WO2012060594A2 - Anti-inflammatory composition containing a thiourea compound and a pharmaceutically acceptable salt thereof as an active ingredient - Google Patents

Abstract

【Abstract】 The present invention relates to a pharmaceutical composition for preventing or treating inflammatory diseases, wherein the pharmaceutical composition contains, as an active ingredient, a thiourea compound represented by the following Formula 1 or a pharmaceutically acceptable salt thereof. The thiourea compound or pharmaceutically acceptable salt thereof, according to the present invention, reduces the activity of NF-kB that causes inflammatory diseases in a fibroblast-type synoviocyte and a mouse model of collagen induced arthritis, and effectively inhibits edema and pain. Thus, the composition of the present invention can be usefully used in preventing and treating inflammatory diseases such as rheumatoid arthritis.

Description

 【Specification】

[Name of invention]

 Anti-inflammatory composition containing a thiourea compound or its pharmaceutically acceptable salt as an active ingredient.

<1> The present invention relates to an anti-inflammatory composition containing a thiourea-based compound or a pharmaceutically acceptable salt thereof as an active ingredient.

<2>

Background Art

<3> Rheumatoid arthritis (RA) is a chronic, progressive autoimmune disease in which inflammatory cells penetrate into the synovial membrane tissue and destroy joints.It is a chronic inflammatory systemic disease that causes irreversible joint damage, chronic pain, stiffness and functional damage ( Smolen JS, Aletaha D, Koeller M, Weisman MH, Emery P (2007) New thera ies for treatment of rheumatoid arthritis.Lancet 370 (9602): 1861-1874).

<4>

 <5> One of the major transcription pathways involved in joint inflammation is nuclear factor- κ

B (NF-B) pathway (Mclnnes IB, Schett G (2007) Cytokines in the pathogenesis of rheumatoid arthritis. Nat Rev Immunol 7 (6): 429-442).

Specifically, fibroblast-like synoviocytes (FLS) and inflammatory cells in the joints produce inflammatory cytokines such as interleukin- 1β (IL ᅳ 1β) and tumor necrosis factor-α (TNFa). do. These cytokines activate NF-κΒ It intensifies inflammation, thereby increasing the expression of cytokines.

Examples of inflammatory mediators include cytokines, RANTES and chemokines such as ENA-78 and adhesion molecules.

 <8>

 In addition, NF-κΒ activity in FLS affects the symptoms of rheumatoid arthritis by increasing the expression of matrix metalloproteinases (XP).

 <ιο> More than 20 MMPs have been reported, of which P-1 and P-3 are known as major enzymes for tissue destruction (Campbell IK, Gerondakis S, O'Donnell K, Wicks IP (2000) Distinct roles for the NF-κ Bl (p50) and c— Rel transcription factors in infla atory arthritis.J Clin Invest 105 (12): 1799-1806).

<ii> NF-κΒ has been reported to increase activity in synovial membrane tissues of rheumatoid arthritis patients and collagen-induced arthritis (CIA) mice compared to the normal control synovial membrane (Handel ML, McMorrow LB, Gravallese). EM (1995) Nuclear factor- B in rheumatoid synovium.Local izat ion of p50 and p65.Arthritis Rheum 38 (12): 1762-1770; Hah YS, Lee YR, Jun JS, Lim HS, Kim HO, Jeong YG et al (2010) A20 suppresses inflammatory responses and bone destruction in f ibroblast-1 ike synoviocytes and col lagen-induced arthritic mice.Arthritis Rheum 62 (8): 2313-2321), and the activation of these NF-κΒ is currently used clinically. Are inhibited by several anti-rheumatic drugs such as glucocorticoids, aspirin, gold salts, leflunomide, methotrexate (Tomita T, Takeuchi E, Tomita N, Mor ishita R, Kaneko, Yamamoto K et al. 1999) Suppressed severity of col lagen- induced arthritis by in vivo transfect ion of nuclear factor Β decoy o 1 i godeoxynuc 1 eot i des as a gene therapy. Arthritis Rheum 42 (12): 2532-2542).

 <i2> In addition, injection of adenovirus gene transfer of NF-κΒ decoy olionucleotide or dominant negative I κ kinase β in the joints may result in collagen-induced arthritis ( CIA) inhibits (Hah YS, Lee YR, Jun JS, Lim HS, Kim HO, Jeong YG et al. (2010) A20 suppresses inflammatory responses and bone destruct ion in fibroblast ᅳ like synoviocytes and col lagen-induced arthritic mice. Arthritis Rheum 62 (8): 2313-2321).

 Therefore, it is possible to effectively treat or ameliorate inflammatory diseases including rheumatoid arthritis by inhibiting the activity of NF-κΒ involved in inflammatory cell activation as a major transcription pathway involved in joint inflammation.

 <14>

 Thus, the present inventors, while studying to develop a compound that inhibits the activity of NF-κΒ, 1- (4- (2— (10— (10-H-phenoxazine-10-yl) ethoxy) phenyl) NF-κΒ-related inflammatory diseases by confirming the activity of reducing NF-κΒ and inhibiting edema and pain in mouse models of -3-methylthiourea islet cell-type synovial membrane cells (FLS) and collagen-induced arthritis To find out that it can be useful for the prevention or treatment of the present invention was completed.

16> [Content of invention]

 [Technical problem]

 An object of the present invention to provide a pharmaceutical composition for the prevention and treatment of inflammatory diseases containing a thiourea-based compound or a pharmaceutically acceptable salt thereof as an active ingredient.

 Another object of the present invention to provide a health food composition for the prevention and improvement of inflammatory diseases containing a thiourea-based compound or a pharmaceutically acceptable salt thereof as an active ingredient.

 <19>

 Technical Solution

 In order to achieve the above object, the present invention provides a pharmaceutical composition for the prevention and treatment of inflammatory diseases containing a thiourea-based compound represented by the following formula (1) or a pharmaceutically acceptable salt thereof as an active ingredient to provide.

 In addition, the present invention provides a health food composition for the prevention and improvement of inflammatory diseases containing a thiourea-based compound represented by the formula (1) as an active ingredient.

 <22> [Formula 1]

【유리한 효과】

Advantageous Effects

 The thiourea-based compounds or pharmaceutically acceptable salts thereof according to the present invention reduce the activity of NF-κΒ, which causes inflammatory diseases, and reduce edema and pain in fibroblast-like synovial cell and collagen-induced arthritis mouse models. By effectively inhibiting, it can be useful for preventing and treating inflammatory diseases such as rheumatoid arthritis.

 <26>

 [Brief Description of Drawings]

 1 is a graph of NF-κΒ DNA binding inhibitory activity of the compound according to an embodiment of the present invention by an electrophoretic mobiloty shift assay (EMSA).

 2 shows inhibition of the binding of p65 and p50 and Ικ of a compound according to an embodiment of the present invention.

 Western blot analysis showing βα degradation inhibitory activity.

<2> Figure 3 is a Western blot analysis showing the phosphorylation inhibitory activity of ΙΚΚα and ΐΚΚβ of the compound according to an embodiment of the present invention.

Figure 4 is the result of the instantaneous MMP-1 and ΜΜΡ-3 inhibitory activity of the compound according to an embodiment of the present invention immediately by the enzyme-linked immunosorbent assay (ELISA) method.

5 is a result of measuring the inhibitory activity of the ENA-78 and RANTES of the compound according to an embodiment of the present invention by the enzyme-linked immunosorbent assay (ELISA) method. Figure 6 is a result of measuring the fibroblast-like synovial cell (FLS) proliferation inhibitory activity of the compound according to an embodiment of the present invention using a BrdU incorporation assay hole.

 Figure 7 is a photograph showing the effect of improving arthritis symptoms in a mouse model of collagen-induced arthritis mouse according to an embodiment of the present invention.

 8 is a result of arthritis incidence showing the effect of improving arthritis symptoms in a mouse model of collagen-induced arthritis according to an embodiment of the present invention.

 Figure 9 is a clinical score showing the effect of improving the symptoms of arthritis in the mouse model of collagen-induced arthritis according to an embodiment of the present invention. Figure 10 is the result of measuring the edema of the hind paws showing the effect of improving arthritis symptoms in the collagen-induced arthritis mouse model of the compound according to an embodiment of the present invention.

 Figure 11 is a Western blot analysis showing the inhibitory activity of MMP-1 and ffiP-3 in collagen-induced arthritis mouse model of the compound according to an embodiment of the present invention.

 12 is a radiograph showing the effect of improving arthritis symptoms in the collagen-induced arthritis mouse model of the compound according to an embodiment of the present invention.

 Figure 13 is a radiographic score graph showing the effect of improving arthritis symptoms in the collagen-induced arthritis mouse model of the compound according to an embodiment of the present invention.

 Figure 14 is a histopathological picture showing the effect of improving arthritis symptoms in the collagen-induced arthritis mouse model of the compound according to an embodiment of the present invention.

Figure 15 is a photograph showing the osteoclast differentiation of collagen-induced arthritis mouse model of the compound according to an embodiment of the present invention. 16 is a graph counting the number of osteoclasts in the collagen-induced arthritis mouse model of the compound according to an embodiment of the present invention.

FIG. 17 is a graph of NF-κΒ DNA binding inhibitory activity of collagen-induced arthritis mouse models of compounds according to an embodiment of the present invention by electrophoretic shift analysis.

FIG. 18 is a result of Western blot analysis showing the inhibition of p65 and p50 binding and κκα degradation in collagen-induced arthritis mouse models of compounds according to an embodiment of the present invention. _,

19 is a result showing the inhibitory activity of cytokine production in the joint tissue in the collagen-induced arthritis mouse model of the compound according to an embodiment of the present invention.

 20 is a graph showing the inhibitory activity of cytokine production in serum in collagen-induced arthritis mouse models of compounds according to an embodiment of the present invention.

 <47>

 [Best form for implementation of the invention]

 Hereinafter, the present invention will be described in detail.

 <49>

The present invention provides a pharmaceutical composition for the prevention and treatment of inflammatory diseases containing a thiourea-based compound represented by Formula 1 or a pharmaceutically acceptable salt thereof as an active ingredient: <51> [Formula 1]

 The general chemical name of the compound is 1-methyl-3- [4- (2-phenoxazine-10-ylethoxy) phenyl] thiourea (1-methyl—3— [4 一 (2-phenoxaz i n_ 10— y 1 ethoxy) pheny 1] thi our ea).

 <53>

The compound of formula 1 of the present invention may be used in the form of a pharmaceutically acceptable salt, and as the salt, an acid addition salt formed by a pharmaceutically acceptable free acid is useful. Acid addition salts include inorganic acids such as hydrochloric acid, nitric acid, phosphoric acid, sulfuric acid, hydrobromic acid, hydrobromide and nitrous acid or phosphorous acid, aliphatic mono and dicarboxylates, phenyl-substituted alkanoates, hydroxy alkanoates and Obtained from non-toxic organic acids such as alkanedioates, aromatic acids, aliphatic and aromatic sulfonic acids. These pharmaceutically nontoxic salts include sulfate, pyrosulfate, bisulfate, sulfite, bisulfite, nitrate, phosphate, monohydrogen phosphate, dihydrogen phosphate, metaphosphate, pyrophosphate chloride, bromide, and iodine. Dioxide, Fluoride, Acetate, Propionate, Decanoate, Caprylate, Acrylate, Formate, Isobutyrate, Caprate, Heptanoate, Propiolate, Oxalate, Malonate succinate, Suverate, sebacate, fumarate, maleate, butyne— 1,4-dioate, nucleic acid-1,6_dioate, benzoate, chlorobenzo Eate, methylbenzoate, dinitro benzoate, hydroxybenzoate, methoxybenzoate, phthalate, terephthalate, benzenesulfonate, toluenesulfonate, chlorobenzenesulfonate, xylenesulfonate, phenylacetate, phenylpropion Nate, Phenyl Butyrate, Citrate, Lactate, β-hydroxybutyrate, Glycolate, Maleate, Tartrate, Methanesulfonate, Propanesulfonate, Naphthalene-1-sulfonate, Naphthalene-2-sulfonate Or mandelate.

 The acid addition salts according to the invention can be prepared by conventional methods, for example, by dissolving a compound of formula 1 in an excess of an aqueous solution of an acid and dissolving the salt with a miscible organic solvent such as methanol, ethane, acetone or acetonitrile It can be prepared by precipitation using. It may also be prepared by evaporating the solvent or excess acid from this mixture and then drying or by suction filtration of the precipitated salt.

Bases can also be used to make pharmaceutically acceptable metal salts. Alkali metal or alkaline earth metal salts are obtained, for example, by dissolving the compound in an excess of alkali metal hydroxide or alkaline earth metal hydroxide solution, filtering the compound salt unintentionally, and evaporating and drying the filtrate. At this time, as the metal salt, it is pharmaceutically suitable to prepare sodium, potassium, or champ salt. In addition, silver salts thereof are obtained by reacting an alkali metal or alkaline earth metal salt with a suitable silver salt (for example, silver nitrate). The compound of formula 1 according to the present invention includes not only a pharmaceutically acceptable salt thereof, but also an isomer thereof or a possible solvate or hydrate that can be prepared therefrom. <59>

 In addition, the compound of Chemical Formula 1 according to the present invention may be commercially available or synthesized using conventional synthetic methods known in the art of organic synthesis.

 <6i> As an example, the compounds according to the invention are described in Cheon YJ, Gim HJ, Jang HR, Ryu JH,

 J eon R (2010) Synthesis of heterocyte-1 inked thioureas and their inhibitory activities of NO product ion in LPS activated macrophages. Bull Korean Chem Soc 31 (1): 27-30.], But is not limited thereto.

<62>

 A pharmaceutical composition containing the compound of Formula 1 or a pharmaceutically acceptable salt thereof according to the present invention as an active ingredient reduces the activity of NF ᅳ κΒ, which is one of the major signaling systems of inflammatory diseases, and degrades ΙκΒα. It can be usefully used for the prevention or treatment of inflammatory diseases by reducing the activity and suppressing various cytokine production.

 In this case, the inflammatory disease may be any one selected from the group consisting of asthma, periodontitis, stomatitis, peritonitis, gastritis, enteritis, arthritis, rheumatoid arthritis, nephritis, hepatitis and degenerative diseases, preferably rheumatoid arthritis .

 <65>

In addition, the present invention provides a method for treating a metabolic disease or complication thereof, comprising administering a thiourea compound represented by Formula 1 or a pharmaceutically acceptable salt thereof to a patient in need thereof. . Furthermore, the present invention provides a thiourea compound represented by Formula 1 or a pharmaceutically acceptable salt thereof used in the prevention or treatment of metabolic diseases or complications thereof. The pharmaceutical composition for the prevention and treatment of inflammatory diseases containing the compound of Formula 1 or a pharmaceutically acceptable salt thereof according to the present invention as an active ingredient may be formulated in various oral or parenteral dosage forms as follows. It is not limited.

Formulations for oral administration include, for example, tablets, pills, hard / soft capsules, solutions, suspensions, emulsifiers, syrups, granules, elixirs, and the like. Fillers ᅳ One or more types of diluents or excipients, such as extenders, wetting agents, disintegrants, lubricants, binders, surfactants and the like can be used. As the disintegration, agar, starch, alginic acid or its sodium salt, calcium monohydrogen phosphate may be used, and lubricant, silica, talc, stearic acid or its magnesium salt or calcium salt, polyethylene glycol, etc. may be used. As the binder, magnesium aluminum silicate, starch paste, gelatin, tragacanth, methylsalose, sodium carboxymethylcellose, polyvinylpyridine, low-substituted hydroxypropylcelose and the like can be used. have. In addition, lactose, dextrose, sucrose, mannose, sorbide, cellulose and glycine may be used as diluents, and in some cases, commonly known boiling mixtures, absorbents, coloring agents, flavoring agents and sweeteners. Etc. can be used together. In addition, the pharmaceutical composition according to the present invention can be administered parenterally, and parenteral administration is by the method of injecting subcutaneous injection, intravenous injection, intramuscular injection or intramuscular injection. In this case, the compound of Formula 1 or a pharmaceutically acceptable salt thereof is mixed in water together with a stabilizer or a laxative to prepare a parenteral formulation, and prepared as a solution or suspension, which is administered in unit doses of ampoules or vials. It can be manufactured in a mold.

 <73>

 The composition may be sterilized or contain preservatives, stabilizers, hydrating agents or low emulsification promoting agents, salts for controlling osmotic pressure, auxiliaries such as buffers, and other therapeutically useful substances, and are common methods. ， Can be formulated according to the granulation or nose bath method. If necessary, the composition according to the present invention may be administered in combination with other drugs, such as other therapeutic agents.

 <75>

When formulated in the unit dosage form of the composition of the present invention, it is preferable that the active ingredient contains a compound of Formula 1 or a pharmaceutically acceptable salt thereof in a unit dose of about 0.1 to 1,500 mg. Dosage depends on the doctor's prescription depending on factors such as the patient's weight, age and the specific nature and severity of the disease. However, the dosage required for adult treatment typically ranges from about 1 to 500 mg per day, depending on the frequency and intensity of administration. Intramuscular or intravenous administration to adults will be divided into single doses, usually a total dose of about 5 to 300 mg per day, but a higher daily dose may be desirable for some patients. -^

<77>

 The composition of the present invention may further comprise suitable carriers, excipients and diluents commonly used in the manufacture of a medicament.

The composition according to the present invention may be formulated in the form of powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols and the like, oral preparations, suppositories, and sterile injectable solutions, respectively, according to a conventional method. Can be used. Carriers, excipients, and diluents that may be included in the compositions of the present invention include lactose, dextrose, sucrose, sorbbi, manny, xili, erythris, malty, starch, acacia rubber, alginate, gelatin, Calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil. have.

When formulated, it is prepared by using a diluent or an excipient such as a laminating agent, an extender binder, a humectant, a disintegrating agent, and a surfactant, which are commonly used. Solid preparations for oral administration include tablets, pills, powders, granules, capsules, and the like. ^' , Such solid preparations include at least one excipient in the composition of the present invention, e.

It is prepared by mixing (calcium carbonate), sucrose or lactose, gelatin, etc. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Oral liquid preparations include suspending agents, solution solutions, emulsions, and syrups, and may include various excipients, such as wetting agents, sweeteners, fragrances, and preservatives, in addition to commonly used simple diluents such as water and liquid paraffin. . Preparations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, Suppositories are included. As non-aqueous solvents and suspending agents, propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl acrylate may be used. As the base of the suppository, witepsol, macrogol, tween 61, cacao butter, laurin butter, glycerol gelatin and the like can be used. The composition of the present invention can be administered orally or parenterally, can be used in any case of parenteral administration, systemic or topical administration is possible, systemic administration is more preferred, intravenous administration is most preferred desirable.

 Preferred dosages of the compositions of the present invention vary depending on the condition and weight of the patient, the severity of the disease, the form of the drug, the route of administration and the duration, and may be appropriately selected by those skilled in the art. However, for the desired effect, the composition of the present invention is preferably administered at 0.0001 to 0.03 g / kg, preferably 0.001 to 8 mg / kg per day. Administration may be once a day or may be divided several times. The dosage does not limit the scope of the invention in any aspect.

 <83>

 In addition, it provides a health food composition for the prevention and improvement of inflammatory diseases containing the compound of formula (1) represented by the formula (1) as an active ingredient.

The composition according to the present invention exhibits an activity of reducing the activity of NF-ΚΒ, one of the major signaling systems of inflammatory diseases, reducing the degradation of ΙκΒα and inhibiting various cytokine production, thereby preventing and preventing inflammatory diseases. For the purpose of improvement, the compound of Formula 1 may be added to health supplements such as food and beverages.

<86> There is no particular limitation on the type of the food. Examples of foods to which the above substances can be added include dairy products, including drinks, meat, sausage bread, biscuits, rice cakes, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, ¾, ice cream, and various soups. ， Beverages, alcoholic beverages and vitamin complexes, dairy products and dairy products, etc., and includes all the health functional foods in the ordinary sense.

The compound of formula 1 of the present invention may be added to a food as it is or used with other food or food ingredients, and may be appropriately used according to a conventional method. The combined amount of the active ingredient may be suitably determined depending on the purpose of use (prevention or improvement). In general, the amount of the compound in the health ^food, of the total weight of the food

0.1 to 90 parts by weight may be added. However, in the case of long-term intake for health and hygiene or health control, the amount may be below the above range, and the active ingredient may be used in an amount above the above range because there is no problem in terms of safety. .

The health functional beverage composition of the present invention is not particularly limited to other ingredients except for containing the compound as an essential ingredient in the indicated ratios, and contains various flavors or natural carbohydrates as additional ingredients, such as ordinary drinks. can do. Examples of the above described natural carbohydrates include monosaccharides such as glucose, fructose and the like; Disaccharides, for example maltose, sucrose and the like; And sugars such as conventional sugars such as polysaccharides such as dextrin, cyclotextin and the like, and xylyl, sorbitol, erythritol and the like. As flavoring agents other than those mentioned above, natural flavoring agents (tautin, stevia extracts (e.g., Rebaudioside A, glycyrgins, etc.) and synthetic flavoring crabs (saccharin, a Spartame and the like) can be advantageously used. The proportion of the natural carbohydrate is generally about 1 to 20 g, preferably about 5 to 12 g per 100 compositions of the present invention. In addition to the above, the compound of formula 1 of the present invention may be used in various nutrients, vitamins, minerals (electrolytes), synthetic flavors and natural flavoring agents, colorants and neutralizing agents (such as cheese, chocolate), pectic acid and salts thereof , alginic acid and its salts, organic acid, protective colloidal increase jeomjeo 1, _P H adjusting agents, stabilizers, preservatives, glycerin, alkoeul, may contain a carbonate agent used in carbonated drinks. In addition, the compound of formula 1 of the present invention may contain a fruit flesh for the production of natural fruit juice and fruit juice beverage and vegetable beverage.

 These components can be used independently or in combination. The proportion of such additives is not so critical but is generally selected in the range of 0.1 to about 20 parts by weight per 100 parts by weight of the compound of the present invention.

[Form for implementation of invention]

 Hereinafter, the present invention will be described in more detail with reference to Examples.

 However, the following examples are merely to illustrate the present invention, but the content of the present invention is not limited by the following examples.

Preparation Example 1 1-Methyl-3- [4- (2-phenoxazin-10-yl) phenyl] thiourea (1-methyl-3- [4- (2-phenoxazin-10-ylethoxy) phenyl] thiourea) ^)

Compounds are described in Cheon YJ, Gim HJ, Jang HR, Ryu JH, J eon R (2010) Synthesis of heterocyte one linked thioureas and their inhibitory activities of NO product ion in LPS activated macrophages. Bull Korean Chem Soc 31 (1): 27-30.

 Specifically, the corresponding mesylate was obtained by N-hydroxyethylation of the starting molecule, phenoxazine, and mesylation of the resulting alcohol.

 The alkylation reaction of mesylate and P-nitrophenyl is carried out in the presence of a base to obtain a nitro compound which is reduced to an amine by hydrogenation reaction using a palladium catalyst under hydrogen pressure. Thereafter, the amine was condensed with methyl isothiocyanate to obtain the target compound of Chemical Formula 1.

The obtained compound of Formula 1 was dissolved in dimethyl sulfoxide at 50 mM and used.

 <10)>

 Example 1 Measurement of NF-KB Activity in Rheumatoid Arthritis-Fibroblastoid Synovial Cells (RA-FLS)

 In order to examine the effect of the compound according to the invention on the NF-KB signal transduction in cultured RA-FLS was carried out as follows.

 <104>

 <i05> (1) Isolation and cultivation of a single body RA-FLS

<i06> RA-FLS was isolated from primary synovial tissue of 12 patients who had met the revised criteria of the US Department of Rheumatology with rheumatoid arthritis undergoing total joint replacement surgery or synovial resection. Cells were cultured after further incubation. to eight). Cells were incubated with 10% fetal bovine serum (FBS), 2 mM glutamine, 100 units / ml penicillin, 100 yg / ml straptomycin, and 2.5 yg / ml amphotericin in an incubator containing 37 ^° C. and C0 ₂ . Incubated in high glucose-containing Dulbecco's modified Eagle's medium with B added

 The informed consent was obtained from all patients, and the research plan was approved by the Ethics Committee of Chonbuk National University Hospital.

 <108>

 (2) Measurement of TNFa-induced NF-κΒ Activity

<ιιο> FLS (2><10^& ) isolated from rheumatoid arthritis patients was pretreated with 10 μΜ of the compound prepared in Preparation Example 1 and then treated with TNFa (10 ng / ml). <ιιι> Electrophoretic mobility shift analysis of NF-κΒ DNA binding activity after incubation for 3 hours

 Analysis by (EMSA).

Specifically, the RA-FLS (2 × 10 ⁶ ) was placed in PBS, washed twice by centrifugation, and compressed into 500 g of pellets for 5 minutes. The pellet was then dissolved in CytoBuster protein extraction buffer (Novagen, Madison, Wis., USA). Cellular and nuclear extracts were prepared using NE ᅳ PER nuclear and cytoplasmic extractants (Pierce, Rockford, IL, USA).

Nuclear extracts prepared from <H3> RA-FLS were treated with proteinase inhibitor cocktails (Calbiochem, San Diego, CA, USA) to inhibit endogenous protein activity. After <Π4>, oligonucleotides containing K-chain binding sites (5'-

CCGGTTMCAGAGGGGGCnTCCGAG-3 ') was synthesized and used as a probe for gel retardation analysis. The two corresponding strands were then heat treated and labeled with α-32Ρ-dCTP. Labeled oligonucleotide, nuclear extract of 10, and binding complete layer (10 mM Tris-HCl, pH 7.6, 500 mM KC1, 10 mM EDTA, 50% glycerol, 100 ng Poly [dI-dC], 1 mM dithio Tray) was mixed to a final volume of 20 μI and then incubated at room temperature for 30 minutes. The reaction mixture was then analyzed by electrophoresis on 4% polyacrylamide gel. The gel was dried and measured by radiograph. Characterization of DNA-protein interactions for NF-κΒ was confirmed by competition analysis using more than 50-fold unlabeled oligonucleotides.

 The measurement results are shown in FIG. 1.

 As shown in FIG. 1, the binding activity of the p65 subunit to the NF-κΒ consensus was increased in TNFa-induced RA-FLS compared to cells without TNFa stimulation. On the contrary, the nuclear extract prepared from RA-FLS treated with the compound according to the present invention was found to significantly reduce nuclear shifting and DNA binding of NF-κΒ.

 Therefore, the thiourea compound according to the present invention activates inflammatory cells.

 Since it effectively inhibits DNA binding of NF-κΒ, it can be usefully used for the prevention and treatment of inflammatory diseases such as rheumatoid arthritis caused by NF-κΒ activity.

 <118>

Example 2 Multiple Stages in Rheumatoid Arthritis-Fibroblastoid Synovial Cells (RA-FLS) Activity measurement of white matter

 <i2 ()> In order to determine the effect of the compound according to the invention on the ΙκΒα and ΙΚΚ protein in cultured RA-FLS was performed as follows.

FLS (2 × 10 ⁶ ) isolated from patients with rheumatoid arthritis was pretreated with 10 μM of the compound prepared in Preparation Example 1 for 1 hour and then treated with TNFa (10 ng / ml).

After incubation for 3 hours, the binding activity of p65, p50, I κΒα, IKK a, ΙΚΚβ, ρ-ΙΚΚα, ρ-ΙΚΚβ, 醒 Ρ-1, and ΜΜΡ-3 proteins were analyzed by Western blot.

Specifically, RA-FLS (2X10 ⁶ ) is a protein extraction solution (Pro-Prep, Intron

Biotechnology, Sungnam, Korea) and homogenized with a protease and phosphate inhibitor. Homogenates containing 30 proteins were separated by 10% digested dodecyl sulfate-poly acrylamide gel electrophoresis and transferred to a polyvinylidene difluoride membrane. β-actin and proliferative cell nuclear antigen (PCNA) were used as loading controls for cytosolic proteins and nuclear proteins, respectively.

P50, p65, I κΒα, PCNA, and β-actin (all manufactured by Santa Cruz Biotechnology,

 Santa Cruz, CA, USA), blot analysis using 1 yg / ml primary antibodies against IKK α, ΙΚΚβ, p-IKKa, and p—Ι Κβ (all of Cell Signaling, Beverly, MA, USA) It was.

 The analysis results are shown in FIGS.

FIG. 2 shows Western blot analysis results for p50, _P 65 and IicBa, and FIG. 3 shows Western blot analysis results for IKK α, ΙΚΚβ, ρ-ΙΚΚα, and ρ-ΙΚΚβ. <i27> Unlike ΙκΒβ, ΙκΒα mainly participates in cytokine-induced NF-κΒ activation in RA-FLS (Lee et al., 2009). Therefore, the present inventors observed ΙκΒα protein degradation in the cytoplasmic part following TNF α treatment.

 As a result, as shown in Fig. 2, TNFa compared to the cells without TNFa stimulation.

 Induced RA-FLS increased nuclear migration of p65 and p50 subunits and increased I κ Βα degradation in the cytoplasm. However, the nuclear extract prepared from the RA-FLS treated with the compound according to the present invention was shown to significantly inhibit the degradation of ΙκΒα protein.

 <129>

 <13 (»Next, the inventors analyzed Western blots for ΙΚΚ activation required for ΙκΒ phosphorylation, and as shown in FIG. 3, the compounds according to the present invention had no effect on IKK activation in cells without TNFa stimulation. TNFa-induced RA-FLS was found to reduce the phosphorylation of ΙΚΚα and ΙΚΚβ.

 <13!> Therefore, the compounds of the present invention inhibit the degradation of ΙκΒα protein, the ΙΚΚα and ΙΚΚβ

 By inhibiting the activity of NF-κΒ it is believed to inhibit the activity.

<132>

 Example 3 Accumulation of MMP Activation and Chemokine Production in Rheumatoid Arthritis-Fibroblastoid Synovial Cells (RA-FLS)

 In order to determine the effect of the compounds according to the invention on the production of Li P activity and chemokine in cultured RA-FLS, the following experiment was performed. .

<135> 10 μΜ of the compound prepared in Preparation Example 1 was added to FLS from patients with rheumatoid arthritis. After pretreatment for hours, it was exposed for 24 hours with TNFa (10 ng / ml).

 The presence of MMP-1 and MMP-3, and ENA-78 and RANTES in cell-free culture supernatants was assessed by protein coupled immunosorbent method (ELISA).

Specifically, Quantikine measures 謹 TES, ENA-78, and 匪 P-3 levels in the supernatant.

 Was measured using an ELISA kit (manufactured by R & D Systems, Minneapolis, MN, USA), and 醒 P-1 was determined by the manufacturer using a fluorocaine E human active MMP-1 fluorescence assay kit (manufactured by R & D Systems). By fluorescence.

 ##

 The measured value is expressed as mean 土 standard error (SEM), where p <0.01 vs. Untreated control; "pO.01 vs. TNFa. The measurement results are shown in FIGS. 4 and 5.

As shown in FIG. 4, when the RA-FLS was incubated with TNFa, the production of 匪 P-1 in the supernatant was increased by 3.4 times, and the production of 應 P-3 was increased by 3.5 times. Pretreatment of the compounds according to the invention reduced TNFa mediated MMP-1 and MMP-3 production by 68.4% and 77.8%, respectively.

<i4i> In addition, in the chemokine production, as shown in Figure 5, together with TNFa

 When RA-FLS was cultured, the production of RANTES in the supernatant was increased by 5.1 times and the production of ENA-78 was increased by 13.3 times. However, TNFa-mediated chemokine production was remarkable when the compound according to the present invention was pretreated. It has been shown to decrease.

Therefore, the thiourea compound according to the present invention significantly inhibits TNFa-mediated 薩 P group and chemokine production, thereby preventing and treating inflammatory diseases such as rheumatoid arthritis. It can be usefully used for treatment.

 <143>

 Example 4 Measurement of TNFa-induced Cell Proliferation in Rheumatoid Arthritis-Fibroblastoid Synovial Cells (RA-FLS)

 <i45> TNFa promotes the proliferation of RA-FLS under in vitro conditions and is known to act via a signaling pathway including NF-κΒ (Youn et al., 2002).

 Therefore, the following experiment was conducted to investigate the effect of the compound according to the present invention on TNFa-induced cell proliferation in RA-FLS. ·

FLS 1 × 10 ⁵ ) from patients with rheumatoid arthritis was pretreated with 10 μΜ of the compound prepared in Preparation Example 1 for 1 hour and then exposed to TNFa (10 ng / ml) for 72 hours.

<] 48 Cell proliferation was measured using BrdU incorporation assay, which is an immunosorbent assay linked to cell proliferation enzymes (BrdU kit; Amersham Biosciences, Piscat away, NJ, USA), according to the method recommended by the manufacturer. It was used to measure the incorporation of BrdU during DNA synthesis.

Specifically, after treatment with TNFa for 72 hours, BrdU (10 μΜ) was added to the culture medium for 2 hours to fix BrdU labeled cells, and DNA was added to the adhesive solution for 30 minutes at room temperature. And denatured. The cells were then incubated with peroxidase bound anti BrdU antibody for 2 hours at room temperature. The resulting immunocomplex was detected using 3,3 ', 5,5',-tetramethylbenzidine substrate reaction and the absorbance was measured at 405 nm. It was.

<i50> measurements are expressed as mean 土 standard error (SEM), where p <0.01 vs. Untreated control; "pO.Ol vs. TNFa. The measurement results are shown in FIG.

 As shown in FIG. 6, the cell proliferation activity after TNFa treatment for 72 hours was significantly increased to 141.3 土 2.4% compared to the control group (ρθ.01). However, it can be seen that the addition of the compound according to the present invention reduces the proliferative activity of TNFa-treated RA-FLS to 110.6 ± 4.7%. In addition, the compound according to the present invention was found to have no effect on TNFa induced proliferation activity.

 Therefore, the compound according to the present invention inhibits the activity of NF-κΒ in TNF-induced A-FLS, thereby reducing the proliferative activity of RA-FLS, and thus inflammatory diseases such as rheumatoid arthritis induced by NF-κΒ. Can be effectively used for prevention or treatment.

 <154>

 Example 5 Effect of Thiourea Compounds According to the Present Invention in a Mouse Model of Collagen-Induced Arthritis

 In order to examine the effect of the compound according to the present invention on inflammatory diseases in vivo, the following experiment was performed.

 <157>

(158) (1) collagen-induced arthritis mouse manufacture <i59> Pathogen-free male DBA / 1 mice were purchased from Orient Bio (Seoul, Korea). Mice were housed in a laminar flow cabinet with a 12 hour light / dark cycle, maintaining free feeding on standard laboratory feed. All experiments used in this experiment were approved by the Research Animal Care and Use Committee at Chonbuk National University.

 After this, male DBA / 1 mice (7-9 weeks of age) were placed in 150 bovine Π collagen.

 (collagen) and emulsified with the same amount of complete Freund's adjuvant (Chondrex, Redmond, WA, USA).

 The primary immunization date was defined as 0 days.

 The mice were then treated on compound 20 (100 y l of corn oil) on day 20.

 5 mg / kg body weight) or carrier in a single intraperitoneal injection. Then, on day 21, it was activated by bovine Π collagen emulsified with the same amount of Freund's adjuvant.

<163>

 (2) Observation of Arthritis Symptoms in Collagen-Induced Arthritis Mice

 After the observation of arthritis symptoms in mice over time is shown in Figure 7 and 8.

 Figure 7 is a picture of the symptoms of arthritis after or without administration of the compound according to the present invention, Figure 8 is arthritis of the mouse from day 21 to day 44 after administration or no administration of the compound according to the present invention It is a graph showing the cumulative occurrence rate of as a percentage.

As shown in FIG. 7, the carrier-injected mouse induced symptoms of arthritis due to collagen, swelling, erythema, and ankle stiffness. Mice injected with these compounds did not show symptoms of arthritis like normal mice.

 In addition, as shown in FIG. 8, the carrier-injected mice induced symptoms of arthritis by collagen, and more than 90% of arthritis occurred after 44 days, but the mice to which the compound according to the present invention was administered were treated. Has a 20% cumulative incidence of arthritis. Found to be very low.

 <169>

 (3) Measurement of Arthritis Index in Collagen-Induced Arthritis Mice

 <i7i> Clinical arthritis scores from day 21 to day 44 are shown in FIG. 9.

 The clinical score was determined in the range of 0-3 for each node: class 0, no swelling;

 1st grade, swollen erythema; 2 points, significantly swollen; Level 3, severe swelling and / or joint stiffness.

 In addition, the location where the hind foot was located across the ankle joint at the widest part was measured with an electric caliper. The increase in the diameter of arthritic ankles after a specific time at day 0 was defined as a foot thickness index, and this measure is expressed as a percentage. Foot thickness indices from day 21 to day 44 are shown in FIG. 10.

As shown in FIGS. 9 and 10, the carrier-injected mice induced symptoms of arthritis by collagen, and the clinical arthritis scores increased over time, and the foot thickness index also increased significantly. Appeared to be. However, mice administered the compound according to the present invention have been shown to alleviate the symptoms of arthritis.

<175>

(176) Histology of collagen-induced periarthritis mice On day 45, mice treated with the carrier or the compound according to the present invention were regenerated. Then, the lubricous membrane tissue of the hind paw was recovered from each animal to the end of the tissue, and the following experiment was performed.

 1) MMP group measurement

 <! 79> MMP- of the joint tissue of the mouse administered with the carrier or the compound according to the present invention

The binding activity of 1 and δ P-3 proteins was analyzed by Western blot.

 <! 80> Specifically, the joint tissue is a protein extraction solution (Pro-Prep, Intron

 Biotechnology, Sungnam, Korea) and homogenized with a protease and phosphate inhibitor. Homogenates containing 30 yg of protein were separated by 10% sodium dodecyl sulfate-poly acrylamide gel electrophoresis and transferred to a polyvinylidene difluoride membrane. β-actin was used as a loading control for cytoplasmic proteins.

<i8i> Blots were analyzed using 1 yg / ml primary antibody against MMP-1 and ΜΜΡ-3 proteins (both from R & D Systems).

The analysis results are shown in FIG. 11.

 As shown in FIG. 11, the addition of the compounds according to the present invention is performed by MMP # 1 and MMP-3.

 It has been shown to inhibit the binding of proteins.

 <184>

 2) Foot Observation in Collagen-Induced Arthritis Mice

 On day 45, the hind paws of mice administered with the carrier or the compound according to the present invention were cut and photographed by radiographs, as shown in FIG. 12.

The room image is based on a direct detection flat panel array design. A mammography imager (Mamiranat Novat i onDR, Siemens Medical Solutions, Er 1 angen, Germany) was used to obtain planar radiographs of the feet at exposure settings of 30 kVp and 90 mA.

 <188> All images are described in the score system described above (Joosten LA, He 1 sen 廳, Saxne T, van De).

 Loo FA, Heinegard D, van Den Berg WB (1999) IL ᅳ 1 α β blockade prevents cartilage and bone destruction in murine type II col lagen- induced arthritis, whereas TNF- a blockade only ameliorates joint inflammation. J Immunol 163 (9): 5049-5055.).

 The degree of joint destruction and bone erosion was assessed within 0-5 points, where 0 = no damage; 1 = weak bone damage as measured by one light spot; 2 = moderate change with 2-4 points in one area; 3 = significant change with 2-4 points in many areas; 4 = severe corrosion affecting the joints, and 5 = complete corrosion of the joints.

The average radioactivity score is shown in FIG. 13.

 As shown in FIGS. 12 and 13, the carrier-injected mice showed typical collagen-induced arthritis in which joint destruction, joint variability, and irreversible bone proliferation occurred over the entire ankle range. In the case of injection, bone destruction was significantly reduced and the average radiological score was also significantly reduced.

 <192>

 <193> 3) lubricating membrane tissue staining

At day 45, the hind paws of mice treated with the carrier or the compound according to the invention were cut to hematoxylin and eosin (H & E), safranin-0 or TRAP for fluorescence microscopy observation. Stained with.

 The stained portion is shown in Figure 14 by observing with a fluorescence microscope.

 In FIG. 14, the square region of each safranin 0 stained portion was enlarged at a higher magnification in the TRAP stained portion, where TB is tibia and TL is atalus. Indicates. The magnification of the fluorescence microscope is 100 times for hemaroxylin and eosin (H & E) staining and safranin-0 staining and 200 times for TRAP staining.

 As shown in FIG. 14, the ankle joint of the carrier-injected mouse showed lubricating fluid growth, cartilage damage, pannus formation, and bone corrosion, but when the compound according to the present invention was injected. There was a marked improvement in arthritis.

 <198>

 4) Osteoclast Differentiation

 Specifically, the joint tissues were incubated for 4 days with BMM in M-CSF (30 ng / ml) and RANKL (50 ng / ml) at the indicated concentration of the compound of formula 1 of the present invention. Cells were fixed with 3.7% formalin, permeabilized with 0.1% Triton X-100, and stained with TRAP solution.

 Osteoclasts are visualized by discoloration with the activity of tartrate resistant acid phosphate (TRAP). This was observed with a fluorescence microscope and is shown in FIG. 15, and TRAP-positive cells were counted as osteoclasts and shown in FIG. 16.

Measured values are expressed as mean standard error (SEM), where p <0.01 vs. Beecher Lee control.

 Osteoclasts are produced from mouse B cells in the presence of M-CSF and RANKL. However, as shown in Figure 15 and Figure 16, it can be seen that the osteoclast differentiation is reduced depending on the concentration when injecting the compound according to the present invention.

 <204>

<2 ⁽⁾ 5><Example6> collagen-induced arthritis mouse model NF-κΒ signaling inhibition measured in ahgye thio urea compound according to the invention in

 Mice received a single intraperitoneal injection of a compound of formula 1 (5 mg / kg body weight in 100 μl corn oil) or carrier on day 20. Then, on day 21, it was activated by bovine type II collagen emulsified with the same amount of Freund's adjuvant. Thereafter, joint homogenate was prepared on day 24.

 The NF-κΒ DNA binding activity with the joint homogenate was analyzed by electrophoretic mobility shift analysis (EMSA) in the same manner as in Example 1, and is shown in FIG. 17, and the p65, p50, and ΙκΒα proteins. Was shown in Figure 18 by the Western blot in the same manner as in Example 2. PCNA and β-actin were used as loading controls for nuclear and cytosolic proteins.

 As shown in FIGS. 17 and 18, in the in vivo experiments, arthritis occurs in the mice administered with the carrier as in the in vitro experiments, NF-KB activity increases, and degradation of ΙκΒα occurs. It can be seen that administration of the compound according to the present invention reduces NF-KB activity and degradation of ΙκΒα.

<209> <Example 7> Measurement of inhibition of proinflammatory cytokine production of thiourea compounds according to the present invention in a mouse model of collagen-induced arthritis

 Mice received a single intraperitoneal injection of a compound of formula 1 (5 mg / kg body weight in 100 μl corn oil) or carrier on day 20. Then, on day 21, it was activated by bovine type II collagen emulsified with the same amount of Freund's adjuvant. After 45 days, the hind paws and serum of the mice were collected, and the homogenate and various cytokines (TNF α, IL-Ιβ, RANKL, IL-17, etc.) in the serum were analyzed by specific enzyme-linked immunosorbent assay (ELISA). Measured as shown in Figure 19 and 20, respectively. -

As shown in FIG. 19, a significant decrease in TNFa, IL-Ιβ, RANKL and IL-17 was observed in the articular tissues of the mice administered the compound according to the present invention compared to the carrier-administered collagen-induced arthritis mice. Rheumatoid arthritis is a systemic autoimmune disease. As shown in Fig. 20, TNFa and IL-Ιβ levels were also measured in the serum, and the serum of the mice to which the compound according to the present invention was administered significantly reduced TNFa and IL-Ιβ levels as shown in the joint tissue. Can be.

Therefore, the compound according to the present invention effectively inhibits the production of cytokines such as TNFa and IL-Ιβ, and thus may be useful for the prevention and treatment of inflammatory diseases such as rheumatoid arthritis.

 <214>

 Preparation Example 1 Preparation of Tablet (Direct Pressurization)

 5.0 mg of the compound of Formula 1

<2i7> lactose 14.1 rag <2i> Crospovidone USNF 0.8 mg

 <2i> Magnesium Stearate 0.1 mg

 In accordance with a conventional method for preparing tablets, the tablets were prepared by adding the above ingredients in the amounts indicated and uniformly mixing and pressing.

 <221>

 Preparation Example 2 Preparation of Tablet (Wet Granulation)

 5.0 rag of the compound of Formula 1

 <224> lactose 16.0 mg

 <225> starch 4.0 mg

 <226> Presorbate 80 0.3 mg

 <227> silicon dioxide 2.7 mg

 <228> magnesium stearate 2.0 mg

 After sifting the compound of Formula 1, lactose and starch were mixed. Then, after dissolving polysorbate in pure water, an appropriate amount was added to the active ingredient, lactose and starch mixture and then granulated. After drying, the granules were granulated and then mixed with colloidal silicon dioxide and magnesium stearate. The granules were pressed to produce tablets.

 <230>

 Preparation Example 3 Preparation of Capsule

 <232>

5.0 mg of the compound of Formula 1 <234> Lactose 14.8 Illg

 <235> polyvinylpyridone 10.0 nig

 <236> magnesium stearate 0.2 lllg

 According to the conventional method for preparing a capsule, the above-mentioned ingredients were added to the contents of the formulations described above, and then uniformly mixed with each other, and then layered on a gelatin accelerator of appropriate size to prepare a desired capsule.

 <238>

 Preparation Example 4 Preparation of Injection

 <240> 100 nig of the compound of Formula 1

 <24i> 180 mg of Manni

_{<242> Na 2 HP0 4 ·} 12H 2 0 26 mg

<243> 2974 mg of distilled water

 According to a conventional method for preparing an injectable, an injectable was prepared by containing the above components in the contents shown.

 <245>

 Preparation Example 5 Preparation of Health Food

 <247> 1000 mg of a compound of Formula 1

 <248> Vitamin Complex

 <249> Vitamin A Acetate 70! g

<250> vitamin E 1.0 rag Vitamin 0.13 rag

 Vitamin B2 0.15 rag

 Vitamin B6 0.5 mg

 Vitamin B12 0.2 μ

 Vitamin C 10 nig

 Biotin 10 ig

 Nicotinamide 1.7 mg

 Folic acid 50 mg

Calcium Pantothenate 0.5 mg

Mineral Mixtures Proper

Ferrous Sulfate 1.75 mg-Zinc Oxide 0.82 mg

Magnesium carbonate 25.3 rag

15 nig of potassium monophosphate

Dicalcium Phosphate 55 rag

Potassium Citrate 90 nig

Calcium Carbonate 100 mg

Magnesium chloride 24.8 nig

The composition ratio of the vitamin and mineral mixtures described above is relatively suitable for health foods ¬¬combined in the preferred examples, but the composition ratio may be arbitrarily modified. After mixing, the above ingredients are mixed according to a conventional health food production method, and then granules are prepared, and can be used for preparing a health food composition according to a conventional method.

 <271>

 <272> <Example 6> Preparation of a health drink

 <273> 1000 nig of a compound of Formula 1

 <274> citric acid 1000 mg

 <275> 100 g oligosaccharides

 <276> plum concentrate 2 g

 <277> taurine 1 g-

<278> 900 mi total purified water

 <279>

 After mixing the above components in accordance with a conventional method for preparing a healthy beverage, and stirring and heating at 85 for about 1 hour, the resulting solution is filtered and obtained in a sterile 21 container, sealed sterilization and stored in storage It was then used to prepare a healthy beverage composition.

Although the composition ratio is a combination of relatively suitable ingredients for a preferred beverage as a preferred embodiment, the composition ratio may be arbitrarily modified according to regional and ethnic preferences such as demand hierarchy, demand country, and usage.

 <282>

 <Example 2> Preparation of Other Health Foods

 <284> 7-1. Manufacture of beverages

<285> honey 522 nig 05 thioctoamide 5 nig

 Nicotinic Acid 10 mg

 Riboflavin Sodium Hydrochloride 3 mg

 Pyridoxine hydrochloride 2 nig

 Inosi 30 mg

Orthoic acid 50 mg

0.48-1.28 mg of compound of Formula 1

Water 200 nd

With the above composition and content, drinks were prepared using conventional methods.

7-2. Preparation of Chewing Gum

Gum base 20%

Sugar 76.36-76.76%

Compound of Formula 1 0.24 0.64%

Fruit flavored 1 ¾>

Water 2%

Chew ¾ol was prepared using conventional methods using the above composition and content.

7-3. Candy

50-60% sugar <306> Starch syrup 39.26-49.66%

 0.24-0.64% of a compound of Formula 1

 <308> 0.1% of orange flavor

 Candy was prepared using a conventional method with the above composition and content.

<310>

 <3ΐι> 7-4. Manufacture of Flour Food

 0.5 to 5 parts by weight of the compound of Formula 1 was added to 100 parts by weight of flour, and bread, cake, cookies, crackers, and noodles were prepared using the mixture to prepare foods for health promotion.

 <313>

 <314> 7-5. Manufacture of dairy oroducts

 5 to 10 parts by weight of the compound of Formula 1 was added to 100 parts by weight of milk, and various dairy products such as butter and ice cream were prepared using the milk.

<316>

 <317> 7-6. Manufacture of wire

 Brown rice, barley, red rice, and yulmu were alphad by a known method to distribute the dried ones, and then prepared into a powder having a particle size of 60 mesh. Black beans, black sesame seeds, and sesame seeds were also roasted by steaming in a known manner, and then prepared into a powder having a particle size of 60 mesh. Cereals and seeds prepared above and the compound of formula 1 of the present invention was prepared by combining the following ratio.

<319> <320> 30%

<321> 15%

<322> Barley 20%

 <324> 7% black beans

<325> black sesame 7%

<326> Compound 3 of Formula 1

<327> Young 0.5%

 <329>

Claims

Claims Claim 11 A pharmaceutical composition for the prophylaxis and treatment of an inflammatory disease containing a thiourea compound represented by the formula (1) or a pharmaceutically acceptable salt thereof as an active ingredient:  [Formula 1]

[Claim 2]  According to claim 1, wherein the compound represented by Formula 1 is a pharmaceutical composition for the prevention and treatment of inflammatory diseases, characterized in that for reducing the activity of NF-i B. [Claim 3] The pharmaceutical composition for preventing and treating inflammatory diseases according to claim 1, wherein the compound represented by Formula 1 reduces the activity of ΙΚΚα and ΙΚΚ and the degradation of I _K Ba. [Claim 4] According to claim 1, wherein the compound represented by the formula (1) is TNFa mediated 醒 P-1 and 丽 pharmaceutical composition for the prevention and treatment of inflammatory diseases, characterized by inhibiting P-3 production. [Claim 5]  The method of claim 1, wherein the inflammatory disease is any one selected from the group consisting of asthma, periodontitis, stomatitis, peritonitis, gastritis, enteritis, arthritis, rheumatoid arthritis, nephritis, hepatitis and degenerative diseases. And pharmaceutical compositions for treatment, [Claim 6]  According to claim 5, The inflammatory disease is a salt composition for preventing and treating salt diseases, characterized in that rheumatoid arthritis. [Claim 7]  Health food composition for the prevention and improvement of inflammatory diseases containing a thiourea-based compound represented by the formula (1) or a pharmaceutically acceptable salt thereof as an active ingredient:  [Formula 1]

(Claim 8)  The method of claim 7, wherein the compound represented by the formula (1) is a health food composition for the prevention and improvement of inflammatory diseases, characterized in that for reducing the activity of NF—κΒ. [Claim 9]  The method of claim 7, wherein the compound represented by the formula (1) is a health food composition for the prevention and improvement of inflammatory diseases, characterized in that to reduce the activity of ΙΚΚα and ΙΚΚβ activity and degradation of ΙκΒα. [Claim 10]  According to claim 7, wherein the compound represented by the formula (1) is a healthy food composition for the prevention and improvement of inflammatory diseases, characterized in that TNFa-mediated 匪 P-1 and 丽 P-3 production is inhibited. [Claim 11] 8. The prevention of inflammatory diseases according to claim 7, wherein the inflammatory disease is any one selected from the group consisting of asthma, periodontitis, stomatitis, peritonitis, gastritis, enteritis, arthritis, rheumatoid arthritis, nephritis, hepatitis and degenerative diseases. And improving health food compositions. [Claim 12]  The method of claim 11, wherein the inflammatory disease is inflammatory disease prevention and improvement health food composition, characterized in that the rheumatoid arthritis. [Claim 13]  A method of treating a metabolic disease or complication thereof comprising administering a thiourea compound represented by Formula 1 or a pharmaceutically acceptable salt thereof to a patient in need thereof.  [Formula 1]

[Claim 14]  Thiourea compound represented by the following formula (1) or a pharmaceutically acceptable salt thereof used for the prevention or treatment of metabolic diseases or complications thereof.  [Formula 1]