WO2017023047A1 - Composition for preventing or treating inflammatory disease or cancer containing aripiprazole as an active ingredient - Google Patents

Composition for preventing or treating inflammatory disease or cancer containing aripiprazole as an active ingredient Download PDF

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WO2017023047A1
WO2017023047A1 PCT/KR2016/008401 KR2016008401W WO2017023047A1 WO 2017023047 A1 WO2017023047 A1 WO 2017023047A1 KR 2016008401 W KR2016008401 W KR 2016008401W WO 2017023047 A1 WO2017023047 A1 WO 2017023047A1
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cancer
aripiprazole
cells
inflammatory disease
preventing
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PCT/KR2016/008401
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French (fr)
Korean (ko)
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조재열
백광수
김용
김은지
박재광
유슬기
김유림
정성구
김여진
김미선
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성균관대학교산학협력단
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Priority claimed from KR1020160086079A external-priority patent/KR20170026115A/en
Priority claimed from KR1020160086077A external-priority patent/KR20170016275A/en
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Publication of WO2017023047A1 publication Critical patent/WO2017023047A1/en

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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
    • A61K31/47—Quinolines; Isoquinolines
    • A61K31/4704—2-Quinolinones, e.g. carbostyril
    • 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
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/496—Non-condensed piperazines containing further heterocyclic rings, e.g. rifampin, thiothixene
    • 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
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/4965—Non-condensed pyrazines
    • A61K31/497—Non-condensed pyrazines containing further heterocyclic rings

Definitions

  • the present invention relates to a composition for preventing or treating inflammatory diseases or cancer containing aripiprazole as an active ingredient.
  • Inflammatory disease refers to the pathological condition of an abscess formed by the invasion of bacteria.
  • Gram-positive cocci cause various bacterial infections such as skin infections, purulent, pneumonia, and sepsis, and when the inflammation occurs, peptidoglycan (PGN) is released into the extracellular membrane in the process of removing Gram-positive cocci from the body. Is released.
  • PPN peptidoglycan
  • Inflammation is an important biological defense response to pathogens, foreign bodies and tissue damage, accompanied by systemic symptoms such as fever, weakness, loss of appetite and chills, or local symptoms such as redness, swelling, pain and dysfunction.
  • Inflammation is classified according to duration into acute inflammation, subacute inflammation and chronic inflammation.
  • Acute inflammation occurs in response to blood vessels, and neutrophils are primarily involved. In particular, in the case of purulent inflammation, an increase in neutrophils is marked.
  • Chronic inflammation is inflammation that lasts for weeks or months, with simultaneous damage and healing of tissues, persistent infections that cause delayed hypersensitivity reactions (eg, tuberculosis, syphilis, fungal infections), persistent endotoxins (eg, , Exposure to increased plasma lipids or exotoxins (eg, silica, asbestos, tar, surgical sutures), or autoimmune responses to autologous tissues (eg, rheumatoid arthritis, systemic lupus erythematosus, multiple sclerosis) , As a result of psoriasis, indicates a latent process that progresses over time.
  • Subacute inflammation refers to acute and chronic intermediate inflammation.
  • nitric oxide synthase NOS
  • COX cyclooxygenase
  • the NOS is an enzyme for producing NO (nitric oxide) from L-arginine, bNOS (brain NOS) in the brain, nNOS (neuronal NOS) in the nervous system and eNOS (endothelial NOS) in the vascular system are present in the body Always expressed at a certain level, a small amount of NO produced by these plays an important role in maintaining the homeostasis of the normal body, such as inducing neurotransmission or vasodilation.
  • iNOS induced NOS
  • iNOS induced NOS
  • cytokines or external stimulants is known to cause excessive toxicity and rapidly cause cytotoxicity and various inflammatory reactions.
  • COX is an enzyme involved in synthesizing prostaglandins from arachidonic acid, and there are two types of COX-1 and COX-2.
  • COX-1 is always present in the cell and exhibits the action of synthesizing the prostaglandin required for cytoprotective action, whereas COX-2 is known to play an important role in causing an inflammatory reaction by rapidly increasing in the cell during the inflammatory response.
  • NSAIDs nonsteroidal anti-inflammatory drugs having anti-inflammatory, antipyretic and analgesic effects, including bradykinin antagonists, COX inhibitors, and the like, have been widely used as anti-inflammatory agents.
  • nonsteroidal anti-inflammatory drugs can cause serious side effects such as gastrointestinal peptic ulcer bleeding, secondary anemia, inhibition of platelet function, inhibition of labor, side effects on kidneys, hepatic damage, and hypersensitivity. Therefore, in order to overcome the side effects of these drugs, there is an urgent need for the development of a therapeutic agent having a low side effect and excellent anti-inflammatory effect on the human body.
  • Cancer is one of the most incurable diseases that all human beings must overcome and can be said to be one of the most common causes of mortality in Korea.
  • In order to conquer cancer progress has been made in the search for new targets including control of cell cycle or apoptosis, and oncogenic and cancer suppressor genes, but the incidence of cancer is still increasing.
  • many methods such as incision, chemotherapy, radiotherapy, and incision have been developed to treat cancer, but radiation therapy and incisions are effective only when the initial diagnosis of cancer is performed. Esau is of no use and can only be treated with chemotherapy. Since chemotherapy introduced in cancer treatment since the 1940s has the advantage of being relatively easy to apply regardless of the timing of cancer, a lot of attention is now focused, and various anti-cancer chemotherapy agents are being developed.
  • the anticancer agents have a disadvantage in that when repeated long-term administration or cancer recurs, cancer cells lose the therapeutic effect by obtaining resistance to the anticancer agent.
  • most anticancer drugs have an effect by inhibiting the synthesis of nucleic acid in the cell or by directly binding to the nucleic acid and impairing its function.
  • These anticancer agents not only act selectively on cancer cells but also on normal cells, especially tissue cells with active cell division. Because of the damage, there are many side effects such as decreased bone marrow function, damage to the gastrointestinal tract, hair loss, and the like.
  • Apoptosis is known for programmed cell death, which is a genetic regulatory mechanism that plays an important role in maintaining cell homeostasis.
  • Apoptosis can generally be induced through the exogenous (killing receptor) pathway or the endogenous (within the mitochondria) pathway.
  • ligation of the TNF / Fas-receptor by its ligand causes cleavage of caspase-8 initiators and directly activate effector caspase-3, or Bcl-2 After inducing cleavage of the family member Bid, a potential of Bax is induced into the mitochondrial membrane.
  • cytochrome c and apoptosis-inducing factor are released from mitochondria, which factors are caspase-dependent and independent. Is involved in apoptosis. Cytochrome c binds with Apaf-1 to form a structure called apoptosome, activates caspase-9, then activates caspase-3, and finally causes apoptotic cell death .
  • AIF is a sign of caspase-independent apoptosis, which is translocated into the nucleus following apoptotic stimulation, leading to DNA fragmentation.
  • Aripiprazole is a quinolinone derivative, which is 7- ⁇ 4- [4- (2,3-dichlorophenyl) -1-piperazinyl] -butoxy ⁇ -3,4-dihydrocarbostyryl It is named.
  • Aripiprazole is a therapeutic agent for psychotropic diseases and is known to be useful for the treatment of schizophrenia characterized by delusions, hallucinations and excessive atrophy from others.
  • Aripiprazole is a potent partial agonist of the dopamine D2 receptor and the 5-HT1A receptor, and is called a dopamine-serotonin-based stabilizer.
  • Partial agonists are substances that block receptors when they are excessively stimulated, and activate the receptors when they are lacking.
  • Aripiprazole is a schizophrenic (schizophrenia) drug developed by Otsuka Pharmaceutical Co., Ltd. and is marketed under the brand name Abilify TM, and is used for the treatment or improvement of schizophrenia, the treatment of acute mania associated with bipolar disorder, and the treatment of major depression. It is used for the purpose.
  • aripiprazole As described above, the pharmacological action of aripiprazole has been limited to the central nervous system and neurological diseases, and the technique has been studied only to increase the solubility and absorption rate of the drug. Little is known about the inflammatory or anticancer effects, and there is little research on this.
  • aripiprazole While we studied the anti-inflammatory effects of aripiprazole, we found that aripiprazole has no cytotoxicity in RAW264.7 cells, and concentration-dependently inhibits NO production, prostaglandin E 2 in RAW264.7 cells treated with peptidoglycan. Inhibition of the production of (PGE 2 ) and secretion of cytokines (COX-2 and TNF- ⁇ ) was excellent, it was confirmed that the effect of inhibiting gastric damage in the in vivo inflammation model that caused acute inflammation.
  • the present invention is to provide a pharmaceutical composition for preventing or treating inflammatory diseases or cancer containing aripiprazole as an active ingredient.
  • the present invention is to provide a method for preventing or treating an inflammatory disease or cancer comprising administering aripiprazole to a subject.
  • the present invention provides a pharmaceutical composition for preventing or treating inflammatory diseases or cancer containing aripiprazole as an active ingredient.
  • the aripiprazole may inhibit the production of nitric oxide (NO) and prostaglandin E2 (PGE2).
  • NO nitric oxide
  • PGE2 prostaglandin E2
  • the aripiprazole may inhibit NF- ⁇ B and AP-1 (activator protein-1) activity.
  • the aripiprazole may inhibit the expression of iNOS, COX-2 and IFN- ⁇ .
  • the inflammatory disease is acute or chronic gastritis, edema, dermatitis, allergy, atopic, asthma, conjunctivitis, periodontitis, rhinitis, otitis media, sore throat, tonsillitis, pneumonia, hepatitis, esophagitis, gastric ulcer, enteritis, pancreatitis , Duodenal ulcer, colitis, hemorrhoids, gout, ankylosing spondylitis, rheumatic fever, lupus, fibromyalgia, psoriatic arthritis, osteoarthritis, rheumatoid arthritis, periarthritis, tendinitis, hay salt, tendonitis, myositis, cystitis, nephritis, multiple sclerosis and At least one member selected from the group consisting of sepsis.
  • the inflammatory disease may be acute or chronic gastritis.
  • the aripiprazole may induce apoptosis by increasing the activity of Caspase-3 protein in cancer cells.
  • the aripiprazole may inhibit Src kinase activity.
  • the aripiprazole may inhibit the expression of Bcl2, MMP2 and MMP9.
  • the cancer is glioma, thyroid cancer, parathyroid cancer, bone marrow cancer, rectal cancer, throat cancer, laryngeal cancer, lung cancer, esophageal cancer, pancreatic cancer, colon cancer, gastric cancer, tongue cancer, skin cancer, brain tumor, uterine cancer, head or neck cancer , Gallbladder cancer, oral cancer, colon cancer, proximal anal cancer, liver cancer, and colorectal cancer.
  • the cancer may be glioma, gastric cancer, breast cancer or brain tumor.
  • the present invention also provides a method for preventing or treating an inflammatory disease or cancer, comprising administering aripiprazole to a subject.
  • the present invention also provides a use for the prevention or treatment of inflammatory disease or cancer of aripiprazole.
  • Aripiprazole according to the present invention shows little decrease in cell viability in RAW264.7 cells and thus has no cytotoxicity, and has a concentration-dependent inhibition of NO production, prostaglandin E 2 (Peptidoglycan) in RAW264.7 cells.
  • PGE 2 is excellent in inhibiting the production and secretion of cytokines (COX-2 and TNF- ⁇ ) and gastric damage suppression effect in the in vivo inflammation model that caused acute inflammation.
  • aripiprazole according to the present invention exhibits an excellent cancer cell proliferation inhibitory effect in C6, U251, LN-428 and MDA-MB-231 cells, and is a marker of apoptosis depending on aripiprazole concentration in C6 cells.
  • the effects of blebbing, increased production of apoptotic bodies, accelerated cancer cell death, and increased amount of Active-Caspase were identified.
  • the present invention may be usefully used as an active ingredient of a pharmaceutical composition for preventing, ameliorating or treating an inflammatory disease or cancer.
  • FIG. 1 is a diagram showing the effect of aripiprazole on cell survival in RAW264.7 cells.
  • Figure 2 shows the effect of aripiprazole on the production of nitric oxide (NO) in peptidoglycan treated RAW264.7 cells.
  • FIG. 3 is a diagram showing the effect of aripiprazole on the production ability of prostaglandin E 2 (PGE 2 ) in peptidoglycan treated RAW264.7 cells.
  • FIG. 4 is a diagram showing the effect of aripiprazole on the mRNA expression of cytokines in RAW264.7 cells treated with peptidoglycan.
  • Figure 5 shows the effect of aripiprazole on mRNA expression of iNOS in peptidoglycan treated RAW264.7 cells.
  • Figure 6 shows the effect of aripiprazole on mRNA expression of COX-2 in peptidoglycan treated RAW264.7 cells.
  • Figure 7 shows the effect of aripiprazole on mRNA expression of IFN- ⁇ in peptidoglycan treated RAW264.7 cells.
  • FIG. 8 is a diagram showing the effect on the AP-1 promoter activity by MyD88 when aripiprazole was treated to HEK293 cells.
  • FIG. 9 is a diagram showing the effect on the NF- ⁇ B promoter activity by MyD88 when aripiprazole was treated to HEK293 cells.
  • FIG. 10 is a diagram showing the effect of TRIF on AP-1 promoter activity when aripiprazole was treated to HEK293 cells.
  • FIG. 11 is a diagram showing the effect on TRF-induced NF- ⁇ B promoter activity when aripiprazole was treated to HEK293 cells.
  • FIG. 12 is a diagram showing the effect of aripiprazole in RAW264.7 cells on the nuclear migration of inflammation-related nuclear proteins p50, p65, c-Fos and c-Jun by peptidoglycan.
  • FIG. 13 is a diagram showing the effect on the phosphorylation of the signaling proteins Src, Syk, AKT and p85 by peptidoglycan when aripiprazole is treated in RAW264.7 cells.
  • FIG. 14 is a diagram showing the gastric damage inhibition effect of Aripiprazole in the in vivo inflammation model after acute gastritis of ICR mice with ethanol / hydrochloric acid.
  • 15 is a diagram showing the effect of inhibiting the gastric damage of aripiprazole in in vivo inflammation model after acute gastritis of ICR mice with aspirin.
  • Figure 16 shows the effect of aripiprazole on cancer cell proliferation in C6 cells.
  • Figure 18 shows the effect of aripiprazole on cancer cell proliferation in LN428 cells.
  • 19 is a diagram showing the effect of aripiprazole on cancer cell proliferation in MDA-MB-231 cells.
  • 20 is a diagram showing the effect of aripiprazole on cancer cell proliferation in MKN-1 cells.
  • 21 shows the results of confirming the change in cell morphology by aripiprazole treatment in C6 cells through an optical microscope.
  • Figure 23 shows the effect of aripiprazole on migration of cells in U251 cells.
  • Fig. 24 shows the results of confirming nuclear contraction by aripiprazole treatment in C6 cells.
  • Figure 25 shows the results of confirming Actin disruption by aripiprazole treatment in C6 cells.
  • Fig. 26 shows the results of apoptosis of cancer cells by aripiprazole treatment in C6 cells by flow cytometer.
  • FIG. 27 shows the results of confirming that cell division cycle is interrupted by aripiprazole in C6 cells.
  • FIG. 28 is a diagram showing the effect on the mRNA expression of Bcl-2 by aripiprazole treatment in C6 cells.
  • 29 is a diagram showing the effect on the mRNA expression of MMP2 and MMP 9 by Aripiprazole treatment in U251 cells.
  • Figure 30 shows the effect on the protein expression of MMP2 and MMP 9 by Aripiprazole treatment in U251 cells.
  • FIG. 34 shows the results of confirming the effect of inhibiting phosphorylated Src expression activity in Src (HA-Src CA) which is continuously activated in HEK 293 cells.
  • Figure 37 shows the results of confirming the tumor size of the mouse orally administered aripiprazole in an animal model induced colon cancer cells.
  • Figure 38 shows the results of confirming the tumor volume increase of the oral administration of aripiprazole in the animal model induced colon cancer cells.
  • 39 shows the tumor weights of mice administered orally with aripiprazole in an animal model inducing colorectal cancer cells.
  • the present invention provides a composition for improving or treating inflammatory diseases or cancer prevention containing aripiprazole as an active ingredient.
  • the composition comprises a pharmaceutical composition.
  • Aripiprazole according to the present invention shows little decrease in cell viability in RAW264.7 cells and thus has no cytotoxicity, and has a concentration-dependent inhibition of NO production, prostaglandin E 2 (Peptidoglycan) in RAW264.7 cells.
  • PGE 2 is excellent in inhibiting the production and secretion of cytokines (COX-2 and TNF- ⁇ ) and gastric damage suppression effect in the in vivo inflammation model that caused acute inflammation. Therefore, aripiprazole according to the present invention can be used as a medicament useful for the prevention or treatment of inflammatory diseases.
  • the inflammatory diseases include acute or chronic gastritis, edema, dermatitis, allergies, atopic, asthma, conjunctivitis, periodontitis, rhinitis, otitis media, sore throat, tonsillitis, pneumonia, hepatitis, esophagitis, gastric ulcer, enteritis, pancreatitis, duodenal ulcer, colitis, hemorrhoids, Include, but are not limited to, gout, ankylosing spondylitis, rheumatic fever, lupus, fibromyalgia, psoriatic arthritis, osteoarthritis, rheumatoid arthritis, periarthritis, tendinitis, hay salt, peritonitis, myositis, cystitis, nephritis, multiple sclerosis and sepsis It doesn't work.
  • aripiprazole according to the present invention exhibits an excellent cancer cell proliferation inhibitory effect in C6, U251, LN-428 and MDA-MB-231 cells, and is a marker of apoptosis depending on aripiprazole concentration in C6 cells.
  • the effects of blebbing, increased production of apoptotic bodies, accelerated cancer cell death, and increased amount of Active-Caspase were identified. Therefore, aripiprazole according to the present invention can be used as a medicament useful for the prevention or treatment of cancer.
  • the cancer is glioma, thyroid cancer, parathyroid cancer, bone marrow cancer, rectal cancer, throat, larynx cancer, lung cancer, esophageal cancer, pancreatic cancer, colon cancer, stomach cancer, tongue cancer, skin cancer, brain tumor, uterine cancer, head or neck cancer, gallbladder cancer, oral cancer, colon cancer, near the anus Cancer, liver cancer, colon cancer, and the like.
  • composition of the present invention may contain at least one known active ingredient having anti-inflammatory or anticancer effect together with aripiprazole.
  • 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.
  • Pharmaceutically acceptable carriers may be used in combination with saline, sterile water, Ringer's solution, buffered saline, dextrose solution, maltodextrin solution, glycerol, ethanol and one or more of these components, if necessary, antioxidants, buffers And other conventional additives such as bacteriostatic agents can be added.
  • Diluents, dispersants, surfactants, binders and lubricants may also be added in addition to formulate into injectable formulations, pills, capsules, granules or tablets such as aqueous solutions, suspensions, emulsions and the like.
  • it can be preferably formulated according to each disease or component using appropriate methods in the art.
  • composition of the present invention can 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 and health of the patient. The range varies depending on the diet, the time of administration, the method of administration, the rate of excretion and the severity of the disease.
  • the daily dose of aripiprazole is about 0.0001 to 100 mg / kg, preferably about 0.001 to 50 mg / kg, preferably administered once to several times a day.
  • compositions of the present invention can be used alone or in combination with methods for using surgery, hormone therapy, drug therapy and biological response modifiers for the prevention or treatment of inflammatory diseases or cancer.
  • the present invention also provides a method for preventing or treating an inflammatory disease or cancer comprising administering aripiprazole to a subject.
  • the subject is a human or non-human mammal, and non-human mammals include, but are not limited to, mice, rats, dogs, cats, horses, cattle, sheep, goats, pigs, rabbits, and the like.
  • Example 1-1 Cell culture
  • RAW264.7 cells a murine macrophage line
  • RAW264.7 cells were 70-80% in 100 mm cell culture dishes using RPMI1640 medium containing penicillin (100 IU / ml), streptomycin (100 ⁇ g / ml) and 10% FBS.
  • Human cell line HEK293 cells were cultured at a density of 70 and cultured in 100 mm cell culture dishes using DMEM medium containing penicillin (100 IU / mL), streptomycin (100 ⁇ g / mL) and 10% FBS. Incubated at a density of ⁇ 80%.
  • aripiprazole showed little decrease in cell viability in RAW264.7 cells, confirming that there was no cytotoxicity.
  • RAW264.7 cells a mouse macrophage line
  • RPMI1640 medium containing penicillin (100 IU / mL), streptomycin (100 ⁇ g / mL) and 10% FBS.
  • 96 well plates were inoculated and pre-incubated at 5% CO 2 and 37 ° C. for 18 hours. The medium was then removed and incubated with aripiprazole (5, 10, 20 ⁇ M) and a medium containing 10 ⁇ g / ml of peptidoglycan simultaneously.
  • aripiprazole in RAW264.7 cells treated with peptidoglycan decreased NO production with increasing concentration. Therefore, it can be seen that aripiprazole has an excellent ability to inhibit NO production in a concentration-dependent manner.
  • RAW264.7 cells a mouse macrophage line
  • RPMI1640 medium containing penicillin (100 IU / mL), streptomycin (100 ⁇ g / mL) and 10% FBS.
  • 96 well plates were inoculated and pre-incubated at 5% CO 2 and 37 ° C. for 18 hours. The medium was then removed and incubated with aripiprazole (10, 20 ⁇ M) and a medium containing 10 ⁇ g / ml of peptidoglycan simultaneously. After 24 hours, the supernatant was transferred to another 96 well plate at 100 ⁇ l, and the concentration of PGE 2 in the supernatant was measured by Griess reagent and EIA kit.
  • aripiprazole in peptidoglycan treated RAW264.7 cells decreased the production of prostaglandin E 2 (PGE 2 ) at increasing concentrations. Therefore, it can be seen that aripiprazole is excellent in inhibiting the production of prostaglandin E 2 (PGE 2 ) in a concentration-dependent manner.
  • the extracted total RNA was prepared cDNA using the first strand cDNA synthesis kit (Thermo scientific), and then the same amount of cDNA was amplified by PCR.
  • the sense and antisense primer sequences of the cytokines used at this time were prepared with reference to the existing literature, and GAPDH was used as a control gene (see Table 1).
  • PCR amplification was performed by using the Hipi PCR kit (Elpis biotech) to sense and antisense primers of cDNA and cytokines (COX-2 and TNF- ⁇ ) and control GAPDH primers of each experimental group, dNTP 250 ⁇ M, Tris-HCl (pH 8.3). 20 ⁇ l of a Hipi PCR kit containing 10 mM, KCl 50 mM, NgCl 2 1.5 mM. PCR was performed under conditions of denaturation at 95 ° C. for 45 seconds, annealing at 55 ° C.
  • TNF- ⁇ lossy factor alpha
  • HEK 293 cell lines were dispensed into 24-well plates using Opti-MEM, and then pre-incubated in a 37 ° C. 5% CO 2 cell incubator.
  • the cells When cultured to 50% density, the cells were co-transfected with AP-1 or NF- ⁇ B Luciferase DNA, MyD88 or TRIF DNA and ⁇ -galactosidase DNA using the PEI method.
  • the PEI method 1 ⁇ g of DNA and 3 ⁇ g of PEI were diluted in Opti-MEM, followed by incubation at room temperature for 20 minutes, followed by mixing the diluted solutions for 20 minutes.
  • the mixed solution was placed in a 24-well plate in which cells were divided, and after 6 hours, the mixture was replaced with cell culture medium [10% FBS, 1% penicillin / streptomycin in DMEM], and after 24 hours, aripiprazole was treated by concentration. After 9 hours incubation. Thereafter, the cells were lysed using lysis buffer, reacted 1: 1 with the substrate, and the absorbance was immediately measured using a luminometer. In the case of ⁇ -galactosidase DNA, reacted with X-gal (substrate) 1: 1 and incubated for 5 minutes in a 37 ° C. incubator, and then measured absorbance at 405 nm.
  • FIGS. 8 and 9 AP-1 and NF- ⁇ B-mediated luciferase activity by MyD88 was inhibited by aripiprazole treatment, and as shown in FIGS. 10 and 11, TRIF was treated by aripiprazole treatment. AP-1 and NF- ⁇ B-mediated luciferase activity was inhibited. Thus, the activity of inflammation-related genes was also inhibited by aripiprazole.
  • cells cultured with a mouse macrophage RAW264.7 cell using RPMI 1640 medium containing penicillin (100 IU / ml) and streptomycin (100 ⁇ g / ml) and 10% FBS were pre-incubated in a dish of 100 mm at a density of 90%, pretreated with aripiprazole for 1 hour and stimulated with stimuli (peptidoglycan), followed by washing with ice-cold PBS after a certain time according to the drug, homogenization Cells are collected using 300 ⁇ l of buffer A [20 mM Tris-HCl pH8.0, 10 mM EGTA, 2 mM EDTA, 2 mM DTT, 1 mM PMSF, 25 ⁇ g / ml Aprotinin, 10 ⁇ g / ml Leupeptin].
  • buffer A [20 mM Tris-HCl pH8.0, 10 mM EGTA, 2 mM EDTA, 2 mM DTT, 1 mM
  • the cells were crushed at the intensity of Output 4 using a Sonicator, and the supernatant (cytoplasmic fraction) was separated by centrifugation at 8000 rpm for 15 minutes at 4 ° C.
  • the pellet (nucleus fraction) was suspended in 300 ⁇ l homogenization buffer B [1% TritonX-100 in Homogenization buffer A], and the cells were disrupted at the intensity of Output 4 using a sonicator.
  • the obtained fractions were assessed by nuclear transfer level of the transcription factor using SDS-PAGE and Western plotting.
  • cells cultured with mouse macrophage RAW264.7 cells using RPMI 1640 medium containing penicillin (100 IU / mL) and streptomycin (100 ⁇ g / mL) and 10% FBS were prepared. Pre-incubate in a 60 mm dish at a concentration of 10 6 cells / ml. Subsequently, each fraction was treated and stimulated with stimuli (peptidoglycan) after a certain time, and after a certain time according to the drug, cells were collected and the cells were crushed using lysis buffer and sonicator to obtain western specimens. The protein concentration of each sample was measured using BSA as a standard.
  • the SDS-PAGE was performed with each sample volume that is the protein concentration based on the obtained values, and the proteins were blotting with PVDF membrane using Western blotting. After that, the membrane was blocked using 5% non-fat dried milk (Bio-rad), and the target protein antibody and the signaling protein antibody (p-Src, p-Syk, p-AKT, p-p85, ⁇ -actin) The solution is first treated and then washed again after the second antibody solution. In the dark room, ECL solution (Amersham, England) was evenly distributed on the membrane, and the resultant was exposed to X-ray film. The results are shown in FIG. 13.
  • aripiprazole inhibited phosphorylation of Src, Syk, AKT and p85, which are signaling proteins by PGN.
  • Example 7 In vivo inflammation model Aripiprazole Stomach Damage Inhibitory Effect
  • Acute gastritis of ICR mice was induced with ethanol / hydrochloric acid or acetylsalicylic acid according to known methods. Specifically, the fasted ICR mice (30 mice) were grouped into 6 groups, followed by 0.5% CMC (carboxymethyl cellulose) solution in 2 groups, aripiprazole (1 and 10 mg / kg) in 2 groups, and The other two groups were orally administered with the positive control ranitidine (40 mg / kg) and styrene (8 mg / kg) for 3 days each.
  • CMC carboxymethyl cellulose
  • RPMI1640 medium or DMEM medium containing C6, U251, LN-428, MKN-1, MDA-MB-231, and HEK 293 cells containing penicillin (100 IU / ml) and streptomycin (100 ⁇ g / ml) and 10% FBS Were cultured in a 100 mm cell culture dish at a density of 70-80%.
  • MTT stop solution (10% sodium dodecyl sulfate in 0.01M HCl) was added to each well.
  • Cell viability was calculated from the amount of MTT reduced to formazan through the OD value obtained by measuring the absorbance at 570 nm, and the results are shown in FIGS. 16 to 20.
  • Aripiprazole significantly reduced the viability of C6, U251, LN-428, MKN-1 and MDA-MB-231 cells. Therefore, it can be seen that the treatment of aripiprazole inhibits the proliferation of cancer cells in a concentration-dependent manner.
  • C6 cells were incubated at a concentration of 5 ⁇ 10 4 cells / ml in 24-well plates, and then the aripiprazole was treated by concentration (25, 50, and 100 ⁇ M). Then, the shape was photographed using an optical microscope at time zones (0, 3, 6, 12, and 24 h), and the results are shown in FIG. 21.
  • C6 cells were incubated at a concentration of 5 ⁇ 10 5 cells / ml in a 12-well plate, and then aripiprazole was treated by concentration (25, 50, and 100 ⁇ M). After 24 hours, cells were collected using PBS, and only cells were separated by centrifugation. Thereafter, the cells were suspended in 1X FACS buffer, treated with Annexin V and PI dyes, stained for 15 minutes, and fluorescence was observed using a flow cytometer. Staurosporin (2.5 ⁇ M) was used as a comparative experimental group.
  • each sample was treated for a certain time, and total RNA was extracted using TRIzol reagent.
  • the extracted total RNA was prepared cDNA using the first strand cDNA synthesis kit (Thermo scientific), and then the same amount of cDNA was amplified by PCR.
  • the sense and antisense primer sequences of the target protein used were prepared with reference to the existing literature, GAPDH was used as a control gene (see Table 3).
  • PCR amplification was performed by using the Hipi PCR kit (Elpis biotech) to detect the sense and antisense primers of the cDNA and target proteins of each experimental group, and control GAPDH primers with dNTP 250 ⁇ M, Tris-HCl (pH 8.3) 10 mM, KCl 50 mM, NgCl2 1.5. 20 ⁇ l of a Hipi PCR kit containing mM was performed. PCR was performed under conditions of denaturation at 95 ° C. for 45 seconds, annealing at 55 ° C. for 45 seconds, and extension at 72 ° C. for 1 minute, and a total of 30 cycles were performed. DNA amplified by PCR was electrophoresed on 1.5% agarose gel, and the intensity of the fractionated DNA band was measured. The results are shown in FIGS. 28 and 29.
  • Example 10 Aripiprazole Measurement of signaling protein phosphorylation inhibition efficacy
  • C6 cells were cultured using RPMI 1640 medium containing penicillin (100 IU / ml) and streptomycin (100 ⁇ g / ml) and 10% FBS at a concentration of 7 ⁇ 10 6 cells / ml. Pre-incubate in mm dish. After that, each sample was processed and after a predetermined time, the cells were collected and the cells were crushed using lysis buffer and sonicator to obtain western specimens. The protein concentration of each sample was measured using BSA as a standard. The SDS-PAGE was performed with each sample volume that is the protein concentration based on the obtained values, and the proteins were blotting with PVDF membrane using Western blotting.
  • the membrane was blocked with 5% non-fat dried milk (Bio-rad), and the target protein antibody and signaling protein antibody (MMP2,9 / pcaspase 3,8,9 / cleaved caspase 3,8,9 / Bcl -2 / Bax / p53 / pSrc, pStat3, pAKT, pPI3K, HA, ⁇ -actin) solution, followed by primary treatment, followed by a second antibody solution after the washing step and washing.
  • ECL solution Anamersham, England
  • aripiprazole in C6 cells reduced the activity of cell survival signal-related proteins, and also confirmed that it targets Src, which is a representative carcinogen.
  • Example 10-1 Based on the results of Example 10-1, in order to further confirm whether the activity of Src, a cell survival signal related protein of aripiprazole, was directly inhibited, the following experiment was performed.
  • HEK 293 cell lines were dispensed into 6-well plates using Opti-MEM, and then pre-cultured in a 37 ° C. 5% CO 2 cell incubator. When the cells became 50% density after culture, the cells were transfected with pcDNA, HA-Src, HA-Src CA, HA-Src KD, HA-Src SH2, and HA-Src SH3 DNA using the PEI method.
  • 1 ⁇ g of DNA and 3 ⁇ g of PEI were diluted in Opti-MEM, followed by incubation at room temperature for 20 minutes, followed by mixing the diluted solutions for 20 minutes.
  • the mixed solution was placed in a 6-well plate in which cells were dispensed, and after 6 hours, the cells were replaced with cell culture medium [10% FBS, 1% penicillin / streptomycin in DMEM], and after 24 hours, aripiprazole was treated by concentration. After 12 hours incubation. After this, perform Western blot.
  • Example 10-1 Based on the results of Example 10-1, in order to further confirm whether the activity of Src, a cell survival signal related protein of aripiprazole, was directly inhibited, the following experiment was performed.
  • the kinase profiler service of Millipore, USA was used. More specifically, the final solution volume was 25 ⁇ l, and when Syk and Src 1 to 5 mU were incubated with the reaction buffer, MgATP was obtained. The reaction was initiated by addition, and reacted at room temperature for 40 minutes, followed by addition of 5 ml of a 3% phosphoric acid solution. Thereafter, 10 ⁇ l of the solution was dropped onto the P30 filtermat, washed three times with 75 mM phosphoric acid three times, dried over methanol, and measured with a scintillation counter.
  • CT 26 which is a colorectal cancer cell, is transplanted into 1 ⁇ 10 6 cells, and the day after transplantation, oral administration of 1 mg / kg of aripiprazole for 17 days.
  • the tumor size was measured every three days during oral administration, and after 18 days of oral administration, the tumor was sacrificed and the tumor weight was measured.
  • mice orally administered aripiprazole As a result, as shown in Figure 37, it was confirmed that the tumor size of the mice orally administered aripiprazole is smaller, and as shown in Figure 38, mice orally administered to aripiprazole confirmed that the tumor grows less than the control group It was.
  • tablets were prepared by tableting according to a conventional method for producing tablets.
  • the capsule was prepared by filling in gelatin capsules according to the conventional method for producing a capsule.
  • Injectables were prepared by mixing the above ingredients per ampoule (2 ml) according to the usual method for preparing injectables.
  • Aripiprazole according to the present invention shows little decrease in cell viability in RAW264.7 cells, and thus has no cytotoxicity, and a concentration-dependent NO production inhibitory effect, prostaglandin E2 (PGE2) in peptidoglycan treated RAW264.7 cells.
  • PGE2 prostaglandin E2
  • aripiprazole according to the present invention exhibits an excellent cancer cell proliferation inhibitory effect in C6, U251, LN-428 and MDA-MB-231 cells, and is a marker of apoptosis depending on aripiprazole concentration in C6 cells.
  • the effects of blebbing, increased production of apoptotic bodies, accelerated cancer cell death, and increased amount of Active-Caspase were identified.
  • the present invention may be usefully used as an active ingredient of a pharmaceutical composition for preventing, ameliorating or treating an inflammatory disease or cancer.

Abstract

The present invention relates to a composition for preventing or treating inflammatory disease or cancer, the composition containing aripiprazole as an active ingredient. The aripiprazole according to the present invention exhibits hardly any reduction in cell survival and is not cytotoxic in RAW264.7 cells, and, in RAW264.7 cells treated with peptidoglycan, exhibits an outstanding concentration-dependent NO production suppressing ability, prostaglandin E2(PGE2) production suppressing ability and cytokine (COX-2 and TNF-α) secretion suppressing ability, and had an outstanding stomach damage suppressing effect in an in vivo inflammatory model involving the induction of acute inflammation. Also, the aripiprazole according to the present invention has been confirmed to exhibit an outstanding cancer cell proliferation suppressing effect in C6, U251, LN-428 and MDA-MB-231 cells, and, in C6 cells, to increase the occurrence of blebbing and apoptotic bodies which are markers of apoptosis in a fashion dependent on the aripiprazole concentration, and to have an effect in promoting cancer cell apoptosis and in increasing the amount of active caspase. Hence, the present invention can be used as a medicinal product which is useful in preventing or treating inflammatory disease or cancer.

Description

아리피프라졸을 유효성분으로 함유하는 염증성 질환 또는 암 예방 또는 치료용 조성물Inflammatory disease or cancer prevention or treatment composition containing aripiprazole as an active ingredient
본 발명은 아리피프라졸을 유효성분으로 함유하는 염증성 질환 또는 암 예방 또는 치료용 조성물에 관한 것이다.The present invention relates to a composition for preventing or treating inflammatory diseases or cancer containing aripiprazole as an active ingredient.
염증성 질환은 세균의 침입에 의하여 형성되는 농양의 병리적 상태를 의미한다. 세균들 중 그람 양성 구균은 피부감염, 화농, 폐렴, 패혈증 등 다양한 세균 감염 질환을 유발하고, 염증이 일어났을 때 그람 양성 구균이 체내에서 제거되는 과정에서 세포외막으로 펩티도글리칸(PGN)이 방출된다. 이 펩티도글리칸은 그람 양성 구균의 세포 외벽에 대다수를 차지하고 있으며, 추가적인 염증반응을 일으킨다.Inflammatory disease refers to the pathological condition of an abscess formed by the invasion of bacteria. Among the bacteria, Gram-positive cocci cause various bacterial infections such as skin infections, purulent, pneumonia, and sepsis, and when the inflammation occurs, peptidoglycan (PGN) is released into the extracellular membrane in the process of removing Gram-positive cocci from the body. Is released. The peptidoglycans make up the majority of the outer wall of Gram-positive cocci and cause additional inflammatory reactions.
염증은 병원체, 이물질 및 조직 손상에 대한 중요한 생체의 방어 반응으로, 열, 쇠약감, 식욕감퇴 및 오한 등의 전신적 증상, 또는 발적, 부종, 동통 및 기능장애 등의 국소적 증상을 동반한다.Inflammation is an important biological defense response to pathogens, foreign bodies and tissue damage, accompanied by systemic symptoms such as fever, weakness, loss of appetite and chills, or local symptoms such as redness, swelling, pain and dysfunction.
염증은 지속 기간에 따라 급성 염증, 아급성 염증 및 만성 염증으로 분류된다. 급성 염증은 혈관을 기준으로 반응이 일어나며, 호중구(neutrophil)가 주로 관여한다. 특히, 화농성 염증의 경우에는 호중구의 증가가 현저히 나타난다. 만성 염증은 수주 또는 수개월 동안 지속되는 염증으로서, 조직의 손상 및 치유가 동시에 일어나며, 지연된 과민감성 반응을 일으키는 계속적인 감염 (예를 들어, 결핵, 매독, 진균 감염), 지속적인 내독소 (예를 들어, 증가된 혈장 지질) 또는 외독소 (예를 들어, 실리카, 석면, 타르, 수술 봉합사)에 대한 노출, 또는 자가 조직에 대한 자가면역 반응 (예를 들어, 류마티스성 관절염, 전신성 홍반성 낭창, 다발성 경화증, 건선)의 결과로서, 시간이 경과됨에 따라 진행되는 잠행성 과정을 나타낸다. 아급성 염증은 급성과 만성의 중간 단계의 염증을 말한다.Inflammation is classified according to duration into acute inflammation, subacute inflammation and chronic inflammation. Acute inflammation occurs in response to blood vessels, and neutrophils are primarily involved. In particular, in the case of purulent inflammation, an increase in neutrophils is marked. Chronic inflammation is inflammation that lasts for weeks or months, with simultaneous damage and healing of tissues, persistent infections that cause delayed hypersensitivity reactions (eg, tuberculosis, syphilis, fungal infections), persistent endotoxins (eg, , Exposure to increased plasma lipids or exotoxins (eg, silica, asbestos, tar, surgical sutures), or autoimmune responses to autologous tissues (eg, rheumatoid arthritis, systemic lupus erythematosus, multiple sclerosis) , As a result of psoriasis, indicates a latent process that progresses over time. Subacute inflammation refers to acute and chronic intermediate inflammation.
염증의 발생에는 다양한 생화학적 물질 및 현상이 관여하고 있으며, 특히 산화질소 합성효소(nitric oxide synthase; NOS) 및 시클로옥시게나제 (cyclooxygenase; COX)는 염증반응을 매개하는데 있어 중요한 역할을 하는 것으로 알려져 있다.The development of inflammation involves a variety of biochemicals and phenomena, especially nitric oxide synthase (NOS) and cyclooxygenase (COX) are known to play an important role in mediating the inflammatory response. have.
상기 NOS는 L-아르기닌으로부터 NO(nitric oxide)를 생성시키는 효소로, 뇌에 존재하는 bNOS(brain NOS), 신경계에 존재하는 nNOS(neuronal NOS) 및 혈관계에 존재하는 eNOS(endothelial NOS)는 체내에서 항상 일정수준으로 발현되고 있으며, 이들에 의해 소량 생성되는 NO는 신경전달이나 혈관확장을 유도하는 등 정상적인 신체의 항상성 유지에 중요한 역할을 한다. 하지만, iNOS(induced NOS)는 각종 사이토카인이나 외부 자극 물질에 의해 유도되어 과량의 NO를 급격히 발생시켜 세포독성이나 각종 염증반응을 일으키는 것으로 알려져 있다.The NOS is an enzyme for producing NO (nitric oxide) from L-arginine, bNOS (brain NOS) in the brain, nNOS (neuronal NOS) in the nervous system and eNOS (endothelial NOS) in the vascular system are present in the body Always expressed at a certain level, a small amount of NO produced by these plays an important role in maintaining the homeostasis of the normal body, such as inducing neurotransmission or vasodilation. However, iNOS (induced NOS) is induced by various cytokines or external stimulants, and is known to cause excessive toxicity and rapidly cause cytotoxicity and various inflammatory reactions.
상기 COX는 아라키돈산으로부터 프로스타글란딘류를 합성하는데 관여하는 효소로, COX-1 및 COX-2의 2종류가 있다. COX-1은 세포 내에 항상 존재하여 세포보호작용에 필요한 프로스타글란딘을 합성하는 작용을 나타내는 반면, COX-2는 염증 반응시 세포 내에서 급격히 증가되어 염증반응을 일으키는데 있어서 중요한 역할을 수행하는 것으로 알려져 있다.COX is an enzyme involved in synthesizing prostaglandins from arachidonic acid, and there are two types of COX-1 and COX-2. COX-1 is always present in the cell and exhibits the action of synthesizing the prostaglandin required for cytoprotective action, whereas COX-2 is known to play an important role in causing an inflammatory reaction by rapidly increasing in the cell during the inflammatory response.
따라서, NO의 생성 및 COX-2의 발현을 억제하면 염증성 질환을 효과적으로 예방 또는 치료할 수 있을 것으로 생각된다.Therefore, it is thought that inhibiting the production of NO and expression of COX-2 can effectively prevent or treat inflammatory diseases.
최근, 염증의 생성을 줄일 수 있는 방법에 관한 연구가 활발히 진행되고 있으며, 지금까지의 연구결과로는 자극에 의해 조직이나 혈장에서 활성화되는 브라디키닌과 같은 키닌(kinins) 등에 의해 또는 COX 경로에 의해 형성된 다양한 사이토카인, 프로스타글란딘 E2(prostaglandin E2) 등이 부종 형성과 혈관 확장에 관여하여 염증의 원인이 된다는 것이 알려져 있다. 따라서, 항염증제로서 브라드키닌 길항제, COX 억제제 등을 포함하는, 항염증, 해열 및 진통 효과를 갖는 비스테로이드성 항염증제(nonsteroidal anti-inflammatory drugs, NSAIDs)가 많이 사용되어 왔다. 그러나 비스테로이드성 항염증제를 장기간 복용하게 되면 위장관계의 소화성 궤양출혈로 인한 이차적 빈혈 초래, 혈소판 기능 억제, 분만 유도 억제, 신장에 대한 부작용, 간장 손상, 과민 반응 등의 심각한 부작용을 초래한다. 따라서, 이러한 약물의 부작용을 극복하기 위하여 인체에 부작용이 적고 항염증 효과가 우수한 치료제의 개발에 대한 필요성이 절실히 요구되고 있다.In recent years, studies on ways to reduce the production of inflammation have been actively conducted. Until now, studies have been conducted on kinins such as bradykinin activated in tissues or plasma by stimulation, or on the COX pathway. to a variety of cytokines, such as prostaglandin E 2 (prostaglandin E 2) which is formed by that the cause of inflammation involved in edema formation and vasodilation are known. Therefore, nonsteroidal anti-inflammatory drugs (NSAIDs) having anti-inflammatory, antipyretic and analgesic effects, including bradykinin antagonists, COX inhibitors, and the like, have been widely used as anti-inflammatory agents. However, long-term use of nonsteroidal anti-inflammatory drugs can cause serious side effects such as gastrointestinal peptic ulcer bleeding, secondary anemia, inhibition of platelet function, inhibition of labor, side effects on kidneys, hepatic damage, and hypersensitivity. Therefore, in order to overcome the side effects of these drugs, there is an urgent need for the development of a therapeutic agent having a low side effect and excellent anti-inflammatory effect on the human body.
암은 전인류가 극복해야 할 난치병 중 하나로서 우리나라에서도 전체 사망률의 가장 많은 원인 중의 하나라고 할 수 있다. 암을 정복하기 위해 세포주기나 세포사멸의 조절과 발암유전자나 암 억제 유전자들을 포함하는 새로운 표적을 모색함으로써 발전을 거듭해 왔으나 암의 발생률은 여전히 증가 추세에 있다. 현재, 암을 치료하기 위한 방법으로 절개술, 화학요법, 방사선요법, 절개술 등의 많은 방법이 개발되었지만 방사선 요법이나 절개술 등은 주로 암의 초기진단이 이루어졌을 때에만 효과가 있을 뿐 말기암으로 진행된 상태에서는 소용이 없으며 화학요법을 주로 사용하여 치료할 수 밖에 없다. 1940년대부터 암치료에 도입된 화학요법은 암의 시기와 관계없이 비교적 쉽게 적용할 수 있다는 장점이 있어서 현재 많은 관심이 집중되고 있으며, 여러 가지 항암 화학요법제가 개발되고 있다.Cancer is one of the most incurable diseases that all human beings must overcome and can be said to be one of the most common causes of mortality in Korea. In order to conquer cancer, progress has been made in the search for new targets including control of cell cycle or apoptosis, and oncogenic and cancer suppressor genes, but the incidence of cancer is still increasing. Currently, many methods such as incision, chemotherapy, radiotherapy, and incision have been developed to treat cancer, but radiation therapy and incisions are effective only when the initial diagnosis of cancer is performed. Esau is of no use and can only be treated with chemotherapy. Since chemotherapy introduced in cancer treatment since the 1940s has the advantage of being relatively easy to apply regardless of the timing of cancer, a lot of attention is now focused, and various anti-cancer chemotherapy agents are being developed.
하지만, 상기 항암제들은 반복적으로 장기간 투여되거나 암이 재발된 경우에는 암세포가 항암제에 대한 내성을 획득함으로써 치료 효과를 상실하는 단점이 있다. 또한, 대부분의 항암제는 세포 내 핵산의 합성을 억제하거나 핵산에 직접 결합하여 그 기능을 손상시킴으로써 효과를 나타내는데, 이들 항암제는 암세포에만 선택적으로 작용하는 것이 아니라 정상세포, 특히 세포분열이 활발한 조직 세포에도 손상을 입히기 때문에 골수 기능 저하, 위장관 점막 손상, 탈모 등 여러 부작용이 나타나는 단점이 있다.However, the anticancer agents have a disadvantage in that when repeated long-term administration or cancer recurs, cancer cells lose the therapeutic effect by obtaining resistance to the anticancer agent. In addition, most anticancer drugs have an effect by inhibiting the synthesis of nucleic acid in the cell or by directly binding to the nucleic acid and impairing its function. These anticancer agents not only act selectively on cancer cells but also on normal cells, especially tissue cells with active cell division. Because of the damage, there are many side effects such as decreased bone marrow function, damage to the gastrointestinal tract, hair loss, and the like.
한편, 아폽토시스(apoptosis)는 프로그램된 세포 사멸로 알려져 있으며, 이는 세포 항상성 유지에서 중요한 역할을 수행하는 유전적 조절 메카니즘이다. 아폽토시스는 일반적으로 외인성(사멸 수용체) 경로 또는 내인성(미토콘드리아 내) 경로를 통해 유도될 수 있다. 외인성 경로에서, 그 리간드(ligand)에 의한 TNF/Fas-수용체의 라이게이션(ligation)은 카스파제-8 개시자의 절단을 유발하고, 효과자 카스파제-3을 직접적으로 활성화시키거나, Bcl-2 패밀리 멤버인 Bid의 절단을 유도한 후, 미토콘드리아 막으로 Bax의 전위를 유도한다. 반면, 내인성 경로는 미토콘드리아에 의해 매개되고, 아톱토틱 자극에 대한 반응에서, 미토콘드리아로부터 시토크롬 c 및 아폽토시스-유도 인자(apoptosis-inducing factor, AIF)가 방출되며, 이러한 인자들은 카스파제-의존적 및 비의존적 아폽토시스에 관여한다. 시토크롬(cytochrome) c는 Apaf-1과 결합하여 아폽토좀(apoptosome)으로 불리는 구조를 형성하고, 카스파제-9를 활성화시키고, 이어서 카스파제-3를 활성화시킨 다음, 최종적으로 아폽토틱 세포 사멸을 일으킨다. AIF는 카스파제 비의존적 아폽토시스의 징표로, 아폽토틱 자극 후에, 핵내로 전위되어, DNA 단편화(fragmentation)를 유도한다.Apoptosis, on the other hand, is known for programmed cell death, which is a genetic regulatory mechanism that plays an important role in maintaining cell homeostasis. Apoptosis can generally be induced through the exogenous (killing receptor) pathway or the endogenous (within the mitochondria) pathway. In the exogenous pathway, ligation of the TNF / Fas-receptor by its ligand causes cleavage of caspase-8 initiators and directly activate effector caspase-3, or Bcl-2 After inducing cleavage of the family member Bid, a potential of Bax is induced into the mitochondrial membrane. In contrast, the endogenous pathway is mediated by mitochondria, and in response to atopotic stimulation, cytochrome c and apoptosis-inducing factor (AIF) are released from mitochondria, which factors are caspase-dependent and independent. Is involved in apoptosis. Cytochrome c binds with Apaf-1 to form a structure called apoptosome, activates caspase-9, then activates caspase-3, and finally causes apoptotic cell death . AIF is a sign of caspase-independent apoptosis, which is translocated into the nucleus following apoptotic stimulation, leading to DNA fragmentation.
한편, 아리피프라졸(aripiprazole)은 퀴놀리논 유도체로서, 7-{4-[4-(2,3-디클로로페닐)-1-피페라지닐]-부톡시}-3,4-디히드로카보스티릴로 명명된다. 아리피프라졸은 향정신병 치료제로서, 망상, 환각 및 다른 이들로부터의 과도한 위축감을 특징으로 하는 정신분열증의 치료에 유용한 것으로 알려져 있다. 아리피프라졸은 도파민 D2 수용체와 5-HT1A 수용체의 강력한 부분효능제 (partial agonist)로, '도파민-세로토닌계 안정제'라고 불린다. 부분효능제란 수용체가 지나치게 자극을 받으면 수용체를 차단하고, 자극이 부족한 상황에서는 수용체를 활성화하는 물질을 말한다. 이러한 아리피프라졸은 일본 오츠카제약이 개발한 정신분열증(조현병) 치료제로 Abilify™라는 상품명으로 시판되고 있으며, 정신분열증의 치료 또는 개선, 양극성 장애와 관련된 급성 조증의 치료, 주요 우울장애 치료의 부가요법 등의 목적으로 사용되고 있다.Aripiprazole, on the other hand, is a quinolinone derivative, which is 7- {4- [4- (2,3-dichlorophenyl) -1-piperazinyl] -butoxy} -3,4-dihydrocarbostyryl It is named. Aripiprazole is a therapeutic agent for psychotropic diseases and is known to be useful for the treatment of schizophrenia characterized by delusions, hallucinations and excessive atrophy from others. Aripiprazole is a potent partial agonist of the dopamine D2 receptor and the 5-HT1A receptor, and is called a dopamine-serotonin-based stabilizer. Partial agonists are substances that block receptors when they are excessively stimulated, and activate the receptors when they are lacking. Aripiprazole is a schizophrenic (schizophrenia) drug developed by Otsuka Pharmaceutical Co., Ltd. and is marketed under the brand name Abilify ™, and is used for the treatment or improvement of schizophrenia, the treatment of acute mania associated with bipolar disorder, and the treatment of major depression. It is used for the purpose.
상기한 바와 같이, 지금까지 아리피프라졸에 대한 약리작용으로는 중추신경계 및 신경질환에 대해서만 한정되어 연구되어왔으며, 그 기술 또한 약물의 용해도 및 흡수율을 높이는데에만 초점을 맞추어 연구되어 있을 뿐, 아리피프라졸의 항염증 또는 항암 효과에 대해서는 잘 알려져 있지 않으며, 이에 대한 연구도 전무한 상태이다.As described above, the pharmacological action of aripiprazole has been limited to the central nervous system and neurological diseases, and the technique has been studied only to increase the solubility and absorption rate of the drug. Little is known about the inflammatory or anticancer effects, and there is little research on this.
따라서, 아리피프라졸의 항염증 효과에 대한 연구 및 개발의 필요성이 절실히 요구되고 있으며, 또한, 세포사멸의 유도에 따른 항암 치료 효과에 대한 관심의 증가와 함께 현재 사용되고 있는 항암제의 단점들을 극복한 암 치료제의 개발이 주요한 과제의 대상이 되고 있고, 이에 대한 연구(한국공개번호 10-2010-0036145호 등)가 이루어지고 있으나, 아직 미비한 실정이다.Therefore, there is an urgent need for the research and development of the anti-inflammatory effect of aripiprazole, and in addition to the increasing interest in anti-cancer therapeutic effects due to induction of cell death, Development has been the subject of major challenges, and research on this (Korea Publication No. 10-2010-0036145, etc.) has been conducted, but it is still inadequate.
본 발명자들은 아리피프라졸의 항염증 효과에 대해 연구하던 중, 아리피프라졸이 RAW264.7 세포 내에서 세포독성이 없으며, 펩티도글리칸을 처리한 RAW264.7 세포 내에서 농도 의존적으로 NO 생성 억제능, 프로스타글란딘 E2(PGE2)의 생성 억제능 및 사이토카인 (COX-2 및 TNF-α)의 분비 억제능이 우수하고, 급성 염증이 유발된 생체 내 염증 모델에서 위 손상 억제 효과가 우수함을 확인하였다.While we studied the anti-inflammatory effects of aripiprazole, we found that aripiprazole has no cytotoxicity in RAW264.7 cells, and concentration-dependently inhibits NO production, prostaglandin E 2 in RAW264.7 cells treated with peptidoglycan. Inhibition of the production of (PGE 2 ) and secretion of cytokines (COX-2 and TNF-α) was excellent, it was confirmed that the effect of inhibiting gastric damage in the in vivo inflammation model that caused acute inflammation.
또한, 아리피프라졸의 항암 효과에 대해 연구하던 중, C6 , U251, LN-428 및 MDA-MB-231 세포 내에서 아리피프라졸의 우수한 암 세포 증식 억제 효과를 확인하였으며, C6 세포 내에서 아리피프라졸 농도 의존적으로 세포사멸(Apoptosis)의 마커인 blebbing, apoptotic bodies의 생성 및 암세포 사멸을 촉진시키고, Active-Caspase의 양을 증가시킴을 확인하고, 본 발명을 완성하였다.In addition, while studying the anti-cancer effect of aripiprazole, it was confirmed that the excellent cancer cell proliferation inhibitory effect of aripiprazole in C6, U251, LN-428 and MDA-MB-231 cells, apoptosis concentration-dependent cell death in C6 cells Promotes blebbing, apoptotic bodies and apoptosis, and increases the amount of Active-Caspase, a marker of apoptosis, and completed the present invention.
따라서, 본 발명은 아리피프라졸을 유효성분으로 함유하는 염증성 질환 또는 암 예방 또는 치료용 약학적 조성물을 제공하고자 한다.Accordingly, the present invention is to provide a pharmaceutical composition for preventing or treating inflammatory diseases or cancer containing aripiprazole as an active ingredient.
또한, 본 발명은 아리피프라졸을 피험자에게 투여하는 단계를 포함하는 염증성 질환 또는 암 예방 또는 치료 방법을 제공하고자 한다.In addition, the present invention is to provide a method for preventing or treating an inflammatory disease or cancer comprising administering aripiprazole to a subject.
본 발명은 아리피프라졸을 유효성분으로 함유하는 염증성 질환 또는 암 예방 또는 치료용 약학적 조성물을 제공한다.The present invention provides a pharmaceutical composition for preventing or treating inflammatory diseases or cancer containing aripiprazole as an active ingredient.
본 발명의 다른 구현예로서, 상기 아리피프라졸은 산화질소(NO) 및 프로스타글란딘 E2 (PGE2)의 생성을 억제할 수 있다.In another embodiment of the present invention, the aripiprazole may inhibit the production of nitric oxide (NO) and prostaglandin E2 (PGE2).
본 발명의 다른 구현예로서, 상기 아리피프라졸은 NF-κB 및 AP-1(activator protein-1) 활성을 억제할 수 있다.In another embodiment of the present invention, the aripiprazole may inhibit NF-κB and AP-1 (activator protein-1) activity.
본 발명의 다른 구현예로서, 상기 아리피프라졸은 iNOS, COX-2 및 IFN-β의 발현을 억제할 수 있다.In another embodiment of the present invention, the aripiprazole may inhibit the expression of iNOS, COX-2 and IFN-β.
본 발명의 다른 구현예로서, 상기 염증성 질환은 급성 또는 만성 위염, 부종, 피부염, 알레르기, 아토피, 천식, 결막염, 치주염, 비염, 중이염, 인후염, 편도염, 폐렴, 간염, 식도염, 위궤양, 장염, 췌장염, 십이지장궤양, 대장염, 치질, 통풍, 강직성 척추염, 류마티스 열, 루푸스, 섬유근통(fibromyalgia), 건선관절염, 골관절염, 류마티스 관절염, 견관절주위염, 건염, 건초염, 건주위염, 근육염, 방광염, 신장염, 다발성 경화증 및 패혈증으로 이루어진 군으로부터 선택된 1종 이상일 수 있다.In another embodiment, the inflammatory disease is acute or chronic gastritis, edema, dermatitis, allergy, atopic, asthma, conjunctivitis, periodontitis, rhinitis, otitis media, sore throat, tonsillitis, pneumonia, hepatitis, esophagitis, gastric ulcer, enteritis, pancreatitis , Duodenal ulcer, colitis, hemorrhoids, gout, ankylosing spondylitis, rheumatic fever, lupus, fibromyalgia, psoriatic arthritis, osteoarthritis, rheumatoid arthritis, periarthritis, tendinitis, hay salt, tendonitis, myositis, cystitis, nephritis, multiple sclerosis and At least one member selected from the group consisting of sepsis.
본 발명의 또 다른 구현예로서, 상기 염증성 질환은 급성 또는 만성 위염일 수 있다.In another embodiment of the present invention, the inflammatory disease may be acute or chronic gastritis.
본 발명의 다른 구현예로서, 상기 아리피프라졸은 암 세포 내 Caspase-3 단백질의 활성을 증가시켜 세포사멸을 유도할 수 있다. In another embodiment, the aripiprazole may induce apoptosis by increasing the activity of Caspase-3 protein in cancer cells.
본 발명의 다른 구현예로서, 상기 아리피프라졸은 Src 키나아제 활성을 억제할 수 있다.In another embodiment of the present invention, the aripiprazole may inhibit Src kinase activity.
본 발명의 다른 구현예로서, 상기 아리피프라졸은 Bcl2, MMP2 및 MMP9의 발현을 억제할 수 있다.In another embodiment of the present invention, the aripiprazole may inhibit the expression of Bcl2, MMP2 and MMP9.
본 발명의 또 다른 구현예로서, 상기 암은 신경아교종, 갑상선암, 부갑상선암, 골수암, 직장암, 인후암, 후두암, 폐암, 식도암, 췌장암, 대장암, 위암, 설암, 피부암, 뇌종양, 자궁암, 두부 또는 경부암, 담낭암, 구강암, 결장암, 항문 부근암, 간암, 및 대장암으로 이루어진 군으로부터 선택될 수 있다. In another embodiment of the present invention, the cancer is glioma, thyroid cancer, parathyroid cancer, bone marrow cancer, rectal cancer, throat cancer, laryngeal cancer, lung cancer, esophageal cancer, pancreatic cancer, colon cancer, gastric cancer, tongue cancer, skin cancer, brain tumor, uterine cancer, head or neck cancer , Gallbladder cancer, oral cancer, colon cancer, proximal anal cancer, liver cancer, and colorectal cancer.
본 발명의 또 다른 구현예로서, 상기 암은 신경아교종, 위암, 유방암 또는 뇌종양일 수 있다. In another embodiment of the present invention, the cancer may be glioma, gastric cancer, breast cancer or brain tumor.
또한, 본 발명은 아리피프라졸을 피험자에게 투여하는 단계를 포함하는, 염증성 질환 또는 암의 예방 또는 치료 방법을 제공하고자 한다.The present invention also provides a method for preventing or treating an inflammatory disease or cancer, comprising administering aripiprazole to a subject.
또한, 본 발명은 아리피프라졸의 염증성 질환 또는 암 예방 또는 치료 용도를 제공한다.The present invention also provides a use for the prevention or treatment of inflammatory disease or cancer of aripiprazole.
본 발명에 따른 아리피프라졸은, RAW264.7 세포 내에서 세포 생존율의 감소를 거의 나타내지 않아 세포독성이 없으며, 펩티도글리칸을 처리한 RAW264.7 세포 내에서 농도 의존적으로 NO 생성 억제능, 프로스타글란딘 E2(PGE2)의 생성 억제능 및 사이토카인 (COX-2 및 TNF-α)의 분비 억제능이 우수하고, 급성 염증이 유발된 생체 내 염증 모델에서 위 손상 억제 효과가 우수하다.Aripiprazole according to the present invention shows little decrease in cell viability in RAW264.7 cells and thus has no cytotoxicity, and has a concentration-dependent inhibition of NO production, prostaglandin E 2 (Peptidoglycan) in RAW264.7 cells. PGE 2 ) is excellent in inhibiting the production and secretion of cytokines (COX-2 and TNF-α) and gastric damage suppression effect in the in vivo inflammation model that caused acute inflammation.
또한, 본 발명에 따른 아리피프라졸은, C6 , U251, LN-428 및 MDA-MB-231 세포 내에서 우수한 암 세포 증식 억제 효과를 나타내며, C6 세포 내에서 아리피프라졸 농도 의존적으로 세포사멸(Apoptosis)의 마커인 blebbing, apoptotic bodies의 생성 증가, 암세포 사멸 촉진, 및 Active-Caspase의 양 증가 효과를 확인하였다.In addition, aripiprazole according to the present invention exhibits an excellent cancer cell proliferation inhibitory effect in C6, U251, LN-428 and MDA-MB-231 cells, and is a marker of apoptosis depending on aripiprazole concentration in C6 cells. The effects of blebbing, increased production of apoptotic bodies, accelerated cancer cell death, and increased amount of Active-Caspase were identified.
이에, 염증성 질환 또는 암을 예방, 개선 또는 치료하기 위한 약학적 조성물의 활성성분으로 유용하게 사용될 수 있을 것으로 기대된다.Accordingly, it is expected that the present invention may be usefully used as an active ingredient of a pharmaceutical composition for preventing, ameliorating or treating an inflammatory disease or cancer.
도 1은 RAW264.7 세포 내에서 아리피프라졸이 세포 생존에 미치는 영향을 나타낸 도이다.1 is a diagram showing the effect of aripiprazole on cell survival in RAW264.7 cells.
도 2는 펩티도글리칸을 처리한 RAW264.7 세포 내에서 아리피프라졸이 산화질소(NO)의 생성능에 미치는 영향을 나타낸 도이다.Figure 2 shows the effect of aripiprazole on the production of nitric oxide (NO) in peptidoglycan treated RAW264.7 cells.
도 3은 펩티도글리칸을 처리한 RAW264.7 세포 내에서 아리피프라졸이 프로스타글란딘 E2(PGE2)의 생성능에 미치는 영향을 나타낸 도이다.3 is a diagram showing the effect of aripiprazole on the production ability of prostaglandin E 2 (PGE 2 ) in peptidoglycan treated RAW264.7 cells.
도 4는 펩티도글리칸을 처리한 RAW264.7 세포 내에서 아리피프라졸이 사이토카인의 mRNA 발현에 미치는 영향을 나타낸 도이다.4 is a diagram showing the effect of aripiprazole on the mRNA expression of cytokines in RAW264.7 cells treated with peptidoglycan.
도 5는 펩티도글리칸을 처리한 RAW264.7 세포 내에서 아리피프라졸이 iNOS의 mRNA 발현에 미치는 영향을 나타낸 도이다.Figure 5 shows the effect of aripiprazole on mRNA expression of iNOS in peptidoglycan treated RAW264.7 cells.
도 6은 펩티도글리칸을 처리한 RAW264.7 세포 내에서 아리피프라졸이 COX-2의 mRNA 발현에 미치는 영향을 나타낸 도이다.Figure 6 shows the effect of aripiprazole on mRNA expression of COX-2 in peptidoglycan treated RAW264.7 cells.
도 7은 펩티도글리칸을 처리한 RAW264.7 세포 내에서 아리피프라졸이 IFN-β의 mRNA 발현에 미치는 영향을 나타낸 도이다.Figure 7 shows the effect of aripiprazole on mRNA expression of IFN-β in peptidoglycan treated RAW264.7 cells.
도 8은 아리피프라졸을 HEK293 세포에 처리하였을 때, MyD88에 의한 AP-1 프로모터 활성에 미치는 영향을 나타낸 도이다.8 is a diagram showing the effect on the AP-1 promoter activity by MyD88 when aripiprazole was treated to HEK293 cells.
도 9는 아리피프라졸을 HEK293 세포에 처리하였을 때, MyD88에 의한 NF-κB 프로모터 활성에 미치는 영향을 나타낸 도이다.9 is a diagram showing the effect on the NF-κB promoter activity by MyD88 when aripiprazole was treated to HEK293 cells.
도 10은 아리피프라졸을 HEK293 세포에 처리하였을 때, TRIF에 의한 AP-1 프로모터 활성에 미치는 영향을 나타낸 도이다.10 is a diagram showing the effect of TRIF on AP-1 promoter activity when aripiprazole was treated to HEK293 cells.
도 11은 아리피프라졸을 HEK293 세포에 처리하였을 때, TRIF에 의한 NF-κB 프로모터 활성에 미치는 영향을 나타낸 도이다.11 is a diagram showing the effect on TRF-induced NF-κB promoter activity when aripiprazole was treated to HEK293 cells.
도 12는 아리피프라졸을 RAW264.7 cells에 처리하였을 때 펩티도글리칸에 의한 염증관련 핵단백질 p50, p65, c-Fos 및 c-Jun의 핵 내 이동에 미치는 영향을 나타낸 도이다.12 is a diagram showing the effect of aripiprazole in RAW264.7 cells on the nuclear migration of inflammation-related nuclear proteins p50, p65, c-Fos and c-Jun by peptidoglycan.
도 13은 아리피프라졸을 RAW264.7 cells에 처리하였을 때 펩티도글리칸에 의한 신호전달 단백질 Src, Syk, AKT 및 p85의 인산화 억제에 미치는 영향을 나타낸 도이다.13 is a diagram showing the effect on the phosphorylation of the signaling proteins Src, Syk, AKT and p85 by peptidoglycan when aripiprazole is treated in RAW264.7 cells.
도 14는 에탄올/염산으로 ICR 마우스의 급성 위염을 유발한 후, 생체 내 염증 모델에서 아리피프라졸의 위 손상 억제 효과를 나타낸 도이다.14 is a diagram showing the gastric damage inhibition effect of Aripiprazole in the in vivo inflammation model after acute gastritis of ICR mice with ethanol / hydrochloric acid.
도 15는 아스피린으로 ICR 마우스의 급성 위염을 유발한 후, 생체 내 염증 모델에서 아리피프라졸의 위 손상 억제 효과를 나타낸 도이다.15 is a diagram showing the effect of inhibiting the gastric damage of aripiprazole in in vivo inflammation model after acute gastritis of ICR mice with aspirin.
도 16은 C6 세포 내에서 아리피프라졸이 암세포 증식에 미치는 영향을 나타낸 도이다. Figure 16 shows the effect of aripiprazole on cancer cell proliferation in C6 cells.
도 17은 U251 세포 내에서 아리피프라졸이 암세포 증식에 미치는 영향을 나타낸 도이다. 17 shows the effect of aripiprazole on cancer cell proliferation in U251 cells.
도 18은 LN428 세포 내에서 아리피프라졸이 암세포 증식에 미치는 영향을 나타낸 도이다.Figure 18 shows the effect of aripiprazole on cancer cell proliferation in LN428 cells.
도 19는 MDA-MB-231 세포 내에서 아리피프라졸이 암세포 증식에 미치는 영향을 나타낸 도이다.19 is a diagram showing the effect of aripiprazole on cancer cell proliferation in MDA-MB-231 cells.
도 20은 MKN-1 세포 내에서 아리피프라졸이 암세포 증식에 미치는 영향을 나타낸 도이다.20 is a diagram showing the effect of aripiprazole on cancer cell proliferation in MKN-1 cells.
도 21은 C6 세포 내에서 아리피프라졸 처리에 의한 세포 형태의 변화를 광학 현미경을 통하여 확인한 결과이다. 21 shows the results of confirming the change in cell morphology by aripiprazole treatment in C6 cells through an optical microscope.
도 22는 C6 세포 내에서 아리피프라졸이 세포의 이동 (migration)에 미치는 영향을 나타낸 도이다.22 shows the effect of aripiprazole on migration of cells in C6 cells.
도 23은 U251 세포 내에서 아리피프라졸이 세포의 이동 (migration)에 미치는 영향을 나타낸 도이다.Figure 23 shows the effect of aripiprazole on migration of cells in U251 cells.
도 24는 C6 세포 내에서 아리피프라졸 처리에 의한 핵 수축을 확인한 결과를 나타낸 도이다.Fig. 24 shows the results of confirming nuclear contraction by aripiprazole treatment in C6 cells.
도 25는 C6 세포 내에서 아리피프라졸 처리에 의한 Actin disruption을 확인한 결과를 나타낸 도이다.Figure 25 shows the results of confirming Actin disruption by aripiprazole treatment in C6 cells.
도 26은 C6 세포 내에 아리피프라졸 처리에 의한 암세포의 세포사멸을 Flow cytometer를 통하여 확인한 결과이다. Fig. 26 shows the results of apoptosis of cancer cells by aripiprazole treatment in C6 cells by flow cytometer.
도 27은 C6 세포 내에서 아리피프라졸에 의해 세포 분열 주기가 중단되는 것을 확인한 결과이다.FIG. 27 shows the results of confirming that cell division cycle is interrupted by aripiprazole in C6 cells.
도 28은 C6 세포 내에 아리피프라졸 처리에 의한 Bcl-2의 mRNA 발현에 미치는 영향을 나타낸 도이다.28 is a diagram showing the effect on the mRNA expression of Bcl-2 by aripiprazole treatment in C6 cells.
도 29는 U251 세포 내에 아리피프라졸 처리에 의한 MMP2 및 MMP 9의 mRNA 발현에 미치는 영향을 나타낸 도이다.29 is a diagram showing the effect on the mRNA expression of MMP2 and MMP 9 by Aripiprazole treatment in U251 cells.
도 30은 U251 세포 내에 아리피프라졸 처리에 의한 MMP2 및 MMP 9의 단백질 발현에 미치는 영향을 나타낸 도이다.Figure 30 shows the effect on the protein expression of MMP2 and MMP 9 by Aripiprazole treatment in U251 cells.
도 31은 C6 세포 내에 아리피프라졸 처리에 의한 세포사멸 유도 및 억제 인자의 발현 활성 효과를 확인한 결과이다.31 shows the results of confirming the effects of apoptazole treatment on apoptosis induction and expression activity of C6 cells.
도 32는 C6 세포 내에 아리피프라졸 처리에 의한 세포생존신호 관련 단백질 활성 효과를 확인한 결과이다.32 shows the results of confirming the cell survival signal-related protein activity effect by Aripiprazole treatment in C6 cells.
도 33은 HEK 293 세포에서 Src 과 발현시 인산화된 Src 발현 활성 억제 효과를 확인한 결과이다.33 is a result confirming the inhibitory effect of phosphorylated Src expression activity when Src and expression in HEK 293 cells.
도 34는 HEK 293 세포에서 계속해서 활성화되어 있는 Src (HA-Src CA)에서 인산화된 Src 발현 활성 억제 효과를 확인한 결과이다.FIG. 34 shows the results of confirming the effect of inhibiting phosphorylated Src expression activity in Src (HA-Src CA) which is continuously activated in HEK 293 cells.
도 35는 HEK 293 세포에서 SH 2 및 SH 3 도메인을 제거한 Src에서 인산화된 Src 발현 활성 억제 효과를 확인한 결과이다.35 is a result confirming the inhibitory effect of phosphorylated Src expression activity in Src from which the SH 2 and SH 3 domains are removed from HEK 293 cells.
도 36은 아리피프라졸의 인산화효소 활성 테스트 결과를 확인한 결과이다.36 shows the results of confirming the kinase activity test result of aripiprazole.
도 37은 대장암 세포를 유도한 동물 모델에서 아리피프라졸을 경구 투여한 마우스의 종양 크기를 확인한 결과이다.Figure 37 shows the results of confirming the tumor size of the mouse orally administered aripiprazole in an animal model induced colon cancer cells.
도 38은 대장암 세포를 유도한 동물 모델에서 아리피프라졸을 경구 투여한 마우스의 종양 부피 증가량을 확인한 결과이다.Figure 38 shows the results of confirming the tumor volume increase of the oral administration of aripiprazole in the animal model induced colon cancer cells.
도 39는 대장암 세포를 유도한 동물 모델에서 아리피프라졸을 경구 투여한 마우스의 종양 무게를 확인한 결과이다.39 shows the tumor weights of mice administered orally with aripiprazole in an animal model inducing colorectal cancer cells.
본 발명은 아리피프라졸을 유효성분으로 함유하는 염증성 질환 또는 암 예방 개선 또는 치료용 조성물을 제공한다.The present invention provides a composition for improving or treating inflammatory diseases or cancer prevention containing aripiprazole as an active ingredient.
상기 조성물은 약학적 조성물을 포함한다.The composition comprises a pharmaceutical composition.
이하, 본 발명에 대해 상세히 설명한다.Hereinafter, the present invention will be described in detail.
본 발명에 따른 아리피프라졸은, RAW264.7 세포 내에서 세포 생존율의 감소를 거의 나타내지 않아 세포독성이 없으며, 펩티도글리칸을 처리한 RAW264.7 세포 내에서 농도 의존적으로 NO 생성 억제능, 프로스타글란딘 E2(PGE2)의 생성 억제능 및 사이토카인 (COX-2 및 TNF-α)의 분비 억제능이 우수하고, 급성 염증이 유발된 생체 내 염증 모델에서 위 손상 억제 효과가 우수하다. 따라서, 본 발명에 따른 아리피프라졸은 염증성 질환의 예방 또는 치료에 유용한 의약품으로 사용될 수 있다.Aripiprazole according to the present invention shows little decrease in cell viability in RAW264.7 cells and thus has no cytotoxicity, and has a concentration-dependent inhibition of NO production, prostaglandin E 2 (Peptidoglycan) in RAW264.7 cells. PGE 2 ) is excellent in inhibiting the production and secretion of cytokines (COX-2 and TNF-α) and gastric damage suppression effect in the in vivo inflammation model that caused acute inflammation. Therefore, aripiprazole according to the present invention can be used as a medicament useful for the prevention or treatment of inflammatory diseases.
상기 염증성 질환은 급성 또는 만성 위염, 부종, 피부염, 알레르기, 아토피, 천식, 결막염, 치주염, 비염, 중이염, 인후염, 편도염, 폐렴, 간염, 식도염, 위궤양, 장염, 췌장염, 십이지장궤양, 대장염, 치질, 통풍, 강직성 척추염, 류마티스 열, 루푸스, 섬유근통(fibromyalgia), 건선관절염, 골관절염, 류마티스 관절염, 견관절주위염, 건염, 건초염, 건주위염, 근육염, 방광염, 신장염, 다발성 경화증 및 패혈증 등을 포함하나, 이에 한정되지 않는다.The inflammatory diseases include acute or chronic gastritis, edema, dermatitis, allergies, atopic, asthma, conjunctivitis, periodontitis, rhinitis, otitis media, sore throat, tonsillitis, pneumonia, hepatitis, esophagitis, gastric ulcer, enteritis, pancreatitis, duodenal ulcer, colitis, hemorrhoids, Include, but are not limited to, gout, ankylosing spondylitis, rheumatic fever, lupus, fibromyalgia, psoriatic arthritis, osteoarthritis, rheumatoid arthritis, periarthritis, tendinitis, hay salt, peritonitis, myositis, cystitis, nephritis, multiple sclerosis and sepsis It doesn't work.
또한, 본 발명에 따른 아리피프라졸은, C6 , U251, LN-428 및 MDA-MB-231 세포 내에서 우수한 암 세포 증식 억제 효과를 나타내며, C6 세포 내에서 아리피프라졸 농도 의존적으로 세포사멸(Apoptosis)의 마커인 blebbing, apoptotic bodies의 생성 증가, 암세포 사멸 촉진, 및 Active-Caspase의 양 증가 효과를 확인하였다. 따라서, 본 발명에 따른 아리피프라졸은 암의 예방 또는 치료에 유용한 의약품으로 사용될 수 있다.In addition, aripiprazole according to the present invention exhibits an excellent cancer cell proliferation inhibitory effect in C6, U251, LN-428 and MDA-MB-231 cells, and is a marker of apoptosis depending on aripiprazole concentration in C6 cells. The effects of blebbing, increased production of apoptotic bodies, accelerated cancer cell death, and increased amount of Active-Caspase were identified. Therefore, aripiprazole according to the present invention can be used as a medicament useful for the prevention or treatment of cancer.
상기 암은 신경아교종, 갑상선암, 부갑상선암, 골수암, 직장암, 인후암, 후두암, 폐암, 식도암, 췌장암, 대장암, 위암, 설암, 피부암, 뇌종양, 자궁암, 두부 또는 경부암, 담낭암, 구강암, 결장암, 항문 부근암, 간암, 및 대장암 등을 포함하나, 이에 한정되지 않는다. The cancer is glioma, thyroid cancer, parathyroid cancer, bone marrow cancer, rectal cancer, throat, larynx cancer, lung cancer, esophageal cancer, pancreatic cancer, colon cancer, stomach cancer, tongue cancer, skin cancer, brain tumor, uterine cancer, head or neck cancer, gallbladder cancer, oral cancer, colon cancer, near the anus Cancer, liver cancer, colon cancer, and the like.
본 발명의 조성물은 아리피프라졸과 함께 항염증 또는 항암 효과를 갖는 공지의 유효성분을 1종 이상 함유할 수 있다.The composition of the present invention may contain at least one known active ingredient having anti-inflammatory or anticancer effect together with aripiprazole.
본 발명의 조성물은, 투여를 위해서 상기 기재한 유효성분 이외에 추가로 약학적으로 허용가능한 담체를 1종 이상 포함하여 제조할 수 있다. 약학적으로 허용 가능한 담체는 식염수, 멸균수, 링거액, 완충 식염수, 덱스트로오스 용액, 말토덱스트린 용액, 글리세롤, 에탄올 및 이들 성분 중 1 성분 이상을 혼합하여 사용할 수 있으며, 필요에 따라 항산화제, 완충액, 정균제 등 다른 통상의 첨가제를 첨가할 수 있다. 또한 희석제, 분산제, 계면활성제, 결합제 및 윤활제를 부가적으로 첨가하여 수용액, 현탁액, 유탁액 등과 같은 주사용 제형, 환약, 캡슐, 과립 또는 정제로 제제화할 수 있다. 더 나아가 당분야의 적정한 방법을 이용하여 각 질환에 따라 또는 성분에 따라 바람직하게 제제화할 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. Pharmaceutically acceptable carriers may be used in combination with saline, sterile water, Ringer's solution, buffered saline, dextrose solution, maltodextrin solution, glycerol, ethanol and one or more of these components, if necessary, antioxidants, buffers And other conventional additives such as bacteriostatic agents can be added. Diluents, dispersants, surfactants, binders and lubricants may also be added in addition to formulate into injectable formulations, pills, capsules, granules or tablets such as aqueous solutions, suspensions, emulsions and the like. Furthermore, it can be preferably formulated according to each disease or component using appropriate methods in the art.
본 발명의 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. 상기 아리피프라졸의 일일 투여량은 약 0.0001~100 ㎎/㎏, 바람직하게는 약 0.001~50 ㎎/㎏이며, 하루 일회 내지 수회에 나누어 투여하는 것이 바람직하다.The composition of the present invention can 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 and health of the patient. The range varies depending on the diet, the time of administration, the method of administration, the rate of excretion and the severity of the disease. The daily dose of aripiprazole is about 0.0001 to 100 mg / kg, preferably about 0.001 to 50 mg / kg, preferably administered once to several times a day.
본 발명의 조성물은 염증성 질환 또는 암 예방 또는 치료를 위하여 단독으로, 또는 수술, 호르몬 치료, 약물 치료 및 생물학적 반응 조절제를 사용하는 방법들과 병용하여 사용할 수 있다.The compositions of the present invention can be used alone or in combination with methods for using surgery, hormone therapy, drug therapy and biological response modifiers for the prevention or treatment of inflammatory diseases or cancer.
또한, 본 발명은 아리피프라졸을 피험자에게 투여하는 단계를 포함하는 염증성 질환 또는 암 예방 또는 치료 방법을 제공한다.The present invention also provides a method for preventing or treating an inflammatory disease or cancer comprising administering aripiprazole to a subject.
상기 피험자는 인간 또는 비-인간을 포함하는 포유류이며, 비-인간 포유류는 마우스, 랫트, 개, 고양이, 말, 소, 양, 염소, 돼지, 토끼 등을 포함하나 이에 한정되지 않는다.The subject is a human or non-human mammal, and non-human mammals include, but are not limited to, mice, rats, dogs, cats, horses, cattle, sheep, goats, pigs, rabbits, and the like.
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.
실시예Example 1:  One: RAW264RAW264 .7 세포 내에서 Within the cell 아리피프라졸이Aripiprazole 세포 생존에 미치는 영향 Impact on cell survival
RAW264.7 세포 내에서 아리피프라졸이 세포 생존에 미치는 영향을 확인하기 위하여, MTT 어세이를 이용하여 하기와 같은 실험을 수행하였다.In order to confirm the effect of aripiprazole on cell survival in RAW264.7 cells, the following experiment was performed using an MTT assay.
실시예 1-1. 세포배양Example 1-1. Cell culture
쥐과(murine) 대식세포주인 RAW264.7 세포를 페니실린 (100 IU/㎖), 스트렙토마이신 (100㎍/㎖) 및 10% FBS를 함유하는 RPMI1640 배지를 이용하여 100㎜ 세포 배양 접시에서 70~80%의 밀도로 배양하였으며, 사람(Human) 세포주인 HEK293 세포를 페니실린 (100 IU/㎖), 스트렙토마이신 (100㎍/㎖) 및 10% FBS를 함유하는 DMEM 배지를 이용하여 100㎜ 세포 배양 접시에서 70~80%의 밀도로 배양하였다.RAW264.7 cells, a murine macrophage line, were 70-80% in 100 mm cell culture dishes using RPMI1640 medium containing penicillin (100 IU / ml), streptomycin (100 µg / ml) and 10% FBS. Human cell line HEK293 cells were cultured at a density of 70 and cultured in 100 mm cell culture dishes using DMEM medium containing penicillin (100 IU / mL), streptomycin (100 μg / mL) and 10% FBS. Incubated at a density of ˜80%.
실시예 1-2. 세포 생존율Example 1-2. Cell viability
96-웰 플레이트에 1 × 106 cell/㎖의 세포를 플레이팅하고, 각 웰에 아리피프라졸 (5, 10, 20 μM)을 가한 다음, 37℃에서 각 면역실험 조건에 상응하는 배양시간 (6, 24 시간) 동안 CO2 배양기에서 배양하였다. 이후 10㎕의 MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphinyltetrazolium bromide) 용액 (저장 농도: 5 mg/㎖)을 첨가하고 3시간 동안 추가반응을 유도하였다. 반응 종료 및 포마잔 결정을 용해하기 위하여, 각 웰에 100 ㎕의 MTT 정지 용액 (0.01M HCl 내 10% 소듐 도데실 설페이트)을 추가적으로 첨가하였다. 세포 생존율은 MTT가 포마잔으로 환원된 양을 570 nm에서 흡광도를 측정하여 얻어진 OD 값을 통해 산출하였다. Plate 1 × 10 6 cells / ml of cells in a 96-well plate, add aripiprazole (5, 10, 20 μM) to each well, and incubate time (6, 24 hours) in a CO 2 incubator. Then 10 μl of MTT (3- [4,5-dimethylthiazol-2-yl] -2,5-diphinyltetrazolium bromide) solution (storage concentration: 5 mg / ml) was added and further reaction was induced for 3 hours. To complete the reaction and dissolve the formazan crystals, 100 μl of MTT stop solution (10% sodium dodecyl sulfate in 0.01M HCl) was added to each well. Cell viability was calculated through the OD value obtained by measuring the absorbance at 570 nm the amount of MTT reduced to formazan.
결과는 도 1에 나타내었다.The results are shown in FIG.
도 1에 나타난 바와 같이, 아리피프라졸은 RAW264.7 세포 내에서 세포 생존율의 감소를 거의 나타내지 않아 세포독성이 없음을 확인하였다.As shown in FIG. 1, aripiprazole showed little decrease in cell viability in RAW264.7 cells, confirming that there was no cytotoxicity.
실시예 2: RAW264.7 세포 내에서 아리피프라졸이 산화질소(NO)의 생성능에 미치는 영향Example 2: Effect of Aripiprazole on the Production of Nitric Oxide (NO) in RAW264.7 Cells
RAW264.7 세포 내에서 아리피프라졸이 산화질소(NO) 생성능에 미치는 영향을 확인하기 위하여, 하기와 같은 실험을 수행하였다.To determine the effect of aripiprazole on nitric oxide (NO) production in RAW264.7 cells, the following experiment was performed.
마우스 대식세포주인 RAW264.7 세포를 페니실린 (100 IU/㎖), 스트렙토마이신 (100㎍/㎖) 및 10% FBS를 함유하는 RPMI1640 배지를 이용하여 1 × 106 cell/㎖의 농도로 조절한 후, 96 웰 플레이트에 접종하고, 5% CO2 및 37℃에서 18시간 동안 전배양하였다. 이후 배지를 제거하고 아리피프라졸 (5, 10, 20 μM)과 10 ㎍/㎖의 펩티도글리칸을 함유한 배지를 동시에 처리하여 배양하였다. 24시간 후 상층액을 100㎕씩 다른 96 웰 플레이트에 옮기고, 상층액 내 NO 정량은 Griess 용액 (0.5% naphthylethylenediamine dihydrochloride, 5% sulfanilamide, 25% H3PO4)을 이용하여 540 nm에서 흡광도를 측정하였다. 표준물질로 아질산나트륨(sodium nitrite; 0 에서 100 μM)을 사용하여 검량선을 작성하였다.RAW264.7 cells, a mouse macrophage line, were adjusted to a concentration of 1 × 10 6 cells / mL using RPMI1640 medium containing penicillin (100 IU / mL), streptomycin (100 μg / mL) and 10% FBS. , 96 well plates were inoculated and pre-incubated at 5% CO 2 and 37 ° C. for 18 hours. The medium was then removed and incubated with aripiprazole (5, 10, 20 μM) and a medium containing 10 μg / ml of peptidoglycan simultaneously. After 24 hours, the supernatant was transferred to another 96 well plate at 100 μl, and the amount of NO in the supernatant was measured for absorbance at 540 nm using Griess solution (0.5% naphthylethylenediamine dihydrochloride, 5% sulfanilamide, 25% H 3 PO 4 ). It was. A calibration curve was prepared using sodium nitrite (0 to 100 μM) as a standard.
결과는 도 2에 나타내었다.The results are shown in FIG.
도 2에 나타난 바와 같이, 펩티도글리칸을 처리한 RAW264.7 세포 내에서 아리피프라졸은 농도가 증가할수록 NO 생성을 감소시켰다. 따라서, 아리피프라졸은 농도 의존적으로 NO 생성 억제능이 우수함을 알 수 있다.As shown in FIG. 2, aripiprazole in RAW264.7 cells treated with peptidoglycan decreased NO production with increasing concentration. Therefore, it can be seen that aripiprazole has an excellent ability to inhibit NO production in a concentration-dependent manner.
실시예Example 3:  3: RAW264RAW264 .7 세포 내에서 Within the cell 아리피프라졸이Aripiprazole 프로스타글란딘 E2( Prostaglandin E2 ( PGE2PGE2 )의 생성능에 미치는 영향Effect on the ability to produce)
RAW264.7 세포 내에서 아리피프라졸이 프로스타글란딘 E2(PGE2)의 생성능에 미치는 영향을 확인하기 위하여, 하기와 같은 실험을 수행하였다.To determine the effect of aripiprazole on the production of prostaglandin E 2 (PGE 2 ) in RAW264.7 cells, the following experiment was performed.
마우스 대식세포주인 RAW264.7 세포를 페니실린 (100 IU/㎖), 스트렙토마이신 (100㎍/㎖) 및 10% FBS를 함유하는 RPMI1640 배지를 이용하여 1 × 106 cell/㎖의 농도로 조절한 후, 96 웰 플레이트에 접종하고, 5% CO2 및 37℃에서 18시간 동안 전배양하였다. 이후 배지를 제거하고 아리피프라졸 (10, 20 μM)과 10 ㎍/㎖의 펩티도글리칸을 함유한 배지를 동시에 처리하여 배양하였다. 24시간 후 상층액을 100㎕씩 다른 96 웰 플레이트에 옮기고, 상층액 내 PGE2의 농도를 Griess 시약과 EIA 키트로 측정하였다.RAW264.7 cells, a mouse macrophage line, were adjusted to a concentration of 1 × 10 6 cells / mL using RPMI1640 medium containing penicillin (100 IU / mL), streptomycin (100 μg / mL) and 10% FBS. , 96 well plates were inoculated and pre-incubated at 5% CO 2 and 37 ° C. for 18 hours. The medium was then removed and incubated with aripiprazole (10, 20 μM) and a medium containing 10 μg / ml of peptidoglycan simultaneously. After 24 hours, the supernatant was transferred to another 96 well plate at 100 µl, and the concentration of PGE 2 in the supernatant was measured by Griess reagent and EIA kit.
결과는 도 3에 나타내었다.The results are shown in FIG.
도 3에 나타난 바와 같이, 펩티도글리칸을 처리한 RAW264.7 세포 내에서 아리피프라졸은 농도가 증가할수록 프로스타글란딘 E2(PGE2)의 생성을 감소시켰다. 따라서, 아리피프라졸은 농도 의존적으로 프로스타글란딘 E2(PGE2)의 생성 억제능이 우수함을 알 수 있다.As shown in FIG. 3, aripiprazole in peptidoglycan treated RAW264.7 cells decreased the production of prostaglandin E 2 (PGE 2 ) at increasing concentrations. Therefore, it can be seen that aripiprazole is excellent in inhibiting the production of prostaglandin E 2 (PGE 2 ) in a concentration-dependent manner.
실시예 4: RAW264.7 세포 내에서 아리피프라졸이 사이토카인의 mRNA 발현에 미치는 영향Example 4 Effect of Aripiprazole on mRNA Expression of Cytokines in RAW264.7 Cells
RAW264.7 세포 내에서 아리피프라졸이 사이토카인의 mRNA 발현에 미치는 영향을 확인하기 위하여, RT-PCR 및 quantitative realtime RT-PCR을 이용하여 하기와 같은 실험을 수행하였다.In order to confirm the effect of aripiprazole on cytokine mRNA expression in RAW264.7 cells, the following experiments were performed using RT-PCR and quantitative realtime RT-PCR.
사이토카인의 mRNA 발현 정도를 전사 수준에서 측정하기 위하여, 먼저 RAW264.7 세포 (1 × 106 cells/㎖)에 펩티도글리칸 (10 ㎍/㎖)을 처리하거나 또는 처리하지 않고 아리피프라졸 (5, 10, 20 μM)과 함께 6시간 동안 배양하였다. 그 다음, 제조사의 지시에 따라 TRIzol 시약 (Gibco BRL)으로 펩티도글리칸-처리한 RAW264.7 세포로부터 총 RNA를 추출하고 사용 전까지 -70℃에 보관하였다. 추출한 총 RNA를 제 1 가닥 cDNA 합성 키트 (Thermo scientific)를 사용하여 cDNA를 제조한 다음, 동량의 cDNA를 PCR로 증폭하였다. 이때 사용한 사이토카인의 센스 및 안티센스 프라이머 염기서열은 기존문헌을 참조하여 제조하였고, 대조군 유전자로는 GAPDH를 사용하였다 (표 1 참조). PCR 증폭은 Hipi PCR kit (Elpis biotech)를 사용하여 각 실험군의 cDNA와 사이토카인 (COX-2 및 TNF-α)의 센스 및 안티센스 프라이머, 대조군 GAPDH 프라이머를 dNTP 250 μM, Tris-HCl (pH 8.3) 10 mM, KCl 50 mM, NgCl2 1.5 mM를 포함한 Hipi PCR kit 20 ㎕에서 수행하였다. PCR은 95℃에서 45초간 변성, 55℃에서 45초간 어닐링, 및 72℃에서 1분간 신장하는 조건으로 수행하였으며, 총 30 주기를 수행하였다. PCR로 증폭된 DNA는 1.5% 아가로오스 겔에서 전기영동 하였고, 분획된 DNA 밴드의 세기를 측정하였으며, quantitative realtime RT-PCR amplication은 SYBR Premix Ex Taq (Takara Bio)과 realtime thermal cycler (Bio-Rad)를 제조사의 매뉴얼에 따라 수행하였다. 이 때, 사용한 사이토카인의 센스 및 안티센스 프라이머 염기서열은 기존문헌을 참조하여 제조하였고, 대조군 유전자로는 GAPDH를 사용하였다 (표 2 참조). 결과는 도 4에 나타내었다.In order to measure the mRNA expression level of cytokines at the transcription level, RAW264.7 cells (1 × 10 6 cells / ml) were treated with or without treatment with aripiprazole (5, with or without peptidoglycan (10 μg / ml). 10, 20 μM) for 6 hours. Next, total RNA was extracted from peptidoglycan-treated RAW264.7 cells with TRIzol reagent (Gibco BRL) and stored at −70 ° C. until use, according to the manufacturer's instructions. The extracted total RNA was prepared cDNA using the first strand cDNA synthesis kit (Thermo scientific), and then the same amount of cDNA was amplified by PCR. The sense and antisense primer sequences of the cytokines used at this time were prepared with reference to the existing literature, and GAPDH was used as a control gene (see Table 1). PCR amplification was performed by using the Hipi PCR kit (Elpis biotech) to sense and antisense primers of cDNA and cytokines (COX-2 and TNF-α) and control GAPDH primers of each experimental group, dNTP 250 μM, Tris-HCl (pH 8.3). 20 μl of a Hipi PCR kit containing 10 mM, KCl 50 mM, NgCl 2 1.5 mM. PCR was performed under conditions of denaturation at 95 ° C. for 45 seconds, annealing at 55 ° C. for 45 seconds, and extension at 72 ° C. for 1 minute, and a total of 30 cycles were performed. DNA amplified by PCR was electrophoresed on 1.5% agarose gel, and the intensity of the fractionated DNA band was measured, and quantitative realtime RT-PCR amplication was performed with SYBR Premix Ex Taq (Takara Bio) and realtime thermal cycler (Bio-Rad). ) Was performed according to the manufacturer's manual. At this time, the sense and antisense primer sequences of the cytokines used were prepared with reference to the existing literature, and GAPDH was used as a control gene (see Table 2). The results are shown in FIG.
프라이머primer 염기서열 (5‘ → 3’)Sequence (5 '→ 3')
COX-2COX-2 정방향Forward direction CACTACATCCTGACCCACTTCACTACATCCTGACCCACTT
역방향Reverse ATGCTCCTGCTTGAGTATGTATGCTCCTGCTTGAGTATGT
TNF-αTNF-α 정방향Forward direction TTGACCTCAG CGCTGAGTTGTTGACCTCAG CGCTGAGTTG
역방향Reverse CCTGTAGCCC ACGTCGTAGCCCTGTAGCCC ACGTCGTAGC
GAPDHGAPDH 정방향Forward direction ACCACAGTCC ATGCCATCACACCACAGTCC ATGCCATCAC
역방향Reverse CCACCACCCT GTTGCTGTAGCCACCACCCT GTTGCTGTAG
프라이머primer 염기서열 (5‘ → 3’)Sequence (5 '→ 3')
iNOSiNOS 정방향Forward direction GGAGCCTTTAGACCTCAACAGAGGAGCCTTTAGACCTCAACAGA
역방향Reverse TGAACGAGGAGGGTGGTGTGAACGAGGAGGGTGGTG
COX-2COX-2 정방향Forward direction CACTACATCCTGACCCACTTCACTACATCCTGACCCACTT
역방향Reverse ATGCTCCTGCTTGAGTATGTATGCTCCTGCTTGAGTATGT
IFN-βIFN-β 정방향Forward direction CTGGCTTCCATCATGAACAACTGGCTTCCATCATGAACAA
역방향Reverse CATTTCCGAATGTTCGTCCTCATTTCCGAATGTTCGTCCT
GAPDHGAPDH 정방향Forward direction CAATGAATACGGCTACAGCAACCAATGAATACGGCTACAGCAAC
역방향Reverse AGGGAGATGCTCAGTGTTGGAGGGAGATGCTCAGTGTTGG
로시스 펙터 알파 (TNF-α)의 mRNA 발현 정도를 현저히 감소시켰으며, 따라서, 아리피프라졸은 농도 의존적으로 사이토카인 (COX-2 및 TNF-α)의 분비 억제능이 우수함을 알 수 있다. The degree of mRNA expression of lossy factor alpha (TNF-α) was significantly reduced, and thus, it was found that aripiprazole was excellent in inhibiting the secretion of cytokines (COX-2 and TNF-α) in a concentration-dependent manner.
또한, 도 5 내지 7에 나타난 바와 같이, 아리피프라졸을 RAW264.7 세포에 처리하였을 때, 펩티도글리칸에 의한 inducible NO synthase (iNOS), 시클로 옥시게나제 2 (COX-2) 및 인터페론 베타(IFN-β)의 mRNA 발현을 감소시키는 것을 확인하였다. In addition, as shown in FIGS. 5 to 7, when aripiprazole was treated to RAW264.7 cells, inducible NO synthase (iNOS), cyclooxygenase 2 (COX-2), and interferon beta (IFN) by peptidoglycan -β) mRNA expression was reduced.
실시예Example 5 :  5: 아리피프라졸의Aripiprazole 염증관련 유전자 활성억제 효과 Inflammation-related gene activity inhibitory effect
5-1. 전사인자 활성 평가5-1. Transcription Factor Activity Evaluation
아리피프라졸이 염증관련 유전자의 활성에 미치는 영향을 확인하기 위하여, 이들 유전자의 발현을 조절하는 전사인자인 NF-κB, AP-1의 활성을 Luciferase gene promotor 발현 측정법으로 측정하였다.To determine the effect of aripiprazole on the activity of inflammation-related genes, the activity of NF-κB, AP-1, which is a transcription factor that regulates the expression of these genes, was measured by Luciferase gene promotor expression measurement.
보다 구체적으로, HEK 293 세포주를 Opti-MEM을 이용하여 24-well 플레이트에 분주한 후, 37℃ 5% CO2 세포배양기에서 전배양하였다. 배양 후 세포가 50% 밀도가 되었을 때, PEI 법을 이용하여 AP-1 또는 NF-κB Luciferase DNA와 MyD88 또는 TRIF DNA와 β-galactosidase DNA를 co-transfection 하였다. PEI 법은 DNA 1 ㎍과 PEI 3 ㎍을 Opti-MEM에 각각 희석해 준 후, 상온에서 20분 동안 배양한 후, 각각 희석액을 혼합하여 다시 20분 배양하는 방법으로 진행하였다. 배양 후 혼합액을 세포가 분주된 24-well 플레이트에 넣어준 후, 6시간 후 세포배양 배지[10 % FBS, 1 % 페니실린/스트렙토마이신 in DMEM]로 교체해주고, 24시간 배양 후 아리피프라졸을 농도별로 처리한 후 9시간 배양하였다. 이 후, lysis buffer를 이용하여 세포를 용해시키고, 기질과 1:1롤 반응시키고 바로 Luminometer로 흡광도를 측정하였다. β-galactosidase DNA의 경우는 기질인 X-gal과 1:1로 반응시킨 후 37 ℃ 배양기에서 5분 배양한 후 405 ㎚로 흡광도를 측정한다.More specifically, HEK 293 cell lines were dispensed into 24-well plates using Opti-MEM, and then pre-incubated in a 37 ° C. 5% CO 2 cell incubator. When cultured to 50% density, the cells were co-transfected with AP-1 or NF-κB Luciferase DNA, MyD88 or TRIF DNA and β-galactosidase DNA using the PEI method. In the PEI method, 1 μg of DNA and 3 μg of PEI were diluted in Opti-MEM, followed by incubation at room temperature for 20 minutes, followed by mixing the diluted solutions for 20 minutes. After incubation, the mixed solution was placed in a 24-well plate in which cells were divided, and after 6 hours, the mixture was replaced with cell culture medium [10% FBS, 1% penicillin / streptomycin in DMEM], and after 24 hours, aripiprazole was treated by concentration. After 9 hours incubation. Thereafter, the cells were lysed using lysis buffer, reacted 1: 1 with the substrate, and the absorbance was immediately measured using a luminometer. In the case of β-galactosidase DNA, reacted with X-gal (substrate) 1: 1 and incubated for 5 minutes in a 37 ° C. incubator, and then measured absorbance at 405 nm.
그 결과, 도 8 및 도 9에 나타난 바와 같이, 아리피프라졸 처리시 MyD88에 의한 AP-1 및 NF-κB 매개의 루시퍼라제 활성을 억제하고, 도 10 및 도 11에 나타난 바와 같이, 아리피프라졸 처리시 TRIF에 의한 AP-1 및 NF-κB 매개의 루시퍼라제 활성을 억제하였는바, 이에 아리피프라졸에 의해 염증관련 유전자의 활성 역시 억제됨을 확인하였다.As a result, as shown in FIGS. 8 and 9, AP-1 and NF-κB-mediated luciferase activity by MyD88 was inhibited by aripiprazole treatment, and as shown in FIGS. 10 and 11, TRIF was treated by aripiprazole treatment. AP-1 and NF-κB-mediated luciferase activity was inhibited. Thus, the activity of inflammation-related genes was also inhibited by aripiprazole.
5-2. 전사인자 핵내 이동평가5-2. Transcription Factor Nuclear Migration Assessment
아리피프라졸이 염증관련 유전자의 활성에 미치는 영향을 확인하기 위하여, 이들 유전자의 발현을 조절하는 전사인자인 AP-1 및 NF-κB의 핵 내 이동여부를 웨스턴 플랏팅을 통해 측정하였다.In order to confirm the effect of aripiprazole on the activity of inflammation-related genes, we measured the migration of nuclear factors AP-1 and NF-κB in the nucleus by Western plotting.
보다 구체적으로는, 마우스 (Murine) 대식 세포주인 RAW264.7 세포를 페니실린 (100 IU/㎖) 및 스트렙토마이신 (100 ㎍/㎖)과 10%의 FBS를 함유하는 RPMI 1640 배지를 이용해서 배양한 세포를 90%의 밀도로 100 ㎜의 dish에서 전 배양시킨 후, 아리피프라졸을 1시간 동안 전처리하고 stimuli (펩티도글리칸)로 자극한 후, 약물에 따라 일정시간 후 ice-cold PBS로 세척하고, homogenization buffer A [20 mM Tris-HCl pH8.0, 10 mM EGTA, 2 mM EDTA, 2 mM DTT, 1 mM PMSF, 25 μg/ml Aprotinin, 10 μg/ml Leupeptin] 300 μl를 이용하여 세포를 모은다. 다음으로 Sonicator를 이용하여 Output 4의 세기로 세포를 파쇄한 후 8000 rpm으로 15분 동안 4 ℃에서 원심분리하여 상층액 (세포질 분획)을 분리하였다. 펠렛(핵 분획)은 300 ㎕ homogenization buffer B [1% TritonX-100 in Homogenization buffer A]로 부유시킨 후, sonicator를 사용하여 Output 4의 세기로 세포를 파쇄하였다. 얻어진 분획을 SDS-PAGE와 웨스턴 플랏팅을 이용하여, 전사인자의 핵 내 이동 수준을 평가하였다.More specifically, cells cultured with a mouse macrophage RAW264.7 cell using RPMI 1640 medium containing penicillin (100 IU / ml) and streptomycin (100 µg / ml) and 10% FBS Was pre-incubated in a dish of 100 mm at a density of 90%, pretreated with aripiprazole for 1 hour and stimulated with stimuli (peptidoglycan), followed by washing with ice-cold PBS after a certain time according to the drug, homogenization Cells are collected using 300 μl of buffer A [20 mM Tris-HCl pH8.0, 10 mM EGTA, 2 mM EDTA, 2 mM DTT, 1 mM PMSF, 25 μg / ml Aprotinin, 10 μg / ml Leupeptin]. Next, the cells were crushed at the intensity of Output 4 using a Sonicator, and the supernatant (cytoplasmic fraction) was separated by centrifugation at 8000 rpm for 15 minutes at 4 ° C. The pellet (nucleus fraction) was suspended in 300 μl homogenization buffer B [1% TritonX-100 in Homogenization buffer A], and the cells were disrupted at the intensity of Output 4 using a sonicator. The obtained fractions were assessed by nuclear transfer level of the transcription factor using SDS-PAGE and Western plotting.
그 결과, 도 12에 나타난 바와 같이, 핵 분획물에서 아리피프라졸을 처리하였을 때, NF-κB의 서브유닛인 p65, p50와 AP-1의 서브유닛인 c-Fos, c-Jun의 단백질이 감소하였는바, 아리피프라졸에 의해 이들 서브유닛의 핵 내 이동이 억제되었음을 확인하였다.As a result, as shown in Fig. 12, when aripiprazole was treated in the nuclear fraction, proteins of p65, p50, which are subunits of NF-κB, and c-Fos, c-Jun, which are subunits of AP-1, were reduced. It was confirmed that aripiprazole inhibited the migration of these subunits in the nucleus.
실시예Example 6 :  6: 아리피프라졸의Aripiprazole 신호전달 단백질 인산화 억제 효능 측정 Measurement of signaling protein phosphorylation inhibition efficacy
아리피프라졸의 신호전달 단백질 인산화 억제능을 측정하기 위해 하기와 같이 실험을 실시하였다.In order to determine the signaling protein phosphorylation inhibitory activity of aripiprazole was performed as follows.
보다 구체적으로 마우스 (Mureine) 대식 세포주인 RAW264.7 세포를 페니실린 (100 IU/㎖) 및 스트렙토마이신 (100 ㎍/㎖)과 10%의 FBS를 함유하는 RPMI 1640 배지를 이용해서 배양한 세포를 7 × 106 cell/㎖ 농도로 60 ㎜의 dish에서 전 배양시킨다. 이 후, 각 분획들을 처리하고 일정시간 후 stimuli (펩티도글리칸)로 자극하고, 약물에 따라 일정시간 후, 세포들을 모아서 lysis buffer와 sonicator를 사용해 세포를 파쇄하여 western 표본을 얻었다. 그리고 각 표본의 단백질 농도를 BSA를 표준으로 잡고 측정하였으며, 이렇게 얻어진 값을 기준으로 단백질 농도가 되는 각 표본량을 가지고 SDS-PAGE를 실행하고, 웨스턴 블랏팅 방법을 사용해 PVDF membrane으로 단백질을 blotting 시킨 후, membrane을 5% non-fat dried milk (Bio-rad)를 사용해 blocking시키고, 표적 단백질 항체 및 신호전달 단백질 항체 (p-Src, p-Syk, p-AKT, p-p85, β-actin) 용액을 사용해 1차 처리하고, 다시 washing 단계 후 2차 항체 용액을 처리하고 washing한다. 그리고 암실에서 membrane에 ECL 용액 (Amersham, England)을 골고루 분주하여 X-ray film으로 감광하였고, 그 결과를 도 13에 나타내었다.More specifically, cells cultured with mouse macrophage RAW264.7 cells using RPMI 1640 medium containing penicillin (100 IU / mL) and streptomycin (100 μg / mL) and 10% FBS were prepared. Pre-incubate in a 60 mm dish at a concentration of 10 6 cells / ml. Subsequently, each fraction was treated and stimulated with stimuli (peptidoglycan) after a certain time, and after a certain time according to the drug, cells were collected and the cells were crushed using lysis buffer and sonicator to obtain western specimens. The protein concentration of each sample was measured using BSA as a standard. The SDS-PAGE was performed with each sample volume that is the protein concentration based on the obtained values, and the proteins were blotting with PVDF membrane using Western blotting. After that, the membrane was blocked using 5% non-fat dried milk (Bio-rad), and the target protein antibody and the signaling protein antibody (p-Src, p-Syk, p-AKT, p-p85, β-actin) The solution is first treated and then washed again after the second antibody solution. In the dark room, ECL solution (Amersham, England) was evenly distributed on the membrane, and the resultant was exposed to X-ray film. The results are shown in FIG. 13.
도 13에 나타낸 바와 같이, 아리피프라졸은 PGN에 의한 신호전달 단백질인 Src, Syk, AKT 및 p85의 인산화를 억제시킴을 확인할 수 있었다.As shown in FIG. 13, aripiprazole inhibited phosphorylation of Src, Syk, AKT and p85, which are signaling proteins by PGN.
실시예Example 7 : 생체 내 염증 모델에서  7: In vivo inflammation model 아리피프라졸의Aripiprazole 위 손상 억제 효과 Stomach Damage Inhibitory Effect
생체 내 염증 모델에서 아리피프라졸의 위 손상 억제 효과를 확인하기 위하여, 하기와 같은 실험을 수행하였다.In order to confirm the gastric damage inhibitory effect of aripiprazole in an in vivo inflammation model, the following experiment was performed.
공지의 방법에 따라 에탄올/염산 또는 아스피린 (acetylsalicylic acid)으로 ICR 마우스의 급성 위염을 유발시켰다. 구체적으로는, 금식시킨 ICR 마우스 (30마리)를 6개의 군으로 분류한 후, 2개의 군에는 0.5% CMC (carboxymethyl cellulose) 용액, 다른 2개의 군에는 아리피프라졸 (1 및 10 ㎎/㎏), 또 다른 2개의 군에는 양성대조군인 라니티딘 (40 ㎎/㎏) 및 스티렌 (8 ㎎/㎏)을 각각 3일 동안 경구 투여하였다. 30분 후, 0.5% CMC를 주입한 1개의 군을 제외하고, 나머지 5개의 군에 150mM 염산 내 60% 에탄올 400㎕ (또는 아스피린 900 ㎎/㎏)를 경구 투여하여 ICR 마우스의 급성 위염을 유발시켰다. 각각의 실험 동물을 마취시키고 괴사제 투여 1시간 후에 우레탄을 치사량 투여하여 희생시켰다. 마우스의 위를 절개한 후, 위벽 상태를 관찰하였다.Acute gastritis of ICR mice was induced with ethanol / hydrochloric acid or acetylsalicylic acid according to known methods. Specifically, the fasted ICR mice (30 mice) were grouped into 6 groups, followed by 0.5% CMC (carboxymethyl cellulose) solution in 2 groups, aripiprazole (1 and 10 mg / kg) in 2 groups, and The other two groups were orally administered with the positive control ranitidine (40 mg / kg) and styrene (8 mg / kg) for 3 days each. After 30 minutes, acute gastritis of ICR mice was induced by oral administration of 400 μl of 60% ethanol (or aspirin 900 mg / kg) in 150 mM hydrochloric acid to the remaining 5 groups, except one group injected with 0.5% CMC. . Each experimental animal was anesthetized and sacrificed by lethal doses of urethane 1 hour after necrosis. After dissection of the mouse, the gastric wall state was observed.
결과는 도 14 및 도 15에 나타내었다.The results are shown in FIGS. 14 and 15.
도 14 및 도 15에 나타난 바와 같이, 급성 염증이 유발된 생체 내 염증 모델에서 아리피프라졸을 투여한 군의 마우스의 위벽은 아리피프라졸을 투여하지 않은 군에 비해 출혈이 적었고, 출혈의 범위도 적게 나타났다. 따라서, 생체 내에서 아리피프라졸의 위 손상 억제 효과가 우수함을 알 수 있다.As shown in Fig. 14 and 15, in the in vivo inflammation model in which acute inflammation was induced, the stomach wall of the mice treated with aripiprazole showed less bleeding and a smaller range of bleeding compared to the group not receiving aripiprazole. Therefore, it can be seen that the effect of inhibiting gastric damage of aripiprazole in vivo.
실시예Example 8:  8: 아리피프라졸의Aripiprazole 암 세포Cancer cell 증식 억제 효과 확인 Confirmation of growth inhibitory effect
아리피프라졸이 암세포 증식에 미치는 영향을 확인하기 위하여, MTT 어세이를 이용하여 하기와 같은 실험을 수행하였다.In order to confirm the effect of aripiprazole on cancer cell proliferation, the following experiment was performed using an MTT assay.
8-1. 세포배양8-1. Cell culture
C6, U251, LN-428, MKN-1, MDA-MB-231 및 HEK 293 세포를 penicillin (100 IU/ml) 및 streptomycin (100 μg/ml)과 10%의 FBS를 함유하는 RPMI1640 배지 또는 DMEM 배지를 이용하여 100 mm cell culture dish에 70-80%의 밀도로 배양하였다. RPMI1640 medium or DMEM medium containing C6, U251, LN-428, MKN-1, MDA-MB-231, and HEK 293 cells containing penicillin (100 IU / ml) and streptomycin (100 μg / ml) and 10% FBS Were cultured in a 100 mm cell culture dish at a density of 70-80%.
8-2. 세포 생존율8-2. Cell viability
96-웰 플레이트에 1 × 106 cell/ml의 세포를 플레이팅하고, 각 웰에 아리피프라졸(6.25, 12.5, 25, 50, 및 100 μM)을 가한 다음, 37℃에서 각 면역실험 조건에 상응하는 배양시간(6, 24 시간) 동안 CO2 배양기에서 배양하였다. 이후 10㎕의 MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphinyltetrazolium bromide) 용액(저장 농도: 5 mg/㎖)을 첨가하고 3 시간 동안 추가반응을 유도하였다. 반응 종료 및 포마잔 결정(formazan crystal)을 용해하기 위하여, 각 웰에 100 ㎕의 MTT 정지 용액 (0.01M HCl 내 10% 소듐 도데실 설페이트)을 추가적으로 첨가하였다. 세포 생존율은 MTT가 포마잔으로 환원된 양을 570 nm에서 흡광도를 측정하여 얻어진 OD 값을 통해 산출하였고, 그 결과를 도 16 내지 도 20에 나타내었다. Plate 1 × 10 6 cells / ml of cells into 96-well plates, add aripiprazole (6.25, 12.5, 25, 50, and 100 μM) to each well, and then correspond to each immunoassay condition at 37 ° C. The cells were incubated in a CO 2 incubator for 6 (24 hours). Then 10 μl of MTT (3- [4,5-dimethylthiazol-2-yl] -2,5-diphinyltetrazolium bromide) solution (storage concentration: 5 mg / ml) was added and further reaction was induced for 3 hours. To terminate the reaction and dissolve the formazan crystals, 100 μl of MTT stop solution (10% sodium dodecyl sulfate in 0.01M HCl) was added to each well. Cell viability was calculated from the amount of MTT reduced to formazan through the OD value obtained by measuring the absorbance at 570 nm, and the results are shown in FIGS. 16 to 20.
도 16 내지 도 20에 나타낸 바와 같이, 아리피프라졸은 C6, U251, LN-428, MKN-1 및 MDA-MB-231세포의 생존율을 크게 감소시켰다. 따라서, 아리피프라졸을 처리한 경우, 암세포의 증식이 농도 의존적으로 억제됨을 알 수 있다. As shown in FIGS. 16-20, Aripiprazole significantly reduced the viability of C6, U251, LN-428, MKN-1 and MDA-MB-231 cells. Therefore, it can be seen that the treatment of aripiprazole inhibits the proliferation of cancer cells in a concentration-dependent manner.
실시예 9: 아리피프라졸의 세포사멸 유도 효과 확인Example 9: Confirmation of apoptosis inducing effect of aripiprazole
9-1. 세포의 형태변화 확인9-1. Confirmation of cell morphology change
C6 세포 내에서 아리피프라졸이 세포의 형태에 미치는 영향을 확인하기 위하여, 하기와 같은 실험을 수행하였다.In order to determine the effect of aripiprazole on the morphology of the cells in C6 cells, the following experiment was performed.
C6 세포를 24-well plate에 5 × 104 cell/ml 농도로 배양한 후, 아리피프라졸을 농도별(25, 50, 및 100 μM)로 처리하였다. 이후, 시간대별(0, 3, 6, 12, 및 24 h)로 광학현미경을 이용해서 형태를 촬영하였고, 그 결과를 도 21에 나타내었다. C6 cells were incubated at a concentration of 5 × 10 4 cells / ml in 24-well plates, and then the aripiprazole was treated by concentration (25, 50, and 100 μM). Then, the shape was photographed using an optical microscope at time zones (0, 3, 6, 12, and 24 h), and the results are shown in FIG. 21.
도 21에 나타낸 바와 같이, 아리피프라졸을 처리한 경우, Rat glioma cell line인 C6 세포 내에 세포사멸(Apoptosis)의 마커인 blebbing들이 생성되는 것을 확인할 수 있었다.As shown in FIG. 21, when aripiprazole was treated, it was confirmed that blebbings, which are markers of apoptosis, were generated in C6 cells, which are rat glioma cell lines.
9-2. 세포 이동 억제 확인9-2. Confirm cell migration inhibition
아리피프라졸에 의해 세포의 이동 (migration)이 억제되는 것을 확인하기 위하여, 하기와 같은 실험을 수행하였다.In order to confirm that migration of cells is inhibited by aripiprazole, the following experiment was performed.
C6 및 U251 세포를 12-well plate에 1 × 106 cell/㎖ 농도로 배양한 후, 물리적인 방법을 이용하여 세포와 세포사이에 임의의 틈을 만들어준 후, 아리피프라졸을 농도별로 처리하고 시간대별 (9, 24 및 30 h)로 광학현미경을 이용해서 형태를 촬영하였다. 또한, 데이터 산출 프로그램을 이용하여 이를 수치로 나타내었다.After culturing C6 and U251 cells in a 12-well plate at a concentration of 1 × 10 6 cells / ml, and using a physical method to create a random gap between the cells, the treatment with aripiprazole by concentration and time zone Morphology was imaged using an optical microscope at (9, 24 and 30 h). In addition, this is represented numerically by using a data calculation program.
그 결과, 도 22 및 도 23에 나타낸 바와 같이, 아리피프라졸을 처리한 경우, C6 및 U251 세포 내에서 세포의 이동이 억제되는 것을 확인할 수 있었다.As a result, as shown in FIG. 22 and FIG. 23, it was confirmed that when the aripiprazole was treated, cell migration was inhibited in the C6 and U251 cells.
9-3. 형광염색법을 이용한 세포사멸여부 확인9-3. Confirmation of Apoptosis by Fluorescent Staining
C6 세포 내에서 아리피프라졸에 의한 암세포의 세포사멸을 확인하기 위하여, 하기와 같은 실험을 수행하였다.In order to confirm apoptosis of cancer cells by aripiprazole in C6 cells, the following experiment was performed.
보다 구체적으로, C6 세포를 12-well plate에 2.5 × 106 cell/ml 농도로 배양한 후, 아리피프라졸을 농도별로 (25, 50, 및 100 μM) 처리한 후, 원하는 형광염색을 하여 공초점 현미경을 이용해 이를 관찰하였다.More specifically, after culturing C6 cells in a 12-well plate at a concentration of 2.5 × 10 6 cells / ml, and treating aripiprazole by concentration (25, 50, and 100 μM), confocal microscopy was performed with desired fluorescent staining. This was observed using.
그 결과, 도 24 및 도 25에 나타낸 바와 같이, 이리피프라졸을 처리한 경우, 세포사멸(Apoptosis)의 특징인 핵 수축 및 Actin disruption이 발생하는 것을 확인할 수 있었다.As a result, as shown in Figures 24 and 25, when treated with iripiprazole, it was confirmed that the nuclear contraction and Actin disruption that is characteristic of Apoptosis occurs.
9-4. 암세포의 세포사멸여부 확인9-4. Confirmation of apoptosis of cancer cells
C6 세포 내에서 아리피프라졸이 암세포의 세포사멸에 미치는 영향을 확인하기 위하여, 하기와 같은 실험을 수행하였다.In order to determine the effect of aripiprazole on apoptosis of cancer cells in C6 cells, the following experiment was performed.
C6 세포를 12-well plate에 5 × 10 5 cell/ml 농도로 배양한 후, 아리피프라졸을 농도별(25, 50, 및 100 μM)로 처리하였다. 24시간 뒤 PBS를 이용해 세포를 모은 후, 원심분리를 이용해 세포만 분리하였다. 이후, 1X FACS buffer에 Cell을 현탁시킨 후, Annexin V, PI 염색약을 처리하였으며, 15분간 염색시킨 뒤 Flow cytometer를 이용하여 형광을 관찰하였고, 비교 실험군으로 Staurosporin(2.5 μM)을 이용하였다. C6 cells were incubated at a concentration of 5 × 10 5 cells / ml in a 12-well plate, and then aripiprazole was treated by concentration (25, 50, and 100 μM). After 24 hours, cells were collected using PBS, and only cells were separated by centrifugation. Thereafter, the cells were suspended in 1X FACS buffer, treated with Annexin V and PI dyes, stained for 15 minutes, and fluorescence was observed using a flow cytometer. Staurosporin (2.5 μM) was used as a comparative experimental group.
도 26 및 도 27에 나타낸 바와 같이, 아리피프라졸을 처리한 경우, 농도 의존적으로 Annexin V에 염색된 세포가 증가하였으며, 세포 분열 주기가 중단되는 것을 확인할 수 있었다. 따라서, 아리피프라졸은 암세포의 세포사멸을 촉진시킴을 알 수 있었다. As shown in FIG. 26 and FIG. 27, when aripiprazole was treated, cells stained with Annexin V increased in a concentration-dependent manner, and cell division cycles were stopped. Thus, it was found that aripiprazole promotes apoptosis of cancer cells.
9-5. 아리피프라졸이 사이토카인의 mRNA 발현에 미치는 영향 확인9-5. Effect of Aripiprazole on mRNA Expression of Cytokines
대식 세포주 RAW264.7 세포 내에서 아리피프라졸이 사이토카인의 mRNA발현에 미치는 영향을 확인하기 위하여, RT-PCR을 이용하여 하기와 같은 실험을 수행하였다.In order to confirm the effect of aripiprazole on cytokine mRNA expression in macrophage RAW264.7 cells, the following experiment was performed using RT-PCR.
사이토카인의 mRNA 발현 정도를 전사 수준에서 측정하기 위하여, 각 시료를 일정시간 동안 처리하고, TRIzol 시약을 사용하여 total RNA를 추출하였다. 추출한 total RNA를 제1 가닥 cDNA 합성 키트 (Thermo scientific)를 사용하여 cDNA를 제조한 다음, 동량의 cDNA를 PCR로 증폭하였다. 이때, 사용한 표적단백질의 센스 및 안티센스 프라이머 염기서열은 기존문헌을 참조하여 제조하였고, 대조군 유전자로는 GAPDH를 사용하였다 (표 3 참조). PCR 증폭은 Hipi PCR kit (Elpis biotech)를 사용하여 각 실험군의 cDNA와 표적단백질들의 센스 및 안티센스 프라이머, 대조군 GAPDH 프라이머를 dNTP 250 μM, Tris-HCl (pH 8.3) 10 mM, KCl 50 mM, NgCl2 1.5 mM를 포함한 Hipi PCR kit 20 ㎕에서 수행하였다. PCR은 95 ℃에서 45 초간 변성, 55 ℃에서 45 초간 어닐링, 및 72 ℃에서 1분간 신장하는 조건으로 수행하였으며, 총 30 주기를 수행하였다. PCR로 증폭된 DNA는 1.5% 아가로오스 겔에서 전기영동 하였고, 분획된 DNA 밴드의 세기를 측정하였다. 결과는 도 28 및 29에 나타내었다.In order to measure the mRNA expression level of cytokines at the transcription level, each sample was treated for a certain time, and total RNA was extracted using TRIzol reagent. The extracted total RNA was prepared cDNA using the first strand cDNA synthesis kit (Thermo scientific), and then the same amount of cDNA was amplified by PCR. At this time, the sense and antisense primer sequences of the target protein used were prepared with reference to the existing literature, GAPDH was used as a control gene (see Table 3). PCR amplification was performed by using the Hipi PCR kit (Elpis biotech) to detect the sense and antisense primers of the cDNA and target proteins of each experimental group, and control GAPDH primers with dNTP 250 μM, Tris-HCl (pH 8.3) 10 mM, KCl 50 mM, NgCl2 1.5. 20 μl of a Hipi PCR kit containing mM was performed. PCR was performed under conditions of denaturation at 95 ° C. for 45 seconds, annealing at 55 ° C. for 45 seconds, and extension at 72 ° C. for 1 minute, and a total of 30 cycles were performed. DNA amplified by PCR was electrophoresed on 1.5% agarose gel, and the intensity of the fractionated DNA band was measured. The results are shown in FIGS. 28 and 29.
프라이머primer 서열(5' -> 3')Sequence (5 '-> 3')
Bcl2Bcl2 정방향Forward direction CAC CCC TGG CAT CTT CTC CTTCAC CCC TGG CAT CTT CTC CTT
역방향Reverse CAC AAT CCT CCC CCA GTT CAC CCAC AAT CCT CCC CCA GTT CAC C
MMP2MMP2 정방향Forward direction CCC ACT GAG GAG TCC AAC ATCCC ACT GAG GAG TCC AAC AT
역방향Reverse CAT TTA CAC GTC GGA TCTCAT TTA CAC GTC GGA TCT
MMP9MMP9 정방향Forward direction TCC CTG GAG ACC TGA GAA CCTCC CTG GAG ACC TGA GAA CC
역방향Reverse GGC AAG TCT TCC GAG TAG TTTGGC AAG TCT TCC GAG TAG TTT
GAPDHGAPDH 정방향Forward direction GTT ACC AGG GCT GCC TTC TCGTT ACC AGG GCT GCC TTC TC
역방향Reverse GAT GGT GAT GGG TTT CCC GTGAT GGT GAT GGG TTT CCC GT
그 결과, 도 28에 나타낸 바와 같이, C6세포 내에서 아리피프라졸에 의해 세포사멸억제 인자인 Bcl-2의 mRNA 발현 정도가 감소하였으며, 도 29에 나타낸 바와 같이, U251 세포 내에서 아리피프라졸에 의해 세포 이동 관련 인자인 MMP2 및 MMP9의 mRNA 발현이 감소하는 것을 확인하였다. 따라서, 아리피프라졸의 농도 의존적으로 사이토카인의 분비 억제능이 우수함을 알 수 있다.As a result, as shown in FIG. 28, the expression level of Bcl-2, an apoptosis inhibitor, was reduced by Aripiprazole in C6 cells, and as shown in FIG. 29, cell migration-related by Aripiprazole in U251 cells. It was confirmed that the mRNA expression of the factors MMP2 and MMP9 is reduced. Therefore, it can be seen that the concentration inhibition of aripiprazole is excellent in cytokine secretion ability.
실시예Example 10:  10: 아리피프라졸의Aripiprazole 신호전달 단백질 인산화 억제 효능 측정 Measurement of signaling protein phosphorylation inhibition efficacy
10-1. 카스파제(Caspase) 활성 유도 효과 확인10-1. Confirmation of caspase activity induction effect
카스파제 (Caspase)는 세포사멸 (apoptosis)에 필수적인 역할을 수행하므로, C6 세포 내에서 아리피프라졸이 카스파제 활성 유도에 미치는 영향을 확인하기 위하여, 하기와 같은 실험을 수행하였다.Since caspase plays an essential role in apoptosis, the following experiment was performed to confirm the effect of aripiprazole on the induction of caspase activity in C6 cells.
보다 구체적으로, C6 세포를 penicillin (100 IU/ml) 및 streptomycin (100 μg/ml)과 10%의 FBS를 함유하는 RPMI 1640 배지를 이용해서 배양한 세포를 7 × 106 cell/㎖ 농도로 60 ㎜의 dish에서 전 배양시킨다. 이 후, 각 시료들을 처리하고 일정시간 후 세포들을 모아서 lysis buffer와 sonicator를 사용해 세포를 파쇄하여 western 표본을 얻었다. 그리고 각 표본의 단백질 농도를 BSA를 표준으로 잡고 측정하였으며, 이렇게 얻어진 값을 기준으로 단백질 농도가 되는 각 표본량을 가지고 SDS-PAGE를 실행하고, 웨스턴 블랏팅 방법을 사용해 PVDF membrane으로 단백질을 blotting 시킨 후, membrane을 5 % non-fat dried milk (Bio-rad)를 사용해 blocking시키고, 표적 단백질 항체 및 신호전달 단백질 항체 (MMP2,9/ pcaspase 3,8,9/ cleaved caspase 3,8,9/ Bcl-2/ Bax/ p53/ pSrc, pStat3, pAKT, pPI3K, HA, β-actin) 용액을 사용해 1차 처리하고, 다시 세척 단계 후 2차 항체 용액을 처리하고 세척한다. 그리고 암실에서 membrane에 ECL 용액 (Amersham, England)을 골고루 분주하여 X-ray film으로 감광하였고, 그 결과를 도 30 내지 도 32에 나타내었다.More specifically, C6 cells were cultured using RPMI 1640 medium containing penicillin (100 IU / ml) and streptomycin (100 μg / ml) and 10% FBS at a concentration of 7 × 10 6 cells / ml. Pre-incubate in mm dish. After that, each sample was processed and after a predetermined time, the cells were collected and the cells were crushed using lysis buffer and sonicator to obtain western specimens. The protein concentration of each sample was measured using BSA as a standard. The SDS-PAGE was performed with each sample volume that is the protein concentration based on the obtained values, and the proteins were blotting with PVDF membrane using Western blotting. Then, the membrane was blocked with 5% non-fat dried milk (Bio-rad), and the target protein antibody and signaling protein antibody (MMP2,9 / pcaspase 3,8,9 / cleaved caspase 3,8,9 / Bcl -2 / Bax / p53 / pSrc, pStat3, pAKT, pPI3K, HA, β-actin) solution, followed by primary treatment, followed by a second antibody solution after the washing step and washing. In the dark room, ECL solution (Amersham, England) was evenly distributed on the membrane, and the resultant was exposed to X-ray film. The results are shown in FIGS. 30 to 32.
그 결과, 도 30에 나타낸 바와 같이, U251 세포에서 아리피프라졸에 의해 세포이동 관련 인자인 MMP2 및 MMP9의 단백질 발현이 감소하였으며, 도 31에 나타낸 바와 같이, C6 세포에서 아리피프라졸에 의해 세포사멸 유도인자인 Active-Caspase 3, Active-Caspase 8, Active-Caspase 9 및 p53의 양을 증가시키는 것을 확인 하였으며, 세포 사멸이 유도되기 전 단계인 Pro-Caspase 3, Pro-Caspase 8, Pro-Caspase9 및 Bcl-2의 양을 감소시키는 것을 확인하였다.As a result, as shown in FIG. 30, the expression of MMP2 and MMP9, which are cell migration-related factors, was reduced by Aripiprazole in U251 cells, and as shown in FIG. It was confirmed that the amount of -Caspase 3, Active-Caspase 8, Active-Caspase 9 and p53 was increased, Pro-Caspase 3, Pro-Caspase 8, Pro-Caspase9 and Bcl-2 It was confirmed to reduce the amount.
또한, 도 32에 나타낸 바와 같이, C6 세포에서 아리피프라졸은 세포생존신호 관련 단백질의 활성을 감소시켰으며, 또한, 대표적인 발암 유전자인 Src를 표적으로 하는 것을 확인하였다.In addition, as shown in FIG. 32, aripiprazole in C6 cells reduced the activity of cell survival signal-related proteins, and also confirmed that it targets Src, which is a representative carcinogen.
10-2. 아리피프라졸의 Src 단백질 활성의 미치는 영향 확인10-2. Identification of the Effect of Src Protein Activity of Aripiprazole
상기 실시예 10-1의 결과를 토대로 아리피프라졸의 세포생존신호 관련 단백질인 Src의 활성을 직접적으로 억제하는지를 더욱 확인하기 위해서, 하기와 같은 실험을 수행하였다.Based on the results of Example 10-1, in order to further confirm whether the activity of Src, a cell survival signal related protein of aripiprazole, was directly inhibited, the following experiment was performed.
보다 구체적으로, HEK 293 세포주를 Opti-MEM을 이용하여 6-well 플레이트에 분주한 후, 37 ℃ 5 % CO2 세포배양기에서 전 배양하였다. 배양 후 세포가 50% 밀도가 되었을 때, PEI 법을 이용하여 pcDNA, HA-Src, HA-Src CA, HA-Src KD, HA-Src SH2, HA-Src SH3 DNA를 transfection 하였다. PEI 법은 DNA 1 ㎍과 PEI 3 ㎍을 Opti-MEM에 각각 희석해 준 후, 상온에서 20분 동안 배양한 후, 각각 희석액을 혼합하여 다시 20분 배양하는 방법으로 진행하였다. 배양 후 혼합액을 세포가 분주된 6-well 플레이트에 넣어준 후, 6시간 후 세포배양 배지[10 % FBS, 1 % 페니실린/스트렙토마이신 in DMEM]로 교체해주고, 24시간 배양 후 아리피프라졸을 농도별로 처리한 후 12시간 배양하였다. 이 후, Western blot을 수행한다.More specifically, HEK 293 cell lines were dispensed into 6-well plates using Opti-MEM, and then pre-cultured in a 37 ° C. 5% CO 2 cell incubator. When the cells became 50% density after culture, the cells were transfected with pcDNA, HA-Src, HA-Src CA, HA-Src KD, HA-Src SH2, and HA-Src SH3 DNA using the PEI method. In the PEI method, 1 μg of DNA and 3 μg of PEI were diluted in Opti-MEM, followed by incubation at room temperature for 20 minutes, followed by mixing the diluted solutions for 20 minutes. After incubation, the mixed solution was placed in a 6-well plate in which cells were dispensed, and after 6 hours, the cells were replaced with cell culture medium [10% FBS, 1% penicillin / streptomycin in DMEM], and after 24 hours, aripiprazole was treated by concentration. After 12 hours incubation. After this, perform Western blot.
그 결과, 도 33에 나타낸 바와 같이, HEK 293 세포에서 Src를 과 발현시켰을 때, 아리피프라졸에 의해 Src의 활성상태인 인산화된 Src의 발현을 감소시키는 것을 확인하였으며, 도 34에 나타낸 바와 같이, 계속해서 활성화되어 있는 Src(HA-Src CA)에서 아리피프라졸에 의해 Src의 활성상태인 인산화된 Src의 발현을 감소시키는 것을 확인하였다. 또한, 도 35에 나타낸 바와 같이, SH 2 및 SH 3 도메인을 제거한 Src에서도 아리피프라졸에 의해 Src의 활성상태인 인산화된 Src의 발현을 감소시키는 것을 확인하였다.As a result, as shown in FIG. 33, it was confirmed that when Src was overexpressed in HEK 293 cells, the expression of phosphorylated Src, which is the active state of Src, was reduced by aripiprazole, and as shown in FIG. Aripiprazole in activated Src (HA-Src CA) was found to reduce the expression of phosphorylated Src, the active state of Src. In addition, as shown in FIG. 35, Src from which the SH 2 and SH 3 domains were removed also reduced the expression of phosphorylated Src, which is an active state of Src, by aripiprazole.
10-3. 아리피프라졸의 Src 인산화효소 활성 억제 효과 확인10-3. Inhibitory Effect of Aripiprazole on Src Kinase Activity
상기 실시예 10-1의 결과를 토대로 아리피프라졸의 세포생존신호 관련 단백질인 Src의 활성을 직접적으로 억제하는지를 더욱 확인하기 위해서, 하기와 같은 실험을 수행하였다.Based on the results of Example 10-1, in order to further confirm whether the activity of Src, a cell survival signal related protein of aripiprazole, was directly inhibited, the following experiment was performed.
아리피프라졸의 인산화력 억제 효과를 평가하기 위해서, 미국 Millipore사의 kinase profiler service를 이용하였으며, 보다 구체적으로, 최종 용액 부피를 25 ㎕로 하여, Syk와 Src 1 내지 5 mU을 반응버퍼와 배양시, MgATP를 첨가하여 반응을 개시하였으며, 40분간 상온에서 반응시킨 뒤, 3 % 인산 용액을 5 ㎖ 첨가하여 반응을 정시시켰다. 이 후, 상기 용액을 10 ㎕를 덜어서 P30 filtermat에 점적 한 후, 75 mM의 인산으로 5분씩 3회 세척하여 메탄올로 건조시킨 뒤 섬광계수기로 측정하였다.In order to evaluate the inhibitory effect of aripiprazole, the kinase profiler service of Millipore, USA was used. More specifically, the final solution volume was 25 μl, and when Syk and Src 1 to 5 mU were incubated with the reaction buffer, MgATP was obtained. The reaction was initiated by addition, and reacted at room temperature for 40 minutes, followed by addition of 5 ml of a 3% phosphoric acid solution. Thereafter, 10 μl of the solution was dropped onto the P30 filtermat, washed three times with 75 mM phosphoric acid three times, dried over methanol, and measured with a scintillation counter.
그 결과, 도 36에 나타낸 바와 같이, 아리피프라졸이 Src 인산화효소 활성을 감소시킨다는 것을 확인하였다.As a result, as shown in FIG. 36, it was confirmed that aripiprazole reduced Src kinase activity.
실시예Example 11. 대장암 동물 모델을 이용한  11. Using Colon Cancer Animal Models 아리피프라졸의Aripiprazole 암 억제 효과 확인 Confirmation of cancer suppression effect
대장암 동물 모델에서 아리피프라졸의 암 억제 효과를 확인하기 위하여, 하기와 같은 실험을 수행하였다.In order to confirm the cancer inhibitory effect of aripiprazole in the colorectal cancer animal model, the following experiment was performed.
보다 구체적으로, 6주령 수컷 BALB/c 마우스의 등 털을 제모크림을 이용하여 제거한 후, 이틀 뒤 Abertin을 이용하여 전신마취를 시켰다. 이 후, 대장암세포인 CT 26을 1 × 106 cells로 등에 이식하고, 이식 하루 후, 17일 동안 1 ㎎/㎏의 아리피프라졸을 경구 투여한다. 이 때, 경구 투여하는 동안 3일에 한번 씩 종양의 크기를 측정하고, 경구 투여 18일 후, 마우스를 희생시켜 종양의 무게를 측정하였다.More specifically, after removing the back hair of 6-week-old male BALB / c mice using hair removal cream, general anesthesia was performed two days later using Abertin. Thereafter, CT 26, which is a colorectal cancer cell, is transplanted into 1 × 10 6 cells, and the day after transplantation, oral administration of 1 mg / kg of aripiprazole for 17 days. At this time, the tumor size was measured every three days during oral administration, and after 18 days of oral administration, the tumor was sacrificed and the tumor weight was measured.
그 결과, 도 37에 나타난 바와 같이, 아리피프라졸을 경구 투여한 마우스의 종양 크기가 더 작은 것을 확인하였으며, 도 38에 나타난 바와 같이, 아리피프라졸을 경구 투여한 마우스의 경우 대조군에 비해 종양이 덜 자라는 것을 확인하였다.As a result, as shown in Figure 37, it was confirmed that the tumor size of the mice orally administered aripiprazole is smaller, and as shown in Figure 38, mice orally administered to aripiprazole confirmed that the tumor grows less than the control group It was.
또한, 도 39에 나타난 바와 같이, 아리피프라졸을 경구투여한 마우스의 종양의 무게는 대조군에 비해 적게 나가는 것을 확인할 수 있었다. 따라서, 생체 내에서 아리피프라졸의 암 억제 활성 효과가 우수함을 알 수 있다.In addition, as shown in Figure 39, it was confirmed that the tumor weight of the oral administration of aripiprazole mice is less than the control group. Therefore, it can be seen that aripiprazole has excellent cancer inhibitory activity in vivo.
하기에 본 발명의 조성물을 위한 제제예를 예시한다.Examples of preparations for the compositions of the present invention are illustrated below.
제제예Formulation example 1 : 약학적 제제의 제조 1: Preparation of pharmaceutical preparation
1. 산제의 제조1. Preparation of powder
아리피프라졸 200㎎Aripiprazole 200mg
유당 100㎎Lactose 100mg
상기의 성분을 혼합하고 기밀포에 충진하여 산제를 제조하였다.The above ingredients were mixed and filled in airtight cloth to prepare a powder.
2. 정제의 제조2. Preparation of Tablets
아리피프라졸 200㎎Aripiprazole 200mg
옥수수전분 100㎎Corn Starch 100mg
유당 100㎎Lactose 100mg
스테아르산 마그네슘 2㎎2 mg magnesium stearate
상기의 성분을 혼합한 후, 통상의 정제의 제조방법에 따라서 타정하여 정제를 제조하였다.After mixing the above components, tablets were prepared by tableting according to a conventional method for producing tablets.
3. 캡슐제의 제조3. Preparation of Capsule
아리피프라졸 200㎎Aripiprazole 200mg
옥수수전분 100㎎Corn Starch 100mg
유당 100㎎Lactose 100mg
스테아르산 마그네슘 2㎎2 mg magnesium stearate
상기의 성분을 혼합한 후, 통상의 캡슐제의 제조방법에 따라서 젤라틴 캡슐에 충전하여 캡슐제를 제조하였다.After mixing the above components, the capsule was prepared by filling in gelatin capsules according to the conventional method for producing a capsule.
4. 주사제의 제조4. Preparation of Injectables
아리피프라졸 200㎎Aripiprazole 200mg
만니톨 100㎎Mannitol 100mg
Na2HPO4·12H2O 2㎎Na 2 HPO 4 12H 2 O 2mg
주사용 멸균 증류수 적량Appropriate sterile distilled water for injection
통상의 주사제의 제조방법에 따라 1 앰플당(2㎖) 상기의 성분을 혼합하여 주사제를 제조하였다.Injectables were prepared by mixing the above ingredients per ampoule (2 ml) according to the usual method for preparing injectables.
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 아리피프라졸은, RAW264.7 세포 내에서 세포 생존율의 감소를 거의 나타내지 않아 세포독성이 없으며, 펩티도글리칸을 처리한 RAW264.7 세포 내에서 농도 의존적으로 NO 생성 억제능, 프로스타글란딘 E2(PGE2)의 생성 억제능 및 사이토카인 (COX-2 및 TNF-α)의 분비 억제능이 우수하고, 급성 염증이 유발된 생체 내 염증 모델에서 위 손상 억제 효과가 우수하다.Aripiprazole according to the present invention shows little decrease in cell viability in RAW264.7 cells, and thus has no cytotoxicity, and a concentration-dependent NO production inhibitory effect, prostaglandin E2 (PGE2) in peptidoglycan treated RAW264.7 cells. ) And the secretion inhibitory effect of cytokines (COX-2 and TNF-α), and the gastric damage inhibitory effect in the in vivo inflammation model in which acute inflammation was induced.
또한, 본 발명에 따른 아리피프라졸은, C6 , U251, LN-428 및 MDA-MB-231 세포 내에서 우수한 암 세포 증식 억제 효과를 나타내며, C6 세포 내에서 아리피프라졸 농도 의존적으로 세포사멸(Apoptosis)의 마커인 blebbing, apoptotic bodies의 생성 증가, 암세포 사멸 촉진, 및 Active-Caspase의 양 증가 효과를 확인하였다.In addition, aripiprazole according to the present invention exhibits an excellent cancer cell proliferation inhibitory effect in C6, U251, LN-428 and MDA-MB-231 cells, and is a marker of apoptosis depending on aripiprazole concentration in C6 cells. The effects of blebbing, increased production of apoptotic bodies, accelerated cancer cell death, and increased amount of Active-Caspase were identified.
이에, 염증성 질환 또는 암을 예방, 개선 또는 치료하기 위한 약학적 조성물의 활성성분으로 유용하게 사용될 수 있을 것으로 기대된다.Accordingly, it is expected that the present invention may be usefully used as an active ingredient of a pharmaceutical composition for preventing, ameliorating or treating an inflammatory disease or cancer.

Claims (13)

  1. 아리피프라졸을 유효성분으로 함유하는 염증성 질환 또는 암 예방 또는 치료용 약학적 조성물.Pharmaceutical composition for preventing or treating inflammatory diseases or cancer containing aripiprazole as an active ingredient.
  2. 제 1항에 있어서, 상기 아리피프라졸은 산화질소(NO) 및 프로스타글란딘 E2 (PGE2)의 생성을 억제하는 것을 특징으로 하는, 염증성 질환 또는 암 예방 또는 치료용 약학적 조성물.The pharmaceutical composition for preventing or treating inflammatory disease or cancer according to claim 1, wherein the aripiprazole inhibits the production of nitric oxide (NO) and prostaglandin E 2 (PGE 2 ).
  3. 제 1항에 있어서, 상기 아리피프라졸은 NF-κB 및 AP-1(activator protein-1) 활성을 억제하는 것을 특징으로 하는, 염증성 질환 또는 암 예방 또는 치료용 약학적 조성물.The pharmaceutical composition for preventing or treating inflammatory disease or cancer according to claim 1, wherein the aripiprazole inhibits NF-κB and activator protein-1 (AP-1) activity.
  4. 제 1항에 있어서, 상기 아리피프라졸은 iNOS, COX-2 및 IFN-β의 발현을 억제하는 것을 특징으로 하는, 염증성 질환 또는 암 예방 또는 치료용 약학적 조성물.The pharmaceutical composition for preventing or treating inflammatory disease or cancer according to claim 1, wherein the aripiprazole inhibits the expression of iNOS, COX-2 and IFN-β.
  5. 제 1항에 있어서, 상기 염증성 질환은 급성 또는 만성 위염, 부종, 피부염, 알레르기, 아토피, 천식, 결막염, 치주염, 비염, 중이염, 인후염, 편도염, 폐렴, 간염, 식도염, 위궤양, 장염, 췌장염, 십이지장궤양, 대장염, 치질, 통풍, 강직성 척추염, 류마티스 열, 루푸스, 섬유근통(fibromyalgia), 건선관절염, 골관절염, 류마티스 관절염, 견관절주위염, 건염, 건초염, 건주위염, 근육염, 방광염, 신장염, 다발성 경화증 및 패혈증으로 이루어진 군으로부터 선택된 1종 이상인 것을 특징으로 하는, 염증성 질환 또는 암 예방 또는 치료용 약학적 조성물.The method of claim 1, wherein the inflammatory disease is acute or chronic gastritis, edema, dermatitis, allergy, atopic, asthma, conjunctivitis, periodontitis, rhinitis, otitis media, sore throat, tonsillitis, pneumonia, hepatitis, esophagitis, gastric ulcer, enteritis, pancreatitis, duodenum Ulcers, colitis, hemorrhoids, gout, ankylosing spondylitis, rheumatic fever, lupus, fibromyalgia, psoriatic arthritis, osteoarthritis, rheumatoid arthritis, periarthritis, tendinitis, hay salt, peritonitis, myositis, cystitis, nephritis, multiple sclerosis and sepsis A pharmaceutical composition for preventing or treating an inflammatory disease or cancer, characterized in that at least one member selected from the group consisting of.
  6. 제 5항에 있어서, 상기 염증성 질환은 급성 또는 만성 위염인 것을 특징으로 하는, 염증성 질환 또는 암 예방 또는 치료용 약학적 조성물.According to claim 5, The inflammatory disease is characterized in that acute or chronic gastritis, Inflammatory disease or cancer pharmaceutical composition for the prevention or treatment of cancer.
  7. 제 1항에 있어서, 상기 아리피프라졸은 암 세포 내 Caspase-3 단백질의 활성을 증가시켜 세포사멸을 유도하는 것을 특징으로 하는, 염증성 질환 또는 암 예방 또는 치료용 약학적 조성물.The pharmaceutical composition for preventing or treating inflammatory disease or cancer according to claim 1, wherein the aripiprazole induces apoptosis by increasing the activity of Caspase-3 protein in cancer cells.
  8. 제 1항에 있어서, 상기 아리피프라졸은 Src 키나아제 활성을 억제하는 것을 특징으로 하는, 염증성 질환 또는 암 예방 또는 치료용 약학적 조성물.The pharmaceutical composition for preventing or treating inflammatory disease or cancer according to claim 1, wherein the aripiprazole inhibits Src kinase activity.
  9. 제 1항에 있어서, 상기 아리피프라졸은 Bcl2, MMP2 및 MMP9의 발현을 억제하는 것을 특징으로 하는, 염증성 질환 또는 암 예방 또는 치료용 약학적 조성물.The pharmaceutical composition for preventing or treating inflammatory disease or cancer according to claim 1, wherein the aripiprazole inhibits the expression of Bcl2, MMP2 and MMP9.
  10. 제 1항에 있어서, 상기 암은 신경아교종, 갑상선암, 부갑상선암, 골수암, 직장암, 인후암, 후두암, 폐암, 식도암, 췌장암, 대장암, 위암, 설암, 피부암, 뇌종양, 자궁암, 두부 또는 경부암, 담낭암, 구강암, 결장암, 항문 부근암, 간암, 및 대장암으로 이루어진 군으로부터 선택되는 것을 특징으로 하는, 염증성 질환 또는 암 예방 또는 치료용 약학적 조성물.According to claim 1, wherein the cancer is glioma, thyroid cancer, parathyroid cancer, bone marrow cancer, rectal cancer, throat cancer, laryngeal cancer, lung cancer, esophageal cancer, pancreatic cancer, colon cancer, gastric cancer, tongue cancer, skin cancer, brain tumor, uterine cancer, head or neck cancer, gallbladder cancer, Oral cancer, colon cancer, near- anal cancer, liver cancer, and colon cancer, characterized in that selected from the group consisting of, inflammatory diseases or cancer prevention or treatment pharmaceutical composition.
  11. 제 10항에 있어서, 상기 암은 신경아교종, 위암, 유방암 또는 뇌종양인 것을 특징으로 하는, 염증성 질환 또는 암 예방 또는 치료용 약학적 조성물.The pharmaceutical composition for preventing or treating inflammatory disease or cancer according to claim 10, wherein the cancer is glioma, gastric cancer, breast cancer or brain tumor.
  12. 아리피프라졸을 피험자에게 투여하는 단계를 포함하는, 염증성 질환 또는 암 예방 또는 치료 방법.A method for preventing or treating an inflammatory disease or cancer comprising administering aripiprazole to a subject.
  13. 아리피프라졸의 염증성 질환 또는 암 예방 또는 치료 용도.Use for the prevention or treatment of inflammatory disease or cancer of aripiprazole.
PCT/KR2016/008401 2015-08-03 2016-07-29 Composition for preventing or treating inflammatory disease or cancer containing aripiprazole as an active ingredient WO2017023047A1 (en)

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