WO2017026830A1 - Pharmaceutical composition for preventing or treating autoimmune diseases comprising thiourea derivative - Google Patents

Pharmaceutical composition for preventing or treating autoimmune diseases comprising thiourea derivative Download PDF

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WO2017026830A1
WO2017026830A1 PCT/KR2016/008867 KR2016008867W WO2017026830A1 WO 2017026830 A1 WO2017026830 A1 WO 2017026830A1 KR 2016008867 W KR2016008867 W KR 2016008867W WO 2017026830 A1 WO2017026830 A1 WO 2017026830A1
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composition
cells
present
autoimmune diseases
disease
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PCT/KR2016/008867
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French (fr)
Korean (ko)
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이미옥
박형근
조미라
한용현
김현지
박진실
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서울대학교 산학협력단
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Priority claimed from KR1020160102224A external-priority patent/KR101834005B1/en
Application filed by 서울대학교 산학협력단 filed Critical 서울대학교 산학협력단
Priority to US15/752,079 priority Critical patent/US20180235912A1/en
Publication of WO2017026830A1 publication Critical patent/WO2017026830A1/en

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    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00Medicinal preparations containing organic active ingredients
    • A61K31/16Amides, e.g. hydroxamic acids
    • A61K31/17Amides, e.g. hydroxamic acids having the group >N—C(O)—N< or >N—C(S)—N<, e.g. urea, thiourea, carmustine

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  • the present invention relates to a novel use of thiourea derivatives, and more particularly to a pharmaceutical composition for preventing or treating autoimmune diseases comprising a thiourea derivative as an active ingredient.
  • the human immune system protects the body from foreign antigens that invade the body, but does not attack its own tissues because of its self tolerance. However, when the immune system's self-tolerance is destroyed and the protein normally expressed by one's genes is recognized by the immune cell as the target of attack, the antibody is made or the T-cell reaction causes the normal tissue to be destroyed. It is called autoimmune disease.
  • Rheumatoid arthritis is a chronic disease that causes inflammation of the synovial membrane that surrounds the joint, spreads the cartilage and bones around it, destroys the joint, and causes disorders. The cells gather, causing the joint fluid to increase, resulting in pain as the joint swells.
  • Rheumatoid arthritis is a disease mainly caused by autoimmune adverse reactions, and abnormally functioning immune functions cause inflammation. Inflammation reactions in joints are mainly involved in TNF- and secreted TNF- Inflammation is caused by ⁇ , interleukin (IL) -1 ⁇ , IL-6, IL-17.
  • IL interleukin
  • M1 macrophages secreting TNF- ⁇ and IL-1 ⁇ and Th17 cells secreting IL-17 are known to exacerbate rheumatoid arthritis disease.
  • TNF inhibitors Infliximab, Etanercept, etc.
  • IL-1 inhibitors Asakinra, Canakinumab
  • steroids nonsteroidal inflammatory inhibitors
  • NSAIDs nonsteroidal inflammatory inhibitors
  • Actemra cytokine inhibitors
  • signaling JAK3 inhibitors or TNF- ⁇ -related antibody therapies
  • the present invention has been made to solve the above-mentioned problems in the prior art, the inventors of the present invention, the thiourea derivatives, which is an activator of ROR ⁇ inhibits the inflammatory response, inhibits the differentiation and activation of Th17 cells and at the same time generates Treg cells It confirmed that it promotes, and based on this, this invention was completed.
  • an object of the present invention is to provide a pharmaceutical composition for preventing or treating autoimmune diseases, comprising a thiourea derivative or a pharmaceutically acceptable salt thereof as an active ingredient.
  • the present invention provides a pharmaceutical composition for preventing or treating autoimmune diseases, comprising a compound represented by the following formula (1) or a pharmaceutically acceptable salt thereof as an active ingredient.
  • R is a phenoxy group, benzyloxy group or pyridinyl group.
  • the autoimmune disease is rheumatoid arthritis, psoriatic arthritis, psoriasis, systemic scleroderma, sclerosis (sclerosis), multiple sclerosis, inflammatory bowel disease, systemic lupus erythematosus, Crohn's disease ( crohn's disease), sepsis and type I diabetes.
  • the composition may inhibit the production of Th17 cells.
  • the composition may promote the production of Regulatory T cells (Treg).
  • the composition may inhibit M1 macrophage differentiation by inhibiting the expression of TNF- ⁇ , NOS2, IL-1 ⁇ or IL-6 inflammatory genes.
  • the composition may promote expression of anti-inflammatory genes such as IL-10, Arg1, Retnla or CD206 in macrophages to enhance M2 macrophage differentiation.
  • anti-inflammatory genes such as IL-10, Arg1, Retnla or CD206
  • the present invention provides a method for treating autoimmune disease comprising administering a compound represented by Formula 1 or a pharmaceutically acceptable salt thereof to a subject.
  • the present invention provides a method for treating autoimmune disease of a compound represented by Formula 1 or a pharmaceutically acceptable salt thereof.
  • Thiourea derivatives according to the present invention not only inhibit the transcription of inflammatory genes such as TNF- ⁇ , IL-1 ⁇ , NOS2, IL-6 and promote the transcription of anti-inflammatory genes such as IL-10, Arg1, CD206, Pharmaceutical compositions for the prevention, amelioration or treatment of various autoimmune diseases, including rheumatoid arthritis, because they can inhibit the activity or production of Th17 and increase the activity or production of Regulatory T cells (Tregs), It is expected to be useful for health food compositions and the like.
  • Figure 1 shows the results confirmed by Real-time PCR the inhibitory effect of M1 inflammatory gene transcription of JC1-40 in macrophage cell line.
  • Figure 2a and Figure 2b is the result confirmed by the real-time PCR the M1 inflammatory gene transcription inhibition effect of JC1-40 in primary cultured mouse intraperitoneal macrophages.
  • Figure 3 is the result of confirming by flow cytometry the decrease of M1 differentiated macrophages when treated with JC1-40 in primary cultured mouse intraperitoneal macrophages.
  • Figures 4a and 4b is a result confirmed by the real-time PCR the M2 anti-inflammatory gene transcription inhibition effect of JC1-40 in primary cultured mouse intraperitoneal macrophages.
  • Figure 5a is a result confirmed by the real-time PCR the IL-17 gene transcription inhibition effect of JC1-40 in T cell line.
  • Figure 5b is the result confirmed by immunostaining the expression of Treg expression of JC1-40 in T cell line.
  • Figure 6a is the result of confirming the reduction of Th17 differentiated cells by flow cytometry when JC1-40 concentration-treated in primary cultured mouse spleen T cells.
  • Figure 6b is the result of measuring the amount of IL-17 in the supernatant when treated with JC1-40 concentration in primary cultured mouse spleen T cells by ELISA.
  • Figure 7a is a result of confirming the quantitative change of Th17 and Treg differentiated cells by flow cytometry when treated with concentrations of JC1-40 in human peripheral mononuclear cells (PBMC).
  • PBMC peripheral mononuclear cells
  • Figure 7b is the result of measuring the amount of IL-17 in the supernatant when the concentration of JC1-40 in human peripheral mononuclear cell (PBMC) by ELISA.
  • FIGS. 8A and 8B show the results of confirming the effect of inhibiting the expression of osteoclast differentiation-related factors by real-time PCR when JC1-40 was treated in different concentrations in osteoclast precursors differentiated from human peripheral mononuclear cell (PBMC). .
  • PBMC peripheral mononuclear cell
  • the present inventors have conducted research on substances effective in treating autoimmune diseases including rheumatoid arthritis.
  • thiourea derivatives which are activators of ROR ⁇ , not only inhibit the expression of inflammatory cytokines such as TNF- ⁇ , IL-1 ⁇ , IL-6, etc.
  • the present invention was completed by promoting the expression of anti-inflammatory factors such as IL-10, Arg1, CD206, inhibiting or inhibiting the differentiation and activation of Th17 cells, and promoting the production of Treg cells.
  • the present invention provides a pharmaceutical composition for preventing or treating autoimmune diseases, comprising a compound represented by the following formula (1) or a pharmaceutically acceptable salt thereof as an active ingredient.
  • R is a phenoxy group, benzyloxy group or pyridinyl group.
  • prevention means any action that inhibits or delays the onset of autoimmune diseases by administration of a pharmaceutical composition according to the present invention.
  • treatment refers to any action in which symptoms caused by an autoimmune disease are improved or advantageously changed by administration of the pharmaceutical composition according to the present invention.
  • Autoimmune disease a disease to be improved, prevented or treated by the composition of the present invention, when there is a problem in inducing or maintaining self-tolerance, an immune response to its antigen occurs, thereby attacking its own tissue. This phenomenon occurs, which means a disease caused by this process.
  • the type of autoimmune disease in the present invention is not limited thereto, but rheumatoid arthritis, psoriatic arthritis, psoriasis, systemic scleroderma, sclerosis (sclerosis), multiple sclerosis, inflammatory bowel disease, systemic redness And may be selected from the group consisting of seminal lupus, crohn's disease, sepsis and type I diabetes, and preferably rheumatoid arthritis.
  • the compound represented by Chemical Formula 1, which is included as an active ingredient in the composition according to the present invention, is a thiourea derivative having a conventional thiazolidinedione-based compound CGP52608 (called JC1 compound), preferably 1-methyl-3- ( 4- (pyridin-2-yl) benzyl) -thiourea (JC1-38), 1- (4-benzyloxy-benzyl) -3-methyl-thiourea (JC1-40) or 1- (4-phenoxy-benzyl ) -3-methyl-thiourea (JC1-42).
  • JC1 compound thiourea derivative having a conventional thiazolidinedione-based compound CGP52608 (called JC1 compound), preferably 1-methyl-3- ( 4- (pyridin-2-yl) benzyl) -thiourea (JC1-38), 1- (4-benzyloxy-benzyl) -3-methyl-thiourea (JC1-40) or 1- (4-phenoxy-benzyl )
  • composition according to the present invention can exhibit the therapeutic effect of autoimmune diseases by inhibiting the expression of inflammatory genes and inhibiting M1 macrophage differentiation.
  • TNF- ⁇ which is involved in inducing inflammation when treated with JC1-40
  • IL-6 inflammatory cytokines
  • composition according to the present invention may exhibit the therapeutic effect of autoimmune diseases by promoting the expression of IL-10, Arg1, Retnla or CD206 anti-inflammatory gene to increase M2 macrophage differentiation.
  • JC1-40 treated anti-inflammatory control in intraperitoneal macrophage lines It was confirmed that the amount of transcription of IL-10, Arg1, Retnla, and CD206 involved was promoted (see Example 4).
  • composition according to the present invention may exhibit the therapeutic effect of autoimmune diseases by inhibiting the activity or production of Th17 and increasing the activity or production of Regulatory T cells (Treg).
  • JC1-40 As a result of confirming the inhibitory effect of Th17 differentiation and IL-17 secretion of JC1-40 in primary cultured mouse spleen T cells and human peripheral mononuclear cells (PBMC), treatment with JC1-40 As a result, it was confirmed that Th17-differentiated (IL-17 positive) T cells were decreased, while the amount of Treg-differentiated cells (FOXP3 positive) was increased (see Examples 5, 6, and 7).
  • composition according to the present invention was treated to osteoclast precursors differentiated from human peripheral mononuclear cell (PBMC), it was confirmed that the expression of osteoclast differentiation-related factors significantly inhibited (see Example 8).
  • PBMC peripheral mononuclear cell
  • the composition according to the present invention was treated in an animal model of arthritis in vivo to observe the progress of arthritis disease, and showed a better arthritis relief effect than the Enbrel treatment used in the existing clinical practice (see Example 9).
  • the compound represented by Chemical Formula 1 of the present invention may include all salts, hydrates, and solvates that may be prepared by conventional methods, as well as pharmaceutically acceptable salts.
  • acid addition salts formed by pharmaceutically acceptable free acids are useful as salts.
  • Acid addition salts include inorganic acids such as hydrochloric acid, nitric acid, phosphoric acid, sulfuric acid, hydrobromic acid, hydroiodic acid, nitrous acid or phosphorous acid and aliphatic mono and dicarboxylates, phenyl-substituted alkanoates, hydroxy alkanoates and alkanes.
  • non-toxic organic acids such as dioates, aromatic acids, aliphatic and aromatic sulfonic acids.
  • Such pharmaceutically nontoxic salts include sulfate, pyrosulfate, bisulfate, sulfite, bisulfite, nitrate, phosphate, monohydrogen phosphate, dihydrogen phosphate, metaphosphate, pyrophosphate chloride, bromide, and iodide.
  • the acid addition salt according to the present invention is dissolved in a conventional method, for example, the compound represented by Chemical Formula 1 in an excess of aqueous acid solution, and the salt is water-miscible organic solvent such as methanol, ethanol, acetone or aceto. It can be prepared by precipitation using nitrile.
  • the same amount of the compound represented by the formula (1) and acid or alcohol in water may be heated and then the mixture is evaporated to dryness, or the precipitated salt may be produced by suction filtration.
  • Bases can also be used to make pharmaceutically acceptable metal salts.
  • Alkali metal or alkaline earth metal salts are obtained, for example, by dissolving a compound in an excess of alkali metal hydroxide or alkaline earth metal hydroxide solution, filtering the insoluble compound salt, and evaporating and drying the filtrate. At this time, it is pharmaceutically suitable to prepare sodium, potassium or calcium salt as the metal salt.
  • Corresponding silver salts are also obtained by reacting an alkali metal or alkaline earth metal salt with a suitable silver salt (eg, silver nitrate).
  • the pharmaceutical composition of the present invention may further contain one or more known active ingredients having an autoimmune disease therapeutic effect together with the compound represented by Formula 1 or a pharmaceutically acceptable salt thereof.
  • composition of the present invention may further comprise suitable carriers, excipients and diluents commonly used in the manufacture of pharmaceutical compositions.
  • Carriers, excipients and diluents that may be included in the composition include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose , Microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxy benzoate, propylhydroxy benzoate, talc, magnesium stearate and mineral oil.
  • diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrating agents, and surfactants are usually used.
  • composition of the present invention may be formulated and administered in various oral or parenteral dosage forms as described below, but is not limited thereto.
  • Formulations for oral administration include, for example, tablets, pills, hard / soft capsules, solutions, suspensions, emulsifiers, syrups, granules, elixirs, etc. These formulations may contain, in addition to the active ingredients, diluents (e.g., lactose, dextrose). Rose, sucrose, mannitol, sorbitol, cellulose and / or glycine), lubricants such as silica, talc, stearic acid and its magnesium or calcium salts and / or polyethylene glycols.
  • diluents e.g., lactose, dextrose
  • Rose sucrose, mannitol, sorbitol, cellulose and / or glycine
  • lubricants such as silica, talc, stearic acid and its magnesium or calcium salts and / or polyethylene glycols.
  • Tablets may also contain binders such as magnesium aluminum silicate, starch paste, gelatin, methylcellulose, sodium carboxymethylcellulose and / or polyvinylpyrrolidine, and optionally such as starch, agar, alginic acid or its sodium salt. Disintegrant or boiling mixtures and / or absorbents, colorants, flavors, and sweeteners.
  • binders such as magnesium aluminum silicate, starch paste, gelatin, methylcellulose, sodium carboxymethylcellulose and / or polyvinylpyrrolidine, and optionally such as starch, agar, alginic acid or its sodium salt.
  • Parenteral administration may also be by subcutaneous injection, intravenous injection, intramuscular injection, intrathoracic injection or transdermal administration method. It may be formulated as an ointment or cream for topical application, and the compound represented by the formula (1) or a pharmaceutically acceptable salt thereof is mixed with water with a stabilizer or buffer to prepare a solution or suspension, which is ampoule or vial It may be prepared in unit dosage form.
  • the composition may be sterile or contain preservatives, stabilizers, emulsifiers or emulsifiers, auxiliaries such as salts and / or buffers for the control of osmotic pressure, and other therapeutically useful substances, and conventional methods of mixing, granulating or It can be formulated according to the coating method.
  • the pharmaceutical composition according to the invention is administered in a pharmaceutically effective amount.
  • pharmaceutically effective amount means an amount sufficient to treat a disease at a reasonable benefit / risk ratio applicable to medical treatment, and an effective dose level means the type, severity, and activity of the patient's disease. , Sensitivity to the drug, time of administration, route of administration and rate of release, duration of treatment, factors including concurrent use of the drug, and other factors well known in the medical arts.
  • the pharmaceutical compositions according to the present invention may be administered as individual therapeutic agents or in combination with other therapeutic agents, may be administered sequentially or simultaneously with conventional therapeutic agents, and may be single or multiple doses. Taking all of the above factors into consideration, it is important to administer an amount that can obtain the maximum effect in a minimum amount without side effects, which can be easily determined by those skilled in the art.
  • the effective amount of the pharmaceutical composition according to the present invention may vary depending on the age, sex, condition, weight of the patient, the absorption of the active ingredient in the body, the inactivation rate and excretion rate, the type of disease, the drug used in general
  • 0.001 to 150 mg, preferably 0.01 to 100 mg per 1 kg of body weight may be administered daily or every other day or divided into 1 to 3 times a day.
  • the dosage may be increased or decreased depending on the route of administration, the severity of obesity, sex, weight, age, etc., and the above dosage does not limit the scope of the present invention in any way.
  • the present invention provides a method for preventing or treating autoimmune disease, comprising administering to a subject a compound represented by Formula 1 or a pharmaceutically acceptable salt thereof.
  • means a subject in need of treatment for a disease, and more specifically, human or non-human primates, mice, rats, dogs, cats, horses and cattle, etc. Mean mammal.
  • the present invention provides a health functional food composition for preventing or ameliorating autoimmune disease comprising the compound represented by Formula 1 or a pharmaceutically acceptable salt thereof.
  • the compound represented by the formula (1) can be easily utilized in the production of health functional foods, such as the main ingredients, secondary ingredients, food additives, functional foods or beverages having an effect of preventing or improving autoimmune diseases.
  • the "health functional food” is a biological defense rhythm control having a food group or a food composition to which added value to act and express the function of the food to a specific purpose using physical, biochemical, biotechnological methods, etc. It means a food processed and designed to fully express the body control function on disease prevention and recovery to the living body, which may further include food acceptable food additives, suitable carriers, excipients and commonly used It may further comprise a diluent.
  • the health functional food composition of the present invention is a variety of nutrients, vitamins, minerals (electrolytes), flavors such as synthetic flavors and natural flavors, colorants and fillers (such as cheese, chocolate), pectic acid and salts thereof, alginic acid And salts thereof, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohols, carbonation agents used in carbonated beverages, and the like, which components can be used independently or in combination.
  • the mouse macrophage line Raw 264.7 (ATCC TIB-71) was purchased from the American Type Culture Collection (ATCC).
  • Raw264.7 cells (2 ⁇ 10 5 cells / well) were seeded in 6-well culture dishes and incubated for one day in Dulbecco's modified Eagle's medium (DMEM) medium containing 10% FBS (fetal bovine serum).
  • DMEM Dulbecco's modified Eagle's medium
  • FBS fetal bovine serum
  • Raw 264.7 cells were maintained at 37 ° C. in a humidified thermostat maintaining 5% CO 2 and 95% air. After incubation, the cells were treated with 50 ng / ml LPS (Lipopolysaccharide) and 20 ⁇ M of JC1-40 for 24 hours.
  • DMEM Dulbecco's modified Eagle's medium
  • FBS fetal bovine serum
  • Real-time PCR real-time reverse transcriptase-polymerase chain reaction
  • Peritoneal macrophages from 7 to 8 week old C57BL / 6 male mice were isolated and seeded at 1 ⁇ 10 6 cells / well in 24-well dishes and incubated for 4 hours in RPMI1640 medium containing 10% FBS (fetal bovine serum). Peritoneal macrophage cultures were treated with 10 ng / ml LPS and 20 ⁇ M JC1-40 or JC1-42, respectively, for 24 hours as indicated. At this time, peritoneal macrophages were maintained at 37 °C in a humidified thermostat maintaining 5% CO 2 and 95% air.
  • M1 cell changes were incubated with PE-CD80 antibody (ebioscience) for 30 minutes and flow cytometry was performed. As a result, as shown in Figure 3, when the macrophages were treated with JC1-40, it was confirmed that M1 differentiation is suppressed.
  • Peritoneal macrophages from 7 to 8 week old C57BL / 6 male mice were isolated and seeded at 1 ⁇ 10 6 cells / well in 24-well dishes and incubated for 4 hours in RPMI1640 medium containing 10% FBS (fetal bovine serum). Peritoneal macrophage cultures were treated with 20 ng / ml IL-4 and 20 ⁇ M JC1-40 or JC1-42, respectively, as indicated for 24 hours.
  • Real-time reverse transcriptase-polymerase chain reaction (RTM) assay was used to determine the expression levels of mRNA of IL-10, Arg1, Retnla and CD206, which are M2 anti-inflammatory markers that cause rheumatoid arthritis. .
  • RTM reverse transcriptase-polymerase chain reaction
  • Murine T cell EL4 cell lines ( 5 ⁇ 10 5 cells / well) were seeded in 24-well culture dishes and incubated for one day in RPMI1640 medium containing 10% FBS by 24 hours pretreatment of 20 ⁇ M of JC1-40. EL4 cell lines were maintained at 37 ° C. in a humidified thermostat maintaining 5% CO 2 and 95% air.
  • Th17 differentiation conditions 0.5 ⁇ g / mL of anti-CD3 antibody, 1 ⁇ g / mL of anti-CD28 antibody, 2 ng / ml of TGF- ⁇ , 20 ng / ml of IL-6, 10 ⁇ g / ml of anti-IL-4, anti -IFN- ⁇ 10 ⁇ g / ml
  • Th17 differentiation conditions 0.5 ⁇ g / mL of anti-CD3 antibody, 1 ⁇ g / mL of anti-CD28 antibody, 2 ng / ml of TGF- ⁇ , 20 ng / ml of IL-6, 10 ⁇ g / ml of anti-IL-4, anti -IFN- ⁇ 10 ⁇ g / ml
  • CD4 + T cells in the spleen of 7-8 week old C57BL / 6 male mice were isolated and inoculated with 5 ⁇ 10 5 cells / well in 48-well dishes, and JC1-40 (0.1, 0.5, 10 and 50 ⁇ M) by concentration 24 Time pretreatment was incubated for one day in RPMI1640 medium containing 10% FBS (fetal bovine serum).
  • FBS fetal bovine serum
  • the cells were treated with Th17 differentiation conditions (0.5 ⁇ g / mL of anti-CD3 antibody, 1 ⁇ g / mL of anti-CD28 antibody, 2 ng / ml of TGF- ⁇ , 20 ng / ml of IL-6, 10 ⁇ g / ml of anti-IL-4, anti -IFN- ⁇ 10 ⁇ g / ml) for 3 days. Thereafter, cells were collected and washed with FACs buffer for flow cytometry, and then blocked for 15 minutes at 4 ° C to inhibit nonspecific binding, and an antibody to CD4 (anti-CD4-PerCP), which is a cell surface marker, was placed at 4 ° C.
  • Th17 differentiation conditions 0.5 ⁇ g / mL of anti-CD3 antibody, 1 ⁇ g / mL of anti-CD28 antibody, 2 ng / ml of TGF- ⁇ , 20 ng / ml of IL-6, 10 ⁇ g / ml of anti-IL-4
  • the culture supernatants of Th17 cells treated with JC1-40 were collected and examined for the amount of IL-17 using the sandwich ELISA method.
  • the monoclonal anti-IL-17 was first treated in a 96-well plate at 2 ⁇ g / mL and reacted overnight at 4 ° C. After the reaction, nonspecific binding was blocked with a blocking solution (1% BSA / PBST). Thereafter, IL-17 was serially diluted by 1/2, and used as a standard. The cell culture supernatant was added and reacted at room temperature for 2 hours. Thereafter, the biotinylated anti-IL-17 was reacted at room temperature for 2 hours and washed four times.
  • ExtraAvidin-Alkaline Phosphatase conjugate was diluted and added and reacted at room temperature for 2 hours. Thereafter, PNPP / DEA solution was added, followed by color development, and absorbance at 405 nm was measured.
  • Example 7 Human Peripheral Mononuclear In cell cells (PBMC) Thiourea Derivative Th17 Confirmation of Differentiation and Inhibition of IL-17 Secretion
  • PBMC Peripheral Mononuclear In cell cells
  • CD4 + cells were isolated from healthy peripheral blood mononuclear cells (PBMCs) and pretreated with JC1-40 concentrations (10, 20 and 40 ⁇ M), and then under Th17 differentiation conditions (anti-CD3 0.5 ⁇ g / ml, anti-CD28). 0.5 ⁇ g / ml, anti-IFN- ⁇ 10 ⁇ g / ml, anti-IL-4 10 ⁇ g / ml, IL-6 20ng / ml, IL-1 ⁇ 20 ng / ml). Expression of Th17 (anti IL-17 PE) and Treg (Anti Foxp3-FITC) in these cells was confirmed by flow cytometry. In addition, the amount of IL-17 in these supernatants was measured by ELISA.
  • T17 differentiated (IL-17 positive) T cells decreased while Treg differentiated cells (FOXP3 positive). The amount of) was increased.
  • Healthy PBMCs were isolated and stimulated with M-CSF (25 ng / ml) for 3 days to differentiate into osteoclast precursors. After 3 days of medium exchange, M-CSF (25 ng / ml), RANKL (30 ng / ml) and JC1-40 (10, 20, 40 iM) were stimulated by concentration and incubated for 3 days, and the differentiation state of cells Observed the same stimulation after medium exchange every three days.
  • mice Six-week-old DBA male mice were inoculated with bovine CII (chondrex) emulsified with CFA (chondrex) and reinoculated 15 days later to induce rheumatoid arthritis.
  • JC1-40 dissolved in 0.5% CMC (carboxymethyl cellulose) was orally administered at 10 mg / kg for 3 consecutive weeks.
  • the other group received 5 mg / kg Enbrel in the same manner as 0.5% CMC vehicle and positive control.
  • the percentage of individuals with arthritis disease was converted to incidence, and the pathophysiological examination of the arthritis tissues was used to compare the status of rheumatoid arthritis between groups.
  • Clinical scores were assessed using a clinical scoring system that visually monitors the progression of arthritis disease daily by visually examining arthritis lesions in the mouse and swelling and redness of the foot or tail area.
  • JC1-40 administration in the CIA animal model inhibited both the incidence and score of arthritis, which was superior to the Enbrel, a therapeutic agent for rheumatoid arthritis actually used in the clinic.
  • Thiourea derivatives according to the present invention not only inhibit the transcription of inflammatory genes and promote the transcription of anti-inflammatory genes, but also inhibit the activity or production of Th17 and inhibit the activity or production of Regulatory T cells (Tregs). Since it can increase, it can be usefully used in pharmaceutical compositions, health food compositions, etc. for the prevention, improvement or treatment of various autoimmune diseases including rheumatoid arthritis.

Abstract

The present invention relates to a novel use of a thiourea derivative and, more specifically, to a pharmaceutical composition for preventing or treating autoimmune diseases comprising a thiourea derivative as an active ingredient. The thiourea derivative according to the present invention can inhibit the transcription of inflammatory genes such as TNF-α, IL-1β, NOS2 and IL-6, and also can inhibit the activity or production of Th17 and increase the activity or production of a regulatory T cell (Treg). Thus, it is expected that the thiourea derivative may be usefully used in a pharmaceutical composition, a health food composition, etc. for the prevention, improvement or treatment of various autoimmune diseases including rheumatoid arthritis.

Description

티오우레아 유도체를 포함하는 자가면역질환 예방 또는 치료용 약학적 조성물Pharmaceutical composition for preventing or treating autoimmune disease, including thiourea derivatives
본 발명은 티오우레아 유도체의 신규한 용도에 관한 것으로서, 보다 구체적으로는 티오우레아 유도체를 유효성분으로 포함하는 자가면역질환 예방 또는 치료용 약학적 조성물에 관한 것이다.The present invention relates to a novel use of thiourea derivatives, and more particularly to a pharmaceutical composition for preventing or treating autoimmune diseases comprising a thiourea derivative as an active ingredient.
인간의 면역계는 인체에 침입한 외부 항원으로부터 신체를 보호하는 역할을 하나, 자기 관용성(self tolerance)이 있어서 자기 조직을 공격하지는 않는다. 그러나 면역계의 자기 관용성이 파괴되어 자신의 유전자에 의해 정상적으로 발현되는 단백질을 면역세포가 공격대상으로 인식하여 항체를 만들거나 T 세포 반응을 일으켜서 정상조직을 파괴하는 경우를 자가 면역이라 부르며 구체적인 증상이 나타나면 자가면역질환이라고 한다.The human immune system protects the body from foreign antigens that invade the body, but does not attack its own tissues because of its self tolerance. However, when the immune system's self-tolerance is destroyed and the protein normally expressed by one's genes is recognized by the immune cell as the target of attack, the antibody is made or the T-cell reaction causes the normal tissue to be destroyed. It is called autoimmune disease.
이러한 자가면역질환 중 하나인 류마티스 관절염은 관절을 싸고 있는 활막에 염증이 생겨 주위의 연골과 뼈에 염증이 퍼져 관절이 파괴되고 장애를 일으키는 만성적 질환으로서, 관절 내에 있는 활막에 염증이 생기고 혈액 내의 면역 세포들이 모이게 되어, 관절액이 증가하게 되어 관절이 부으면서 통증이 수반되게 된다. 류마티스 관절염은 주로 자가면역 이상반응에 의한 질환이고, 비정상적으로 작동하는 면역기능이 염증을 일으키는 것이며, 관절 내의 염증 반응은 주로 T 세포와 B 세포 및 대식세포 등이 주로 관여하며 이들에서 분비되는 TNF-α, 인터류킨 (IL)-1β, IL-6, IL-17에 의해 염증이 유발된다. 특히, TNF-α와 IL-1β를 분비하는 M1 대식세포와 IL-17을 분비하는 Th17 세포가 류마티스 관절염 질환을 더 악화시키는 것으로 알려져 있다.Rheumatoid arthritis, one of these autoimmune diseases, is a chronic disease that causes inflammation of the synovial membrane that surrounds the joint, spreads the cartilage and bones around it, destroys the joint, and causes disorders. The cells gather, causing the joint fluid to increase, resulting in pain as the joint swells. Rheumatoid arthritis is a disease mainly caused by autoimmune adverse reactions, and abnormally functioning immune functions cause inflammation. Inflammation reactions in joints are mainly involved in TNF- and secreted TNF- Inflammation is caused by α, interleukin (IL) -1β, IL-6, IL-17. In particular, M1 macrophages secreting TNF-α and IL-1β and Th17 cells secreting IL-17 are known to exacerbate rheumatoid arthritis disease.
현재, 류마티스 관절염을 비롯한 자가면역질환 치료는 TNF 억제제(Infliximab, Etanercept 등), IL-1 억제제 (Anakinra, Canakinumab), 스테로이드나, 비스테로이드성 염증억제제 (NSAID), 사이토카인 억제제 (Actemra), 신호전달 억제제 (JAK3 inhibitor), 혹은 TNF-α 관련 항체 치료제가 주종을 이루었다. 하지만, 이러한 치료제들은 가려움, 호흡기 감염 등의 부작용이 따르고, 염증 억제를 통한 통증감소에 초점이 맞춰있기 때문에 완치가 어려워 자가면역질환의 근본적인 치료법은 되지 못하는 실정이다.Currently, the treatment of autoimmune diseases, including rheumatoid arthritis, includes TNF inhibitors (Infliximab, Etanercept, etc.), IL-1 inhibitors (Anakinra, Canakinumab), steroids, nonsteroidal inflammatory inhibitors (NSAIDs), cytokine inhibitors (Actemra), signaling JAK3 inhibitors, or TNF-α-related antibody therapies, were predominant. However, these treatments have side effects such as itching and respiratory infections, and because the focus is on reducing pain through inflammation inhibition, it is difficult to cure them and thus cannot be a fundamental treatment for autoimmune diseases.
상기에서 기재한 바와 같이, 대부분의 자가면역질환은 그 원인 및 발병 기전이 분명하지 않고 매우 다양하여 치료가 어려우며, 현재 사용하고 있는 치료제들의 경우 장기간 투약 시 내성이 생기거나 부작용이 심각하여 새로운 치료제 개발이 절실히 요구되는 실정이고, 이에 대한 연구가 이루어지고 있으나(한국 공개특허 제10-2013-0031229호 등), 아직 미미한 실정이다.As described above, most autoimmune diseases are difficult to treat because their causes and mechanisms of disease are not clear and are variously diverse, and current therapeutic agents develop resistances due to long-term dosing or serious side effects. This situation is desperately required, and research on this has been made (Korea Patent Publication No. 10-2013-0031229, etc.), but it is still insignificant.
본 발명은 상기와 같은 종래 기술상의 문제점을 해결하기 위해 안출된 것으로서, 본 발명자들은 RORα의 활성자인 티오우레아 유도체가 염증 반응을 억제시키고, Th17 세포의 분화 및 활성화를 저해함과 동시에 Treg 세포의 생성을 촉진하는 것을 확인하고, 이에 기초하여 본 발명을 완성하였다.The present invention has been made to solve the above-mentioned problems in the prior art, the inventors of the present invention, the thiourea derivatives, which is an activator of RORα inhibits the inflammatory response, inhibits the differentiation and activation of Th17 cells and at the same time generates Treg cells It confirmed that it promotes, and based on this, this invention was completed.
이에, 본 발명의 목적은 티오우레아 유도체 또는 이의 약학적으로 허용가능한 염을 유효성분으로 포함하는, 자가면역질환 예방 또는 치료용 약학적 조성물을 제공하는 것이다.Accordingly, an object of the present invention is to provide a pharmaceutical composition for preventing or treating autoimmune diseases, comprising a thiourea derivative or a pharmaceutically acceptable salt thereof as an active ingredient.
그러나 본 발명이 이루고자 하는 기술적 과제는 이상에서 언급한 과제에 제한되지 않으며, 언급되지 않은 또 다른 과제들은 아래의 기재로부터 당업자에게 명확하게 이해될 수 있을 것이다.However, the technical problem to be achieved by the present invention is not limited to the above-mentioned problem, another task that is not mentioned will be clearly understood by those skilled in the art from the following description.
상기 목적을 달성하기 위하여, 본 발명은 하기 화학식 1로 표시되는 화합물 또는 이의 약학적으로 허용가능한 염을 유효성분으로 포함하는, 자가면역질환 예방 또는 치료용 약학적 조성물을 제공한다.In order to achieve the above object, the present invention provides a pharmaceutical composition for preventing or treating autoimmune diseases, comprising a compound represented by the following formula (1) or a pharmaceutically acceptable salt thereof as an active ingredient.
[화학식 1][Formula 1]
Figure PCTKR2016008867-appb-I000001
Figure PCTKR2016008867-appb-I000001
여기서, R은 페녹시기, 벤질옥시기 또는 피리디닐기이다.Here, R is a phenoxy group, benzyloxy group or pyridinyl group.
본 발명의 일 구현예로, 상기 자가면역질환은 류마티스 관절염, 건선성 관절염, 건선, 전신성 피부 경화증(systemic scleroderma), 경화증(sclerosis), 다발성 경화증, 염증성 장질환, 전신성 홍반성 루푸스, 크론병(crohn's disease), 패혈증 및 타입 I 당뇨병으로 이루어진 군으로부터 선택될 수 있다.In one embodiment of the invention, the autoimmune disease is rheumatoid arthritis, psoriatic arthritis, psoriasis, systemic scleroderma, sclerosis (sclerosis), multiple sclerosis, inflammatory bowel disease, systemic lupus erythematosus, Crohn's disease ( crohn's disease), sepsis and type I diabetes.
본 발명의 다른 구현예로, 상기 조성물은 Th17 세포의 생성을 억제할 수 있다.In another embodiment of the present invention, the composition may inhibit the production of Th17 cells.
본 발명의 또 다른 구현예로, 상기 조성물은 조절 T 세포(Regulatory T cell: Treg)의 생성을 촉진할 수 있다.In another embodiment of the present invention, the composition may promote the production of Regulatory T cells (Treg).
본 발명의 또 다른 구현예로, 상기 조성물은 TNF-α, NOS2, IL-1β 또는 IL-6 염증 유전자의 발현을 억제하여 M1 대식세포 분화를 억제할 수 있다.In another embodiment of the present invention, the composition may inhibit M1 macrophage differentiation by inhibiting the expression of TNF-α, NOS2, IL-1β or IL-6 inflammatory genes.
본 발명의 또 다른 구현예로, 상기 조성물은 대식세포에서 IL-10, Arg1, Retnla 또는 CD206 등의 항염증 유전자의 발현을 촉진하여 M2 대식세포 분화를 증대시킬 수 있다.In another embodiment of the present invention, the composition may promote expression of anti-inflammatory genes such as IL-10, Arg1, Retnla or CD206 in macrophages to enhance M2 macrophage differentiation.
본 발명은 상기 화학식 1로 표시되는 화합물 또는 이의 약학적으로 허용가능한 염을 개체에 투여하는 단계를 포함하는 자가면역질환 치료방법을 제공한다.The present invention provides a method for treating autoimmune disease comprising administering a compound represented by Formula 1 or a pharmaceutically acceptable salt thereof to a subject.
본 발명은 상기 화학식 1로 표시되는 화합물 또는 이의 약학적으로 허용가능한 염의 자가면역질환 치료 용도를 제공한다.The present invention provides a method for treating autoimmune disease of a compound represented by Formula 1 or a pharmaceutically acceptable salt thereof.
본 발명에 따른 티오우레아 유도체는 TNF-α, IL-1β, NOS2, IL-6 등의 염증 유전자의 전사를 억제하고 IL-10, Arg1, CD206 등의 항염증 유전자의 전사를 촉진시킬 뿐만 아니라, Th17의 활성 또는 생성을 억제하고, 조절 T 세포(Regulatory T cell: Treg)의 활성 또는 생성을 증가시킬 수 있기 때문에, 류마티스 관절염을 비롯한 다양한 자가면역 질환의 예방, 개선 또는 치료를 위한 약학적 조성물, 건강식품 조성물 등에 유용하게 사용할 수 있을 것으로 기대된다.Thiourea derivatives according to the present invention not only inhibit the transcription of inflammatory genes such as TNF-α, IL-1β, NOS2, IL-6 and promote the transcription of anti-inflammatory genes such as IL-10, Arg1, CD206, Pharmaceutical compositions for the prevention, amelioration or treatment of various autoimmune diseases, including rheumatoid arthritis, because they can inhibit the activity or production of Th17 and increase the activity or production of Regulatory T cells (Tregs), It is expected to be useful for health food compositions and the like.
도 1은 대식세포 세포주에서 JC1-40의 M1 염증 유전자 전사 억제 효과를 Real-time PCR을 통해 확인한 결과이다.Figure 1 shows the results confirmed by Real-time PCR the inhibitory effect of M1 inflammatory gene transcription of JC1-40 in macrophage cell line.
도 2a 및 도 2b는 1차 배양된 마우스 복강 내 대식세포에서 JC1-40의 M1 염증 유전자 전사 억제 효과를 Real-time PCR을 통해 확인한 결과이다.Figure 2a and Figure 2b is the result confirmed by the real-time PCR the M1 inflammatory gene transcription inhibition effect of JC1-40 in primary cultured mouse intraperitoneal macrophages.
도 3은 1차 배양된 마우스 복강 내 대식세포에서 JC1-40을 처리하였을 때, M1 분화 대식세포의 감소를 유세포분석법으로 확인한 결과이다.Figure 3 is the result of confirming by flow cytometry the decrease of M1 differentiated macrophages when treated with JC1-40 in primary cultured mouse intraperitoneal macrophages.
도 4a 및 도 4b는 1차 배양된 마우스 복강 내 대식세포에서 JC1-40의 M2 항염증 유전자 전사 억제 효과를 Real-time PCR을 통해 확인한 결과이다.Figures 4a and 4b is a result confirmed by the real-time PCR the M2 anti-inflammatory gene transcription inhibition effect of JC1-40 in primary cultured mouse intraperitoneal macrophages.
도 5a는 T세포 세포주에서 JC1-40의 IL-17 유전자 전사 억제 효과를 Real-time PCR을 통해 확인한 결과이다.Figure 5a is a result confirmed by the real-time PCR the IL-17 gene transcription inhibition effect of JC1-40 in T cell line.
도 5b는 T세포 세포주에서 JC1-40의 Treg의 발현 증가를 면역염색법으로 확인한 결과이다.Figure 5b is the result confirmed by immunostaining the expression of Treg expression of JC1-40 in T cell line.
도 6a는 1차 배양된 마우스 비장 T세포에서 JC1-40을 농도별로 처리하였을 때, Th17 분화 세포의 감소를 유세포염색법으로 확인한 결과이다.Figure 6a is the result of confirming the reduction of Th17 differentiated cells by flow cytometry when JC1-40 concentration-treated in primary cultured mouse spleen T cells.
도 6b는 1차 배양된 마우스 비장 T세포에서 JC1-40을 농도별로 처리하였을 때, 상층액에서 IL-17의 양을 ELISA로 측정한 결과이다.Figure 6b is the result of measuring the amount of IL-17 in the supernatant when treated with JC1-40 concentration in primary cultured mouse spleen T cells by ELISA.
도 7a는 인간 말초 단핵 세포구(PBMC)에서 JC1-40을 농도별로 처리하였을 때, Th17 및 Treg 분화세포의 양적변화를 유세포염색법으로 확인한 결과이다.Figure 7a is a result of confirming the quantitative change of Th17 and Treg differentiated cells by flow cytometry when treated with concentrations of JC1-40 in human peripheral mononuclear cells (PBMC).
도 7b는 인간 말초 단핵 세포구(PBMC)에서 JC1-40을 농도별로 처리하였을 때, 상층액에서 IL-17의 양을 ELISA로 측정한 결과이다.Figure 7b is the result of measuring the amount of IL-17 in the supernatant when the concentration of JC1-40 in human peripheral mononuclear cell (PBMC) by ELISA.
도 8a 및 도 8b는 인간 말초 단핵 세포구(PBMC)에서 분화시킨 파골세포 전구체에 JC1-40을 농도별로 처리하였을 때, 파골세포 분화 관련 인자의 발현 억제 효과를 Real-time PCR을 통해 확인한 결과이다.8A and 8B show the results of confirming the effect of inhibiting the expression of osteoclast differentiation-related factors by real-time PCR when JC1-40 was treated in different concentrations in osteoclast precursors differentiated from human peripheral mononuclear cell (PBMC). .
도 9는 관절염 동물모델 (type II collagen-induced arthritis-CIA)에서 JC1-40의 투여에 따른 관절염 incidence와 score를 산출한 결과이다.9 is a result of calculating the arthritis incidence and score according to the administration of JC1-40 in arthritis animal model (type II collagen-induced arthritis-CIA).
본 발명자들은 류마티스 관절염을 포함한 자가면역질환을 치료하는데 효과적인 물질에 대하여 연구 노력한 결과, RORα의 활성자인 티오우레아 유도체가 TNF-α, IL-1β, IL-6 등의 염증 싸이토카인 발현을 억제시킬 뿐만 아니라 IL-10, Arg1, CD206 등의 항염증 인자 발현을 촉진시키고, Th17 세포의 분화 및 활성화를 저해 또는 억제함과 동시에 Treg 세포의 생성을 촉진하는 것을 확인하고, 이에 기초하여 본 발명을 완성하였다.The present inventors have conducted research on substances effective in treating autoimmune diseases including rheumatoid arthritis. As a result, thiourea derivatives, which are activators of RORα, not only inhibit the expression of inflammatory cytokines such as TNF-α, IL-1β, IL-6, etc. The present invention was completed by promoting the expression of anti-inflammatory factors such as IL-10, Arg1, CD206, inhibiting or inhibiting the differentiation and activation of Th17 cells, and promoting the production of Treg cells.
이하, 본 발명을 상세히 설명한다.Hereinafter, the present invention will be described in detail.
본 발명은 하기 화학식 1로 표시되는 화합물 또는 이의 약학적으로 허용가능한 염을 유효성분으로 포함하는, 자가면역질환 예방 또는 치료용 약학적 조성물을 제공한다.The present invention provides a pharmaceutical composition for preventing or treating autoimmune diseases, comprising a compound represented by the following formula (1) or a pharmaceutically acceptable salt thereof as an active ingredient.
[화학식 1][Formula 1]
Figure PCTKR2016008867-appb-I000002
Figure PCTKR2016008867-appb-I000002
여기서, R은 페녹시기, 벤질옥시기 또는 피리디닐기이다.Here, R is a phenoxy group, benzyloxy group or pyridinyl group.
본 발명에서 사용되는 용어, "예방"이란 본 발명에 따른 약학적 조성물의 투여에 의해 자가면역질환을 억제시키거나 발병을 지연시키는 모든 행위를 의미한다.As used herein, the term "prevention" means any action that inhibits or delays the onset of autoimmune diseases by administration of a pharmaceutical composition according to the present invention.
본 발명에서 사용되는 용어, "치료"란 본 발명에 따른 약학적 조성물의 투여에 의해 자가면역질환에 의한 증세가 호전되거나 이롭게 변경되는 모든 행위를 의미한다.As used herein, the term "treatment" refers to any action in which symptoms caused by an autoimmune disease are improved or advantageously changed by administration of the pharmaceutical composition according to the present invention.
본 발명의 조성물에 의한 개선, 예방 또는 치료 대상 질병인 "자가면역질환" 은 자기관용을 유도하거나 계속 유지하는데 있어서 문제가 생기게 되면 자기 항원에 대하여 면역반응이 일어나게 되고, 이로 인하여 자신의 조직을 공격하는 현상이 발생하는데 이러한 과정에 의해 발생되는 질환을 의미한다. 또한, 본 발명에서 상기 자가면역질환의 종류로는 이에 제한되지는 않으나, 류마티스 관절염, 건선성 관절염, 건선, 전신성 피부 경화증(systemic scleroderma), 경화증(sclerosis), 다발성 경화증, 염증성 장질환, 전신성 홍반성 루푸스, 크론병(crohn's disease), 패혈증 및 타입 I 당뇨병으로 이루어진 군으로부터 선택될 수 있고, 바람직하게는 류마티스 관절염일 수 있다."Autoimmune disease", a disease to be improved, prevented or treated by the composition of the present invention, when there is a problem in inducing or maintaining self-tolerance, an immune response to its antigen occurs, thereby attacking its own tissue. This phenomenon occurs, which means a disease caused by this process. In addition, the type of autoimmune disease in the present invention is not limited thereto, but rheumatoid arthritis, psoriatic arthritis, psoriasis, systemic scleroderma, sclerosis (sclerosis), multiple sclerosis, inflammatory bowel disease, systemic redness And may be selected from the group consisting of seminal lupus, crohn's disease, sepsis and type I diabetes, and preferably rheumatoid arthritis.
본 발명에 따른 조성물에 유효성분으로 포함되는 상기 화학식 1로 표시되는 화합물은 기존의 thiazolidinedione계 화합물 CGP52608을 선도 물질로 하는 thiourea 유도체로써(JC1 화합물 이라 함), 바람직하게는 1-메틸-3-(4-(피리딘-2-일)벤질)-thiourea(JC1-38), 1-(4-벤질옥시-벤질)-3-메틸-thiourea(JC1-40) 또는 1-(4-페녹시-벤질)-3-메틸-thiourea(JC1-42)일 수 있다.The compound represented by Chemical Formula 1, which is included as an active ingredient in the composition according to the present invention, is a thiourea derivative having a conventional thiazolidinedione-based compound CGP52608 (called JC1 compound), preferably 1-methyl-3- ( 4- (pyridin-2-yl) benzyl) -thiourea (JC1-38), 1- (4-benzyloxy-benzyl) -3-methyl-thiourea (JC1-40) or 1- (4-phenoxy-benzyl ) -3-methyl-thiourea (JC1-42).
본 발명에 따른 조성물은 염증 유전자의 발현을 억제하여 M1 대식세포 분화를 억제함으로써 자가면역 질환의 치료효과를 나타낼 수 있다.The composition according to the present invention can exhibit the therapeutic effect of autoimmune diseases by inhibiting the expression of inflammatory genes and inhibiting M1 macrophage differentiation.
본 발명의 일 실시예에 따르면, 대식세포 세포주 및 1차 배양된 마우스 복강 내 대식세포에서 JC1-40의 염증 조절 효과를 확인한 결과, JC1-40을 처리하였을 때 염증 유발에 관여하는 TNF-α, NOS2, IL-1β 및 IL-6 등 염증 싸이토카인의 전사량이 현저히 감소되었고 M1 대식세포의 분화가 억제됨을 확인하였다(실시예 2 및 3 참조).According to one embodiment of the present invention, as a result of confirming the inflammatory control effect of JC1-40 in macrophage cell line and primary cultured mouse intraperitoneal macrophages, TNF-α, which is involved in inducing inflammation when treated with JC1-40, It was confirmed that the amount of transcription of inflammatory cytokines such as NOS2, IL-1β, and IL-6 was significantly reduced and the differentiation of M1 macrophages was suppressed (see Examples 2 and 3).
또한, 본 발명에 따른 조성물은 IL-10, Arg1, Retnla 또는 CD206 항염증 유전자의 발현을 촉진하여 M2 대식세포 분화를 증대시킴으로써 자가면역 질환의 치료효과를 나타낼 수 있다.In addition, the composition according to the present invention may exhibit the therapeutic effect of autoimmune diseases by promoting the expression of IL-10, Arg1, Retnla or CD206 anti-inflammatory gene to increase M2 macrophage differentiation.
본 발명의 다른 실시예에 따르면, 1차 배양된 마우스 복강 내 대식세포에서 JC1-40의 M1 염증 유전자 전사 억제 효과를 확인한 결과, JC1-40을 처리하였을 때 복강 내 대식세포 주에서 항염증 조절에 관여하는 IL-10, Arg1, Retnla 및 CD206의 전사량이 촉진됨을 확인하였다 (실시예 4 참조).According to another embodiment of the present invention, as a result of confirming the inhibitory effect of JC1-40 on M1 inflammatory gene transcription in primary cultured mouse intraperitoneal macrophages, JC1-40 treated anti-inflammatory control in intraperitoneal macrophage lines It was confirmed that the amount of transcription of IL-10, Arg1, Retnla, and CD206 involved was promoted (see Example 4).
더욱이, 본 발명에 따른 조성물은 Th17의 활성 또는 생성을 억제하고, 조절 T 세포(Regulatory T cell: Treg)의 활성 또는 생성을 증가시킴으로써 자가면역 질환의 치료효과를 나타낼 수 있다.Moreover, the composition according to the present invention may exhibit the therapeutic effect of autoimmune diseases by inhibiting the activity or production of Th17 and increasing the activity or production of Regulatory T cells (Treg).
본 발명의 또 다른 실시예에 따르면, 1차 배양된 마우스 비장 T 세포 및 인간 말초 단핵 세포구(PBMC)에서 JC1-40의 Th17 분화 및 IL-17 분비 억제 효과를 확인한 결과, JC1-40으로 처리하였을 때, Th17으로 분화한 (IL-17 양성) T 세포는 감소한 반면, Treg으로 분화한 세포 (FOXP3 양성)의 양은 증가함을 확인하였다(실시예 5, 6 및 7 참조).According to another embodiment of the present invention, as a result of confirming the inhibitory effect of Th17 differentiation and IL-17 secretion of JC1-40 in primary cultured mouse spleen T cells and human peripheral mononuclear cells (PBMC), treatment with JC1-40 As a result, it was confirmed that Th17-differentiated (IL-17 positive) T cells were decreased, while the amount of Treg-differentiated cells (FOXP3 positive) was increased (see Examples 5, 6, and 7).
또한, 본 발명에 따른 조성물을 인간 말초 단핵 세포구(PBMC)에서 분화시킨 파골세포 전구체에 처리하였을 때, 파골세포 분화 관련 인자의 발현이 유의적으로 억제됨을 확인하였다(실시예 8 참조). 뿐만 아니라, 본 발명에 따른 조성물을 in vivo 관절염 동물모델에 처리하여 관절염 질환의 진행과정을 관찰한 결과, 기존 임상에서 사용되고 있는 Enbrel 치료제보다 더 우수한 관절염 완화 효과를 보였다 (실시예 9 참조).In addition, when the composition according to the present invention was treated to osteoclast precursors differentiated from human peripheral mononuclear cell (PBMC), it was confirmed that the expression of osteoclast differentiation-related factors significantly inhibited (see Example 8). In addition, the composition according to the present invention was treated in an animal model of arthritis in vivo to observe the progress of arthritis disease, and showed a better arthritis relief effect than the Enbrel treatment used in the existing clinical practice (see Example 9).
본 발명의 상기 화학식 1로 표시되는 화합물은 약학적으로 허용되는 염뿐만 아니라, 통상의 방법에 의해 제조될 수 있는 모든 염, 수화물 및 용매화물을 모두 포함할 수 있다. 약학적으로 허용 가능한 염의 형태로 사용하는 경우, 염으로는 약학적으로 허용가능한 유리산(free acid)에 의해 형성된 산부가염이 유용하다. 산 부가염은 염산, 질산, 인산, 황산, 브롬화수소산, 요드화수소산, 아질산 또는 아인산과 같은 무기산류와 지방족 모노 및 디카르복실레이트, 페닐-치환된 알카노에이트, 하이드록시 알카노에이트 및 알칸디오에이트, 방향족 산류, 지방족 및 방향족 설폰산류와 같은 무독성 유기산으로부터 얻는다. 이러한 약학적으로 무독한 염류로는 설페이트, 피로설페이트, 바이설페이트, 설파이트, 바이설파이트, 니트레이트, 포스페이트, 모노하이드로겐 포스페이트, 디하이드로겐 포스페이트, 메타포스페이트, 피로포스페이트 클로라이드, 브로마이드, 아이오다이드, 플루오라이드, 아세테이트, 프로피오네이트, 데카노에이트, 카프릴레이트, 아크릴레이트, 포메이트, 이소부티레이트, 카프레이트, 헵타노에이트, 프로피올레이트, 옥살레이트, 말로네이트, 석시네이트, 수베레이트, 세바케이트, 푸마레이트, 말리에이트, 부틴-1,4-디오에이트, 헥산-1,6-디오에이트, 벤조에이트, 클로로벤조에이트, 메틸벤조에이트, 디니트로 벤조에이트, 하이드록시벤조에이트, 메톡시벤조에이트, 프탈레이트, 테레프탈레이트, 벤젠설포네이트, 톨루엔설포네이트, 클로로벤젠설포네이트, 크실렌설포네이트, 페닐아세테이트, 페닐프로피오네이트, 페닐부티레이트, 시트레이트, 락테이트, β-하이드록시부티레이트, 글리콜레이트, 말레이트, 타트레이트, 메탄설포네이트, 프로판설포네이트, 나프탈렌-1-설포네이트, 나프탈렌-2-설포네이트 또는 만델레이트를 포함한다.The compound represented by Chemical Formula 1 of the present invention may include all salts, hydrates, and solvates that may be prepared by conventional methods, as well as pharmaceutically acceptable salts. When used in the form of pharmaceutically acceptable salts, acid addition salts formed by pharmaceutically acceptable free acids are useful as salts. Acid addition salts include inorganic acids such as hydrochloric acid, nitric acid, phosphoric acid, sulfuric acid, hydrobromic acid, hydroiodic acid, nitrous acid or phosphorous acid and aliphatic mono and dicarboxylates, phenyl-substituted alkanoates, hydroxy alkanoates and alkanes. Obtained from non-toxic organic acids such as dioates, aromatic acids, aliphatic and aromatic sulfonic acids. Such pharmaceutically nontoxic salts include sulfate, pyrosulfate, bisulfate, sulfite, bisulfite, nitrate, phosphate, monohydrogen phosphate, dihydrogen phosphate, metaphosphate, pyrophosphate chloride, bromide, and iodide. Id, fluoride, acetate, propionate, decanoate, caprylate, acrylate, formate, isobutyrate, caprate, heptanoate, propiolate, oxalate, malonate, succinate, suverate , Sebacate, fumarate, maleate, butyne-1,4-dioate, hexane-1,6-dioate, benzoate, chlorobenzoate, methylbenzoate, dinitro benzoate, hydroxybenzoate, meth Oxybenzoate, phthalate, terephthalate, benzenesulfonate, toluenesulfonate, chlorobenzenesul Nate, xylenesulfonate, phenylacetate, phenylpropionate, phenylbutyrate, citrate, lactate, β-hydroxybutyrate, glycolate, malate, tartrate, methanesulfonate, propanesulfonate, naphthalene-1- Sulfonates, naphthalene-2-sulfonates or mandelate.
본 발명에 따른 산 부가염은 통상의 방법, 예를 들면, 상기 화학식 1로 표시되는 화합물을 과량의 산 수용액 중에 용해시키고, 이 염을 수혼화성 유기 용매, 예를 들면 메탄올, 에탄올, 아세톤 또는 아세토니트릴을 사용하여 침전시켜서 제조할 수 있다. 동량의 상기 화학식 1로 표시되는 화합물 및 물 중의 산 또는 알코올을 가열하고, 이어서 이 혼합물을 증발시켜서 건조시키거나 또는 석출된 염을 흡입 여과시켜 제조할 수도 있다.The acid addition salt according to the present invention is dissolved in a conventional method, for example, the compound represented by Chemical Formula 1 in an excess of aqueous acid solution, and the salt is water-miscible organic solvent such as methanol, ethanol, acetone or aceto. It can be prepared by precipitation using nitrile. The same amount of the compound represented by the formula (1) and acid or alcohol in water may be heated and then the mixture is evaporated to dryness, or the precipitated salt may be produced by suction filtration.
또한, 염기를 사용하여 약학적으로 허용 가능한 금속염을 만들 수 있다. 알칼리 금속 또는 알칼리 토금속 염은 예를 들면 화합물을 과량의 알칼리 금속 수산화물 또는 알칼리 토금속 수산화물 용액 중에 용해하고, 비용해 화합물 염을 여과하고, 여액을 증발, 건조시켜 얻는다. 이때, 금속염으로는 나트륨, 칼륨 또는 칼슘염을 제조하는 것이 제약상 적합하다. 또한, 이에 대응하는 은 염은 알칼리 금속 또는 알칼리 토금속 염을 적당한 은염(예, 질산은)과 반응시켜 얻는다.Bases can also be used to make pharmaceutically acceptable metal salts. Alkali metal or alkaline earth metal salts are obtained, for example, by dissolving a compound in an excess of alkali metal hydroxide or alkaline earth metal hydroxide solution, filtering the insoluble compound salt, and evaporating and drying the filtrate. At this time, it is pharmaceutically suitable to prepare sodium, potassium or calcium salt as the metal salt. Corresponding silver salts are also obtained by reacting an alkali metal or alkaline earth metal salt with a suitable silver salt (eg, silver nitrate).
한편, 본 발명의 약학적 조성물은 상기 화학식 1로 표시되는 화합물 또는 이의 약학적으로 허용가능한 염과 함께 자가면역질환 치료 효과를 갖는 공지의 유효성분을 1종 이상 더 함유할 수 있다.Meanwhile, the pharmaceutical composition of the present invention may further contain one or more known active ingredients having an autoimmune disease therapeutic effect together with the compound represented by Formula 1 or a pharmaceutically acceptable salt thereof.
본 발명의 조성물은 약학적 조성물의 제조에 통상적으로 사용하는 적절한 담체, 부형제 및 희석제를 더 포함할 수 있다. 상기 조성물에 포함될 수 있는 담체, 부형제 및 희석제로는 락토오스, 덱스트로오스, 수크로오스, 소르비톨, 만니톨, 자일리톨, 에리스리톨, 말티톨, 전분, 아카시아 고무, 알지네이트, 젤라틴, 칼슘 포스페이트, 칼슘 실리케이트, 셀룰로오스, 메틸 셀룰로오스, 미정질 셀룰로오스, 폴리비닐 피롤리돈, 물, 메틸히드록시 벤조에이트, 프로필히드록시 벤조에이트, 탈크, 마그네슘 스테아레이트 및 광물유 등이 있다. 상기 조성물을 제제화할 경우에는 보통 사용하는 충진제, 증량제, 결합제, 습윤제, 붕해제, 계면활성제 등의 희석제 또는 부형제를 사용하여 조제된다.The composition of the present invention may further comprise suitable carriers, excipients and diluents commonly used in the manufacture of pharmaceutical compositions. Carriers, excipients and diluents that may be included in the composition include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose , Microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxy benzoate, propylhydroxy benzoate, talc, magnesium stearate and mineral oil. In formulating the composition, diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrating agents, and surfactants are usually used.
본 발명의 약학적 조성물은 임상투여 시에 다양한 하기의 경구 또는 비경구 투여 형태로 제제화되어 투여될 수 있으나, 이에 한정되는 것은 아니다.The pharmaceutical composition of the present invention may be formulated and administered in various oral or parenteral dosage forms as described below, but is not limited thereto.
경구 투여용 제형으로는 예를 들면 정제, 환제, 경/연질 캅셀제, 액제, 현탁제, 유화제, 시럽제, 과립제, 엘릭시르제 등이 있는데, 이들 제형은 유효성분 이외에 희석제(예: 락토즈, 덱스트로즈, 수크로즈, 만니톨, 솔비톨, 셀룰로즈 및/또는 글리신), 활택제(예: 실리카, 탈크, 스테아르산 및 그의 마그네슘 또는 칼슘염 및/또는 폴리에틸렌 글리콜)를 함유할 수 있다. 정제는 또한 마그네슘 알루미늄 실리케이트, 전분 페이스트, 젤라틴, 메틸셀룰로즈, 나트륨 카복시메틸셀룰로즈 및/또는 폴리비닐피롤리딘과 같은 결합제를 함유할 수 있으며, 경우에 따라 전분, 한천, 알긴산 또는 그의 나트륨 염과 같은 붕해제 또는 비등 혼합물 및/또는 흡수제, 착색제, 향미제, 및 감미제를 함유할 수 있다.Formulations for oral administration include, for example, tablets, pills, hard / soft capsules, solutions, suspensions, emulsifiers, syrups, granules, elixirs, etc. These formulations may contain, in addition to the active ingredients, diluents (e.g., lactose, dextrose). Rose, sucrose, mannitol, sorbitol, cellulose and / or glycine), lubricants such as silica, talc, stearic acid and its magnesium or calcium salts and / or polyethylene glycols. Tablets may also contain binders such as magnesium aluminum silicate, starch paste, gelatin, methylcellulose, sodium carboxymethylcellulose and / or polyvinylpyrrolidine, and optionally such as starch, agar, alginic acid or its sodium salt. Disintegrant or boiling mixtures and / or absorbents, colorants, flavors, and sweeteners.
또한, 비경구 투여는 피하주사, 정맥주사, 근육 내 주사, 흉부 내 주사 또는 경피투여 방법에 의할 수 있다. 국소 적용을 위해서 연고나 크림으로 제형화 할 수 있고, 상기 화학식 1로 표시되는 화합물 또는 이의 약학적으로 허용되는 염을 안정제 또는 완충제와 함께 물에 혼합하여 용액 또는 현탁액으로 제조하고, 이를 앰플 또는 바이알 단위 투여형으로 제조할 수 있다. 상기 조성물은 멸균되거나 방부제, 안정화제, 수화제 또는 유화 촉진제, 삼투압 조절을 위한 염 및/또는 완충제 등의 보조제, 및 기타 치료적으로 유용한 물질을 함유할 수 있으며, 통상적인 방법인 혼합, 과립화 또는 코팅 방법에 따라 제제화할 수 있다.Parenteral administration may also be by subcutaneous injection, intravenous injection, intramuscular injection, intrathoracic injection or transdermal administration method. It may be formulated as an ointment or cream for topical application, and the compound represented by the formula (1) or a pharmaceutically acceptable salt thereof is mixed with water with a stabilizer or buffer to prepare a solution or suspension, which is ampoule or vial It may be prepared in unit dosage form. The composition may be sterile or contain preservatives, stabilizers, emulsifiers or emulsifiers, auxiliaries such as salts and / or buffers for the control of osmotic pressure, and other therapeutically useful substances, and conventional methods of mixing, granulating or It can be formulated according to the coating method.
본 발명에 따른 약학적 조성물은 약제학적으로 유효한 양으로 투여한다. 본 발명에 있어서, "약제학적으로 유효한 양"은 의학적 치료에 적용 가능한 합리적인 수혜/위험 비율로 질환을 치료하기에 충분한 양을 의미하며, 유효용량 수준은 환자의 질환의 종류, 중증도, 약물의 활성, 약물에 대한 민감도, 투여 시간, 투여 경로 및 배출 비율, 치료기간, 동시 사용되는 약물을 포함한 요소 및 기타 의학 분야에 잘 알려진 요소에 따라 결정될 수 있다. 본 발명에 따른 약제학적 조성물은 개별 치료제로 투여하거나 다른 치료제와 병용하여 투여될 수 있고 종래의 치료제와는 순차적 또는 동시에 투여될 수 있으며, 단일 또는 다중 투여될 수 있다. 상기한 요소들을 모두 고려하여 부작용 없이 최소한의 양으로 최대 효과를 얻을 수 있는 양을 투여하는 것이 중요하며, 이는 당업자에 의해 용이하게 결정될 수 있다.The pharmaceutical composition according to the invention is administered in a pharmaceutically effective amount. In the present invention, “pharmaceutically effective amount” means an amount sufficient to treat a disease at a reasonable benefit / risk ratio applicable to medical treatment, and an effective dose level means the type, severity, and activity of the patient's disease. , Sensitivity to the drug, time of administration, route of administration and rate of release, duration of treatment, factors including concurrent use of the drug, and other factors well known in the medical arts. The pharmaceutical compositions according to the present invention may be administered as individual therapeutic agents or in combination with other therapeutic agents, may be administered sequentially or simultaneously with conventional therapeutic agents, and may be single or multiple doses. Taking all of the above factors into consideration, it is important to administer an amount that can obtain the maximum effect in a minimum amount without side effects, which can be easily determined by those skilled in the art.
구체적으로, 본 발명에 따른 약학적 조성물의 유효량은 환자의 연령, 성별, 상태, 체중, 체내에서 활성 성분의 흡수도, 불활성율 및 배설속도, 질병종류, 병용되는 약물에 따라 달라질 수 있으며, 일반적으로는 체중 1㎏ 당 0.001 내지 150 mg, 바람직하게는 0.01 내지 100 mg을 매일 또는 격일 투여하거나 1일 1 내지 3회로 나누어 투여할 수 있다. 그러나 투여 경로, 비만의 중증도, 성별, 체중, 연령 등에 따라서 증감될 수 있으므로 상기 투여량이 어떠한 방법으로도 본 발명의 범위를 한정하는 것은 아니다.Specifically, the effective amount of the pharmaceutical composition according to the present invention may vary depending on the age, sex, condition, weight of the patient, the absorption of the active ingredient in the body, the inactivation rate and excretion rate, the type of disease, the drug used in general For example, 0.001 to 150 mg, preferably 0.01 to 100 mg per 1 kg of body weight may be administered daily or every other day or divided into 1 to 3 times a day. However, the dosage may be increased or decreased depending on the route of administration, the severity of obesity, sex, weight, age, etc., and the above dosage does not limit the scope of the present invention in any way.
본 발명의 다른 양태로서, 본 발명은 상기 화학식 1로 표시되는 화합물 또는 이의 약학적으로 허용가능한 염을 개체에 투여하는 단계를 포함하는 자가면역질환의 예방 또는 치료방법을 제공한다.As another aspect of the present invention, the present invention provides a method for preventing or treating autoimmune disease, comprising administering to a subject a compound represented by Formula 1 or a pharmaceutically acceptable salt thereof.
본 발명에서 "개체"란 질병의 치료를 필요로 하는 대상을 의미하고, 보다 구체적으로는 인간 또는 비-인간인 영장류, 생쥐 (mouse), 쥐 (rat), 개, 고양이, 말 및 소 등의 포유류를 의미한다.As used herein, "individual" means a subject in need of treatment for a disease, and more specifically, human or non-human primates, mice, rats, dogs, cats, horses and cattle, etc. Mean mammal.
본 발명의 또 다른 양태로서, 본 발명은 상기 화학식 1로 표시되는 화합물 또는 이의 약학적으로 허용가능한 염을 포함하는 자가면역질환 예방 또는 개선용 건강기능식품 조성물을 제공한다. 본 발명에서, 상기 화학식 1로 표시되는 화합물은 자가면역질환 예방 또는 개선 효과가 있는 건강기능식품, 예컨대, 식품의 주원료, 부원료, 식품 첨가제, 기능성 식품 또는 음료의 제조에 용이하게 활용할 수 있다.As another aspect of the present invention, the present invention provides a health functional food composition for preventing or ameliorating autoimmune disease comprising the compound represented by Formula 1 or a pharmaceutically acceptable salt thereof. In the present invention, the compound represented by the formula (1) can be easily utilized in the production of health functional foods, such as the main ingredients, secondary ingredients, food additives, functional foods or beverages having an effect of preventing or improving autoimmune diseases.
본 발명에서 상기 "건강기능식품"이란 식품에 물리적, 생화학적, 생물공학적 수법 등을 이용하여 해당 식품의 기능을 특정 목적에 작용, 발현하도록 부가가치를 부여한 식품군이나 식품 조성이 갖는 생체방어리듬조절, 질병방지와 회복 등에 관한 체내조절기능을 생체에 대하여 충분히 발현하도록 설계하여 가공한 식품을 의미하며, 이는 식품학적으로 허용 가능한 식품 보조 첨가제를 더 포함할 수 있으며, 통상적으로 사용되는 적절한 담체, 부형제 및 희석제를 더 포함할 수 있다.In the present invention, the "health functional food" is a biological defense rhythm control having a food group or a food composition to which added value to act and express the function of the food to a specific purpose using physical, biochemical, biotechnological methods, etc. It means a food processed and designed to fully express the body control function on disease prevention and recovery to the living body, which may further include food acceptable food additives, suitable carriers, excipients and commonly used It may further comprise a diluent.
또한, 본 발명의 건강기능식품 조성물은 여러 가지 영양제, 비타민, 광물(전해질), 합성 풍미제 및 천연 풍미제 등의 풍미제, 착색제 및 충진제(치즈, 초콜릿 등), 펙트산 및 그의 염, 알긴산 및 그의 염, 유기산, 보호성 콜로이드 증점제, pH 조절제, 안정화제, 방부제, 글리세린, 알코올, 탄산음료에 사용되는 탄산화제 등을 함유할 수 있으며, 상기 성분은 독립적으로 또는 조합하여 사용할 수 있다.In addition, the health functional food composition of the present invention is a variety of nutrients, vitamins, minerals (electrolytes), flavors such as synthetic flavors and natural flavors, colorants and fillers (such as cheese, chocolate), pectic acid and salts thereof, alginic acid And salts thereof, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohols, carbonation agents used in carbonated beverages, and the like, which components can be used independently or in combination.
이하, 본 발명의 이해를 돕기 위하여 바람직한 실시예를 제시한다. 그러나 하기의 실시예는 본 발명을 보다 쉽게 이해하기 위하여 제공되는 것일 뿐, 하기 실시예에 의해 본 발명의 내용이 한정되는 것은 아니다.Hereinafter, preferred examples are provided to aid in understanding the present invention. However, the following examples are merely provided to more easily understand the present invention, and the contents of the present invention are not limited by the following examples.
[실시예]EXAMPLE
실시예 1. 화합물 제조Example 1. Compound Preparation
1-1. 1-메틸-3-(4-(피리딘-2-일)벤질)-thiourea(JC1-38) 제조1-1. Preparation of 1-methyl-3- (4- (pyridin-2-yl) benzyl) -thiourea (JC1-38)
두 가지 둥근 바닥 플라스크에 4-(2-피리딜)벤잘데하이드(97%, 1000mg, 5.46mmol)와 N-methylthiourea(4921mg, 54.6mmol)을 넣은 후, 감압하여 아르고 기체를 치환시켰다. 그 후 무수 THF(Tetrahydrofuran, 20ml)을 용매로 넣고, 냉동 보관되어 있던 Ti(OiPr)4(2.72ml, 9.28mmol)을 넣어서 환류시켰다. TLC 상에서 기질이 다 없어지면 반응 용기를 천천히 식힌 후, 소디윰보로하이드라이드(103mg, 2.73mmol)를 넣어주었다. 그리하여 노란색의 원하는 프로덕트(512.9mg, 2mmol)를 얻었다. (수율: 37%)4- (2-pyridyl) benzaldehyde (97%, 1000mg, 5.46mmol) and N-methylthiourea (4921mg, 54.6mmol) were added to two round bottom flasks, and argo gas was substituted under reduced pressure. After that, anhydrous THF (Tetrahydrofuran, 20 ml) was added as a solvent, and refrigerated by adding Ti (OiPr) 4 (2.72 ml, 9.28 mmol), which was stored in a frozen state. After the substrate was gone on TLC, the reaction vessel was cooled slowly, and Sodijanborohydride (103 mg, 2.73 mmol) was added thereto. Thus, the desired product in yellow color (512.9 mg, 2 mmol) was obtained. (Yield 37%)
1H-NMR (300MHZ, CDCl3) δ8.67-8.65(d, J = 5.0 Hz, 1H), 7.95-7.92(d, J = 8.2 Hz, 2H), 7.78-7.68(m, 2H), 7.42-7.39(d, J = 8.0 Hz, 2H), 7.21(s, 1H), 4.71(s,2H), 2.99(s, 3H) 1 H-NMR (300MHZ, CDCl 3) δ 8.67-8.65 (d, J = 5.0 Hz, 1H), 7.95-7.92 (d, J = 8.2 Hz, 2H), 7.78-7.68 (m, 2H), 7.42- 7.39 (d, J = 8.0 Hz, 2H), 7.21 (s, 1H), 4.71 (s, 2H), 2.99 (s, 3H)
1-2. 1-(4-벤질옥시-벤질)-3-메틸-thiourea(JC1-40) 제조1-2. Preparation of 1- (4-benzyloxy-benzyl) -3-methyl-thiourea (JC1-40)
메틸아민염화수소 염(1eq)에 DMF 용매 하에 TEA(TriEthylAmine, 1.2eq) 4-벤질옥시벤잘데하이드를 넣은 후, 교반하였다. TLC 상에 기질이 없어짐을 확인 후, DMF를 날렸다. 그 후 에틸아세테이트로 희석한 후 브라인을 사용하여 씻어낸 후 감압증발하여 얻은 잔사로 컬럼 크로마토그래피(헥산:에틸 아세테이트 = 3:1)를 실시하여 노란색의 고체(202.7mg, 0.71mmol)를 얻었다. (수율: 30%)To a methylamine hydrogen chloride salt (1eq) was added TEA (TriEthylAmine, 1.2eq) 4-benzyloxybenzaldehyde with DMF solvent, followed by stirring. After confirming the disappearance of the substrate on the TLC, DMF was blown. Thereafter, the mixture was diluted with ethyl acetate, washed with brine, and then subjected to column chromatography (hexane: ethyl acetate = 3: 1) using a residue obtained by evaporation under reduced pressure to obtain a yellow solid (202.7 mg, 0.71 mmol). (Yield 30%)
1H-NMR (300MHZ, CD3OD) δ7.44-7.28 (m, 5H), 7.23-7.20(d, J = 8.4 Hz, 2H), 6.98-6.94(m, 2H), 5.07(s, 2H), 4.55(s, 2H), 2.82(s, 3H) 1 H-NMR (300MHZ, CD3OD) δ 7.44-7.28 (m, 5H), 7.23-7.20 (d, J = 8.4 Hz, 2H), 6.98-6.94 (m, 2H), 5.07 (s, 2H), 4.55 (s, 2H), 2.82 (s, 3H)
1-3. 1-(4-페녹시-벤질)-3-메틸-thiourea(JC1-42) 제조1-3. Preparation of 1- (4-phenoxy-benzyl) -3-methyl-thiourea (JC1-42)
메틸아민염화수소 염(1eq)에 DMF 용매 하에 TEA(TriEthylAmine, 1.2eq) 4-페녹시벤잘데하이드를 넣은 후, 교반하였다. TLC 상에 기질이 없어짐을 확인 후, DMF를 날렸다. 그 후 에틸아세테이트로 희석한 후 브라인을 사용하여 씻어낸 후 감압증발하여 얻은 잔사로 컬럼 크로마토그래피(헥산:에틸 아세테이트 = 3:1)를 실시하여 노란색의 고체(178.9mg, 0.66mmol)를 얻었다.(수율: 65%)To a methylamine hydrogen chloride salt (1eq) was added TEA (TriEthylAmine, 1.2eq) 4-phenoxybenzaldehyde, under DMF solvent, followed by stirring. After confirming the disappearance of the substrate on the TLC, DMF was blown. Thereafter, the mixture was diluted with ethyl acetate, washed with brine, and then subjected to column chromatography (hexane: ethyl acetate = 3: 1) using a residue obtained by evaporation under reduced pressure to obtain a yellow solid (178.9 mg, 0.66 mmol). (Yield 65%)
1H-NMR (300MHZ, CDCl3) δ7.33-7.23 (m, 4H), 7.11-7.06(m, 1H), 6.98-6.92(m, 4H), 4.62(s, 2H), 2.96-2.94(d, J= 4.6 Hz, 3H) 1 H-NMR (300MHZ, CDCl3) δ7.33-7.23 (m, 4H), 7.11-7.06 (m, 1H), 6.98-6.92 (m, 4H), 4.62 (s, 2H), 2.96-2.94 (d , J = 4.6 Hz, 3H)
실시예Example 2. 대식세포 세포주에서  2. In macrophage cell lines 티오우레아Thiourea 유도체의 염증 유전자 전사 억제 효과 확인 Inhibition of Inflammatory Gene Transcription by Derivatives
쥐 대식세포주 Raw 264.7 (ATCC TIB-71)는 ATCC (American Type Culture Collection)로부터 구입하였다. Raw264.7 세포 (2X105 세포/웰)를 6-웰 배양 디쉬에 접종하고, 10% FBS (fetal bovine serum)를 함유하는 DMEM (Dulbecco's modified Eagle's medium) 배지에서 하루 동안 배양하였다. Raw 264.7 세포는 5% CO2 및 95% 공기를 유지하는 함습 항온기에서 37℃로 유지하였다. 배양 후, 세포를 50 ng/ml의 LPS (Lipopolysaccharide)와 20 μM의 JC1-40을 24 시간 처리하였다. Real-time PCR (real-time reverse transcriptase-polymerase chain reaction; 정량 중합효소 연쇄반응) 분석법으로 류마티스 관절염을 유발시키는 염증 마커들인 TNF-α, NOS2, IL-1β, IL-6의 mRNA의 발현 정도를 측정하였다.The mouse macrophage line Raw 264.7 (ATCC TIB-71) was purchased from the American Type Culture Collection (ATCC). Raw264.7 cells (2 × 10 5 cells / well) were seeded in 6-well culture dishes and incubated for one day in Dulbecco's modified Eagle's medium (DMEM) medium containing 10% FBS (fetal bovine serum). Raw 264.7 cells were maintained at 37 ° C. in a humidified thermostat maintaining 5% CO 2 and 95% air. After incubation, the cells were treated with 50 ng / ml LPS (Lipopolysaccharide) and 20 μM of JC1-40 for 24 hours. Real-time PCR (real-time reverse transcriptase-polymerase chain reaction) assay revealed the expression levels of mRNAs of inflammatory markers that cause rheumatoid arthritis, TNF-α, NOS2, IL-1β and IL-6. Measured.
그 결과, 도 1에 나타낸 바와 같이, JC1-40을 처리하였을 때 Raw 264.7 대식세포주에서 염증 유발에 관여하는 TNF-α, NOS2, IL-1β 및 IL-6의 전사량이 감소되는 것을 확인할 수 있었다.As a result, as shown in Figure 1, when treated with JC1-40 it was confirmed that the amount of transcription of TNF-α, NOS2, IL-1β and IL-6 involved in inflammation in the Raw 264.7 macrophage line reduced.
실시예Example 3. 1차 배양된 마우스 복강 내 대식세포에서  3. In primary cultured mouse intraperitoneal macrophages 티오우레아Thiourea 유도체의 염증 유전자 전사 억제 효과 확인 Inhibition of Inflammatory Gene Transcription by Derivatives
생후 7~8 주령 된 C57BL/6 수컷 마우스의 peritoneal 대식세포를 분리하여 24-웰 디쉬에 1X106 세포/웰로 접종하고, 10% FBS (fetal bovine serum)를 함유하는 RPMI1640 배지에서 4시간 배양하였다. Peritoneal 대식세포 배양액에 10 ng/ml LPS와 20 μM JC1-40 또는 JC1-42를 각각 표시된 바대로 24시간 처리하였다. 이때, peritoneal 대식세포는 5% CO2 및 95% 공기를 유지하는 함습 항온기에서 37℃로 유지하였다.Peritoneal macrophages from 7 to 8 week old C57BL / 6 male mice were isolated and seeded at 1 × 10 6 cells / well in 24-well dishes and incubated for 4 hours in RPMI1640 medium containing 10% FBS (fetal bovine serum). Peritoneal macrophage cultures were treated with 10 ng / ml LPS and 20 μM JC1-40 or JC1-42, respectively, for 24 hours as indicated. At this time, peritoneal macrophages were maintained at 37 ℃ in a humidified thermostat maintaining 5% CO 2 and 95% air.
먼저, Real-time PCR (real-time reverse transcriptase-polymerase chain reaction; 정량 중합효소 연쇄반응) 분석법으로 류마티스 관절염을 유발시키는 염증 마커들인 TNF-α, NOS2, IL-1β, IL-6의 mRNA의 발현 정도를 측정하였다. 그 결과, 도 2에 나타낸 바와 같이, JC1-40 또는 JC1-42를 처리하였을 때 복강 내 대식세포에서 염증 유발에 관여하는 TNF-α, NOS2, IL-1β 및 IL-6의 mRNA 양이 감소됨을 확인할 수 있었다.First, the expression of mRNA of TNF-α, NOS2, IL-1β, IL-6, which are inflammation markers that induce rheumatoid arthritis by real-time PCR (real-time reverse transcriptase-polymerase chain reaction) assay The degree was measured. As a result, as shown in Figure 2, when treated with JC1-40 or JC1-42, the mRNA levels of TNF-α, NOS2, IL-1β and IL-6 involved in inducing inflammation in the intraperitoneal macrophages was reduced. I could confirm it.
추가적으로, 이들 세포에서 M1 세포의 변화를 M1 표지 마커인 PE-CD80 antibody (ebioscience)를 30분간 incubation시킨 후 유세포분석법을 시행하였다. 그 결과, 도 3에 나타낸 바와 같이, 대식세포를 JC1-40으로 처리하였을 때, M1 분화가 억제됨을 확인하였다.In addition, M1 cell changes were incubated with PE-CD80 antibody (ebioscience) for 30 minutes and flow cytometry was performed. As a result, as shown in Figure 3, when the macrophages were treated with JC1-40, it was confirmed that M1 differentiation is suppressed.
실시예Example 4. 1차 배양된 마우스 복강 내 대식세포에서  4. In primary cultured mouse intraperitoneal macrophages 티오우레아Thiourea 유도체의 M2 항염증 유전자 전사 촉진 효과 확인 Confirmation of the M2 Anti-inflammatory Gene Transcription Effect
생후 7~8 주령 된 C57BL/6 수컷 마우스의 peritoneal 대식세포를 분리하여 24-웰 디쉬에 1X106 세포/웰로 접종하고, 10% FBS (fetal bovine serum)를 함유하는 RPMI1640 배지에서 4시간 배양하였다. Peritoneal 대식세포 배양액에 20 ng/ml IL-4와 20 μM JC1-40 또는 JC1-42를 각각 표시된 바대로 24시간 처리하였다. Real-time PCR (real-time reverse transcriptase-polymerase chain reaction; 정량 중합효소 연쇄반응) 분석법으로 류마티스 관절염을 유발시키는 M2 항염증 마커들인 IL-10, Arg1, Retnla 및 CD206의 mRNA의 발현 정도를 측정하였다. 이때, peritoneal 대식세포는 5% CO2 및 95% 공기를 유지하는 함습 항온기에서 37℃로 유지하였다.Peritoneal macrophages from 7 to 8 week old C57BL / 6 male mice were isolated and seeded at 1 × 10 6 cells / well in 24-well dishes and incubated for 4 hours in RPMI1640 medium containing 10% FBS (fetal bovine serum). Peritoneal macrophage cultures were treated with 20 ng / ml IL-4 and 20 μM JC1-40 or JC1-42, respectively, as indicated for 24 hours. Real-time reverse transcriptase-polymerase chain reaction (RTM) assay was used to determine the expression levels of mRNA of IL-10, Arg1, Retnla and CD206, which are M2 anti-inflammatory markers that cause rheumatoid arthritis. . At this time, peritoneal macrophages were maintained at 37 ℃ in a humidified thermostat maintaining 5% CO 2 and 95% air.
그 결과, 도 4a 및 도 4b에 나타낸 바와 같이, JC1-40 또는 JC1-42를 처리하였을 때 복강 내 대식세포주에서 항염증에 관여하는 IL-10, Arg1, Retnla 및 CD206의 전사량이 증가됨을 확인할 수 있었다.As a result, as shown in Figures 4a and 4b, the treatment of JC1-40 or JC1-42, it can be seen that the amount of transcription of IL-10, Arg1, Retnla and CD206 involved in anti-inflammatory in the intraperitoneal macrophage line increased there was.
실시예Example 5. T 세포 세포주에서  5. In T cell cell line 티오우레아Thiourea 유도체의  Derivative Th17Th17 분화 및 IL-17 분비 억제 효과 확인 Confirmation of Differentiation and Inhibition of IL-17 Secretion
쥐 T 세포 EL4 세포주 (5X105 세포/웰)를 24-웰 배양 디쉬에 접종하고, 20 μM의 JC1-40을 24시간 전처리하여 10% FBS를 함유하는 RPMI1640 배지에서 하루 동안 배양하였다. EL4 세포주는 5% CO2 및 95% 공기를 유지하는 함습 항온기에서 37℃로 유지하였다. 배양 후, 세포를 Th17 분화 조건(anti-CD3 항체 0.5μg/mL, anti-CD28 항체 1μg/mL, TGF-β 2ng/ml, IL-6 20ng/ml, anti-IL-4 10μg/ml, anti-IFN-γ 10μg/ml) 하에 48시간 배양하였다. Real-time PCR (real-time reverse transcriptase-polymerase chain reaction; 정량 중합효소 연쇄반응) 분석법으로 JC1-40 활성 핵수용체인 RORα와 류마티스 관절염을 유발시키는 염증 마커인 IL-17의 mRNA의 발현 정도를 측정하였다.Murine T cell EL4 cell lines ( 5 × 10 5 cells / well) were seeded in 24-well culture dishes and incubated for one day in RPMI1640 medium containing 10% FBS by 24 hours pretreatment of 20 μM of JC1-40. EL4 cell lines were maintained at 37 ° C. in a humidified thermostat maintaining 5% CO 2 and 95% air. After incubation, the cells were treated with Th17 differentiation conditions (0.5 μg / mL of anti-CD3 antibody, 1 μg / mL of anti-CD28 antibody, 2 ng / ml of TGF-β, 20 ng / ml of IL-6, 10 μg / ml of anti-IL-4, anti -IFN-γ 10 μg / ml) for 48 hours. Real-time PCR (real-time reverse transcriptase-polymerase chain reaction) assay measures the expression levels of JC1-40 active nuclear receptor RORα and IL-17 mRNA, an inflammation marker that causes rheumatoid arthritis It was.
그 결과, 도 5a에 나타낸 바와 같이, JC1-40을 처리하였을 때 핵수용체 RORα 전사 발현 증가와 IL-17 전사 발현 감소를 확인할 수 있었다. 또한, JC1-40 처리에 의해 Th17 활성을 억제시키는 Treg의 대표 인자 Foxp3의 발현양이 증가하는 것을 위의 조건에서 배양한 세포에 형광이 표지된 항체 (anti-CD4-FITC, anti-CD25-APC, anti-Foxp3-PE) 염색 후 공초점 현미경 관찰을 통해 확인하였다 (도 5b 참조).As a result, as shown in Figure 5a, when treated with JC1-40 it was confirmed that the increase in nuclear receptor RORα transcriptional expression and IL-17 transcriptional expression. In addition, the fluorescently labeled antibodies (anti-CD4-FITC, anti-CD25-APC) were cultured in cells cultured under the above conditions to increase the expression of Foxp3, a representative factor of Tre, that inhibits Th17 activity by JC1-40 treatment. , anti-Foxp3-PE) staining was confirmed by confocal microscopy (see Figure 5b).
실시예Example 6. 1차 배양된 마우스 비장  6. Primary Cultured Mouse Spleen T세포에서In T cells 티오우레아Thiourea 유도체의  Derivative Th17Th17 분화 및 IL-17 분비 억제 효과 확인 Confirmation of Differentiation and Inhibition of IL-17 Secretion
생후 7~8 주령 된 C57BL/6 수컷 마우스의 비장 내 CD4+ T 세포를 분리하여 48-웰 디쉬에 5X105 세포/웰로 접종하고, 농도별 JC1-40 (0.1, 0.5, 10 및 50 μM)을 24시간 전처리하여 10% FBS (fetal bovine serum)를 함유하는 RPMI1640 배지에서 하루 동안 배양하였다. 배양 후, 세포를 Th17 분화 조건 (anti-CD3 항체 0.5μg/mL, anti-CD28 항체 1μg/mL, TGF-β 2ng/ml, IL-6 20ng/ml, anti-IL-4 10μg/ml, anti-IFN-γ 10μg/ml) 하에 3일간 배양하였다. 이후, 세포를 모으고 유세포 분석을 위해 FACs buffer로 세척한 후, 비특이성 결합을 억제하기 위해 4℃에서 15분간 blocking하고, 세포 표면 마커인 CD4에 대한 항체 (anti-CD4-PerCP)를 넣고 4℃에서 30분간 반응한 뒤 perm wash buffer로 세척하였다. Cytofix/cytoperm 과정을 4℃에서 20분간 진행한 후 perm wash buffer로 세척하였다. Anti-IL-17 PE를 넣고 4℃에서 30분간 반응한 뒤 perm wash buffer로 세척하였다. 염색이 끝난 세포의 변화를 유세포 분석법으로 분석하였다.CD4 + T cells in the spleen of 7-8 week old C57BL / 6 male mice were isolated and inoculated with 5 × 10 5 cells / well in 48-well dishes, and JC1-40 (0.1, 0.5, 10 and 50 μM) by concentration 24 Time pretreatment was incubated for one day in RPMI1640 medium containing 10% FBS (fetal bovine serum). After incubation, the cells were treated with Th17 differentiation conditions (0.5 μg / mL of anti-CD3 antibody, 1 μg / mL of anti-CD28 antibody, 2 ng / ml of TGF-β, 20 ng / ml of IL-6, 10 μg / ml of anti-IL-4, anti -IFN-γ 10 μg / ml) for 3 days. Thereafter, cells were collected and washed with FACs buffer for flow cytometry, and then blocked for 15 minutes at 4 ° C to inhibit nonspecific binding, and an antibody to CD4 (anti-CD4-PerCP), which is a cell surface marker, was placed at 4 ° C. After 30 minutes in the reaction was washed with perm wash buffer. Cytofix / cytoperm process was performed for 20 minutes at 4 ℃ and washed with perm wash buffer. Anti-IL-17 PE was added thereto, reacted at 4 ° C. for 30 minutes, and washed with perm wash buffer. Changes in stained cells were analyzed by flow cytometry.
그 결과, 도 6a에 나타낸 바와 같이, JC1-40으로 처리하였을 때, Th17로 분화된 IL-17 양성 세포의 양이 감소됨을 확인할 수 있다.As a result, as shown in Figure 6a, when treated with JC1-40, it can be seen that the amount of IL-17 positive cells differentiated into Th17 is reduced.
추가적으로, JC1-40이 처리된 Th17 세포의 배양 상층액을 모아 sandwich ELISA 방법을 이용하여 IL-17의 양을 조사하였다. 이를 위해 먼저 96웰 플레이트에 단클론성 anti-IL-17을 2μg/mL 으로 처리하여 4℃에서 밤새 반응시켰으며, 반응 후 차단용액(1% BSA/PBST)으로 비특이적 결합을 차단시켰다. 이후 IL-17을 1/2씩 연속 희석하여 standard로 사용하였으며, 세포배양 상층액을 넣고 실온에서 2시간 동안 반응시켰다. 이후, biotinylated anti-IL-17을 2시간 동안 실온에서 반응시킨 후 4회 세척한 다음, ExtraAvidin-Alkaline Phosphatase conjugate를 희석하여 첨가하고 실온에서 2시간 동안 반응시켰다. 이후, PNPP/DEA 용액을 넣고 발색한 후 405 nm 파장에서 흡광도를 측정하였다.In addition, the culture supernatants of Th17 cells treated with JC1-40 were collected and examined for the amount of IL-17 using the sandwich ELISA method. To this end, the monoclonal anti-IL-17 was first treated in a 96-well plate at 2 μg / mL and reacted overnight at 4 ° C. After the reaction, nonspecific binding was blocked with a blocking solution (1% BSA / PBST). Thereafter, IL-17 was serially diluted by 1/2, and used as a standard. The cell culture supernatant was added and reacted at room temperature for 2 hours. Thereafter, the biotinylated anti-IL-17 was reacted at room temperature for 2 hours and washed four times. Then, the ExtraAvidin-Alkaline Phosphatase conjugate was diluted and added and reacted at room temperature for 2 hours. Thereafter, PNPP / DEA solution was added, followed by color development, and absorbance at 405 nm was measured.
그 결과, 도 6b에 나타낸 바와 같이, JC1-40으로 처리하였을 때, 배양액으로 분비된 IL-17 단백질의 양이 감소됨을 확인할 수 있었다.As a result, as shown in Figure 6b, when treated with JC1-40, it was confirmed that the amount of IL-17 protein secreted into the culture medium is reduced.
실시예Example 7. 인간 말초  7. Human Peripheral 단핵Mononuclear 세포구(PBMC)에서In cell cells (PBMC) 티오우레아Thiourea 유도체의  Derivative Th17Th17 분화 및 IL-17 분비 억제 효과 확인 Confirmation of Differentiation and Inhibition of IL-17 Secretion
건강인 PBMC(peripheral blood mononuclear cell)에서 CD4+ cells을 분리하여 JC1-40을 농도별(10, 20 및 40 μM)로 전처리한 뒤, Th17 분화 조건하 (anti-CD3 0.5㎍/ml, anti-CD28 0.5 ㎍/ml, anti-IFN-γ 10 ㎍/ml, anti-IL-4 10 ㎍/ml, IL-6 20ng/ml, IL-1β 20 ng/ml)에 3일간 배양하였다. 이들 세포에서 Th17 (anti IL-17 PE) 및 Treg (Anti Foxp3-FITC)의 발현을 유세포분석법으로 확인하였다. 또한, 이들 상층액에서 IL-17의 양을 ELISA 측정하였다.CD4 + cells were isolated from healthy peripheral blood mononuclear cells (PBMCs) and pretreated with JC1-40 concentrations (10, 20 and 40 μM), and then under Th17 differentiation conditions (anti-CD3 0.5 μg / ml, anti-CD28). 0.5 μg / ml, anti-IFN-γ 10 μg / ml, anti-IL-4 10 μg / ml, IL-6 20ng / ml, IL- 20 ng / ml). Expression of Th17 (anti IL-17 PE) and Treg (Anti Foxp3-FITC) in these cells was confirmed by flow cytometry. In addition, the amount of IL-17 in these supernatants was measured by ELISA.
그 결과, 도 7a 및 7b에 나타낸 바와 같이, 인간 말초 단핵 세포구를 JC1-40으로 처리하였을 때, Th17으로 분화한 (IL-17 양성) T 세포는 감소한 반면, Treg으로 분화한 세포 (FOXP3 양성)의 양은 증가함을 확인할 수 있었다.As a result, as shown in FIGS. 7A and 7B, when human peripheral mononuclear cells were treated with JC1-40, T17 differentiated (IL-17 positive) T cells decreased while Treg differentiated cells (FOXP3 positive). The amount of) was increased.
실시예 8. 티오우레아 유도체의 파골세포 분화 조절 효과 확인Example 8. Confirmation of Osteoblast Differentiation Control Effect of Thiourea Derivatives
건강인 PBMC를 분리하여 M-CSF (25 ng/ml)를 3일간 자극하여 파골세포 전구체로 분화시켰다. 3일 뒤 배지 교환 후, M-CSF (25 ng/ml), RANKL (30 ng/ml) 및 JC1-40 (10, 20, 40 iM)을 농도 별로 자극하여 3일간 배양하였고, 세포의 분화 상태를 관찰하며 3일 마다 배지 교환 후 동일한 자극을 진행하였다.Healthy PBMCs were isolated and stimulated with M-CSF (25 ng / ml) for 3 days to differentiate into osteoclast precursors. After 3 days of medium exchange, M-CSF (25 ng / ml), RANKL (30 ng / ml) and JC1-40 (10, 20, 40 iM) were stimulated by concentration and incubated for 3 days, and the differentiation state of cells Observed the same stimulation after medium exchange every three days.
분화된 세포로부터 RNA를 분리하여 cDNA 합성 후 파골세포 분화 관련 인자인 Cathepsin K와 TRAP mRNA 발현 변화를 real-time PCR로 분석하였다.RNA was isolated from the differentiated cells, and the changes in cathepsin K and TRAP mRNA expression related to osteoclast differentiation after cDNA synthesis were analyzed by real-time PCR.
그 결과, 도 8a 및 도 8b에 나타낸 바와 같이, JC1-40을 처리하였을 때, 파골세포 분화 관련 인자의 발현이 유의적으로 감소됨을 확인할 수 있었다.As a result, as shown in Figures 8a and 8b, when the JC1-40 treatment, it was confirmed that the expression of osteoclast differentiation-related factors significantly decreased.
실시예Example 9. 관절염 동물모델 (type II collagen-induced arthritis-CIA)에서 티오우레아 유도체의 관절염 억제 효과 확인 9. Confirmation of Arthritis Inhibitory Effect of Thiourea Derivatives in Animal Model of Arthritis (type II Collagen-induced Arthritis-CIA)
생후 6 주령 된 DBA 수컷 마우스에 CFA (chondrex)에 emulsified된 bovine CII (chondrex)를 접종하고, 15일 뒤에 다시 재접종하여 류마티스 관절염을 유발하였다. 첫 투여 후 주 3회 지속으로 0.5% CMC (carboxymethyl cellulose)에 녹아있는 JC1-40을 10 mg/kg으로 경구 투여하였다. 나머지 그룹은 0.5% CMC vehicle과 양성 대조군으로 5 mg/kg Enbrel을 같은 방식으로 투여하였다. 관절염 질환이 나타난 개체 수 비율을 incidence로 환산하였고, 관절염의 조직을 병태생리학적으로 검사하여 질환의 진행 정도를 score로 환산하여 각 그룹간의 류마티스 관절염 질환 상태를 비교하였다. 임상수치는 관절염증 질환의 진행 정도를 매일 마우스의 관절염 병변을 육안으로 관찰하여, 발이나 꼬리 부위의 종창 및 발적 여부 대한 각각의 점수를 매기는 임상점수체계를 사용하여 평가하였다.Six-week-old DBA male mice were inoculated with bovine CII (chondrex) emulsified with CFA (chondrex) and reinoculated 15 days later to induce rheumatoid arthritis. After the first administration, JC1-40 dissolved in 0.5% CMC (carboxymethyl cellulose) was orally administered at 10 mg / kg for 3 consecutive weeks. The other group received 5 mg / kg Enbrel in the same manner as 0.5% CMC vehicle and positive control. The percentage of individuals with arthritis disease was converted to incidence, and the pathophysiological examination of the arthritis tissues was used to compare the status of rheumatoid arthritis between groups. Clinical scores were assessed using a clinical scoring system that visually monitors the progression of arthritis disease daily by visually examining arthritis lesions in the mouse and swelling and redness of the foot or tail area.
그 결과, 도 9에 나타낸 바와 같이, CIA 동물 모델에서 JC1-40 투여는 관절염의 incidence와 score를 모두 억제하였으며 이는 임상에서 실제로 사용되는 류마티스 관절염 치료제인 Enbrel보다 우수한 효과를 나타내었다.As a result, as shown in Fig. 9, JC1-40 administration in the CIA animal model inhibited both the incidence and score of arthritis, which was superior to the Enbrel, a therapeutic agent for rheumatoid arthritis actually used in the clinic.
전술한 본 발명의 설명은 예시를 위한 것이며, 본 발명이 속하는 기술분야의 통상의 지식을 가진 자는 본 발명의 기술적 사상이나 필수적인 특징을 변경하지 않고서 다른 구체적인 형태로 쉽게 변형이 가능하다는 것을 이해할 수 있을 것이다. 그러므로 이상에서 기술한 실시예들은 모든 면에서 예시적인 것이며 한정적이 아닌 것으로 이해해야만 한다.The foregoing description of the present invention is intended for illustration, and it will be understood by those skilled in the art that the present invention may be easily modified in other specific forms without changing the technical spirit or essential features of the present invention. will be. Therefore, it should be understood that the embodiments described above are exemplary in all respects and not restrictive.
본 발명에 따른 티오우레아 유도체는 염증 유전자의 전사를 억제하고 항염증 유전자의 전사를 촉진시킬 뿐만 아니라, Th17의 활성 또는 생성을 억제하고, 조절 T 세포(Regulatory T cell: Treg)의 활성 또는 생성을 증가시킬 수 있기 때문에, 류마티스 관절염을 비롯한 다양한 자가면역 질환의 예방, 개선 또는 치료를 위한 약학적 조성물, 건강식품 조성물 등에 유용하게 사용될 수 있다.Thiourea derivatives according to the present invention not only inhibit the transcription of inflammatory genes and promote the transcription of anti-inflammatory genes, but also inhibit the activity or production of Th17 and inhibit the activity or production of Regulatory T cells (Tregs). Since it can increase, it can be usefully used in pharmaceutical compositions, health food compositions, etc. for the prevention, improvement or treatment of various autoimmune diseases including rheumatoid arthritis.

Claims (8)

  1. 하기 화학식 1로 표시되는 화합물 또는 이의 약제학적으로 허용가능한 염을 유효성분으로 포함하는, 자가면역질환 예방 또는 치료용 약학적 조성물.A pharmaceutical composition for preventing or treating autoimmune diseases, comprising a compound represented by the following Formula 1 or a pharmaceutically acceptable salt thereof as an active ingredient.
    [화학식 1][Formula 1]
    Figure PCTKR2016008867-appb-I000003
    Figure PCTKR2016008867-appb-I000003
    여기서, R은 페녹시기, 벤질옥시기 또는 피리디닐기이다.Here, R is a phenoxy group, benzyloxy group or pyridinyl group.
  2. 제1항에 있어서,The method of claim 1,
    상기 자가면역질환은 류마티스 관절염, 건선성 관절염, 건선, 전신성 피부 경화증(systemic scleroderma), 경화증(sclerosis), 다발성 경화증, 염증성 장질환, 크론병(crohn's disease), 패혈증 및 타입 I 당뇨병으로 이루어진 군으로부터 선택되는 것을 특징으로 하는, 조성물.The autoimmune disease is from the group consisting of rheumatoid arthritis, psoriatic arthritis, psoriasis, systemic scleroderma, sclerosis, multiple sclerosis, inflammatory bowel disease, crohn's disease, sepsis and type I diabetes Characterized in that the composition is selected.
  3. 제1항에 있어서,The method of claim 1,
    상기 조성물은 Th17 세포의 생성을 억제하는 것을 특징으로 하는, 조성물.The composition, characterized in that to inhibit the production of Th17 cells.
  4. 제1항에 있어서,The method of claim 1,
    상기 조성물은 조절 T 세포(Regulatory T cell: Treg)의 생성을 촉진하는 것을 특징으로 하는, 조성물.The composition is characterized in that to promote the production of Regulatory T cells (Treg), composition.
  5. 제1항에 있어서,The method of claim 1,
    상기 조성물은 TNF-α, NOS2, IL-1β 또는 IL-6 유전자의 발현을 억제하는 것을 특징으로 하는, 조성물.The composition is characterized by inhibiting the expression of the TNF-α, NOS2, IL-1β or IL-6 gene.
  6. 제1항에 있어서,The method of claim 1,
    상기 조성물은 IL-10, Arg1, Retnla 또는 CD206 유전자의 발현을 촉진시키는 것을 특징으로 하는, 조성물.The composition is characterized in that to promote the expression of IL-10, Arg1, Retnla or CD206 gene.
  7. 제1항의 조성물을 개체에 투여하는 단계를 포함하는, 자가면역 질환의 치료방법.A method of treating autoimmune disease, comprising administering to a subject a composition of claim 1.
  8. 제1항의 조성물의 자가면역 질환 치료 용도.Use of the composition of claim 1 for the treatment of autoimmune diseases.
PCT/KR2016/008867 2015-08-12 2016-08-12 Pharmaceutical composition for preventing or treating autoimmune diseases comprising thiourea derivative WO2017026830A1 (en)

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