JP6692673B2 - Method for evaluating changes in intercellular lipids due to drugs, method for screening drugs, method for evaluating intercellular lipids - Google Patents

Description

  The present invention relates to a method for simply evaluating the fine structure of intercellular lipids.

  The intercellular lipids on the surface of the living body establish a lipid barrier by connecting cells together. For example, intercorneal lipids in the stratum corneum of the skin surface have a role of preventing foreign substances from entering from the outside and maintaining water inside the skin due to their barrier ability.

Information on the fine structure of intercellular lipids has great significance in the fields of biological diagnosis and drug development, and methods for evaluating the fine structure of intercellular lipids have been actively studied.
For example, Patent Document 1 discloses a method for evaluating the structural destruction of intercorneocyte lipids using differential scanning calorimetry.

JP, 2010-237098, A

  However, the above-mentioned conventional method using differential scanning calorimetry has a problem that it is not possible to evaluate the fine structure such as the lamellar structure or the packed structure of intercellular lipids.

  Therefore, it is an object of the present invention to provide a method capable of easily evaluating the fine structure of intercellular lipids.

  There are various methods for evaluating the fine structure of intercellular lipids, and typical examples thereof include X-ray diffraction (XRD) measurement, and more specifically, small-angle / wide-angle X-ray scattering (SWAXS) measurement. Be done. It is said that the small-angle X-ray scattering measurement mainly provides information about the lamellar structure of intercellular lipids, and the wide-angle X-ray scattering measurement mainly provides information about the packed structure of intercellular lipids.

FIG. 1 (a) schematically shows the result of differential scanning calorimetry of a certain object, and FIG. 1 (b) shows that the peak of differential scanning calorimetry shown in (a) is divided by curve fitting by the nonlinear least squares method. 3 schematically shows the three small peaks (A) to (C). In the figure, "x" indicates the peak tops of the small peaks (A) to (C), and the temperatures at the positions of the respective peak tops are indicated by S1 to S3.
FIG. 2A shows the result of the small-angle X-ray scattering measurement of a certain object, and FIG. 2B shows the result of the wide-angle X-ray scattering measurement of the certain object.
In FIG. 2 (a), the "×", the peak position of the peak (X) ~ (Z) ^{(respectively, 0.27nm -1, 0.23nm -1, 0.22nm} -1) indicates, for each position The temperatures T1 to T3 in FIG.
In FIG. 2 (b), the "×" indicates at 25 ° C., a peak (V), the peak position of (W) (respectively, 2.4nm ^-1, 2.7nm ^-1). The peak (V) at 2.4 nm ^-1 is derived from hexagonal crystals and orthorhombic crystals, and the peak (W) at 2.7 nm ^-1 is derived from orthorhombic crystals. When the scattering intensity of the peak (V) is (Sv) and the scattering intensity of the peak (W) is (Sw), the ratio (%) of the orthorhombic crystal to the total of the orthorhombic crystal and the hexagonal crystal is It is calculated by [Sw / {(Sv-2 * Sw) / 3 + Sw}] * 100.

  So far, the area of the peak of the differential scanning calorimetry has a correlation with the integrated value of the intensity of the diffraction peak of the small-angle X-ray scattering measurement, specifically, the latter increases as the former increases or decreases. It is known that the absolute amount of the lamella structure (absolute amount of the orthorhombic and hexagonal crystals) can be evaluated based on the area of the peak of the differential scanning calorimetry. It is known (see FIG. 1 (a) and FIG. 2 (a)).

This time, the inventors tried a new method of dividing the peak of the differential scanning calorimetry of the object into a plurality of small peaks by curve fitting by the nonlinear least square method, and the divided small peaks (A) to At each temperature at the peak position of (C) (indicated by S1 to S3 in FIG. 1B) and at the peak positions of diffraction peaks (X) to (Z) by the temperature scanning small angle X-ray scattering measurement of the target. It has been found that there is a correlation with the temperature (indicated by T1 to T3 in FIG. 2A), specifically, the temperatures S1 to S3 are extremely close to T1 to T3, respectively. (See FIG. 1 (b) and FIG. 2 (a)).
With this discovery, it is possible to obtain information from the peak of differential scanning calorimetry to not only the outline of the fine structure such as the total amount of the lamella structure but also the details of the fine structure such as the ratio of orthorhombic and hexagonal crystals. Sex was shown.

  Then, according to the studies by the inventors, it is speculated that the diffraction peak (Y) by the temperature scanning small-angle X-ray scattering measurement of the object is derived from the lamella structure composed of fatty acid, cholesterol, and ceramides. It became so.

  Therefore, the inventors of the present invention relate to the fine structure of intercellular lipids (for example, the ratio of orthorhombic crystals and hexagonal crystals) which is information obtained from the peaks of differential scanning calorimetry, for example, from wide-angle X-ray scattering measurement. The inventors have come to the conclusion that information may be obtained and have completed the present invention (see FIGS. 1B and 2B).

The gist of the present invention is as follows .
In the present specification, “orthorhombic” and “orthorhombic” have the same meaning.
In one aspect of the method of the present invention, by performing differential scanning calorimetry on the intercellular lipid subject after addition of the drug and the intercellular lipid subject without addition of the drug, the intercellular lipid subject after the addition of the drug and the above Changes in the ultrastructure of the intercellular lipids before and after the drug addition is evaluated between the drug-free intracellular lipid subjects.
The method of the present invention divides the peaks of the differential scanning calorimetry of the intercellular lipid test subject after the drug addition and the intercellular lipid test subject without the drug addition into a plurality of small peaks by the nonlinear least squares method. , A peak division step is included. The method of the present invention comprises the plurality of small peaks of the differential scanning calorimetry of the intercellular lipid subject after the addition of the drug, and the plurality of small peaks of the differential scanning calorimetry of the drug-free intercellular lipid subject. And a comparison step before and after the addition of the drug.
In the method of the present invention, in the comparison step before and after the addition of the drug, the area of the peak of the differential scanning calorimetry of the intercellular lipid subject after the addition of the drug is the differential scanning calorific value of the intercellular lipid subject without the addition of the drug. Compared with the area of the peak of the measurement, by a tetragonal with respect to the sum of a portion with a orthorhombic crystal and a portion with a hexagonal crystal, which is large and among the plurality of small peaks of the differential scanning calorimetry of the intercellular lipid subject. The area of the first rectangular parallelepiped-based small peak in the differential scanning calorimetry of the intercellular lipid subject after the addition of the drug is defined as the first rectangular-based small peak having the largest proportion of the portion. , When compared to the area of the first orthorhombic main small peak in the differential scanning calorimetry of the intercellular lipid subject to which the drug was not added, the drug improved the barrier ability of the intercellular lipid subject when it was large. When It may be price.

A further aspect of the method of the present invention is a method for screening a drug, which uses the method according to one aspect of the method of the present invention.

In another aspect of the method of the present invention, the ratio of orthorhombic and hexagonal in the packed structure of intercellular lipids in the intercellular lipid sample is obtained by performing differential scanning calorimetry on the intercellular lipid sample and the intercellular lipid control. Evaluate.
The method of the present invention includes a peak division step of dividing the peaks of the differential scanning calorimetry of the intercellular lipid sample and the intercellular lipid control into a plurality of small peaks by the nonlinear least squares method. The method of the invention comprises comparing the plurality of small peaks of the differential scanning calorimetry of the intercellular lipid sample with the plurality of small peaks of the differential scanning calorimetry of the intercellular lipid control, a sample-control comparison step. including.
In the method of the present invention, in the sample-control comparison step, as the intercellular lipid control, the differential scanning calorimetry smaller than the area of the plurality of small peaks in the differential scanning calorimetry of the intercellular lipid sample is used. Select those having an area of a plurality of small peaks, from the area of the plurality of small peaks of the differential scanning calorimetry of the intercellular lipid sample, of the plurality of small peaks of the differential scanning calorimetry of the intercellular lipid control The area of the plurality of small peaks obtained by subtracting the area may be regarded as the area of the plurality of small peaks derived from hexagonal crystals in the intercellular lipid sample.
In another aspect of the method of the present invention, the intercellular lipid is preferably an intercellular lipid model containing a mixture of fatty acid, cholesterol and ceramides.

In the method of the present invention , an intercellular lipid model composed of a mixture of palmitic acid, cholesterol and ceramide AS (ceramide 5) is more preferable. In the method of the present invention, the mixing ratio in the mixture is preferably palmitic acid: cholesterol: ceramide AS (ceramide 5) = 45 to 75:00 to 25:10 to 50, and palmitic acid: cholesterol: ceramide AS ( Ceramide 5) = 55 to 65:10 to 15:20 to 40 is particularly preferable.

A further aspect of the method of the invention is the ratio of orthorhombic and hexagonal in the packing structure of intercellular lipids in said intercellular lipid sample by performing differential scanning calorimetry on the intercellular lipid sample and intercellular lipid control. Evaluate.
The method of the present invention includes a sample-control comparison step of comparing the peak of the differential scanning calorimetry of the intercellular lipid sample with the peak of the differential scanning calorimetry of the intercellular lipid control. In the method of the present invention, in the sample-control comparison step, as the intercellular lipid control, the area of the peak of the differential scanning calorimetry is smaller than the area of the peak of the differential scanning calorimetry of the intercellular lipid sample. The area of the peak obtained by subtracting the area of the peak of the differential scanning calorimetry of the intercellular lipid control from the area of the peak of the differential scanning calorimetry of the intercellular lipid sample, It may be regarded as the area of the peak derived from hexagonal crystals in the intercellular lipid sample.

  According to the method of the present invention, the details of the fine structure of intercellular lipids can be easily evaluated.

(A) is a figure which shows typically the result of the differential scanning calorimetry of a certain object, (b) divided the peak of the differential scanning calorimetry shown in (a) by curve fitting by the nonlinear least squares method. It is a figure which shows typically three small peaks (A)-(C). In the figure, "x" indicates the peak tops of the small peaks (A) to (C), and the temperatures at the positions of the respective peak tops are indicated by S1 to S3. (A) is a figure which shows the result of a small angle X-ray scattering measurement of a certain target, (b) is a figure which shows the result of a wide angle X-ray scattering measurement of a certain target. In (a), "×" is the peak position ^{(respectively, 0.27nm -1, 0.23nm -1, 0.22nm} -1) peaks (X) ~ (Z) indicates the temperature at each position T1 -T3 shows a phase transition temperature. (B) in, "×" indicates at 25 ° C., a peak (V), the peak position of (W) (respectively, 2.4nm ^-1, 2.7nm ^-1). It is a schematic diagram showing a method of evaluating the fine structure of intercellular lipid of an embodiment of the present invention. It is a schematic diagram showing a method of evaluating a fine structure of intercellular lipid of a first embodiment of the present invention. Differential scanning calorimetry of intercellular lipid sample (intercellular lipid model) and intercellular lipid control (intercellular lipid model with addition of each drug) in the method of Example which is an example of the method of the first embodiment FIG. 4 is a diagram showing the results of the above with respect to the areas of the DSC peak and three small peaks (first peak, intermediate peak, second peak) obtained by dividing the DSC peak. In a preferred form of the method of the first embodiment, the peak and / or a plurality of small peaks of an intercellular lipid sample (intercellular lipid model) and intercellular lipid control (intercellular lipid model to which each drug is added) And / or a plurality of small peaks in relation to the areas of the DSC peak and three small peaks (first peak, intermediate peak, second peak) obtained by dividing the DSC peak. .. Wide-angle X-ray scattering measurement for making the details of the fine structure known in the intercellular lipid control (the addition of each drug to the intercellular lipid model) in the method of the example, which is an example of the method of the first embodiment. It is a figure which shows the result of. Wide-angle X-ray scattering measurement was not performed on the intercellular lipid sample. In a more preferred form of the method of the first embodiment, a peak and / or a plurality of small peaks of an intercellular lipid sample (intercellular lipid model) and an intercellular lipid control (intercellular lipid model to which each drug is added ) Peak and / or a plurality of small peaks are compared with each other with respect to an area of a DSC peak and three small peaks (first peak, intermediate peak, second peak) obtained by dividing the DSC peak. is there. It is a figure which shows the outline of the result obtained by the more suitable form of the method of 1st embodiment. It is a figure which shows the result of the wide-angle X-ray-scattering measurement for confirming the detail of a microstructure about the intercellular lipid sample (intercellular lipid model) in the method of the Example which is an example of the method of 1st embodiment. It is a schematic diagram showing the method of evaluating the fine structure of the intercellular lipid of a second embodiment of the present invention. In the method of Example, which is an example of the method of the second embodiment, the intercellular lipid test subject after addition of a drug (each drug added to the intercellular lipid model), and the intercellular lipid test subject to which no drug was added ( It is a figure which shows the result of the differential scanning calorimetry of an intercellular lipid model) regarding the area of a DSC peak and three small peaks (1st peak, an intermediate peak, 2nd peak) obtained by dividing it. In a preferred form of the method of the second embodiment, a peak and / or a plurality of small peaks of an intercellular lipid test subject (each intercellular lipid model added with each drug) after addition of the drug, and cells to which no drug is added Inter-lipid subject (intercellular lipid model) peak and / or a plurality of small peaks, DSC peak, and three small peaks (first peak, intermediate peak, second peak) obtained by dividing the DSC peak. It is a figure which shows the result compared with respect to an area. Regarding an intercellular lipid subject (intercellular lipid model) after addition of a drug (N-acetyl-L-glutamine) suitable for improving the barrier ability in the method of Example which is an example of the method of the second embodiment FIG. 3 is a diagram showing the results of wide-angle X-ray scattering measurement for confirming the details of the fine structure. It is a figure which shows the result of the wide-angle X-ray-scattering measurement about the horny layer sheet before and after the addition of the chemical | medical agent (N-acetyl-L-glutamine) suitable for improving a barrier ability about the scattering intensity of an orthorhombic crystal and a hexagonal crystal. It is a figure which shows the result of the wide-angle X-ray-scattering measurement about the horny layer sheet before and after addition of the chemical | medical agent (N-acetyl-L-glutamine) suitable for improving a barrier ability about the ratio of an orthorhombic crystal and a hexagonal crystal. To confirm the details of the fine structure of the intercellular lipid subject (intercellular lipid model) after addition of an amino acid other than N-acetyl-L-glutamine in the method of Example, which is an example of the method of the second embodiment. It is a figure which shows the result of the wide-angle X-ray-scattering measurement.

  Hereinafter, with reference to the drawings, embodiments of the method for evaluating the fine structure of intercellular lipids of the present invention will be described in detail as an example.

  The method for evaluating the microstructure of intercellular lipids of the embodiment of the present invention (hereinafter, also referred to as “method of the present embodiment”) is based on differential scanning calorimetry (hereinafter, also referred to as “DSC measurement”). ..

  In the method of the present embodiment, it is essential to perform the small-angle / wide-angle X-ray scattering (SWAXS) measurement on the object of interest based on the above-described knowledge about the relationship between the DSC measurement result and the XRD measurement result. Without doing so, it is possible to evaluate the fine structure of intercellular lipids by performing differential scanning calorimetry on the object of interest, and thus to easily evaluate the fine structure of intercellular lipids for many objects of interest. To enable that.

Further, according to the method of the present embodiment, it becomes possible to evaluate the details of the fine structure of intercellular lipid.
Furthermore, according to the method of the present embodiment, it is possible to evaluate the change in the fine structure of intercellular lipids before and after the addition of the drug. This makes it possible to screen for cosmetics and drugs that affect the packing structure of intercellular lipids, and evaluate whether they are components that loosen the packing structure of intercellular lipids or components that make the packing structure dense. be able to. As a result, it becomes possible to control the barrier of the stratum corneum and to enhance the transdermal absorption of the active ingredient contained in the cosmetic. Further, by using a component that makes the filling structure dense in the step after the administration of the active ingredient, it is possible to keep the transepidermal water loss in the stratum corneum low and maintain the homeostasis of the skin.

  Intercellular lipids to which the method of the present embodiment can be applied include keratin of skin, cell membrane complex (CMC (Cell Membrane Complex)) portion inside hair, and more specifically, cuticle CMC (CU-CU). , Cuticle-cortex CMC (CU-CO), cortex-cortex CMC (CO-CO), model systems thereof (described later), and the like. Particularly, differential scanning calorimetry in a high lipid concentration state. The model system is preferred because it can be performed and peak detection is easy.

Then, in the method of the present embodiment, it is preferable to perform differential scanning calorimetry on a plurality of objects. By using a plurality of subjects, the results of differential scanning calorimetry can be compared among the subjects, and detailed knowledge about the fine structure of intercellular lipids can be obtained.
Here, the plurality of targets refers to two or more targets, and the target is preferably three or more.

  FIG. 3 shows a schematic diagram of a method for evaluating the fine structure of intercellular lipids according to the embodiment of the present invention.

  In the method of the present embodiment, first, differential scanning calorimetry is performed on a plurality of targets (target A and target B in FIG. 3) (DSC measurement step).

The method of the present embodiment then divides the peaks of the differential scanning calorimetry of the plurality of objects into a plurality of small peaks by the nonlinear least squares method (peak dividing step).
Here, the plurality of small peaks refer to two or more small peaks, and the number of small peaks is preferably three or more.
This step may be performed using a computing device such as a computer, for example.
In the nonlinear least-squares method, examples of functions used for curve fitting with respect to the peak shape include Gaussian functions, Lorentzian functions, Voigt's functions convolving these two, and the like. In particular, Gaussian functions and Lorentzian functions should be used. Is preferred.
Examples of the peak analysis software include Origin and the like.
Examples of algorithms installed in the analysis software include Levenberg-Marquardt (LMA).

The method of the present embodiment continues with the differential scanning calorimetry peak and / or the plurality of small peaks of one of the plurality of objects and the other differential scanning of the plurality of objects corresponding to these peaks. The calorimetric peak and / or a plurality of small peaks are compared (comparing step).
In this step, peaks and / or a plurality of small peaks may be compared in area, peak position, or the like.

  Here, the above-mentioned plurality of subjects are not particularly limited, but those which can be compared and examined are preferable, for example, intercellular lipid sample and intercellular lipid control, intercellular lipid subject after drug addition and drug-free Intercellular lipid subjects, stratum corneum collected from beautiful skin and stratum corneum collected from rough skin, healthy hair, damaged hair, and the like.

Of these plural subjects, the other may be one in which the drug is given.
The drug refers to a drug that can change the fine structure of intercellular lipids (lamella structure, filling structure), and is not particularly limited as long as it is a component or the like to be added to cosmetics and the like. , Active ingredients such as whitening ingredients, anti-skin roughening ingredients, and moisturizing ingredients.

(First embodiment)
FIG. 4 shows a schematic diagram of a method for evaluating the fine structure of intercellular lipids according to the first embodiment of the present invention.
A method for evaluating a fine structure of an intercellular lipid according to the first embodiment of the present invention (hereinafter, also referred to as “method of the first embodiment”) is a method in which an intercellular lipid sample and an intercellular lipid are used for the plurality of subjects described above. As a control, the fine structure of the intercellular lipid in the intercellular lipid sample is evaluated in detail.
In addition, the intercellular lipid sample refers to the intercellular lipid that is a target for evaluating the details of the fine structure, and the intercellular lipid control refers to the intercellular lipid that is a target for comparison with the intercellular lipid sample.

  Here, in the method of Example (which will be described later) which is an example of the method of the first embodiment, the details of the fine structure of the intercellular lipid are the ratio of the orthorhombic crystal and the hexagonal crystal in the packed structure of the intercellular lipid. ..

  In addition, in the method of this example, an intercellular lipid sample is used as an intercellular lipid model, and an intercellular lipid control is used as a drug (ethanol, glycerin, 1,3-butylene glycol (1,3-BG)). , A mixture of these) is added.

  In the method of the first embodiment, first, differential scanning calorimetry is performed on an intercellular lipid sample and an intercellular lipid control (DSC measurement step).

Then, the peaks of the differential scanning calorimetry of the intercellular lipid sample and the intercellular lipid control are divided into a plurality of small peaks by the non-linear least squares method (peak dividing step).
In the example shown here, the DSC peak of the intercellular lipid model and the one in which the drug is added to the intercellular lipid model is divided into three small peaks of the first peak, the intermediate peak, and the second peak (see FIG. 4). ..

  FIG. 5 shows the results of the intercellular lipid sample (intercellular lipid model) and the intercellular lipid control (intercellular lipid model to which each drug was added) in the method of Example which is an example of the method of the first embodiment. The results of the differential scanning calorimetry are shown with respect to the areas of the DSC peak and three small peaks (first peak, intermediate peak, second peak) obtained by dividing the DSC peak.

In the method of the first embodiment, subsequently, a differential scanning calorimetric peak and / or a plurality of small peaks of the intercellular lipid sample and a differential scanning calorimetric peak and / or a plurality of small peaks of the intercellular lipid control are provided. Are compared (sample-control comparison step).
In the example shown here, the DSC peak of the intercellular lipid model, the first peak, the intermediate peak, and the second peak obtained by dividing the DSC peak, the DSC peak of each agent added to the intercellular lipid model, and The first peak, the intermediate peak, and the second peak obtained by dividing it are compared (see FIGS. 6 and 8).

  In particular, in a preferable mode of the method of the first embodiment, the sample-control comparison step described above may be as follows.

  FIG. 6 shows a peak of an intercellular lipid sample (intercellular lipid model) and / or a plurality of small peaks, and an intercellular lipid control (each drug is added to the intercellular lipid model) in a preferred form of the method of the first embodiment. (Added)) and / or a plurality of small peaks in terms of the area of the DSC peak and three small peaks (first peak, intermediate peak, second peak) obtained by dividing the DSC peak. Show.

In this preferred form, first, as an intercellular lipid control, a differential scanning calorimetric peak and / or a plurality of small differential scanning calorimetric peaks and / or a plurality of small differential scanning calorimetric areas are compared with an area of a differential scanning calorimetric peak and / or a plurality of small peaks of an intercellular lipid sample. Select one with a small peak area.
In the example shown here, the area of all DSC peaks in the intercellular lipid model obtained by adding a drug (ethanol, glycerin, 1,3-butylene glycol (1,3-BG), or a mixture thereof) to the intercellular lipid model is , Is smaller than the area of the DSC peak of the intercellular lipid model (see FIG. 6). Based on this, as an intercellular lipid control, all of the intercellular lipid models to which the above-mentioned drug was added are selected.

In this preferred embodiment, the area of the differential scanning calorimetry peak of the intercellular lipid sample and / or the area of the plurality of small peaks is changed to the area of the differential scanning calorimetry peak of the intercellular lipid control and / or the area of the plurality of small peaks. Is regarded as the area of the peak and / or the plurality of small peaks derived from the hexagonal crystal in the intercellular lipid sample.
In the example shown here, from the areas of the first peak, the intermediate peak, and the second peak of the intercellular lipid model, the first peak, the intermediate peak, and the second peak of the intercellular lipid model in which each drug is added to the intercellular lipid model. The area of the small peak obtained by subtracting the area of each of the two peaks, that is, the area of the peak related to the portion indicated by the broken line in FIG. 6, is regarded as the area of the peak derived from the hexagonal crystal in the intercellular lipid model. ing.

  A preferred form of the method of the first embodiment is that when the lamellar structure is lost in the intercellular lipid, the hexagonal crystals are lost in preference to the orthorhombic crystals, that is, the absolute amount of the orthorhombic crystals is large. It is based on the findings of the inventors that the absolute amount of hexagonal crystals decreases while not changing and maintaining in some cases. In other words, the above findings, in other words, when one subject has less lamellar structure than another subject, in the subject, there are less hexagonal rather than orthorhombic, that is, the absolute amount of orthorhombic is above. It is also meant that the absolute amount of orthorhombic crystals is smaller than that of the above-mentioned other object although it is almost the same as that of the other object.

Here, in the method of the first embodiment, from the viewpoint of improving the accuracy of evaluation, for intercellular lipid control, details of the fine structure are known, in this case, the ratio of orthorhombic and hexagonal in the packing structure. It may be preferred to be known.
The ratio of the orthorhombic crystal to the hexagonal crystal in the packed structure can be known by a known method such as the above-described small angle / wide angle X-ray scattering (SWAXS) measurement.

  FIG. 7 shows a wide-angle view for making the details of the fine structure known for the intercellular lipid control (the addition of each drug to the intercellular lipid model) in the method of the example, which is an example of the method of the first embodiment. The result of X-ray scattering measurement is shown. Wide-angle X-ray scattering measurement was not performed on the intercellular lipid sample.

  Further, in a more preferable form of the method of the first embodiment, the following may be adopted in the preferable form of the method of the first embodiment described above.

  FIG. 8 shows a peak and / or a plurality of small peaks of an intercellular lipid sample (intercellular lipid model) and an intercellular lipid control (each drug in the intercellular lipid model) in a more preferable form of the method of the first embodiment. Of the DSC peak and three small peaks (first peak, intermediate peak, second peak) obtained by dividing the DSC peak, and Indicates.

In this more preferable form, first, as the intercellular lipid control, one having an orthorhombic to hexagonal ratio of 80:20 to 100: 0 is selected.
In the example shown here, the intercellular lipid model in which glycerin is added to the intercellular lipid model in which the ratio of orthorhombic to hexagonal is 90:10 is selected as the intercellular lipid control.

And in this more preferred form, the area of the differential scanning calorimetry peak and / or the plurality of small peaks of the intercellular lipid control is (substantially) the peak derived from the orthorhombic crystal in the intercellular lipid sample and / or Alternatively, the ratio of orthorhombic and hexagonal crystals in an intercellular lipid sample can be regarded as the area of multiple small peaks, and the ratio of the differential scanning calorimetry peak of the intercellular lipid control and / or the multiple small peaks can be obtained. Of the hexagonal crystals and / or the areas of a plurality of small peaks in the intercellular lipid sample.
In the example shown here, the area of the DSC peak in the intercellular lipid model to which glycerin was added can be regarded as the area of the peak derived from the orthorhombic crystal in the intercellular lipid model. The ratio of the tetragonal crystals to the hexagonal crystals is the area of the DSC peak of the intercellular lipid model to which glycerin is added, that is, the area of the peak related to the part shown in black in FIG. 8 and the DSC peak of the intercellular lipid model. Is the area of the small peak obtained by subtracting the area of the DSC peak of the intercellular lipid model to which glycerin is added, that is, the ratio to the area of the peak related to the part indicated by the broken line in FIG. I regard it.

  In the example shown here, not only the intercellular lipid model, but also the intercellular lipid model added with a drug other than glycerin, as the intercellular lipid sample, similarly, the ratio of orthorhombic and hexagonal (See FIG. 8).

  A more preferable form of the method of the first embodiment is characterized in that, as the intercellular lipid control, one in which the packing structure is substantially orthorhombic is selected.

FIG. 9 shows an outline of the results obtained by the more preferable form of the method of the first embodiment.
In the example shown here, the ratio between the orthorhombic crystal and the hexagonal crystal in the intercellular lipid model can be obtained as indicated by the boxed line in FIG. 9.

(Second embodiment)
FIG. 11 shows a schematic diagram of a method for evaluating the fine structure of intercellular lipids according to the second embodiment of the present invention.
The method for evaluating the fine structure of intercellular lipids according to the second embodiment of the present invention (hereinafter, also referred to as “method of second embodiment”) is a method of treating a plurality of subjects with an intercellular lipid subject after drug addition and Evaluating the changes in the ultrastructure of the intercellular lipids between the intercellular lipid test subject after drug addition and the intercellular lipid test subject after drug addition before and after drug addition It is a thing.
In addition, the intercellular lipid subject refers to the intercellular lipid to be evaluated for the change in the fine structure before and after the addition of the drug.

  Here, in the method of Example (which will be described later) which is an example of the method of the second embodiment, ethanol, glycerin, and 1,3-butylene glycol (1,3, which were also used in the above-described method of the first embodiment, were used as drugs. -BG), a mixture thereof, and N-acetyl-L-glutamine.

  In the method of the second embodiment, first, differential scanning calorimetry is performed on the intercellular lipid test subject after drug addition and the intercellular lipid test subject without drug addition (DSC measurement step).

In the method of the second embodiment, then, the peaks of the differential scanning calorimetry of the intercellular lipid test subject after the drug addition and the intercellular lipid test subject without the drug addition, by the nonlinear least squares method, into a plurality of small peaks, respectively. Divide (peak dividing step).
In the example shown here, the DSC peaks of the intercellular lipid test subject after drug addition and the intercellular lipid test subject without drug addition are divided into a first peak, an intermediate peak, and a second peak (see FIG. 11). ..

  FIG. 12 shows an intercellular lipid test subject after addition of a drug (an intercellular lipid model to which each drug was added) and a cell without a drug in the method of Example, which is an example of the method of the second embodiment. The results of the differential scanning calorimetry of the lipid subject (intercellular lipid model) are shown with respect to the areas of the DSC peak and three small peaks (first peak, intermediate peak, second peak) obtained by dividing the DSC peak.

In the method of the second embodiment, subsequently, a differential scanning calorimetry peak and / or a plurality of small peaks of the intercellular lipid subject after addition of the drug and a differential scanning calorimetry measurement of the intercellular lipid subject without addition of the drug are performed. And / or a plurality of small peaks (comparison step before and after drug addition).
In the example shown here, the DSC peak of the intercellular lipid test subject to which no drug was added, and the first, intermediate, and second peaks obtained by dividing the DSC peak and the DSC of the intercellular lipid test subject after drug addition The peak and each of the first peak, the intermediate peak, and the second peak obtained by dividing the peak are compared (see FIG. 13).

  In particular, in a preferred form of the method of the second embodiment, in order to evaluate whether or not the drug has improved the barrier ability of the intercellular lipid subject, in the above-mentioned pre- and post-drug comparison step, as described below, Good.

More specifically, in the comparison step before and after drug addition, it is evaluated that the drug improves the barrier ability of the intercellular lipid subject when the following conditions (A) and (B) are satisfied.
(A) The area of the peak of the differential scanning calorimetry of the intercellular lipid subject after addition of the drug is larger than the area of the peak of the differential scanning calorimetry of the intercellular lipid subject without addition of the drug.
(B) Among a plurality of small peaks in the differential scanning calorimetry of the intercellular lipid test subject, the one having the first largest proportion of the orthorhombic portion in the total of the orthorhombic portion and the hexagonal portion. The area of the first orthorhombic main small peak in the differential scanning calorimetry of the intercellular lipid subject after addition of the drug is the area of the first orthorhombic main small peak of the intercellular lipid subject to which no drug is added. The area is large as compared with the area of the first orthorhombic main small peak in the differential scanning calorimetry.

Here, the point (A) is based on the known fact that there is a correlation between the area of the DSC peak and the absolute amount of the lamella structure (see, for example, Netsu Sokutei 34 (4) 159-166). is there.
When the condition of (A) above is satisfied, it means that the absolute amount of lamellar structure in the intercellular lipid subject increased after the addition of the drug.

  The point (B) is the ratio of orthorhombic and hexagonal crystals in the entire peak of the differential scanning calorimetry, and the orthorhombic and hexagonal in each small peak obtained by dividing the peak of the differential scanning calorimetry. The ratio with the crystal is not necessarily the same, a small peak having a large ratio compared to the ratio of orthorhombic and hexagonal in the entire peak, and the ratio of orthorhombic and hexagonal in the entire peak. This is based on the new finding of the inventors that there is a small peak having a small ratio in comparison.

Then, the first orthorhombic main minor peak at the point (B) may be determined by using a known method for a drug-free intracellular lipid subject, for example, a drug-free intracellular lipid. It can be determined by using a more suitable form of the above-described method of the first embodiment with the subject as an intercellular lipid sample and the intercellular lipid subject to which glycerin is added as the intercellular lipid control.
That is, first, as shown in FIG. 13, the ratios of the orthorhombic portion and the hexagonal portion in a plurality of small peaks of the intercellular lipid test subject to which no drug was added were measured for the intercellular lipid test after drug addition. From the areas of the orthorhombic parts in the multiple small peaks of the body and the areas of the multiple small peaks of the intercellular lipid test subject to which no drug was added, the It is considered as the ratio with the area of the small peak obtained by subtracting the area of the portion due to the tetragonal crystal.
In the example shown here, the ratios of the orthorhombic portion and the hexagonal portion in the first peak, the intermediate peak, and the second peak of the intracellular lipid model to which no drug was added, respectively, were determined to be the intracellular lipids to which glycerin was added. Areas of orthorhombic portions in the first peak, intermediate peak, and second peak of the model (shown in black in FIG. 13), and the first peak, intermediate peak, and second peak of the drug-free intracellular lipid model Areas of the first peak, the intermediate peak, and the second peak obtained by subtracting the areas of the orthorhombic portions of the first peak, the intermediate peak, and the second peak of the glycerin-added intercellular lipid model from the area of the peak ( (Indicated by a broken line in FIG. 13).
Next, the first orthorhombic crystal having the highest ratio of the orthorhombic crystal portion in the total of the orthorhombic crystal portion and the hexagonal crystal portion among the plurality of small peaks in the intercellular lipid subject is selected as the first orthorhombic crystal. It is defined as the subject's small peak.
In the example shown here, the ratio of the orthorhombic portion in the total of the orthorhombic portion and the hexagonal portion among the first peak, the intermediate peak, and the second peak of the intercellular lipid model is the first. The intermediate peak is defined as the first orthorhombic main peak.

In a preferred form of the method of the second embodiment in the example shown here, as shown in FIG.
About (A), the area of the peak of the differential scanning calorimetry of the intercellular lipid model which added N-acetyl-L-glutamine compared with the area of the peak of the differential scanning calorimetry of the drug-free intercellular lipid subject. And is getting bigger.
Also, as shown in FIG.
Regarding (B), the orthorhombic crystal in the total of the orthorhombic crystal and the hexagonal crystal among the first peak, the intermediate peak, and the second peak of the differential scanning calorimetry of the intercellular lipid model to which N-acetyl-L-glutamine was added. The intermediate peak in which the proportion of the portion due to is the first largest is an orthorhombic-dominant small peak, and the area of the intermediate peak in the differential scanning calorimetry of the intercellular lipid subject after addition of N-acetyl-L-glutamine is It is larger than the area of the intermediate peak of the differential scanning calorimetry of the non-added intercellular lipid test subject.
As described above, the intercellular lipid model to which N-acetyl-L-glutamine is added satisfies the conditions (A) and (B), and therefore N-acetyl-L-glutamine increases the barrier ability of the intercellular lipid subject. It is evaluated as improved. And N-acetyl-L-glutamine can be evaluated as a suitable agent for improving the barrier ability of an intercellular lipid subject.

  In a preferred mode of the method of the second embodiment, in the case where the aforementioned (A) or (B) (one of (A) and (B)) is not satisfied in the above-mentioned comparison step before and after drug addition, Alternatively, it may be evaluated that the drug does not improve the barrier ability of the intercellular lipid subject.

In the example shown here, as shown in FIG.
The area of the differential scanning calorimetry peak of the glycerin-added intercellular lipid model is smaller than the area of the differential scanning calorimetry peak of the drug-free intercellular lipid subject, and the aforementioned ( A) is not satisfied. Therefore, it is evaluated that glycerin does not improve the barrier ability of intercellular lipid subjects. Then, glycerin can be evaluated as a drug that is not suitable for improving the barrier ability of an intercellular lipid subject.
Further, as in the case of glycerin, ethanol, 1,3-butylene glycol (1,3-BG), and a mixture of these and glycerin also do not satisfy the above (A), and these drugs are not Intermediate lipids can be evaluated as drugs that are not suitable for improving the barrier ability of a subject.

Further, in a preferred mode of the method of the second embodiment, the above condition (B) may be the following condition (B ′).
(B ′) Among a plurality of small peaks in the differential scanning calorimetry of the intercellular lipid subject, the ratio of the orthorhombic portion in the total of the orthorhombic portion and the hexagonal portion is the intercellular lipid subject. The difference between the orthorhombic and hexagonal in the total peak of the differential scanning calorimetry of the orthorhombic part in the total peak of the orthorhombic crystal is larger than the ratio of the orthorhombic part in the total peak. The area of the orthorhombic-dominant small peak in the differential scanning calorimetry of the lipid subject is larger than the area of the orthorhombic-dominant small peak in the differential scanning calorimetry of the drug-free intercellular lipid subject.
Here, the orthorhombic main small peak may be one or plural.
Further, the proportion of the orthorhombic portion in the total of the orthorhombic portion and the hexagonal portion in the orthorhombic main small peak is determined depending on the intercellular lipid subject to be used.
For example, if the percentage of the total peaks is 28% and the percentages of the three small peaks are 45%, 30%, 25%, then the small peaks have a percentage of 45% and the percentages of 30%. The small peak may be an orthorhombic-based small peak.
The ratio may be, for example, 0: 100 to 50:50, 50:50 to 100: 0, or 70:30 to 100: 0.

  The method of the second embodiment of the invention described above can be used to screen for agents that may or may not be suitable for improving the barrier capacity of an intercellular lipid subject. That is, the present invention can also provide a method for screening a drug, which uses the method of the second embodiment of the present invention.

  As the intercellular lipid model suitably used in the method for evaluating the fine structure of intercellular lipids of the embodiment of the present invention, from the viewpoint of forming orthorhombic and hexagonal crystals, a mixture of fatty acids, cholesterol and ceramides is used. Those containing are preferable, and those comprising the mixture are more preferable.

  The fatty acid used in the intercellular lipid model is preferably a saturated or unsaturated fatty acid having 14 to 22 carbon atoms, more preferably 16 to 18 carbon atoms. More specifically, as the fatty acid, palmitic acid, stearic acid, linoleic acid, and linolenic acid are preferable. You may use these salts as a fatty acid.

The ceramides used in the intercellular lipid model include compounds having a basic structure of a structure in which sphingoids and fatty acids are amide-bonded (hereinafter, also referred to as "basic ceramide"), derivatives of the basic ceramide (hereinafter, "ceramide"). Also referred to as “derivative”), an analog synthesized by imitating the above basic ceramide and its derivative (hereinafter, also referred to as “ceramide analog”), and a sphingoid.
The ceramides in the present invention may be derived from synthetic products, natural products (eg plant extracts) and the like.

--- Basic ceramide ---
The basic structure of the basic ceramide includes a structure represented by the following formula (S1).
In addition, among the basic ceramides represented by the formula (S1), ceramides 1 to 10 are ceramides (skin ceramides) contained in the stratum corneum of humans.
In fact, in ceramides derived from humans and other animals, there are various modifications regarding the alkyl chain lengths of the sphingoids and fatty acids represented by the above formula (S1). The basic ceramide includes a compound having the same skeleton as the compound represented by the formula (S1) except the alkyl chain length.
Further, as the above-mentioned basic ceramide, a natural type (D (-) form) optically active substance or a non-natural type (L (+) form) optically active substance may be used. A mixture with a non-natural type may be used. The relative configuration of a plurality of asymmetric carbons in the structure of the basic ceramide may be a natural configuration or a non-natural configuration other than that, or may be a mixture thereof. Good.

Examples of sphingoid moieties contained in ceramides include natural sphingosine and its analogs. Specific examples of the natural sphingosine include sphingosine, sfujidihydrosphingosine, phytosphingosine, 6-hydroxysphingosine (above, represented by the above formula (S1)), sphingadienin, dehydrosphingosine, dehydrophytosphingosine, and these. Examples thereof include derivatives such as N-alkylated products (for example, N-methylated products) and acetylated products, and sphingosine is preferred.
Examples of the fatty acid moiety contained in the ceramides include saturated or unsaturated fatty acids having 14 to 34 carbon atoms and having no hydroxyl group, α-hydroxy fatty acids, ω-hydroxy fatty acids, and derivatives thereof. .Alpha.-hydroxy fatty acids of .about.34 are preferred.
The sphingoid moiety and fatty acid moiety contained in the ceramides may be derived from synthetic products, natural products (for example, plant extracts) and the like.

--- Ceramide derivative ---
Examples of the ceramide derivative include a ceramide compound (hereinafter also referred to as “sugar ceramide”) that is modified in the molecule with a saccharide. Examples of saccharides used for sugar ceramide include monosaccharides such as glucose and galactose; disaccharides such as lactose and maltose; oligosaccharides and polysaccharides in which these monosaccharides and disaccharides are polymerized by glucosidic bonds. Is mentioned. Further, as another ceramide derivative, in the above sugar ceramide, a ceramide compound modified intramolecularly with a saccharide analog in which a hydroxyl group contained in a sugar unit of a saccharide is substituted with another functional group (hereinafter, referred to as “sugar ceramide Also referred to as "analogue"). Examples of the saccharide analog used for the sugar ceramide analog include glucosamine, glucuronic acid, N-acetylglucosamine and the like.
The number of sugar units in the sugar ceramide and the sugar ceramide analog is preferably 1 to 5 from the viewpoint of dispersion stability in the composition, and 1 or 2 (in the case of sugar ceramide, the sugar is glucose or lactose, respectively). ) Is more preferable, and 1 is particularly preferable.
The sugar ceramide and sugar ceramide analog may be obtained by chemical synthesis or may be obtained by purchasing a commercial product.
Examples of commercially available sugar ceramides include (brand name) “Plant Sphingo Solution FR1” manufactured by Okayasu Shoten Co., Ltd.

--- Ceramide analog ---
Examples of the ceramide analog include a ceramide compound represented by the following formula (S2).

--- Sphingoids ---
Further, the ceramides in the present invention also include sphingoids. Examples of the sphingoids include those used for the sphingoids part contained in the above-mentioned ceramides.
Specific examples of phytosphingosine preferably used in the present invention include (Phytosphingosine) (trade name) manufactured by Evonik Goldschmidt GmbH.

  What was illustrated as said ceramides may be used individually by 1 type, and may be used in combination of 2 or more type.

  As ceramides used in the intercellular lipid model, ceramide AS (ceramide 5), ceramide NS (ceramide 2), and ceramide EOS (ceramide 1), which have a relatively large abundance in natural intercellular lipids, are preferable. Among these, ceramide EOS (ceramide 1), which tends to form a long-period lamella structure having higher barrier ability, is preferable, and ceramide AS (ceramide 5), ceramide NS has a particularly large abundance ratio in natural intercellular lipids. (Ceramide 2) is preferred.

When a mixture of fatty acids, cholesterol and ceramides is used as an intercellular lipid model, the mixing ratio of each component is preferably 45 to 75: 0 to 25:10 to 50 in this order, and 55 to 65: It is more preferably 10 to 15:20 to 40.
In particular, when a mixture of palmitic acid, cholesterol and ceramide AS (ceramide 5) is used as an intercellular lipid model, the mixing ratio of each component is particularly preferably 59.6: 13.9: 26.5. ..

  Hereinafter, the present invention will be described in more detail with reference to Examples, but the present invention is not limited to the following Examples.

  In this example, the mixture was composed of palmitic acid: cholesterol: ceramide AS (ceramide 5) = 59.6: 13.9: 26.5, and the ratio of orthorhombic and hexagonal was 31:69. A confirmed intercellular lipid model was used.

In addition, in the present example, the following agents were used in order to control the intercellular lipid for the intercellular lipid sample and to prepare the intercellular lipid test subject after the drug addition to the intercellular lipid test subject to which no drug was added.
・ Ethanol (Nippon Alcohol)
・ Glycerin (made by Sakamoto Yakuhin Kogyo Co., Ltd.)
・ 1,3-Butylene glycol (manufactured by Daicel)
-N-acetyl-L-glutamine (manufactured by Nissay Chemical Co., Ltd.)

And the object in this example was prepared as follows.
An intercellular lipid model solution 200 mL (lipid concentration 10 mmol / L) was prepared (hereinafter, the target in this case is also referred to as “Blank”).
Further, a drug solution was added so that the intracellular lipid model solution had a volume of 200 mL, and the temperature was raised to a phase transition temperature or higher. Then, ultrasonic treatment was performed, and then the cell model was stored in a thermostat bath at 35 ° C. for 24 hours. Then, by removing the water content of this solution, a thin film of an intercellular lipid model to which a drug was added was obtained.
As the drug solution during the above operation, 25.12 mL of 10% aqueous solution of ethanol (hereinafter, the target in this case is also referred to as “ethanol”), 16 mL of 10% aqueous solution of glycerin (hereinafter, the target in this case is also referred to as “glycerin”). .), 1,3-butylene glycol 10% aqueous solution (hereinafter, the target in this case is also referred to as “1,3-BG”), 20 mL. Further, a mixed solution of 12.56 mL of 5% aqueous solution of ethanol, 25.12 mL of 10% aqueous solution of glycerin, and 20 mL of 10% aqueous solution of 1,3-butylene glycol (hereinafter, the target in this case is also referred to as “mixing”), N-acetyl. 20 mL of a 10% L-glutamine aqueous solution (hereinafter, the object in this case is also referred to as "N-acetyl-L-glutamine") was used.

  The analysis method used in the examples will be described below.

(1) Differential Scanning Calorimeter Measurement Using a differential scanning calorimeter (DSC6200 manufactured by Seiko Instruments Inc.), the temperature range was 5 to 95 ° C., and the temperature rising rate was 3 ° C./min.
As software for analyzing peaks in differential scanning calorimetry, Origin, manufactured by LightStone, was used.

(2) Small-angle / wide-angle X-ray scattering (SWAXS) measurement Using SPring-8 / BL40B2, temperature scanning: 25 to 85 ° C., heating rate: 0.83 ° C./min, 2.5 ° C. Data was acquired for each.

The method for evaluating the microstructure of intercellular lipids of the present invention was carried out according to the following procedure.
-Differential scanning calorimetry was performed according to (1) for the five objects of "Blank", "ethanol", "glycerin", "1,3-BG", and "mixing" prepared above (see FIG. 5). ..

Firstly, using "Blank" as an intercellular lipid sample, an attempt was made to evaluate the details of the fine structure of the intercellular lipid in the intercellular lipid sample (ratio of orthorhombic and hexagonal in the packed structure).

-Next, when each of the 5 DSC peaks of interest was divided into small peaks according to (1), it was divided into three small peaks of the first peak, the intermediate peak, and the second peak (see Fig. 5).
-Here, the wide-angle X-ray-scattering measurement was performed according to (2) with respect to four objects except "Blank" among the five objects (see FIG. 7). As a result, as shown in FIG. 7, the ratios of orthorhombic and hexagonal in “ethanol”, “glycerin”, “1,3-BG”, and “mixed” were 65:35, 90:10, and It was found to be 70:30 and 90:10.
-Subsequently, "glycerin" having a smaller DSC peak area than that of "Blank" and an orthorhombic to hexagonal ratio of 80:20 to 100: 0 was selected. It was selected as an intercellular lipid control.
Then, the area of the peak obtained by subtracting the area of the DSC peak of "glycerin" from the area of the DSC peak of "Blank" (indicated by the broken line in FIG. 8) is the peak derived from the hexagonal crystal in "Blank". The ratio of the orthorhombic crystal and the hexagonal crystal in “Blank” is the area of the DSC peak of “glycerin” (shown in black in FIG. 8) and the ratio of the DSC peak of “Blank”. It was regarded as a ratio with the area of the peak obtained by subtracting the area of the DSC peak of "glycerin" from the area.
As a result, as shown in FIG. 9, it was evaluated that the ratio of orthorhombic crystals and hexagonal crystals in “Blank” was 35:65 (shown by a box in FIG. 9). The ratios of the orthorhombic crystal and the hexagonal crystal in “ethanol”, “1,3-BG” and “mixed” were also evaluated to be 65:35, 70:30 and 100: 0.

-Here, also for "Blank", for confirmation, wide-angle X-ray scattering measurement was performed according to (2) (see FIG. 10).
FIG. 10 shows the results of wide-angle X-ray scattering measurement for confirming the fine structure details of the intercellular lipid sample (intercellular lipid model) in the method of Example which is an example of the method of the first embodiment. ..
As a result, as shown in FIG. 10, it was found that the ratio of orthorhombic crystals and hexagonal crystals in “Blank” was 31:69.

  From the above, the evaluation of the fine structure details (ratio of orthorhombic crystals and hexagonal crystals in the packed structure) of the intercellular lipids in the intercellular lipid sample obtained by the method of the present example was performed by the analysis of the fine structure details. It was demonstrated to have a high correlation with the evaluation by wide-angle X-ray scattering measurement, which is a method established in.

-Secondly, "Blank" was used as an intercellular lipid test subject to which no drug was added, and five subjects except "Blank" were used as intercellular lipid test subjects after drug addition, before and after drug addition of the microstructure of intercellular lipids. We attempted to evaluate the changes in.

-Here, in addition to "Blank", "ethanol", "glycerin", "1,3-BG", and "mixed" prepared above, also for "N-acetyl-L-glutamine", (1) Differential scanning calorimetry was performed according to (see FIG. 12).
Further, when the DSC peak of "N-acetyl-L-glutamine" was divided into small peaks according to (1), respectively, it was divided into three small peaks of a first peak, an intermediate peak, and a second peak (Fig. 12).

-Here, the ratio of the orthorhombic portion and the hexagonal portion in the first peak, the intermediate peak, and the second peak of "Blank" is 35:65, respectively, as described above, as shown in FIG. It is evaluated as 55:45 and 20:80. From this result, in this system, the intermediate peak was defined as the first orthorhombic main small peak.

At this time, the area of the DSC peak of "N-acetyl-L-glutamine" is larger than the area of the DSC peak of "Blank", and the intermediate peak of "N-acetyl-L-glutamine" is It was found that the area is large compared to the area of the intermediate peak of "Blank".
From this, it was evaluated that the barrier ability of the intercellular lipid model was improved by the addition of N-acetyl-L-glutamine (see FIG. 13).

-Here, the "N-acetyl-L-glutamine" was also subjected to wide-angle X-ray scattering measurement according to (2) for confirmation (see Fig. 14).
FIG. 14 shows an intercellular lipid subject (intercellular) after addition of a drug (N-acetyl-L-glutamine) suitable for improving the barrier ability in the method of Example which is an example of the method of the second embodiment. The results of wide-angle X-ray scattering measurement for confirming the details of the fine structure are shown for the lipid model).
As a result, as shown in FIG. 14, the ratio of the orthorhombic crystal and the hexagonal crystal in “N-acetyl-L-glutamine” was 50:50, which was compared with that in “Blank” (31:69). It turned out to be getting bigger.

  From the above, an evaluation of the change in the microstructure of intercellular lipids before and after the addition of a drug (improvement of the barrier ability of intercellular lipids by the drug) obtained by the method of this example was established in the analysis of the fine structure. It has been proved to have a high correlation with the evaluation by the wide-angle X-ray scattering measurement, which is a conducted method.

  The present inventors focused on the areas of the DSC peaks of “Blank” and “N-acetyl-L-glutamine” and the ratio of orthorhombic and hexagonal crystals shown in FIG. A new finding has been obtained that when (orthorhombic and hexagonal) increases, there is a tendency to maintain at least the ratio of orthorhombic to hexagonal (sometimes increase the orthorhombic ratio).

Next, in order to deepen the above-mentioned findings and to confirm the effect of N-acetyl-L-glutamine for improving the barrier ability of intercellular lipids, the present inventors have developed a stratum corneum sheet instead of the intercellular lipid model. Wide angle X-ray scattering measurement was performed on the horny layer sheet before and after the addition of the drug.
As the stratum corneum sheet, a stratum corneum sheet (manufactured by BIOPREDIC International (France)) obtained by trypsinizing the skin separated from the skin of the human chest was used.
As the drug, the above-mentioned N-acetyl-L-glutamine and water as its control were used.
Here, as shown in FIG. 15, the stratum corneum sheet before addition of N-acetyl-L-glutamine and the stratum corneum sheet after addition of N-acetyl-L-glutamine (1.5 hours after addition, 24 hours after addition) Wide-angle X-ray scattering measurement was carried out). The same applies to water (purified water).

The specific experimental terms are as follows.
A stratum corneum sheet (manufactured by BIOPREDIC International (France)) (hereinafter, also simply referred to as “corneal stratum sheet”) prepared by trypsinizing the skin separated from the skin of the human chest was prepared.
Five sheets each having a size of 3 × 3 mm ² were cut out from the stratum corneum sheet, and these were laminated to obtain a sample for wide-angle X-ray scattering measurement.
A 2% aqueous solution of N-acetyl-L-glutamine (NAG) was used as a drug solution, and purified water was used as a control solution.

The sample of the prepared stratum corneum sheet was sandwiched between washers for SWAXS measurement.
Here, small-angle / wide-angle X-ray scattering (SWAXS) measurement was performed under the following conditions (hereinafter the same); measuring device: SPring-8 beamline BL40B2, exposure time: 180 seconds / time, measuring temperature 32 ° C., single Double irradiation, X-ray wavelength: 0.083 nm (15 keV), camera length: 539.281 (mm), detector: imaging plate. After obtaining the scattering image, the circular average was calculated to obtain one-dimensional scattering profile data. Analysis was performed by fitting the obtained diffraction peak to a Gaussian function.
20 μL of a 2% NAG aqueous solution was added from the center of the washer of the sample, and the sample was stored at room temperature (20 ° C.). During storage, water was volatilized from the drug solution while allowing the sample to absorb the drug in the drug solution.
1.5 hours after the addition, the sample of the stratum corneum sheet was in a wet state. At this time, the wide-angle X-ray scattering measurement was performed again.
Twenty-four hours after the addition, the stratum corneum sheet sample was dry. Here, further wide-angle X-ray scattering measurement was performed.

  FIG. 15 shows the results of wide-angle X-ray scattering measurement of the horny layer sheet before and after the addition of the agent (N-acetyl-L-glutamine) suitable for improving the barrier ability, regarding the orthorhombic and hexagonal scattering intensity. ..

  FIG. 16 shows the results of wide-angle X-ray scattering measurement of the horny layer sheet before and after the addition of a drug (N-acetyl-L-glutamine) suitable for improving the barrier ability, with respect to the ratio of orthorhombic and hexagonal crystals. ..

  As shown in FIG. 15, it was confirmed that the addition of N-acetyl-L-glutamine increased the lamellar structure (orthorhombic and hexagonal) in the horny layer sheet.

Further, as shown in FIG. 16, the ratio of the orthorhombic crystal and the hexagonal crystal in the stratum corneum sheet before the addition of N-acetyl-L-glutamine was 60:40, and the addition of N-acetyl-L-glutamine was 1.5. The ratio of orthorhombic crystals and hexagonal crystals in the stratum corneum sheet after 56 hours was 56:44, and the ratio of orthorhombic crystals and hexagonal crystals in the stratum corneum sheet 24 hours after the addition of N-acetyl-L-glutamine was , 57:43.
In the case of control water (purified water), the ratio of orthorhombic crystal and hexagonal crystal was 55:45 before addition, 53:47 1.5 hours after addition, and 24 hours after addition. At 51:49.
From this result, it was confirmed that the ratio of orthorhombic crystals and hexagonal crystals was maintained in the stratum corneum sheet by the addition of N-acetyl-L-glutamine.

  In addition, using amino acids other than N-acetyl-L-glutamine as a drug, it was also attempted to evaluate changes in the fine structure of intercellular lipids before and after drug addition.

The intercellular lipid model solution (lipid concentration 10 mmol / L) and N-acetyl-L-glutamine 10% aqueous solution used for preparing the intercellular lipid test subject after drug addition were as described above.
As amino acids other than N-acetyl-L-glutamine, phenylalanine (manufactured by Junsei Chemical Co., Ltd.), tyrosine (manufactured by Junsei Chemical Co., Ltd.), tryptophan (manufactured by Junsei Chemical Co., Ltd.), N-acetyl-L-glutamic acid (Japan Protein Co., Ltd.) Company), glycine (Junsei Kagaku Co., Ltd.), glutamine (Junsei Kagaku Co., Ltd.), and aspartic acid (Junsei Kagaku Co., Ltd.) were used. Then, the concentration of the amino acid is set as high as possible in consideration of the solubility of each amino acid as a drug solution, and a 1% aqueous solution of phenylalanine, 0.01% aqueous solution of tyrosine, 1% aqueous solution of tryptophan, and 2% aqueous solution of N-acetyl-L-glutamic acid. , 20% each of glycine 15% aqueous solution, glutamine 4% aqueous solution, and aspartic acid 0.1% aqueous solution (hereinafter, the target in each case is "phenylalanine", "tyrosine", "tryptophan", "N-". Also referred to as "acetyl-L-glutamic acid", "glycine", "glutamine", and "aspartic acid").

Here, the above-prepared "Blank", "N-acetyl-L-glutamine", "phenylalanine", "tyrosine", "tryptophan", "N-acetyl-L-glutamic acid", "glycine", "glutamine". Also for "aspartic acid", differential scanning calorimetry was performed according to (1) (see FIG. 17).
Moreover, when the DSC peak of the target to which each amino acid solution was added was divided into small peaks according to (1), it was divided into three small peaks of the first peak, the intermediate peak, and the second peak (see FIG. 17). ).

  As described above, the ratios of the orthorhombic portion and the hexagonal portion in the first, intermediate, and second peaks of “Blank” are 35:65 and 35:65, respectively, as described above, as shown in FIG. It is evaluated as 55:45 and 20:80. From this result, in this system, the intermediate peak was defined as the first orthorhombic main small peak.

At this time, the area of the DSC peak of "N-acetyl-L-glutamine" is larger than the area of the DSC peak of "Blank", in contrast to "phenylalanine", "tyrosine", and "tryptophan". , "N-acetyl-L-glutamic acid", "glycine", "glutamine", "aspartic acid" DSC peak area was smaller than that of "Blank" DSC peak area (Fig. 17). reference).
Thus, in contrast to the addition of N-acetyl-L-glutamine, addition of phenylalanine, tyrosine, tryptophan, N-acetyl-L-glutamic acid, glycine, glutamine, and aspartic acid resulted in the intracellular lipid model. It was evaluated that the effect of improving the barrier ability was not found. (See Figure 17).

According to the method of the present invention, it is possible to easily evaluate the fine structure of intercellular lipids, specifically, to evaluate the fine structure of intercellular lipids, and to evaluate the change of intercellular lipids before and after the addition of a fine structure drug. Can be evaluated.
The method of the present invention can screen for agents that affect the packing structure of intercellular lipids. By using the drug, it is possible to control the barrier of the stratum corneum, enhance the transdermal absorption of the drug compounded in external preparations such as cosmetics and skin external preparation, and keep the transdermal water loss in the stratum corneum low. , Has industrial applicability in that it allows maintaining homeostasis of the skin.

Claims (9)

By performing the differential scanning calorimetry for intercellular lipid subject and intercellular lipids subject drug was not added after the addition of drug agents, intercellular lipids subject of the agent is not added and intercellular lipids subject after the drug addition A method for evaluating the change in the microstructure of the intercellular lipid before and after the addition of a drug between the body and the body,
The peak of the differential scanning calorimetry of the intercellular lipid test subject after the drug addition and the intercellular lipid test subject without the drug addition is divided into a plurality of small peaks by the nonlinear least squares method, and a peak division step,
Comparing the plurality of small peaks of the differential scanning calorimetry of the intercellular lipid subject after the addition of the drug, and the plurality of small peaks of the differential scanning calorimetry of the drug-free intercellular lipid subject Including a comparison step before and after addition,
In the comparison step before and after drug addition,
The area of the peak of the differential scanning calorimetry of the intercellular lipid test subject after the drug addition, as compared with the area of the peak of the differential scanning calorimetry of the drug-free intercellular lipid test subject, large,
and
Of the plurality of small peaks of the differential scanning calorimetry of the intercellular lipid test subject, the one having the first largest proportion of the portion of the orthorhombic crystal to the total of the portion of the orthorhombic crystal and the portion of the hexagonal crystal is the first one. The area of the first rectangular parallelepiped-based small peak in the differential scanning calorimetry of the intercellular lipid subject after addition of the drug is the differential scan of the drug-free intercellular lipid subject when the crystal-based small peak is used. Larger than the area of the first orthorhombic main small peak in calorimetry
In this case, it is evaluated that the drug improves the barrier ability of the intercellular lipid subject,
Method. A method for screening a drug, which comprises using the method according to claim 1 . By performing differential scanning calorimetry on intercellular lipid sample and intercellular lipid control is a method of evaluating the ratio of orthorhombic and hexagonal in the packed structure of intercellular lipid in the intercellular lipid sample ,
Peaks of the differential scanning calorimetry of the intercellular lipid sample and the intercellular lipid control are each divided into a plurality of small peaks by a nonlinear least squares method, a peak division step,
Comparing the plurality of small peaks of the differential scanning calorimetry of the intercellular lipid sample with the plurality of small peaks of the differential scanning calorimetry of the intercellular lipid control, a sample-control comparison step,
Including,
In the sample-control comparison step,
As the intercellular lipid control, select one having an area of the plurality of small peaks of the differential scanning calorimetry smaller than the area of the plurality of small peaks of the differential scanning calorimetry of the intercellular lipid sample,
From the area of the plurality of small peaks of the differential scanning calorimetry of the intercellular lipid sample, the area of the plurality of small peaks of the differential scanning calorimetry of the intercellular lipid control, of the plurality of small peaks obtained by subtracting Area is regarded as the area of the plurality of small peaks derived from hexagonal crystals in the intercellular lipid sample,
Method. As the intercellular lipid control, one having a ratio of orthorhombic crystals and hexagonal crystals of 80:20 to 100: 0 is selected,
Ratio of Akira Nogata and hexagonal in the intercellular lipid sample, and the area of the front Symbol plurality of small peaks of differential scanning calorimetry of the intercellular lipid control, before from hexagonal in the intercellular lipid sample SL Considered as the ratio with the area of multiple small peaks,
The method of claim 3 . The method according to claim 3 or 4 , wherein the ratio of the orthorhombic crystal and the hexagonal crystal in the intercellular lipid control is evaluated in advance by wide-angle X-ray scattering measurement. The method according to any one of claims 3 to 5 , wherein the intercellular lipid control is the intercellular lipid sample given a drug. The method according to any one of claims 1 to 6 , wherein the intercellular lipid is an intercellular lipid model containing a mixture of fatty acid, cholesterol and ceramides. The method according to claim 7 , wherein the mixing ratio in the mixture is palmitic acid: cholesterol: ceramide AS (ceramide 5) = 45 to 75: 0 to 25:10 to 50. By performing differential scanning calorimetry on intercellular lipid sample and intercellular lipid control is a method of evaluating the ratio of orthorhombic and hexagonal in the packed structure of intercellular lipid in the intercellular lipid sample,
Comparing the peak of the differential scanning calorimetry of the intercellular lipid sample with the peak of the differential scanning calorimetry of the intercellular lipid control, a sample-control comparison step
Including,
In the sample-control comparison step,
As the intercellular lipid control, select one having an area of the peak of the differential scanning calorimetry smaller than the area of the peak of the differential scanning calorimetry of the intercellular lipid sample,
From the area of the peak of the differential scanning calorimetry of the intercellular lipid sample, the area of the peak obtained by subtracting the area of the peak of the differential scanning calorimetry of the intercellular lipid control, in the intercellular lipid sample Considered as the area of the peak derived from hexagonal crystal,
Method.