JP2896321B2 - Method for activating β-glucocerebrosidase - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a method for activating β-glucocerebrosidase in epidermis. More specifically, the present invention relates to a method for activating β-glucocerebrosidase in epidermis by β-galactosylceramide applied externally to the skin.

[0002]

BACKGROUND OF THE INVENTION Rough skin generally refers to dry skin in which exfoliation of keratinocytes is observed. Such rough skin elutes intercellular lipids such as cholesterol, ceramide, and fatty acids, and forms a horny layer permeation barrier due to degeneration of keratinocytes and disruption of the proliferation and keratinization balance of epidermal cells due to ultraviolet rays and detergents. It is caused by failure or the like. Focusing on the fact that intercellular lipids are an essential component of the stratum corneum permeation barrier for the purpose of preventing or healing this rough skin, supplying a lipid component of the stratum corneum or a synthetic lipid component similar to it, Or EG
Researches such as administration of epidermal growth and keratinizing substances such as epidermal growth factor (F) have been conducted.

The stratum corneum intercellular lipid is formed by lamellar granules biosynthesized in the cells of the spinous layer and the stratum granulosum, which are released between the cells just below the stratum corneum and extended to form a lamellar structure. , Spread between cells. The lamellar granules are composed of glucosylceramide, cholesterol, ceramide, phospholipid, etc., but glucosylceramide is scarcely contained in the stratum corneum intercellular lipid. That is, glucosylceramide in the lamellar granules undergoes hydrolysis by β-glucocerebrosidase and is converted into ceramide, and as a result of this ceramide taking a lamellar structure, the formation of a stratum corneum permeation barrier as a stratum corneum intercellular lipid. It is thought to improve and act as a barrier for rough skin protection. For example, pathologically rough skin is observed in patients with Gaucher disease type 2 in which β-glucocerebrosidase is genetically completely deficient, and histological studies on the epidermis show abnormal lamellar structures of intercellular lipids in the stratum corneum. Has been recognized. Also, a correlation between abnormal lamellar structure of stratum corneum intercellular lipids and rough skin has been observed in transgenic mice artificially deficient in β-glucocerebrosidase. Furthermore, experimentally, when β-glucocerebrosidase is inhibited, abnormalities in the lamellar structure of lipids between rough skin and stratum corneum are observed. From these facts, it is said that glucosylceramide needs to be hydrolyzed to ceramide by β-glucocerebrosidase to form a normal stratum corneum permeation barrier. Therefore, it is thought that the activation of β-glucocerebrosidase improves the formation of the stratum corneum permeation barrier, and as a result, can improve rough skin.

[0004] Against this background, SAP-2 discovered from guinea pig spleen and A1a and saposin C discovered from human Gaucher disease spleen are conventionally known as activators of β-glucocerebrosidase. .

However, these activators are proteins, and using them externally to activate epidermal β-glucocerebrosidase poses a serious problem in terms of transdermal absorbability and safety. In addition, it is extremely difficult to isolate these proteins for industrial use.

[0006] From the above, a simple method for activating β-glucocerebrosidase in the epidermis has been demanded. Then, the present inventors, in view of the above circumstances, as a result of intensive research on a method for solving the conventional problems, surprisingly found that β-glucocerebrosidase in the epidermis can be extremely easily activated by the method described below, The present invention has been completed.

[0007]

, There is provided a solution for the] That is, the present onset Ming, β- moth
A method for activating β-glucocerebrosidase in epidermis, characterized by using lactosylceramide .

Hereinafter, the configuration of the present invention will be described in detail. The method of activating β-glucocerebrosidase in the epidermis of the present invention is achieved by applying an external preparation for skin such as a lotion containing β-galactosylceramide to the skin.

The amount of these β-galactosylceramides in the external preparation for skin is 0.005 to 5% based on the total amount.
5.0% by weight is preferred. If the amount is less than each of the lower limits, the intended effect of the present invention is not sufficient, while if the amount exceeds the upper limit, the effect corresponding to the increase is not improved.

In general, the β-glucocerebrosidase activity of the epidermis is measured using a homogenate of the epidermis as an enzyme source and, as a substrate, radiolabeled β-glucosylceramide or a synthetic substrate, 4-methylumbelliferyl β-glucopyranoside. Is done.

Hereinafter, the present invention will be proved based on examples and comparative examples.

[0012]

【Example】

Example 1, Comparative Example 1 A lotion of a propylene glycol / ethanol mixture (7: 3) containing 1.0% of β-galactosylceramide (derived from bovine brain, Sigma, USA) was prepared and used as a sample for one day. Was applied to the back skin of a group of 5 hairless mice (10 weeks old) at a single frequency for 10 days continuously. These experiments, which were performed at different concentrations of β-galactosylceramide, are referred to as Example 1. In addition, a lotion containing only a propylene glycol / ethanol mixture containing no β-galactosylceramide was applied in the same manner. This experiment is referred to as Comparative Example 1.

The skin was collected 24 hours after the final application of the sample in Example 1 and Comparative Example 1, and the subcutaneous adipose tissue was removed as much as possible, and then left in a 2N aqueous sodium bromide solution at 37 ° C. for 2 hours. did. Next, the epidermis was peeled off with tweezers, and a homogenate was prepared in a 10-fold volume of ice-cold phosphate buffered saline using a Polytron homogenizer (Kinematica, Switzerland).

The β-glucocerebrosidase activity is determined by the method of Miel and Van der Fluck (British Journal of Dermatology, Vol. 95, pp. 271-27).
4, 1976). That is, 500 μl of 100 mM citric acid-200 mM phosphate buffer (pH 5.6) and 500 μl of 10 mM taurocholate-100 mM citrate-200 mM phosphate buffer (pH 5.6) were added to 50 μl of the homogenate, and the mixture was added at 37 ° C. for 10 minutes. Heated. Then, 50 μl of 0.5 mM 4-methylumbelliferyl-β-D-glucoside (Sigma, USA) was added, and the mixture was heated at 37 ° C. for 60 minutes. Thereafter, a 200 mM sodium carbonate-sodium bicarbonate buffer (pH 10.
5) was added, and the excitation wavelength was 360 nM and the absorption wavelength was 450 nM.
Was used to measure the fluorescence intensity. The enzyme activity was calculated based on a calibration curve prepared from the fluorescence intensity of a standard product, 4-methylumbelliferone (Sigma, USA).

Comparative Examples 2, 3 In order to prove the specificity of β-glucocerebrosidase activity, conjugater-β-epoxide known as a specific inhibitor of β-glucocerebrosidase (Sigma, USA ) Was added to a final concentration of 50 μM, and the enzyme activity was measured in the same manner as described above. That is, the first embodiment
Comparative Example 2 was obtained by adding conjugater-β-epoxide to the enzyme sample of Comparative Example 1, and Comparative Example 3 was obtained by adding conjugater-β-epoxide to the enzyme sample of Comparative Example 1.

FIG. 1 shows the measurement results of β-glucocerebrosidase activity in the epidermis in Example 1 and Comparative Examples 1, 2, and 3.
Shown in

In these results, a comparison between the lotion containing β-galactosylceramide (Example 1) and the lotion not containing it (Comparative Example 1) showed that the risk factor between Example 1 and Comparative Example 1 was 1%. A significant difference was confirmed below, and β
-Galactosylceramide was found to increase β-glucocerebrosidase activity in the epidermis. Also, conjugater, a specific inhibitor of β-glucocerebrosidase,
In Comparative Examples 2 and 3 to which -β-epoxide was added, almost no enzyme activity was detected. This means that the β-glucocerebrosidase activity was specifically measured under the measurement conditions.

Examples 2 and 3, Comparative Examples 4 and 5 A propylene glycol / ethanol mixture (7%) containing 0.1% or 1.0% β-galactosylceramide (derived from bovine brain, Sigma, USA). : 3) A lotion based on 3) was prepared and used as samples of Examples 2 and 3.
Further, a lotion containing only a mixture of propylene glycol and ethanol containing no β-galactosylceramide was prepared and used as a sample of Comparative Example 4. Furthermore, propylene glycol containing 1.0% ceramide (Sigma, USA)
A lotion of ethanol mixture (7: 3) was prepared and used as a sample of Comparative Example 4. These samples were applied to the back skin of 5 hairless rats (9 weeks old) once a day at a frequency of 30 times a day.
0 μL was applied for 10 days. Thereafter, β-glucocerebrosidase activity was examined in the same manner as in Examples 1 and 2. The result is shown in FIG.

From the results, a comparison was made between a lotion containing β-galactosylceramide (Examples 2 and 3) and a lotion not containing it (Comparative Example 4) or a lotion containing ceramide (Comparative Example 5). A significant difference was confirmed between Example 2, Example 3 and Comparative Example 4 or Comparative Example 5 at a risk factor of 0.1% or less, and β-galactosylceramide increased β-glucocerebrosidase activity in epidermis;
Ceramide was found not to enhance its activity. Such a method for increasing β-glucocerebrosidase activity has shown a new method for preventing and protecting rough skin.

[0020]

As described above, the present invention relates to
-It is clear that it provides a simple and excellent method of activating glucocerebrosidase. Also, the present method enables new prevention and protection of rough skin.

[Brief description of the drawings]

FIG. 1 shows the results of measuring β-glucocerebrosidase activity in Example 1 and Comparative Examples 1, 2, and 3.

FIG. 2 shows the results of measuring β-glucocerebrosidase activity in Examples 2, 3 and Comparative Examples 4, 5.

Claims (1)

(57) [Claims] 1. A method for activating β-glucocerebrosidase in epidermis, which comprises using β-galactosylceramide (excluding medical practice) .