WO2015126048A1 - Anti-inflammatory composition comprising loratadine - Google Patents

Anti-inflammatory composition comprising loratadine Download PDF

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WO2015126048A1
WO2015126048A1 PCT/KR2014/012422 KR2014012422W WO2015126048A1 WO 2015126048 A1 WO2015126048 A1 WO 2015126048A1 KR 2014012422 W KR2014012422 W KR 2014012422W WO 2015126048 A1 WO2015126048 A1 WO 2015126048A1
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loratadine
inflammation
cells
composition
inflammatory
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French (fr)
Korean (ko)
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조재열
백광수
양우석
정덕
박재광
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성균관대학교산학협력단
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    • 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
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
    • A61K31/445—Non condensed piperidines, e.g. piperocaine
    • A61K31/4523—Non condensed piperidines, e.g. piperocaine containing further heterocyclic ring systems
    • A61K31/4545—Non condensed piperidines, e.g. piperocaine containing further heterocyclic ring systems containing a six-membered ring with nitrogen as a ring hetero atom, e.g. pipamperone, anabasine
    • 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]

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  • the present invention relates to loratadine (Loratadine, Ethyl 4- (8-chloro-5,6-dihydro-11H-benzo [5,6] cyclohepta [1,2-b] pyridin-11-ylidene) -1-piperidinecarboxylate) It relates to an anti-inflammatory composition containing as an active ingredient.
  • Inflammatory diseases are reactions in the human body, including infection and non-infection. Symptoms of inflammation include fever, redness, edema, and pain. Late in the inflammatory process, the migration of white blood cells from the inflammatory site and cytokines, degradative enzymes, and bioactive lipid intermediates. Intracellular changes occur, such as transient reactive oxygen species and the production of sensitized lmphocytes. In addition, in chronic inflammatory diseases, cell activation and cell death occur due to infiltration of white blood cells. Prostaglandins and nitric oxide (NO) are important mediators for the development of carcinogenesis and inflammation.
  • NO nitric oxide
  • Nonsteroidal antiinflammatory drugs one of the drugs that control inflammation and pain, have pharmacological effects by inhibiting COX, which produces prostaglandin (PG) in the body.
  • COX has been shown to exist in two isoforms, COX-1 and COX-2, of which COX-1 produces PGs that are involved in cellular protection and regulation in the gastrointestinal mucosa, platelets, and kidneys under normal conditions.
  • COX-2 is very low in normal cells, COX-2 is induced by various stimuli (cytokine, endotoxin, mitogen) in inflammation-related cells and is involved in the production of PGs that are involved in pain or inflammation.
  • Nitric oxide is also involved in the regulation of vascular tone, neurotransmission, killing of bacteria and cancer cells. High levels of NO also occur in physiological reactions such as circulatory shock, inflammation, and carcinogenesis. Neuronal NO and endothelial NO are also present in normal conditions, whereas iNOS (NOS type 2) is induced and expressed by LPS, cytokines, and the like.
  • iNOS NOS type 2
  • LPS low-denspasmodase
  • cytokines cytokines
  • the nonsteroidal inflammatory diseases also inhibit the production of PG related to gastric mucosa protection and platelet function in addition to PGs involved in inflammatory reactions in the human body, and also have side effects such as gastrointestinal disorders and bleeding. Will appear together. Therefore, the development of a therapeutic agent for inflammatory diseases without side effects is required.
  • loratadine is a drug commercially available as an anti-histamine, and has been used for the treatment of rhinitis and urticaria of the drug.
  • Loratadine has been only limitedly studied for inhibiting the expression of histamine (Korean Patent Publication No. 10-1992-7000033), and the effect on acute inflammation and the like has not been studied.
  • the present invention has been made to overcome the limitations of the conventional anti-inflammatory agents, and to provide a pharmaceutical composition, food composition for preventing or treating inflammatory diseases comprising Loratadine or a pharmaceutically acceptable salt thereof as an active ingredient. It is done.
  • the present invention relates to loratadine (Loratadine, Ethyl 4- (8-chloro-5,6-dihydro-11H-benzo [5,6] cyclohepta [1,2-b] pyridin-11-ylidene) -1-piperidinecarboxylate) or It provides a pharmaceutical composition for preventing or treating inflammatory diseases comprising the pharmaceutically acceptable salt thereof as an active ingredient.
  • the composition is characterized in that the production of nitric oxide (Nitric Oxide, NO) or PGE 2 (Prostaglandin E 2 ) to suppress the production.
  • nitric oxide Nitric Oxide, NO
  • PGE 2 Prostaglandin E 2
  • the inflammation is selected from the group consisting of gastric ulcer, duodenal ulcer, hepatitis, esophagitis, gastritis, enteritis, pancreatitis, colitis, peritonitis, nephritis, systemic edema and local edema.
  • the present invention is a loratadine (Loratadine, Ethyl 4- (8-chloro-5,6-dihydro-11H-benzo [5,6] cyclohepta [1,2-b] pyridin-11-ylidene) -1-piperidinecarboxylate
  • a pharmaceutically acceptable salt thereof as an active ingredient can be provided a food composition for preventing or improving inflammatory diseases.
  • the present invention is a loratadine (Loratadine, Ethyl 4- (8-chloro-5,6-dihydro-11H-benzo [5,6] cyclohepta [1,2-b] pyridin-11-ylidene) -1-piperidinecarboxylate Or a pharmaceutically acceptable salt thereof, may be provided to a subject for the prevention or treatment of an inflammatory disease.
  • loratadine Liratadine, Ethyl 4- (8-chloro-5,6-dihydro-11H-benzo [5,6] cyclohepta [1,2-b] pyridin-11-ylidene) -1-piperidinecarboxylate
  • a pharmaceutically acceptable salt thereof may be provided to a subject for the prevention or treatment of an inflammatory disease.
  • the present invention is a loratadine (Loratadine, Ethyl 4- (8-chloro-5,6-dihydro-11H-benzo [5,6] cyclohepta [1,2-b] pyridin-11-ylidene) -1-piperidinecarboxylate ) Or a pharmaceutically acceptable salt thereof.
  • Anti-inflammatory pharmaceutical composition, food composition and cosmetic composition comprising Loratadine of the present invention as an active ingredient has an excellent inflammation treatment effect by reducing the production of inflammation-related factors PGE 2 , NO.
  • the composition of the present invention can be safely applied to all medicines, foods and cosmetics. Therefore, it is expected that it can be importantly applied as an anti-inflammatory agent.
  • FIG. 2 is a diagram showing the effect of Ranitidine, an inflammatory drug, on the production of nitric oxide (NO) in RAW264.7 cells treated with LPS.
  • Figure 3 shows the effect of Loratidine on PGE 2 production in LPS treated RAW264.7 cells.
  • Figure 5 is a photograph of the erosive mucosal erosion after harvesting the stomach after administration of Loratidine or Ranitidine in the gastritis-induced mice.
  • FIG. 6 is a diagram illustrating the comparison of erosive mucosal erosion lesion area (mm 2) measured by pixel count after administration of Loratadine or Ranitidine, respectively, in mice causing gastritis.
  • Figure 7 is a diagram comparing the amount of mucus secretion by dissecting the stomach after administration of Loratadine or Sucralfate, respectively, in rats induced inflammation with anhydrous ethanol.
  • FIG. 8 is a diagram comparing and comparing the inhibitory effect of the digestive fluid by resection of the stomach after pyloric ligation in rats administered Loratadine or Sucralfate, respectively.
  • FIG. 9 is a diagram comparing the amount of nitric oxide in body fluids intraperitoneally after administration of Loratadine or Loratadine vehicle in mice induced peritonitis.
  • AST aspartate aminotransferase
  • FIG. 11 is a diagram measuring iNOS mRNA expression according to Loratadine concentration (0, 20, 30, 40 ⁇ M) in RAW264.7 cells.
  • FIG. 12 is a diagram measuring the amount of COX-2 mRNA expression according to Loratadine concentration in RAW264.7 cells.
  • FIG. 13 is a diagram of IL-1 ⁇ mRNA expression according to Loratadine concentration in RAW264.7 cells.
  • FIG. 14 is a diagram measuring the amount of IL-6 mRNA expression according to the concentration of Loratadine in RAW264.7 cells.
  • NF- ⁇ B Luciferase activity is a diagram measuring NF- ⁇ B Luciferase activity according to Loratadine concentration in HEK 293 cells treated with NF- ⁇ B Luciferase (0.8 ⁇ g) and beta-galactosidase (0.1 ⁇ g).
  • 16 is a diagram measuring NF- ⁇ B Luciferase activity according to Loratadine concentration in HEK 293 cells treated with NF- ⁇ B Luciferase (0.3 ⁇ g), TRIF (0.3 ⁇ g), and beta-galactosidase (0.1 ⁇ g).
  • FIG. 17 is a diagram measuring NF- ⁇ B Luciferase activity according to Loratadine concentration in HEK 293 cells treated with NF- ⁇ B Luciferase (0.3 ⁇ g), MyD88 (0.3 ⁇ g), and beta-galactosidase (0.1 ⁇ g).
  • FIG. 18 is a diagram measuring AP-1 Luciferase activity according to Loratadine concentration in HEK 293 cells treated with AP-1 Luciferase (0.8 ⁇ g) and beta-galactosidase (0.1 ⁇ g).
  • 19 is a diagram measuring AP-1 Luciferase activity according to Loratadine concentration in HEK 293 cells treated with AP-1 Luciferase (0.3 ⁇ g), TRIF (0.3 ⁇ g), and beta-galactosidase (0.1 ⁇ g).
  • FIG. 20 is a diagram measuring AP-1 Luciferase activity according to Loratadine concentration in HEK 293 cells treated with AP-1 Luciferase (0.3 ⁇ g), MyD88 (0.3 ⁇ g), and beta-galactosidase (0.1 ⁇ g).
  • 21 is a diagram confirming the expression of target protein antibodies (p65, p50, c-Jun, Lamin A / C) by Loratadine treatment in the Nuclear fraction of RAW264.7 cells through Western blot.
  • Loratadine, C 22 H 23 ClN 2 O 2 , Ethyl 4- (8-chloro-5,6-dihydro-11H-benzo [5,6] cyclohepta [1,2-b] pyridin-11-ylidene -1-piperidinecarboxylate, CAS number 79794-75-5) is represented by the formula (1).
  • LPS Lipopolysaccharide
  • an object of the present invention is Loratadine, Ethyl 4- (8-chloro-5,6-dihydro-11H-benzo [5,6] cyclohepta [1,2-b] pyridin-11-ylidene) -1. -piperidinecarboxylate) or a pharmaceutically acceptable salt thereof as an active ingredient to provide a pharmaceutical composition for preventing or treating inflammatory diseases.
  • the loratadine is characterized in that it inhibits the production of nitric oxide (Nitric Oxide, NO) or PGE 2 (Prostaglandin E 2 ).
  • the loratadine is characterized by inhibiting the activity of NF- ⁇ B or AP-1.
  • the type of inflammation may be gastric ulcer, duodenal ulcer, hepatitis, esophagitis, gastritis, enteritis, pancreatitis, colitis, peritonitis, nephritis, gastric cancer, systemic edema or local edema, but the type of inflammation is not limited thereto.
  • composition of the present invention may be prepared by including one or more pharmaceutically acceptable carriers in addition to the above-described active ingredients for administration.
  • compositions of the present invention may be administered orally or parenterally (eg, applied intravenously, subcutaneously, intraperitoneally or topically) according to the desired method, and the dosage is based on the weight, age, sex, The range varies depending on the state of health, diet, time of administration, method of administration, rate of excretion and the severity of the disease. For example, from 0.1 mg / kg (body weight) to 500 mg / kg (body weight), 0.1 mg / kg (body weight) to 400 mg / kg body weight or 1 mg / kg body weight based on a daily active ingredient To 300 mg / kg body weight, and may be administered once or divided into several.
  • the dosage does not limit the scope of the invention in any aspect.
  • the present invention is a loratadine (Loratadine, Ethyl 4- (8-chloro-5,6-dihydro-11H-benzo [5,6] cyclohepta [1,2-b] pyridin-11-ylidene) -1-piperidinecarboxylate
  • a pharmaceutically acceptable salt thereof as an active ingredient can be provided a food composition for preventing or improving inflammatory diseases.
  • Loratadine Ethyl 4- (8-chloro-5,6-dihydro-11H-benzo [5,6] cyclohepta [1,2-b] pyridin-11-ylidene) -1-piperidinecarboxylate) or its It can provide a cosmetic composition for preventing or improving an inflammatory disease comprising a pharmaceutically acceptable salt as an active ingredient.
  • composition of the present invention may be added to a dietary supplement for the purpose of preventing and improving inflammation.
  • Mixing amounts may be appropriately determined depending on the purpose of use (prevention, health or therapeutic treatment). In general, it can be added in an amount of up to 15% by weight, preferably up to 10% by weight based on the raw materials in the manufacture of food or beverage.
  • 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. There are no particular restrictions on the type of food.
  • composition of the present invention may be added to the cosmetic for the purpose of preventing and improving inflammation.
  • the cosmetic composition may be provided for use in basic cosmetics, makeup cosmetics, body cosmetics, hair cosmetics, scalp cosmetics, shaving cosmetics or oral cosmetics.
  • the active ingredient may be included in an amount of 0.001 to 50% by weight based on the total weight of the cosmetic composition, preferably 0.01 to 20% by weight, but the content is the content of ingredients other than the active ingredient contained in the formulation or cosmetic composition. According to the present invention, the amount of the active ingredient included in the present invention is not limited.
  • the present invention is a loratadine (Loratadine, Ethyl 4- (8-chloro-5,6-dihydro-11H-benzo [5,6] cyclohepta [1,2-b] pyridin-11-ylidene) -1-piperidinecarboxylate ) Or a pharmaceutically acceptable salt thereof can be administered to provide a method for preventing or treating inflammatory diseases.
  • loratadine Liratadine, Ethyl 4- (8-chloro-5,6-dihydro-11H-benzo [5,6] cyclohepta [1,2-b] pyridin-11-ylidene) -1-piperidinecarboxylate
  • a pharmaceutically acceptable salt thereof can be administered to provide a method for preventing or treating inflammatory diseases.
  • the present invention can provide a new use of loratadine, for the treatment of inflammatory diseases.
  • Example 1 confirms the effect of Loratadine on NO production. As a result, it was confirmed that there is an excellent NO production inhibitory effect compared to Ranitidine which is a conventional inflammatory treatment.
  • NO is one of the factors involved in inflammation that plays a role in dilating blood vessels.
  • NO nitric oxide
  • iNOS inducible nitric oxide synthase
  • Example 2 confirms the effect of Loratadine on PGE 2 . As a result, it was confirmed that the addition of Loratadine can rapidly decrease the concentration of PGE 2 even at low concentrations.
  • Prostaglandin E 2 is a substance in which arachidonic acid liberated by phospholipase A2 is catalyzed by an enzyme called cyclooxygenaes (COXs). PGE 2 is known to be deeply involved in the development of cancer, including inflammatory reactions such as pain, fever, immune responses, and angiogenesis.
  • Example 3 confirms the effect of Loratadine on cell viability, and since Loratadine treatment has no effect on cell viability, it was confirmed that it can be used as a safe composition for inflammation treatment.
  • Example 4 confirms the inflammatory treatment effect of Loratadine in an animal model that causes inflammation using ethanol / hydrochloric acid, and confirms that Loratadine significantly reduces gastric damage.
  • Example 5 confirmed the effect of Loratadine on mucus secretion.
  • the side effect of reducing the amount of gastric mucus that protects the gastric mucosa shows a side effect.
  • the results of Example 5 compared to the conventional gastritis, Sucralfate, when using Loratidine of the present invention mucus The secretion of the effect is not reduced.
  • Example 6 confirmed the effect of Loratadine on digestive juice secretion. According to the results of Example 6, compared with conventional antihistamine Cimetidine, which has an inhibitory effect on gastric acid secretion, when Loratidine of the present invention is used, it has a superior gastric acid secretion inhibitory effect.
  • Examples 7 to 8 confirmed the therapeutic effect of Loratadine in peritonitis or hepatitis animal model, and confirmed the reduction of the amount of nitric oxide in the intraperitoneal liquor and the decrease of the blood aspartate aminotransferase (AST) concentration by Loratadine treatment.
  • AST blood aspartate aminotransferase
  • Examples 9 to 11 confirm the mechanism of the anti-inflammatory mechanism of Loratadine, by reducing the inflammation-related factors iNOS, COX-2, IL-1 ⁇ , and IL-6 by Loratadine treatment and NF- ⁇ B which is an inflammation-related transcription factor Or inhibition of activity of AP-1 was confirmed.
  • Rat macrophage cell lines (RAW264.7 cells) were maintained in RPMI1640 medium supplemented with 100 U / ml penicillin, 100 ⁇ g / ml streptomycin and 10% FBS. Cells were incubated in 37 ° C., 5% CO 2 humid air.
  • the cultured cells were treated with Loratadine (0, 20, 30, 40 ⁇ M) for 60 minutes, and then LPS (1 ⁇ g / ml). And further incubated for 24 hours. The supernatant was collected and the concentration of nitric oxide (NO) in the supernatant was measured with Griess reagent. DMSO, a vehicle of Loratadine, was injected as a control.
  • Loratadine inhibited the increased NO production by LPS treatment, and the 20 ⁇ M treatment reduced NO production by about 50% compared to the control without any treatment, and continued concentration-dependently. By reducing the NO content, it was confirmed that more than 90% when treated with 40 uM.
  • Ranitidine was used to confirm the inhibitory effects of nitric oxide production. After incubating RAW264.7 cells (1 ⁇ 10 5 cells / well) for 24 hours, the cultured cells were treated with Ranitidine (0, 10, 20, 40, 80, 160 ⁇ M) for 60 minutes and then LPS (1 Incubated for 24 h more). The supernatant was collected and the concentration of nitric oxide (NO) in the supernatant was measured with Griess reagent. DMSO, a vehicle of Loratadine, was injected as a control.
  • Loratadine (5, 10, 20, 30, 40 ⁇ M) was added to RAW264.7 cells (1 ⁇ 10 5 cells / well) and incubated for 24 hours.
  • DMSO a vehicle of Loratadine
  • the cytotoxic effect was then measured by the general MTT assay. That is, 10 ml of MTT solution (10 mg / ml in phosphate buffer, pH 7.4) was added 3 hours before the end of the culture, and the cells were continuously cultured until the end of the assay.
  • Ethanol / hydrochloric acid induced inflammation of the stomach of ICR mice according to a known method [Lee et al., 2010; Okabe et al.]. Fasted ICR mice (7 mice) received oral administration of Loratadine (10 and 4 mg / kg) or Ranitidine (40 mg / kg) twice daily for 3 days. 0.5% CMC, Loratadine's vehicle, was injected as a control. After 30 minutes of the last administration of loratadine, 400 ⁇ l of 60% ethanol was added to 150 mM hydrochloric acid, and the mice were orally administered. Each experimental animal was anesthetized and sacrificed by lethal doses of urethane 1 hour after necrosis.
  • the stomach was incised and then rinsed carefully with running tap water. After opening the stomach along the Taiwan and spreading on the board, the area (mm 2) of the erosive mucosal corrosion lesion was measured using a pixel-counter. In addition, photographs of mucosal erosion lesions were taken and visually observed.
  • Loratadine 50 mg / kg
  • Sucrafate 375 mg / kg
  • gastric lesions were induced with anhydrous ethanol.
  • 0.5% CMC Loratadine's vehicle
  • Rats fasted for 24 hours prior to the experiment were administered with Loratadine (10 mg / kg) and Cimetidine (250 mg / kg) in the duodenum. 0.5% CMC, Loratadine's vehicle, was injected as a control.
  • CMC Loratadine's vehicle
  • the rats were sacrificed, and the stomach was excised and centrifuged at 1050 xg for 10 minutes.
  • the total amount, pH and gastric acid (mEq / mL) of the digestion solution in the centrifuged solution was measured by titrating the digestion solution at 0.1 M NaOH using methyl orange as an indicator.
  • Loratadine of the present invention shows a better effect in the treatment or prevention of gastritis.
  • mice were divided into three groups (Normal, LPS, LPS + Loratadine), and 1 mL of 3% Thioglycolate was injected intraperitoneally.
  • two groups (Normal and LPS) received oral administration of Loratadine 0.5% CMC, and the other group (LPS + Loratadine) orally administered Loratadine for 5 days.
  • One of the vehicle-treated groups did not cause peritonitis, and the other two groups (LPS, LPS + Loratadine) induced peritonitis by intraperitoneal injection of LPS (lipopolysaccharide).
  • mice Three groups of mice (Normal, LPS, LPS + Loratadine) were sacrificed to collect the intraperitoneal sap, and the amount of nitric oxide generated by peritonitis was measured in order to confirm the treatment or preventive effect of peritonitis. More specifically, absorbance was measured at 540 nm using Griess solution (0.5% naphthylethylenediamine dihydrochloride, 5% sulfanilamide, 25% H 3 PO 4 ), and a calibration curve was prepared using sodium nitrite (0 to 100 ⁇ M) as a standard material. It was.
  • the amount of nitric oxide was significantly increased in the intraperitoneal body fluids of two groups (LPS, LPS + Loratadine) that caused peritonitis, and the group treated with Loratadine was more effective in peritonitis compared to the control group (LPS). It was confirmed that the amount of nitric oxide caused by the decrease.
  • Loratadine of the present invention shows an excellent effect on the treatment or prevention of peritonitis.
  • ICR mice were classified into three groups (Normal, LPS + D-gel, LPS + D-gel + Loratatine), and then two groups (Normal, LPS + D-gel) were used as Loratadine's vehicle 0.5% CMC, The other group (LPS + D-gel + Loratatine) received oral administration of Loratadine for 5 days.
  • One of the vehicle treated groups (Normal) did not cause hepatitis
  • two groups LPS + D-gel, LPS + D-gel + Loratatine were injected intraperitoneally with LPS (lipopolysaccharide) and D-gel.
  • LPS lipopolysaccharide
  • AST aspartate aminotransferase
  • Murine macrophage lines RAW264.7 cells, were 70-80% density in 100 mm cell culture dish using RPMI 1640 medium containing penicillin (100 IU / ml) and streptomycin (100 ⁇ g / ml) and 10% FBS. Incubated with Afterwards, the cultured RAW264.7 cells (1 ⁇ 10 5 cells / well) were treated with Loratadine (0, 20, 30, 40 ⁇ M) for 60 minutes, followed by LPS (1 ⁇ g / mL) for 6 hours. Incubated.
  • PCR amplication was performed using SYBR Premix Ex Taq (Takara Bio) and realtime thermal cycler (Bio-Rad) according to the manufacturer's manual, GAPDH was used as a control gene.
  • Loratadine of the present invention has an anti-inflammatory effect by inhibiting the expression of inflammation-related factors at the transcription level.
  • Example 10 Inhibitory Effect of Loratadine Transcription Factor NF- ⁇ B / AP-1 Activity
  • HEK 293 cell line After dispensing HEK 293 cell line into a 24 well plate using Opti-MEM and pre-cultured at 37 °C, 5% CO 2 cell incubator, the experiment was performed when the cells became 50% density.
  • NF- ⁇ B, AP-1 Luciferase DNA, TRIF, MyD88 DNA and beta-galactosidase DNA were co-transfected, respectively.
  • 1 ⁇ g of DNA and 3 ⁇ g of PEI were respectively diluted in Opti-MEM, incubated at room temperature for 20 minutes, and then mixed with DNA dilution and PEI dilution for 20 minutes at room temperature.
  • the mixed solution was placed in a 24 well plate in which cells were dispensed. After 6 hours, the mixture was replaced with cell culture medium [10% FBS, 1% penicillin / streptomycin in DMEM] and incubated for 24 hours. After that, Loratadine was treated by concentration (0, 20, 30, 40 ⁇ M), and then incubated for another 30 minutes.
  • NF- ⁇ B or AP-1 Luciferase was evaluated by measuring the absorbance with a luminometer. In the case of beta-galactosidase, it was reacted 1: 1 with X-gal, incubated for 5 minutes in a 37 ° C. incubator, and the absorbance was measured at 405 nm.
  • NF- ⁇ B Luciferase (0.8 ⁇ g) and beta-galactosidase (0.1 ⁇ g) treated group Fig. 15
  • NF- ⁇ B Luciferase (0.3 ⁇ g) NF- ⁇ B Luciferase activity was decreased by Loratadine, and as the concentration of Loratadine increased, NF- ⁇ B Luciferase activity was further decreased.
  • AP-1 Luciferase (0.8 ⁇ g) and beta-galactosidase (0.1 ⁇ g) -treated group Fig. 18
  • AP-1 Luciferase (0.3 ⁇ g), TRIF as above. 0.3 ⁇ g
  • group treated with beta-galactosidase 0.1 ⁇ g
  • FIG. 19 group treated with AP-1 Luciferase (0.3 ⁇ g), MyD88 (0.3 ⁇ g), and beta-galactosidase (0.1 ⁇ g).
  • Loratadine of the present invention has an anti-inflammatory effect by inhibiting the activity of inflammation-related transcription factor NF- ⁇ B / AP-1.
  • Murine macrophage RAW264.7 cells were cultured in RPMI 1640 medium containing penicillin (100 IU / ml) and streptomycin (100 ⁇ g / ml) and 10% FBS. Pre-incubated in dish. Thereafter, Loratadine (1 ⁇ g / ml) was pretreated for 30 minutes, stimulated with stimuli (Lipopolysaccharide, LPS), and then washed with ice-cold PBS after a certain time, followed by Homogenization buffer A [20 mM Tris-HCl pH8.0, Cells were collected using 300 ⁇ l of 10 mM EGTA, 2 mM EDTA, 2 mM DTT, 1 mM PMSF, 25 ⁇ g / ml Aprotinin, 10 ⁇ g / ml Leupeptin].
  • stimuli Lipopolysaccharide, LPS
  • Protein concentration of each sample was measured using BSA (Bovine Serum Albumin) as a standard. SDS-PAGE was carried out using each sample amount, which was the protein concentration.
  • BSA Bovine Serum Albumin
  • SDS-PAGE was carried out using each sample amount, which was the protein concentration.
  • the membrane was blocked with 5% non-fat dried milk (Bio-rad). Thereafter, the primary protein antibody (p65, p50, c-Jun, Lamin A / C) solution was first treated, and after the washing step, the secondary antibody solution was treated and washed. In the dark room, ECL solution (Amersham, England) was evenly distributed on the membrane, and the resultant was exposed to X-ray film. Lamin A / C was used as a control protein.
  • the present invention relates to loratadine (Loratadine, Ethyl 4- (8-chloro-5,6-dihydro-11H-benzo [5,6] cyclohepta [1,2-b] pyridin-11-ylidene) -1-piperidinecarboxylate)
  • the present invention relates to an anti-inflammatory pharmaceutical composition and a food composition containing the active ingredient, even when the composition of the present invention is treated at a high concentration, as well as showing no cytotoxicity. Excellent anti-inflammatory effects were confirmed through in vivo or ex vivo experiments.
  • the methods or compositions of the present invention can be used to effectively treat anti-inflammatory diseases.

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Abstract

The present invention relates to an anti-inflammatory pharmaceutical composition, food composition and cosmetic composition comprising loratadine (ethyl 4-(8-chloro-5,6-dihydro-11H-benzo[5,6]cyclohepta[1,2-b]pyridin-11-ylidene)-1-piperidinecarboxylate) as an active ingredient. The compositions of the present invention have an excellent anti-inflammatory effect by reducing the generation of NO and PGE2 which are inflammation-related factors. Also, the compositions show an effect of suppressing the secretion of mucus or digestive juices even when treated at a low concentration, and show no cytotoxicity even when treated at a high concentration, and therefore can be safely applied to pharmaceuticals, food and cosmetics. Accordingly, it is expected that the composition may be importantly applied as an anti-inflammatory agent.

Description

로라타딘을 포함하는 항염증용 조성물 Anti-inflammatory composition containing loratadine
본 발명은 로라타딘(Loratadine, Ethyl 4-(8-chloro-5,6-dihydro-11H-benzo[5,6]cyclohepta[1,2-b]pyridin-11-ylidene)-1-piperidinecarboxylate)을 유효성분으로 함유하는 항염증용 조성물에 관한 것이다. The present invention relates to loratadine (Loratadine, Ethyl 4- (8-chloro-5,6-dihydro-11H-benzo [5,6] cyclohepta [1,2-b] pyridin-11-ylidene) -1-piperidinecarboxylate) It relates to an anti-inflammatory composition containing as an active ingredient.
염증성질환은 감염 및 비감염을 포함한 인체 내의 반응이다. 염증의 증후로는 발열, 홍조, 부종, 통증 등이 있으며, 염증과정의 후반에는 염증부위로부터 백혈구의 이주 및 사이토카인(cytokine), 분해효소(degradative enzyme), 생활성지질 중간체(bioactive lipid intermediate), 일과성 반응적인 산화물질(transient reactive oxygen species), 림프구(sensitized lmphocyte)의 생성 등 세포내의 변화가 일어난다. 더불어 만성적인 염증 질환에서는 백혈구의 침윤에 의해 세포활성(cell activation) 및 세포사멸이 일어난다. 염증반응의 화학매개물 중 프로스타글란딘(prostaglandins)과 산화 질소(nitric oxide, NO)는 발암 및 염증의 진행과정에 중요한 매개물질이다.Inflammatory diseases are reactions in the human body, including infection and non-infection. Symptoms of inflammation include fever, redness, edema, and pain. Late in the inflammatory process, the migration of white blood cells from the inflammatory site and cytokines, degradative enzymes, and bioactive lipid intermediates. Intracellular changes occur, such as transient reactive oxygen species and the production of sensitized lmphocytes. In addition, in chronic inflammatory diseases, cell activation and cell death occur due to infiltration of white blood cells. Prostaglandins and nitric oxide (NO) are important mediators for the development of carcinogenesis and inflammation.
염증이나 통증을 조절하는 약물 중 하나인 비스테로이드계 염증질환제 (nonsteroidal antiinflammatory drugs)는 체내에 프로스타글란딘(prostaglandin, PG)을 생성하는 COX를 저해함으로써 약리작용을 나타낸다. COX는 COX-1과 COX-2의 두 가지 이성체(isoform)으로 존재한다는 것이 밝혀졌는데, 이중 COX-1은 정상상태에서 위장관 점막과 혈소판, 신장에서 세포 보호 작용과 조절 작용에 관여하는 PG류 생성에 관여하는 효소인데 반해, COX-2는 정상세포에서는 그 농도가 매우 낮으나 염증관련세포에서 여러 자극(cytokine, endotoxin,mitogen)에 의해 유도 발현되어 통증이나 염증에 관여하는 PG류 생성에 관여한다. Nonsteroidal antiinflammatory drugs, one of the drugs that control inflammation and pain, have pharmacological effects by inhibiting COX, which produces prostaglandin (PG) in the body. COX has been shown to exist in two isoforms, COX-1 and COX-2, of which COX-1 produces PGs that are involved in cellular protection and regulation in the gastrointestinal mucosa, platelets, and kidneys under normal conditions. Although COX-2 is very low in normal cells, COX-2 is induced by various stimuli (cytokine, endotoxin, mitogen) in inflammation-related cells and is involved in the production of PGs that are involved in pain or inflammation.
또한 산화질소(nitric oxide, NO)는 혈관의 긴장도(vascular tone), 신경전달(neurotransmission), 세균 및 암세포의 사멸 조절에 관여한다. 또한 높은 농도의 NO는 순환기 쇼크(circulatory shock), 염증(inflammation), 발암(carcinogenesis) 등의 생리반응에서 나타난다. 신경성 NO(Neuronal NO)와 내피성 NO(endothelial NO)는 정상적인 상태에서도 존재하며, 반면 iNOS(NOS type2)는 LPS, 사이토카인(cytokine) 등에 의해 유도되어 발현된다. 염증이나 통증을 조절하는 약물 중 상기 비스테로이드계 염증질환제 는 인체 내에서 염증 반응에 관여하는 PG류 이외에 위점막 보호, 혈소판기능과 관련된 PG의 생성도 억제하여, 위장관 장애나 출혈 등의 부작용도 함께 나타나게 된다. 따라서 부작용이 없는 염증성 질환의 치료제의 개발이 요구되고 있는 실정이다.Nitric oxide (NO) is also involved in the regulation of vascular tone, neurotransmission, killing of bacteria and cancer cells. High levels of NO also occur in physiological reactions such as circulatory shock, inflammation, and carcinogenesis. Neuronal NO and endothelial NO are also present in normal conditions, whereas iNOS (NOS type 2) is induced and expressed by LPS, cytokines, and the like. Among the drugs that control inflammation and pain, the nonsteroidal inflammatory diseases also inhibit the production of PG related to gastric mucosa protection and platelet function in addition to PGs involved in inflammatory reactions in the human body, and also have side effects such as gastrointestinal disorders and bleeding. Will appear together. Therefore, the development of a therapeutic agent for inflammatory diseases without side effects is required.
한편, 로라타딘(Loratadine)은 항히스타민제(Anti-histamine)로 시판중인 약물로서, 기존에는 약물의 비염 및 두드러기의 치료 목적으로 이용되었다. 종래 기술에서, Loratadine의 약리작용은 히스타민의 발현 억제에 대해서만 한정적으로 연구되어 왔고(한국 특허공개번호 10-1992-7000033), 급성염증 등에 대한 효과에 대해서는 연구된 바 없다. Meanwhile, loratadine is a drug commercially available as an anti-histamine, and has been used for the treatment of rhinitis and urticaria of the drug. In the prior art, the pharmacological action of Loratadine has been only limitedly studied for inhibiting the expression of histamine (Korean Patent Publication No. 10-1992-7000033), and the effect on acute inflammation and the like has not been studied.
본 발명은 종래 항염증 제제들의 한계를 극복하기 위해 안출된 것으로, Loratadine 또는 그 약제학적으로 허용되는 염을 유효성분으로 포함하는 염증질환 예방 또는 치료용 약학적 조성물, 식품 조성물을 제공하는 것을 그 목적으로 한다. The present invention has been made to overcome the limitations of the conventional anti-inflammatory agents, and to provide a pharmaceutical composition, food composition for preventing or treating inflammatory diseases comprising Loratadine or a pharmaceutically acceptable salt thereof as an active ingredient. It is done.
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.
본 발명은 로라타딘(Loratadine, Ethyl 4-(8-chloro-5,6-dihydro-11H-benzo[5,6]cyclohepta[1,2-b]pyridin-11-ylidene)-1-piperidinecarboxylate) 또는 그 약제학적으로 허용되는 염을 유효성분으로 포함하는 염증질환 예방 또는 치료용 약학적 조성물을 제공한다. The present invention relates to loratadine (Loratadine, Ethyl 4- (8-chloro-5,6-dihydro-11H-benzo [5,6] cyclohepta [1,2-b] pyridin-11-ylidene) -1-piperidinecarboxylate) or It provides a pharmaceutical composition for preventing or treating inflammatory diseases comprising the pharmaceutically acceptable salt thereof as an active ingredient.
본 발명의 일구현예로서, 상기 조성물은 일산화질소(Nitric Oxide, NO) 또는 PGE2(Prostaglandin E2)의 생성을 억제하는 것을 특징으로 한다.In one embodiment of the present invention, the composition is characterized in that the production of nitric oxide (Nitric Oxide, NO) or PGE 2 (Prostaglandin E 2 ) to suppress the production.
본 발명의 다른 구현예로서, 상기 염증은 위궤양, 십이지장궤양, 간염, 식도염, 위염, 장염, 췌장염, 대장염, 복막염, 신장염, 전신부종 및 국소부종으로 이루어진 군에서 선택되는 것을 특징으로 한다.In another embodiment of the present invention, the inflammation is selected from the group consisting of gastric ulcer, duodenal ulcer, hepatitis, esophagitis, gastritis, enteritis, pancreatitis, colitis, peritonitis, nephritis, systemic edema and local edema.
또한, 본 발명은 로라타딘(Loratadine, Ethyl 4-(8-chloro-5,6-dihydro-11H-benzo[5,6]cyclohepta[1,2-b]pyridin-11-ylidene)-1-piperidinecarboxylate) 또는 그 약제학적으로 허용가능한 염을 유효성분으로 포함하는 염증질환 예방 또는 개선용 식품 조성물을 제공할 수 있다.In addition, the present invention is a loratadine (Loratadine, Ethyl 4- (8-chloro-5,6-dihydro-11H-benzo [5,6] cyclohepta [1,2-b] pyridin-11-ylidene) -1-piperidinecarboxylate Or a pharmaceutically acceptable salt thereof as an active ingredient can be provided a food composition for preventing or improving inflammatory diseases.
또한, 본 발명은 로라타딘(Loratadine, Ethyl 4-(8-chloro-5,6-dihydro-11H-benzo[5,6]cyclohepta[1,2-b]pyridin-11-ylidene)-1-piperidinecarboxylate) 또는 그 약제학적으로 허용가능한 염을 개체에 투여하는 단계를 포함하는, 염증질환의 예방 또는 치료방법을 제공할 수 있다. In addition, the present invention is a loratadine (Loratadine, Ethyl 4- (8-chloro-5,6-dihydro-11H-benzo [5,6] cyclohepta [1,2-b] pyridin-11-ylidene) -1-piperidinecarboxylate Or a pharmaceutically acceptable salt thereof, may be provided to a subject for the prevention or treatment of an inflammatory disease.
아울러, 본 발명은 로라타딘(Loratadine, Ethyl 4-(8-chloro-5,6-dihydro-11H-benzo[5,6]cyclohepta[1,2-b]pyridin-11-ylidene)-1-piperidinecarboxylate) 또는 그 약제학적으로 허용되는 염의 염증질환의 치료용도를 제공할 수 있다. In addition, the present invention is a loratadine (Loratadine, Ethyl 4- (8-chloro-5,6-dihydro-11H-benzo [5,6] cyclohepta [1,2-b] pyridin-11-ylidene) -1-piperidinecarboxylate ) Or a pharmaceutically acceptable salt thereof.
본 발명의 Loratadine을 유효성분으로 포함하는 항염증용 약학적 조성물, 식품 조성물 및 화장료 조성물은 염증관련 인자인 PGE2, NO의 생산을 감소시켜 우수한 염증 치료 효과를 가진다. 또한 고농도로 처리한 경우에도 세포독성을 보이지 않고, 소화액의 분비를 감소시키거나, 위 점막을 손상시키지 않는 것을 확인하였으므로, 본 발명의 조성물은 의약품, 식품 및 화장료에 모두 안전하게 적용할 수 있다. 따라서 항염증제로서 중요하게 응용될 수 있을 것으로 기대된다.Anti-inflammatory pharmaceutical composition, food composition and cosmetic composition comprising Loratadine of the present invention as an active ingredient has an excellent inflammation treatment effect by reducing the production of inflammation-related factors PGE 2 , NO. In addition, even when treated at a high concentration, it does not show cytotoxicity, and it is confirmed that it does not reduce the secretion of digestive juices or damage the gastric mucosa, the composition of the present invention can be safely applied to all medicines, foods and cosmetics. Therefore, it is expected that it can be importantly applied as an anti-inflammatory agent.
도 1은 로라타딘(Loratadine, C22H23ClN2O2, Ethyl 4-(8-chloro-5,6-dihydro-11H-benzo[5,6]cyclohepta[1,2-b]pyridin-11-ylidene)-1-piperidinecarboxylate, CAS number 79794-75-5)이 LPS를 처리한 RAW264.7 세포내에서 일산화질소(NO) 생성에 미치는 영향을 나타낸 도이다.1 is Loratadine, C 22 H 23 ClN 2 O 2 , Ethyl 4- (8-chloro-5,6-dihydro-11H-benzo [5,6] cyclohepta [1,2-b] pyridin-11 -ylidene) -1-piperidinecarboxylate, CAS number 79794-75-5), shows the effect of NO on the production of LPS-treated RAW264.7 cells.
도 2는 종래 염증치료제인 Ranitidine이 LPS를 처리한 RAW264.7 세포내에서 일산화질소(NO) 생성에 미치는 영향을 나타낸 도이다.2 is a diagram showing the effect of Ranitidine, an inflammatory drug, on the production of nitric oxide (NO) in RAW264.7 cells treated with LPS.
도 3은 Loratidine이 LPS를 처리한 RAW264.7 세포내에서 PGE2 생성에 미치는 영향을 나타낸 도이다.Figure 3 shows the effect of Loratidine on PGE 2 production in LPS treated RAW264.7 cells.
도 4는 Loratidine이 세포 생존률에 미치는 영향을 나타낸 도이다.4 shows the effect of Loratidine on cell viability.
도 5는 위염을 유발시킨 마우스에서 Loratidine 또는 Ranitidine을 투여한 후 위를 적출하여 미란성 점막 부식 병소를 관찰한 사진이다.Figure 5 is a photograph of the erosive mucosal erosion after harvesting the stomach after administration of Loratidine or Ranitidine in the gastritis-induced mice.
도 6은 위염을 유발시킨 마우스에서 Loratadine 또는 Ranitidine을 각각 투여한 후 위를 적출하여 미란성 점막 부식 병소 넓이(㎟)를 픽셀-계수기로 측정하여 비교한 도이다. FIG. 6 is a diagram illustrating the comparison of erosive mucosal erosion lesion area (mm 2) measured by pixel count after administration of Loratadine or Ranitidine, respectively, in mice causing gastritis.
도 7은 무수에탄올로 염증을 유발시킨 랫트에서 Loratadine 또는 Sucralfate를 각각 투여한 후 위를 절제하여 점액분비량을 측정하여 비교한 도이다.Figure 7 is a diagram comparing the amount of mucus secretion by dissecting the stomach after administration of Loratadine or Sucralfate, respectively, in rats induced inflammation with anhydrous ethanol.
도 8은 Loratadine 또는 Sucralfate를 각각 투여한 랫트를 대상으로 유문결찰한 후 위를 절제하여 소화액의 억제 효과를 측정하여 비교한 도이다.8 is a diagram comparing and comparing the inhibitory effect of the digestive fluid by resection of the stomach after pyloric ligation in rats administered Loratadine or Sucralfate, respectively.
도 9는 복막염을 유발시킨 마우스에서, Loratadine 또는 Loratadine의 vehicle을 투여한 후, 복강 내 체액에서의 산화질소 양을 측정하여 비교한 도이다. 9 is a diagram comparing the amount of nitric oxide in body fluids intraperitoneally after administration of Loratadine or Loratadine vehicle in mice induced peritonitis.
도 10은 간염을 유발시킨 마우스에서, Loratadine 또는 D-gel을 투여한 후, 혈액 내에서의 AST(aspartate aminotransferase)의 농도를 측정하여 비교한 도이다. 10 is a diagram comparing and measuring the concentration of aspartate aminotransferase (AST) in blood after administration of Loratadine or D-gel in hepatitis-induced mice.
도 11은 RAW264.7 세포에서, Loratadine 농도(0, 20, 30, 40μM)에 따른 iNOS mRNA 발현량을 측정한 도이다. 11 is a diagram measuring iNOS mRNA expression according to Loratadine concentration (0, 20, 30, 40μM) in RAW264.7 cells.
도 12는 RAW264.7 세포에서, Loratadine 농도에 따른 COX-2 mRNA 발현량을 측정한 도이다. 12 is a diagram measuring the amount of COX-2 mRNA expression according to Loratadine concentration in RAW264.7 cells.
도 13은 RAW264.7 세포에서, Loratadine 농도에 따른 IL-1β mRNA 발현량을 측정한 도이다. FIG. 13 is a diagram of IL-1β mRNA expression according to Loratadine concentration in RAW264.7 cells. FIG.
도 14는 RAW264.7 세포에서, Loratadine 농도에 따른 IL-6 mRNA 발현량을 측정한 도이다. 14 is a diagram measuring the amount of IL-6 mRNA expression according to the concentration of Loratadine in RAW264.7 cells.
도 15는 NF-κB Luciferase(0.8μg) 및 beta-galactosidase(0.1μg)를 처리한 HEK 293 세포에서, Loratadine 농도에 따른 NF-κB Luciferase 활성을 측정한 도이다. 15 is a diagram measuring NF-κB Luciferase activity according to Loratadine concentration in HEK 293 cells treated with NF-κB Luciferase (0.8 μg) and beta-galactosidase (0.1 μg).
도 16은 NF-κB Luciferase(0.3μg), TRIF(0.3μg), 및 beta-galactosidase(0.1μg)를 처리한 HEK 293 세포에서, Loratadine 농도에 따른 NF-κB Luciferase 활성을 측정한 도이다. 16 is a diagram measuring NF-κB Luciferase activity according to Loratadine concentration in HEK 293 cells treated with NF-κB Luciferase (0.3 μg), TRIF (0.3 μg), and beta-galactosidase (0.1 μg).
도 17은 NF-κB Luciferase(0.3μg), MyD88(0.3μg), 및 beta-galactosidase(0.1μg)를 처리한 HEK 293 세포에서, Loratadine 농도에 따른 NF-κB Luciferase 활성을 측정한 도이다. 17 is a diagram measuring NF-κB Luciferase activity according to Loratadine concentration in HEK 293 cells treated with NF-κB Luciferase (0.3 μg), MyD88 (0.3 μg), and beta-galactosidase (0.1 μg).
도 18은 AP-1 Luciferase(0.8μg) 및 beta-galactosidase(0.1μg)를 처리한 HEK 293 세포에서, Loratadine 농도에 따른 AP-1 Luciferase 활성을 측정한 도이다.18 is a diagram measuring AP-1 Luciferase activity according to Loratadine concentration in HEK 293 cells treated with AP-1 Luciferase (0.8 μg) and beta-galactosidase (0.1 μg).
도 19는 AP-1 Luciferase(0.3μg), TRIF(0.3μg), 및 beta-galactosidase(0.1μg)를 처리한 HEK 293 세포에서, Loratadine 농도에 따른 AP-1 Luciferase 활성을 측정한 도이다.19 is a diagram measuring AP-1 Luciferase activity according to Loratadine concentration in HEK 293 cells treated with AP-1 Luciferase (0.3 μg), TRIF (0.3 μg), and beta-galactosidase (0.1 μg).
도 20은 AP-1 Luciferase(0.3μg), MyD88(0.3μg), 및 beta-galactosidase(0.1μg)를 처리한 HEK 293 세포에서, Loratadine 농도에 따른 AP-1 Luciferase 활성을 측정한 도이다.20 is a diagram measuring AP-1 Luciferase activity according to Loratadine concentration in HEK 293 cells treated with AP-1 Luciferase (0.3 μg), MyD88 (0.3 μg), and beta-galactosidase (0.1 μg).
도 21은 RAW264.7 세포의 Nuclear 분획에서, Loratadine 처리에 의한 표적 단백질 항체 (p65, p50, c-Jun, Lamin A/C)의 발현을 Western blot을 통하여 확인한 도이다. 21 is a diagram confirming the expression of target protein antibodies (p65, p50, c-Jun, Lamin A / C) by Loratadine treatment in the Nuclear fraction of RAW264.7 cells through Western blot.
로라타딘(Loratadine, C22H23ClN2O2, Ethyl 4-(8-chloro-5,6-dihydro-11H-benzo[5,6]cyclohepta[1,2-b]pyridin-11-ylidene)-1-piperidinecarboxylate, CAS number 79794-75-5)의 구조식은 하기 화학식 1과 같다.Loratadine, C 22 H 23 ClN 2 O 2 , Ethyl 4- (8-chloro-5,6-dihydro-11H-benzo [5,6] cyclohepta [1,2-b] pyridin-11-ylidene -1-piperidinecarboxylate, CAS number 79794-75-5) is represented by the formula (1).
[화학식 1][Formula 1]
Figure PCTKR2014012422-appb-I000001
Figure PCTKR2014012422-appb-I000001
본 발명자들은 Loratadine이 염증과 관련된 다양한 인자들에 미치는 영향을 분석하여 항염효과를 확인하였다. RAW 264.7 세포에 LPS를 처리하여 염증관련 단백질들의 생산을 증가시킨 후 Loratadine의 영향을 확인하였다. LPS(lipopolysaccharide)는 그람 음성균의 세포벽 구성성분으로서 내독소로 작용하고, 대식세포를 활성화시키는 물질이다.The inventors confirmed the anti-inflammatory effect by analyzing the effects of Loratadine on various factors related to inflammation. LPS treatment of RAW 264.7 cells increased the production of inflammation-related proteins and confirmed the effects of Loratadine. Lipopolysaccharide (LPS) is a cell wall component of Gram-negative bacteria that acts as an endotoxin and activates macrophages.
즉, 본 발명의 목적은 로라타딘(Loratadine, Ethyl 4-(8-chloro-5,6-dihydro-11H-benzo[5,6]cyclohepta[1,2-b]pyridin-11-ylidene)-1-piperidinecarboxylate) 또는 그 약제학적으로 허용되는 염을 유효성분으로 포함하는 염증질환 예방 또는 치료용 약학적 조성물을 제공하는 것에 있다. That is, an object of the present invention is Loratadine, Ethyl 4- (8-chloro-5,6-dihydro-11H-benzo [5,6] cyclohepta [1,2-b] pyridin-11-ylidene) -1. -piperidinecarboxylate) or a pharmaceutically acceptable salt thereof as an active ingredient to provide a pharmaceutical composition for preventing or treating inflammatory diseases.
본 발명의 일구현예로, 상기 로라타딘은 일산화질소(Nitric Oxide, NO) 또는 PGE2(Prostaglandin E2)의 생성을 억제하는 것을 특징으로 한다.In one embodiment of the present invention, the loratadine is characterized in that it inhibits the production of nitric oxide (Nitric Oxide, NO) or PGE 2 (Prostaglandin E 2 ).
본 발명의 일구현예로, 상기 로라타딘은 NF-κB 또는 AP-1의 활성을 억제하는 것을 특징으로 한다. In one embodiment of the present invention, the loratadine is characterized by inhibiting the activity of NF-κB or AP-1.
상기 염증의 종류는 위궤양, 십이지장궤양, 간염, 식도염, 위염, 장염, 췌장염, 대장염, 복막염, 신장염, 위암, 전신부종 또는 국소부종일 수 있으나, 상기 염증의 종류는 이에 제한되지 않는다.The type of inflammation may be gastric ulcer, duodenal ulcer, hepatitis, esophagitis, gastritis, enteritis, pancreatitis, colitis, peritonitis, nephritis, gastric cancer, systemic edema or local edema, but the type of inflammation is not limited thereto.
본 발명의 조성물은, 투여를 위해서 상기 기재한 유효성분 이외에 추가로 약학적으로 허용가능한 담체를 1종 이상 포함하여 제조할 수 있다. The composition of the present invention may be prepared by including one or more pharmaceutically acceptable carriers in addition to the above-described active ingredients for administration.
본 발명의 약학적 조성물은 목적하는 방법에 따라 경구 투여하거나 비경구 투여(예를 들어, 정맥 내, 피하, 복강 내 또는 국소에 적용)할 수 있으며, 투여량은 환자의 체중, 연령, 성별, 건강상태, 식이, 투여시간, 투여방법, 배설율 및 질환의 중증도 등에 따라 그 범위가 다양하다. 일 예로 1일 유효성분을 기준으로 하였을 때 0.1 mg/kg(체중)내지 500 mg/kg(체중), 0.1 mg/kg(체중) 내지 400 mg/kg(체중) 또는 1 mg/kg(체중)내지 300 mg/kg(체중)으로 투여할 수 있으며, 1회 또는 수회로 나누어 투여할 수 있다. 상기 투여량은 어떠한 면으로든 본 발명의 범위를 한정하는 것은 아니다.The pharmaceutical compositions of the present invention may be administered orally or parenterally (eg, applied intravenously, subcutaneously, intraperitoneally or topically) according to the desired method, and the dosage is based on the weight, age, sex, The range varies depending on the state of health, diet, time of administration, method of administration, rate of excretion and the severity of the disease. For example, from 0.1 mg / kg (body weight) to 500 mg / kg (body weight), 0.1 mg / kg (body weight) to 400 mg / kg body weight or 1 mg / kg body weight based on a daily active ingredient To 300 mg / kg body weight, and may be administered once or divided into several. The dosage does not limit the scope of the invention in any aspect.
또한, 본 발명은 로라타딘(Loratadine, Ethyl 4-(8-chloro-5,6-dihydro-11H-benzo[5,6]cyclohepta[1,2-b]pyridin-11-ylidene)-1-piperidinecarboxylate) 또는 그 약제학적으로 허용가능한 염을 유효성분으로 포함하는 염증질환 예방 또는 개선용 식품 조성물을 제공할 수 있다.In addition, the present invention is a loratadine (Loratadine, Ethyl 4- (8-chloro-5,6-dihydro-11H-benzo [5,6] cyclohepta [1,2-b] pyridin-11-ylidene) -1-piperidinecarboxylate Or a pharmaceutically acceptable salt thereof as an active ingredient can be provided a food composition for preventing or improving inflammatory diseases.
아울러, 로라타딘(Loratadine, Ethyl 4-(8-chloro-5,6-dihydro-11H-benzo[5,6]cyclohepta[1,2-b]pyridin-11-ylidene)-1-piperidinecarboxylate) 또는 그 약제학적으로 허용가능한 염을 유효성분으로 포함하는 염증질환 예방 또는 개선용 화장료 조성물을 제공할 수 있다.In addition, Loratadine, Ethyl 4- (8-chloro-5,6-dihydro-11H-benzo [5,6] cyclohepta [1,2-b] pyridin-11-ylidene) -1-piperidinecarboxylate) or its It can provide a cosmetic composition for preventing or improving an inflammatory disease comprising a pharmaceutically acceptable salt as an active ingredient.
즉, 본 발명의 조성물은 염증의 예방 및 개선을 목적으로 건강기능식품에 첨가될 수 있다. 혼합양은 사용 목적(예방, 건강 또는 치료적 처치)에 따라 적합하게 결정될 수 있다. 일반적으로, 식품 또는 음료의 제조시 원료에 대하여 15 중량% 이하, 바람직하게는 10 중량% 이하의 양으로 첨가될 수 있다. 그러나, 건강 및 위생을 목적으로 하거나 또는 건강 조절을 목적으로 하는 장기간의 섭취의 경우 상기 양은 상기 범위 이하일 수 있으며, 안전성 면에서 아무런 문제가 없기 때문에 유효성분은 상기 범위 이상의 양으로도 사용될 수 있다. 식품의 종류에는 특별한 제한은 없다. In other words, the composition of the present invention may be added to a dietary supplement for the purpose of preventing and improving inflammation. Mixing amounts may be appropriately determined depending on the purpose of use (prevention, health or therapeutic treatment). In general, it can be added in an amount of up to 15% by weight, preferably up to 10% by weight based on the raw materials in the manufacture of food or beverage. However, in the case of long-term intake for health and hygiene or for health control, the amount may be below the above range, and the active ingredient may be used in an amount above the above range because there is no problem in terms of safety. There are no particular restrictions on the type of food.
또한, 본 발명의 조성물은 염증의 예방 및 개선을 목적으로 화장료에 첨가될 수 있다. 상기 화장료 조성물은 기초 화장료, 메이크업 화장료, 바디 화장료, 두발용 화장료, 두피용 화장료, 면도용 화장료 또는 구강용 화장료의 용도로 제공될 수 있다. 상기 유효성분은 상기 화장료 조성물 총 중량을 기준으로 0.001 내지 50 중량%로 포함될 수 있으며, 바람직하기로는 0.01 내지 20 중량%일 수 있으나, 상기 함량은 제형 또는 화장료 조성물에 함유되는 유효성분 외의 성분의 함량에 따라 적절히 조절할 수 있으며, 상기 함량에 의해 본 발명에 포함되는 유효성분의 함량이 제한되는 것은 아니다.In addition, the composition of the present invention may be added to the cosmetic for the purpose of preventing and improving inflammation. The cosmetic composition may be provided for use in basic cosmetics, makeup cosmetics, body cosmetics, hair cosmetics, scalp cosmetics, shaving cosmetics or oral cosmetics. The active ingredient may be included in an amount of 0.001 to 50% by weight based on the total weight of the cosmetic composition, preferably 0.01 to 20% by weight, but the content is the content of ingredients other than the active ingredient contained in the formulation or cosmetic composition. According to the present invention, the amount of the active ingredient included in the present invention is not limited.
따라서, 본 발명은 로라타딘(Loratadine, Ethyl 4-(8-chloro-5,6-dihydro-11H-benzo[5,6]cyclohepta[1,2-b]pyridin-11-ylidene)-1-piperidinecarboxylate) 또는 그 약제학적으로 허용되는 염을 유효성분을 투여하여, 염증질환을 예방 또는 치료 방법을 제공할 수 있다. Therefore, the present invention is a loratadine (Loratadine, Ethyl 4- (8-chloro-5,6-dihydro-11H-benzo [5,6] cyclohepta [1,2-b] pyridin-11-ylidene) -1-piperidinecarboxylate ) Or a pharmaceutically acceptable salt thereof can be administered to provide a method for preventing or treating inflammatory diseases.
또한, 본 발명은 로라타딘의 새로운 용도로, 염증질환 치료 용도를 제공할 수 있다.In addition, the present invention can provide a new use of loratadine, for the treatment of inflammatory diseases.
실시예 1은 Loratadine이 NO 생산에 미치는 영향을 확인한 것이다. 그 결과, 종래 염증치료제인 Ranitidine에 비해 뛰어난 NO 생성 억제 효과가 있다는 것을 확인하였다.Example 1 confirms the effect of Loratadine on NO production. As a result, it was confirmed that there is an excellent NO production inhibitory effect compared to Ranitidine which is a conventional inflammatory treatment.
NO는 혈관을 확장시키는 역할을 하는 대표적인 염증에 관련된 인자 중 하나이다. 대식세포가 자극을 받으면 iNOS(inducible nitric oxide synthase)라는 효소에 의해 L-알기닌이 L-시트룰린으로 변하는 과정에서 일산화질소(NO)가 생성됨으로써 대식세포로부터 NO가 생성된다. NO is one of the factors involved in inflammation that plays a role in dilating blood vessels. When macrophages are stimulated, NO is produced from macrophages by the production of nitric oxide (NO) in the process of converting L-arginine into L-citrulline by an enzyme called inducible nitric oxide synthase (iNOS).
실시예 2는 Loratadine이 PGE2에 미치는 영향을 확인한 것이다. 그 결과, Loratadine을 첨가한 경우 저농도에서도 PGE2의 농도를 급격하게 감소시킬 수 있다는 것을 확인하였다.Example 2 confirms the effect of Loratadine on PGE 2 . As a result, it was confirmed that the addition of Loratadine can rapidly decrease the concentration of PGE 2 even at low concentrations.
프로스타글란딘 E2(PGE2)는 포스포리파제 A2(phospholipase A2)의해 유리된 아라키돈산이 COXs(cyclooxygenaes)라고 불리우는 효소의 촉매작용을 받아 형성되는 물질이다. PGE2는 통증, 발열 등의 염증반응, 면역반응, 그리고 혈관생성(angiogenesis)을 촉진하는 등 암 발생에도 깊이 관여하고 있는 것으로 알려져 있다.Prostaglandin E 2 (PGE 2 ) is a substance in which arachidonic acid liberated by phospholipase A2 is catalyzed by an enzyme called cyclooxygenaes (COXs). PGE 2 is known to be deeply involved in the development of cancer, including inflammatory reactions such as pain, fever, immune responses, and angiogenesis.
실시예 3은 Loratadine이 세포생존률에 미치는 영향을 확인한 것으로, Loratadine의 처리가 세포 생존률에 미치는 영향이 없으므로, 염증치료시 안전한 조성물로 이용될 수 있음을 확인하였다.Example 3 confirms the effect of Loratadine on cell viability, and since Loratadine treatment has no effect on cell viability, it was confirmed that it can be used as a safe composition for inflammation treatment.
실시예 4는 에탄올/염산을 이용하여 염증을 유발시킨 동물 모델에서 Loratadine의 염증치료 효과를 확인한 것으로서, Loratadine이 위 손상을 현저히 감소시킨다는 것을 확인하였다.Example 4 confirms the inflammatory treatment effect of Loratadine in an animal model that causes inflammation using ethanol / hydrochloric acid, and confirms that Loratadine significantly reduces gastric damage.
실시예 5는 Loratadine이 점액 분비에 미치는 영향을 확인한 것이다. 일반적으로, 염증치료제를 투여할 경우, 위 점막을 보호하는 위의 점액의 발생량을 줄이는 부작용을 보이는데, 실시예 5의 결과에 따르면 종래 위염치료제인 Sucralfate에 비교하여, 본 발명의 Loratidine을 사용할 경우 점액의 분비가 줄어들지 않는 효과를 보인다.Example 5 confirmed the effect of Loratadine on mucus secretion. In general, when the inflammatory treatment is administered, the side effect of reducing the amount of gastric mucus that protects the gastric mucosa shows a side effect. According to the results of Example 5, compared to the conventional gastritis, Sucralfate, when using Loratidine of the present invention mucus The secretion of the effect is not reduced.
실시예 6은 Loratadine이 소화액 분비에 미치는 영향을 확인한 것이다. 실시예 6의 결과에 따르면 종래 위산 분비 억제 작용을 가진 항히스타민제인 Cimetidine과 비교하여, 본 발명의 Loratidine을 사용할 경우 보다 우수한 위산 분비 억제 작용을 가진다.Example 6 confirmed the effect of Loratadine on digestive juice secretion. According to the results of Example 6, compared with conventional antihistamine Cimetidine, which has an inhibitory effect on gastric acid secretion, when Loratidine of the present invention is used, it has a superior gastric acid secretion inhibitory effect.
실시예 7 내지 8은 복막염 또는 간염 동물모델에서, Loratadine의 치료효과를 확인한 것으로서, Loratadine 처리에 의한 복강 내 채액에서의 산화질소 양의 감소및 혈중 AST(aspartate aminotransferase) 농도의 감소를 확인하였다. Examples 7 to 8 confirmed the therapeutic effect of Loratadine in peritonitis or hepatitis animal model, and confirmed the reduction of the amount of nitric oxide in the intraperitoneal liquor and the decrease of the blood aspartate aminotransferase (AST) concentration by Loratadine treatment.
실시예 9 내지 11은 Loratadine의 항염증의 기전을 확인한 것으로서, Loratadine 처리에 의하여, 염증 관련 인자인 iNOS, COX-2, IL-1β, 및 IL-6의 감소 및 염증 관련 전사인자인 NF-κB 또는 AP-1의 활성 억제를 확인하였다.Examples 9 to 11 confirm the mechanism of the anti-inflammatory mechanism of Loratadine, by reducing the inflammation-related factors iNOS, COX-2, IL-1β, and IL-6 by Loratadine treatment and NF-κB which is an inflammation-related transcription factor Or inhibition of activity of AP-1 was confirmed.
이하, 본 발명의 이해를 돕기 위하여 바람직한 실시예를 제시한다. 그러나 하기의 실시예는 본 발명을 보다 쉽게 이해하기 위하여 제공되는 것일 뿐, 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 examples.
실시예 1 : RAW264.7 세포내에서 NO의 생성 측정Example 1 Measurement of NO Production in RAW264.7 Cells
1-1. 세포배양1-1. Cell culture
쥐의 대식세포 세포주(RAW264.7 세포)를 100 U/㎖ 페니실린, 100㎍/㎖ 스트렙토마이신과 10% FBS로 보충된 RPMI1640 배지 내에서 유지시켰다. 세포는 37℃, 5% CO2 습한 공기에서 배양하였다.Rat macrophage cell lines (RAW264.7 cells) were maintained in RPMI1640 medium supplemented with 100 U / ml penicillin, 100 μg / ml streptomycin and 10% FBS. Cells were incubated in 37 ° C., 5% CO 2 humid air.
1-2. RAW264.7 세포 내에서 Loratadine의 일산화질소(NO)의 생성 억제 효과 1-2. Inhibitory Effect of Loratadine on Nitric Oxide Production in RAW264.7 Cells
RAW264.7 세포(1×105 cells/well) 를 24시간 동안 배양시킨 후, 배양된 세포를 Loratadine(0, 20, 30, 40μM)으로 60분 동안 처리한 다음, LPS(1㎍/㎖)와 함께 24시간 동안 더 배양하였다. 상등액을 모으고, 상등액 내 일산화질소(NO)의 농도를 Griess 시약으로 측정하였다. 대조군으로는 Loratadine의 vehicle인 DMSO를 주입하였다.After incubating the RAW264.7 cells (1 × 10 5 cells / well) for 24 hours, the cultured cells were treated with Loratadine (0, 20, 30, 40 μM) for 60 minutes, and then LPS (1 μg / ml). And further incubated for 24 hours. The supernatant was collected and the concentration of nitric oxide (NO) in the supernatant was measured with Griess reagent. DMSO, a vehicle of Loratadine, was injected as a control.
결과는 도 1에 나타내었다. NO 생성 억제 효과는 아무런 시료를 첨가하지 않은 대조군(control)에 대한 백분율로 표시하였다.The results are shown in FIG. The NO production inhibitory effect was expressed as a percentage of the control without adding any sample.
도 1에 나타난 바와 같이, Loratadine은 LPS 처리에 의해 증가된 NO 생성을 억제하였고, 20 uM 처리한 경우 아무 처리도 하지 않은 대조군과 비교하여 약 50% 정도의 NO 생성량이 감소되었으며, 농도 의존적으로 계속하여 NO 함량이 감소하여, 40 uM를 처리한 경우 90% 이상 감소한 것을 확인 하였다. As shown in FIG. 1, Loratadine inhibited the increased NO production by LPS treatment, and the 20 μM treatment reduced NO production by about 50% compared to the control without any treatment, and continued concentration-dependently. By reducing the NO content, it was confirmed that more than 90% when treated with 40 uM.
1-3. 비교실험 : RAW264.7 세포 내에서 Ranitidine의 일산화질소(NO)의 생성 억제 효과 1-3. Comparative Experiment: Ranitidine Inhibitory Effects of Ranitidine Production in RAW264.7 Cells
Loratadine과의 효과를 비교하기 위해서, Ranitidine을 사용하여 일산화질소의 생성 억제 효과를 확인해 보았다. RAW264.7 세포(1×105 cells/well) 를 24시간 동안 배양시킨 후, 배양된 세포를 Ranitidine(0, 10, 20, 40, 80, 160μM)으로 60분 동안 처리한 다음, LPS(1㎍/㎖)와 함께 24시간 동안 더 배양하였다. 상등액을 모으고, 상등액 내 일산화질소(NO)의 농도를 Griess 시약으로 측정하였다. 대조군으로는 Loratadine의 vehicle인 DMSO를 주입하였다.In order to compare the effects with loratadine, Ranitidine was used to confirm the inhibitory effects of nitric oxide production. After incubating RAW264.7 cells (1 × 10 5 cells / well) for 24 hours, the cultured cells were treated with Ranitidine (0, 10, 20, 40, 80, 160 μM) for 60 minutes and then LPS (1 Incubated for 24 h more). The supernatant was collected and the concentration of nitric oxide (NO) in the supernatant was measured with Griess reagent. DMSO, a vehicle of Loratadine, was injected as a control.
결과는 도 2에 나타내었다. NO 생성 억제 효과는 아무런 시료를 첨가하지 않은 대조군(control)에 대한 백분율로 표시하였다.The results are shown in FIG. The NO production inhibitory effect was expressed as a percentage of the control without adding any sample.
도 2에 나타난 바와 같이, Ranitidine은 NO 생성을 전혀 억제하지 못한다는 것을 확인하였다.As shown in Figure 2, Ranitidine was confirmed that does not inhibit the NO production at all.
실시예 2. RAW264.7 세포내에서 프로스타글란딘 EExample 2. Prostaglandin E in RAW264.7 Cells 22 (PGE(PGE 22 )의 생성 억제 효과) Inhibitory effect
RAW264.7 세포(1×105 cells/well) 를 24시간 동안 전배양시킨 후, 전배양된 세포를 Loratadine 40μM 및 Ranitidine 160μM으로 60분 동안 처리하였다. 상등액을 모으고, 상등액 내 PGE2의 농도를 EIA 키트로 측정하였다. 대조군으로는 Loratadine의 vehicle인 DMSO를 주입하였다.After pre-culture of RAW264.7 cells (1 × 10 5 cells / well) for 24 hours, the pre-cultured cells were treated with Loratadine 40 μM and Ranitidine 160 μM for 60 minutes. The supernatant was collected and the concentration of PGE 2 in the supernatant was measured with an EIA kit. DMSO, a vehicle of Loratadine, was injected as a control.
결과는 도 3에 나타내었다. PGE2 생성 억제 효과는 LPS만을 첨가한 대조군(control)에 대한 백분율로 표시하였다.The results are shown in FIG. PGE 2 production inhibitory effect was expressed as a percentage of the control (control) added only LPS.
도 3에 나타난 바와 같이, Loratadine을 첨가한 경우 저농도에서도 PGE2의 농도를 급격하게 감소시킬 수 있다는 것을 확인할 수 있다.As shown in Figure 3, it can be seen that the addition of Loratadine can sharply reduce the concentration of PGE 2 even at low concentrations.
실시예 3. RAW264.7 세포에서의 세포 독성 측정Example 3. Cytotoxicity Measurements in RAW264.7 Cells
RAW264.7 세포(1×105 cells/well) 에 상기 Loratadine(5, 10, 20, 30, 40μM)를 가하여, 24시간 동안 배양하였다. 대조군으로는 Loratadine의 vehicle인 DMSO를 주입하였다. 그 다음 세포독성 효과를 일반적인 MTT 어세이로 측정하였다. 즉, 배양 종료 3시간 전에 10㎖의 MTT 용액(인산염 완충액 중 10㎎/㎖, pH 7.4)을 가하고 분석 종료까지 세포를 계속 배양하였다. 15% SDS(sodium dodecyl sulphate)를 각 웰에 가하고 포마잔을 녹여 배양을 중지시킨 후, Spectramax 250 마이크로플레이트 리더를 사용하여 570~630㎚(OD570-630)에서 흡광도를 측정하였다.Loratadine (5, 10, 20, 30, 40 μM) was added to RAW264.7 cells (1 × 10 5 cells / well) and incubated for 24 hours. DMSO, a vehicle of Loratadine, was injected as a control. The cytotoxic effect was then measured by the general MTT assay. That is, 10 ml of MTT solution (10 mg / ml in phosphate buffer, pH 7.4) was added 3 hours before the end of the culture, and the cells were continuously cultured until the end of the assay. After 15% SDS (sodium dodecyl sulphate) was added to each well and the formazan was dissolved to stop the culture, the absorbance was measured at 570-630 nm (OD570-630) using a Spectramax 250 microplate reader.
결과는 도 4에 나타내었다. 세포의 생존율은 아무런 시료를 첨가하지 않은 대조군(control)에 대한 백분율로 표시하였다.The results are shown in FIG. Cell viability was expressed as a percentage of the control with no sample added.
도 4에 나타난 바와 같이 Loratadine을 처리한 경우에도 LPS를 처리한 RAW264.7 세포 생존률의 감소가 거의 나타나지 않아 세포독성이 없음을 확인하였다.As shown in Figure 4, even when treated with Loratadine it was confirmed that there is almost no decrease in the survival rate of RAW264.7 cells treated with LPS, there is no cytotoxicity.
실시예 4 : 생체 내 에탄올/염산-유도 염증 모델에서 Loratadine의 항염증 활성 확인Example 4 Confirmation of the Anti-inflammatory Activity of Loratadine in an In Vivo Ethanol / HCl-Induced Inflammation Model
4-1. 위염 유발 및 Loratadine 및 Ranitidine 투여4-1. Gastritis Induction and Administration of Loratadine and Ranitidine
공지된 방법에 따라 에탄올/염산으로 ICR 마우스의 위의 염증을 유발하였다 [Lee et al., 2010; Okabe et al.]. 금식시킨 ICR 마우스 (7마리)에게 Loratadine(10 및 4 ㎎/㎏) 또는 Ranitidine (40 ㎎/㎏)을 3일 동안 1일 2회 경구 투여하였다. 대조군으로는 Loratadine의 vehicle인 0.5% CMC를 주입하였다. Loratadine의 마지막 투여 30분 후, 150mM의 염산에 60% 에탄올 400㎕ 넣어 마우스에게 경구 투여하였다. 각각의 실험 동물을 마취시키고 괴사제 투여 1시간 후에 우레탄을 치사량 투여하여 희생시켰다. Ethanol / hydrochloric acid induced inflammation of the stomach of ICR mice according to a known method [Lee et al., 2010; Okabe et al.]. Fasted ICR mice (7 mice) received oral administration of Loratadine (10 and 4 mg / kg) or Ranitidine (40 mg / kg) twice daily for 3 days. 0.5% CMC, Loratadine's vehicle, was injected as a control. After 30 minutes of the last administration of loratadine, 400 μl of 60% ethanol was added to 150 mM hydrochloric acid, and the mice were orally administered. Each experimental animal was anesthetized and sacrificed by lethal doses of urethane 1 hour after necrosis.
4-2. 병소 부위 관찰4-2. Lesion site observation
위를 절개한 후 조심스럽게 흐르는 수돗물로 헹구어내었다. 대만부를 따라 위를 열고 보드 위에 펼친 후, 미란성 점막 부식 병소의 넓이(㎟)를 픽셀-계수기를 이용하여 측정하였다. 또한, 점막 부식 병소의 사진을 찍어 육안으로 관찰하였다.The stomach was incised and then rinsed carefully with running tap water. After opening the stomach along the Taiwan and spreading on the board, the area (mm 2) of the erosive mucosal corrosion lesion was measured using a pixel-counter. In addition, photographs of mucosal erosion lesions were taken and visually observed.
결과는 도 5 및 도 6에 나타내었다. 도 5 및 도 6에 나타난 바와 같이, Loratadine은 위염이 유발된 마우스에서 에탄올/염산에 의한 위 손상을 현저히 감소시켰고, Ranitidine과 비교하여도 우수한 위 손상 감소 효과가 있음을 확인하였다.The results are shown in FIGS. 5 and 6. As shown in Figure 5 and 6, Loratadine significantly reduced the gastric damage caused by ethanol / hydrochloric acid in the gastritis-induced mice, it was confirmed that there is an excellent gastric damage reduction effect compared to Ranitidine.
실시예 5 : 생체 내 무수에탄올-유도 염증 모델에서 Loratadine의 점막 보호 효과 확인Example 5 Confirmation of the Mucosal Protective Effect of Loratadine in In Vivo Anhydrous Ethanol-Induced Inflammation Model
5-1. 위염 유발 및 Loratadine 및 Sucralfate 투여5-1. Gastritis Induction and Administration of Loratadine and Sucralfate
실험에 앞서 24시간동안 금식시킨 랫트(자유 급수)를 대상으로 Loratadine(50 mg/kg) 및 Sucrafate(375 mg/kg)을 경구 투여하였다. 30분 후에, 무수 에탄올로 위 병변을 유발시켰다. 대조군으로는 Loratadine의 vehicle인 0.5% CMC를 주입하였다. 한시간 후 랫트를 희생시키고, 위의 점액분비량을 측정하여 도 7에 나타내었다.Before rats, Loratadine (50 mg / kg) and Sucrafate (375 mg / kg) were orally administered to rats fasted for 24 hours. After 30 minutes, gastric lesions were induced with anhydrous ethanol. 0.5% CMC, Loratadine's vehicle, was injected as a control. After one hour, the rats were sacrificed, and the amount of mucus secretion was measured and shown in FIG. 7.
도 7에 나타난 바와 같이, Loratadine을 저용량으로 투여하더라도, 종래 점막 보호 약물로 알려진 Sucralfate에 비해 점액 분비량이 줄어들지 않는다는 것을 확인하였다.As shown in Figure 7, it was confirmed that even when administered at a low dose of Loratadine, mucus secretion is not reduced compared to Sucralfate known as a conventional mucosal protective drug.
실시예 6 : 생체 내 무수에탄올-유도 염증 모델에서 Loratadine의 위액 분비 확인Example 6 Confirmation of Gastric Secretion of Loratadine in In Vivo Anhydrous Ethanol-Induced Inflammation Model
위염 유발 및 Loratadine 및 Cimetidine 투여Gastritis Induction and Administration of Loratadine and Cimetidine
실험에 앞서 24시간동안 금식시킨 랫트를 대상으로 Loratadine(10 mg/kg) 및 Cimetidine(250 mg/kg)을 십이지장 내에 투여하였다. 대조군으로는 Loratadine의 vehicle인 0.5% CMC를 주입하였다. 유문결찰(pyloric ligation) 시키고 4시간 후, 랫트를 희생시키고, 위를 절제하여 10분 동안 1050 xg로 원심분리하였다. 상기 원심분리된 용액에서 소화액의 총 량, pH 및 위산량(mEq/mL)은 methyl orange를 지시약으로 사용하여 0.1M NaOH에서 소화액을 적정하여 측정한 결과를 도 8에 나타내었다. Rats fasted for 24 hours prior to the experiment were administered with Loratadine (10 mg / kg) and Cimetidine (250 mg / kg) in the duodenum. 0.5% CMC, Loratadine's vehicle, was injected as a control. Four hours after pyloric ligation, the rats were sacrificed, and the stomach was excised and centrifuged at 1050 xg for 10 minutes. The total amount, pH and gastric acid (mEq / mL) of the digestion solution in the centrifuged solution was measured by titrating the digestion solution at 0.1 M NaOH using methyl orange as an indicator.
도 8에 나타난 바와 같이, Loratadine을 저용량으로 투여하여도 보다 뛰어난 위산분비 억제 효과를 가지므로, 본 발명의 Loratadine이 위염 치료 또는 예방에 보다 우수한 효과를 보인다는 것을 확인할 수 있다.As shown in Figure 8, even when administered at a low dose of Loratadine has a superior gastric acid secretion inhibitory effect, it can be seen that Loratadine of the present invention shows a better effect in the treatment or prevention of gastritis.
실시예 7 : 복막염 동물모델에서 Loratadine의 복막염 치료효과 확인Example 7 Confirmation of Treatment Effect of Loratadine on Peritonitis in Animal Models of Peritonitis
7-1. 복막염 유발 및 Loratadine 투여7-1. Peritonitis and Loratadine Administration
우선, C57BL/6 mouse를 세 군 (Normal, LPS, LPS+Loratadine)으로 분류한 뒤, 1mL의 3% 티오클리콜레이트(Thioglycolate)를 복강 내로 주사하였다. 이 후, 두 군(Normal, LPS)은 Loratadine의 vehicle인 0.5% CMC을, 나머지 한 군(LPS+Loratadine)은 Loratadine을 5일간 경구 투여하였다. vehicle을 처리한 군 중 한 군(Normal)은 복막염을 유발시키지 않았으며, 나머지 두 군(LPS, LPS+Loratadine)은 LPS(lipopolysaccharide)를 복강 내 주사하여 복막염을 유발시켰다. First, C57BL / 6 mice were divided into three groups (Normal, LPS, LPS + Loratadine), and 1 mL of 3% Thioglycolate was injected intraperitoneally. Afterwards, two groups (Normal and LPS) received oral administration of Loratadine 0.5% CMC, and the other group (LPS + Loratadine) orally administered Loratadine for 5 days. One of the vehicle-treated groups (Normal) did not cause peritonitis, and the other two groups (LPS, LPS + Loratadine) induced peritonitis by intraperitoneal injection of LPS (lipopolysaccharide).
7-2. 복강 내 체액에서의 산화질소 양 측정 7-2. Determination of Nitric Oxide in Abdominal Fluids
세 군의 마우스(Normal, LPS, LPS+Loratadine)를 희생시켜 복강 내 채액을 채취하였으며, 복막염의 치료 또는 예방효과를 확인하기 위하여, 복막염에 의해 발생한 산화질소 양을 측정하였다. 보다 구체적으로 Griess 용액 (0.5% naphthylethylenediamine dihydrochloride, 5% sulfanilamide, 25% H3PO4)을 이용하여 540 nm에서 흡광도를 측정하였으며, 표준 물질로 sodium nitrite (0 에서 100 μM) 를 사용하여 검량선을 작성하였다. Three groups of mice (Normal, LPS, LPS + Loratadine) were sacrificed to collect the intraperitoneal sap, and the amount of nitric oxide generated by peritonitis was measured in order to confirm the treatment or preventive effect of peritonitis. More specifically, absorbance was measured at 540 nm using Griess solution (0.5% naphthylethylenediamine dihydrochloride, 5% sulfanilamide, 25% H 3 PO 4 ), and a calibration curve was prepared using sodium nitrite (0 to 100 μM) as a standard material. It was.
도 9에 나타낸 바와 같이, 복막염을 유발시킨 두 군(LPS, LPS+Loratadine)의 복강 내 체액에서 산화질소의 양이 현저하게 증가하였으며, Loratadine을 처리한 군은 대조군(LPS)과 비교하여 복막염에 의해 유발된 산화질소 양이 감소함를 확인하였다. 상기 결과는 본 발명의 Loratadine이 복막염의 치료 또는 예방에 우수한 효과를 보인다는 것을 의미한다.  As shown in FIG. 9, the amount of nitric oxide was significantly increased in the intraperitoneal body fluids of two groups (LPS, LPS + Loratadine) that caused peritonitis, and the group treated with Loratadine was more effective in peritonitis compared to the control group (LPS). It was confirmed that the amount of nitric oxide caused by the decrease. The results indicate that Loratadine of the present invention shows an excellent effect on the treatment or prevention of peritonitis.
실시예 8 : 간염 동물모델에서 Loratadine의 간염 치료효과 확인Example 8 Confirmation of the Hepatitis Treatment Effect of Loratadine in Hepatitis Animal Model
8-1. 간염 유발 및 Loratadine 투여8-1. Hepatitis Induction and Loratadine Administration
우선, ICR mouse를 세 군 (Normal, LPS+D-gel, LPS+D-gel+Loratatine)으로 분류한 뒤, 두 군(Normal, LPS+D-gel)은 Loratadine의 vehicle인 0.5% CMC를, 나머지 한 군(LPS+D-gel+Loratatine)은 Loratadine을 5일간 경구 투여하였다. vehicle을 처리한 군 중 한 군(Normal)은 간염을 유발시키지 않았으며, 두 군(LPS+D-gel, LPS+D-gel+Loratatine)은 LPS(lipopolysaccharide)와 D-gel을 복강 내 주사하여 급성 간염을 유발시켰다. First, ICR mice were classified into three groups (Normal, LPS + D-gel, LPS + D-gel + Loratatine), and then two groups (Normal, LPS + D-gel) were used as Loratadine's vehicle 0.5% CMC, The other group (LPS + D-gel + Loratatine) received oral administration of Loratadine for 5 days. One of the vehicle treated groups (Normal) did not cause hepatitis, and two groups (LPS + D-gel, LPS + D-gel + Loratatine) were injected intraperitoneally with LPS (lipopolysaccharide) and D-gel. Caused acute hepatitis.
8-2. 혈액 내에서의 AST의 농도 측정 8-2. Determination of AST Concentration in Blood
세 군의 마우스(Normal, LPS+D-gel, LPS+D-gel+Loratatine)를 희생시켜 혈액을 채취한 후, 혈청만을 분리하였다. 간염의 치료 또는 예방효과를 확인하기 위하여, 간염의 지표인 AST(aspartate aminotransferase)의 농도를 Roche Modular spectrophotometric autoanalyzer를 이용하여 측정하였다.Blood was collected at the expense of three groups of mice (Normal, LPS + D-gel, LPS + D-gel + Loratatine), and only serum was isolated. In order to confirm the therapeutic or prophylactic effect of hepatitis, the concentration of aspartate aminotransferase (AST), which is an indicator of hepatitis, was measured using a Roche Modular spectrophotometric autoanalyzer.
도 10에 나타낸 바와 같이, 간염을 유발시킨 두 군(LPS+D-gel, LPS+D-gel+Loratatine)의 혈액에서 AST의 농도가 현저하게 증가하였으며, Loratadine을 처리한 군은 대조군(LPS+D-gel)과 비교하여 혈중 AST의 농도가 절반 이하로 크게 감소함을 확인하였다. 상기 결과는 본 발명의 Loratadine이 간염의 치료 또는 예방에 우수한 효과를 보인다는 것을 의미한다.  As shown in Figure 10, the concentration of AST in the blood of the two groups (LPS + D-gel, LPS + D-gel + Loratatine) induced hepatitis significantly increased, the group treated with Loratadine was the control (LPS + Compared with D-gel), the concentration of blood AST was significantly reduced to less than half. The results indicate that Loratadine of the present invention shows an excellent effect on the treatment or prevention of hepatitis.
실시예 9 : Loratadine의 전사 수준에서의 염증반응 억제효과Example 9 Inhibitory Effect of Loratadine on Transcription Level
대식세포로부터 cytokine 분비능 측정Measurement of cytokine secretion from macrophages
Murine 대식세포주인 RAW264.7 세포를 penicillin (100 IU/ml) 및 streptomycin (100 μg/ml)과 10%의 FBS를 함유하는 RPMI 1640 배지를 이용하여 100 mm cell culture dish에 70-80%의 밀도로 배양하였다. 이 후, 배양된 RAW264.7 세포(1×105 cells/well)를 Loratadine(0, 20, 30, 40μM)으로 60분 동안 처리한 다음, LPS(1㎍/㎖)와 함께 6시간 동안 더 배양하였다.Murine macrophage lines, RAW264.7 cells, were 70-80% density in 100 mm cell culture dish using RPMI 1640 medium containing penicillin (100 IU / ml) and streptomycin (100 μg / ml) and 10% FBS. Incubated with Afterwards, the cultured RAW264.7 cells (1 × 10 5 cells / well) were treated with Loratadine (0, 20, 30, 40 μM) for 60 minutes, followed by LPS (1 μg / mL) for 6 hours. Incubated.
사이토카인의 발현 정도를 전사수준에서 조사하기 위해 Trizol reagent를 사용하여 total RNA를 추출하였다. 추출한 total RNA를 First strand cDNA synthesis kit (Thermo scientific)를 사용하여 cDNA를 제조한 다음, 동량의 cDNA를 Realtime-PCR을 통해 증폭함으로써, 염증과 관련된 인자인 iNOS, COX-2, IL-1β, 및 IL-6의 발현 정도를 비교하였다. 본 실시예에서 사용된 프라이머는 하기 표 1에 나타내었다. To investigate the expression level of cytokines at the transcription level, total RNA was extracted using Trizol reagent. The extracted total RNA was prepared using a First strand cDNA synthesis kit (Thermo scientific) to prepare cDNA, and then amplified the same amount of cDNA through Realtime-PCR, iNOS, COX-2, IL-1β, which are factors related to inflammation, and The degree of expression of IL-6 was compared. Primers used in this example are shown in Table 1 below.
PCR amplication은 SYBR Premix Ex Taq (Takara Bio)과 realtime thermal cycler (Bio-Rad)를 제조사의 매뉴얼에 따라 수행하였으며, 대조 유전자로 GAPDH를 사용하였다. PCR amplication was performed using SYBR Premix Ex Taq (Takara Bio) and realtime thermal cycler (Bio-Rad) according to the manufacturer's manual, GAPDH was used as a control gene.
[표 1]TABLE 1
Figure PCTKR2014012422-appb-I000002
Figure PCTKR2014012422-appb-I000002
도 11 내지 도 14에 나타낸 바와 같이, LPS에 의해 유도된 염증 관련 인자인 iNOS, COX-2, IL-1β, 및 IL-6의 증가된 발현량이 Loratadine 처리에 의해서 감소하였으며, Loratadine의 농도가 증가할수록 대체적으로 발현량이 더욱 감소함을 확인하였다. 상기 결과는 본 발명의 Loratadine은 전사 수준에서 염증 관련 인자의 발현을 억제함으로서, 항염증효과를 가짐을 의미한다. As shown in Figures 11 to 14, the increased expression of the inflammation-related factors iNOS, COX-2, IL-1β, and IL-6 induced by LPS was reduced by Loratadine treatment, the concentration of Loratadine was increased As a result, it was confirmed that the amount of expression was further reduced. The results indicate that Loratadine of the present invention has an anti-inflammatory effect by inhibiting the expression of inflammation-related factors at the transcription level.
실시예 10 : Loratadine의 전사인자 NF-κB/ AP-1 활성 억제효과Example 10: Inhibitory Effect of Loratadine Transcription Factor NF-κB / AP-1 Activity
Luciferase gene promotor 발현 측정법Luciferase gene promotor expression assay
HEK 293 세포주를 Opti-MEM을 이용하여 24 well plate에 분주한 후 37 ℃, 5% CO2 세포배양기에서 전 배양한 후, 세포가 50% 밀도가 되었을 때 실험을 진행하였다. PEI 법을 이용하여 NF-κB, AP-1 Luciferase DNA와 TRIF, MyD88 DNA, beta-galactosidase DNA를 각기 co-transfection하였다. 구체적으로, DNA 1 μg과 PEI 3 μg을 Opti-MEM에 각각 희석해 주고, 상온에서 20분 동안 배양 한 후, DNA 희석액과 PEI 희석액을 혼합하여 다시 상온에서 20분 배양하였다. After dispensing HEK 293 cell line into a 24 well plate using Opti-MEM and pre-cultured at 37 ℃, 5% CO 2 cell incubator, the experiment was performed when the cells became 50% density. Using the PEI method, NF-κB, AP-1 Luciferase DNA, TRIF, MyD88 DNA and beta-galactosidase DNA were co-transfected, respectively. Specifically, 1 μg of DNA and 3 μg of PEI were respectively diluted in Opti-MEM, incubated at room temperature for 20 minutes, and then mixed with DNA dilution and PEI dilution for 20 minutes at room temperature.
배양 후 혼합액을 세포가 분주된 24 well plate에 넣어 주었으며, 6시간 후 세포배양 배지[10 % FBS, 1 % penicillin/streptomycin in DMEM]로 교체해주고, 24시간 배양하였다. 이 후, Loratadine을 농도별(0, 20,30, 40 μM)로 처리한 다음, 다시 30분간 배양하였다. After incubation, the mixed solution was placed in a 24 well plate in which cells were dispensed. After 6 hours, the mixture was replaced with cell culture medium [10% FBS, 1% penicillin / streptomycin in DMEM] and incubated for 24 hours. After that, Loratadine was treated by concentration (0, 20, 30, 40 μM), and then incubated for another 30 minutes.
이 후, NF-κB, AP-1 DNA를 활성화 시키는 stimuli (100 nM PMA)를 처리한 후 6-18 시간동안 배양하였다. 배양 후 lysis buffer를 이용하여 세포를 용해시키고 substrate와 1:1로 반응시켰으며, Luminometer로 흡광도를 측정하여 NF-κB 또는 AP-1 Luciferase의 활성을 평가 하였다. beta-galactosidase의 경우, X-gal과 1:1로 반응시키고, 37 ℃ 배양기에서 5분 배양한 후, 405 nm로 흡광도를 측정하였다. Subsequently, stimuli (100 nM PMA), which activates NF-κB and AP-1 DNA, was incubated for 6-18 hours. After incubation, the cells were lysed using lysis buffer and reacted 1: 1 with the substrate. The activity of NF-κB or AP-1 Luciferase was evaluated by measuring the absorbance with a luminometer. In the case of beta-galactosidase, it was reacted 1: 1 with X-gal, incubated for 5 minutes in a 37 ° C. incubator, and the absorbance was measured at 405 nm.
도 15 내지 도 17에 나타낸 바와 같이, NF-κB Luciferase(0.8μg) 및 beta-galactosidase(0.1μg)를 처리한 군(도 15), NF-κB Luciferase(0.3μg), TRIF(0.3μg), 및 beta-galactosidase(0.1μg)을 처리한 군(도 16), NF-κB Luciferase(0.3μg), MyD88(0.3μg), 및 beta-galactosidase(0.1μg)를 처리한 군(도 17)에서, NF-κB Luciferase 활성이 Loratadine에 의하여 감소하였으며, Loratadine의 농도가 증가할수록 NF-κB Luciferase 활성이 더욱 감소함을 확인하였다. As shown in Figure 15 to 17, NF-κB Luciferase (0.8μg) and beta-galactosidase (0.1μg) treated group (Fig. 15), NF-κB Luciferase (0.3μg), TRIF (0.3μg), And in the group treated with beta-galactosidase (0.1 μg) (FIG. 16), NF-κB Luciferase (0.3 μg), MyD88 (0.3 μg), and beta-galactosidase (0.1 μg) (FIG. 17). NF-κB Luciferase activity was decreased by Loratadine, and as the concentration of Loratadine increased, NF-κB Luciferase activity was further decreased.
또한, 도 18 내지 도 20에 나타낸 바와 같이, 상기와 마찬가지로 AP-1 Luciferase(0.8μg) 및 beta-galactosidase(0.1μg)를 처리한 군(도 18), AP-1 Luciferase(0.3μg), TRIF(0.3μg), 및 beta-galactosidase(0.1μg)를 처리한 군(도 19), 및 AP-1 Luciferase(0.3μg), MyD88(0.3μg), 및 beta-galactosidase(0.1μg)를 처리한 군(도 20)에서, AP-1 Luciferase 활성이 Loratadine에 의하여 감소하였으며, Loratadine의 농도가 증가할수록 AP-1 Luciferase 활성이 더욱 감소함을 확인하였다. 상기 결과는 본 발명의 Loratadine은 염증관련 전사인자인 NF-κB/ AP-1의 활성을 억제함으로써, 항염증효과를 가짐을 의미한다. In addition, as shown in Fig. 18 to Fig. 20, AP-1 Luciferase (0.8 μg) and beta-galactosidase (0.1 μg) -treated group (Fig. 18), AP-1 Luciferase (0.3 μg), TRIF as above. (0.3 μg), and group treated with beta-galactosidase (0.1 μg) (FIG. 19), and group treated with AP-1 Luciferase (0.3 μg), MyD88 (0.3 μg), and beta-galactosidase (0.1 μg). In FIG. 20, AP-1 Luciferase activity was decreased by Loratadine, and as the concentration of Loratadine was increased, AP-1 Luciferase activity was further decreased. The results indicate that Loratadine of the present invention has an anti-inflammatory effect by inhibiting the activity of inflammation-related transcription factor NF-κB / AP-1.
실시예 11 : Loratadine의 전사인자 NF-κB/ AP-1의 핵내 이동 억제효과Example 11 Inhibition of Intranuclear Migration of Loratadine Transcription Factor NF-κB / AP-1
Murine 대식 세포주인 RAW264.7 세포를 penicillin (100 IU/ml) 및 streptomycin (100μg/ml)과 10%의 FBS를 함유하는 RPMI 1640 배지에서 배양하였으며, 상기 배양한 세포를 90%의 밀도로 100mm의 dish에서 전 배양시켰다. 이 후 Loratadine(1μg/ml)를 30분 동안 전 처리하고, stimuli (Lipopolysaccharide, LPS)로 자극한 후, 일정 시간 후 ice-cold PBS로 washing 하고 Homogenization buffer A [20 mM Tris-HCl pH8.0, 10 mM EGTA, 2 mM EDTA, 2 mM DTT, 1 mM PMSF, 25μg/ml Aprotinin, 10μg/ml Leupeptin] 300μl를 이용하여 세포를 모았다. Sonicator를 사용하여 Output 4의 세기로 세포를 파쇄한 후, 8000 rpm으로 15분 동안 4 ℃에서 원심분리 하여 상층액 (Cytosol 분획)을 분리하였다. Pellet (Nuclear 분획)은 300μl Homogenization buffer B [1% TritonX-100 in Homogenization buffer A]로 부유시킨 후, sonicator를 사용하여 Output 4의 세기로 세포를 파쇄하여 수득하였으며, 얻어진 분획을 SDS-PAGE와 western blotting 법을 이용하여 전사인자의 핵 내 이동 수준을 평가하였다.Murine macrophage RAW264.7 cells were cultured in RPMI 1640 medium containing penicillin (100 IU / ml) and streptomycin (100 μg / ml) and 10% FBS. Pre-incubated in dish. Thereafter, Loratadine (1 μg / ml) was pretreated for 30 minutes, stimulated with stimuli (Lipopolysaccharide, LPS), and then washed with ice-cold PBS after a certain time, followed by Homogenization buffer A [20 mM Tris-HCl pH8.0, Cells were collected using 300 μl of 10 mM EGTA, 2 mM EDTA, 2 mM DTT, 1 mM PMSF, 25 μg / ml Aprotinin, 10 μg / ml Leupeptin]. Cells were disrupted at the intensity of Output 4 using a Sonicator, and the supernatant (Cytosol fraction) was separated by centrifugation at 4 ° C. for 15 minutes at 8000 rpm. Pellet (Nuclear fraction) was obtained by suspending with 300μl Homogenization buffer B [1% TritonX-100 in Homogenization buffer A] and then crushing the cells at the intensity of Output 4 using a sonicator. The level of nuclear transfer of transcription factors was assessed using blotting.
각 표본의 단백질 농도를 BSA(Bovine Serum Albumin)를 표준으로 하여 측정였다. 단백질 농도가 되는 각 표본량을 이용하여 SDS-PAGE를 실시하고, Western blotting 방법을 사용해 PVDF membrane으로 단백질을 blotting시킨 후, membrane을 5 % non-fat dried milk (Bio-rad)를 사용해 blocking시켰다. 이 후, 표적 단백질 항체 (p65, p50, c-Jun, Lamin A/C) 용액을 사용해 1차 처리하고, 다시 washing 단계 후, 2차 항체 용액을 처리하고 washing하였다. 그리고 암실에서 membrane에 ECL 용액 (Amersham, England)을 골고루 분주하여 X-ray film으로 감광하였으며, 대조 단백질로 Lamin A/C를 사용하였다.Protein concentration of each sample was measured using BSA (Bovine Serum Albumin) as a standard. SDS-PAGE was carried out using each sample amount, which was the protein concentration. After protein blotting with PVDF membrane using Western blotting method, the membrane was blocked with 5% non-fat dried milk (Bio-rad). Thereafter, the primary protein antibody (p65, p50, c-Jun, Lamin A / C) solution was first treated, and after the washing step, the secondary antibody solution was treated and washed. In the dark room, ECL solution (Amersham, England) was evenly distributed on the membrane, and the resultant was exposed to X-ray film. Lamin A / C was used as a control protein.
도 21 에 나타낸 바와 같이, Nuclear 분획에서, 시간의 경과에 따른 Loratadine에 의한 표적 단백질 항체 (p65 및 p50 및 c-Jun) 단백질의 감소를 확인하였으며, 상기 결과는 본 발명의 Loratadine은 NF-κB/ AP-1 유전자의 전사인자인 p65 및 p50 및 c-JUN의 핵내 이동을 억제함으로써, 항염증효과를 가짐을 의미한다.As shown in FIG. 21, in the Nuclear fraction, the decrease of target protein antibody (p65 and p50 and c-Jun) protein by Loratadine over time was confirmed, and the result shows that Loratadine of the present invention is NF-κB / By inhibiting the nuclear transfer of the transcription factors p65 and p50 and c-JUN of the AP-1 gene, it means that it has an anti-inflammatory effect.
전술한 본 발명의 설명은 예시를 위한 것이며, 본 발명이 속하는 기술 분야의 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 이해해야 한다.The above 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 발명은 로라타딘(Loratadine, Ethyl 4-(8-chloro-5,6-dihydro-11H-benzo[5,6]cyclohepta[1,2-b]pyridin-11-ylidene)-1-piperidinecarboxylate)을 유효성분으로 함유하는 항염증용 약학적 조성물, 식품 조성물에 관한 것으로서, 본 발명의 조성물을 고농도로 처리한 경우에도 세포 독성을 보이지 않았을 뿐만 아니라. 우수한 항염증 효과를 생체 내 또는 생체 외 실험을 통하여 확인하였다. 따라서, 본 발명의 방법 또는 조성물을 이용하여, 항염증 질환을 효과적으로 치료 할 수 있다.The present invention relates to loratadine (Loratadine, Ethyl 4- (8-chloro-5,6-dihydro-11H-benzo [5,6] cyclohepta [1,2-b] pyridin-11-ylidene) -1-piperidinecarboxylate) The present invention relates to an anti-inflammatory pharmaceutical composition and a food composition containing the active ingredient, even when the composition of the present invention is treated at a high concentration, as well as showing no cytotoxicity. Excellent anti-inflammatory effects were confirmed through in vivo or ex vivo experiments. Thus, the methods or compositions of the present invention can be used to effectively treat anti-inflammatory diseases.
<210> 1<210> 1
<211> 22<211> 22
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<213> Artificial Sequence<213> Artificial Sequence
<220><220>
<223> iNOS Forward primer<223> iNOS Forward primer
<400> 1<400> 1
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<210> 2<210> 2
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<213> Artificial Sequence<213> Artificial Sequence
<220><220>
<223> iNOS Reverse primer<223> iNOS Reverse primer
<400> 2<400> 2
tgaacgagga gggtggtg 18tgaacgagga gggtggtg 18
<210> 3<210> 3
<211> 21<211> 21
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<213> Artificial Sequence<213> Artificial Sequence
<220><220>
<223> COX-2 Forward primer<223> COX-2 Forward primer
<400> 3<400> 3
ccagcacttc acccatcagt t 21ccagcacttc acccatcagt t 21
<210> 4<210> 4
<211> 20<211> 20
<212> DNA<212> DNA
<213> Artificial Sequence<213> Artificial Sequence
<220><220>
<223> COX-2 Reverse primer<223> COX-2 Reverse primer
<400> 4<400> 4
acccaggtcc tcgcttatga 20 acccaggtcc tcgcttatga 20
<210> 5<210> 5
<211> 21<211> 21
<212> DNA<212> DNA
<213> Artificial Sequence<213> Artificial Sequence
<220><220>
<223> IL-1beta Forward primer<223> IL-1beta Forward primer
<400> 5<400> 5
gttgacggac cccaaaaaga t 21gttgacggac cccaaaaaga t 21
<210> 6<210> 6
<211> 20<211> 20
<212> DNA<212> DNA
<213> Artificial Sequence<213> Artificial Sequence
<220><220>
<223> IL-1beta Reverse primer<223> IL-1beta Reverse primer
<400> 6<400> 6
cctcatcctg gaaggtccac 20 cctcatcctg gaaggtccac 20
<210> 7<210> 7
<211> 19<211> 19
<212> DNA<212> DNA
<213> Artificial Sequence<213> Artificial Sequence
<220><220>
<223> IL-6 Forward primer<223> IL-6 Forward primer
<400> 7<400> 7
aacgatgatg cattgcaga 19aacgatgatg cattgcaga 19
<210> 8<210> 8
<211> 20<211> 20
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<220><220>
<223> IL-6 Reverse primerIL-6 Reverse primer
<400> 8<400> 8
gagcattgga aattggggta 20 gagcattgga aattggggta 20
<210> 9<210> 9
<211> 20<211> 20
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<213> Artificial Sequence<213> Artificial Sequence
<220><220>
<223> GAPDH Forward primer<223> GAPDH Forward primer
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caatgaatac ggctacagca 20 caatgaatac ggctacagca 20
<210> 10<210> 10
<211> 20<211> 20
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<213> Artificial Sequence<213> Artificial Sequence
<220><220>
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agggagatgc tcagtgttgg 20 agggagatgc tcagtgttgg 20

Claims (10)

  1. 로라타딘(Loratadine, Ethyl 4-(8-chloro-5,6-dihydro-11H-benzo[5,6]cyclohepta[1,2-b]pyridin-11-ylidene)-1-piperidinecarboxylate) 또는 그 약제학적으로 허용되는 염을 유효성분으로 포함하는 염증질환 예방 또는 치료용 약학적 조성물.Loratadine, Ethyl 4- (8-chloro-5,6-dihydro-11H-benzo [5,6] cyclohepta [1,2-b] pyridin-11-ylidene) -1-piperidinecarboxylate) or pharmaceuticals thereof Inflammatory disease prevention or treatment pharmaceutical composition comprising a salt as an active ingredient.
  2. 제 1항에 있어서, The method of claim 1,
    상기 조성물은 일산화질소(Nitric Oxide, NO) 또는 PGE2(Prostaglandin E2)의 생성을 억제하는 것을 특징으로 하는, 약학적 조성물.The composition is characterized in that to inhibit the production of nitric oxide (Nitric Oxide, NO) or PGE 2 (Prostaglandin E 2 ), pharmaceutical composition.
  3. 제 1항에 있어서,The method of claim 1,
    상기 조성물은 NF-κB 또는 AP-1의 활성을 억제하는 것을 특징으로 하는, 약학적 조성물.The composition is characterized in that to inhibit the activity of NF-κB or AP-1, pharmaceutical composition.
  4. 제 1항에 있어서, The method of claim 1,
    상기 염증은 위궤양, 십이지장궤양, 간염, 식도염, 위염, 장염, 췌장염, 대장염, 복막염, 신장염, 전신부종 및 국소부종으로 이루어진 군에서 선택되는 것을 특징으로 하는, 약학적 조성물.The inflammation is selected from the group consisting of gastric ulcer, duodenal ulcer, hepatitis, esophagitis, gastritis, enteritis, pancreatitis, colitis, peritonitis, nephritis, systemic edema and local edema.
  5. 제 4항에 있어서, The method of claim 4, wherein
    상기 염증은 위염, 간염, 또는 복막염인 것을 특징으로 하는, 약학적 조성물.The inflammation is characterized in that gastritis, hepatitis, or peritonitis, pharmaceutical composition.
  6. 로라타딘(Loratadine, Ethyl 4-(8-chloro-5,6-dihydro-11H-benzo[5,6]cyclohepta[1,2-b]pyridin-11-ylidene)-1-piperidinecarboxylate) 또는 그 약제학적으로 허용가능한 염을 유효성분으로 포함하는 염증질환 예방 또는 개선용 식품 조성물.Loratadine, Ethyl 4- (8-chloro-5,6-dihydro-11H-benzo [5,6] cyclohepta [1,2-b] pyridin-11-ylidene) -1-piperidinecarboxylate) or pharmaceuticals thereof Inflammatory disease prevention or improvement food composition comprising a salt as an active ingredient.
  7. 제 6항에 있어서,The method of claim 6,
    상기 염증은 위궤양, 십이지장궤양, 간염, 식도염, 위염, 장염, 췌장염, 대장염, 복막염, 신장염, 전신부종 및 국소부종으로 이루어진 군에서 선택되는 것을 특징으로 하는, 식품 조성물.The inflammation is selected from the group consisting of gastric ulcer, duodenal ulcer, hepatitis, esophagitis, gastritis, enteritis, pancreatitis, colitis, peritonitis, nephritis, systemic edema and local edema.
  8. 제 7항에 있어서,The method of claim 7, wherein
    상기 염증은 위염, 간염, 또는 복막염인 것을 특징으로 하는, 식품 조성물.The inflammation is gastritis, hepatitis, or peritonitis, characterized in that the food composition.
  9. 로라타딘(Loratadine, Ethyl 4-(8-chloro-5,6-dihydro-11H-benzo[5,6]cyclohepta[1,2-b]pyridin-11-ylidene)-1-piperidinecarboxylate) 또는 그 약제학적으로 허용되는 염을 개체에 투여하는 단계를 포함하는, 염증질환의 예방 또는 치료방법.Loratadine, Ethyl 4- (8-chloro-5,6-dihydro-11H-benzo [5,6] cyclohepta [1,2-b] pyridin-11-ylidene) -1-piperidinecarboxylate) or pharmaceuticals thereof A method for preventing or treating an inflammatory disease, comprising administering to a subject an acceptable salt.
  10. 로라타딘(Loratadine, Ethyl 4-(8-chloro-5,6-dihydro-11H-benzo[5,6]cyclohepta[1,2-b]pyridin-11-ylidene)-1-piperidinecarboxylate) 또는 그 약제학적으로 허용되는 염의 염증질환의 치료 용도.Loratadine, Ethyl 4- (8-chloro-5,6-dihydro-11H-benzo [5,6] cyclohepta [1,2-b] pyridin-11-ylidene) -1-piperidinecarboxylate) or pharmaceuticals thereof Use of the salt for the treatment of inflammatory diseases.
PCT/KR2014/012422 2014-02-24 2014-12-16 Anti-inflammatory composition comprising loratadine WO2015126048A1 (en)

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