WO2016056769A1 - Pharmaceutical composition for preventing or treating inflammatory diseases, containing, as active ingredient, cerulenin or cerulenin derivative - Google Patents

Abstract

The present invention relates to an anti-inflammatory composition containing, as an active ingredient, cerulenin, a cerulenin derivative or a pharmaceutically acceptable salt thereof. Cerulenin or a cerulenin derivative of the present invention exhibits excellent prevention, treatment and alleviation effects on inflammatory diseases, and thus could be applied to a composition for preventing or treating inflammatory diseases and to a material for related food or cosmetic products.

Description

Pharmaceutical composition for preventing or treating inflammatory diseases containing cerulein or cerulein derivative as an active ingredient

The present invention relates to the use of cerulenin, cerulenin derivatives or pharmaceutically acceptable salts thereof.

Inflammation is a localized immune response that minimizes damage and restores damaged areas when cells or tissues are damaged or destroyed by various factors such as harmful substances or organisms from outside. It is a useful defense mechanism to remove products produced by tissue damage. Several factors that cause inflammation include physical factors caused by trauma, burns, frostbite, radioactivity, chemical factors such as acids, and immunological factors caused by antibody reactions. It can also occur.

Inflammation normally acts to restore the normal structure and function by neutralizing or eliminating the onset factors and regenerating the upper tissues through the inflammatory response in vivo, but the degree of inflammation becomes more than a certain level or becomes chronic. This is a problem if you progress to the same disease state.

Enzymes involved in the inflammatory response are produced by immune cells, which travel through the blood vessels to the damaged area with the help of histamine, nitric oxide (NO) or prostaglandin E2 (PGE2). Initiate an inflammatory response. Immune cells migrated to the damaged area were tumor necrosis factor-α (TNF-α), interleukin-1β (interleukin-1β, IL-1β) or interleukin-6 (interleukin-6, IL-6). Cytokines, macrophage inflammatory proteins (MIP-1), interleukin-8 (IL-8), monocyte chemotactic protein-1, Chemokine (MCP-1), etc. secrete chemokine (chemokine) to destroy direct external invasion or other immune cells to initiate an inflammatory response. Induced nitric oxide synthase when exposed to pro-inflammatory interferon-γ, lipoichoic acid, lipopolysaccharide (LPS), or various inflammatory cytokines synthase (iNOS) and cyclooxygenase-2 (COX-2) are expressed, resulting in excess production of NO and PGE2. Several of these inflammatory initiation factors (iNOS, COX-2, TNFα, IL-6, etc.) are promoted by activated nuclear factor-κB (NF-κB), which causes shock to occur when NO is generated more than necessary. Caused by vasodilation, tissue damage caused by an inflammatory response, mutagenesis, and damage to nerve tissue. In addition, overexpression of COX-2 causes an inflammatory response.

Steroidal anti-inflammatory drugs used to treat chronic inflammatory diseases, such as conventional acute or rheumatoid arthritis, cause side effects such as glaucoma, cataracts, hypertension, mood swings, weight gain, diabetes and osteoporosis. Therefore, there is a need for the development of a substance that can effectively inhibit inflammation with a component derived from a natural substance having little or no risk for such side effects or cytotoxicity.

Serulenin inhibits sterol biosynthesis by inhibiting HMG-CoA reductase activity, and inhibits the activity of fatty acid biosynthetic enzymes by forming covalent bonds with fatty acid biosynthesis by reacting with cysteine in the region responsible for condensation reaction It is known. In addition, cerulenin also inhibits the biosynthesis of polyketides, such as leucomycin, methylsalicylic acid, candicidin, flavanone, and alternari, which have a polyketide biosynthesis pathway. All of them are known to inhibit biosynthesis by cerulenin. However, the study of the industrialization is still insignificant, and the degree of the sebum production inhibitory effect of cerulein is disclosed in Korean Patent Publication No. 10-2007-0023730 related to "method generation and pore size reduction method".

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

[Preceding technical literature]

(Patent Document 1) Republic of Korea Patent Publication No. 10-2007-0023730

Accordingly, it is an object of the present invention to provide a composition for the prevention or treatment of inflammatory diseases, and the use thereof, comprising a natural substance-derived component as an active ingredient.

Another object of the present invention to provide a composition for improving inflammation, containing a natural substance-derived component as an active ingredient.

In order to achieve the above object, the present invention provides a pharmaceutical composition for the prevention or treatment of inflammatory diseases, containing as cerulein, cerulenin derivatives or pharmaceutically acceptable salts thereof as an active ingredient.

In order to achieve the above object, the present invention provides a method for preventing or treating an inflammatory disease comprising administering to a mammal a cerulein, a cerulein derivative or a pharmaceutically acceptable salt thereof.

In order to achieve the above object, the present invention provides the use of cerulenin, cerulenin derivatives or pharmaceutically acceptable salts thereof for use in the manufacture of a medicament for the prevention or treatment of inflammatory diseases.

In order to achieve the above another object, the present invention provides a food and cosmetic composition for improving inflammation containing cerulein, a cerulein derivative or a pharmaceutically acceptable salt thereof as an active ingredient.

The composition containing cerulenin, cerulenin derivatives or pharmaceutically acceptable salts thereof according to the present invention as an active ingredient is derived from natural products, and MTT analysis shows no possibility of side effects due to lack of cytotoxicity, thereby improving inflammatory diseases. It can be used very effectively for prevention, or treatment.

1 is a graph confirming the inhibition of NO production of cerulenin and comparing with BAY11-7082, where the horizontal axis represents the treatment concentration of cerulenin or BAY11-7082, and the vertical axis represents the amount of NO produced.

2 is a graph confirming the cytotoxicity of cerulein and compared with BAY11-7082, the horizontal axis of the graph means the treatment concentration of cerulenin or BAY11-7082, the vertical axis shows the cell viability (%) Indicates.

Figure 3 is a graph confirming that the cerulein inhibits the mRNA expression of inflammatory factors iNOS, COX-2, TNFα, IL-6, MCP-1 and IL-1, Con on the horizontal axis of the graphs treated LPS Lng 100ng / ml represents the group which treated LPS with 100ng / ml. In addition, the vertical axis represents the degree of mRNA expression relative to CypA mRNA expression of each inflammation-related factor.

Figure 4 is an electrophoresis picture confirming that the cerulein inhibits protein expression of inflammation-related factors iNOS and COX-2, the number above the figure is the concentration of LPS (ng / ㎖), the left is specific to each protein A protein whose expression was confirmed using a primary antibody of interest is shown.

5 is a graph confirming the inhibition of NF-κB transcriptional activity of cerulenin, wherein the horizontal axis of the graph shows the treatment and concentration of LPS, the vertical axis of the NF-κB reporter activity (renilla) value of The degree of transcriptional activity of NF-κB corrected by the ratio is shown.

6 is a graph confirming that cerulein inhibits the increase in serum TNF-α concentration caused by LPS administered to mice, wherein the horizontal axis shows whether cerulein and LPS are administered, and the vertical axis shows serum concentration of TNF-α ( ng / ml).

7 is a graph confirming the inhibition of NO production of cerulenin derivative C75 and comparing it with cerulenin, wherein the horizontal axis of the graph represents the treatment concentration of the samples, and the vertical axis represents the amount of NO production.

8 is a graph confirming the cytotoxicity of the cerulein derivative C75 and compared with the cerulein, wherein the horizontal axis represents the treatment concentration of the samples, the vertical axis represents the cell viability (%).

9 is a graph confirming that the cerulein derivative C75 inhibits mRNA expression of inflammation-related factors and compared with cerulein, wherein the horizontal axis of the graph shows the treatment and concentration of LPS, and the vertical axis shows the CypA of each inflammation-related factor. The degree of mRNA expression relative to mRNA expression is shown.

10 is a graph confirming that cerulein increases the survival rate of animals in the sepsis animal model, the horizontal axis of the graph shows the time course after galactosamine N and LPS administration for sepsis induction, the vertical axis of the animal Survival rate.

EMBODIMENT OF THE INVENTION Hereinafter, this invention is demonstrated in detail.

The present invention provides a composition for preventing or treating inflammatory diseases containing cerulenin, cerulenin derivatives or pharmaceutically acceptable salts thereof as an active ingredient.

The cerulenin or cerulenin derivatives may be represented by the following Chemical Formula 1 or 2, respectively:

[Formula 1]

, 또는

, or

[Formula 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 is cephalosporium carulens ( Cephalosporium) caerulens ) or may be prepared by methods known in the art.

The molecular weight of the cerulein derivative C75 (C ₁₄ H ₂₂ O _{4 ;} 4-Methylene-2-octyl-5-oxotetrahydrofuran-3-carboxylic acid) is 254.32 (g / mol).

Serulenin or a cerulenin derivative, an active ingredient of the composition according to the present invention, can be used for the prevention and treatment of inflammatory diseases by inhibiting the transcriptional activity of NF-κB, the production of nitric oxide and the expression of inflammation-related factors.

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

The composition according to the invention shows a 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 cerulein was found to inhibit the production of NO without toxicity (Examples 1-2, FIGS. 1 and 2).

The composition according to the present invention exhibits expression inhibitory activity of iNOS, COX-2, TNF-α, IL-6, MCP-1β and IL-1β. Specifically, according to one embodiment of the present invention, mRNA expression of iNOS, COX-2, TNF-α, IL-6, MCP-1 and IL-1β induced by LPS in the experimental group treated with 10 μM of cerulein is It was confirmed to be significantly suppressed (Examples 1-3, see FIG. 3).

The composition according to the present invention shows the expression inhibitory activity of iNOS and COX-2. Specifically, according to one embodiment of the present invention, it was confirmed that protein expression of iNOS and COX-2 induced by LPS is significantly suppressed in the experimental group treated with 10 μM of cerulein (Examples 1-4 and FIG. 4). Reference).

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

The composition according to the present invention exhibits TNF-α blood concentration inhibitory activity. Specifically, according to one embodiment of the present invention, it was confirmed that cerulenin significantly inhibits the secretion of TNF-α by LPS (see Examples 1-6 and FIG. 6).

The composition according to the invention shows a concentration dependent NO production inhibitory activity without toxicity. Specifically, according to one embodiment of the present invention, 1, 5 and 10 μM of cerulein derivative C75 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 the 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 is significantly suppressed in the experimental group treated with 10 μM of cerulein derivative C75 (Example 2, see FIG. 9).

The composition according to the invention increases the survival of sepsis animal models. 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 cerulein at 15, 30 or 60 mg / kg is significantly increased. (Example 3, see FIG. 10).

The inflammatory disease may be a chronic inflammatory disease, an acute inflammatory disease or other inflammation-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 a rejection of sepsis, shock or organ transplantation.

The other inflammation-related diseases may be ophthalmic diseases, bronchitis, dermatitis, allergy, systemic lupus erythematosus, 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 allergy, food allergy or drug allergy.

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

Preferred salt forms of the compounds according to the invention include salts with inorganic or organic acids. At this time, the inorganic acid may be used hydrochloric acid, sulfuric acid, nitric acid, phosphoric acid, perchloric acid, bromic acid and the like. Organic acids include 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, tartaric acid, salicylic acid, malic acid, Oxalic acid, benzoic acid, embonic acid, aspartic acid, glutamic acid and the like can be used. Organic bases that can be used for the preparation of organic base addition salts are tris (hydroxymethyl) methylamine, dicyclohexylamine and the like. Amino acids that can be used to prepare amino acid addition bases are natural amino acids such as alanine, glycine and the like.

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

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

Also included in the scope of the present invention is the form of a hydrate or solvate of the heterocycle derivative of formula (1). Such hydrates or solvates may be prepared by known methods, and are preferably nontoxic and water soluble, and are hydrates or solvates having 1 to 5 molecules of water or alcoholic solvents (especially ethanol, etc.) bound together. It is preferable.

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 on the composition of the present invention. The known material may be a COX-2 inhibitor or a nitrogen monoxide (NO) inhibitor, but is not limited thereto.

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

The compositions according to the invention can each be formulated and used according to conventional methods. In particular, methods known in the art may be employed and formulated to provide rapid, sustained or delayed release of the active ingredient after administration to a mammal.

The method of administering the composition according to the present invention can be easily selected according to the dosage form, and can be administered orally or parenterally. For example, it may be used through intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, nasal, epidural and oral routes, but is not limited thereto.

Solid preparations for oral administration may be, but are not limited to, tablets, pills, soft or hard capsules, pills, powders or granules. On the other hand, the form for parenteral administration may be in the form of creams, lotions, ointments, warnings, solutions, patches or injections, but is not limited thereto.

The dosage of the composition according to the present invention may vary depending on the age, sex, weight, degree of disease, and route of administration of the patient, but is generally in an amount of 5 to 500 mg / kg, preferably 100 to 250 mg / kg. The amount may be administered once to three times daily. However, the dosage does not limit the scope of the invention in any aspect.

The present invention provides a method for preventing or treating an inflammatory disease, comprising administering to a mammal a cerulein, a cerulein derivative or a pharmaceutically acceptable salt thereof.

The present invention also provides the use of a cerulenin, a cerulenin derivative or a pharmaceutically acceptable salt thereof for use in the manufacture of a medicament for the prevention or treatment of an inflammatory disease.

The cerulenin and cerulenin derivatives may be represented by Formula 1 or 2, and the cerulein, cerulenin derivatives or pharmaceutically acceptable salts thereof are as described above.

Inflammatory diseases according to the present invention may be chronic inflammatory diseases, acute inflammatory diseases or other inflammation-related diseases, specific examples of inflammatory diseases are as described above.

In addition, the present invention provides a food composition for improving inflammation containing cerulenin, a cerulein derivative or a pharmaceutically acceptable salt thereof.

The cerulenin or cerulenin derivatives may be represented by Formula 1 or 2, respectively.

Food composition for improving inflammation of the present invention is a 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% by weight of the total composition Include.

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

The health beverage 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 is no particular limitation on the liquid component except for containing the compound as an essential ingredient in the indicated ratio, and various flavors or natural carbohydrates, etc. may be added as additional ingredients, as in general beverages. Examples of the above-mentioned natural carbohydrates include monosaccharides such as glucose, fructose and other disaccharides such as maltose and sucrose polysaccharides such as conventional sugars such as dextrin and cyclodextrin, and Sugar alcohols such as xylitol, sorbitol, and erythritol. As flavoring agents other than those mentioned above, natural flavoring agents (tauumatin, stevia extract (e.g., Rebaudioside A, glycyrazine, etc.) and synthetic flavoring agents (saccharin, aspartame, etc.) can be advantageously used. Can be. The ratio of the natural carbohydrate is generally about 1-20 g, preferably about 5-12 g per 100 mg of the compound of the present invention.

In addition to the above, the food composition for improving inflammation of the present invention includes various nutrients, vitamins, minerals (electrolytes), synthetic flavors and natural flavors such as flavoring agents, colorants and neutralizing agents (such as cheese and chocolate), pectic acid and salts thereof. , Alginic acid and salts thereof, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohols, carbonation agents used in carbonated drinks and the like. Others may contain pulp for the production of natural fruit juices, fruit juice drinks and vegetable drinks. These components can be used independently or in combination.

Inflammation-improving foods to which the compound represented by Formula 1 or 2 of the present invention, or a pharmaceutically acceptable salt thereof may be added, for example, various foods, beverages, gums, vitamin complexes, health supplements, etc. There is this.

In another aspect, the present invention provides a cosmetic composition for improving inflammation containing cerulenin, cerulenin derivatives or pharmaceutically acceptable salts thereof.

The cerulenin or cerulenin derivatives may be represented by Formula 1 or 2, respectively.

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

The cosmetic composition may be formulated into a cosmetic formulation commonly prepared in the art. The cosmetic composition is formulated, for example, in solutions, suspensions, emulsions, pastes, gels, creams, lotions, powders, veins, surfactant-containing cleansing, oils, powder foundations, emulsion foundations, wax foundations and sprays, and the like. However, the present invention is not limited thereto. More specifically, it may be formulated in the form of a flexible lotion, nutrition lotion, nutrition cream, massage cream, essence, eye cream, cleansing cream, cleansing foam, cleansing water, pack, spray or powder.

When the formulation of the cosmetic composition of the present invention is a paste, cream or gel, animal oil, vegetable oil, wax, paraffin, starch, tragacanth, cellulose derivative, polyethylene glycol, silicone, bentonite, silica, talc, zinc oxide and mixtures thereof It may include a carrier component selected from the group consisting of.

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

When the formulation of the cosmetic composition of the present invention is a solution or emulsion, it may include a carrier component selected from the group consisting of solvents, solvating agents, emulsions and mixtures thereof. Examples thereof include water, ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylglycol oil, glycerol aliphatic esters, polyethylene glycols, sorbitan fatty acid esters, mixtures thereof, and the like. Can be mentioned.

When the formulation of the cosmetic composition of the present invention is a suspension, a liquid diluent such as water, ethanol or propylene glycol, suspending agents such as ethoxylated isostearyl alcohol, polyoxyethylene sorbitol ester and polyoxyethylene sorbitan ester, microcrystal Carrier cellulose, aluminum metahydroxy, bentonite, agar, tragacanth and mixtures thereof.

When the formulation of the cosmetic composition of the present invention is a surfactant-containing cleansing, aliphatic alcohol sulfate, aliphatic alcohol ether sulfate, sulfosuccinic acid monoester, isethionate, imidazolinium derivative, methyltaurate, sarcosinate, fatty acid amide ether And a carrier component selected from the group consisting of sulfates, alkylamidobetaines, aliphatic alcohols, fatty acid glycerides, fatty acid diethanolamides, vegetable oils, lanolin derivatives, ethoxylated glycerol fatty acid esters, and mixtures thereof.

The "carrier component" in the cosmetic composition of the present invention is a compound or composition already known and used that may be included in a cosmetic preparation, and is a component that is not toxic, unstable, or irritating beyond contact with the human body.

In addition to the carrier component, the cosmetic composition of the present invention may further include an adjuvant selected from the group consisting of antioxidants, stabilizers, solubilizers, humectants, pigments, perfumes, sunscreens, colorants, surfactants, and combinations thereof. The adjuvant is not limited to use as long as the adjuvant commonly used in the preparation of the cosmetic composition.

Hereinafter, the present invention will be described in more detail with reference to the following examples. However, the following examples are only for illustrating the present invention, and the scope of the present invention is not limited thereto.

EXAMPLE

Example 1. Confirmation of anti-inflammatory activity of cerulenin

Example 1-1. Preparation of Samples, Cells, and Mice

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

Dulbecco's modified Eagles medium (DMEM), Fetal bovine serum (FBS) and penicillin / streptomycin are from Hyclone (Thermo Scientific Inc., Germany), SYBR green from Roche (Switzerland), Dual 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 Cell Signaling (USA), and anti-HSC70 monoclonal antibody was purchased from Rockland (USA). Cerulenin, BAY11-7082, MTT and the remaining reagents were purchased from Sigma Aldrich Co. (USA).

Experiments for measuring the anti-inflammatory activity of cerulenin were used macrophage line RAW264.7 purchased from ATCC and peritoneal macrophages isolated from mice. RAW264.7 and peritoneal macrophages were treated at 37 ° C., 5% CO ₂ , using glucose high glucose DMEM with 10% FBS and penicillin (100 U / ml) / streptomycin (100 μg / ml). Cultured in the incubator.

C57 / BL6 mouse species were used to determine the anti-inflammatory activity of cerulenin in animal models. The diet used solid feed and mice and diet were purchased from Hyochang Science (Korea). The breeding of the experimental animals was carried out under a temperature condition of 20 ℃ to 24 ℃, humidity conditions of 60% to 70% and day and night lighting conditions of 12 hours cycle, water and diet was to be ingested freely.

The anti-inflammatory activity of cerulenin was measured by measuring mRNA expression, protein expression, transcriptional activity and NO production of factors related to the inflammatory response using the cells, mice, reagents and devices. Experimental results for identifying anti-inflammatory activity were expressed as mean ± S.D., and in relation to statistical significance, defined as * (p <0.05), the p <0.05 was considered to be statistically significant.

Example 1-2. Inhibition of NO and production of NO

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

RAW264.7 cells, a mouse macrophage line, were dispensed in 96-well plates at 5 × 10 ⁴ cells / well and stabilized for 16 hours, followed by 1 hour of concentration of cerulein and BAY11-7082 (1, 5, 10, 20 μM). Treated during. Thereafter, LPS was treated at 100 ng / ml as an inflammatory derivative and incubated for 16 hours. Grease reagent was added to the culture to quantify NO content. Specifically, 50 μl of the cultured cell culture solution and 50 μl of the grease reagent were mixed and reacted at room temperature for about 5 minutes, and then the absorbance was measured at 540 nm using a microplate reader (Tecan). After obtaining a standard curve using 0.1M nitrite standard solution, the NO concentration of the cell culture solution was calculated. The calculated NO value is shown in FIG. 1.

In addition, to confirm the toxicity of cerulenin, MTT (3- (4,5-dimethylthiazol-2-yl) -2,5-diphenyl tetrazolium bromide) solution (5 mg / ml) in a 96-well plate in which the cells were cultured. 20 μl was added and incubated for 1 hour. Thereafter, all of the culture solution was removed, and the resulting formazan crystal was dissolved in 150 µl of DMSO, and then the absorbance was measured at 570 nm using a microplate reader. The measurement results are shown in FIG. 2, and cell viability is expressed as a percentage of a control treated with LPS only.

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

Example 1-3. Inhibition of mRNA expression of inflammation-related factors

In order to confirm the anti-inflammatory activity of cerulenin, it was analyzed whether the mRNA expression of inflammation-related factors induced by LPS in primary cells (peritoneal macrophages). That is, real-time PCR of mRNA expression of NF-κB target genes, iNOS, COX-2, TNF-α, IL-6, MCP-1 and IL-1β, which are known to be important transcription regulators in the inflammatory response Confirmed by law.

Specifically, in order to obtain the peritoneal macrophages, 1 ml of 3% thioglycollate was intraperitoneally administered to male mice (C57 / BL6) weighing 20 g to 25 g, and 4 days after administration, The macrophages were obtained. The obtained peritoneal macrophages were incubated in DMEM medium for 24 hours, and cultured for 24 hours by dispensing 200 × 10 ⁴ cells / well in 6-well plates. Thereafter, cerulein was pretreated at 10 μM for 1 hour, treated with LPS (100 ng / ml) for 1 hour to confirm expression of TNF-α, and incubated 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 PBS, lysed with 500 µl of trizol reagent (Life technologies), and then 100 µl of chloroform was added and vortexed. After incubation at room temperature for 10 minutes, the mixture was centrifuged at 13,000 rpm for 15 minutes to obtain 400 µl supernatant, and incubated with 500 µl of isopropanol for 10 minutes at room temperature. Thereafter, pellets were obtained by centrifugation for 15 minutes at 13,000 rpm. The obtained pellet was washed with 1 ml of 75% ethanol and dried at room temperature to separate RNA.

The isolated RNA is 0.1% DEPC (Diethyl pyrocarbonate) It was dissolved in 20 μl to 30 μl of solution and used for cDNA synthesis. 4 g of the isolated RNA was aliquoted to synthesize cDNA using M-MLV reverse transcriptase (promega), and real-time PCR analysis was performed to measure mRNA content of inflammatory factors.

Sequences and experimental conditions of the primers for measuring the contents of inflammation-related factors iNOS, COX-2, TNFα, IL-6, MCP-1 and IL-1β are shown in Table 1 below.

The mRNA expression level of the inflammation-related factors is shown in FIG. 3, and the mRNA expression level is corrected and expressed as a ratio with respect to the amount of the control group CypA mRNA.

As a result, as shown in Figure 3, in the control group not treated with LPS mRNA expression of iNOS, COX-2, TNF-α, IL-6, MCP-1 and IL-1β is low, while when 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 inhibited in the experimental group treated with 10 μM of cerulein. appear.

Example 1-4. Inhibition of protein expression of inflammation-related factors

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

Primary cells were obtained by raising experimental animals in the same manner as in Example 1-3, and then cultured in DMEM medium for 24 hours, then divided into 6 well plates at 200 × 10 ⁴ cells / well and incubating for 24 hours. It was. Thereafter, cerulein was pretreated at 10 μM for 1 hour and treated with LPS (100 ng / ml or 1000 ng / ml) 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 PBS, and then PBS was removed and 0.01 M Tris-HCl (pH 7.4), 0.15 M NaCl, 0.001 M EDTA, 0.001 M EGTA, 1% Triton X-100, 200 μl of RIPA buffer containing 0.002M PMSF (phenylmethanesulfonyl fluoride) and a protease inhibitor were added, vortexed, placed on ice for 30 minutes, and centrifuged for 10 minutes at 13,000 rpm. Then, the supernatant was obtained, and the protein content was quantified using BCA reagent (pierce biotechnology).

The quantitated protein samples were dispensed in equal amounts, mixed with 5 × SDS sample buffer, boiled at 95 for 5 minutes, and then subjected to SDS-PAGE using a 7% gel. The protein of the electrophoresis was transferred to the nitrocellulose membrane, and pretreated with 5% skim milk for 1 hour at room temperature to inhibit nonspecific antibody binding. Thereafter, anti-iNOS polyclonal antibody, anti-COX-2 polyclonal antibody and anti-HSC70 monoclonal antibody were added to 5% skim milk to react with the membrane for 4 to 24 hours depending on the type of antibody. After the reaction, the mixture was washed three times with PBST for 10 minutes, and reacted with a secondary antibody at room temperature for 1 hour. Then, the membrane was washed three times with PBST for 10 minutes, and then reacted with ECL western blotting detection reagent (GE healthcare) to confirm the expression level of each protein with LAS2000 (GE healthcare).

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

Example 1-5. Inhibition of transcriptional activity of NF-κB

In order to confirm the anti-inflammatory activity of cerulenin, it was confirmed by luciferase assay whether the inhibition of NF-κB transcriptional activity increased by LPS in RAW264.7 cells.

Specifically, the RAW264.7 cells were transfected with NF-κB reporter vector and Renilla vector using lipofectamine 2000 (life technologies) to 40 × in a 24-well plate with high glucose DMEM medium. Aliquots were made to 10 ⁴ cells / well. After 6 hours of incubation, the medium was replaced and incubated for another 16 hours. After the incubation, cerulein or BAY11-7082 was added at 10 μM, and after 1 hour, LPS was treated with 100ng / ml for 8 hours and 100 μl of lysis buffer was added. 50 μl of the cell lysate was dispensed into 96-well white plates, and the degree of NF-κB transcriptional activity was measured using a dual-luciferase reporter system (promega), and the results are shown in FIG. 5. At this time, the degree of NF-κB transcriptional activity was expressed by adjusting the ratio of the Renilla value which is a control of each sample.

As a result, as shown in Figure 5, in the control group not treated with LPS, NF-κB transcriptional activity was low, but it was confirmed that the transcriptional activity increased when the LPS treatment. In addition, it was confirmed that NF-κB transcriptional activity increased by LPS was inhibited by cerulein, and the degree of inhibition of NF-κB transcriptional activity of cerulein was superior to BAY11-7082. That is, cerulein was shown to have excellent anti-inflammatory activity by inhibiting NF-κB transcriptional activity.

Example 1-6. Decreased Plasma Levels of TNF-α Induced by LPS in Mice

In order to confirm the suppression of the secretion of inflammatory cytokines of cerulenin, it was confirmed whether the suppression of serum TNF-α concentration induced by LPS in C57 / BL6 mouse species.

Specifically, 8 weeks-old C57 / BL6 male mice each administered nothing, a group administered with cerulein 60 mg / kg intraperitoneally, a group administered with LPS 50 mg / kg, and cerulein 60 mg / kg 1 hour after the administration of LPS 50mg / kg divided into the group was administered to the experiment. One hour after the administration of LPS, 200 μl of blood was obtained from the mice, and serum was separated by centrifugation at 13,000 rpm for 3 minutes with a BD microtainer. After diluting the separated serum 50-fold, serum TNF-α concentration was measured using a TNF-α ELISA kit (R & D system).

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

Example 2. Confirmation of anti-inflammatory activity of cerulenin derivative C75

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

Specifically, the inhibition of NO production was confirmed by treating C75 and cerulein with 1, 5, and 10 μM in RAW264.7 cells in the same manner as in Example 1-2 above, and the result was confirmed in FIG. 7 by cytotoxicity. The results are shown in FIG. In addition, in the same manner as in Example 1-3, C75 and cerulein was confirmed in the inhibition of mRNA expression of inflammation-related factors induced by LPS is shown in Figure 9 shown.

As a result, as shown in Figures 7, 8, C75 and cerulenin was shown to inhibit NO production, without toxicity at each concentration (1, 5, 10μM). In addition, as shown in Figure 9, C75 and cerulein was shown to inhibit the mRNA expression of iNOS, COX-2 and IL-6.

Example 3. Confirmation of the Sepsis Treatment Effect of Serulenin in a Mouse Model

In order to confirm whether cerulein has a therapeutic effect of an inflammatory disease in an animal model, the following experiment was performed by treating cerulein with a sepsis mouse model.

Specifically, eight mice with a body weight of about 25 g of 8-week-old C57 / BL6 mice were conducted in one group. First, cerulein dissolved in 40% DMSO solution 1 hour before induction of sepsis was administered to each group of mice intraperitoneally at concentrations of 15, 30 and 60 mg / kg, respectively, and only 40% DMSO solution was administered to mice as a control. . Then, galactosamine N and LPS were dissolved in PBS at a concentration of 700 mg / kg and 150 µg / kg, respectively, and intraperitoneally administered to induce sepsis. Was measured. Survival measurement was calculated by checking every hour after induction of sepsis.

As a result, the relative value based on the survival rate of the mouse 1 hour after induction of sepsis is shown in Figure 10.

As shown in FIG. 10, the group of mice administered with cerulein at a concentration of 30 mg / kg had a sharp survival rate from 6 hours after induction of sepsis, and the group of mice administered with a concentration of 15 mg / kg with cerulein after 8 hours. After 12 hours, the survival rate was about 10% for the 15 mg / kg group and about 25% for the 30 mg / kg group. On the other hand, the cerulein 60 mg / kg administration group showed a sharp decrease in survival rate of about 60% after 7 hours of induction of sepsis, but after about 12% after about 12 hours, the survival rate was about 40%. On the other hand, in the solvent control group, the survival rate decreased to 20% after 7 hours of induction of sepsis, and decreased to 10% after 12 hours.

From the above results, it was confirmed that the cerulein of the present invention, which inhibits the expression and activity of inflammatory factors, has an effect of preventing and treating sepsis by increasing the survival rate in sepsis-generating animal models, and the cerulein derivatives showing the same activity also have the same effect. It can be seen that there is.

Claims (12)

A pharmaceutical composition for preventing or treating inflammatory diseases, which comprises cerulein, a cerulein derivative, or a pharmaceutically acceptable salt thereof represented by Formula 1 or 2, respectively, as an active ingredient: [Formula 1]

, or [Formula 2]

. The method of claim 1, The composition is characterized by inhibiting the transcriptional activity of NF-κB, the production of nitric oxide or expression of inflammation-related factors. The method of claim 2, The inflammation-related factors include inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), interleukin-6 (IL-6), monocyte chemotactic protein-1 (MCP-1), interleukin- 1β (IL-1β) and tumor necrosis factor-α (TNF-α). The method of claim 1, The inflammatory disease is a composition, characterized in that a chronic inflammatory disease, acute inflammatory disease or inflammation-related diseases. The method of claim 4, wherein The chronic inflammatory disease is a composition, characterized in that selected from the group consisting of rheumatoid arthritis, arteriosclerosis, diabetes, osteoporosis, Alzheimer's disease, Parkinson's disease, lupus and multiple sclerosis. The method of claim 4, wherein The acute inflammatory disease is composition, characterized in that selected from the group consisting of sepsis, shock and rejection of organ transplantation. The method of claim 4, wherein The inflammation-related disease is characterized in that the eye disease, bronchitis, dermatitis, allergic, systemic lupus erythematosus, retinitis, gastritis, hepatitis, enteritis, pancreatitis and nephritis. The method of claim 7, wherein The dermatitis or allergy is selected from the group consisting of hypersensitivity, allergic rhinitis, asthma, allergic conjunctivitis, allergic dermatitis, atopic dermatitis, contact dermatitis, urticaria, insect allergy, food allergy and drug allergy . A food composition for improving inflammation containing cerulenin, a cerulenin derivative or a pharmaceutically acceptable salt thereof represented by Formula 1 or 2, respectively: [Formula 1]

, or [Formula 2]

. A cosmetic composition for improving inflammation containing cerulenin, a cerulenin derivative or a pharmaceutically acceptable salt thereof represented by Formula 1 or 2, respectively: [Formula 1]

, or [Formula 2]

. A method of preventing or treating an inflammatory disease comprising administering to a mammal a cerulein, a cerulein derivative or a pharmaceutically acceptable salt thereof represented by Formula 1 or 2, respectively. Use of a cerulein, a cerulenin derivative or a pharmaceutically acceptable salt thereof represented by Formula 1 or 2, respectively, for use in the manufacture of a medicament for the prevention or treatment of an inflammatory disease.

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