JP2010208992A - Anti-inflammatory agent - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a new anti-inflammatory agent for preventing or treating skin inflammation, especially inflammation caused by the irradiation of UV rays. <P>SOLUTION: There are provided an anti-inflammatory agent for preventing or treating skin inflammation, containing apelin, and an anti-inflammatory agent for preventing or treating skin inflammation, containing a vector containing a gene encoding apelin. <P>COPYRIGHT: (C)2010,JPO&INPIT

Description

  The present invention relates to an anti-inflammatory agent characterized by containing apelin or a vector comprising a gene encoding apelin for preventing or treating skin inflammation.

  Physical or chemical stimulation of the skin induces angiogenesis and vascular permeability such as by VEGF-A, resulting in tissue fluid retention and edema. In particular, it is known that constant ultraviolet irradiation causes skin damage such as wrinkle formation, degradation of dermal matrix polymers such as collagen, and fibrosis (Non-patent Document 1). It is also known that skin inflammation such as erythema, thickening of the epidermis, increased vascular permeability, edema, etc. occurs in the skin by a single exposure to ultraviolet B (hereinafter referred to as UVB) (wavelength 290 nm to 320 nm) ( Non-patent document 2).

  Apelin is a molecule isolated from bovine stomach cell extract in 1998 as a binding factor for APJ, a seven-transmembrane G protein-coupled receptor that has long been an orphan receptor. The apelin cDNA encodes 77 amino acids, but the precursor forms a long form (42-77 amino acids) and a short form (65-77 amino acids). Both apelins are known to induce APJ activation. So far, APJ expression has been reported in the cardiovascular system and central nervous system, suggesting that it is involved in the regulation mechanism of body fluids, such as controlling myocardial contraction in the heart and vasopressin expression in the nervous system. It is coming. APJ is also a receptor that is attracting a lot of attention as a target for drug discovery from various viewpoints because it is involved in infection as a receptor for AIDS virus. APJ expression is expressed in vascular endothelial cells and mural cells in the vascular system, and the apelin / APJ system plays an essential role in vascular development in Xenopus gene knockdown experiments. In addition, since the expression of this receptor is also observed in endothelial cells in mice and humans, it has been expected to participate in angiogenesis in mammals (Non-patent Document 3). In recent years, it has been reported that apelin secreted when vascular endothelial cells are stimulated with Ang1 controls the diameter of blood vessels through analysis of apelin knockout mice and in vitro vascular system analysis (non-) Patent Document 4).

  However, it has not been clarified so far that apelin has a protective effect on the skin.

Kligman AM. The treatment of photoaged human skin by topical tretinoin. Drugs 1989; 38: 1-8. Cox NH, Diffey BL, Farr PM.The relationship between chronological age and the erythemal response to ultraviolet B radiation.Br J Dermatol 1992; 126: 315-9. Experimental Medicine Vol. 26, No.9 (2008), pp. 1380-1383 Kidoya et al, (2008) Spatial and temporal role of the apelin / APJ system in the caliber size regulation of blood vessels during angiogenesis.EMBO J 27: 522-534

  This time, in order to investigate the relationship between apelin and skin condition, the present inventor compared the mouse skin condition after irradiation with ultraviolet rays in a mouse and a wild type mouse into which a gene that highly expresses apelin was introduced. In wild-type mice, edema of the dermis, thickening of the epidermis, and increase of macrophages, which are skin inflammatory cells, were observed after UV irradiation, but significant changes were observed in mice with high expression of apelin epidermis. Not observed. These results show the surprising effect that apelin is extremely useful for preventing or treating skin inflammation.

  Accordingly, an object of the present invention is to provide a novel anti-inflammatory agent for preventing or treating skin inflammation, preferably skin inflammation caused by ultraviolet irradiation.

This application includes the following inventions:
1. An anti-inflammatory agent containing apelin for preventing or treating skin inflammation.
2. An anti-inflammatory agent comprising a vector comprising a gene encoding apelin for preventing or treating skin inflammation.
3. The anti-inflammatory agent as described above, wherein the skin inflammation is an inflammation caused by ultraviolet irradiation.
4). A cosmetic method for improving or preventing inflammation, comprising applying any one of the anti-inflammatory agents of 1 to 3.

  The present invention makes it possible to prevent or treat skin inflammation, in particular inflammation caused by ultraviolet irradiation.

It is a photograph which shows the change of the skin thickness by irradiation of an ultraviolet-ray in an apelin epidermal high expression mouse | mouth and a wild type mouse. It is a graph which shows the skin thickness in FIG. 1 quantitatively. It is a photograph which shows the macrophage dyeing | staining before and behind ultraviolet irradiation in an apelin epidermal high expression mouse | mouth and a wild type mouse | mouth.

  Human apelin has been reported to be expressed in various sites such as heart, lung, kidney, fat, stomach, brain, adrenal gland, and endothelium, and is a ligand of 36 amino acid apelin APJ receptor derived from 77 amino acid precursor protein. (Kawamata Y et al, (2001) Molecular properties of apelin: tissue distribution and receptor binding. BIOCHMICA ET BIOPHYSICA ACTA. 2-3: 162-171). Also known as apelin are mutants lacking the N-terminal and / or C-terminal amino acids, such as apelin-13 (polypeptide consisting of the 24th to 36th amino acid sequences of apelin). When referring to “Apelin” in the book, not only full-length human apelin consisting of 36 amino acids, but also an apelin variant in which one or several amino acids have been deleted, substituted, or added as long as it has the activity of apelin, and fragments thereof Include. Apelin includes not only human but also non-human primates and rodents such as rats, mice and rabbits. Furthermore, the gene encoding apelin as used herein is a polypeptide having one or several nucleotides deleted, substituted or added in addition to the gene encoding apelin and having apelin activity. And a gene encoding a polypeptide having apelin activity that hybridizes under high stringency conditions to a gene encoding apelin. Here, “apelin activity” is defined as the phosphorylation of intracellular signaling molecule Akt or the reduction of intracellular cAMP concentration by binding to the receptor APJ. The Highly stringent hybridization conditions refer to conditions including, for example, a sodium concentration of about 10 to 40 mM, preferably about 20 mM, and a temperature of about 50 to 70 ° C., preferably about 60 to 65 ° C.

  The APJ receptor, also called AGTRL1 (Angiotensin receptor-like 1), is a GPCR and consists of 380 amino acids. This transmembrane region shows 40-50% homology with the angiotensin (AT1) receptor. The apelin / APJ signal is a downstream signal of ANG1 / Tie2 in blood vessels and has been reported to be involved in myocardial contraction, blood pressure and blood flow, and angiogenesis (Kidoya et al, (2008) Spatial and temporal role of the apelin / APJ system in the caliber size regulation of blood vessels during angiogenesis. EMBO J 27: 522-534 etc.).

  However, the protective action of apelin on the skin has not been clarified so far.

  Accordingly, in one embodiment of the invention, an anti-inflammatory agent containing apelin is provided for preventing or treating skin inflammation. The anti-inflammatory agent of the present invention is particularly effective for skin inflammation caused by ultraviolet irradiation, and is also considered effective for dermatitis such as atopic dermatitis, redness, rosacea, and psoriasis.

  The dosage, usage, and dosage form of the anti-inflammatory agent of the present invention can be appropriately determined according to the purpose of use. For example, the dosage form of the anti-inflammatory agent of the present invention may be oral, parenteral, external use and the like. Examples of the dosage form include oral preparations such as tablets, powders, capsules, granules, extracts, and syrups, or parenteral preparations such as injections, drops, and suppositories, ointments, creams, emulsions, and lotions. And external preparations such as packs and bath preparations.

  The blending amount of apelin in the anti-inflammatory agent of the present invention can be appropriately determined according to the use, but is generally 0.00001 to 20.0% by mass, preferably 0.00001 to 10.0% by mass, based on the total amount of the anti-inflammatory agent.

  Further, in the anti-inflammatory agent of the present invention, in addition to apelin as an active ingredient, for example, pharmaceutically acceptable excipients, moisture-proofing agents, preservatives, reinforcing agents, thickeners, emulsifiers, antioxidants , Sweeteners, acidulants, seasonings, colorants, fragrances, whitening agents, moisturizers, oily ingredients, UV absorbers, surfactants, thickeners, alcohols, powder ingredients, colors, etc. An agent, an aqueous component, water, various skin nutrients, and the like can be appropriately blended as necessary.

  Further, when the anti-inflammatory agent of the present invention is used as an external preparation for skin, auxiliary agents commonly used for external preparations for skin, such as disodium edetate, trisodium edetate, sodium citrate, sodium polyphosphate, sodium metaphosphate, glucone Sequestering agents such as acids, caffeine, tannin, verapamil, tranexamic acid and its derivatives, licorice extract, grabrizine, hot water extract of karin fruit, various herbal medicines, tocopherol acetate, glycyrrhizic acid and its derivatives or salts thereof, etc. Whitening agents such as vitamin C, magnesium ascorbate phosphate, glucoside ascorbate, arbutin, kojic acid, sugars such as glucose, fructose, mannose, sucrose, trehalose, retinoic acid, retinol, retinol acetate, retinol palmitate Etc. Vita Such emission A compound also can be appropriately blended.

  In addition, when a gene encoding apelin (apelin gene) in a subject is in an inactive state or a silenced state, and as a result, the cell is in an apelin-deficient state, the apelin gene itself is introduced into the cell. Can be used. In the vector, it is preferable to arrange a regulatory sequence that enhances the expression of the apelin gene, such as a promoter or an enhancer, at a position operable with respect to the apelin gene.

  As a method for introducing an apelin gene into a cell, a gene introduction method using a viral vector or a non-viral gene introduction method (Nikkei Science, April 1994, pp. 20-45, Experimental Medicine Extra Number, 12 ( 15) (1994), experimental medicine separate volume "Basic technology of gene therapy", Yodosha (1996)) can be applied. As a gene transfer method using a viral vector, for example, a DNA virus such as a retrovirus, an adenovirus, an adeno-associated virus, a herpes virus, a vaccinia virus, a pox virus, a polio virus, a symbis virus, or an RNA virus, a DNA encoding apelin The method of introducing by incorporating is mentioned. Of these, methods using retroviruses, adenoviruses, adeno-associated viruses, and vaccinia viruses are particularly preferred. Non-viral gene transfer methods include a method in which an expression plasmid is directly administered intramuscularly (DNA vaccine method), a liposome method, a lipofectin method, a microinjection method, a calcium phosphate method, an electroporation method, etc. The vaccine method and the liposome method are preferred. In addition, in order for the above gene to actually act as a medicine, an in vivo method in which DNA is directly introduced into the body, and certain cells from a human are taken out and introduced into the cells outside the body, and the cells are returned to the body. There is an ex vivo method (Nikkei Science, April 1994, 20-45 pages, monthly pharmacy, 36 (1), 23-48 (1994), experimental medicine extra, 12 (15) (1994)). In vivo methods are more preferred. When administered by an in vivo method, it can be administered by an appropriate administration route according to the disease, symptoms and the like. For example, it can be administered intravenously, artery, subcutaneously, intradermally, intramuscularly. When administered by the in vivo method, it is generally an injection or the like, and a conventional carrier may be added if necessary. Moreover, when it is made into the form of a liposome or a membrane fusion liposome (Sendai virus (HVJ) -liposome or the like), it can be made into a liposome preparation such as a suspension, a freezing agent, and a centrifugal concentrated freezing agent.

  Accordingly, in a further embodiment of the present invention there is provided an anti-inflammatory agent comprising a vector comprising a gene encoding apelin for preventing or treating skin inflammation. In this case, the gene encoding apelin is preferably operably linked to a regulatory sequence that enhances expression of the apelin gene.

  The amount of the vector comprising the gene encoding apelin in the anti-inflammatory agent of the present invention can be appropriately determined according to the use, but is generally 0.00001 to 20.0% by mass, preferably 0.00001, based on the total amount of the anti-inflammatory agent. ~ 10.0 mass%.

The present invention further provides a method of screening for skin anti-inflammatory agents. This method is characterized in that a drug that enhances the expression of endogenous apelin in skin cells is selected as a skin anti-inflammatory agent. Enhancement of endogenous apelin expression can be determined, for example, by directly measuring the amount of apelin in skin cells. Skin cells, particularly epidermal cells, include keratinocytes, granule cells, spinous cells and the like, and may be derived from humans or other animals such as rats, mice, rabbits, and the like. Preferably, this measurement uses an antibody specific for apelin, and is a method well known in the art, such as immunostaining using a fluorescent substance, dye, enzyme, etc., Western blotting, immunoassay such as ELISA, Various methods such as RIA can be used. It can also be determined by extracting RNA from skin cells and measuring the amount of mRNA encoding apelin. Extraction of mRNA and measurement of the amount thereof are also well known in the art. For example, RNA is quantified by quantitative polymerase chain reaction (PCR). In addition to the above, the expression level of apelin can also be measured by measuring the known biological activity of apelin. Alternatively, apelin expression can be determined through in situ hybridization and measurement of its biological activity.
For example, if the expression of endogenous apelin in skin cells is increased by 30% or more, preferably 50% or more, more preferably 70% or more, and most preferably 100% or more compared to the control value, it is judged as a “skin anti-inflammatory agent” Yes, you can. Control values include, but are not limited to, for example, a statistically significant number (for example, 10 or more, preferably 100 or more) of healthy cells corresponding to the endogenous apelin of skin cells at the corresponding site. It may be an average expression level.

  In a preferred embodiment, the screening method further comprises applying the candidate drug having the enhancement ability to a model animal, for example, an apelin-deficient animal or an animal in which skin is inflamed due to ultraviolet irradiation, etc. Comprising confirming.

  The following examples further illustrate the present invention. Note that the present invention is not limited to this.

Example 1. Skin hematoxylin and eosin (HE) staining of epidermal apelin-expressing mice C57 / BL6 mice (12-week-old) (hereinafter referred to as apelin TG) with high expression of apelin in the epidermis under the control of the K14 promoter ultraviolet light with a wavelength of 280 to 340 nm For a total of 200 mJ / cm2. The auricular skin of this apelin TG mouse was collected and embedded in Tissue-Tech OCT compound (Sakura Finetechnical, Tokyo, Japan). Thereafter, frozen sections were sliced from the embedding block into 6 μm using a cryostat. The prepared tissue specimens were fixed with 10% formalin and stained with HE. As controls, tissue slices prepared from the skin of the auricles of C57 / BL6 mice (hereinafter referred to as WT) that were similarly irradiated with ultraviolet rays, WT that were not irradiated with ultraviolet rays, and apelin TG mice were used. Thereafter, the stained specimen was observed using an optical microscope, and the thickness of the skin was quantified using IP-Lab software.
As a result, in WT mice, the skin thickness was significantly increased by UVB irradiation, whereas in apelin TG mice, the skin thickness did not change even after UVB irradiation (FIGS. 1 and 2). From this, it was clarified that apelin has an effect of suppressing dermal edema.

Example 2. Immunostaining of skin inflammatory cells (macrophages) of epidermal apelin-expressing mice As described above, the auricular skin of apelin TG mice irradiated with ultraviolet rays was sliced and frozen sections were prepared. The prepared tissue specimen was fixed with acetone, and stained with a rat anti-CD11b antibody (BD Biosciences, Pharmingen, San Diego, Calif.) As a primary antibody. As the secondary antibody, Alexa Fluor488 rabbit anti-rat antibody (Molecular Probes, Eugene, OR) was used. As a control, WT similarly irradiated with ultraviolet rays, WT not irradiated with ultraviolet rays, and tissue sections prepared from auricular skin of apelin TG mice were used. After staining, inflammatory cells were detected using a fluorescence microscope.
As a result, in WT mice, macrophages were increased by UVB irradiation, but in TG mice, macrophages were not increased by UVB irradiation (FIG. 3). This revealed that apelin has a skin inflammation inhibitory effect.

Claims (3)

  An anti-inflammatory agent containing apelin for preventing or treating skin inflammation.   An anti-inflammatory agent comprising a vector comprising a gene encoding apelin for preventing or treating skin inflammation.   The anti-inflammatory agent according to claim 1 or 2, wherein the skin inflammation is an inflammation caused by ultraviolet irradiation.