WO2022025482A1 - Composition pour la prévention et le traitement du cancer colorectal, comprenant de la streptonigrine et un inhibiteur de point de contrôle immunitaire en tant que principes actifs - Google Patents

Composition pour la prévention et le traitement du cancer colorectal, comprenant de la streptonigrine et un inhibiteur de point de contrôle immunitaire en tant que principes actifs Download PDF

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WO2022025482A1
WO2022025482A1 PCT/KR2021/008910 KR2021008910W WO2022025482A1 WO 2022025482 A1 WO2022025482 A1 WO 2022025482A1 KR 2021008910 W KR2021008910 W KR 2021008910W WO 2022025482 A1 WO2022025482 A1 WO 2022025482A1
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streptonigrin
colorectal cancer
immune checkpoint
present
checkpoint inhibitor
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PCT/KR2021/008910
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English (en)
Korean (ko)
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김태원
탁은영
김형돈
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재단법인 아산사회복지재단
울산대학교 산학협력단
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Priority claimed from KR1020210089583A external-priority patent/KR102658984B1/ko
Application filed by 재단법인 아산사회복지재단, 울산대학교 산학협력단 filed Critical 재단법인 아산사회복지재단
Publication of WO2022025482A1 publication Critical patent/WO2022025482A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • 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
    • A61K31/4709—Non-condensed quinolines and containing further heterocyclic rings
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents

Definitions

  • the present invention relates to a composition for preventing and treating colon cancer comprising streptonigrin and an immune checkpoint inhibitor as active ingredients.
  • Colorectal cancer is a mass made up of cancer cells in the large intestine, and more than 90% of colorectal cancers are adenocarcinomas in which adenocarcinomas of the pancreatic ducts are formed.
  • cystic tumors of the colon There are several types of tumors of the colon, the most common being benign cystic tumors, serous and mucinous cystic tumors, intraductal papillary mucinous tumors, solid pseudopapillary tumors, and lymphoid epithelial cysts. and mesenchymal tumors such as cystic teratoma, and malignant tumors include neuroendocrine tumors in addition to exocrine ductal adenocarcinoma and acinar cell carcinoma. Among cystic tumors, there are also malignant tumors, and initially benign tumors may become malignant.
  • the treatment method for colorectal cancer is one or a combination of several methods in some cases, considering the size, location, stage, age and health of the patient.
  • Chemotherapy is used to treat advanced colorectal cancer or postoperative colorectal cancer.
  • Advanced colorectal cancer refers to locally advanced or systemically advanced colorectal cancer.
  • the purpose of chemotherapy in the treatment of advanced colorectal cancer is to suppress the progression of cancer to improve the patient's symptoms, improve the quality of life, and ultimately prolong the patient's survival period.
  • the cancer mass (tumor mass) tissue of colorectal cancer is mainly composed of fibrous tissue, and there are only a few cancer cells, so it is difficult to evaluate the therapeutic response to cancer after chemotherapy. Because it is known as cancer, chemotherapy for colon cancer has not been actively implemented for a long time. However, as recent studies have revealed that chemotherapy for colorectal cancer is more effective than temporary treatment, chemotherapy is being actively used to treat advanced colorectal cancer.
  • the present invention has been devised to solve the above prior art needs, and the present inventors have confirmed that the combination of streptonigrin and an immune checkpoint inhibitor, anti-PD-1 antibody, effectively inhibits colorectal cancer, based on this Thus, the present invention was completed.
  • an object of the present invention is streptonigrin or a pharmaceutically acceptable salt thereof; And to provide a pharmaceutical composition for preventing or treating colon cancer comprising an immune checkpoint inhibitor as an active ingredient.
  • the present invention provides streptonigrin or a pharmaceutically acceptable salt thereof; And it provides a pharmaceutical composition for preventing or treating colon cancer comprising an immune checkpoint inhibitor as an active ingredient.
  • the streptonigrin may be represented by the following formula (1).
  • the streptonigrin and the immune checkpoint inhibitor may be mixed in a concentration ratio (w/v%) of 1:0.01 to 500, but is not limited thereto.
  • the immune checkpoint inhibitor may be a PD-L1 inhibitor or a PD-1 inhibitor, but is not limited thereto.
  • the PD-L1 inhibitor is atezolizumab, avelumab, duvalumab, envafolimab, cosibelimab, AUNP12, It may be selected from the group consisting of CA-170 and BMS-986189, but is not limited thereto.
  • the PD-1 inhibitor is nivolumab, pembrolizumab, semiplimab, spartalizumab, camrelizumab, syn It may be selected from the group consisting of tilimab (sintilimab), tislelizumab, toripalimab (toripalimab), dostalimab (dostarlimab), INCMGA00012, AMP-224 and AMP-514, but is not limited thereto. .
  • the streptonigrin and the immune checkpoint inhibitor may be administered simultaneously, but the present invention is not limited thereto.
  • the streptonigrin and the immune checkpoint inhibitor may be administered sequentially, but the present invention is not limited thereto.
  • the composition may increase the expression of one or more proteins selected from the group consisting of PD-L1, STAT1 and IRF1, but is not limited thereto.
  • the present invention is streptonigrin or a pharmaceutically acceptable salt thereof; And it provides a method for preventing or treating colon cancer, comprising administering to the subject a composition comprising an immune checkpoint inhibitor as an active ingredient.
  • the present invention is streptonigrin or a pharmaceutically acceptable salt thereof; And it provides a preventive or therapeutic use of a composition comprising an immune checkpoint inhibitor as an active ingredient.
  • the present invention provides streptonigrin or a pharmaceutically acceptable salt thereof for the preparation of a medicament for the prevention or treatment of colorectal cancer; and immune checkpoint inhibitors.
  • the present inventors confirmed that streptonigrin not only significantly reduced the survival rate of colorectal cancer cell lines, but also increased the expression of PD-L1 protein in colorectal cancer cell lines. This indicates that streptonigrin will restore the immune sensitivity of colorectal cancer cell lines and promote the effect of the anti-PD-1 antibody. .
  • FIG. 5 shows the results of measuring PD-L1, p-STAT1 and IRF1 protein expression 24 hours after co-treatment with streptonigrin and IFN- ⁇ in HT29 colorectal cancer cell line.
  • FIG. 7 shows the results of measuring the expression of apoptosis-related proteins (Caspase 3, PARP, cytochrome C) by concentration (0 nM, 50 nM, 100 nM, 150 nM, 200 nM) according to streptonigrin treatment in HT29 colorectal cancer cell line.
  • apoptosis-related proteins Caspase 3, PARP, cytochrome C
  • FIG. 8 shows the results of measuring the changes in PD-L1 and MHC-I protein expression according to streptonigrin treatment in HT29 colorectal cancer cell line by flow cytometry.
  • FIG. 9 shows the results of measuring the expression change of antigen presentation mechanism related proteins (TAP1, TAP2, PSMB8, PSMB9, PSMB10) after co-treatment with streptonigrin and IFN- ⁇ in HT29 colorectal cancer cell line.
  • TAP1, TAP2, PSMB8, PSMB9, PSMB10 antigen presentation mechanism related proteins
  • FIG. 10 shows the results of measuring changes in protein expression of NK activating ligand ULBP1 and MIC A/B when streptonigrin was treated in HT29 and WiDr colorectal cancer cell lines.
  • FIG. 11 shows the cytotoxic effect of the combined treatment with streptonigrin and anti-PD-1 antibody when the NK92 cell line and the HT29 colorectal cancer cell line are simultaneously cultured at 1:1 and 2:1 ratios, respectively.
  • FIG. 12 is a schematic diagram of the mechanism of operation of NK cells and cancer cells.
  • the present inventors observed that streptonigrin not only significantly reduced the survival rate of colorectal cancer cell lines, but also increased the expression of PD-L1 protein in colorectal cancer cell lines. As it was confirmed that it would promote the effect of the PD-1 antibody, the present invention was completed (see Examples of the present invention).
  • the present invention relates to streptonigrin or a pharmaceutically acceptable salt thereof; And it relates to a pharmaceutical composition for preventing or treating colon cancer comprising an immune checkpoint inhibitor as an active ingredient.
  • the streptonigrin may be represented by the following formula (1).
  • the molecular weight of streptonigrin is 506.46, and IUPAC name 5-Amino-6-(7-amino-6-methoxy-5,8-dioxo-5,8-dihydroquinolin-2-yl)-4- (2-hydroxy-3,4-dimethoxyphenyl)-3-methylpyridine-2-carboxylic acid may also be named, but is not limited thereto.
  • the streptonigrin and the immune checkpoint inhibitor are 1:0.01 to 500, 1:0.01 to 400, 1:0.01 to 300, 1:0.01 to 250, 1:0.01 to 230, 1:0.01 to 210, 1 :0.1 to 500, 1:0.1 to 400, 1:0.1 to 300, 1:0.1 to 250, 1:0.1 to 230, 1:0.1 to 210, 1:1 to 500, 1:1 to 400, 1:1 to 300, 1:1 to 250, 1:1 to 230, 1:1 to 210, 1:10 to 500, 1:10 to 400, 1:10 to 300, 1:10 to 250, 1:10 to 230 , 1:10 to 210, 1:50 to 500, 1:50 to 400, 1:50 to 300, 1:50 to 250, 1:50 to 230, 1:50 to 210, 1:100 to 500, 1 :100 to 400, 1:100 to 300, 1:100 to 250, 1:100 to 230, 1:100 to 210, 1:150 to 500, 1:150 to 400, 1:150 to 300, 1:150 to 250, 1:150 to 250, 1:1
  • the immune checkpoint inhibitor may be a PD-L1 inhibitor or a PD-1 inhibitor, but is not limited thereto.
  • the PD-L1 inhibitor may be an anti-PD-L1 antibody, but is not limited thereto.
  • the PD-1 inhibitor may be an anti-PD-1 antibody, but is not limited thereto.
  • the PD-L1 inhibitor is atezolizumab, avelumab, duvalumab, envafolimab, cosibelimab, AUNP12, CA-170 and BMS It may be selected from the group consisting of -986189, but is not limited thereto.
  • the PD-1 inhibitor is nivolumab, pembrolizumab, semiplimab, spartalizumab, camrelizumab, sintilimab) , but may be selected from the group consisting of tislelizumab, toripalimab, dostarlimab, INCMGA00012, AMP-224 and AMP-514, but is not limited thereto.
  • the streptonigrin and the immune checkpoint inhibitor may be administered simultaneously, but is not limited thereto.
  • the streptonigrin and the immune checkpoint inhibitor may be sequentially administered, but the present invention is not limited thereto.
  • the composition may increase the expression of one or more proteins selected from the group consisting of PD-L1, STAT1 and IRF1, but is not limited thereto.
  • the composition may increase the expression of one or more apoptosis-related proteins selected from the group consisting of cleaved Caspase 3, cleaved PARP, and cytochrome C, but is not limited thereto.
  • the composition may increase the expression of one or more NK activating ligand proteins selected from the group consisting of ULBP1 and MIC A / B, but is not limited thereto.
  • the present invention may also include a pharmaceutically acceptable salt of streptonigrin as an active ingredient.
  • pharmaceutically acceptable salt includes salts derived from pharmaceutically acceptable inorganic acids, organic acids, or bases.
  • acids examples include hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, perchloric acid, fumaric acid, maleic acid, phosphoric acid, glycolic acid, lactic acid, salicylic acid, succinic acid, toluene-p-sulfonic acid, tartaric acid, acetic acid, citric acid, methanesulfonic acid, formic acid , benzoic acid, malonic acid, gluconic acid, naphthalene-2-sulfonic acid, benzenesulfonic acid, and the like.
  • Acid addition salts can be prepared by conventional methods, for example, by dissolving the compound in an aqueous solution of an excess of acid, and precipitating the salt using a water-miscible organic solvent such as methanol, ethanol, acetone or acetonitrile. It can also be prepared by heating an equimolar amount of the compound and an acid or alcohol in water and then evaporating the mixture to dryness, or by suction filtration of the precipitated salt.
  • a water-miscible organic solvent such as methanol, ethanol, acetone or acetonitrile.
  • Salts derived from suitable bases may include, but are not limited to, alkali metals such as sodium and potassium, alkaline earth metals such as magnesium, and ammonium.
  • the alkali metal or alkaline earth metal salt can be obtained, for example, by dissolving the compound in an excess alkali metal hydroxide or alkaline earth metal hydroxide solution, filtering the undissolved compound salt, and then evaporating and drying the filtrate.
  • a metal salt it is pharmaceutically suitable to prepare a sodium, potassium or calcium salt, and the corresponding silver salt can be obtained by reacting an alkali metal or alkaline earth metal salt with a suitable silver salt (eg, silver nitrate).
  • the content of streptonigrin in the composition of the present invention can be appropriately adjusted depending on the symptoms of the disease, the degree of progression of the symptoms, the condition of the patient, etc., for example, 0.0001 to 99.9% by weight, or 0.001 to 50% by weight based on the total weight of the composition %, but is not limited thereto.
  • the content ratio is a value based on the dry amount from which the solvent is removed.
  • the total effective amount of the checkpoint inhibitor of the present invention may be administered as a single dose, or may be administered by a fractionated treatment protocol in which multiple doses are administered for a long period of time.
  • the pharmaceutical composition of the present invention may vary the content of the active ingredient (PD-L1 inhibitor or PD-1 inhibitor of the present invention) depending on the degree and/or purpose of the disease, but typically 0.01 ⁇ g to 10000 mg when administered once , Preferably, it may be administered repeatedly several times a day at an effective dose of 0.1 ⁇ g to 1000 mg.
  • the dosage of the pharmaceutical composition is determined by considering various factors such as the formulation method, administration route, and number of treatments, as well as the patient's age, weight, health status, sex, severity of disease, diet and excretion rate, etc., the effective dosage for the patient is determined. Therefore, in consideration of this point, those of ordinary skill in the art will be able to determine an appropriate effective dosage of the composition of the present invention.
  • the pharmaceutical composition according to the present invention is not particularly limited in its formulation, administration route and administration method as long as the effect of the present invention is exhibited.
  • the pharmaceutical composition according to the present invention may further include suitable carriers, excipients and diluents commonly used in the preparation of pharmaceutical compositions.
  • the excipient may be, for example, at least one selected from the group consisting of a diluent, a binder, a disintegrant, a lubricant, an adsorbent, a humectant, a film-coating material, and a controlled-release additive.
  • the pharmaceutical composition according to the present invention can be prepared according to a conventional method, respectively, in powders, granules, sustained-release granules, enteric granules, liquids, eye drops, elsilic, emulsions, suspensions, alcohols, troches, fragrances, and limonaade.
  • tablets, sustained release tablets, enteric tablets, sublingual tablets, hard capsules, soft capsules, sustained release capsules, enteric capsules, pills, tinctures, soft extracts, dry extracts, fluid extracts, injections, capsules, perfusates, Warnings, lotions, pasta, sprays, inhalants, patches, sterile injection solutions, or external preparations such as aerosols can be formulated and used, and the external preparations are creams, gels, patches, sprays, ointments, warning agents , lotion, liniment, pasta, or cataplasma.
  • Carriers, excipients and diluents that may be included in the pharmaceutical composition according to the present invention include lactose, dextrose, sucrose, oligosaccharide, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum acacia, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil.
  • formulation it is prepared using commonly used diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrants, and surfactants.
  • water diluted hydrochloric acid, diluted sulfuric acid, sodium citrate, monostearate sucrose, polyoxyethylene sorbitol fatty acid esters (Twinester), polyoxyethylene monoalkyl ethers, lanolin ethers, Lanolin esters, acetic acid, hydrochloric acid, aqueous ammonia, ammonium carbonate, potassium hydroxide, sodium hydroxide, prolamine, polyvinylpyrrolidone, ethyl cellulose, sodium carboxymethyl cellulose, etc.
  • water diluted hydrochloric acid, diluted sulfuric acid, sodium citrate, monostearate sucrose, polyoxyethylene sorbitol fatty acid esters (Twinester), polyoxyethylene monoalkyl ethers, lanolin ethers, Lanolin esters, acetic acid, hydrochloric acid, aqueous ammonia, ammonium carbonate, potassium hydroxide, sodium hydroxide, prolamine, polyvinylpyrrolidone,
  • sucrose solution other sugars or sweeteners may be used, and if necessary, a fragrance, colorant, preservative, stabilizer, suspending agent, emulsifying agent, thickening agent, etc. may be used.
  • Purified water may be used in the emulsion according to the present invention, and if necessary, an emulsifier, preservative, stabilizer, fragrance, etc. may be used.
  • Suspension agents according to the present invention include acacia, tragacantha, methylcellulose, carboxymethylcellulose, sodium carboxymethylcellulose, microcrystalline cellulose, sodium alginate, hydroxypropylmethylcellulose (HPMC), HPMC 1828, HPMC 2906, HPMC 2910, etc.
  • An agent may be used, and a surfactant, a preservative, a stabilizer, a colorant, and a fragrance may be used as needed.
  • Injectables according to the present invention include distilled water for injection, 0.9% sodium chloride injection, ring gel injection, dextrose injection, dextrose + sodium chloride injection, PEG (PEG), lactated ring gel injection, ethanol, propylene glycol, non-volatile oil-sesame oil , solvents such as cottonseed oil, peanut oil, soybean oil, corn oil, ethyl oleate, isopropyl myristate, and benzene benzoate; Solubilizing aids such as sodium benzoate, sodium salicylate, sodium acetate, urea, urethane, monoethyl acetamide, butazolidine, propylene glycol, tweens, nijeongtinamide, hexamine, and dimethyl acetamide; Weak acids and their salts (acetic acid and sodium acetate), weak bases and their salts (ammonia and ammonium acetate), organic compounds, proteins, buffers such as albumin
  • the suppository according to the present invention includes cacao fat, lanolin, witepsol, polyethylene glycol, glycerogelatin, methyl cellulose, carboxymethyl cellulose, a mixture of stearic acid and oleic acid, Subanal, cottonseed oil, peanut oil, palm oil, cacao butter + Cholesterol, Lecithin, Lanet Wax, Glycerol Monostearate, Tween or Span, Imhausen, Monolene (Propylene Glycol Monostearate), Glycerin, Adeps Solidus, Butyrum Tego -G), Cebes Pharma 16, Hexalide Base 95, Cotomar, Hydroxote SP, S-70-XXA, S-70-XX75 (S-70-XX95), Hydro Hydrokote 25, Hydrokote 711, Idropostal, Massa estrarium, A, AS, B, C, D, E, I, T, Massa-MF, Masupol, Masupol-15, Neos
  • Solid preparations for oral administration include tablets, pills, powders, granules, capsules, etc., and these solid preparations include at least one excipient in the extract, for example, starch, calcium carbonate, sucrose ) or lactose, gelatin, etc.
  • excipients for example, starch, calcium carbonate, sucrose ) or lactose, gelatin, etc.
  • lubricants such as magnesium stearate and talc are also used.
  • Liquid formulations for oral administration include suspensions, internal solutions, emulsions, syrups, etc.
  • various excipients such as wetting agents, sweeteners, fragrances, and preservatives may be included.
  • Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, and suppositories.
  • Non-aqueous solvents and suspending agents include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate.
  • composition according to the present invention is administered in a pharmaceutically effective amount.
  • pharmaceutically effective amount means an amount sufficient to treat a disease at a reasonable benefit/risk ratio applicable to medical treatment, and the effective dose level is determined by the type, severity, drug activity, and type of the patient's disease; Sensitivity to the drug, administration time, administration route and excretion rate, treatment period, factors including concurrent drugs and other factors well known in the medical field may be determined.
  • the pharmaceutical composition according to the present invention may be administered as an individual therapeutic agent or may be administered in combination with other therapeutic agents, may be administered sequentially or simultaneously with conventional therapeutic agents, and may be administered singly or multiple times. In consideration of all of the above factors, it is important to administer an amount capable of obtaining the maximum effect with a minimum amount without side effects, which can be easily determined by a person skilled in the art to which the present invention pertains.
  • the pharmaceutical composition of the present invention may be administered to an individual by various routes. Any mode of administration can be contemplated, for example, oral administration, subcutaneous injection, intraperitoneal administration, intravenous injection, intramuscular injection, paraspinal space (intrathecal) injection, sublingual administration, buccal administration, rectal insertion, vaginal It can be administered according to internal insertion, ocular administration, ear administration, nasal administration, inhalation, spraying through the mouth or nose, skin administration, transdermal administration, and the like.
  • the pharmaceutical composition of the present invention is determined according to the type of drug as an active ingredient along with several related factors such as the disease to be treated, the route of administration, the patient's age, sex, weight, and the severity of the disease.
  • “individual” means a subject in need of treatment for a disease, and more specifically, human or non-human primates, mice, rats, dogs, cats, horses, cattle, etc. It may be a mammal of, but is not limited thereto.
  • administration means providing a given composition of the present invention to a subject by any suitable method.
  • prevention means any action that inhibits or delays the onset of a desired disease
  • treatment means that the desired disease and metabolic abnormalities are improved or It means any action that is advantageously changed
  • improvement means any action that reduces a parameter related to a desired disease, for example, the degree of a symptom by administration of the composition according to the present invention.
  • colorectal cancer cell lines were cultured in a medium containing penicillin, streptomycin, and 10% inactivated fetal bovine serum (FBS), 5% carbon dioxide, in a cell incubator at 37°C. Cells were grown by dividing the cells into 1 x 10 6 plates, and then subcultured for 48 hours to stabilize them. After that, only the protein was extracted using a protein degradation buffer, heated at 95° C. for 5 minutes, electrophoresed on 12% SDS-polyacrylamide gel for 2 hours, and the protein was transferred to a nitrocellulose membrane.
  • FBS inactivated fetal bovine serum
  • the membrane was blocked with TBS-T containing 5% skim milk for 30 minutes, and primary antibodies (anti-PD-L1 antibody, anti-p-STAT1 antibody and anti- ⁇ -actin antibody) diluted 1:1000 were added. After targeting overnight at 4°C, unbound antibody was removed by washing 3 times with TBS-T. HRP-conjugated secondary antibody was reacted at a ratio of 1:10,000 at room temperature for 1 hour, and then washed 3 times with TBS-T for 10 minutes to remove unbound antibody. After that, the protein was photosensitized to confirm the expression of TG2, PD-L1, and beta-actin proteins, respectively.
  • WiDr and HT-29 cell lines were selected and the experiment was carried out.
  • WiDr and HT-29 cells were seeded in a 96-well plate at 2 x 10 4 cells/mL and then cultured for 12, 24, and 48 hours, respectively. Then, 50 ⁇ l of MTT (1 mg/mL) was added to each well, and the medium was completely removed by inhalation after an additional incubation for 1 hour. After washing the cultured cells with PBS buffer, 150 ⁇ l of MDSO was dispensed into each well and absorbance was measured at a wavelength of 540 nm. The absorbance of the experimental group with respect to the absorbance of the control group was converted into % survival, and the concentration (IC 50 value) capable of inhibiting the proliferation of cells by 50% was used as the standard.
  • TG2 is known to promote cancer growth, induce resistance to anticancer drugs and radiation therapy, promote cancer angiogenesis, and inhibit cancer cell death. Accordingly, to determine whether streptonigrin reduces the TG2 protein, and to confirm the possibility of combination with the anti-PD-L1 antibody, the PD-L1 protein expression level was measured.
  • Example 2 After treatment with streptonigrin for 24 hours, only protein was extracted using a proteolysis buffer and heated at 95° C. for 5 minutes. Thereafter, the same western blot test method as in Example 1 was used. By photosensitizing the protein, the expression levels of TG2, PD-L1, and beta-actin were confirmed.
  • IFN- ⁇ known as a factor that activates immune T cells
  • HT29 cells a colorectal cancer cell line
  • streptonigrin (0, 250, 500 nM) and IFN- ⁇ (10 IU/mL). was heated for 5 minutes.
  • IFN- ⁇ (10 IU/mL). was heated for 5 minutes.
  • the protein was transferred to a nitrocellulose membrane.
  • the membrane was blocked with TBS-T containing 5% skim milk for 30 minutes, and primary antibodies (anti-PD-L1 antibody, anti-p-STAT1 antibody, anti-IRF1 antibody and anti- ⁇ -actin antibody) was targeted at 4° C. overnight and then washed 3 times with TBS-T to remove unbound antibody.
  • HRP-conjugated secondary antibody was reacted at a ratio of 1:10,000 at room temperature for 1 hour, and then washed 3 times with TBS-T for 10 minutes to remove unbound antibody. Thereafter, the protein was photosensitized to determine its effect on the expression of PD-L1, STAT1 and IRF1 proteins.
  • the STAT1 factor is activated in cancer cells, and through the activation and elevation of IRF1, it is bound to the promoter part of PD-L1, expresses the corresponding PD-L1 gene, and is translated into protein to induce an increase in PD-L1.
  • IRF1 and PD-L1 can Therefore, when streptonigrin is co-treated with IFN- ⁇ , an in vivo-like immune environment is created and STAT1 is activated in colorectal cancer cells, thereby increasing IRF1 and PD-L1.
  • apoptosis-related proteins (Caspase 3, PARP, cytochrome C) was measured in the HT29 colorectal cancer cell line according to the streptonigrin treatment by concentration (0 nM, 50 nM, 100 nM, 150 nM, 200 nM).
  • a total of 500,000 HT29 cells were treated with streptonigrin at different concentrations (0, 250, 500 nM) and incubated for 24 hours after which the surface marker was converted to an anti-PD-L1 antibody conjugated with Pacific Blue dye (BD Bioscience and APC). (allophycocyanin) and anti-MHC-I antibody (Invitrogen) conjugated was used for staining, and a BD FACSCanto II (BD Bioscience) flow cytometer was used to analyze 10,000 cases in 500,000 cells/500 ⁇ l PBS.
  • Example 7 Confirmation of protein expression related to antigen presentation mechanism according to combined treatment with streptonigrin and IFN- ⁇
  • IFN- ⁇ known as a factor that activates immune T cells
  • HT29 cells a colorectal cancer cell line
  • streptonigrin (0, 250, 500 nM) and IFN- ⁇ (10 IU/mL). was heated for 5 minutes.
  • IFN- ⁇ (10 IU/mL). was heated for 5 minutes.
  • the protein was transferred to a nitrocellulose membrane.
  • the membrane was blocked with TBS-T containing 5% powdered skim milk for 30 minutes and diluted 1:1000 with TAP1 (Abcam), TAP2 (Abcam), PSMB8 (Abcam), PSMB9 (Abcam), and PSMB10 (Abcam)
  • Unbound antibody was removed by targeting the primary antibody against the target overnight at 4° C. and then washing 3 times with TBS-T.
  • HRP-conjugated secondary antibody was reacted at a ratio of 1:10,000 at room temperature for 1 hour, and then washed 3 times with TBS-T for 10 minutes to remove unbound antibody. Thereafter, the protein was photosensitized to determine its effect on the expression of TAP1, TAP2, PSMB8, PSMB9, and PSMB10 proteins.
  • NKG2D ligand is one of the activating receptors expressed on the surface of NK cells and plays a very important role in the elimination of target cells.
  • NKG2D ligands MICA, MICB, ULBP1, ULBP2, ULBP3
  • MICA/B MHC class I-related chain A and B
  • ULBPs binding proteins
  • protein expression changes of ULBP1, an NK activating ligand, and MIC A/B were measured when streptonigrin was treated in HT29 and WiDR colorectal cancer cell lines. It was carried out using the same western blot test method as in Example 1. For Western blotting, primary antibodies against ULBP1 (Abcam) and MIC A/B (Cell Signaling Technology) were used.
  • Example 9 Confirmation of colon cancer cell line killing effect according to combined treatment with streptonigrin and anti-PD-1 antibody
  • colorectal cancer cells HT29 were treated with 0, 50, and 100 nM of streptonigrin, respectively, and 24 hours later, co-cultured with the NK92 cell line at 1:1 and 2:1 number ratios for 4 hours.
  • streptonigrin (0, 50, 100 nM) and 10 ⁇ g/mL anti-Igg isotype control antibody (BioLegend) or 10 ⁇ g/mL anti-PD-1 antibody (BioLegend) were treated alone or in combination.
  • Cell damage was measured by measuring lactate dehydrogenase (LDH) present in the culture medium after culture.
  • LDH Cytotoxicity Detection Kit (TAKARA) was used to measure the concentration of lactate dehydrogenase, and it was quantified by measuring the absorbance at 492 nm through an ELISA reader. The degree of cell damage was calculated by correcting the experimental value to the maximum value obtained by treating the untreated control group (target spontaneous) and 2% triton X-100 (target maximum).
  • streptonigrin not only reduces the survival rate of colorectal cancer cells, but also has a synergistic effect when combined with the anti-PD-1 antibody, which is an immune checkpoint inhibitor.
  • the streptonigrin of the present invention and an immune checkpoint inhibitor can be used in combination to prevent or treat colorectal cancer.
  • the present inventors confirmed that streptonigrin not only significantly reduced the survival rate of colorectal cancer cell lines, but also increased the expression of PD-L1 protein in colorectal cancer cell lines. This indicates that streptonigrin will restore the immune sensitivity of colorectal cancer cell lines and promote the effect of the anti-PD-1 antibody. There is availability.

Abstract

La présente invention concerne une composition pour la prévention et le traitement du cancer colorectal, comprenant de la streptonigrine et un inhibiteur de de point de contrôle immunitaire comme principes actifs. Les présents inventeurs ont confirmé que la streptonigrine non seulement réduit de manière significative le taux de survie d'une lignée de cellules du cancer colorectal, mais augmente également l'expression de la protéine PD-L1 dans la lignée de cellules du cancer colorectal. Ledit résultat montre que la streptonigrine peut restaurer la sensibilité immunitaire de la lignée de cellules du cancer colorectal, ce qui favorise l'effet d'un anticorps anti-PD-1, et ainsi la présente invention est censée permettre un traitement efficace du cancer colorectal au moyen de la co-administration de streptonigrine et de l'anticorps anti-PD-1.
PCT/KR2021/008910 2020-07-29 2021-07-12 Composition pour la prévention et le traitement du cancer colorectal, comprenant de la streptonigrine et un inhibiteur de point de contrôle immunitaire en tant que principes actifs WO2022025482A1 (fr)

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KR1020210089583A KR102658984B1 (ko) 2020-07-29 2021-07-08 스트렙토니그린 및 면역관문 억제제를 유효성분으로 포함하는 대장암 예방 및 치료용 조성물
KR10-2021-0089583 2021-07-08

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WO2018129559A1 (fr) * 2017-01-09 2018-07-12 Tesaro, Inc. Procédés de traitement du cancer à l'aide d'anticorps anti-pd-1
KR20190045827A (ko) * 2017-10-24 2019-05-03 국립암센터 스트렙토니그린 및 라파마이신을 유효성분으로 포함하는, 암 예방 또는 치료용 약학적 조성물

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KR20190045827A (ko) * 2017-10-24 2019-05-03 국립암센터 스트렙토니그린 및 라파마이신을 유효성분으로 포함하는, 암 예방 또는 치료용 약학적 조성물

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