Patent Description:
The epidermis is the outermost layer of the skin, and the stratum corneum is the outermost layer of the epidermis. When the stratum corneum, composed of keratinocytes, is in normal state, it can protect the skin from various external irritants and retain the moisture released from the body. The keratinocytes proliferate in the basal layer, which is the innermost skin layer, and differentiate gradually as they pass through the spinous layer and the granular layer. Through this keratinization process, the keratinocytes produce natural moisturizing factors (NMFs) and lipids (ceramides, cholesterols or fatty acids) and form the stratum corneum. The skin barrier function can work properly only when the stratum corneum is formed normally.

Skin diseases and troubles such as atopy, psoriasis or acne occur when the stratum corneum does not maintain its normal function due to various factors. They mainly exhibit skin barrier destruction and skin xerosis. Currently, immunosuppressants, etc. are mainly used for the treatment of atopy and psoriasis, and ceramides are mainly used for strengthening the skin barrier and treating skin xerosis. However, they have disadvantages that they neither have a noticeable effect nor effect a permanent cure.

In one aspect, an object of the present invention is to provide a non-therapeutic use of a composition comprising a material inhibiting the expression or activity of JAB-<NUM> for moisturizing skin or strengthening skin barrier function by promoting the differentiation of skin cells.

Document <NPL> discloses a nucleic acid which inhibits JAB-<NUM> expression, and that the knockdown of Jun activation domain-binding protein <NUM> (JAB1) results in the reduction of IFNR level and IFN-Induced gene expression. It discloses also and antibody which specifically binds to JAB-<NUM>.

Document <NPL>, discloses a nucleic acid which inhibits JAB-<NUM> expression, and that the knockdown of Jun activation domain-binding protein <NUM> (JAB1) results in the reduction of IFNR level and IFN-Induced gene expression. It discloses also and antibody which specifically binds to JAB-<NUM>.

Document <CIT>, discloses JAB-<NUM> inhibiting ligands such as antisense nucleic acids, and their formulation as pharmaceutical compositions.

In one aspect, the present invention provides a non-therapeutic use of a composition for moisturizing skin or strengthening skin barrier function by promoting differentiation of skin cells comprising a material inhibiting the expression or activity of JAB-<NUM> (Jun activator binding-<NUM>) as an active ingredient.

The composition for promoting differentiation of skin cells used in the present invention inhibits the expression of JAB-<NUM> (Jun activator binding-<NUM>) or inhibits the binding between JAB-<NUM> and psoriasin, thereby inhibiting the migration of psoriasin into the cell nucleus so as to enable the differentiation of skin cells to be effectively promoted. Therefore, the composition is effective in skin moisturization or skin barrier function strengthening.

JAB-<NUM> (Jun activator binding-<NUM> or Jun activation domain binding protein-<NUM>) is the first known protein that serves as a coactivator of AP-<NUM> protein. Although JAB-<NUM>'s mechanism in the proliferation of cancer cells is known, its mechanism in the differentiation of skin cells is not known yet.

The present inventors have found that psoriasin migrates into the nucleus after reaction with JAB-<NUM> and that the expression levels of JAB-<NUM> and psoriasin are high, particularly in atopic patients and patients with psoriasis. Based on the findings, the present inventors have found that it is possible to promote the differentiation of skin cells by inhibiting the expression of JAB-<NUM>. Specifically, the present inventors have found the following: psoriasin reacts with JAB-<NUM> and then migrates into the cell nucleus. The psoriasin that has migrated into the cell nucleus inhibits the differentiation of skin cells. However, it is possible to promote the differentiation of skin cells by inhibiting the migration of psoriasin into the cell nucleus by inhibiting the expression of JAB-<NUM>. Psoriasin, which is a protein also called S100A7 (S100 calcium-binding protein A7), is encoded by the S100A7 gene.

In one aspect, the present invention relates to the non-therapeutic use of a composition for moisturizing skin or strengthening skin barrier function by promoting the differentiation of skin cells comprising a material inhibiting the expression or activity of JAB-<NUM> (Jun activator binding-<NUM>) as an active ingredient.

In another aspect, the present specification relates to materials inhibiting the expression or activity of JAB-<NUM> for moisturizing skin or strengthening skin barrier function by promoting differentiation of skin cells.

In another aspect, the present specification discloses the non-therapeutic use of materials inhibiting the expression or activity of JAB-<NUM> as active ingredients for moisturizing skin or strengthening skin barrier function by promoting the differentiation of skin cells.

In another aspect, the present specification discloses the use of materials inhibiting the expression or activity of JAB-<NUM> for the preparation of a composition for moisturizing skin or strengthening skin barrier function by promoting differentiation of skin.

As used herein, the expression "inhibiting the activity of JAB-<NUM>" means inhibiting the binding between JAB-<NUM> and psoriasin.

As used herein, the term "skin" refers to tissues covering the surface of an animal body. It encompasses not only tissues covering the surface of a face or a body but also the scalp and hair. The skin as used herein includes not only the skin of a living body but also an artificial skin or a skin mimic mimicking the skin of a living body.

In one aspect of the present invention, the skin cells comprise keratinocytes. In another aspect of the present invention, the skin cells comprise human keratinocytes. In yet another aspect of the present invention, the skin cells comprise normal human keratinocytes.

As used herein, the expression "differentiation of skin cells" may mean a process in which keratinocytes functionally differentiate in the basal layer, which is the innermost skin layer, and gradually form the spinous layer, the granular layer and the stratum corneum.

As used herein, the expression "relative expression level" may mean the expression level of JAB-<NUM> in skin cells treated with a test material compared with the expression level of JAB-<NUM> in skin cells not treated with the test material. The expression level may include an expression amount and an expression quality.

In the above aspect, the material inhibiting the expression or activity of JAB-<NUM> is an RNA nucleic acid molecule.

In the composition for promoting the differentiation of skin cells according to one aspect of the present invention, the RNA nucleic acid molecule is at least one of siRNA, shRNA, and miRNA which bind to the mRNA of JAB-<NUM>.

siRNA, shRNA and miRNA are RNA fragments that induce RNA interference (RNAi). RNA interference means the degradation of a target mRNA by a double stranded RNA (dsRNA), resulting in downregulation of the expression of a specific gene.

miRNA is an endogenous small RNA. It is derived from a DNA that does not synthesize proteins and generated from a hairpin-shaped transcript. The miRNA binds to the complementary sequence of the <NUM>'-UTR of the target mRNA to induce the inhibition of the translation or the destabilization of the mRNA, ultimately serving as a repressor to inhibit protein synthesis of the target mRNA. This action is called RNAi. It is known that one miRNA can target several mRNAs, and mRNA can also be regulated by several miRNAs. Other RNAs that induce RNAi include short interfering RNA (siRNA), which is a short RNA of about <NUM> to <NUM> mer, and shRNA with a short hairpin structure. In general, siRNA and shRNA are artificially introduced into cells to induce RNAi, while miRNA is naturally present in cells.

In the above aspect, the siRNA may comprise sequences that are each capable of hybridizing with them or sequences that are each complementary to them.

As used herein, the expression "capable of hybridizing" means capable of binding to a specific single stranded RNA to form a double stranded RNA.

The composition for promoting the differentiation of skin cells according to one aspect of the present invention may inhibit the migration of psoriasin into the cell nucleus. Specifically, psoriasin binds to JAB-<NUM> and then migrates into the cell nucleus to inhibit cell differentiation, but the composition can inhibit this mechanism.

In one embodiment, the skin cells may be keratinocytes.

In the above aspect, the composition is a composition for moisturizing the skin or strengthening the skin barrier function by promoting differentiation of skin cells. If the differentiation of skin cells is not performed properly, the stratum corneum cannot perform its normal function, which leads to reduction of the water holding capacity of the skin and deterioration of the skin barrier function. Further, skin diseases such as atopy and psoriasis occur when the stratum corneum does not maintain its normal function due to various factors. The major symptoms of the diseases are skin inflammation and skin xerosis. Thus, materials promoting the differentiation of skin
cells can exert the effects of skin moisturization, and skin barrier function strengthening.

The composition for promoting the differentiation of skin cells is a cosmetic composition, or a health food composition.

The cosmetic composition may be provided in any formulation suitable for topical application, for example, a solution, an oil-in-water emulsion, a water-in-oil emulsion, a suspension, a solid, a gel, a powder, a paste, a foam, or an aerosol composition. These formulations may be prepared according to methods commonly employed in the art.

The cosmetic composition may comprise other ingredients which do not negatively affect the main effect of the present invention, and which preferably provide a synergic effect to the main effect. The cosmetic composition of the present invention may comprise a material selected from the group consisting of vitamins, polypeptides, polysaccharides, and sphingolipids. Also, the cosmetic composition of the present invention may comprise a humectant, an emollient, a surfactant, a UV absorbent, a preservative, a sterilizer, an antioxidant, a pH adjuster, an organic or inorganic pigment, a fragrance, a cooling agent, an antiperspirant or the like. The amount of these ingredients may be easily determined by those skilled in the art within a range not negatively affecting the purpose and effect of the present invention. The content of the ingredients may be <NUM>-<NUM>% by weight, specifically, <NUM>-<NUM>% by weight, based on the total weight of the composition.

The health food may refer to a food prepared with raw materials or ingredients (functional raw materials) having a nutrient likely to be deficient in normal diets or a function useful for the human body, and which helps to maintain and improve health by maintaining the normal function or activating the physiological function of the human body, although not limited thereto. The health food may be prepared and processed in the form of a tablet, a capsule, a powder, a granule, a liquid, a pill or the like. However, the health food is not limited thereto and may be prepared and processed in any form according to the law.

The health beverage composition according to one aspect of the present invention has no particular limitation on other ingredients, except that a predetermined amount of the above compound is included as an essential ingredient. It may comprise various flavoring agents, natural carbohydrates or the like as additional ingredients, as do common beverages. Examples of the natural carbohydrates include conventional sugars such as monosaccharides, polysaccharides, and cyclodextrins and sugar alcohols such as xylitol, sorbitol, and erythritol. Also, natural flavoring agents (thaumatin, stevia extract (for example, rebaudioside A, glycyrrhizin, etc.)) and synthetic flavoring agents (for example, saccharin, aspartame, etc.) may be used as the flavoring agents.

In general, the daily dose of the active ingredient of the health food composition may be <NUM>/kg/day to about <NUM>/kg/day, preferably <NUM>/kg/day to <NUM>/kg/day. It may be administered once a day or in several divided doses per day.

In the composition for promoting differentiation of skin cells according to one aspect of the present invention, the material inhibiting the expression or activity of JAB-<NUM> may be included at a concentration of <NUM> to <NUM>.

In addition, the content of the material inhibiting the expression or activity of JAB-<NUM> may be <NUM> to <NUM>% by weight, for example, <NUM> to <NUM>% by weight, <NUM> to <NUM>% by weight, <NUM> to <NUM>% by weight or <NUM> to <NUM>% by weight, preferably <NUM> to <NUM>% by weight, based on the total weight of the composition.

The present specification relates to a method for screening for materials promoting the differentiation of skin cells, comprising the steps of: treating skin cells with test materials; and identifying the relative expression levels of JAB-<NUM> in the skin cells before and after treatment with the test materials.

The screening method may further comprise the step of, if the expression level of JAB-<NUM> after treatment with test materials is lower than the level before the treatment, determining that the test materials are materials promoting the differentiation of skin cells. In one embodiment, the screening method may further comprise the step of, if the expression level of JAB-<NUM> after treatment with test materials is at least <NUM>% lower than the level before the treatment, determining that the test materials are materials promoting the differentiation of skin cells. For example, if the expression level of JAB-<NUM> after treatment with test materials is at least <NUM>%, at least <NUM>%, at least <NUM>%, at least <NUM>%, at least <NUM>%, at least <NUM>% or at least <NUM>% lower than the level before the treatment, the test materials can be determined to be materials promoting the differentiation of skin cells.

For example, if the expression level of JAB-<NUM> in keratinocytes treated with test materials is higher than the expression level of JAB-<NUM> in keratinocytes not treated with the test materials, it can be determined that the test materials increased the expression level of JAB-<NUM>. On the contrary, if the expression level of JAB-<NUM> in skin cells treated with test materials is lower than or similar to the expression level of JAB-<NUM> in skin cells not treated with the test materials, it can be determined that the test materials did not increase the expression level of JAB-<NUM>. As described above, if test materials reduce the expression level of JAB-<NUM> by at least <NUM>% compared to the level before the treatment, the test materials can be determined to be materials promoting the differentiation of skin cells.

The expression level of JAB-<NUM> may be identified by a known technology, for example, reverse transcription-polymerase chain reaction (RT-PCR), ELISA, western blot or immune blot, although not limited thereto.

It can also be visually identified whether or not the expression level of JAB-<NUM> has decreased.

The screening method may further comprise the step of identifying the relative expression level of at least one of keratin <NUM> and keratin <NUM> before and after treatment with the test materials. The method may further comprise the step of, if the expression level of at least one of keratin <NUM> and keratin <NUM> after treatment with test materials is higher than the level before the treatment, determining that the test materials are materials promoting the differentiation of skin cells. In one embodiment, the screening method may further comprise the step of, if the expression level of at least one of keratin <NUM> and keratin <NUM> after treatment with test materials is at least <NUM>% higher than the level before the treatment, determining that the test materials are materials promoting the differentiation of skin cells. For example, if the expression level of keratin <NUM> or keratin <NUM> after treatment with test materials is at least <NUM>%, at least <NUM>%, at least <NUM>%, at least <NUM>%, at least <NUM>%, at least <NUM>%, at least <NUM>% or at least <NUM>% higher than the level before the treatment, the test materials can be determined to be materials promoting the differentiation of skin cells.

In another aspect, the present specification disclosed that keratin <NUM> and keratin <NUM> can serve as markers of differentiation of skin cells. If keratin <NUM> and keratin <NUM> are highly expressed, it can be determined that skin cell differentiation is active.

In one embodiment, the skin cells may be keratinocytes, although not limited thereto.

Hereinafter, the constitution and effects of the present invention will be described in more detail through examples. However, the following examples are provided for illustrative purposes only to facilitate understanding of the present invention, and the scope of the present invention is not limited thereto.

The expression levels of JAB-<NUM> in the skin tissues of an atopic patient and a normal human were identified. The skin tissues of an atopic patient were obtained from the dermatology department of Chung-Ang University.

<NUM>-mm epidermis biopsy samples were obtained from the normal group (age <NUM>, male) and the atopy group (age <NUM>, male). The samples were then embedded in a frozen section compound (FSC <NUM>®, Leica Microsystems, USA) and cut into <NUM> sections. The tissue slides were immunostained using a reagent for cell nuclear staining, DAPI (<NUM>',<NUM>-diamidino-<NUM>-phenylindole) and an anti-JAB-<NUM> antibody. From the results, it was seen that in the normal group, blue (less bright portion) stained nuclei were found throughout the epidermal layer while no stained JAB-<NUM> (green; brighter portion) was observed (<FIG>). In contrast, in the atopic tissues, green (brighter portion) stained JAB-<NUM> increased in the upper part of the epidermis and stained nuclei were found (<FIG>). Thus, it can be understood that the expression level of JAB-<NUM> was higher in the skin
(epidermis) of the atopic patient.

Keratinocytes were purchased from Lonza. Each cell was cultured in a CO<NUM> incubator under the conditions of <NUM> and <NUM>% CO<NUM>. The cell culture was performed according to Lonza's guidelines. The medium used in the cell culture was prepared by adding <NUM> of a KGM-<NUM> bullet kit (bovine pituitary extract (BPE)), <NUM> of human epidermal growth factor (hEGF), <NUM> of insulin, <NUM> of hydrocortisone, <NUM> of transferrin, <NUM> of epinephrine, and <NUM> of gentamycin sulfate and amphotericin-B (GA-<NUM>) to <NUM> of KGM-<NUM> (Clonetics CC-<NUM>) medium. The keratinocytes were cultured in a <NUM> culture dish to a confluency of <NUM>-<NUM>%. Then, in order to investigate the expression level of S100A7, which is a protein binding to JAB-<NUM>, and the expression level of JAB-<NUM> when treated with an inflammatory cytokine IL-<NUM> (<NUM>/ml), immunostaining was performed using an anti-S100A7 antibody to determine the expression level of S100A7 (<FIG>) and western blot was performed to determine the expression level of JAB-<NUM> (<FIG>).

From the results, it was found that the group treated with IL-<NUM> showed higher expression levels of S100A7 and JAB-<NUM>, stained in green (slightly bright portions in the cells), than the group not treated with IL-<NUM> (<FIG>). Interestingly, it can be seen that a large amount of S100A7 is found in the nuclei of the group with increased expression of JAB-<NUM>. The yellow portions (very bright portions inside the circles) in <FIG> resulted from increased nuclear translocation of S100A7.

Cells were treated with IL-<NUM> according to the same method as Example <NUM> and transfected with siRNA of the JAB-<NUM> gene (<NUM>, ON-TARGET plus siRNA Reagents purchased from Dharmacon) using lipofectamine to knock down JAB-<NUM>. Then, it was observed whether S100A7 migrated into the nucleus.

Propidium iodide was used for nuclear staining, and a fluorescence labeled anti-S100A7 antibody (Abcam ab13680) was used for staining S100A7. The stained nuclei were red and the stained S100A7 was green. If S100A7 migrates into the nucleus, red and green are combined to give yellow. From the results, it was found that in the group treated with IL-<NUM>, green S100A7 was present as yellow portions in the nuclei (the brightest portions in the third figure in <FIG>), but in the case where JAB-<NUM> was knocked down, yellow was hardly observed, indicating that almost no S100A7 migrated into the nucleus (<FIG>).

After treatment with IL-<NUM> and JAB-<NUM> knockdown according to the same method as Example <NUM>, the mRNA expression levels of keratin <NUM> and keratin <NUM>, which are epidermal differentiation markers, were measured. From the results, it was found that the expression levels of mRNAs of keratin <NUM> and keratin <NUM> increased when JAB-<NUM> was knocked down, and that the epidermal differentiation markers expression reduced by IL-<NUM> was restored when JAB-<NUM> was knocked down (<FIG>).

Two days after knocking down JAB-<NUM> according to the same method as Example <NUM>, the cells were cultured, the medium was removed, <NUM> of Trizol (Invitrogen) was added and RNA was isolated according to the RNA isolation method of Invitrogen for comparison with the control group (non-targeting RNA, <NUM>). RNA was quantified using an ultraviolet detector (Hewlett Packard) at <NUM> and subjected to reverse transcription-polymerase chain reaction (RT-PCR). For gene analysis of each sample, normalization was performed with the complementary gene RPL13A.

From the results, it was found that in the JAB-<NUM> knockdown group, the gene expression level of JAB-<NUM> decreased by about <NUM>-<NUM>% in comparison with the control group.

Hereinafter, formulation examples of the composition according to one aspect of the present invention will be described. However, it may be formulated into various other forms. These examples are for illustrative purposes only and are not intended to limit the scope of the present invention.

A milk lotion can be prepared according to a conventional method with the composition shown in Table <NUM> below.

A nourishing lotion can be prepared according to a conventional method with the composition shown in Table <NUM> below.

A nourishing cream can be prepared according to a conventional method with the composition shown in Table <NUM> below.

A pack can be prepared according to a conventional method with the composition shown in Table <NUM> below.

An ointment can be prepared according to a conventional method with the composition shown in Table <NUM> below.

An injection can be prepared according to a conventional method with the composition shown in Table <NUM> below.

Claim 1:
A non-therapeutic use of a composition comprising a material inhibiting the expression or activity of JAB-<NUM> (Jun activator binding-<NUM>) for moisturizing skin or strengthening skin barrier function by promoting differentiation of skin cells,
wherein the material inhibiting the expression or activity of JAB-<NUM> is an RNA nucleic acid molecule which is at least one of siRNA, shRNA, and miRNA which bind to the mRNA of JAB-<NUM>, and
wherein the material inhibiting the expression or activity of JAB-<NUM> is formulated in a form of a composition which is a cosmetic composition or a health food composition.