[go: up one dir, main page]

WO2012060594A2 - Composition anti-inflammatoire contenant un composé thiourée et un sel pharmaceutiquement acceptable de celui-ci en tant que principe actif - Google Patents

Composition anti-inflammatoire contenant un composé thiourée et un sel pharmaceutiquement acceptable de celui-ci en tant que principe actif Download PDF

Info

Publication number
WO2012060594A2
WO2012060594A2 PCT/KR2011/008205 KR2011008205W WO2012060594A2 WO 2012060594 A2 WO2012060594 A2 WO 2012060594A2 KR 2011008205 W KR2011008205 W KR 2011008205W WO 2012060594 A2 WO2012060594 A2 WO 2012060594A2
Authority
WO
WIPO (PCT)
Prior art keywords
formula
present
prevention
compound represented
arthritis
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/KR2011/008205
Other languages
English (en)
Korean (ko)
Other versions
WO2012060594A3 (fr
Inventor
전라옥
류재하
천예진
박병현
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Industry Academic Cooperation Foundation of Chonbuk National University
Sookmyung Womens University SWU
Original Assignee
Industry Academic Cooperation Foundation of Chonbuk National University
Sookmyung Womens University SWU
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Industry Academic Cooperation Foundation of Chonbuk National University, Sookmyung Womens University SWU filed Critical Industry Academic Cooperation Foundation of Chonbuk National University
Publication of WO2012060594A2 publication Critical patent/WO2012060594A2/fr
Publication of WO2012060594A3 publication Critical patent/WO2012060594A3/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

Links

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/535—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with at least one nitrogen and one oxygen as the ring hetero atoms, e.g. 1,2-oxazines
    • A61K31/5375—1,4-Oxazines, e.g. morpholine
    • A61K31/538—1,4-Oxazines, e.g. morpholine ortho- or peri-condensed with carbocyclic ring systems
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P11/00—Drugs for disorders of the respiratory system
    • A61P11/06—Antiasthmatics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/02—Drugs for skeletal disorders for joint disorders, e.g. arthritis, arthrosis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]

Definitions

  • Anti-inflammatory composition containing a thiourea compound or its pharmaceutically acceptable salt as an active ingredient.
  • the present invention relates to an anti-inflammatory composition containing a thiourea-based compound or a pharmaceutically acceptable salt thereof as an active ingredient.
  • Rheumatoid arthritis 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).
  • fibroblast-like synoviocytes FLS and inflammatory cells in the joints produce inflammatory cytokines such as interleukin- 1 ⁇ (IL ⁇ 1 ⁇ ) and tumor necrosis factor- ⁇ (TNFa). do. These cytokines activate NF- ⁇ It intensifies inflammation, thereby increasing the expression of cytokines.
  • IL ⁇ 1 ⁇ interleukin- 1 ⁇
  • TNFa tumor necrosis factor- ⁇
  • inflammatory mediators include cytokines, RANTES and chemokines such as ENA-78 and adhesion molecules.
  • NF- ⁇ activity in FLS affects the symptoms of rheumatoid arthritis by increasing the expression of matrix metalloproteinases (XP).
  • XP matrix metalloproteinases
  • NF- ⁇ 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).
  • ⁇ i2> injection of adenovirus gene transfer of NF- ⁇ decoy olionucleotide or dominant negative I ⁇ kinase ⁇ 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 ⁇ like synoviocytes and col lagen-induced arthritic mice. Arthritis Rheum 62 (8): 2313-2321).
  • the present inventors while studying to develop a compound that inhibits the activity of NF- ⁇ , 1- (4- (2— (10— (10-H-phenoxazine-10-yl) ethoxy) phenyl) NF- ⁇ -related inflammatory diseases by confirming the activity of reducing NF- ⁇ and inhibiting edema and pain in mouse models of -3-methylthiourea islet cell-type synovial membrane cells (FLS) and collagen-induced arthritis was completed.
  • FLS -3-methylthiourea islet cell-type synovial membrane cells
  • 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.
  • 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.
  • 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.
  • the thiourea-based compounds or pharmaceutically acceptable salts thereof according to the present invention reduce the activity of NF- ⁇ , 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.
  • ESA electrophoretic mobiloty shift assay
  • Figure 3 is a Western blot analysis showing the phosphorylation inhibitory activity of ⁇ and ⁇ of the compound according to an embodiment of the present invention.
  • Figure 4 is the result of the instantaneous MMP-1 and ⁇ -3 inhibitory activity of the compound according to an embodiment of the present invention immediately by the enzyme-linked immunosorbent assay (ELISA) method.
  • ELISA enzyme-linked immunosorbent assay
  • FIG. 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.
  • FLS fibroblast-like synovial cell
  • 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.
  • 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.
  • FIG. 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- ⁇ 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 ⁇ 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.
  • 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 ⁇ (2-phenoxaz i n_ 10— y 1 ethoxy) pheny 1] thi our ea).
  • 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.
  • 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.
  • a miscible organic solvent such as methanol, ethane, acetone or acetonitrile
  • 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.
  • the metal salt it is pharmaceutically suitable to prepare sodium, potassium, or champ salt.
  • 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.
  • 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,
  • 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 ⁇ ⁇ , which is one of the major signaling systems of inflammatory diseases, and degrades ⁇ . It can be usefully used for the prevention or treatment of inflammatory diseases by reducing the activity and suppressing various cytokine production.
  • 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 .
  • 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.
  • 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 ⁇
  • diluents or excipients such as extenders, wetting agents, disintegrants, lubricants, binders, surfactants and the like can be used.
  • 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.
  • magnesium aluminum silicate, starch paste, gelatin, tragacanth, methylsalose, sodium carboxymethylcellose, polyvinylpyridine, low-substituted hydroxypropylcelose and the like can be used.
  • 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.
  • 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.
  • 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.
  • 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.
  • the active ingredient 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. - ⁇
  • composition of the present invention may further comprise suitable carriers, excipients and diluents commonly used in the manufacture of a medicament.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • composition according to the present invention exhibits an activity of reducing the activity of NF- ⁇ , one of the major signaling systems of inflammatory diseases, reducing the degradation of ⁇ and inhibiting various cytokine production, thereby preventing and preventing inflammatory diseases.
  • 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.
  • 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, 3 ⁇ 4, 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • additives 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.
  • 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.
  • 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 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 ⁇ 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.
  • 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 ⁇ -32 ⁇ -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 ⁇ I 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- ⁇ was confirmed by competition analysis using more than 50-fold unlabeled oligonucleotides.
  • the binding activity of the p65 subunit to the NF- ⁇ consensus was increased in TNFa-induced RA-FLS compared to cells without TNFa stimulation.
  • 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- ⁇ .
  • the thiourea compound according to the present invention activates inflammatory cells.
  • RA-FLS (2X10 6 ) is a protein extraction solution (Pro-Prep, Intron
  • FIG. 2 shows Western blot analysis results for p50, P 65 and IicBa
  • FIG. 3 shows Western blot analysis results for IKK ⁇ , ⁇ , ⁇ - ⁇ , and ⁇ - ⁇ .
  • ⁇ i27> Unlike ⁇ , ⁇ mainly participates in cytokine-induced NF- ⁇ activation in RA-FLS (Lee et al., 2009). Therefore, the present inventors observed ⁇ protein degradation in the cytoplasmic part following TNF ⁇ treatment.
  • TNFa compared to the cells without TNFa stimulation.
  • RA-FLS Induced RA-FLS increased nuclear migration of p65 and p50 subunits and increased I ⁇ ⁇ degradation in the cytoplasm.
  • 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 ⁇ protein.
  • the compounds of the present invention inhibit the degradation of ⁇ protein, the ⁇ and ⁇
  • ⁇ 135> 10 ⁇ 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).
  • MMP-1 and MMP-3, and ENA-78 and RANTES in cell-free culture supernatants were assessed by protein coupled immunosorbent method (ELISA).
  • Quantikine measures ⁇ TES, ENA-78, and ⁇ P-3 levels in the supernatant.
  • the measured value is expressed as mean ⁇ standard error (SEM), where p ⁇ 0.01 vs. Untreated control; "pO.01 vs. TNFa.
  • SEM standard error
  • the thiourea compound according to the present invention significantly inhibits TNFa-mediated ⁇ P group and chemokine production, thereby preventing and treating inflammatory diseases such as rheumatoid arthritis. It can be usefully used for treatment.
  • TNFa promotes the proliferation of RA-FLS under in vitro conditions and is known to act via a signaling pathway including NF- ⁇ (Youn et al., 2002).
  • BrdU incorporation assay 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.
  • BrdU (10 ⁇ ) 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 ⁇ standard error (SEM), where p ⁇ 0.01 vs. Untreated control; "pO.Ol vs. TNFa. The measurement results are shown in FIG.
  • the cell proliferation activity after TNFa treatment for 72 hours was significantly increased to 141.3 ⁇ 2.4% compared to the control group ( ⁇ .01).
  • the addition of the compound according to the present invention reduces the proliferative activity of TNFa-treated RA-FLS to 110.6 ⁇ 4.7%.
  • the compound according to the present invention was found to have no effect on TNFa induced proliferation activity.
  • the compound according to the present invention inhibits the activity of NF- ⁇ in TNF-induced A-FLS, thereby reducing the proliferative activity of RA-FLS, and thus inflammatory diseases such as rheumatoid arthritis induced by NF- ⁇ . Can be effectively used for prevention or treatment.
  • mice Male DBA / 1 mice (7-9 weeks of age) were placed in 150 bovine ⁇ collagen.
  • the primary immunization date was defined as 0 days.
  • mice were then treated on compound 20 (100 y l of corn oil) on day 20.
  • 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.
  • 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.
  • 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.
  • the clinical score was determined in the range of 0-3 for each node: class 0, no swelling;
  • foot thickness index 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.
  • mice 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.
  • 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.
  • the binding activity of 1 and ⁇ P-3 proteins was analyzed by Western blot.
  • the joint tissue is a protein extraction solution (Pro-Prep, Intron
  • the addition of the compounds according to the present invention is performed by MMP # 1 and MMP-3.
  • 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.
  • the average radioactivity score is shown in FIG. 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.
  • bone destruction was significantly reduced and the average radiological score was also significantly reduced.
  • 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.
  • 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.
  • 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.
  • 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.
  • TRAP tartrate resistant acid phosphate
  • 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.
  • mice received a single intraperitoneal injection of a compound of formula 1 (5 mg / kg body weight in 100 ⁇ l 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.
  • a compound of formula 1 (5 mg / kg body weight in 100 ⁇ l 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- ⁇ 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 ⁇ proteins.
  • ESA electrophoretic mobility shift analysis
  • FIG. 17 The NF- ⁇ 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 ⁇ proteins.
  • ESA electrophoretic mobility shift analysis
  • PCNA and ⁇ -actin were used as loading controls for nuclear and cytosolic proteins.
  • mice received a single intraperitoneal injection of a compound of formula 1 (5 mg / kg body weight in 100 ⁇ l 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 ⁇ , IL- ⁇ , 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. -
  • TNFa, IL- ⁇ , RANKL and IL-17 were 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.
  • TNFa and IL- ⁇ 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- ⁇ levels as shown in the joint tissue. Can be.
  • the compound according to the present invention effectively inhibits the production of cytokines such as TNFa and IL- ⁇ , and thus may be useful for the prevention and treatment of inflammatory diseases such as rheumatoid arthritis.
  • the tablets were prepared by adding the above ingredients in the amounts indicated and uniformly mixing and pressing.
  • 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.
  • 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.
  • an injectable was prepared by containing the above components in the contents shown.
  • Vitamin B6 0.5 mg
  • Vitamin B12 0.2 ⁇
  • 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.
  • 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.
  • 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.
  • 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.
  • drinks were prepared using conventional methods.
  • Chew 3 ⁇ 4ol was prepared using conventional methods using the above composition and content.
  • 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.

Landscapes

  • Health & Medical Sciences (AREA)
  • General Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Veterinary Medicine (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Medicinal Chemistry (AREA)
  • Animal Behavior & Ethology (AREA)
  • Public Health (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • General Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Engineering & Computer Science (AREA)
  • Rheumatology (AREA)
  • Pulmonology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Orthopedic Medicine & Surgery (AREA)
  • Pain & Pain Management (AREA)
  • Physical Education & Sports Medicine (AREA)
  • Immunology (AREA)
  • Epidemiology (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
  • Acyclic And Carbocyclic Compounds In Medicinal Compositions (AREA)

Abstract

La présente invention concerne une composition pharmaceutique pour la prévention ou le traitement de maladies inflammatoires, la composition pharmaceutique contenant, en tant que principe actif, un composé thiourée représenté par la Formule 1 suivante ou un sel pharmaceutiquement acceptable de celui-ci. Le composé thiourée ou son sel pharmaceutiquement acceptable, selon la présente invention, réduit l'activité de NF-kB qui provoque des maladies inflammatoires dans un synoviocyte de type fibroblastique et chez un modèle murin d'arthrite induite par le collagène, et inhibe efficacement l'œdème et la douleur. Ainsi, la composition de la présente invention peut être utilisée efficacement dans la prévention et le traitement de maladies inflammatoires, telles que la polyarthrite rhumatoïde.
PCT/KR2011/008205 2010-11-05 2011-10-31 Composition anti-inflammatoire contenant un composé thiourée et un sel pharmaceutiquement acceptable de celui-ci en tant que principe actif Ceased WO2012060594A2 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
KR1020100109793A KR20120048250A (ko) 2010-11-05 2010-11-05 티오우레아계 화합물 또는 이의 약학적으로 허용 가능한 염을 유효성분으로 함유하는 항염증 조성물
KR10-2010-0109793 2010-11-05

Publications (2)

Publication Number Publication Date
WO2012060594A2 true WO2012060594A2 (fr) 2012-05-10
WO2012060594A3 WO2012060594A3 (fr) 2012-06-28

Family

ID=46024928

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/KR2011/008205 Ceased WO2012060594A2 (fr) 2010-11-05 2011-10-31 Composition anti-inflammatoire contenant un composé thiourée et un sel pharmaceutiquement acceptable de celui-ci en tant que principe actif

Country Status (2)

Country Link
KR (1) KR20120048250A (fr)
WO (1) WO2012060594A2 (fr)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN111830254A (zh) * 2020-07-29 2020-10-27 武汉生之源生物科技股份有限公司 一种基质金属蛋白酶-3测定试剂盒及其制备方法

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR101639599B1 (ko) * 2015-11-09 2016-07-14 서울대학교산학협력단 펩타이드 싸이오우레아 유도체, 이를 포함하는 방사성 동위원소 표지 화합물 및 이를 유효 성분으로 함유하는 전립선암 치료 또는 진단용 약학적 조성물

Family Cites Families (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CA2468302C (fr) * 2001-11-29 2012-08-14 Theracos, Inc. Composes servant au traitement d'une inflammation, des diabetes et des troubles associes
CN101910163A (zh) * 2007-10-29 2010-12-08 先灵公司 杂环脲和硫脲衍生物及其用法

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN111830254A (zh) * 2020-07-29 2020-10-27 武汉生之源生物科技股份有限公司 一种基质金属蛋白酶-3测定试剂盒及其制备方法
CN111830254B (zh) * 2020-07-29 2022-07-26 武汉生之源生物科技股份有限公司 一种基质金属蛋白酶-3测定试剂盒及其制备方法

Also Published As

Publication number Publication date
WO2012060594A3 (fr) 2012-06-28
KR20120048250A (ko) 2012-05-15

Similar Documents

Publication Publication Date Title
US11571435B2 (en) S1PR4-targeting composition for preventing or treating non-alcoholic steatohepatitis
US20140010899A1 (en) Pharmaceutical composition for preventing or treating inflammatory diseases comprising trachelospermi caulis extract and paeonia suffruticosa andrews extract, and method for preparing the same
US20230082624A1 (en) Composition, containing quisqualis indica extract, for preventing or treating prostatic hyperplasia
US20200223824A1 (en) Pharmaceutical composition containing indirubin derivative as active ingredient
US11931324B2 (en) Pharmaceutical composition for prevention or treatment of osteoarthritis comprising obtusifolin, derivative thereof, or pharmaceutically acceptable salt thereof as active ingredient
KR102338415B1 (ko) 3,4-디카페오일퀴닉산을 포함하는 골관절염 예방 또는 치료용 약학 조성물
CN117715634A (zh) 包含化合物的混合物的组合物及其用途
KR101458061B1 (ko) 항암용 조성물
JP7591283B2 (ja) ブドウ膜黒色腫の治療のための併用療法
WO2012060594A2 (fr) Composition anti-inflammatoire contenant un composé thiourée et un sel pharmaceutiquement acceptable de celui-ci en tant que principe actif
KR20130107533A (ko) 죽여 추출물을 유효성분으로 함유하는 만성폐쇄성 폐질환 예방 및 치료용 약학적 조성물
KR101875705B1 (ko) 디아미노디페닐술폰을 포함하는 근육 소모 관련 질환의 예방 또는 치료용 약학적 조성물
KR102790960B1 (ko) 커큐민 유도체와 TGF-β 수용체 억제제의 병용투여를 통한 비알코올성 지방간염 치료 방법
KR101539978B1 (ko) 크로멘-4-온 유도체 또는 이의 약학적으로 허용가능한 염을 유효성분으로 포함하는 골형성 촉진용 약학적 조성물
KR101492213B1 (ko) 폴리코사놀을 유효성분으로 하는 히스타민 수용체 길항제 조성물
KR101898610B1 (ko) ＰＰＡＲδ 활성물질의 태자 재프로그래밍 용도
KR100676761B1 (ko) 신남알데히드 유도체 화합물을 함유하는 염증성 질환의 예방 및 치료용 약학조성물
KR101457442B1 (ko) 감마오리자놀을 유효성분으로 하는 히스타민 수용체 길항제 조성물
JP7671091B2 (ja) シクロ－ｌ－フェニルアラニル－ｌ－プロリンジペプチドを含む筋線維形成の促進または破骨細胞の分化抑制メカニズムによるサルコペニアまたは骨粗鬆症の治療用組成物
KR20130112236A (ko) 부티르산 나트륨을 유효성분으로 함유하는 고 글루코즈로 유도된 염증 억제용 조성물
AU2020205863B2 (en) Pharmaceutical composition for treatment of dementia and cerebrovascular disorders
KR101832351B1 (ko) 신코닌 또는 이의 약학적으로 허용 가능한 염을 유효성분으로 함유하는 골질환의 예방 또는 치료용 약학적 조성물
KR101255939B1 (ko) 4-(1ｈ-인다졸-6-일아미노)-ｎ-아이소프로필-2-(메틸티오)피리미딘-5-카르복스아미드 화합물 또는 이의 약학적으로 허용가능한 염을 포함하는 당뇨병 예방 또는 치료용 조성물
KR20250046438A (ko) Frangulin A를 유효성분으로 포함하는 섬유화증 억제용 조성물
KR20240029547A (ko) 퀸산을 유효성분으로 함유하는 근육 질환 개선, 치료 또는 예방용, 또는 근 기능 개선용 조성물

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 11838199

Country of ref document: EP

Kind code of ref document: A2

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 11838199

Country of ref document: EP

Kind code of ref document: A2