JP2009120513A - Method for measuring in vivo percutaneous absorption and method for evaluating sensitive skin - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for evaluating sensitive skin by carrying out in vivo and non-invasive measurement of percutaneous absorbency (skin barrier function) of a substance from the external world in the sensitive skin, and to further provide a method for screening a drug for the sensitive skin. <P>SOLUTION: There is provided a method for judging the in vivo percutaneous absorbency or the skin barrier function which uses an image according to fluorescent video microscope observation of the skin coated with a fluorescent substance. <P>COPYRIGHT: (C)2009,JPO&INPIT

Description

  The present invention relates to an in vivo transdermal absorbability or skin barrier function determination method, sensitive skin evaluation method, and sensitive skin drug evaluation method. The in vivo transdermal absorbability or skin barrier function judgment method and sensitive skin evaluation method are suitable for evaluating sensitive skin that is susceptible to irritation because external stimulants easily penetrate the skin. Useful from a scientific point of view. In addition, by evaluating drugs for sensitive skin using this evaluation method, it is possible to screen for drugs that enhance the skin barrier function of sensitive skin, which is useful for research and development of effective cosmetic ingredients.

  Conventionally, the necessity of a method for evaluating the skin barrier function in sensitive skin has been considered, but the mechanism and definition regarding sensitive skin have not been clarified. In general, transepidermal water loss (TEWL) is measured as a skin barrier function, and the evaluation of the barrier function has been substituted by the amount of water that transpires from the inside of the skin to the outside.

  On the other hand, research on the transdermal absorption of substances from the outside to the inside of the skin is also underway, and in fact, studies have been made mainly using animal skin. Evaluation is carried out by creating or quantifying the amount of target substance by grinding the collected skin. However, these methods require skin collection.

  In recent years, an increasing number of people complain of sensitive skin, and the development of a skin external preparation suitable for sensitive skin has been demanded. Therefore, there is a need for a method for measuring transdermal absorbability (skin barrier function) in sensitive skin. However, it is difficult for a person with actual sensitive skin to perform measurement using a method that requires collecting skin. Therefore, a method for evaluating the percutaneous absorbability (skin barrier function) of a person with sensitive skin in a non-invasive manner without collecting the skin is an aesthetic point of view and research and development of effective cosmetic ingredients. is important. As a non-invasive percutaneous absorption measurement method, there is a method using a photoacoustic measurement device disclosed in JP-A-1-191040, or a percutaneous absorption measurement method of a drug disclosed in JP-A-5-107232. However, since the measuring device is large, it is expensive, not versatile, and cannot be carried, so it is difficult to use in actual tests.

JP-A-1-191040 JP-A-5-107232

  An object of the present invention is to provide a method for determining transdermal absorbability or skin barrier function, a method for evaluating sensitive skin, and a method for evaluating a drug for sensitive skin by a noninvasive method.

  In view of the above problems, the present inventors have investigated a substance to be percutaneously absorbed and evaluated a device. The inventors have found that measurement can be performed in an invasive manner, and have completed the present invention.

Accordingly, this application includes the following inventions:
(1) A method for determining in vivo transdermal absorbability or skin barrier function from an image obtained by fluorescent videomicroscope observation of a skin coated with a fluorescent substance.
(2) The method according to (1), wherein the fluorescent substance is fluorescein or a salt thereof.
(3) A method for evaluating sensitive skin based on an image obtained by fluorescent video microscope observation of a skin to which a fluorescent material is applied.
(4) The method according to (3), wherein the fluorescent substance is fluorescein or a salt thereof.
(5) A sensitive skin drug evaluation method using the method of (3) or (4) in order to measure the effect of the sensitive skin drug.

  ADVANTAGE OF THE INVENTION According to this invention, the determination method of percutaneous absorbability or a skin barrier function by a non-invasive means, the sensitive skin evaluation method, and the sensitive skin drug evaluation method are provided.

Hereinafter, the configuration of the present invention will be described in detail.
The fluorescent substance used in the present invention is not particularly limited as long as it emits fluorescence, has low toxicity, and has been conventionally used in clinical practice, and is preferably fluorescein or a salt thereof. Although it does not specifically limit as a salt, For example, a sodium salt, magnesium salt, calcium salt, ammonium salt, an isothiocyanate salt, etc. are mentioned.
By using such a fluorescent substance, transdermal absorption can be measured non-invasively without causing harm to the human body or human skin.

  In the present invention, a particularly preferred fluorescent moiety is sodium fluorescein, which is a substance that emits fluorescence having a wavelength of 515 nm (green) when irradiated with excitation light having a wavelength of 490 nm (blue). It is a water-soluble substance having a molecular weight of 376.27 (fluorescein simple substance is 332.31), and is used as a typical example for measuring transdermal absorption due to its nature. Fluorescein is used in clinical practice, mainly in ophthalmology outpatients for fluorescent fundus angiography, and for diagnostic tests such as diabetic retinopathy, central serous chorioretinopathy, central retinal vein occlusion. However, there is no known method for non-invasive use in vivo for measuring the skin stratum corneum barrier function.

  In addition, measurement using other fluorescent materials such as fluorescein isothiocyanate (FITC) and dansyl chloride is also possible. Furthermore, fluorescently labeled substances such as FITC-labeled IgG can be used.

  A video microscope is an apparatus that magnifies and observes an object by bringing a small probe close to an object to be observed (in the present invention, skin). Therefore, it is possible to perform measurement at an arbitrary place by moving the probe. Unlike a microscope, it is not necessary to fix an observation object on the stage, and it is possible to observe a large object that is not placed on the stage, which is suitable for skin measurement. The entire device is also small and can be carried by a person.

  The video microscope is a system that projects an image observed by a CCD camera on a monitor. A normal video microscope irradiates an observation target with visible light and obtains an enlarged image from all reflected light. As the fluorescent video microscope used in the present invention, an ordinary video microscope modified so as to emit excitation light having a specific wavelength and detect reflected light of a specific wavelength can be used. The video microscope suitably used in the present invention irradiates the skin with a blue LED in a state where it is shielded from other light, and captures a fluorescent image by photographing with a CCD camera through a green filter. Obtainable.

  The fluorescent substance in the present invention is 0.0001 to 5.0% by mass, preferably 0.01 to 0.5% by mass, in the aqueous solution when applied. In addition to water, ethanol or water-containing ethanol can also be used as the solvent, and it can also be formulated into gels, creams, ointments and the like. In addition to direct application on the skin, the coating method can be applied by occlusive application using a patch test adhesive bandage or the like. And the form which can take the transdermal absorbability (skin barrier function) determination method and sensitive skin evaluation method of this invention is not limited to these dosage forms and forms.

  As a result of analyzing the image obtained by fluorescent video microscope observation of the skin to which the fluorescent material is applied, the fluorescent material is absorbed into the skin and the transdermal absorbability of the skin is high enough, or the skin It can be determined that the barrier function is low. In addition, since skin having such high skin absorbability or low skin barrier function is generally considered as “sensitive skin”, the image obtained here is used for determination of sensitive skin.

  When the present invention is applied, the fluorescent material absorbed to the inside of the skin can be observed more clearly by removing the outermost stratum corneum by tape stripping after applying the fluorescent material. Further, by analyzing an image photographed with a fluorescent video microscope, it is possible to pinpoint a site having high transdermal absorbability, in other words, a poor barrier function. It is also possible to analyze by digitizing the luminance of the image. And the form which the transdermal absorbability (skin barrier function) determination method and sensitive skin evaluation method of this invention obtain is not limited to these methods and forms.

  Next, although the anti-aging agent of this invention is demonstrated in detail based on an Example, this invention is not limited only to this Example. Prior to the examples, test methods and results relating to the measurement method / evaluation method of the present invention will be described.

1. Comparison with percutaneous absorption amount Using a commercially available Yucatan minipig skin, a comparison experiment with percutaneous absorption observation using a frozen section that has been conventionally used was conducted. In order to create skin with different transdermal absorbability (skin barrier function), a part of Yucatan minipig skin was degreased with acetone. A 0.1% aqueous solution of sodium fluorescein was applied openly for 5 minutes to both the site treated with acetone and the site not treated. After application, the skin sample was washed with water, and observed with a fluorescent video microscope as well as with frozen sections.

The result is shown in FIG. From the frozen section photograph, it can be seen that there is more percutaneous absorption of fluorescein at the site where the percutaneous absorption was artificially increased with acetone (the barrier function was deteriorated) (FIG. 1 (a)). When this is compared with the result observed with a fluorescent video microscope, it can be seen that the same tendency is observed (FIG. 1B).
Further, when the luminance is calculated by analyzing this image, it is 22.2 at the barrier destruction site and 8.5 (each n = 6 average) at the control site (FIG. 1 (c)), and the barrier destruction side is more It was found to be significantly higher.

2. Comparison with transcutaneous water transpiration A measurement was performed using human forearm skin and skin that had been barrier-disrupted with acetone and normal skin. A 0.1% aqueous sodium fluorescein solution was occluded for 5 minutes using a patch test adhesive bandage. After the application, the skin sample was washed with water, and the outermost stratum corneum was peeled off by tape stripping, followed by observation with a fluorescent video microscope. The brightness of the photographed image was calculated by image analysis software (Photoshop). At the same time, transepidermal water loss (TEWL) was measured at the same site using a Tewameter (Vapometer), and the two were compared. The result is shown in FIG. 2, and it can be seen that TEWL and luminance are correlated.

3. Example of measurement on face Using human forearm skin, a 0.1% aqueous solution of fluorescein sodium was occluded for 5 minutes using a patch test adhesive bandage. After application, the skin sample was washed with water. Thereafter, the outermost stratum corneum was removed by tape stripping, and observation was performed with a fluorescent video microscope. The result is shown in FIG. It was observed that even when the outermost stratum corneum was peeled off by tape stripping after the application of fluorescein, fluorescence remained in the pores and was further percutaneously penetrated into the skin. Thus, it was possible by this invention to identify the site | part with high transdermal absorbability (site | part with a bad barrier function) within the same image.

4). Oleic acid rough skin test using the forearm The forearm of 7 healthy men was used. 30% oleic acid (solvent: ethanol) was applied to one person's forearm using a patch test adhesive bandage (Torii Pharmaceutical), and the other forearm was untreated. Oleic acid treatment was carried out once a day for 3 hours once for 3 consecutive days. One day, 100 μl of 0.1% fluorescein sodium was applied on the fifth day using a patch test bandage (Torii Pharmaceutical) for 5 minutes. After peeling, the surface horny layer was peeled off twice with cello tape (registered trademark). Then, it observed with the fluorescence video microscope. For the observed image, the average luminance of the entire image was obtained by image processing software (Photoshop). The results are shown in FIG. 4, and it can be seen that percutaneous absorbability is significantly increased (skin barrier function is significantly deteriorated) by oleic acid treatment.

  Separately, the TEWL of the oleic acid-treated and untreated forearm was measured with a Vapometer, and the correlation with the luminance (VMS luminance) obtained with the fluorescent video microscope was examined. The result is shown in FIG. 5, and it can be seen that there is a correlation between the VMS luminance and the TEWL value (correlation coefficient R = 0.75).

5). DP formulation application test One month with d-program products (lotion (lotion 2), emulsion (emulsion 2), cream (cream AD)) on half face, 0.1% fluorescein sodium on both cheeks after continuous use 100 μl occlusion was applied for 1 minute using a patch test adhesive bandage (Torii Pharmaceutical Co., Ltd.) After peeling, washed with water, the surface stratum corneum was peeled twice with a tape, and then observed with a fluorescent video microscope. It can be seen that skin care suppresses transdermal absorbability and enhances the barrier function.

  Since the present invention is effective for the determination of transdermal absorbability, skin barrier function and sensitive skin, it is useful in the beauty industry and the cosmetics industry.

The percutaneous absorption of fluorescein in Yucatan minipig skin is shown by comparing the results of observation with a frozen section and fluorescence videomicroscope. The barrier function in human forearm skin is shown by comparing TEWL with values measured from percutaneous absorption of fluorescein. The image which measured the percutaneous absorption of the fluorescein in the human cheek skin with the fluorescence microscope is shown. A comparison of the fluorescence videomicroscope brightness of human forearm treated with oleic acid and untreated is shown. The correlation between the fluorescence videomicroscope brightness of the human forearm and TEWL is shown. It shows a decrease in in vitro transdermal absorbability of skin fluorescent substance with enhanced skin barrier function by continuous use of DP preparation.

Claims (5)

  A method for determining in vivo transdermal absorbability or skin barrier function from an image obtained by fluorescent videomicroscope observation of skin coated with a fluorescent substance.   The method according to claim 1, wherein the fluorescent substance is fluorescein or a salt thereof.   A method for evaluating sensitive skin using images obtained by fluorescent videomicroscope observation of skin coated with a fluorescent substance.   The method according to claim 3, wherein the fluorescent substance is fluorescein or a salt thereof.   A method for evaluating a sensitive skin drug using the method according to claim 3 or 4 in order to measure the effect of the sensitive skin drug.