KR20140052293A - Pharmaceutical composition for prevention or treatment of tlr-mediated immune diseases comprising retinol - Google Patents

Abstract

The present invention relates to a novel use of retinol for prevention or treatment of toll-like receptor (TRL)-mediated immune diseases, and provided are a pharmaceutical composition for prevention or treatment of toll-like receptor-mediated immune diseases, a health food, and a cosmetic composition alleviating skin inflammatory diseases, comprising retinol as an active ingredient. Since retinol of the present invention can suppress the expression of various inflammatory mediators (TNF-alpha, IL-6, IL-12, IFN-beta, IP-10, RANTES, iNOS, CXCL9 and IL-27), when toll-like receptor 2 (TLR2), toll-like receptor 3 (TLR3) and toll-like receptor 4 (TLR4) are activated, toll-like receptor-mediated immune diseases can be ultimately alleviated through this mechanism. Accordingly, the composition of the present invention comprising retinol as an active ingredient can be used as medical supplies such as a pharmacological composition as well as health foods and cosmetic products so that the value of the present invention is expected to be enhanced when being used in food industries, medicine industries and cosmetic industries.

Description

[0001] The present invention relates to a pharmaceutical composition for prevention or treatment of a toll-like receptor mediated immune disease comprising retinol as an active ingredient,

The present invention relates to a novel use of retinol-like receptor-mediated immunological disease-mediated immunological disease, and more particularly to a pharmaceutical composition for preventing or treating immune diseases mediated by tol-like receptor mediated by retinol as an active ingredient, And to a cosmetic composition for improving disease.

An immune response is a series of reactions caused by activated immune cells against adventitious and endogenous substances (antigens). When a microorganism including a bacterium, a virus, or a foreign body of a living body enters the living body, the host cell secretes a factor such as cytokine which causes the inflammation reaction to overcome the infection and induces an inflammatory reaction. When these microorganisms and living organisms are recognized by the immune cells, the immune cells are activated. In the activated immune cells, many factors that cause the inflammation reaction are secreted to induce the inflammation reaction.

The development of molecular biology and genetic engineering has revealed molecular targets in inflammation related to the inflammation. Especially in the nonspecific congenital immune response at the early stage of the infection, a gene that recognizes a specific part of the pathogen has been identified, Molecular mechanistic analysis is being done. Recently, Toll-like receptors (TLRs) have been recognized as pathogen-recognizing genes in the early stages of inflammation, including lipopolysaccharide (LPS) and nucleic acid constructs (ds RNA, single strand RNA, GpG DNA, etc.), studies on TLR have been actively conducted.

TLR is a type 1 transmembrane signal molecule, a type 1 transmembrane signal molecule, which is mainly expressed in cells responsible for the innate immune system. In these cells, TLRs recognize pathogen-associated molecular patterns (PAMPs) or microorganism-associated molecular patterns (MAMPs) through the extracellular domain and consequently induce inflammatory cell activation in innate immune cells. Since TLRs have been identified, efforts have been made to find PAMPs that bind to TLRs, and up to now, one or more ligands have been identified, with the exception of TLR10, TLR12, and TLR13 (Kawai T. et al., Ann. NY Acad. , 1143, 1-20). TLRs differ, but share a signaling mechanism (Liew F. Y., et al., Nat. Rev. Immunol., 2005, 5 (6), 446-458). One common feature of signaling by TLRs is the activation of NFkB through the Toll / interleukin-1 receptor-like domain (TIR) domain.

To briefly describe the ligands of TLRs known to date, TLR1 is a tri-acyl lipopeptide of bacteria; TLR2 is a microbial lipoprotein (such as peptidoglycan, lipoarabinomannan, glycoinositolphospholipids, glycolipids, porins, zymosan, etc.); TLR3 is the dsRNA of the virus; TLR4 includes LPS and Taxol; TLR5 is a bacterial flagellin; TLR6 is the di-acyl lipopeptides of mycoplasma; TLRs are synthetic compounds (imidazoquinolin, loxoribine, bropirimine) and ssRNA of the virus; TLR8 is an imidazole quinoline and a viral ssRNA; TLR9 is the bacterial or viral CpG ODN and malaria pigment hemozoin.

The signaling pathways of TLRs due to the stimulation of these TLRs ligands can be largely divided into MyD88-dependent pathways and TRIF-dependent pathways. Both TLR2 and TLR4 have MyD88 as the adapter molecule, whereas the TRIF-dependent pathway does not have TLR2 and TLR4 . Activation of the MyD88-dependent pathway begins with the binding of MyD88 to IRAK-4 and IRAK-1, IRAK-1 is phosphorylated and binds TRAF6, which in turn binds to TAK1 and TAK1 activates the IKKβ complex. The IKKβ complex phosphorylates IκBα, facilitating the entry of NFκB into the nucleus, and entering the nucleus, NFκB promotes the synthesis of cytokines and enzymes involved in immunity. On the other hand, TRIF activates NFkB by activating RIP1 in combination with TRAF6. It also activates IKKe and TBK1 by binding to TRAF3, thereby activating IRF3 transcription factors, thereby inducing the production of type I interferons and chemokines. Thus, activation of TLR-2 and TLR4 induces the production of immunomodulatory cytokines and chemokines, thereby activating the natural immune system and also playing an important role in the activation of the acquired immune system.

On the other hand, retinol is a kind of vitamin A naturally present in the human body, and it exists in intestinal mucosal cells of animals and is abundant in greenish-yellow plants. It is also known as a fat-soluble vitamin that is essential for the differentiation and growth of epithelial tissue, which is transformed into the active form retinoic acid. It is nutritionally effective for growth and blindness. These retinol promotes cell differentiation by expressing RNA in the cell nuclei present in skin cells, promoting the biosynthesis of collagen, elastin, which is a light protein that exists as a fibrous solid between animal cells, and elastic fiber It has been known that it has an effect of reducing wrinkles and increasing skin elasticity, and has been developed as an effective ingredient of cosmetics since the early days of wrinkles and skin aging.

In addition, retinol has been reported to affect the function of innate immune cells such as macrophages, neutrophils, dendritic cells and NK cells, and retinol has been reported to play an important role in the regulation of the immune system. Several studies reported so far have shown that phagocytic capacity and killing activity of bacteria in the peritoneal macrophages and neutrophils of vitamin A deficient rats were reduced in these activities [Wiedermann, U., A. Tarkowski, T. Bremell, LA Hanson, H. Kahu, and UIDahlgren. 1996. Vitamin A deficiency predisposes to Staphylococcus aureus infection. Infect Immun 64: 209-214. / Twining, S. S., D. P. Schulte, P. M. Wilson, B. L. Fish, and J. E. Moulder. 1997. Vitamin A deficiency alters rat neutrophil function. J Nutr 127: 558-565 .; The cytolytic activity of NK cells in the spleen of vitamin A deficient mice was reduced [Ross, A. C., and C. B. Stephensen. 1996. Vitamin A and retinoids in antiviral responses. FASEB J 10: 979-985.]; The expression of IL-1α and IL-1β was up-regulated in injured corneas of vitamin A deficient mice [Shams, N. B., Reddy, K. Watanabe, S. A. Elgebaly, L. A. Hanninen, and K. R. Kenyon. 1994. Increased interleukin-1 activity in the injured vitamin A-deficient cornea. Cornea 13: 156-166.].

However, how retinol specifically regulates the activity of TLRs has not yet been elucidated. In particular, it has been suggested that the mechanisms by which TLRs are inhibited by retinol depend on the signaling pathways MyD88 and TRIF pathways, or whether they occur independently It has not been studied to date.

The present inventors first examined whether retinol could inhibit the activity of TLRs, and in order to investigate how TLRs inhibit the activity of MyD88 and the TRIF pathway, the present inventors examined retinol treatment in MyD88- or STAT1-deficient macrophages The present inventors have completed the present invention.

Korea Patent No. 10-0271315 Korean Patent Publication No. 10-2006-0091829

It is therefore an object of the present invention to provide pharmaceutical compositions for the prevention or treatment of toll-like receptor mediated immune diseases.

It is another object of the present invention to provide a health functional food for preventing or ameliorating toll-like receptor mediated immune diseases.

It is still another object of the present invention to provide a cosmetic composition for improving a toll-like receptor mediated skin inflammation disease.

In order to achieve the object of the present invention, the present invention provides a pharmaceutical composition for preventing or treating immune diseases mediated by tol-like receptors comprising retinol as an active ingredient.

In one embodiment of the present invention, the immunological disease may be an autoimmune disease or an inflammatory disease.

In one embodiment, the autoimmune disease is selected from the group consisting of rheumatoid arthritis, systemic scleroderma, systemic lupus erythematosis, diabetes, atopic dermatitis, alopecia areata, psoriasis, pemphigus, asthma, aphthas stomatitis, chronic thyroiditis, ulcerative colitis, Multiple sclerosis, multiple sclerosis, autoimmune hemolytic anemia, autoimmune encephalomyelitis, fibromyalgia, and temporal arteritis.

In one embodiment of the present invention, the inflammatory disease may be selected from the group consisting of airway inflammation, asthma, chronic inflammation, acute inflammation, chronic prostatitis, inflammatory bowel disease, ulcerative colitis, and conjunctivitis.

In one embodiment of the invention, the retinol is selected from the group consisting of various inflammatory mediators in bone marrow-derived macrophages stimulated with ligands of tol-like receptor 2 (TLR2), toll-like receptor 3 (TLR3) or toll- Expression-inhibiting mechanisms can prevent or treat toll-like receptor mediated immune diseases.

In one embodiment of the present invention, the inflammatory mediator may be selected from the group consisting of TNF- [alpha], IL-6, IL-12, IFN- [beta], IP-10, RANTES, iNOS, CXCL9 and IL- have.

In one embodiment of the invention, the retinol is capable of preventing or treating a toll-like receptor mediated immune disease through a mechanism that inhibits the activity of a toll-like receptor independent of MyD88 and STAT1.

The present invention also provides a tole-like receptor mediated health functional food for preventing or ameliorating an immune disease comprising retinol as an active ingredient.

In one embodiment of the present invention, the food is selected from the group consisting of beverage, meat, chocolate, foods, confectionery, pizza, ram noodles, gums, candy, ice cream, alcoholic beverages, .

In addition, the present invention provides a cosmetic composition for improving a toll-like receptor mediated skin inflammation disease comprising retinol as an active ingredient.

In one embodiment of the present invention, the skin inflammatory disease may be acne or atopic dermatitis.

In one embodiment of the present invention, the composition may be a skin lotion, a skin softener, a skin toner, an astringent, a lotion, a milk lotion, a moisturizing lotion, a nutrition lotion, a massage cream, a nutritive cream, And may be formulated into one selected from the group consisting of a pack, a soap, a shampoo, a cleansing foam, a cleansing lotion, a cleansing cream, a body lotion, a body cleanser, a latex, a lipstick, a makeup base, a foundation, a press powder and a loose powder .

The retinol of the present invention is useful for the treatment of various inflammatory mediators (TNF-a, IL-6, IL-12, and IL-12) when TLR2, TLR3 and TLR4 are activated, Such as IFN-beta, IP-10, RANTES, iNOS, CXCL9, and IL-27, can ultimately improve toll-like receptor mediated immune diseases. Therefore, the composition of the present invention containing retinol as an active ingredient can be utilized not only as a pharmaceutical such as a pharmacological composition but also as a health functional food and a cosmetic, and thus is highly valuable when applied to the food industry, the pharmaceutical industry and the cosmetics industry .

Figure 1 shows the results of pretreatment of macrophages with retinol followed by stimulation with various TLR ligands (TLR2 ligands: MALP-2, TLR3 ligand: poly (I: C), TLR4 ligand: LPS) 6, IL-12, IFN- ?, IP-10, RANTES, iNOS, CXCL9 and IL-27.
FIG. 2 shows results of immunoblotting analysis showing the amount of protein expression of MAPKs and transcription factors induced after pretreatment of retinol with macrophages and stimulation with TLR ligand.
FIG. 3 shows the inhibitory effect of the lower signaling pathway (CREB, AP-1, NFκB, IFN-?) Of TLRs of retinol through the gene reporter assay.
FIG. 4 shows the results of RT-PCR analysis showing the expression levels of proinflammatory cytokines (IP-10, CXCL9, IL-27) after pretreatment with retinol in MyD88-deficient macrophages and stimulating with LPS.
FIG. 5 shows the results of RT-PCR analysis showing the expression levels of proinflammatory cytokines (TNF-α, IFN-β, IP-10 and RANTES) after pretreatment of retinol with STAT1-deficient macrophages and stimulation with LPS.

The present invention is characterized by providing a pharmaceutical composition for preventing or treating immune diseases mediated by tol-like receptors comprising retinol as an active ingredient.

The retinol of the present invention is a kind of vitamin A naturally present in the human body and exists in intestinal mucosal cells of animals and is abundant in greenish-yellow plants. It is also known as a fat-soluble vitamin essential for the differentiation and growth of epithelial tissue, which is transformed into the active form retinoic acid. It is nutritionally effective for promoting growth and blindness.

Retinol has been reported to affect the function of innate immune cells such as macrophages, neutrophils, dendritic cells and NK cells and has also been shown to play an important role in the regulation of the immune system.

However, it has not yet been elucidated how to specifically regulate the activity of TLRs, and in particular, whether the mechanism of TLRs inhibition of retinol is dependent on the signaling pathways MyD88 and TRIF pathways, or whether they occur independently, It is not.

The present inventors have found that the retinol of the present invention is useful for the treatment of various inflammatory mediators (TNF-α, IL-6, IL, and IL-4 when TLR2, TLR3 and TLR4 are activated, -12, IFN- [beta], IP-10, RANTES, iNOS, CXCL9 and IL-27) and that this mechanism ultimately leads to improvement of toll-like receptor mediated immune diseases He said. In addition, we first elucidated that the inhibitory mechanism of retinol TLRs is independent of the signaling pathways MyD88 and TRIF.

In the following Example 1 of the present invention, TLR2, TLR3 and TLR4 activity of retinol was inhibited. As a result, pretreatment of retinol was performed on mouse macrophages and various TLR ligands (TLR2 ligands: MALP-2 and TLR3 ligands: poly IL-6, IL-12, IFN-β, IP-10, RANTES, iNOS, CXCL9, and IL-27 in the macrophage when stimulated with the TLR4 ligand: Was suppressed (see Fig. 1).

In the following Example 2 of the present invention, the effect of regulating the activation of the lower signal pathway of TLRs of retinol was examined. As a result, when pretreating retinol in mouse macrophages and stimulating macrophages with TLR ligands, phosphorylation of MAPKs and IRF3 And activation of NFkB, IFN-beta. This confirms that retinol does not affect the activation of the lower signaling pathway of TLRs (see FIGS. 2 and 3).

In Example 3 of the present invention, it was found that the inhibitory effect of retinol on TLR activation is dependent on MyD88 and STAT1, which play a central role in the TLR signaling pathway. As a result, MyD88-deficient macrophages and STAT1- (IP-10, CXCL9, IL-27, TNF-α, IFN-β, IP-10 and RANTES) were still suppressed in the pretreated experimental group. Independent (independent of STAT1).

These results suggest that various inflammatory mediators (TNF-a, IL-2, IL-4, IL-6, IL-6, IL-12, IFN-β, IP-10, RANTES, iNOS, CXCL9 and IL-27), and inhibits TLRs activation Lt; RTI ID = 0.0 > MyD88 < / RTI > and STAT1.

Therefore, the composition of the present invention comprising retinol as an active ingredient can effectively prevent or treat toll-like receptor mediated immune diseases.

The retinol according to the present invention can be used in the form of a salt, preferably a pharmaceutically acceptable salt. The salt is preferably an acid addition salt formed by a pharmaceutically acceptable free acid, and the free acid may be an organic acid or an inorganic acid. The organic acids include, but are not limited to, citric, acetic, lactic, tartaric, maleic, fumaric, formic, propionic, oxalic, trifluroacetic, benzoic, gluconic, methosulfonic, glycolic, succinic, Glutamic acid and aspartic acid. The inorganic acid includes, but is not limited to, hydrochloric acid, bromic acid, sulfuric acid, and phosphoric acid.

Retinol according to the present invention may be a commercially available product, or may be prepared from a natural product or produced by a chemical synthetic method known in the art.

The term "toll-like receptor mediated immune disease" of the present invention means that NF-κB activated by activation of TLR-2, TLR-3 and TLR-4 belonging to a toll-like receptor (TLR) Refers to diseases in which various cytokines and cell junction molecule-related genes are expressed in the nucleus. The toll-like receptor mediated immune diseases include, but are not limited to, autoimmune diseases; arthritis; Atopic dermatitis; allergy; asthma; Arteriosclerosis; osteoporosis; Graft rejection; Graft versus host disease; ssRNA and dsRNA viral infections; blood poisoning; cancer; Immune deficiency; And diseases which are induced by increased production of TNF- [alpha], IL-6, MCP-1, IFN- [beta], RANTES or ICAM-1.

The disease caused by the increase of the production of TNF-α, IL-6, MCP-1, IFN-β, RANTES or ICAM-1 is not particularly limited, , Dermatitis, eczema, systemic lupus erythematosus, gout, periodontal disease, multiple myelopathy, Alzheimer's disease, Behcet's syndrome, cachexia, obesity, edema, superimmunopathologic globulinemia, vasculitis and Kawasaki disease, Meniere's disease, myocardial infarction, Acute and chronic infections of germs and fungi, diabetic retinopathy, diabetic nephropathy, autoimmune pancreatitis, tumor metastasis, cancerous cachexia, transplant rejection, pain, irritable pulmonary disease, vasculitis, endocrine ophthalmic disease, multiple sclerosis, glomerulonephritis, Acute and chronic bronchitis, upper respiratory infections including generalized cold, influenza infections, etc. Preferably, the TLR-mediated diseases are osteoporosis, arthritis, arthritis, Piseong can be dermatitis, psoriasis, etc. Particularly, osteoporosis, arthritis, atopic dermatitis and psoriasis belong to autoimmune diseases such as TLR and IL-6 mediated diseases, and the pharmaceutical composition comprising the retinol compound of the present invention or a pharmaceutically acceptable salt thereof as an active ingredient Can exhibit a therapeutic effect by a mechanism different from that caused by the action mechanism of the conventional antioxidant.

The composition of the present invention is a pharmaceutical composition comprising retinol as an active ingredient, and can be prepared using pharmaceutically acceptable and physiologically acceptable adjuvants in addition to the active ingredients. Examples of the adjuvants include excipients, disintegrants, sweeteners, A binder, a coating agent, a swelling agent, a lubricant, a lubricant or a flavoring agent.

The pharmaceutical composition may be formulated into a pharmaceutical composition containing at least one pharmaceutically acceptable carrier in addition to the above-described active ingredients for administration.

The pharmaceutical composition may be in the form of granules, powders, tablets, coated tablets, capsules, suppositories, liquids, syrups, juices, suspensions, emulsions, drops or injectable solutions. For example, for formulation into tablets or capsules, the active ingredient may be combined with an oral, non-toxic pharmaceutically acceptable inert carrier such as ethanol, glycerol, water, and the like. Also, if desired or necessary, suitable binders, lubricants, disintegrants and coloring agents may also be included as a mixture. Suitable binders include, but are not limited to, natural and synthetic gums such as starch, gelatin, glucose or beta-lactose, natural sugars such as corn sweeteners, acacia, tracker candles or sodium oleate, sodium stearate, magnesium stearate, Benzoate, sodium acetate, sodium chloride, and the like. Disintegrants include, but are not limited to, starch, methyl cellulose, agar, bentonite, xanthan gum and the like. Acceptable pharmaceutical carriers for compositions that are formulated into a liquid solution include sterile water and sterile water suitable for the living body such as saline, sterile water, Ringer's solution, buffered saline, albumin injection solution, dextrose solution, maltodextrin solution, glycerol, One or more of these components may be mixed and used. If necessary, other conventional additives such as an antioxidant, a buffer, and a bacteriostatic agent may be added. In addition, diluents, dispersants, surfactants, binders, and lubricants may be additionally added to formulate into injectable solutions, pills, capsules, granules or tablets such as aqueous solutions, suspensions, emulsions and the like. Further, it can be suitably formulated according to each disease or ingredient, using the method disclosed in Remington's Pharmaceutical Science, Mack Publishing Company, Easton PA as an appropriate method in the field.

In one embodiment of the present invention, the retinol of the present invention may be contained in the composition at a concentration of 1 to 300 μM, and the retinol of the present invention may be contained in an amount of 0.1 to 95% by weight based on the total weight of the composition.

The present invention also provides the use of a composition comprising retinol as an active ingredient for the manufacture of a medicament for the prevention or treatment of immune diseases mediated by toll-like receptor. The composition of the present invention comprising retinol as an active ingredient can be used for the preparation of a medicament for preventing or treating a toll-like receptor mediated immune disease.

The invention also provides a method of preventing or treating a toll-like receptor mediated immune disease comprising administering retinol to a mammal.

The term "mammal " as used herein refers to a mammal that is the subject of treatment, observation or experimentation, preferably a human.

The term "therapeutically effective amount " as used herein refers to the amount of active ingredient or pharmaceutical composition that elicits a biological or medical response in a tissue system, animal or human, as contemplated by a researcher, veterinarian, The amount that induces the relief of the symptoms of the disease or disorder being treated. It will be apparent to those skilled in the art that the therapeutically effective dose and the number of administrations of the active ingredient of the present invention will vary depending on the desired effect. The optimal dosage to be administered can therefore be readily determined by those skilled in the art and will depend upon the nature of the disease, the severity of the disease, the amount of active and other ingredients contained in the composition, the type of formulation, and the age, , Sex and diet, time of administration, route of administration and rate of administration of the composition, duration of treatment, concurrent administration of the drug, and the like. In the treatment method of the present invention, in the case of an adult, it is preferable to administer the retinol of the present invention at a dose of 0.01 mg / kg to 250 mg / kg once to several times a day.

In the therapeutic method of the present invention, the composition comprising retinol as an active ingredient of the present invention can be administered orally, rectally, intravenously, intraarterally, intraperitoneally, intramuscularly, intrasternally, transdermally, topically, ≪ / RTI >

The present invention also provides a health functional food for preventing or improving a toll-like receptor mediated immune disease comprising retinol as an active ingredient.

The health functional food of the present invention can be manufactured and processed in the form of tablets, capsules, powders, granules, liquids, and circles for the purpose of preventing and improving toll-like receptor mediated immune diseases.

In the present invention, the term " health functional food " refers to foods manufactured and processed using raw materials or ingredients having useful functions in accordance with Law No. 6727 on Health Functional Foods. Or to obtain a beneficial effect in health use such as physiological action.

The health functional foods of the present invention may contain conventional food additives and, unless otherwise specified, whether or not they are suitable as food additives are classified according to the General Rules for Food Additives approved by the Food and Drug Administration, Standards and standards.

Examples of the items listed in the above-mentioned "food additives" include chemical compounds such as ketones, glycine, calcium citrate, nicotinic acid, and cinnamic acid; Natural additives such as persimmon extract, licorice extract, crystalline cellulose, high color pigment and guar gum; L-glutamic acid sodium preparations, noodle-added alkalis, preservative preparations, tar coloring preparations and the like.

For example, the health functional food in tablets may be prepared by granulating a mixture of retinol, an active ingredient of the present invention, with an excipient, a binder, a disintegrant, and other additives in a conventional manner, , The mixture can be directly compression molded. In addition, the health functional food of the tablet form may contain a mating agent or the like if necessary.

The hard capsule may be prepared by filling a normal hard capsule with a mixture of retinol, an active ingredient of the present invention, with an additive such as an excipient. The soft capsule may be prepared by mixing retinol with an excipient such as an excipient Can be prepared by filling a mixture into a capsule base such as gelatin. The soft capsule may contain a plasticizer such as glycerin or sorbitol, a coloring agent, a preservative and the like, if necessary.

The ring-shaped health functional food can be prepared by molding a mixture of retinol, an active ingredient of the present invention, an excipient, a binder and a disintegrant in a conventionally known method, and if necessary, Or the surface may be coated with a material such as starch, talc.

The granular health functional food may be prepared by granulating a mixture of retinol, an active ingredient of the present invention, an excipient, a binder, a disintegrant, and the like into a granular form by a known method, and adding a flavoring agent, ≪ / RTI >

The health functional food may be a beverage, a meat, a chocolate, a food, a confectionery, a pizza, a ramen, a noodle, a gum, a candy, an ice cream, an alcoholic beverage, a vitamin complex and a health supplement food.

In addition, the present invention provides a cosmetic composition for improving a toll-like receptor mediated skin inflammation disease comprising retinol as an active ingredient.

When the composition of the present invention is prepared with a cosmetic composition, the composition of the present invention may contain not only the above-mentioned retinol but also components commonly used in cosmetic compositions, such as antioxidants, stabilizers, And customary adjuvants such as fragrances, and carriers.

In addition, the composition of the present invention may be mixed with an anti-inflammatory agent which has been used in the past so long as it does not impair the skin protecting effect by reacting with retinol in addition to retinol.

Examples of products to which the cosmetic composition of the present invention can be added include cosmetics such as astringent lotion, softening longevity lotion, nutrition lotion, various creams, essences, packs, foundation and the like, cleansing, cleanser, soap, .

As a specific formulation of the cosmetic composition of the present invention, there may be mentioned a skin lotion, a skin softener, a skin toner, an astringent, a lotion, a milk lotion, a moisturizing lotion, a nutrition lotion, a massage cream, It includes formulations such as soap, shampoo, cleansing foam, cleansing lotion, cleansing cream, body lotion, body cleanser, latex, lipstick, makeup base, foundation, press powder, loose powder, eye shadow.

According to a preferred embodiment of the present invention, the retinol content of the present invention is 0.0001-20% by weight, preferably 0.5-20% by weight, more preferably 1.0-20% by weight based on the total weight of the composition. If the content of retinol is less than 0.0001% by weight, the effect of improving retinol-induced inflammation is greatly reduced. If the content of retinol is more than 20% by weight, skin irritation may be caused.

Toll-like receptor mediated skin inflammation diseases that can exhibit the skin inflammatory disease-improving effect of the cosmetic composition of the present invention include acne, hypersensitive dermatitis, atopic dermatitis, insect allergies, food allergies, drug allergies and urticaria ), But the present invention is not limited thereto.

Meanwhile, the cosmetic composition according to the present invention may be formulated by stabilizing retinol by incorporating retinol inside the nanoliposome. When the retinol is contained in the nanoliposome, the components of retinol are stabilized, thereby solving problems such as precipitation formation, discoloration, and deterioration upon formulation, and can increase the solubility and transdermal absorption rate of the components, Can be maximally expressed.

In the present invention, nanoliposome refers to a liposome having a typical liposome form and having an average particle diameter of 10 to 500 nm. According to a preferred embodiment of the present invention, the average particle diameter of the nanoliposome is 50 to 300 nm. When the average particle diameter of the nanoliposome is more than 300 nm, improvement of skin penetration and improvement of formulation stability is very weak among technological effects to be achieved in the present invention.

The nanoliposomes used to stabilize retinol in accordance with the present invention may be prepared by a mixture comprising a polyol, an oily component, a surfactant, a phospholipid, a fatty acid and water.

The polyol used in the nanoliposome of the present invention is not particularly limited and is preferably a polyol such as propylene glycol, dipropylene glycol, 1,3-butylene glycol, glycerin, methylpropanediol, isopropylene glycol, pentylene glycol, erythritol, , Sorbitol, and mixtures thereof. The amount thereof is 10 to 80% by weight, preferably 30 to 70% by weight, based on the total weight of the nanoliposome.

The oil component used in the preparation of the nanoliposome of the present invention may be selected from a variety of oils known in the art and is preferably a hydrocarbon oil such as hexadecane and paraffin oil, Vegetable oils such as silicon oil such as dimethicone and cyclomethicone, sunflower oil, corn oil, soybean oil, avocado oil, sesame oil and fish oil, ethoxylated alkyl ether oils, propoxylated alkyl ether oils ₄₀ is a fatty alcohol, and mixtures thereof -, phytosphingosine, sphingosine, and scan non-ping the same scan pinggo cannabinoid lipid, cholesterol side-by celebrity, sitosterol cholesteryl sulfate, cholesteryl sulfate, cytokines, C _10. The amount thereof may be 1.0 to 30.0% by weight, and preferably 3.0 to 20.0% by weight based on the total weight of the nanoliposome.

Any surfactant known in the art may be used as the surfactant used in the preparation of the nanoliposome of the present invention. For example, anionic surfactants, cationic surfactants, amphoteric surfactants and nonionic surfactants can be used. Preferred are anionic surfactants and nonionic surfactants. Specific examples of anionic surfactants include alkyl acyl glutamates, alkyl phosphates, alkyl lactylates, dialkyl phosphates and trialkyl phosphates. Specific examples of nonionic surfactants include alkoxylated alkyl ethers, alkoxylated alkyl esters, alkyl polyglycosides, polyglyceryl esters and sugar esters. Particularly preferred surfactants are polysorbates belonging to nonionic surfactants. The amount thereof may be 0.1 to 10% by weight, preferably 0.5 to 5.0% by weight, based on the total weight of the nanoliposome.

The phospholipid, another component used in the preparation of the nanoliposomes of the present invention, is a lipophilic lipid that is used with both affinity lipids and is composed of natural phospholipids (e.g., egg yolk lecithin or soy lecithin, sphingomyelin) Dipalmitoyl phosphatidylcholine or hydrogenated lecithin), preferably lecithin. In particular, unsaturated lecithin or saturated lecithin derived from natural origin extracted from soybean or egg yolk is preferable. Normally, the amount of phosphatidylcholine is 23 to 95% and the amount of phosphatidylethanolamine is 20% or less in natural derived lecithin. In the production of the nanoliposome of the present invention, the amount of the phospholipid to be used is 0.5 to 20.0% by weight, preferably 2.0 to 8.0% by weight based on the total weight of the nanoliposome.

Fatty acid to be used in nano-liposome preparation of the present invention is a higher fatty acid, preferably a C ₁₂ - ₂₂ as a saturated or unsaturated fatty acid of the alkyl chain, e.g., lauric acid, myristic acid, palmitic acid, stearic acid, oleic acid and linoleic acid . The amount thereof may be 0.05 to 3.0% by weight, preferably 0.1 to 1.0% by weight, based on the total weight of the nanoliposome.

The water used in the preparation of the nanoliposome of the present invention is generally deionized distilled water, and the amount thereof may be 5.0-40 wt% based on the total weight of the nanoliposome.

The preparation of nanoliposomes can be accomplished through a variety of methods known in the art, but most preferably is made by applying a mixture comprising the ingredients to a high pressure homogenizer. The preparation of a nanoliposome by a high pressure homogenizer can be carried out under various conditions (for example, pressure, number of times, etc.) depending on a desired particle size, and preferably at a high pressure homogenizer So that the nanoliposome can be produced.

The cosmetic composition for improving skin-like receptor mediated skin inflammation disease according to the present invention may contain 0.1 to 20.0% by weight of the retinol on the basis of the weight of the nanoliposome.

Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are for further illustrating the present invention, and the scope of the present invention is not limited to these examples.

< Example  >

Cell culture

Murine macrophage cells RAW264.7 (ATCC TIB-71) were obtained from the American Type Culture Collection (Manassas, Va.). Bone marrow cells were isolated from C57BL / 6 mice (Orient Bio, Seoul, Korea) and differentiated into macrophages. Immortalized macrophages, derived from wild-type or MyD88 knockdown mice, Lt; / RTI &gt; from Howard A. Young (NCI-Frederick, Frederick, Md.). STAT1 knockdown mice were received from R. Schreiber (Washington University School of Medicine, St. Louis, Mo.). Cells 10% (v / v) yeolbul activated fetal calf serum (Invitrogen, Carlsbad, CA), 100 units / ml penicillin and 100μg / ml streptomycin (Invitrogen) 37 ℃ in supplemented DMEM medium, 5% CO ₂ / air.

Reagents and plasmids

Retinol was purchased from Sigma-Aldrich (St. Louis, Mo.); LPS purified from E. coli was purchased from List Biological Laboratories Inc. (Campbell, CA); MALP-2 was purchased from Alexis Biochemicals (San Diego, CA); Poly (I: C) was purchased from Amersham Biosciences (Pittsburgh, Pa.). Antibodies to phospho-ERK, ERK, phospho-JNK, JNK, and phospho-IRF3 were purchased from Cell Signaling (Boston, MA). Anti-IRF3 antibodies were purchased from Invitrogen. Antibodies to IκBα and actin were purchased from SantaCruz Biotechnology (Santa Cruz, Calif.). The NFkB (2x) -luciferase reporter construct is a &lt; RTI ID = 0.0 &gt; Donated from Frank Mercurio (Signal Pharmaceuticals, San Diego, Calif.); The heat shock protein 70- [beta] -galactosidase plasmid was obtained from Dr. Donated by Robert Modlin (University of California, Los Angeles, CA); The IFN- [beta] PRDIII-I-luciferase plasmid was obtained from Dr. Katherine Fitzgerald (University of Massachusetts Medical School, Worcester, MA). DNA constructs for transfection were also prepared using the Endofree Plasmid Maxi kit (Qiagen, Valencia, Calif.). All other reagents used in the experiments were purchased from Sigma-Aldrich.

statistics

All results were expressed as mean ± SD. One-way ANOVA (Systat Inc., Evaston, IL, USA) was performed to analyze the results. A p-value <0.05 was considered statistically significant.

< Example  1>

Retinol TLR2 , TLR3  And TLR4 The inhibitory effect of

In order to evaluate whether retinol can regulate TLRs activity, RAW264.7, a mouse macrophage, was treated with various TLR ligands (TLR2 ligand: MALP-2, TLR3 ligand: poly (I: C), TLR4 ligand: LPS) . The expression of the proinflammatory genes was analyzed by RT-PCR analysis.

(TLR2 ligand: MALP-2 [50 ng / ml], TLR3 ligand: poly (I: C) [30 μg / ml] was added to each bone marrow- derived macrophage after pretreatment with 50 μM retinol for 1 hour ], And TLR4 ligand: LPS [100 ng / ml]) and reacted for 4 hours. Total RNA of the cultured cells was extracted with Welprep ^TM reagent (Jeil Biotechservice Inc., Daegu, Korea). The extracted RNAs were reverse transcribed using ImProm-IIT reverse transcriptase (Promega, Madison, Wis.) And amplified with Taq DNA polymerase using IQTM5 (Bio-Rad, Hercules, Calif.). PCR was carried out using the following primers (see Table 1 and Table 2 below). The PCR product was separated by electrophoresis on 1.5% agarose gel at 100 V for 10 minutes. The gel was stained with 1 mg / ml EtBr and photographed under UV illumination using AlphaEase gel image analysis software (Alpha Innotech., San Leandro, Calif., USA).

As a result, as shown in Fig. 1, proinflammatory genes (TNF-a, IL-6, IL-12, IFN-beta, IP-10, RANTES, iNOS, CXCL9 and IL-27) in the test group treated with the retinol of the present invention, whereas the gene expression of all of them was decreased in the retinol-treated experimental group of the present invention. This suggests that retinol can inhibit TLR activation by inhibiting the expression of TLR2, TLR3 and TLR4 proinflammatory genes.

RT-PCR conditions Temperature time 42 1 hour 30 minutes 95 5 minutes 4 ∞

The primer sequences used in RT-PCR gene Primer sequence TNF-a Forward 5'-AAAATTCGAGTGACAAGCCTGTAG-3 ' Reverse 5'-CCCTTGAAGAGAACCTGGGAGTAG-3 ' IL-6 Forward 5'-TTCCTCTCTGCAAGAGACT-3 ' Reverse 5'-TGTATCTCTCTGAAGGACT-3 ' IL12p40 Forward 5'-GAAGTTCAACATCAAGAGCAGTAG-3 ' Reverse 5'-AGGGAGAAGTAGGAATGGGG-3 ' IFN-beta Forward 5'-TCCAAGAAAGGACGAACATTCG-3 ' Reverse 5'-TGCGGACATCTCCCACGTCAA-3 ' IP-10 Forward 5'-TCCTGCTGGGTCTGAGTG-3 ' Reverse 5'-ATTCTTGATGGTCTTAGATTCCG-3 ' RANTES Forward 5'-GCCCACGTCAAGGAGTATTTCTAC-3 ' Reverse 5'-AGGACTAGAGCAAGCGATGACAG -3 ' iNOS Forward 5'-GATGTTGAACTATGTCCTATCTCC-3 ' Reverse 5'-AACACCACTTTCACCAAGAC-3 ' CXCL9 Forward 5'-TCCTTTTGGGCATCATCTTC-3 ' Reverse 5'-TGAACGACGACGACTTTGG-3 ' IL-27 Forward 5'-CTCTGCTTCCTCGCTACCAC-3 ' Reverse 5'-GGGGCAGCTTCTTTTCTTCT-3 ' Actin Forward 5'-TCATGAAGTGTGACGTTGACATCCGT-3 ' Reverse 5'-TTGCGGTGCACGATGGAGGGGCCGGA-3 '

< Example  2>

Retinol TLRs The lower part of Signal path  Activation modulating effect

In Example 1, it was confirmed that retinol could inhibit the activation of TLR2, TLR3 and TLR4. In this experiment, immunolabeling and immunogenicity analysis of how retinol affects the activation of the lower signaling pathway of TLR And examined by a transfection and luciferase assay.

<2-1> Immunoblotting ( Immunoblotting )

For immunoblotting, RAW264.7 and BMDM cells were pretreated with retinol (10, 20, 50 μM) for 1 hour, and RAW264.7 cells were treated with 10 ng / ml LPS for 30 minutes BMDM cells were treated with 10 ng / ml LPS and reacted for 1 hour. The lysates of the cells were first separated by SDS-PAGE for immunoblot and then transferred to polyvinylidene difluoride (PVDF, Bio-Rad Laboratories, Hercules, Calif.) Membrane. Then, the membrane was reacted with anti-phospho-ERK, anti-ERK, anti-phospho-JKN, anti-JKN, anti-IKBA, anti-phospho-IRF3, anti-IRF3 and anti-actin. The reaction band was visualized with an enhanced chemiluminescence system (Amersham Bioscience) and the degree of expression of the proteins was confirmed.

As a result, as shown in Fig. 2, it was found that the phosphorylation of MAPKs such as ERK and JNK induced by LPS was not inhibited in the experimental group pretreated with retinol in RAW264.7 cells (Fig. 2A) and RAW264.7 In the experimental group pretreated with retinol in cell and bone marrow-derived macrophages (BMDM), the activation of NFκB induced by LPS was not inhibited (Fig. 2B), and phosphorylation of IRF3 was not weakened (Fig. 2C).

<2-2> Genetic Reporter Essay

This analysis used the previously described method (Youn, HS, YS Kim, ZY Park, SY Kim, NY Choi, SM Joung, JA Seo, KM Lim, MK Kwak, DH Hwang, and JY Lee, 2010. Sulforaphane suppresses oligomerization of TLR4 in a thiol-dependent manner. J Immunol 184: 411-419.). Briefly, a SuperFect transfection reagent was used. That is, the IFN-beta promoter of the cAMP response element (CRE), AP-1 biding site, NFκB binding site or IFN- RAW264.7 cells were transformed with a luciferase reporter plasmid containing the domain (IFN-beta PRD III-I) and a beta-galactosidase expression plasmid as an internal control. The luciferase and beta-galactosidase enzyme activities were then analyzed using the Luciferase Assay System and the beta-galactosidase Enzyme System (Promega) according to the manufacturer's instructions.

As a result, as shown in Fig. 3, activation of CREB and AP-1 was not inhibited in the experimental group pretreated with retinol in RAW264.7 cells (Figs. 3A and 3B), and RAW264.7 cells were pretreated with retinol In one experimental group, activation of NFκB induced by LPS or MALP-2 was not inhibited (FIG. 3C and 3D). In addition, activation of IFN-β induced by ploy (1: C) was not inhibited in RAW264.7 cells pretreated with retinol (FIG. 3E).

These results indicate that retinol inhibits TLR activation by inhibiting the expression of TLR2, TLR3 and TLR4 proinflammatory genes, but does not affect the activation of the lower signaling pathway of TLRs.

< Example  3>

Retinol TLR In the activation suppression effect of MyD88  And STAT1 In the signal path Rain Dignity

In Example 1, it was confirmed that retinol could inhibit the activation of TLR2, TLR3 and TLR4. In this experiment, it was shown that the inhibitory effect of retinol on TLR activation was dependent on MyD88 and TRIF Were investigated through MyD88-deficient and STAT1-deficient macrophage cell lines.

<3-1> Retinol TLRs  In the activity inhibition effect MyD88 In the signal path Non-dependence

First, the inhibitory effect of MyD88-deficient macrophages on the expression of MyD88 gene and the inhibitory effect of the MyD88-deficient macrophages on the expression of inflammatory cytokines following pretreatment with retinol were investigated by RT-PCR analysis.

For RT-PCR analysis, each bone marrow-derived macrophage (BMDM) obtained from wild-type and MyD88-deficient mice was pretreated with 50 μM of retinol for 1 hour, ml of LPS and allowed to react for 8 hours. The RT-PCR analysis using the cultured total cells was carried out in the same manner as in Example 1, and the primer sequences used in the amplification of the MyD88 gene used in the RT-PCR were shown in Table 3 below.

The MyD88 gene primer sequence used in RT-PCR gene Primer sequence MyD88 Forward 5'-TCTACAGAGCAAGGAATGTGACT-3 ' Reverse 5'-TGAACCGCAGGATACTGGGAA-3 '

As shown in Fig. 4, it was confirmed that MyD88-deficient macrophages did not express MyD88 mRNA (Fig. 4A). In the test group pretreated with MyD88-deficient macrophages, LPS- 10, CXCL9, and IL-27 were still inhibited (Fig. 4B). Thus, it was possible to demonstrate that the mechanism of TLRs inhibition of retinol is independent (independent) in the MyD88 signaling pathway.

&Lt; 3-2 > TLRs  In the activity inhibition effect STAT1 In the signal path Non-dependence

The activity of the TRIF-dependent signal pathway in the TLR3 and TLR4 signals results in the production of type 1 enteropone. Type 1 interferons act on the initiation of activation of the secondary signal of their own receptors and on interferon inducible genes (such as IP-10 and RANTES). JAK and STAT are key constituents in these secondary signals (mediating the expression of type 1 interferon inducible genes). Therefore, we investigated whether STAT1 is required for the inhibitory effect of retinol on TLRs activity by using STAT1 deficient BMDM macrophages.

Each bone marrow-derived macrophage obtained from wild type and STAT1-deficient mice was pretreated with 50 μM of retinol for 1 hour, treated with 100 ng / ml of LPS, and reacted for 4 hours. RT-PCR analysis using the cultured total cells was carried out in the same manner as in Example 1 above.

As a result, as shown in FIG. 5, it was confirmed that LPS-induced TNF- ?, IFN- ?, IP-10 and RANTES expression were still inhibited in the pretreatment group of STAT1-deficient macrophages pretreated with retinol. Thus, it could be shown that the mechanism of TLRs inhibition of retinol is independent of STAT1.

The present invention has been described with reference to the preferred embodiments. It will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention as defined by the appended claims. Therefore, the disclosed embodiments should be considered in an illustrative rather than a restrictive sense. The scope of the present invention is defined by the appended claims rather than by the foregoing description, and all differences within the scope of equivalents thereof should be construed as being included in the present invention.

TLR: Toll-like receptor
LPS: lipopolysaccharide
BMDM: Bone marrow-derived macrophage

Claims (12)

A pharmaceutical composition for preventing or treating immunological diseases mediated by tol-like receptors, which comprises retinol as an active ingredient. The method according to claim 1,
Wherein said immune disease is an autoimmune disease or an inflammatory disease. 3. The method of claim 2,
Wherein said autoimmune disease is selected from the group consisting of rheumatoid arthritis, systemic scleroderma, systemic lupus erythematosus, diabetes, atopic dermatitis, alopecia areata, psoriasis, pemphigus, asthma, aphthas stomatitis, chronic thyroiditis, ulcerative colitis, multiple myositis, Anemia, autoimmune encephalomyelitis, fibromyalgia, and temporal arteritis. &Lt; RTI ID = 0.0 &gt; 11. &lt; / RTI &gt; 3. The method of claim 2,
Wherein said inflammatory disease is selected from the group consisting of airway inflammation, asthma, chronic inflammation, acute inflammation, chronic prostatitis, inflammatory bowel disease, ulcerative colitis, and conjunctivitis. The method according to claim 1,
The retinol has been shown to inhibit the expression of inflammatory mediators in bone marrow-derived macrophages stimulated with ligands of toll-like receptor 2 (TLR2), toll-like receptor 3 (TLR3) or toll-like receptor 4 (TLR4) A pharmaceutical composition characterized by preventing or treating a mediated immune disease. 6. The method of claim 5,
Wherein said inflammatory mediator is selected from the group consisting of TNF-a, IL-6, IL-12, IFN- ?, IP-10, RANTES, iNOS, CXCL9 and IL-27. The method according to claim 1,
Wherein the retinol is to prevent or treat toll-like receptor mediated immune diseases through a mechanism that inhibits the activity of the toll-like receptor independent of MyD88 and STAT1. A health functional food for preventing or improving a toll-like receptor mediated immune disease comprising retinol as an active ingredient. 9. The method of claim 8,
Wherein said food is selected from the group consisting of beverage, meat, chocolates, foods, confectionery, pizza, ramen, other noodles, gums, candy, ice cream, alcoholic beverages, vitamin complexes and health supplement foods. Claims 1. A cosmetic composition for improving skin-like receptor mediated skin inflammation disease comprising retinol as an active ingredient. 11. The method of claim 10,
Wherein the skin inflammatory disease is acne or atopic dermatitis. 11. The method of claim 10,
The composition may be in the form of a skin lotion, a skin softener, a skin toner, an astringent, a lotion, a milk lotion, a moisturizing lotion, a nutrition lotion, a massage cream, a nutrition cream, a moisturizing cream, a hand cream, Wherein the composition is one selected from the group consisting of a cleansing lotion, a cleansing cream, a body lotion, a body cleanser, an emulsion, a lipstick, a makeup base, a foundation, a press powder and a loose powder.

Images