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WO2017023047A1 - Composition pour la prévention ou le traitement d'une maladie inflammatoire ou du cancer contenant de l'aripiprazole en tant qu'ingrédient actif - Google Patents

Composition pour la prévention ou le traitement d'une maladie inflammatoire ou du cancer contenant de l'aripiprazole en tant qu'ingrédient actif Download PDF

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Publication number
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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Korean (ko)
Inventor
조재열
백광수
김용
김은지
박재광
유슬기
김유림
정성구
김여진
김미선
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Sungkyunkwan University
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Sungkyunkwan University
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Priority claimed from KR1020160086079A external-priority patent/KR20170026115A/ko
Priority claimed from KR1020160086077A external-priority patent/KR20170016275A/ko
Application filed by Sungkyunkwan University filed Critical Sungkyunkwan University
Publication of WO2017023047A1 publication Critical patent/WO2017023047A1/fr
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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 or sparfloxacin
    • 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.

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Abstract

La présente invention concerne une composition pour la prévention ou le traitement d'une maladie inflammatoire ou du cancer, la composition contenant de l'aripiprazole en tant qu'ingrédient actif. L'aripiprazole, selon la présente invention, ne présente quasiment pas de réduction de la survie cellulaire et n'est pas cytotoxique dans des cellules RAW264.7, et, dans des cellules RAW264.7 traitées par du peptidoglycane, présente de remarquables aptitude à la suppression de la production de NO dépendant de la concentration, aptitude à la suppression de la production de prostaglandine E2(PGE2) et aptitude à la suppression de la sécrétion de cytokine (COX-2 et TNF-α), et présente un excellent effet de suppression de lésion de l'estomac dans un modèle inflammatoire in vivo impliquant l'induction d'une inflammation aiguë. En outre, il a été démontré que l'aripiprazole, selon la présente invention, présente un excellent effet de suppression de la prolifération de cellules cancéreuses en C6, U251, LN-428 et dans des cellules MDA-MB-231, et dans des cellules C6, augmente l'apparition de phénomène de boursoufflures et de corps apoptotiques qui sont des marqueurs de l'apoptose d'une manière dépendant de la concentration d'aripiprazole, et présente un effet favorisant l'apoptose de cellules cancéreuses et augmentant la quantité de caspase actif. Par conséquent, la présente invention peut être utilisée en tant que produit médicinal qui est utile dans la prévention ou le traitement d'une maladie inflammatoire ou du cancer.
PCT/KR2016/008401 2015-08-03 2016-07-29 Composition pour la prévention ou le traitement d'une maladie inflammatoire ou du cancer contenant de l'aripiprazole en tant qu'ingrédient actif Ceased WO2017023047A1 (fr)

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KR1020160086079A KR20170026115A (ko) 2015-08-26 2016-07-07 아리피프라졸을 유효성분으로 함유하는 암 예방 또는 치료용 조성물
KR10-2016-0086079 2016-07-07
KR1020160086077A KR20170016275A (ko) 2015-08-03 2016-07-07 아리피프라졸을 유효성분으로 함유하는 염증성 질환의 예방 또는 치료용 조성물

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KR20220036878A (ko) * 2020-09-16 2022-03-23 한국원자력의학원 아리피프라졸을 유효성분으로 함유하는 bcl2 저해제의 항암 효과 증진용 조성물

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WO2018162522A1 (fr) * 2017-03-07 2018-09-13 University Of Copenhagen Utilisation d'un agoniste du récepteur de la dopamine pour le traitement de cancers colorectaux
CN112074273A (zh) * 2018-05-04 2020-12-11 韩国原子力医学院 包含阿立哌唑作为活性成分的辐射敏感性增强组合物
CN112074273B (zh) * 2018-05-04 2023-06-30 韩国原子力医学院 包含阿立哌唑作为活性成分的辐射敏感性增强组合物
KR20220036878A (ko) * 2020-09-16 2022-03-23 한국원자력의학원 아리피프라졸을 유효성분으로 함유하는 bcl2 저해제의 항암 효과 증진용 조성물
KR102683903B1 (ko) 2020-09-16 2024-07-12 한국원자력의학원 아리피프라졸을 유효성분으로 함유하는 bcl2 저해제의 항암 효과 증진용 조성물
CN114073702A (zh) * 2021-03-05 2022-02-22 中以海德人工智能药物研发股份有限公司 喹诺酮类化合物在治疗或预防乙型肝炎中的应用
CN114073702B (zh) * 2021-03-05 2023-12-12 中以海德人工智能药物研发股份有限公司 喹诺酮类化合物在治疗或预防乙型肝炎中的应用

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