KR101705253B1 - Pharmaceutical composition for prevention or treatment of inflammatory diseases comprising cerulenin or cerulenin derivative as an active ingredient - Google Patents

Abstract

The present invention relates to an anti-inflammatory composition comprising cerulenin, a cerulenin derivative or a pharmaceutically acceptable salt thereof as an active ingredient. The cerulenin or cerulenin derivative of the present invention exhibits excellent preventive, therapeutic and ameliorative effects on inflammatory diseases and can be applied not only to compositions for the prevention or treatment of inflammatory diseases but also as materials for related foods or cosmetics.

Description

TECHNICAL FIELD [0001] The present invention relates to a pharmaceutical composition for preventing or treating an inflammatory disease, which contains cerulenin or a cerulenin derivative as an active ingredient. The present invention relates to a pharmaceutical composition for preventing or treating an inflammatory disease,

The present invention relates to the use of cerulenin, a cerulenin derivative or a pharmaceutically acceptable salt thereof.

Inflammation is a localized immune response that minimizes the damage of a cell or tissue when it is damaged or destroyed by various factors, such as harmful substances or organisms from the outside, and restores the damaged area to the original state. And is a useful defense mechanism to remove the products produced by tissue damage. Several factors that cause inflammation include physical factors due to trauma, burns, frostbite, radiation, chemical factors caused by chemicals such as acid, and immunological factors due to antibody reaction. In addition, .

Inflammation is normal in case of in vivo inflammation reaction by neutralizing or eliminating the cause of the cause and regenerate the upper tissue to restore normal structure and function, but the degree of inflammation is more than a certain level or chronic inflammation and chronic inflammation It becomes a problem when proceeding to the same disease state.

The enzymes involved in the inflammatory response are produced by immune cells that migrate through the blood vessels to the affected area with the help of histamine, nitric oxide (NO) or prostaglandin E2 (PGE2) Initiates an inflammatory reaction. The immune cells migrated to the injured area were treated with TNF-α, interleukin-1β, interleukin-6, interleukin-6, IL-6, ), Such as cytokine or macrophage inflammatory protein (MIP-1), interleukin-8 (IL-8), monocyte chemotactic protein- MCP-1) to kill direct external invaders or to collect other immune cells to initiate an inflammatory response. When exposed to various inflammation-inducing cytokines such as interferon-γ, lipoteichoic acid, lipopolysaccharide (LPS) and the like, inflammatory inducers inducible nitric oxide synthase synthase, iNOS) and cyclooxygenase-2 (COX-2), resulting in excessive production of NO and PGE2. These inflammatory initiators (iNOS, COX-2, TNFα, IL-6, etc.) are promoted by activated NF-κB (nuclear factor-κB) , Tissue damage caused by inflammatory reaction, mutagenesis, and damage to nerve tissue. In addition, COX-2 overexpression causes an inflammatory reaction.

Steroidal anti-inflammatory drugs used in the treatment of chronic inflammatory diseases such as acute or rheumatoid arthritis are known to cause side effects such as glaucoma, cataract, hypertension, bipolar disorder, weight gain, diabetes and osteoporosis. Therefore, there is a demand for development of a substance capable of effectively inhibiting inflammation, which is free from the risk of such side effects or cytotoxicity or is derived from a small amount of natural substances.

Cerulenine inhibits the activity of HMG-CoA reductase to inhibit sterol biosynthesis. When fatty acid biosynthesis occurs, it inhibits the activity of fatty acid biosynthesis enzyme by reacting with the cysteine in the fatty acid biosynthetic enzyme to form a covalent bond . In addition, cerulenin inhibits the biosynthesis of polyketide, and is also useful as a biosurfactant for the production of polyketide biosynthesis pathway, such as Leucomycin, Methylsalicylic acid, Candicidin, Flavanone, Alternaria and the like are all known to be inhibited by cerulenin. However, research on its industrialization has not yet been found. In Korean Patent Laid-Open No. 10-2007-0023730 concerning "sebum production and pore size reduction method", the effect of inhibiting sebum production of cerulenin is disclosed.

Accordingly, the present inventors have completed the present invention by discovering that cerulenin or cerulenin derivatives have excellent anti-inflammatory activity without side effects.

Korean Patent Publication No. 10-2007-0023730

Accordingly, an object of the present invention is to provide a composition for preventing or treating inflammatory diseases, which comprises cerulenin, a cerulenin derivative or a pharmaceutically acceptable salt thereof as an active ingredient.

Another object of the present invention is to provide a composition for improving inflammation, which comprises cerulenin, a cerulenin derivative or a pharmaceutically acceptable salt thereof as an active ingredient.

In order to achieve the above object, the present invention provides a pharmaceutical composition for preventing or treating inflammatory diseases, which comprises cerulenin, a cerulenin derivative or a pharmaceutically acceptable salt thereof as an active ingredient.

To achieve these and other objects, the present invention provides an inflammation-improving food and cosmetic composition containing cerulenin, a cerulenin derivative or a pharmaceutically acceptable salt thereof as an active ingredient.

The composition containing the cerulenin, cerulenin derivative or a pharmaceutically acceptable salt thereof according to the present invention as an active ingredient is derived from a natural substance, and as a result of MTT analysis, there is no possibility of occurrence of side effects due to no cytotoxicity, , Can be used very effectively for prevention or treatment.

Fig. 1 is a graph showing inhibition of NO formation by cerulenin and comparison with BAY11-7082. In the graph, the abscissa represents the treatment concentration of cerulenin or BAY11-7082, and the ordinate represents the amount of NO produced.
FIG. 2 is a graph showing cellulolytic cytotoxicity and comparison with BAY11-7082. The abscissa in the graph indicates the treatment concentration of cerulenin or BAY11-7082, and the ordinate indicates cell viability (%) .
FIG. 3 is a graph showing that cerulenin inhibits mRNA expression of inflammation-related factors iNOS, COX-2, TNF ?, IL-6, MCP-1 and IL-1. And 100 ng / ml of LPS represents the group treated with 100 ng / ml of LPS. In addition, the vertical axis indicates the degree of mRNA expression relative to CypA mRNA expression of each inflammation-related factor.
Fig. 4 is an electrophoresis image showing that cerulenin inhibits the expression of iNOS and COX-2, which are inflammation-related factors. The numbers on the figure are the concentrations of LPS (ng / ml) Lt; RTI ID = 0.0 > primary < / RTI > antibody.
FIG. 5 is a graph showing inhibition of NF-κB transcriptional activity of cerulenin. The abscissa of the graph indicates the treatment and concentration of LPS and the ordinate indicates the reporter activity of the NF-κB reporter activity. Lt; RTI ID = 0.0 > NF-kB < / RTI >
FIG. 6 is a graph showing that cerulenin inhibits the increase of serum TNF-alpha concentration by LPS administered to mice. The abscissa of the graph indicates whether cerulenin and LPS are administered, and the ordinate axis indicates the serum concentration of TNF- ng / ml).
FIG. 7 is a graph showing inhibition of the formation of NO of the cerulenin derivative C75 and comparison with cerulenine. The abscissa of the graph represents the treatment concentration of the samples and the ordinate represents the amount of NO produced.
FIG. 8 is a graph comparing the cellulolytic activity of cerulenin derivative C75 with that of cerulenin. In FIG. 8, the abscissa of the graph represents the treatment concentration of the sample and the ordinate represents the cell viability (%).
FIG. 9 is a graph showing that the cerulenin derivative C75 inhibits mRNA expression of inflammation-related factors and is compared with cerulenin. The abscissa of the graph indicates the treatment and concentration of LPS and the ordinate indicates CypA mRNA expression relative to mRNA expression.
FIG. 10 is a graph showing that cerulenin increases the survival rate of animals in an animal model of sepsis. In the graph, the horizontal axis shows the time elapsed after administration of galactosamine N and LPS for inducing sepsis, Lt; / RTI >

Hereinafter, the present invention will be described in detail.

The present invention provides a composition for preventing or treating an inflammatory disease containing cerulenin, a cerulenin derivative or a pharmaceutically acceptable salt thereof as an active ingredient.

The cerulenin or cerulenin derivative may be represented by the following formula (1) or (2), respectively:

[Chemical Formula 1]

, 또는

, or

(2)

.

.

The cerulenin (C ₁₂ H ₁₇ NO ₃ ; The molecular weight of (2R, 3S) -3 - [(4E, 7E) -nona-4,7-dienoyl] oxirane-2-carboxamide is 223.2 (g / mol).

The cerulenin may be extracted from Cephalosporium caerulens or may be prepared by a method known in the art.

A molecular weight of 254.32 (g / _mol); the serul renin derivative C75 (4-Methylene-2- octyl-5-oxotetrahydrofuran-3-carboxylic acid C 14 H 22 O 4).

The cerulenin or cerulenin derivative which is an active ingredient of the composition according to the present invention can be used for prevention and treatment of inflammatory diseases by inhibiting the transcription activity of NF-κB, the production of nitrogen monoxide and the expression of inflammation-related factors.

The inflammatory-related factor may be selected from the group consisting of inducible nitric oxide synthases (iNOS), cyclooxygenase-2, COX-2, interleukin-6, IL-6, In the group consisting of monocyte chemotactic protein-1, MCP-1, interleukin-1β, IL-1β and tumor necrosis factor-α Can be selected.

The composition according to the present invention exhibits concentration-dependent NO production inhibitory activity without toxicity. Specifically, according to one embodiment of the present invention, each composition containing 1, 5, 10 and 2 μM of cerulenin has been found to inhibit NO production without toxicity (Examples 1-2, Figures 1 and 2) 2).

The composition according to the present invention shows an inhibitory activity on the expression of iNOS, COX-2, TNF- ?, IL-6, MCP-1? And IL-1 ?. Specifically, according to one embodiment of the present invention, mRNA expression of iNOS, COX-2, TNF-a, IL-6, MCP-1 and IL-1β induced by LPS in the experimental group treated with cerulenin (Examples 1-3, see Fig. 3).

The composition according to the present invention shows the inhibitory activity of iNOS and COX-2. Specifically, according to one embodiment of the present invention, it was confirmed that the expression of proteins of iNOS and COX-2 induced by LPS was remarkably inhibited in the experimental group treated with 10 μM of cerulenin (Examples 1-4, Fig. 4 Reference).

The composition according to the present invention exhibits NF-kB transcriptional activity inhibitory activity. Specifically, according to one embodiment of the present invention, it was confirmed that LPS-induced NF-κB transcriptional activity was remarkably inhibited in the experimental group treated with 10 μM of cerulenin (see Examples 1-5, FIG. 5).

The composition according to the present invention exhibits TNF-α plasma concentration-inhibiting activity. Specifically, according to one embodiment of the present invention, it was confirmed that cerulenin significantly inhibits the secretion of TNF-? By LPS (see Examples 1-6, Fig. 6).

The composition according to the present invention exhibits concentration-dependent NO production inhibitory activity without toxicity. Specifically, according to one embodiment of the present invention, the cerulenin derivative C75 of 1, 5 and 10 μM was found to inhibit the production of NO by LPS without toxicity (see Example 2, FIGS. 7 and 8).

The composition according to the present invention shows mRNA expression inhibitory activity of iNOS, COX-2 and IL-6. Specifically, according to one embodiment of the present invention, it was confirmed that mRNA expression of iNOS, COX-2 and IL-6 induced by LPS was remarkably inhibited in the experimental group treated with 10 μM of cerulenin derivative C75 (Example 2, Fig. 9).

The composition according to the present invention increases the survival rate of an animal model of sepsis. Specifically, according to one embodiment of the present invention, the survival rate for sepsis induced by galactosamine N and LPS in the experimental group treated with cerulenin at 15, 30 or 60 mg / kg was remarkably increased (Example 3, see Fig. 10).

The inflammatory disease may be chronic inflammatory disease, acute inflammatory disease or other inflammatory-related diseases, but is not limited thereto.

The chronic inflammatory disease may be rheumatoid arthritis, arteriosclerosis, diabetes, osteoporosis, Alzheimer's disease, Parkinson's disease, lupus or multiple sclerosis.

The acute inflammatory disease may be sepsis, shock or rejection of organ transplantation.

The other inflammatory diseases may be ophthalmic diseases, bronchitis, dermatitis, allergy, systemic lupus erythematosis, retinitis, gastritis, hepatitis, enteritis, pancreatitis or nephritis.

The dermatitis or allergy may be hypersensitivity, allergic rhinitis, asthma, allergic conjunctivitis, allergic dermatitis, atopic dermatitis, contact dermatitis, urticaria, insect allergies, food allergies or drug allergies.

The present invention also provides a pharmaceutically acceptable salt of cerulenin or a cerulenin derivative represented by the above formula (1) or (2). Pharmaceutically acceptable salts should be low in toxicity to humans and should not adversely affect the biological activity and physicochemical properties of the parent compound. Pharmaceutically acceptable salts include salts of acid addition salts, alkali metal salts (such as sodium salts) and alkaline earth metal salts (such as calcium salts) of a basic compound of the formula (1) with pharmaceutically usable free acids, organic bases and carboxylic acids of the formula Organic base addition salts, amino acid addition salts, and the like.

Preferred salts of the compounds according to the invention include salts with inorganic or organic acids. The inorganic acid may be hydrochloric acid, sulfuric acid, nitric acid, phosphoric acid, perchloric acid, bromic acid, or the like. In addition, the organic acid may be acetic acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, fumaric acid, maleic acid, malonic acid, phthalic acid, succinic acid, lactic acid, citric acid, citric acid, gluconic acid, Oxalic acid, benzoic acid, embonic acid, aspartic acid, glutamic acid, and the like. Organic bases that can be used to prepare organic base addition salts include tris (hydroxymethyl) methylamine, dicyclohexylamine, and the like. Amino acids that can be used in the production of amino acid addition bases are natural amino acids such as alanine and glycine.

Such salts may be prepared by conventional methods. For example, the compound of formula (1) or (2) can be prepared by dissolving the compound of formula (1) or (2) in a solvent which can be mixed with water such as methanol, ethanol, acetone or 1,4-dioxane and then adding a free acid or a free base .

In addition, the compounds according to the present invention may have asymmetric carbon centers and therefore may exist as R or S isomers, racemic compounds, individual enantiomers or mixtures, individual diastereomers or mixtures thereof, All stereoisomers and mixtures are included within the scope of the present invention.

Also included within the scope of the present invention are the forms of the hydrates or solvates of the heterocycle derivatives of formula (I). Such a hydrate or solvate may be prepared by a known method and is preferably non-toxic and water-soluble, and may be a hydrate or a solvate in which one to five molecules of water or an alcohol-based solvent (particularly, ethanol etc.) .

The composition according to the present invention has an anti-inflammatory effect and may further contain one or more substances known to have an anti-inflammatory effect in the composition of the present invention. The known substances may be COX-2 inhibitors or nitrogen monoxide (NO) inhibitors, but are not limited thereto.

The composition according to the present invention may further contain one or more pharmaceutically acceptable additives selected from the group consisting of excipients, lubricants, wetting agents, sweeteners, fragrances and preservatives.

Each of the compositions according to the present invention can be formulated and used according to a conventional method. Can be formulated employing methods known in the art to provide rapid, sustained or delayed release of the active ingredient, especially after administration to the mammal.

The method of administration of the composition according to the present invention can be easily selected according to the formulation, and can be administered orally or parenterally. But are not limited to, intravenous, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, intrathecal, and oral routes.

Solid form preparations for oral administration may be, but are not limited to, tablets, pills, soft or hard capsules, pills, powders or granules. Meanwhile, the form for parenteral administration may be in the form of a cream, a lotion, an ointment, a warning agent, a liquid agent, a patch or an injection, but is not limited thereto.

The dosage of the composition according to the present invention may vary depending on the patient's age, sex, weight, degree of pathology, and route of administration, but is generally in the range of 5 to 500 mg / kg, preferably 100 to 250 mg / The dose can be administered once or three times a day. However, the dosages do not in any way limit the scope of the invention.

In addition, the present invention provides an inflammation-improving food composition comprising cerulenin, a cerulenin derivative or a pharmaceutically acceptable salt thereof.

The cerulenin or cerulenin derivative may be represented by the above formula (1) or (2), respectively.

The food composition for improving inflammation of the present invention comprises the compound represented by the formula (1) or (2) of the present invention, or a pharmaceutically acceptable salt thereof in an amount of 0.1 to 100% by weight, preferably 20 to 80% .

The inflammation-improving food composition of the present invention may be in the form of powder, granules, tablets, capsules or beverages, and may further contain other natural or synthetic substances for health function and additives for commercialization.

The health drink of the present invention may contain 0.1 to 50 g, preferably 1 to 10 g, of the compound represented by Formula 1 or 2, or a pharmaceutically acceptable salt thereof, based on 100 ml. In addition, there are no particular limitations on the liquid components other than those containing the above-mentioned compounds as essential components in the indicated ratios, and may contain various flavors or natural carbohydrates as additional components such as ordinary beverages. Examples of the above-mentioned natural carbohydrates include monosaccharides, polysaccharides such as disaccharides such as glucose and fructose, such as maltose and sucrose, and the like, such as dextrin, cyclodextrin and the like, and Xylitol, sorbitol, and erythritol. As a flavoring agent other than the above, a natural flavoring agent (tautatin, stevia extract (e.g., rebaudioside A, glycyrrhizin etc.)) and a synthetic flavoring agent (saccharin, aspartame etc.) . The ratio of the natural carbohydrate is generally about 1 to 20 g, preferably about 5 to 12 g per 100 mg of the compound of the present invention.

In addition to the above, the inflammation-improving food composition of the present invention may further contain flavoring agents such as various nutrients, vitamins, minerals (electrolytes), synthetic flavors and natural flavors, coloring agents and thickening agents such as cheese and chocolate, , Alginic acid and its salts, organic acids, protective colloid thickeners, pH adjusting agents, stabilizers, preservatives, glycerin, alcohols, carbonating agents used in carbonated drinks and the like. It may also contain flesh for the production of natural fruit juices, fruit juice drinks and vegetable drinks. These components may be used independently or in combination.

Examples of the inflammation-improving food to which the compound represented by Formula 1 or 2 of the present invention or the pharmaceutically acceptable salt thereof can be added include various foods, beverages, gums, vitamins complexes, health supplements, etc. .

The present invention also provides a cosmetic composition for improving inflammation comprising cerulenin, a cerulenin derivative or a pharmaceutically acceptable salt thereof.

The cerulenin or cerulenin derivative may be represented by the above formula (1) or (2), respectively.

The cosmetic composition of the present invention can be directly applied to skin for the purpose of improving inflammation.

The cosmetic composition may be formulated into cosmetic formulations conventionally produced in the art. The cosmetic composition may be formulated, for example, as a solution, suspension, emulsion, paste, gel, cream, lotion, powder, However, the present invention is not limited thereto. More specifically, it can be formulated into a formulation of a soft lotion, a nutritional lotion, a nutritional cream, a massage cream, an essence, an eye cream, a cleansing cream, a cleansing foam, a cleansing water, a pack, a spray or a powder.

When the formulation of the cosmetic composition of the present invention is a paste, a cream or a gel, it may be an animal oil, vegetable oil, wax, paraffin, starch, tragacanth, cellulose derivative, polyethylene glycol, silicone, bentonite, silica, talc, zinc oxide, , ≪ / RTI > and the like.

When the formulation of the cosmetic composition of the present invention is a powder or a spray, it may contain a carrier component selected from the group consisting of lactose, talc, silica, aluminum hydroxide, calcium silicate, polyamide powder and mixtures thereof, , It may further include chlorofluorohydrocarbons, propane / butane, dimethyl ether, and the like.

When the formulation of the cosmetic composition of the present invention is a solution or an emulsion, it may contain a carrier component selected from the group consisting of a solvent, a solubilizing agent, an emulsifying agent and a mixture thereof. Examples thereof include water, ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butyl glycol oil, glycerol aliphatic ester, polyethylene glycol, sorbitan fatty acid ester, .

When the formulation of the cosmetic composition of the present invention is a suspension, it may be diluted with water, a liquid diluent such as ethanol or propylene glycol, a suspension such as ethoxylated isostearyl alcohol, polyoxyethylene sorbitol ester and polyoxyethylene sorbitan ester, Hydroxypropylmethylcellulose, hydroxypropylmethylcellulose, hydroxypropylcellulose, hydroxypropylmethylcellulose, hydroxypropylmethylcellulose, hydroxypropylmethylcellulose, hydroxypropylcellulose, hydroxypropylcellulose, hydroxypropylmethylcellulose,

When the formulation of the cosmetic composition of the present invention is a surfactant-containing cleansing, the surfactant-containing cleansing agent may be selected from the group consisting of aliphatic alcohol sulfate, aliphatic alcohol ether sulfate, sulfosuccinic acid monoester, isethionate, imidazolinium derivative, methyl taurate, sarcosinate, A fatty acid diethanolamide, a vegetable oil, a lanolin derivative, an ethoxylated glycerol fatty acid ester, and a mixture thereof.

The "carrier component" in the cosmetic composition of the present invention is a known or used compound or composition which can be contained in a cosmetic preparation, and which is free from toxicity, instability or irritation which can be adapted to human body upon contact with skin.

The cosmetic composition of the present invention may further comprise, in addition to the carrier component, an adjuvant selected from the group consisting of antioxidants, stabilizers, solubilizers, moisturizers, pigments, fragrances, ultraviolet screening agents, coloring agents, surfactants and combinations thereof. The adjuvant is not limited as long as it is an adjuvant commonly used in the production of a cosmetic composition.

Hereinafter, the present invention will be described in more detail with reference to the following examples. However, the following examples are for illustrative purposes only and are not intended to limit the scope of the present invention.

[ Example ]

Example  One. Cerulenine  Identify anti-inflammatory activity

Example  1-1. Preparation of samples, cells and mice

To measure anti-inflammatory activity of cerulenin, the following samples, cells and mice were prepared.

(Dulbecco's modified Eagles medium), Fetal bovine serum (FBS) and penicillin / streptomycin in Hyclone (Thermo Scientific Inc., Germany), SYBR green in Roche The luciferase reporter system and the Griess reagent system were purchased from promega (USA), and the TNF-α ELISA kit was purchased from the R & D system (USA). In addition, anti-iNOS polyclonal antibody was purchased from BD bioscience (USA), anti-COX2 polyclonal antibody was purchased from Cell Signaling (USA), and anti-HSC70 monoclonal antibody was purchased from Rockland (USA). Cerulenine, BAY11-7082, MTT and the remaining reagents were purchased from Sigma Aldrich Co. (USA).

Experiments to determine the anti-inflammatory activity of cerulenin used the macrophage cell line RAW264.7 purchased from ATCC and peritoneal macrophages isolated from mice. In the RAW264.7 cells and restore gangdaesik 10% FBS and penicillin (100U / ㎖) / streptomycin (100㎍ / ㎖) a high content of glucose (high glucose) using DMEM 37 ℃, 5% CO ₂ conditions, the addition of Lt; / RTI >

C57 / BL6 mouse species were used for the measurement of anti-inflammatory activity of cerulenin in animal models. Diets were fed with solid diet and mice and diets were purchased from Hyochang Science (Korea). Breeding of the experimental animals was carried out at a temperature of 20 to 24 캜, a humidity condition of 60% to 70% and a daylighting condition of a 12-hour period, and water and diet were freely ingested.

Anti-inflammatory activity of cerulenin was measured by measuring the mRNA expression, protein expression, transcription activity and NO production of the factors involved in inflammation using the above cells, mouse, reagent and apparatus. Experimental results for confirming anti-inflammatory activity were expressed as mean ± SD. With respect to statistical significance, p <0.05 was considered to be statistically significant, defined as * (p <0.05).

Example  1-2. Suppression of nitrogen monoxide (NO) formation and toxicity

In order to confirm the anti-inflammatory activity of cerulenin, inhibition of NO production was analyzed. For comparison of anti-inflammatory activity, BAY11-7082, an NF-κB inhibitor known to have anti-inflammatory activity, was used.

Mouse macrophages RAW264.7 cells were seeded at 5 × 10 ⁴ cells / well in a 96-well plate and stabilized for 16 hours. Then, cerulenin and BAY11-7082 were cultured for 1 hour (1, 5, 10, 20 μM) Lt; / RTI &gt; Thereafter, LPS was treated as an inflammatory reaction derivative at a concentration of 100ng / ml and cultured for 16 hours. A grease reagent was added to the culture solution to quantify the NO content. Specifically, 50 占 퐇 of the cultured cell culture solution and 50 占 퐇 of a grease reagent were mixed and allowed to react at room temperature for 5 minutes, and the absorbance was measured at 540 nm using a microplate reader (Tecan). A standard curve was obtained using 0.1 M nitrite standard solution, and the NO concentration of the cell culture fluid was calculated. The calculated NO value is shown in Fig.

In order to confirm the toxicity of cerulenin, a solution of MTT (3- (4,5-dimethylthiazol-2-yl) -2,5-diphenyl tetrazolium bromide) (5 mg / ) Was added and then cultured for 1 hour. Thereafter, all of the culture medium was removed, and the formazan crystal thus formed was dissolved in 150 mu l of DMSO, and the absorbance was measured at 570 nm using a microplate reader. The results of the measurement are shown in FIG. 2, and the cell viability was expressed as a percentage of the control group treated with LPS alone.

As a result, as shown in Fig. 1 and Fig. 2, it was confirmed that cerulenin inhibits NO production at each concentration (1, 5, 10, 20 μM) without toxicity. On the other hand, BAY11-7082 showed a similar inhibitory effect on the production of NO, similar to cerulenin, but showed considerable toxicity. Specifically, cerulenin inhibited increased NO production by LPS treatment, decreased NO content significantly from 1 μM treatment, and decreased NO content in a concentration - dependent manner. In addition, BAY11-7082 showed toxicity, but cerulenin did not cause toxicity to cells even when treated at a concentration of 20 μM.

Example  1-3. Inflammatory factors mRNA  Confirmation of expression inhibition

In order to confirm the antiinflammatory activity of cerulenin, the inhibition of mRNA expression of inflammatory factors induced by LPS in primary cells, a primary cell, was analyzed. In other words, mRNA expression of iNOS, COX-2, TNF-α, IL-6, MCP-1 and IL-1β, the target genes of NF-κB, .

Specifically, to obtain the above-described peritoneal macrophages, 1 ml of 3% thioglycollate was intraperitoneally administered to a male mouse (C57 / BL6) having a body weight of 20 g to 25 g, and after 4 days from administration, The granular cells were obtained. The obtained cells were cultured in DMEM medium for 24 hours, and cultured in a 6-well plate at 200 × 10 ⁴ cells / well for 24 hours. Thereafter, cerulenin was pretreated with a concentration of 10 μM for 1 hour, treated with LPS (100 ng / ml) for 1 hour to confirm the expression of TNF-α, and cultured for 6 hours for other cytokines. After the LPS treatment, RNA was isolated from each cell.

Isolation of the RNA was carried out in the following manner. The cultured cells were washed twice with 1 ml of PBS and lysed with 500 μl of trizol reagent (Life technologies), followed by addition of 100 μl of chloroform and vortexing. The mixture was incubated at room temperature for 10 minutes and centrifuged at 13,000 rpm for 15 minutes to obtain 400 μl of supernatant. The supernatant was then incubated with 500 μl of isopropanol for 10 minutes at room temperature. Thereafter, the mixture was centrifuged at 13,000 rpm for 15 minutes to obtain a pellet. The obtained pellet was washed with 1 ml of 75% ethanol and dried at room temperature to isolate RNA.

The isolated RNA was purified using 0.1% DEPC (Diethyl pyrocarbonate) Was dissolved in 20 ㎕ to 30 용액 of the solution and used for cDNA synthesis. CDNA was synthesized using M-MLV reverse transcriptase (promega) by dividing 4 g of the separated RNA, and real-time PCR analysis was performed for measuring mRNA content of inflammatory factors using the cDNA.

The sequences of the primers and the experimental conditions for measuring the contents of the inflammation-related factors iNOS, COX-2, TNF ?, IL-6, MCP-1 and IL-1?

The degree of mRNA expression of the inflammation-related factors is shown in FIG. 3, and the degree of mRNA expression was corrected by the ratio to the amount of CypA mRNA as a control group.

As a result, mRNA expression of iNOS, COX-2, TNF-α, IL-6, MCP-1 and IL-1β was low in the control group not treated with LPS, mRNA expression of iNOS, COX-2, TNF-α, IL-6, MCP-1 and IL-1β was increased and the increase in mRNA expression induced by LPS was significantly suppressed in the group treated with cerulenin 10 μM appear.

Example  1-4. Confirmation of inhibition of protein expression by inflammation-related factors

In order to confirm the anti-inflammatory activity of cerulenin, inhibition of protein expression of iNOS and COX-2, which are inflammation-related factors induced by LPS, in the primary cell, peritoneal macrophages, was confirmed by immunoblot analysis .

The primary cells were obtained by raising the experimental animals in the same manner as in Example 1-3, cultured in DMEM medium for 24 hours, and then divided into 6-well plates at 200 × 10 ⁴ cells / well and cultured for 24 hours Respectively. Thereafter, cerulenin was pretreated at a concentration of 10 μM for 1 hour, and treated with LPS (100 ng / ml or 1000 ng / ml) and cultured for 16 hours. After the LPS treatment, proteins were separated from each cell.

Protein separation was carried out in the following manner. First, the cultured cells were washed twice with 1 ml of PBS, and then PBS was removed, and the cells were washed with 0.01 M Tris-HCl (pH 7.4), 0.15 M NaCl, 0.001 M EDTA, 0.001 M EGTA, 1% Triton X- 200 μl of RIPA buffer containing 0.002 M PMSF (phenylmethanesulfonyl fluoride) and a protease inhibitor was added, and the mixture was vortexed, placed on ice for 30 minutes, and then centrifuged at 13,000 rpm for 10 minutes. The supernatant was then obtained and the protein content was quantified using BCA reagent (pierce biotechnology).

The quantified protein samples were equally divided, mixed with a 5 x SDS sample buffer, boiled at 95 for 5 minutes, and subjected to SDS-PAGE using 7% gel. Proteins from the electrophoresed gels were transferred to a nitrocellulose membrane and pretreated with 5% skim milk for 1 hour at room temperature to inhibit nonspecific antibody binding. Subsequently, anti-iNOS polyclonal antibody, anti-COX-2 polyclonal antibody and anti-HSC70 monoclonal antibody were added to 5% skim milk and reacted with the membrane for 4 to 24 hours depending on the type of antibody. After the reaction, the cells were washed 3 times with PBST for 10 minutes and then reacted with the secondary antibody at room temperature for 1 hour. The membranes were then washed three times for 10 minutes with PBST and reacted with ECL Western Blotting Detection Reagent (GE healthcare) to confirm the expression of each protein with LAS2000 (GE healthcare).

As a result, as shown in FIG. 4, the expression of iNOS and COX-2 was not observed in the control group not treated with LPS, but increased when treated with 100 ng / ml or 1000 ng / ml of LPS. In addition, it was confirmed that protein expression of iNOS and COX-2 increased by LPS was significantly inhibited by cerulenin. That is, cerulenin has excellent anti-inflammatory activity.

Example  1-5. NF Confirmation of transcriptional activity inhibition of -KB

In order to confirm anti-inflammatory activity of cerulenin, inhibition of NF-κB transcription activity by LPS in RAW264.7 cells was confirmed by Luciferase assay.

Specifically, the RAW264.7 cells were transfected with NF-κB reporter vector and Renilla vector using lipofectamine 2000 (life technologies), and the cells were cultured in a 24 well plate with glucose-rich DMEM medium at 40 × 10 ⁴ cells / well. After 6 hours of incubation, the medium was changed and incubated again for 16 hours. After the incubation, cerulenin or BAY11-7082 was added at 10 μM, and after 1 hour, 100 ng / ml of LPS was treated and cultured for 8 hours, and 100 μl of lysis buffer was added. The above cell lysate was dispensed into a 96-well white plate in an amount of 50, and the degree of NF-κB transcription activity was measured using a dual-luciferase reporter system (promega). The results are shown in FIG. At this time, the degree of NF-κB transcription activity was corrected by the ratio of the Renilla value, which is a control group of each sample.

As a result, as shown in FIG. 5, it was confirmed that the transcription activity of NF-κB was low in the control group not treated with LPS, but increased when LPS was treated. In addition, it was confirmed that the NF-κB transcriptional activity increased by LPS was inhibited by cerulenin and that the inhibition of NF-κB transcriptional activity of cerulenin was superior to that of BAY11-7082. In other words, cerulenin showed excellent anti-inflammatory activity by inhibiting NF-κB transcription activity.

Example  1-6. From the mouse LPS Induced by TNF -a decrease in blood concentration

In order to confirm secretion inhibition of inflammatory cytokines of cerulenin, it was confirmed whether inhibition of LPS-induced serum TNF-a concentration increase in C57 / BL6 mouse species.

Specifically, 8-week-old C57 / BL6 male mice were divided into groups in which none of 4 rats were injected, 60 mg / kg of cerulenin, 50 mg / kg of LPS, and 60 mg / kg of cerulenin And 1 hour after the administration of LPS (50 mg / kg). One hour after administration of LPS, 200 [mu] l of blood was obtained from the mice, and the serum was separated by centrifugation at 13,000 rpm for 3 minutes with a BD micro-container (BD microtainer). After the separated serum was diluted 50-fold, serum TNF-α concentration was measured using a TNF-α ELISA kit (R & D system).

As a result, as shown in FIG. 6, although the serum concentration of TNF-α was low in the control group to which LPS was not administered, it was confirmed that the serum concentration of TNF-α was increased when LPS was administered. In addition, it was confirmed that the serum concentration of TNF-alpha increased by LPS was inhibited by cerulenin. In other words, cerulenin significantly inhibited the secretion of TNF-α by LPS.

Example  2. Cerulenine  derivative C75 Of anti-inflammatory activity

The inhibition of NO production induced by LPS of cerulenin and inhibition of mRNA expression of inflammation-related factors confirmed in Examples 1-2 and 1-3 were confirmed by using cerulenin derivative C75.

Specifically, RAW264.7 cells were treated with C75 and cerulenin at 1, 5, and 10 mu M in the same manner as in Example 1-2, and inhibition of NO production was confirmed. The results are shown in Fig. 7, The results are shown in Fig. In addition, the inhibition of mRNA expression of inflammatory factors induced by LPS by C75 and cerulenin was confirmed in the same manner as in Example 1-3, and is shown in FIG.

As a result, as shown in Figs. 7 and 8, C75 and cerulenin inhibited NO production at each concentration (1, 5, 10 μM) without toxicity. In addition, as shown in Fig. 9, C75 and cerulenin inhibited mRNA expression of iNOS, COX-2 and IL-6.

Example  3. In the mouse model Cerulenine  Confirmation of sepsis treatment effect

In order to confirm that cerulenin shows therapeutic effects of inflammatory diseases in animal models, cerulenin was treated in a sepsis mouse model and the following experiment was conducted.

Specifically, experiments were carried out on 8 mice of 8 weeks old C57 / BL6 mice, each having a body weight of about 25 g, as one group. First, cerulenin dissolved in 40% DMSO solution was administered intraperitoneally to mice of each group at a concentration of 15, 30 and 60 mg / kg, respectively, 1 hour before the induction of sepsis, and only 40% DMSO solution was administered to the mice as a control group . Then, in order to induce sepsis, galactosamine N and LPS were dissolved in PBS to a concentration of 700 mg / kg and 150 μg / kg, respectively, and the mice were intraperitoneally administered. The survival rate Were measured. Survival was assessed every hour after induction of sepsis.

As a result, a relative value based on the survival rate of the mouse 1 hour after the induction of sepsis was shown in FIG.

As shown in Fig. 10, in the mouse group to which cerulenin was administered at a concentration of 30 mg / kg, the survival rate rapidly increased from 6 hours after the induction of sepsis to 8 hours after the administration of cerulenin at the concentration of 15 mg / After 12 hours, the survival rate was about 10% in the 15 mg / kg group and about 25% in the 30 mg / kg group. On the other hand, the survival rate of cerulenin 60mg / kg group rapidly decreased after 7 hours of induction of sepsis to 60%, but after that, the survival rate was gradually decreased after 12 hours. On the other hand, the solvent control group showed a 20% reduction in survival rate after 7 hours of induction of sepsis and a 10% decrease after 12 hours.

From the above results, it was confirmed that cerulenin of the present invention, which inhibits the expression and activity of inflammatory factors, has an effect of preventing and treating septicemia by increasing the survival rate in an animal model for producing sepsis, .

<110> UNIST Academy-Industry Research Corporation <120> PHARMACEUTICAL COMPOSITION FOR PREVENTION OR TREATMENT OF          INFLAMMATORY DISEASES COMPRISING CERULENIN OR CERULENIN          DERIVATIVE AS AN ACTIVE INGREDIENT <130> FPD / 201508-0087 / C <150> KR 2014/134560 <151> 2014-10-06 <160> 14 <170> Kopatentin 2.0 <210> 1 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> sense primer for iNOS <400> 1 gctcatgcgg cctccttt 18 <210> 2 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> Antisense primer for iNOS <400> 2 cctggtacgg gcattgct 18 <210> 3 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> sense primer for COX-2 <400> 3 tgctgtacaa gcagtggcaa 20 <210> 4 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Antisense primer for COX-2 <400> 4 agggctttca attctgcagc ca 22 <210> 5 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> sense primer for TNF-alpha <400> 5 tgggacagtg acctggactg t 21 <210> 6 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Antisense primer for TNF-alpha <400> 6 ttcggaaagc ccatttgagt 20 <210> 7 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> sense primer for IL-6 <400> 7 acttcacaag tcggaggctt 20 <210> 8 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Antisense primer for IL-6 <400> 8 ctgaaggact ctggctttgt ct 22 <210> 9 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> sense primer for MCP-1 <400> 9 aactgcatct gccctaaggt 20 <210> 10 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Antisense primer for MCP-1 <400> 10 agtgcttgag gtggttgtgg aa 22 <210> 11 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> sense primer for IL-1beta <400> 11 gggctgcttc caaacctttg ac 22 <210> 12 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Antisense primer for IL-1beta <400> 12 atgggaacgt cacacaccag ca 22 <210> 13 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> sense primer for CypA <400> 13 cagccatggt caaccccacc g 21 <210> 14 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Antisense primer for CypA <400> 14 ctgctgtctt tggaactttg tctg 24

Claims (10)

A pharmaceutical composition for preventing or treating inflammatory diseases, comprising cerulenin represented by the following formula (1) or a pharmaceutically acceptable salt thereof as an active ingredient,
Wherein the inflammatory disease is selected from the group consisting of rheumatoid arthritis, sepsis, rejection of organ transplantation, dermatitis, allergy, retinitis, gastritis, hepatitis, enteritis, pancreatitis and nephritis.
[Chemical Formula 1]

.
The method according to claim 1,
Wherein said composition inhibits the transcriptional activity of NF-kB, the production of nitrogen monoxide or the expression of inflammatory-related factors.
3. The method of claim 2,
The inflammation-related factor is selected from the group consisting of inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), interleukin-6 (IL-6), monocyte chemotactic protein- 1 &lt; / RTI &gt; beta (IL-1 beta) and tumor necrosis factor-alpha (TNF-alpha).
delete delete delete delete delete CLAIMS 1. A composition for improving inflammation comprising cerulenin or a salt thereof represented by the following formula (1): &lt; EMI ID =
[Chemical Formula 1]

.
A cosmetic composition for improving inflammation containing cerulenin or a salt thereof represented by the following formula (1): &lt; EMI ID =
[Chemical Formula 1]

.

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