JP4383427B2 - Skin moisturizer and dermatitis treatment agent - Google Patents

Description

  The present invention relates to a dermatitis therapeutic agent and a skin moisturizer containing ceramide-derived ceramide as an active ingredient.

  Dermatitis is an inflammatory reaction of the skin to various causes, atopic dermatitis, seborrheic dermatitis, contact dermatitis, hand eczema, hives, sebum deficiency dermatitis, pruritus, psoriasis, polymorphism Exudative erythema is known. Steroids and non-steroidal anti-inflammatory agents are mainly used for the treatment of such dermatitis, and antihistamines are also used for the treatment of atopic dermatitis. However, all of these treatment methods are only symptomatic treatments, and there are many cases in which recurrence is repeated. In addition, since side effects are associated with steroids, their long-term use has been limited.

Therefore, there is a need for a new dermatitis therapeutic agent that has few side effects and can be used for a long time.
WO98 / 44928 Gombert M, et al. The Journal of Immunology, 2005, 174: 5082-5091 Vestergaard C, et al. Experimental Dermatology, 2004, 13: 551-557 Makiura M, et al. The Journal of International Medical Research. 2004, 32: 392-399

  The inventors of the present invention have found that ceramide derived from Tamogi mushroom has a high moisturizing effect and a suppressive effect on the onset and symptoms of dermatitis. That is, the present invention provides a dermatitis therapeutic agent and a skin moisturizing agent containing ceramide-derived ceramide as an active ingredient. The dermatitis therapeutic agent and skin moisturizer of the present invention can be used for various types of dermatitis including atopic dermatitis (seborrheic dermatitis, contact dermatitis, hand eczema, hives, sebum-deficient dermatitis, skin pruritus). It is useful for prevention and treatment of symptom, psoriasis, polymorphic exudative erythema).

  Tamogi mushroom is a mushroom belonging to the genus Oyster mushroom and is widely used for food mainly in Hokkaido. Tamamogi contains a large amount of glycosphingolipid, a glycolipid structure characteristic of mushrooms, and its main components are 9-methyl-4-trans-8-trans-sphingadienin and 2 -Monoglucosylceramide composed of hydroxypalmitic acid. Glycosphingolipid, a so-called ceramide component, is an important physiological substance for cell membrane stabilization, not only cell membrane stabilization but also various activities such as cell-cell interactions, certain virus receptors, and apoptosis-inducing molecules. Have been reported.

  It is well known that ceramide components derived from rice bran and konjac have a skin moisturizing effect by inducing cell membrane stabilization. Regarding immune regulation, it has been reported that sponge-derived ceramide glycolipid selectively activates only some immune cells (NKT cells) and exhibits an antitumor effect (International Publication No. WO98 / 44928). . However, the effect of ceramide directly suppressing the onset mechanism of allergy and the effect of suppressing the onset of dermatitis has not been known. The present invention is based on the discovery that ceramide derived from Tamogi koji has the effect of suppressing the production of dermatitis-induced chemokines and can suppress the onset of dermatitis in model animals.

  In order to extract ceramide from rice candy, any of known ceramide extraction methods can be used. First, the fruit body or stone bump of Tamogi mushroom is dried as it is or after removing water-soluble components with water or hot water. As a method, any of conventional drying methods such as air drying, heat drying, and vacuum drying may be used, and the water content after drying can be appropriately selected in consideration of the subsequent steps. Next, the dried sample is crushed into powder and ceramide is extracted. As the extraction solvent, any organic solvent that can dissolve lipids, such as alcohols such as ethanol, methanol, propanol, and isopropanol, hexane, and chloroform, can be used. Moreover, a mixture of water and alcohols may be used, or an alkaline ethanol solution may be used. Alternatively, it may be extracted with carbon dioxide by a supercritical extraction method.

  The effectiveness of the ceramide derived ceramide obtained in this way is evaluated by examining the inhibitory effect of dermatitis-induced chemokine production from cells, or by examining the moisturizing action and inflammation inhibitory effect in dermatitis model animals. can do.

  Langerhans cells are cells in the subcutaneous dermis layer that are involved in the immune response to allergic antigens that infiltrate the skin. Abnormal activation of Langerhans cells causes allergic reactions such as atopic dermatitis. It is thought to exacerbate. It is known that this abnormal activation mainly consists of overproduction of CCL1 (also known as I-309, TCA-3), which is one of chemokines (cell chemotactic factors) (Gombert M, et al. The The Journal of Immunology, 2005, 174: 5082-5091). High expression of the CCL1 gene has been confirmed in patients with atopic dermatitis, and it is known that Langerhans cells stimulated with an antigen are cells that produce this CCL1. Therefore, it is expected that a substance having an effect of inhibiting the production of CCL1 has an antiallergic effect.

  On the other hand, epidermal keratinocytes are a group of cells that are important for the skin barrier that protects living organisms from foreign pathogens, and damage and inflammation of keratinocytes are considered to cause allergic reactions. In particular, the cell chemotactic factor CCL27 (also known as CTACK, ALP, ILC ESkine) produced by keratinocytes is a chemokine whose production is enhanced when skin epidermal cells are damaged, and induces T cell chemotaxis in the skin epidermis. Helps to eliminate (Vestergaard C, et al. Experimental Dermatology, 2004, 13: 551-557). However, since the overproduction induces chronic dermatitis, it is expected that a substance that regulates production of CCL27 can exert an anti-dermatitis effect.

  From the above background, it can be presumed that components that negatively control the functions of Langerhans cells and keratinocytes have antiallergic effects. Therefore, anti-dermatitis treatment is performed using the effect of ceramide derived from potato on these cells as an index. The effect of ceramide derived ceramide as a medicine can be confirmed.

  Furthermore, the dermatitis development inhibitory effect can be evaluated using HR-1 mice which are dermatitis development model mice. The HR-1 mouse is one of the hairless (nude) mice whose hair root development is inhibited. When a special diet with a low content of magnesium, which is a mineral, is mixed with the mouse, it is about 2 weeks old. The skin moisturizing action is reduced, and dry skin with itching is induced. This mouse is used as a model mouse for atopic dermatitis that develops various symptoms such as skin inflammation, cell infiltration, and increased serum IgE level (Makiura M, et al. The Journal of International Medical Research. 2004, 32 : 392-399). This low-mineral special fertilizer is blended with ceramide-derived ceramide to allow mice to freely ingest ceramide, measure moisture transpiration from the skin, and analyze pathological sections of the skin tissue to analyze the skin of ceramide-derived ceramide. The usefulness as a moisturizing agent and the effectiveness as a preventive and therapeutic agent for dermatitis can be evaluated.

  The dermatitis therapeutic agent / skin moisturizing agent of the present invention can be made into a pharmaceutical preparation by methods known to those skilled in the art. For oral administration, the ceramide-derived ceramide prepared as described above is mixed with a pharmaceutically acceptable carrier well known in the art to produce tablets, pills, dragees, capsules, liquids , Gels, syrups, slurries, suspensions and the like. Alternatively, the dermatitis therapeutic agent / skin moisturizing agent of the present invention may be prepared as a food additive. For parenteral administration, ceramide derived ceramide is a pharmaceutically acceptable carrier or excipient well known in the art, such as sterile water or saline, vegetable oil, emulsifier, suspension, interface. Can be formulated by combining with active agents, stabilizers, flavoring agents, excipients, vehicles, preservatives, binders, etc. as appropriate and admixed in unit dosage forms generally accepted in pharmaceutical practice . In particular, the dermatitis therapeutic agent / skin moisturizing agent of the present invention can be formulated as a skin external preparation in the form of a lotion, ointment, cream, pack, patch or the like.

  The dermatitis therapeutic agent / skin moisturizing agent of the present invention is preferably administered orally or added to foods, but can also be administered locally to the affected area of the skin. The dose of the dermatitis therapeutic agent / skin moisturizing agent of the present invention varies depending on symptoms, administration route, patient weight and age, other drugs used in combination, etc., but the amount of ceramide is, for example, 0.6 to 3 mg per day. It can be administered one to several times a day.

  EXAMPLES The present invention will be described below in more detail with reference to examples, but the present invention is not limited to these examples.

Induction of Langerhans cells Langerhans cells were induced from human peripheral blood monocytes according to a report by Pivarcsi et al. (Pivarcsi A, et al. Journal of Immunology, 2004, 173: 5810-5817). An overview is shown in FIG. Using a syringe to which heparin (manufactured by Shimizu Pharmaceutical Co., Ltd.) was added, 15-20 ml of blood was collected from the left arm vein of a healthy person and diluted with an equal volume of PBS (phosphate buffer) under aseptic conditions. The same-diluted blood was overlaid on 15 ml of Lymphosepar (manufactured by IBL) prepared in advance in a sterile plastic tube, and centrifuged at 1500 rpm for 30 minutes. After centrifugation, the mononuclear cell layer on Lymphosepar was recovered, and the cells were washed twice by centrifugation with PBS. Mononuclear cells are finally diluted in RPMI1640 medium and 10% fetal bovine serum (Sigma), and CD14 positive monocytes contained in about 10% of mononuclear cells are converted into magnetic beads (AutoMACS, Monocyte Isolation kit II). , Miltenyi Biotec) and collected with a purity of about 95%. The CD14 monocytes were cultured for 7 days in a culture solution to which GM-CSF, IL-4, and TGF-beta1 were added at a concentration of 50 ng / ml. As a result, it was possible to recover floating cells having large dendrites. The cells were stained with an anti-E cadherin antibody and an anti-langerin antibody and analyzed by flow cytometry. As a result, E-cadherin and Langerin-positive Langerhans cells were induced, unlike dendritic cells.

Effect of ceramide-derived ceramide on the production of dermatitis-induced chemokine CCL1 from Langerhans cells (1 × 10 ⁵ cells / ml) Tobacco-derived ceramide was added at various concentrations (1 to 10 μg / ml) to Langerhans cells. The ceramide derived ceramide was extracted from freeze-dried rice coconut bodies with chloroform / methanol, treated with alkali, washed several times with water / chloroform / methanol, and purified by flash column and HPLC. Since ceramide was dissolved in a 70% ethanol solution at 1 mg / ml, ethanol at the same final concentration was added to the culture solution as a negative control. After 24 hours of culture, LPS (bacterial component, Sigma) or CpG oligonucleic acid (virus component, Sigma-Genosys) was added at a final concentration of 1 μg / ml as antigen stimulation. After 36 hours, the Langerhans cell culture supernatant was collected, and the amount of chemokine CCL1 produced was measured by ELISA (R & D).

  When ceramide-derived ceramide was added to the culture and cultured, and Langerhans cells were observed with a microscope, no obvious cell death, growth inhibition, or morphological changes were observed. The results of measuring the amount of chemokine produced in the culture supernatant after 24 hours are shown in FIG. Langerhans cells produce a little CCL1 production of 70 ± 5 pg / ml without antigen stimulation, but LPS antigen stimulation produces 422 ± 17 pg / ml and CpG oligonucleic acid stimulation produces 131 ± 14 pg / ml. Induced. When ceramide-derived ceramide was added to these antigen-stimulated cells at a final concentration of 1 μg / ml, CCL1 chemokine production was 270 ± 14 pg / ml (43% inhibition) and 85 ± 4 pg / ml (inhibition rate 75, respectively). %) Was significantly suppressed.

  Next, Langerhans cells treated in the same manner were collected in a plastic tube, centrifuged and washed with RPMI1640 medium, then PE-labeled anti-human CD80 antibody, anti-human CD86 antibody, anti-human CD83 antibody, anti-human CCR7 antibody (all of which are eBioscience) 5 μl each) was added and reacted at 4 ° C. for 1 hour, and then the expression change of the cell surface antigen was analyzed by FACS (BD). As a result, the expression of cell surface antigens CD80, CD83, CD86, and CCR7 necessary for antigen presentation was not changed (light line-ceramide non-added: dark line-ceramide added). Since the expression of these surface molecules is equally induced by LPS stimulation, the effect of this ceramide-derived ceramide is not due to non-specific suppression on Langerhans cells but specifically on chemokine production. It was suggested that there is.

Effects of ceramide derived ceramide on the production of dermatitis-induced chemokine CCL27 from keratinocytes Human skin keratinocytes were purchased from Sanko Junyaku, and the culture medium was also a culture medium for keratinocyte culture specified by Sanko Junyaku After thawing, the cells were cultured. Add ceramide-derived ceramide to cultured keratinocytes (1x10 ⁵ cells / ml) at various concentrations (1-10 μg / ml), and after 24 hours of culture, inflammatory cytokine TNFα (R & D) is used as a final antigen concentration Added at 50 ng / ml. After 36 hours, the keratinocyte culture supernatant was collected and the amount of chemokine CCL27 produced was measured by ELISA (R & D).

  As a result of culturing epidermal keratinocytes and adding ceramide-derived ceramide to the culture medium and observing with a microscope, no obvious cell death, growth inhibition, or morphological change was observed. The results of measuring the amount of chemokine produced in the culture supernatant are shown in FIG. The amount of CCL27 produced from epidermal keratinocytes was 31.7 ± 0.3 pg / ml, but the production of 61.5 ± 0.5 pg / ml was increased by about 2 times by stimulation with inflammatory cytokine TNF. When ceramide-derived ceramide was added to the TNF-stimulated keratinocytes at a final concentration of 1 μg / ml, the production of CCL27 chemokine was significantly suppressed to 50.5 ± 1.3 pg / ml (inhibition rate 37%).

  Based on the above results, ceramide derived from Tamogi mushroom significantly suppresses the production of two chemokines CCL1 and CCL27 that play an important role in the development of dermatitis. It was suggested to be effective as a therapeutic agent.

Pathological analysis of HR-1 mouse dermatitis The effectiveness of the dermatitis therapeutic agent / skin moisturizer of the present invention was evaluated using HR-1 mice, which are model animals of dermatitis. Three week old Hos: HR-1 male mice were bred for one week and then used for the experiment. The body weight at the start of the experiment was 10.5-16.9 g. The untreated group was inoculated with normal feed and tap water, and the control group and the test substance-administered group were inoculated with special feed (refined feed for HR-AD, powder, manufactured by Nippon Agricultural Industrial Co., Ltd.) and tap water freely. The animals were reared under the rearing conditions set at 223 ° C., humidity 50 20%, lighting time 8: 00-20: 00, and ventilation rate 10-17 times / hour.

  Four-week-old HR-1 mice were assigned to each group by stratified continuous randomization using body weight as an index. After grouping, except for the normal feed group (A group), B to D groups were fed special feed. Furthermore, each of the groups C and D was allowed to freely ingest a special feed to which a test substance was added. The ceramide produced in the same manner as in Example 1 was used as the ceramide-derived ceramide, and the commercially available ceramide (Nippon Ceramide RPS, manufactured by Nisshin Flour Milling Co., Ltd.) was used as the rice bran-derived ceramide.

Moisturizing effect of ceramide derived from bamboo shoots During the breeding with a special feed, the water transpiration (TEWL) of the back skin was measured once a week. The measurement was performed according to a standard method using Tewameter TM300 (Courage + Khazaka). The results are shown in FIG. In the control group fed with the special diet, TEWL increased with time, but in the ceramide-derived ceramide group, a significant inhibitory effect on TEWL increase was observed, and on days 28 and 35, it was almost the same as the untreated group. Value. On the other hand, the rice bran-derived ceramide group showed no significant difference from the control group. During the test period, feed intake was almost constant, and there was no difference in body weight gain for each group. From these results, it was shown that, in HR-1 mice, ceramide-derived ceramide has a significantly higher moisturizing effect than rice bran-derived ceramide.

Changes in back skin condition due to ingestion of ceramide-derived ceramide In 6 weeks (10 weeks of age), the skin condition of all the cases was observed with a digital camera. HR-1 mice do not develop dermatitis associated with dry skin when fed with normal diet, but as a result of ingesting a low mineral special diet for 6 weeks, as shown in the lower left of FIG. Prominent and wrinkle count increased. On the other hand, the dry skin state was reduced in the mice (in FIG. 6) fed with ceramide derived ceramide, and no obvious difference was observed with the normal feed-ingested mice on the right in FIG.

HR-1 mice (10 weeks of age) that were allowed to freely take a special diet for skin pruritus behavior for 6 weeks and mice in each ceramide combination diet group were observed with a video camera for 30 minutes, and pruritus behavior (scratching behavior, grooming behavior, licking behavior) The results are shown in Fig. 7. Grooming behavior and licking behavior are also observed in healthy mice and do not increase in mice fed special feed. Scratch behavior) was significantly increased by the combination of special feed, indicating that the mouse has reached a state of atopic dermatitis with itching. % Of the special feed-ingested mice group (Group C), which had a significant decrease in the number of behaviours, and almost the same number of behaviors as normal mice. Scratch behavior was also reduced in the ingested mice, but this was considered because the mice in this group (Group D) were weaker than those in the other groups, and the overall behavioral ability was reduced.

Analysis of dermatitis onset suppression effect using skin pathological tissue After euthanizing HR-1 mice (10 weeks old) fed with special diet for 6 weeks and mice in each ceramide-containing diet group under ether anesthesia, The skin was incised into a rectangular size of 1 cm × 5 cm, and the skin tissue (keratin-epithelium-dermis-fat layer) was peeled from the back muscle layer and collected. The collected skin tissue was immersed in a neutral buffered 10% formalin solution (Wako Pure Chemical Industries) and fixed at 4 ° C. overnight or longer. The fixed skin tissue was embedded in paraffin, and the exfoliated pathological section was adsorbed on a slide glass, followed by hematoxylin / eosin staining (HE staining). Stained pathological sections were observed with a microscope (manufactured by NIKON), and 200 and 400 magnification images were taken with a CCD camera and stored as digital files.

  When normal feed is ingested by HR-1 hairless mice (Group A), the skin structure is normal as shown in FIGS. As a feature, the stratum corneum is thin from the surface side → the cell layer is one epidermal layer (average thickness 15 μm) → dermis layer → fat layer. Langerhans-like cells are mixed in the dermis layer, but there is no bias in cell distribution, and a normal skin tissue image is exhibited. On the other hand, as shown in FIGS. 10 and 11, in the mice fed with a low mineral special diet (group B), as shown in FIGS. 10 and 11, hyperkeratinization (stratification of stratum corneum), keratinocyte death, and epidermal hyperplasia. (Thickening of the skin) was observed, and the skin was in a dry skin state where moisture retention was not maintained. The average epidermal layer thickness was 60 μm, which was about 4 times that of normal mice. Furthermore, in the dermis, a large amount of inflammatory cell infiltration and extravascular exudation of erythrocytes are observed everywhere at the boundary with the epithelium, exhibiting typical skin inflammation.

  On the other hand, as shown in FIGS. 12 and 13, the thickness of the epidermal cell layer is slightly increased in the mice (group C) ingested with the diet containing the ceramide derived from potato paste in the special feed, as shown in FIGS. Inflammatory findings such as moisturization inhibition and cell infiltration due to increased keratinization were not observed, and the onset of dermatitis was clearly suppressed. As shown in FIGS. 14 and 15, in the special feed-ingested mice (group D) containing ceramide derived from rice bran at the same concentration as the object of comparative study, the skin inflammation was not improved at all, and the tendency was worse. It turned out to be. In particular, severe keratinization and inflammatory cells infiltrated not only into the dermis layer but also into the epidermis layer. Red blood cell exudation was also observed everywhere.

  As described above, the behavioral analysis and the pathological analysis results revealed that ceramide-derived ceramide has an effect of suppressing the onset of dermatitis. On the other hand, the ceramide derived from rice bran, which is the comparison target, has no dermatitis inhibitory effect, and this dermatitis inhibitory effect is a unique effect of ceramide derived from ceramide. Combined with the experimental results of Examples 2 and 3 in which ceramide-derived ceramide suppresses dermatitis-induced chemokine production, tamagi-derived ceramide has various dermatitis (seborrheic skin, including atopic dermatitis). It can be applied to prevention and treatment for inflammation, contact dermatitis, hand eczema, hives, sebum-deficient dermatitis, pruritus, psoriasis, polymorphic exudative erythema).

  The dermatitis therapeutic agent / skin moisturizing agent of the present invention containing ceramide-derived ceramide as an active ingredient is effective as a therapeutic agent for dermatitis including atopic dermatitis.

FIG. 1 shows a method for inducing Langerhans cells from human peripheral blood. FIG. 2 shows the inhibitory effect of ceramide-derived ceramide on the production of chemokine CCL1 from Langerhans cells. FIG. 3 shows an analysis of changes in the surface antigen expression of Langerhans cells by adding ceramide derived ceramide. FIG. 4 shows the inhibitory effect of ceramide-derived ceramide on chemokine CCL27 production from epidermal keratinocytes. FIG. 5 shows skin moisture transpiration in dermatitis-induced HR-1 mice. FIG. 6 shows the state of the back skin of dermatitis-induced HR-1 mice. FIG. 7 shows pruritus behavior of dermatitis-induced HR-1 mice. FIG. 8 shows the skin tissue of normal HR-1 mice. FIG. 9 shows the skin tissue of normal HR-1 mice. FIG. 10 shows the skin tissue of dermatitis-induced HR-1 mice. FIG. 11 shows the skin tissue of dermatitis-induced HR-1 mice. FIG. 12 shows skin tissue when ceramide-derived ceramide is administered to dermatitis-induced HR-1 mice. FIG. 13 shows skin tissue when ceramide-derived ceramide is administered to dermatitis-induced HR-1 mice. FIG. 14 shows the skin tissue when ceramide derived from rice bran was administered to dermatitis-induced HR-1 mice. FIG. 15 shows the skin tissue when ceramide derived from rice bran was administered to dermatitis-induced HR-1 mice.

Claims (3)

A therapeutic agent for dermatitis containing ceramide derived from potato rice cake as an active ingredient. The dermatitis therapeutic agent of Claim 1 whose dermatitis is atopic dermatitis. A skin moisturizer containing ceramide derived ceramide as an active ingredient.

Images