present invention relates to a skin behavior evaluation method.

Animal or human skin plays a role such as a protective action such as invasion of a foreign body, a shock-absorbing action of an impact or temperature, and the like.
In recent years, drugs such as highly permeable vitamins having enhanced permeability to the epidermis, dermis, etc., and transdermal patches that allow the drug to be directly absorbed into blood vessels through the skin have been used. At this time, it is necessary to adjust the drug delivery so that the necessary drug can be delivered to and stored in the necessary layer in order to efficiently exert the drug effect.
However, since the skin has a multi-layered structure of the stratum corneum, epidermis, and dermis, in order to maximize the effects of the external preparation for skin, base ingredients and medicinal ingredients (hereinafter referred to as “test substances”) are used in animals. Test substances have been measured in various systems to determine whether or not they have effectively reached or stored at a target site in human skin, and the kinetics of the test substance in the skin have been evaluated.
For example, when using isolated skin of animals, humans, etc., there is a lack of consistency with a living body and it is difficult to say that the dynamics in the skin can be evaluated with high accuracy.
In addition, in invasive measurements such as biopsies, the skin behavior of the test substance can be directly grasped, but in humans the mental and physical burden on the subject is significantly large, while in animals it is not compatible with humans, There is also a problem from the point of animal welfare.
In addition, in noninvasive measurements such as IR, Raman, and fluorescence detection, although the behavior of the test substance in the skin can be directly grasped (for example, Patent Documents 1 and 2), since signals other than the test substance are picked up together, precise measurement is performed. There is a problem that the skin dynamics cannot be evaluated with high accuracy.

JP 2008-188302 A International Publication No. 2007/026325 Pamphlet

Furthermore, since the skin has a multi-layer structure with the stratum corneum, epidermis, and dermis, and the properties of these layers are also different, it is desirable to be able to understand how the test substance reaches and is stored in each layer. Specifically, it is desirable that the amount of the test substance or its metabolite applied to each skin layer can be accurately measured.
Thus, a method capable of highly accurately evaluating the skin dynamics of a test substance in the skin has been desired.
Therefore, the present invention provides a method capable of evaluating the kinetics of a test substance in the skin with high accuracy.

  As a result of intensive studies, the present inventor has increased the skin dynamics of the test substance by measuring the behavior of the test substance in the skin with an accelerator mass spectrometry system using the test substance containing the radionuclide on the skin. The inventors have found that it can be evaluated with accuracy, and have completed the present invention.

That is, the present invention provides a method for evaluating the behavior of a test substance in the skin, in which the distribution of the test substance containing a radionuclide in the thickness direction of the skin is measured by an accelerator mass spectrometry system.
The present invention also provides a method for evaluating the behavior of a test substance in the skin, wherein a time-dependent change in the skin of a radiolabeled test substance is measured with an accelerator mass spectrometry system.

  ADVANTAGE OF THE INVENTION According to this invention, the method which can evaluate the skin dynamics of a test substance with high precision can be provided.

FIG. 1 is a diagram showing the behavior of lidocaine hydrochloride in pig auricle skin. The vertical axis represents the concentration (μg / ml) of the test substance, and the horizontal axis represents the depth (x / L) in the thickness direction of the skin. The left figure shows the results of AMS measurement showing the behavior of lidocaine hydrochloride labeled with the radioisotope 14C. The right figure shows the results of LC-MS / MS measurement showing the behavior of unlabeled lidocaine hydrochloride. FIG. 2 is a diagram showing a stratum corneum-donor distribution coefficient K in AMS measurement and LC-MS / MS measurement. FIG. 3 is a schematic diagram of the change over time of the test substance in the skin.

  In the method for evaluating the behavior of a test substance in the skin of the present disclosure, (a) distribution in the skin thickness direction of a test substance containing a radionuclide, or (b) change over time in the skin is measured with an accelerator mass spectrometry system. It is characterized by measuring.

Examples of cases that can be specifically considered as “evaluation of behavior in the skin” include, for example, the following (1) to (9), which are “skin in the thickness direction” and / or “identical” at regular time intervals. It is possible to collect “skin on the plane”, measure the RI concentration, and analyze the relationship between the thickness (Z axis) and the same plane (XY axis), where T is the concentration of the test substance containing the radionuclide. It is. Moreover, you may display the relationship of these density | concentration, thickness, and a plane with a graph. However, the evaluation of the behavior in the skin is not limited to these (1) to (9).
(1) Check the penetration of the test substance at “depth-concentration” (time fixed, sampling position fixed).
(2) Check the storage, metabolism, decomposition, and disappearance of the test substance in “Time-concentration” (depth fixed, sampling position shifted to the next).
(3) Under “Position-Concentration” (fixed depth, fixed time), observe the diffusion of the test substance in the same plane.
(4) A plurality of (1) “depth-concentration” at different times are compared.
(5) A plurality of (1) “depth-concentration” at different positions are compared.
(6) A plurality of (1) “depth-concentration” having different depths are compared.
(7) A plurality of the above (2) “time-concentrations” having greatly different positions are compared.
(8) A plurality of (3) “position-density” having different depths are compared.
(9) A plurality of (3) “position-concentration” at different times are compared.

In the method of the present disclosure, a kinetic parameter of a test substance may be used when performing the above-described analysis, and this parameter can be calculated by a least square method (for example, the following (Equation 1)). It is.
As a method for calculating pharmacokinetic parameters, the following formula 1 can be used. Here, in Formula 1, C _X is the drug concentration at depth x, C _veh is the drug concentration in the donor, L is the thickness of the stratum corneum, D is the diffusion constant of the drug in the stratum corneum, and K is the stratum corneum − The donor distribution coefficient, t, is the donor application time.

  In the method of the present disclosure, measurement is performed using a radionuclide present in a test substance in a measurement sample. Thereby, it becomes possible to evaluate the behavior of the test substance in the skin. Preferably, a test substance labeled with a radionuclide is used.

The test substance for evaluating the behavior in the skin in the present disclosure is not particularly limited, and may be either an inorganic compound or an organic compound. Examples of the organic compound include hydrocarbons; basic heterocyclic systems; halogen derivatives; compounds containing atoms selected from oxygen, sulfur, and nitrogen. More specific organic compounds are known to have useful effects on the skin, such as amino acids, peptides, proteins, lipids, polysaccharides, oligosaccharides, ceramides, vitamins, and derivatives thereof. Etc.
Among these, ingredients that prevent acne and acemo; ingredients that smooth the skin; ingredients that keep the skin smooth; ingredients that prevent rough skin; ingredients that moisturize the skin; ingredients that moisturize the skin; moisture in the skin; Ingredients: Ingredients that keep the skin soft; Ingredients that protect the skin; Ingredients that prevent dryness of the skin; Ingredients that soften the skin; Ingredients that give the skin elasticity; Ingredients that give the skin a glossy; Ingredients to prevent sunburn; Ingredients to prevent stains and freckles due to sunburn; Ingredients to make small wrinkles inconspicuous by drying are preferred, and ingredients used as active ingredients of pharmaceutical ingredients or quasi drugs are particularly preferred.
The test substance may be any of an extract (for example, derived from a plant, an animal, a microorganism, etc.) and a synthetic product (for example, chemical synthesis).

If the test substance in the measurement sample contains a radionuclide with a concentration measurable by accelerator mass spectrometry (for example, ¹⁴ C), the test substance containing this radionuclide is considered from the viewpoint of work efficiency and measurement cost. Can be used for measurement.
Furthermore, in order to evaluate the behavior of the test substance with higher accuracy, it is desirable to add a test substance labeled with a radionuclide to the measurement sample to increase the concentration of the test substance containing the radionuclide in the measurement sample. As described above, it is desirable to measure a measurement sample to which a labeled substance obtained by labeling a test substance with a radionuclide is added with an accelerator mass spectrometry system because a stable measurement result can be obtained.

The radionuclide used in the present disclosure is not particularly limited as long as it is a radionuclide that can be measured by an accelerator mass spectrometry system (hereinafter also referred to as “AMS system”). For example, radioactive beryllium (beryllium 7,10 ^{(7 Be,} 10 Be), etc.), radioactive carbon (carbon 14 ⁽¹⁴ C), etc.), radioactive aluminum (aluminum ^{26 (26} Al), etc.), radioactive silicon (silicon 32 ⁽³² Si), etc.), radioactive chlorine (chlorine ^{36 (36} Cl), etc.), radioactive calcium (calcium ^{41 (41} Ca), etc.), radioactive manganese (manganese ^{53 (53} Mn), etc.), radioactive iodine (iodine ^{129 (129} I) Etc.), radioactive hydrogen (tritium ( ³ H), etc.) and the like, and one or more of these can be used.

  When obtaining a test substance labeled with a radionuclide, it is possible to use a known production method such as organic synthesis and microbial production by incorporating the radionuclide into the raw material.

Moreover, it is also possible to evaluate the influence of the test substance on the skin based on the relationship between the concentration of the test substance containing the radionuclide and the thickness direction of the skin tissue.
Here, “influence on skin” includes effects due to penetration, storage, metabolism, decomposition, etc. of the test substance.
Moreover, it is accurate and reproducible that a test substance containing a radionuclide is contained in 1 mL or 1 g of a measurement sample, preferably 0.2 Bq to 0.002 Bq, more preferably 0.1 Bq to 0.001 Bq. From the viewpoint of sex, it is preferable.
Moreover, the mass ratio of the test substance that structurally contains the radionuclide and the substance that does not structurally contain the same radionuclide is preferably 100000 to 1: 1, more preferably 10,000: 1 to 100: 1.
In the present disclosure, “containing” of “a test substance including a radionuclide” means that it is included in the chemical structure of the test substance, and is also referred to as “a test substance structurally including a radionuclide”.
A liquid scintillation counter is mentioned as an apparatus for measuring the concentration of a test substance containing a radionuclide in a measurement sample.
It is preferable to evaluate the influence of the test substance on the skin from the concentration of the test substance and the behavior in the thickness direction of the skin tissue of the test substance structurally containing such a radionuclide. It is possible to understand the behavior.

The “skin” used in the present disclosure is not particularly limited, and examples thereof include those derived from mammals such as humans, monkeys, pigs, dogs, cats, mice, rats, rabbits, guinea pigs, cows and horses. Among these, humans, monkeys, and pigs are preferable in that the behavior of the drug on human skin is confirmed. Furthermore, pigs that are readily available and close to human skin are preferred.
Moreover, the site | part to be used is not specifically limited, For example, a face, a foreskin, an ear | edge, an abdomen, a back part, a flank, a bevel part, a shoulder, a buttocks, etc. are mentioned. The site to be used may be selected according to the purpose of evaluation, but it is desirable to use the skin of the site whose properties are close to the site for which the pharmacokinetics of the drug is desired. Moreover, the site | part which can be obtained may be sufficient and a human newborn foreskin and the ear pin of the pig which can be obtained in large quantities easily are suitable.

The skin may be artificial skin, and artificial skin that is thick and has a layered shape is desirable. For example, a three-dimensional skin model, etc. can be mentioned. As this production method, epidermal keratinocytes are seeded on a liquid-permeable membrane of a culture insert, and if necessary, produced by combining immersion culture, gas-liquid culture, etc. And so on. The epidermal cells to be seeded at this time are preferably those grown (initially) to about 5 × 10 ^{2 to} 5 × 10 ⁶ cells / cm ² . In addition, dermal fibroblasts can be added to create a three-dimensional skin model.

  The test substance-containing measurement sample can be selected from solid, semi-solid, and liquid forms depending on the expected use state of the test substance. For example, if it is assumed to be used with a liquid with high fluidity such as lotion, the measurement reagent is used when the measurement sample is assumed to be a liquid or used in a patch or pack. May be semi-solid (for example, gel).

In the method of the present disclosure, a test substance containing the radionuclide is measured by an accelerator mass spectrometry system (hereinafter also referred to as “AMS system”).
An accelerator mass spectrometer system is a mass spectrometer with an accelerator (for example, Hiroyuki Matsuzaki, “Principle of Accelerator Mass Spectrometry”, J. Vac. Soc. Jpn (vacuum), Vol. 50 ( 2007) No. 7. January 1, 2008).
In the present disclosure, the radionuclide is measured with an AMS system after the measurement sample is oxidized and made glassfight.

  In the method of the present disclosure, “(a) distribution of test substance in skin thickness direction” and / or “(b) change of test substance in skin over time” is measured by the AMS system.

By measuring “(a) distribution of test substance containing radionuclide in the thickness direction of skin”, the distribution of a single or a plurality of positions of the test substance in the thickness direction can be clarified. The “thickness direction” is also referred to as the layer direction of the stratum corneum, epidermis, or dermis.
More specifically, a test substance that structurally contains a radionuclide is brought into contact with the skin. The contact portion is not particularly limited as long as it is skin, and the stratum corneum may be peeled off and the test substance may be brought into contact with the epidermis or dermis. Preferably, a test substance containing a radionuclide is brought into contact with the outermost layer (preferably the stratum corneum) of the skin. Examples of the “contact” include application, spraying, coating, and sticking.

Then, the contacted skin is divided into multiple sections in the thickness direction, and the presence and amount of a test substance containing a radionuclide in the one section of skin can be clarified by an AMS system. Further, the presence and amount of a test substance containing a radionuclide can be clarified in the AMS system for other categories as well.
At this time, it is preferable to use a skin obtained by collecting one or a plurality of skins from a plurality of skins in the thickness direction. And the test substance containing the radionuclide in the collected skin in one section is measured by the AMS system.

Thereby, the distribution of the test substance in the thickness direction can be clarified from the existence and the amount of the test substance including the radionuclide in each section, and thereby the behavior of the test substance in the skin can be evaluated.
Similarly, according to the method of the present disclosure, it is possible to precisely measure the skin kinetics such as the absolute amount of the test substance in the thickness direction, the penetration, metabolism, and degradation of the test substance, and evaluate such skin kinetics. be able to.

By measuring “(b) Time-dependent change of test substance in skin”, a test substance that changes with time can be clarified.
More specifically, a test substance containing a radionuclide is brought into contact with the above-described skin. The contact portion is not particularly limited as long as it is skin, and the stratum corneum may be peeled off and the test substance may be brought into contact with the epidermis or dermis. Preferably, a test substance containing a radionuclide is brought into contact with the outermost layer (preferably the stratum corneum) of the skin. Examples of the “contact” include application, spraying, coating, and sticking.

In addition, for example, in a two-dimensional plot of “skin depth−test substance concentration”, when the test substance permeates every time until a deep section of the skin, linearity increases in proportion to the application time. (Line = steady state). In this way, by obtaining data of two-dimensional plots of various test substances, it is possible to predict the application time to reach a certain section of skin in a known test substance or to reach within the application time The required concentration of the test substance can be predicted.
For example, the concentration of a test substance in a certain category, for example, when the skin depth is 0.4, can be obtained with respect to time.
At this time, it is preferable to use skin collected at regular time intervals. The “fixed time interval” is not particularly limited, and can be determined as appropriate by the measurer, for example, every one minute interval.

As the skin to be collected, skin obtained by collecting one or a plurality of skins in one thickness direction may be used. Thereby, it becomes possible to understand the temporal change of the test substance in the depth direction.
Further, the collected skin may be a skin obtained by collecting one or more parts of the skin on the same plane. This makes it possible to understand changes over time when the test substance diffuses or penetrates on the same plane.
Therefore, the skin in the thickness direction or the skin on the same plane can be measured and the change with time of the skin can be observed. By measuring the skin in the thickness direction, it is possible to understand the permeability of the test substance.
In addition, by measuring a plurality of portions of the skin on the same plane at regular intervals, it is possible to understand a change with time in a specific skin portion of the test substance.

As a more preferred procedure of the method for evaluating the behavior of a test substance in the skin of the present disclosure, (a) contacting the skin with a test substance containing a radionuclide, (b) from multiple categories in the thickness direction of the contacted skin, Collecting one or more skins of a section, (c) measuring the test substance containing the collected radionuclide in the section of skin with an accelerator mass spectrometry system, (d) the test substance containing the radionuclide Obtaining a distribution in the thickness direction of the skin.
Thereby, the distribution of the test substance in the thickness direction of the skin can be easily and accurately understood.

As a more preferable procedure of the method for evaluating the behavior of a test substance in the skin of the present disclosure, (a) after contacting a test substance containing a radionuclide, collecting the skin at regular time intervals; (b) collected A method comprising measuring the skin with an accelerator mass spectrometry system and (c) evaluating the behavior of the test substance containing the radionuclide over time in the skin is suitable.
Thereby, it is possible to easily and accurately understand the change over time of the test substance in the skin (preferably skin in the thickness direction or skin on the same plane).

Generally, when evaluating the drug delivery ability of a test substance, apparatuses and methods such as confocal Raman spectroscopy, LC / MS / MS measurement method, fluorescence measurement method, scintillation counter, and the like are used.
However, in infrared absorption spectroscopy and confocal Raman spectroscopy (for example, Patent Documents 1 and 2), although the behavior of the test substance in the skin can be directly grasped, the measurement blurring is large and the sensitivity is low. Accurate measurement is not possible because signals other than are picked up together.
In addition, it is conceivable that the behavior of the test substance in the skin can be evaluated by the fluorescence measurement method, but it is possible to distinguish the amount of drug applied to each layer from the test substance present in the living body. However, since the fluorescent substance may be removed from the metabolite, stable quantification is difficult.
In addition, it is conceivable to evaluate the behavior of the test substance in the skin using a scintillation counter. However, in this case, since the sensitivity is low, it is necessary to increase the amount of the test substance and the skin area to which the test substance is applied. This is disadvantageous in terms of efficiency, manufacturing cost, detection sensitivity, and detection accuracy.
In addition, as shown in Examples below, although the amount of drugs and metabolic degradation products can be measured with high sensitivity for each skin layer by LC / MS / MS measurement method, compounds without standard substances cannot be analyzed. If the structure is changed, identification becomes impossible and traceability is lacking, and it is necessary to set an analysis method for each test substance such as pretreatment, separation, and detection, which is complicated.
In addition, the AMS system is known to be used in microdose clinical trials because it can be measured with ultra-high sensitivity. However, it is difficult to perform sample preparation and special correction because of the ultra-high sensitivity. Therefore, various trials and errors are necessary depending on the purpose of evaluation.

As described above, the time-dependent change and distribution of the test substance in the thickness direction can be determined from the existence and amount of the test substance including the radionuclide in each category, and the application time. The behavior of the substance in the skin can be evaluated.
Thus, the method of the present disclosure makes it possible to precisely measure the absolute amount of the test substance that changes over time, the skin dynamics such as the penetration, metabolism, and degradation of the test substance that changes over time. Such skin dynamics can be evaluated with high accuracy. In addition, it is possible to evaluate drug drug delivery in the skin, and to contribute to the development of drugs that regulate drug drug delivery.

Further, it is preferable to collect the section of skin by tape stripping. This tape stripping uses a sampling tape that can collect a section of skin. By combining this tape stripping and AMS system, it is possible to carry out highly accurate measurement with a simple sample processing procedure.
As for the thickness of one section of the skin, it is preferable to divide the skin stratum corneum, epidermis and dermis into 2 to 50 sections. More specifically, in the case of the stratum corneum, it is preferably 0.4 to 10 μm, in the case of the epidermis, preferably 4 to 100 μm, and in the case of the dermis, preferably 40 to 1000 μm.
Then, with the sampling tape, a section of skin is sequentially collected from the side in contact with the skin. The skin collected at this time is preferably the stratum corneum. Since the stratum corneum has a structure that prevents the drug substance from penetrating into the body, it is important to understand the test substance in the stratum corneum and the changes over time in the skin of the test substance. It is.

The “collecting tape” used in the “tape stripping” of the present disclosure is not particularly limited, and examples thereof include an adhesive tape obtained by applying an adhesive to a thin sheet tape such as a plastic film, paper, cloth, and aluminum foil. . An adhesive tape is suitable as the sampling tape.
The base material of the plastic film can be classified into a plant type and a petroleum type. Petroleum is preferable because it can reduce noise during AMS measurement.
Moreover, as a base material of a plastic film, for example, polyolefins such as triacetyl cellulose (TAC), polyethylene, polypropylene, poly (4-methylpentene-1), amides such as polyimide, polyimide amide, polyether imide, and polyamide; Ketones such as polyetheretherketone and polyetherketone; Organic sulfurs such as polyketone sulfide, polyethersulfone, polysulfone and polyphenylene sulfide; Phthalates such as polyethylene terephthalate, polybutylene terephthalate and polyethylene naphthalate; Polyphenylene oxide, polyacetal, Polycarbonate, polyarylate, acrylic resin, polyvinyl alcohol, cellulosic plastics, epoxy resin, phenol resin Etc.
Of these substrates, polypropylene is preferred.
An expanded polypropylene (OPP) obtained by further stretching this propylene in the longitudinal and transverse biaxial directions is preferable as the substrate, and this expanded polypropylene can be suitably used for the tape substrate.
Furthermore, the polypropylene tape substrate (polypropylene film) preferably has a tensile strength of 10 to 120 N / cm, more preferably 30 to 90 N / cm. This tensile strength prevents the tape from being distorted or cut during tape stripping.
The tensile strength was measured under the conditions of tape width: 25 mm, initial chuck distance: 100 mm, and tensile speed: 300 mm / min.

The thickness of the tape base material is preferably 0.03 to 0.4 mm, particularly preferably 0.04 to 0.1 mm, from the viewpoint of handling without causing cutting during tape stripping.
The tape thickness was measured with a dial gauge having a contact surface diameter of 5 mm.

Examples of the polymer used as the pressure-sensitive adhesive include rubber, acrylic, silicone, and urethane. These may be used alone or in combination. Of these, rubber-based and / or acrylic-based are preferable. This is because these polymers are often prepared without using a solvent, and are used in many sanitary products, and also do not roughen the skin when applied to the skin.
Examples of the rubber polymer include natural rubber, isoprene, chloroprene rubber, styrene-butadiene rubber, styrene-butadiene rubber latex, styrene-isoprene-styrene copolymer (SIS), and styrene-butadiene. -A styrene copolymer (SBS) type, a nitrile rubber type, a butadiene type, etc. are mentioned. These may be used alone or in combination.
As the acrylic polymer, an acrylic resin such as an acrylic ester resin or a methacrylic ester resin is preferably used.

  As the form of the pressure-sensitive adhesive, the polymer is dissolved in a solvent (for example, an organic solvent used in an extraction solvent described later) (solvent type), dispersed in water (solvent-free type, emulsion type, or (Also referred to as latex), and polymers that are cured by chemically reacting a polymer with a curing agent or water (reactive curing type). From the viewpoint of safety when applied to the skin, a solventless type is preferable among these, and a solventless type adhesive using a rubber polymer is particularly preferable.

The adhesive strength of the adhesive tape is preferably 1 to 10 N / cm, more preferably 4 to 7 N / cm, from the viewpoint that the skin can be collected uniformly and efficiently.
Adhesive strength measurement conditions Substrate: Stainless steel plate polished with water-resistant abrasive paper Tape width: 25 mm
Crimping: 2kg rubber roller 1 reciprocation Curing: Room temperature 20-40min Tensile strength: 30mm / min Tensile direction: 180 degree direction

  By making the type and pressure-sensitive adhesive of the pressure-sensitive adhesive more suitable, it becomes possible to peel the skin of one section for AMS measurement well from the skin tissue. If the peeling becomes poor, the amount of the skin in one category is likely to be inaccurate, but by adjusting the adhesive as in the present disclosure, the distribution of the test substance and the results of changes over time can be made accurate. Thereby, the distribution of the test substance on the skin and the change over time can be measured accurately and satisfactorily.

Furthermore, it is preferable to dissolve the test substance in a solvent when collecting the test substance containing the radionuclide on the structure from the skin attached to the adhesive tape. Thereby, noise can be reduced and a more accurate result can be obtained.
The container in which the adhesive tape to which the skin adheres during dissolution is put in need only have chemical resistance in the solvent, and examples thereof include plastic and glass.
Examples of physical means for dissolving include stirring, shaking, and ultrasonic waves.
The temperature conditions for dissolution are not particularly limited, and may be appropriately set according to the thermal stability and solubility of the test substance, but are preferably room temperature of about 4 to 40 ° C. from the viewpoint of work efficiency. . What is necessary is just to adjust the processing time at this time suitably.
A sample containing a test substance containing a radionuclide from which the extraction solvent has been removed may be measured by an AMS system, or a test substance containing a radionuclide structurally contained in the extraction solvent may be measured using an AMS system. You may measure.

The extraction solvent is not particularly limited and may be appropriately selected depending on the solubility of the test substance. However, since the test substance applied to the skin is generally easily dissolved in an organic solvent, it is preferably used for normal extraction. It is an organic solvent.
Examples of the extraction solvent include alcohols (for example, methanol, ethanol, isopropanol, etc.), saturated or cyclic hydrocarbons (hexane, cyclohexane, etc.), ester (for example, ethyl acetate, butyl acetate, etc.), aromatic hydrocarbons ( Examples thereof include toluene, xylene, etc.), ketones (eg, acetone, methyl isobutyl ketone, etc.), ethers (eg, diethyl ether, petroleum ether, etc.), dimethyl sulfoxide, dimethylformamide, chloroform, acetonitrile, tetrahydrofuran, and the like. These may be used alone or in combination.
Among these, preferably, from the viewpoint of solubility of the test substance from the tape or the tape adhesive, methanol, ethanol, isopropanol, acetonitrile, tetrahydrofuran, dimethyl sulfoxide, hexane, chloroform, ethyl acetate, butyl acetate, cyclohexane, xylene, Dimethylformamide, diethyl ether, methyl isobutyl ketone and petroleum ether.
Further preferred are water-soluble solvents, among which alcohols having 1 to 3 carbon atoms (for example, methanol, ethanol, isopropanol etc.), acetonitrile, tetrahydrofuran and dimethyl sulfoxide.
These may be used alone or in combination.

  The amount of the extraction solvent to be used is not particularly limited, but is preferably 0.1 to 5 mL with respect to 0.01 to 1 mg of skin. More preferably, noise during AMS measurement can be reduced, and the extraction solvent is removed. Is easily 0.5 to 2 mL.

Hereinafter, a more specific procedure of the method of the present disclosure will be described.
In addition, the “skin” used in the present disclosure cuts the skin from the site to be used, removes the hair and the fat layer from the cut skin, and cuts it to a size used for measurement. A tank for adding PBS or the like is formed inside by fixing the cut skin to the fixing member and fixing the fixing member to the support.
PBS (phosphate buffered saline) is added to the fixed skin to hydrate the skin. The treatment temperature at this time is preferably room temperature of about 4 to 40 ° C., and the treatment time is preferably about 5 to 30 minutes.

  Then, after removing the PBS, a measurement sample containing a test substance containing a radionuclide is brought into contact with the hydrated skin and treated with the measurement sample for a predetermined time. The treatment temperature at this time is preferably about 4 to 40 ° C., and the treatment time is preferably about 0.5 to 3 hours.

After the measurement sample is treated for a predetermined time after coming into contact with the skin, the remainder of the measurement sample and the moisture in the skin are appropriately removed, and the skin is washed with PBS. After washing, air-dry at room temperature of about 4-40 ° C. for about 1-10 minutes.
The initial value of transdermal moisture transpiration of the treated skin is measured with a moisture transpiration measuring device.
Remove the treated skin from the support.
Adhere the tape on the treated skin with constant pressure. After the tape is adhered, it is peeled off at a constant speed, and the skin is stripped. This stripping causes the skin layer to adhere to the tape. Also, the amount of skin obtained by stripping is measured by measuring the mass of the tape before and after stripping.
The transdermal moisture transpiration is measured each time stripping is performed one or more times. This predetermined number is preferably 2 to 4 times.
Further, the stripping is preferably performed at a fixed number of 10 to 20 times or until the water content reaches 50 g / m 2 h.

Furthermore, it is preferable to extract the test substance from the tape obtained by stripping. Thereby, it is possible to perform the measurement by the acceleration mass spectrometry system with high accuracy.
After putting the tape obtained by stripping into a container, a solvent is added to the container. Shake after addition. The shaking time is preferably about 0.5 to 2 hours, and the shaking temperature is preferably 10 to 30 ° C. When storing an extract containing a test substance, a low temperature (1 to 4 ° C.) is desirable.

The above-described method of the present disclosure can provide an effective medicinal component, preparation, and means for developing an administration method for improving skin conditions and treating skin diseases.
In addition, according to the method of the present disclosure, for example, the skin dynamics of a test substance when a substance to be applied to the skin such as an external preparation for skin, cosmetics, or quasi drugs is administered can be evaluated. Thereby, the presence or absence of the influence of the test substance on the skin can be determined from the viewpoint of safety as well as the effectiveness.
In addition, according to the method of the present disclosure, a test substance that has penetrated a specific thickness of skin is measured, and the value is compared among multiple persons or by comparing values at different time points among individuals. Skin functions such as functions and metabolic functions can be evaluated.
With the method of the present disclosure, various skin functions can be evaluated, and the evaluation criteria may be appropriately examined. For example, the following evaluation criteria (1) to (5) For example, permeability, storage, safety, barrier function, metabolic function, etc.).
(1) It can be evaluated that “the test substance has high permeability” when the test substance penetrates to a certain depth within a certain time or penetrates to a certain depth within a short time.
(2) If the test substance has penetrated to a certain depth and remains at that depth without being metabolized, lost, penetrated, or diffused, it can be evaluated as “highly test substance storage”. .
(3) A test substance whose safety is concerned when it reaches the dermis can be evaluated as “the test substance is highly safe” when it can be confirmed that the test substance has not reached the dermis.
(4) When evaluating that “the barrier function of the skin is high”, it is carried out as follows: First, the test substance is applied to human skin having a plurality of healthy barrier functions, and general skin behavior is observed. To grasp. Thereafter, the skin function data obtained by applying the test substance to the target human skin can be compared with the known skin behavior to evaluate the barrier function. For example, if the permeability is higher than that of a human having a healthy barrier function, it can be evaluated that the human barrier function is low.
(5) When it is evaluated that “the metabolic function of the skin is low”, it is carried out as follows: First, the test substance is applied to human skin having a plurality of healthy metabolic functions, and general skin behavior To figure out. Thereafter, the in-skin behavior data obtained by applying the test substance to the subject's human skin can be compared with known in-skin behavior to evaluate metabolic function. For example, if the substance is slower in metabolism / disappearance than a human having a healthy metabolic function, it can be evaluated that the human metabolic function is low.

In addition, this technique can also employ | adopt the following structures.
[1] A method for evaluating the behavior of a test substance in the skin, wherein the distribution of the test substance containing a radionuclide in the thickness direction of the skin is measured with an accelerator mass spectrometry system.
[2] bringing a test substance containing a radionuclide into contact with the skin;
From one or more samples of one section of skin, from multiple sections in the thickness direction of the contacted skin,
Measuring a test substance containing radionuclides in a sample of collected skin with an accelerator mass spectrometry system;
Obtaining a distribution in the skin thickness direction of a test substance containing a radionuclide,
The method for evaluating the behavior of a test substance in the skin according to [1], comprising:
[3] A method for evaluating the behavior of a test substance in the skin, wherein a time-dependent change in the skin of a test substance containing a radionuclide is measured with an accelerator mass spectrometry system.
[4] After contact with a test substance containing a radionuclide, collect skin at regular time intervals;
Measuring the collected skin with an accelerator mass spectrometry system;
Assessing the behavior of the test substance containing radionuclides over time in the skin;
The method for evaluating the behavior of a test substance in the skin according to [3], comprising:
[5] The method for evaluating the behavior of a test substance in the skin according to [4], wherein the skin collected at a certain time interval is skin in the thickness direction or skin on the same plane.

[6] The method for evaluating the behavior of a test substance in the skin according to [2], [4] or [5], wherein the collection of the skin of the one category is performed by tape stripping.
An adhesive tape is suitable for the tape used for tape stripping. More preferably, the pressure-sensitive adhesive tape is composed of a polypropylene tape base material and a rubber-based pressure-sensitive adhesive. More preferably, the adhesive strength of the adhesive tape is 1 to 10 N / cm.
After the tape stripping, the test substance in the skin is collected with the extraction solvent from the tape to which the skin obtained by tape stripping is attached, and the test substance structurally containing the radionuclide is measured with the AMS system. Is preferred.
As the extraction solvent, alcohols having 1 to 3 carbon atoms such as methanol are suitable.

[7] The method for evaluating the behavior of a test substance in the skin according to any one of [2], [4] to [6], wherein a section of skin is sequentially collected from the contacted side.
[8] The method for evaluating the behavior of a test substance in the skin according to any one of [2], [4] to [7], wherein a test substance containing a radionuclide is brought into contact with the outermost layer of the skin.
[9] The method for evaluating the behavior of a test substance in the skin according to any one of [2], [4] to [8], wherein the collected skin is a stratum corneum portion.
[10] The method for evaluating the behavior of a test substance in the skin according to any one of [2], [4] to [9], wherein a test substance containing a radionuclide is extracted from the skin of the category by extraction with an organic solvent. .
[11] Any one of [2], [4] to [10], wherein the influence of the test substance on the skin is evaluated based on the relationship between the concentration of the test substance containing the radionuclide and the thickness direction of the skin tissue. A method for evaluating the behavior of the test substance in the skin.

  EXAMPLES Next, although an Example is given and this technique (this invention) is demonstrated in more detail, this technique (this invention) is not restrict | limited to these Examples at all.

[Test example]
Intradermal behavior of radiolabeled lidocaine hydrochloride was evaluated using porcine auricular skin.

Preparation of donor solution:
Lidocaine hydrochloride (reagent special grade, manufactured by SIGMA) 10 μg / mL (in phosphate buffer (PBS; pH 7.4)) and [ ¹⁴ C] -lidocaine hydrochloride ethanol solution (American Radiolabeled Chemicals, 2.2 × 10 ¹²⁾ Bq / mol) was mixed to prepare 10 ng / mL, 4.4 × 10 ⁷ Bq / mol of [ ¹⁴ C] -lidocaine hydrochloride solution (in PBS) to obtain a donor solution.

Donor application:
After removing the hair and fat layer of pig auricular skin (purchased from the National Federation of Agricultural Cooperatives) and cutting it into an appropriate size, the donor-applied cell was firmly attached to the stratum corneum with double-sided tape. Phosphate buffer (PBS; pH 7.4) was added into the cell, and the skin was hydrated at room temperature (20-30 ° C.) for 15 minutes. After removing PBS, 10 mL of donor solution was applied to the cell and allowed to stand at room temperature for 1 hour.

Stripping:
The donor in the cell was partially collected into a glass vial using a Pasteur pipette, and the residue was removed. The moisture of the skin was wiped off with Kimwipe, and the skin was washed 3 times with PBS and then air-dried at room temperature for about 5 minutes. TEWL was measured using a TEWL meter (VAPOSCAN AS-VT100RS, manufactured by Asahi Techno Lab), and the initial value was measured. Tape for stripping using tweezers (manufactured by 3M, Scotch (registered trademark) 375SN, tape substrate: polypropylene film, adhesive: solventless rubber system, adhesive strength: 5.6 N / cm, tensile strength: 67 N / cm, tape thickness: 0.077 mm) was peeled off from the support, and the tape was adhered to the skin with a constant pressure, and then peeled off at a constant speed to strip the skin. The tape was weighed with a microbalance before and after stripping, and the amount of skin obtained by stripping was measured. The stripped tape was placed in a glass vial. TEWL was measured after every two strips. Stripping was performed a fixed number of times 10 to 20 times or until TEWL reached 50 g / m ² h.

Extract adjustment:
After 1 mL of methanol was added to the glass vial, the mixture was shaken for 1 hour at room temperature (20 to 30 ° C.) with a soaking machine. Thereafter, the tape in the vial was removed and stored at 5 ° C. until measurement. In addition, 1 mL of methanol is added to 0.01 to 1 mg of stripped skin.

Sample oxidation and graphitization:
First, sodium benzoate (2.5 mg equivalent carbon) was fractionated into a preparative quartz tube. Thereafter, 10 μL of the extract (methanol) was collected using a micropipette and added to a preparative quartz tube. After the sample was dried at room temperature, it was placed in an oxidation quartz tube together with about 1 g of linear copper oxide, and melted and sealed under vacuum. Thereafter, the oxidation quartz tube was heated at 850 ° C. in a muffle furnace to oxidize the sample.
The oxidized sample was taken out from the muffle furnace and attached to a vacuum line, and CO ₂ produced by the oxidation treatment was isolated, purified and quantified. About 2.1-fold moles of H ₂ gas was added to the purified CO ₂ gas and reduced by heating to 650 ° C. under about 2.5 mg of iron catalyst to produce graphite. About half of the produced graphite (about 1 mg) was filled in an aluminum cathode.

AMS measurement:
A cathode filled with graphite was loaded into an AMS measurement wheel and attached to the ion source (MS-SNICS) of a tandem electrostatic accelerator mass spectrometer (1.5SDH-1 type 0.6MV Pelletron AMS system, manufactured by NEC). One measurement was performed at 1000 cycles (about 100 sec), and the isotope ratio was calculated from three data. At the same time, a standard sample for AMS was loaded on an AMS measurement wheel and measured. Standard samples for AMS include oxalic acid (NIST Oxalic Acid II 4990C, NIST), carbon powder (Wako Pure Chemical Industries) and iron powder (Aldrich Chemical Company) mixed in the same weight, sucrose (IAEA C6, IAEA) was used.

analysis:
Calculate the isotope ratio (14C / 12C; pMC) using analysis software (abc, ver.6.0, manufactured by NEC), and calculate the 14C concentration (Sample 14C, dpm / mL) of the sample using the following formula. Calculated.
100 pMC (Percentage of Modern Carbon) = 13.56 dpm / gC
Graphite 14C (dpm) = A (pMC) × 0.1356 (dpm / gC / pMC) × C amount (gC)
Sample 14C (dpm / mL) = (Graphite14C (dpm)) / Aliquot (mL)

  The result of plotting the drug concentration (drug amount / horny layer weight) collected on the tape against x / L is shown in FIG.

LC-MS / MS measurement:
As the donor solution, 10 μg / mL (in PBS) of lidocaine hydrochloride (reagent special grade, manufactured by SIGMA) without radiolabeling was used. Lidocaine hydrochloride was quantified by LC-MS / MS under the following measurement conditions.

Separation column: Shodex 5 μm, 2.0 mm i. d. × 50mm (Showa Denko)
Guard column: YMC ODS-AM 23 × 4.0 mm i. d. S-5μm12nm
Eluent: 10 mM acetic acid: acetonitrile = 7: 3
Flow rate: 0.2 mL / min Column temperature: 40 ° C
Sample solution injection volume: 10 μL
Runtime: 7.5 minutes

Mass spectrometry conditions MS: API4000 (Applied Biosystems Japan)
Ionization method: ESI
Polarity: positive Mass measured: 235.21 and 86.10.
Vcap voltage: 5000 (V)
Nebulizer pressure: 40
Drying gas temperature: 500
Dry gas flow rate: 20

Calculation of pharmacokinetic parameters: The diffusion of the drug transferred from the donor to the skin in the stratum corneum is expressed by the following equation 1 using Fick's second law. Where C _X is the drug concentration at depth x, C _veh is the drug concentration in the donor, L is the stratum corneum thickness, D is the diffusion constant of the drug in the stratum corneum, K is the stratum corneum-donor partition coefficient, t is the application time of the donor. According to Formula 1, the drug concentration (drug amount / horny layer weight) collected on the tape was plotted against x / L, and the diffusion constant D and the distribution coefficient K of the drug were determined by the least square method.

  As shown in FIG. 1, the drug concentration vs. stratum corneum depth plot by AMS measurement had high linearity. This was the same result as a plot of drug concentration and stratum corneum depth by LC-MS / MS measurement. Moreover, as shown in FIG. 2, the stratum corneum-donor distribution coefficient K obtained by AMS measurement was close to K obtained by LC-MS / MS measurement. From these results, it was shown that the intradermal behavior of radiolabeled lidocaine hydrochloride can be evaluated by using the method of the present invention. Moreover, it turned out that the method of this invention is highly sensitive rather than the method by the existing LC-MS / MS measurement.

Claims (4)

A method for evaluating the behavior of a test substance in a skin, including the following (A) to (D), wherein a distribution of a test substance including a radionuclide in a thickness direction of the skin is measured with an accelerator mass spectrometry system.
(A) bringing a test substance containing a radionuclide into contact with the outermost layer of the skin;
(B) One or more samples of a plurality of sections in the thickness direction in the contacted skin are sequentially collected from the contacted side by stripping using a petroleum-based plastic film-based adhesive tape. ,
(C) measuring the test substance containing the radionuclide extracted from the adhesive tape and the collected skin of one section with an alcohol system having 1 to 3 carbon atoms with an accelerator mass spectrometry system; and
(D) Obtaining the distribution of the test substance containing the radionuclide in the thickness direction of the skin. A method for evaluating the behavior of a test substance in the skin, comprising the following (A) to (C) , wherein a time-dependent change in the skin of a test substance containing a radionuclide is measured with an accelerator mass spectrometry system.
(A) After contacting a test substance containing a radionuclide with the outermost layer of the skin,
(A-1) The skin in the thickness direction is divided into multiple sections, and one or a plurality of sections of multiple sections to one section are collected by tape stripping using a petroleum-based plastic film base adhesive tape at regular time intervals. Or
(A-2) One or more samples of a part of the skin in a plurality of locations on the same plane by tape stripping using an adhesive tape of a petroleum-based base material at regular time intervals,
(B) Accelerator mass spectrometry of a test substance containing a radionuclide extracted from the adhesive tape and the collected one section or from the adhesive tape and a part of the collected skin with an alcohol system having 1 to 3 carbon atoms. Measuring in the system, and
(C) To evaluate the behavior of the test substance containing the radionuclide over time in the skin. The method for evaluating the behavior of a test substance in the skin according to claim 1 or 2 , wherein the collected skin is a stratum corneum. After measuring the test substance containing the extracted radionuclide with an accelerator mass spectrometry system , the effect of the test substance on the skin is determined based on the relationship between the concentration of the test substance containing the radionuclide and the thickness direction of the skin tissue or on the same plane. The method for evaluating in-skin behavior of a test substance according to claim 1 to be evaluated.

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