KR20220014818A - Composition for prevention and treatment of colorectal cancer comprising streptonigrin and immune checkpoint inhibitor as an active ingredient - Google Patents

Abstract

The present invention relates to a composition for preventing and treating colorectal cancer, comprising streptonigrin and an immune checkpoint inhibitor as active ingredients. The present inventors have confirmed that streptonigrin not only significantly reduces the survival rate of a colorectal cancer cell line, but also increases the expression of the PD-L1 protein in the colorectal cancer cell line. The present invention shows that streptonigrin can restore the immune sensitivity of the colorectal cancer cell line, thereby promoting an effect of an anti-PD-1 antibody. Therefore, the present invention is expected to enable effective treatment of colorectal cancer by means of the co-administration of streptonigrin and the anti-PD-1 antibody.

Description

Composition for prevention and treatment of colorectal cancer comprising streptonigrin and immune checkpoint inhibitor as an active ingredient

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.

According to the data released by the Central Cancer Registry published in 2015, there were 225,343 cancer cases in Korea in 2013. Among them, colorectal cancer took the 8th place with 5,511 cases of both men and women, accounting for 2.4% of the total cancer incidence. The crude incidence rate per 10,000 people (the number of new cases in the target population during the observation period. The standard for calculating the mortality rate is the same) is 10.9 cases. The male to female ratio was 1.2 : 1, and the number of cases was 2,982 in males, ranking ninth among male cancers, and 2,529 cases in females, ranking ninth among female cancers. Combining men and women by age group, those in their 70s accounted for the most at 33.6%, followed by those in their 60s with 24.8%, and those in their 50s and 80s with 17.3%.

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 most effective treatment for colorectal cancer is complete surgical excision. However, such radical surgery (surgery for a cure) is possible only in about 20-25% of colorectal cancer patients, and in reality, it is mostly limited to patients with a tumor in the colon head, where jaundice is an early symptom. The average survival period of patients with colorectal cancer that cannot be surgically resected is about 6 months, and the main purpose of treatment for these patients is to alleviate the patient's symptoms and improve the quality of life during survival.

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.

However, the possibility of treating colorectal cancer through the combination of streptonigrin and an immune checkpoint inhibitor has not been known so far.

Republic of Korea Patent Publication No. 10-2019-0001574

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.

Accordingly, 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.

However, the technical problem to be achieved by the present invention is not limited to the above-mentioned problems, and other problems not mentioned will be clearly understood by those skilled in the art from the following description.

In order to achieve the object of the present invention as described above, 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.

In one embodiment of the present invention, the streptonigrin may be represented by the following formula (1).

[Formula 1]

In another embodiment of the present invention, 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.

In another embodiment of the present invention, the immune checkpoint inhibitor may be a PD-L1 inhibitor or a PD-1 inhibitor, but is not limited thereto.

In another embodiment of the present invention, 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.

In another embodiment of the present invention, 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. .

In another embodiment of the present invention, the streptonigrin and the immune checkpoint inhibitor may be administered simultaneously, but the present invention is not limited thereto.

In another embodiment of the present invention, the streptonigrin and the immune checkpoint inhibitor may be administered sequentially, but the present invention is not limited thereto.

In another embodiment of the present invention, 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.

In addition, 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.

In addition, 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.

In addition, the present invention is streptonigrin or a pharmaceutically acceptable salt thereof for the preparation of a colorectal cancer drug; 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. .

1 shows the results of confirming the expression of TG-2 and PD-L1 for each colorectal cancer cell line.
2 shows the viability of WiDr and HT29 cell lines according to time (0h, 12h, 24h, 48h) according to streptonigrin treatment.
3 shows the results of measuring TG2 and PD-L1 protein expression according to streptonigrin treatment.
4 shows the results of measuring PD-L1 protein expression 24 hours after the HT29 colorectal cancer cell line was treated with streptonigrin and IFN-γ in combination.
FIG. 5 shows the results of measuring PD-L1, p-STAT1 and IRF1 protein expression 24 hours after co-treatment with streptonigrin and IFN-γ in HT29 colorectal cancer cell line.
6 shows the mechanism of operation of immune cells and cancer cells.
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.
FIG. 8 shows the results of measuring changes in PD-L1 and MHC-I protein expression according to streptonigrin treatment in HT29 colorectal cancer cell line by flow cytometry.
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-γ in HT29 colorectal cancer cell line.
10 shows the results of measuring the protein expression changes of ULBP1 and MIC A/B, which are NK activating ligands, when streptonigrin is treated in HT29 and WiDr colorectal cancer cell lines.
11 shows the cytotoxic effect of the combined treatment with streptonigrin and anti-PD-1 antibody when NK92 cell line and HT29 colorectal cancer cell line are simultaneously cultured at 1:1 and 2:1 ratios, respectively.
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).

Accordingly, 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.

Hereinafter, the present invention will be described in detail.

In the present invention, the streptonigrin may be represented by the following formula (1).

[Formula 1]

In the present invention, 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.

In the present invention, 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 230, 1:150 to 210, 1:180 to 230, 1:190 to 210, or may be mixed in a concentration ratio (w/v%) of about 1:200, but limited thereto no. In addition, the streptonigrin and the immune checkpoint inhibitor may be administered individually in the above concentration ratio, but is not limited thereto.

In the present invention, the immune checkpoint inhibitor may be a PD-L1 inhibitor or a PD-1 inhibitor, but is not limited thereto.

In the present invention, the PD-L1 inhibitor may be an anti-PD-L1 antibody, but is not limited thereto.

In the present invention, the PD-1 inhibitor may be an anti-PD-1 antibody, but is not limited thereto.

In the present invention, 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.

In the present invention, 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.

In the present invention, the streptonigrin and the immune checkpoint inhibitor may be administered simultaneously, but is not limited thereto.

In the present invention, the streptonigrin and the immune checkpoint inhibitor may be sequentially administered, but the present invention is not limited thereto.

In the present invention, 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.

In the present invention, 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.

In the present invention, 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. As used herein, the term “pharmaceutically acceptable salt” includes salts derived from pharmaceutically acceptable inorganic acids, organic acids, or bases.

Examples of suitable acids 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.

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. In this case, as 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 μg to 10000 mg when administered once , Preferably, it may be administered repeatedly several times a day at an effective dose of 0.1 μg to 1000 mg. However, 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.

In the case of formulation, it is prepared using commonly used diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrants, and surfactants.

Corn starch, potato starch, wheat starch, lactose, sucrose, glucose, fructose, di-mannitol, precipitated calcium carbonate, synthetic aluminum silicate, phosphoric acid as additives for tablets, powders, granules, capsules, pills, and troches according to the present invention Calcium monohydrogen, calcium sulfate, sodium chloride, sodium hydrogen carbonate, purified lanolin, microcrystalline cellulose, dextrin, sodium alginate, methyl cellulose, sodium carboxymethyl cellulose, kaolin, urea, colloidal silica gel, hydroxypropyl starch, hydroxypropyl methyl excipients such as cellulose (HPMC), HPMC 1928, HPMC 2208, HPMC 2906, HPMC 2910, propylene glycol, casein, calcium lactate, and Primogel; Gelatin, gum arabic, ethanol, agar powder, cellulose acetate phthalate, carboxymethylcellulose, calcium carboxymethylcellulose, glucose, purified water, sodium caseinate, glycerin, stearic acid, sodium carboxymethylcellulose, sodium methylcellulose, methylcellulose, microcrystalline cellulose, dextrin , hydroxycellulose, hydroxypropyl starch, hydroxymethylcellulose, purified shellac, starch powder, hydroxypropylcellulose, hydroxypropylmethylcellulose, polyvinyl alcohol, polyvinylpyrrolidone, etc. Hydroxypropylmethylcellulose, corn starch, agar powder, methylcellulose, bentonite, hydroxypropyl starch, sodium carboxymethylcellulose, sodium alginate, calcium carboxymethylcellulose, calcium citrate, sodium lauryl sulfate, silicic anhydride, 1-hydroxy Propyl cellulose, dextran, ion exchange resin, polyvinyl acetate, formaldehyde treated casein and gelatin, alginic acid, amylose, guar gum, sodium bicarbonate, polyvinylpyrrolidone, calcium phosphate, gelled starch, gum arabic, Disintegrants such as amylopectin, pectin, sodium polyphosphate, ethyl cellulose, sucrose, magnesium aluminum silicate, di-sorbitol solution, light anhydrous silicic acid; Calcium stearate, magnesium stearate, stearic acid, hydrogenated vegetable oil, talc, lycopodite, kaolin, petrolatum, sodium stearate, cacao fat, sodium salicylate, magnesium salicylate, polyethylene glycol (PEG) 4000, PEG 6000, liquid paraffin, hydrogen Added soybean oil (Lubri wax), aluminum stearate, zinc stearate, sodium lauryl sulfate, magnesium oxide, macrogol, synthetic aluminum silicate, silicic anhydride, higher fatty acid, higher alcohol, silicone oil, paraffin oil, polyethylene glycol fatty acid ether, A lubricant such as starch, sodium chloride, sodium acetate, sodium oleate, dl-leucine, light anhydrous silicic acid; may be used.

As additives for the liquid formulation according to the present invention, 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. can be used.

In the syrup according to the present invention, a 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.

The injection according to the present invention includes distilled water for injection, 0.9% sodium chloride injection solution, ring gel injection solution, dextrose injection solution, dextrose + sodium chloride injection solution, PEG (PEG), lactated ring gel injection solution, 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, peptone, gum; isotonic agents such as sodium chloride; Stabilizers such as sodium bisulfite (NaHSO ₃ ) carbon dioxide gas, sodium metabisulfite (Na ₂ S ₂ O ₅ ), sodium sulfite (Na ₂ SO ₃ ), nitrogen gas (N ₂ ), ethylenediaminetetraacetic acid; sulphating agents such as sodium bisulfide 0.1%, sodium formaldehyde sulfoxylate, thiourea, disodium ethylenediaminetetraacetate, acetone sodium bisulfite; analgesic agents such as benzyl alcohol, chlorobutanol, procaine hydrochloride, glucose, and calcium gluconate; suspending agents such as SiMC sodium, sodium alginate, Tween 80, or aluminum monostearate.

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, Neosupostal-N, Paramound-B, Suposiro (OSI, OSIX, A, B, C, D, H, L), Suppository IV type (AB, B, A, BC, BBG, E, BGF, C, D, 299), supostal (N, Es), Wecobi (W, R, S, M, Fs), tester triglyceride base (TG-95, MA, 57) and The same mechanism may be used.

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. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used.

Liquid formulations for oral administration include suspensions, internal solutions, emulsions, syrups, etc. In addition to water and liquid paraffin, which are commonly used simple diluents, various excipients such as wetting agents, sweeteners, fragrances, and preservatives may be included. have. 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.

The pharmaceutical composition according to the present invention is administered in a pharmaceutically effective amount. In the present invention, "pharmaceutically effective amount" means an amount sufficient to treat a disease at a reasonable benefit/risk ratio applicable to medical treatment, and 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.

In the present invention, "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.

In the present invention, "administration" means providing a given composition of the present invention to a subject by any suitable method.

In the present invention, "prevention" means any action that inhibits or delays the onset of a desired disease, and "treatment" means that the desired disease and metabolic abnormalities are improved or It means all actions that are advantageously changed, and "improvement" means all actions that reduce the parameters related to the desired disease, for example, the degree of symptoms by administration of the composition according to the present invention.

All documents mentioned herein are incorporated herein by reference as if their contents were set forth herein. When introducing an element of the present invention or preferred aspect(s) thereof, the articles “a”, “an”, “the” and “said” refer to one or more of the elements. is intended to mean that there is The terms “comprising,” “including,” and “having” are intended to be inclusive and mean that there may be additional elements other than the listed elements. Although the invention has been described with respect to specific aspects or aspects, it should not be construed as limiting the details of these aspects.

Hereinafter, preferred examples are presented to help the understanding of the present invention. However, the following examples are only provided for easier understanding of the present invention, and the contents of the present invention are not limited by the following examples.

Example 1. Selection of colorectal cancer cell lines

To confirm the effect of streptonigrin on the PD-L1 protein, Western blotting was performed to select colon cancer cell lines that express TG2 (Transglutaminase 2, TGase2) but have low PD-L1 expression.

Specifically, 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. After that, 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-β-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. Thereafter, the protein was photosensitized to confirm the expression of TG2, PD-L1, and beta-actin proteins, respectively.

As a result, as shown in FIG. 1 , it was confirmed that TG2 was well expressed in WiDr and HT-29 cell lines, but PD-L1 was poorly expressed.

Therefore, in the Examples of the present invention, WiDr and HT-29 cell lines were selected and the experiment was carried out.

Example 2. Confirmation of the effect of streptonigrin on the survival rate of colorectal cancer cell lines

The survival rate inhibitory effect of streptonigrin on the colorectal cancer cell line selected in Example 1 was confirmed.

Specifically, 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 μl 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 μl 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.

As a result, as shown in FIG. 2 , it was confirmed that the survival rate of colorectal cancer cell lines was significantly reduced when 250 nM of streptonigrin was treated.

Example 3. Confirmation of the effect of streptonigrin on TG2 and PD-L1 protein expression

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.

Specifically, 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.

As a result, as shown in FIG. 3 , it was confirmed that streptoligrin only affects the function of TG2 and has a tendency not to clearly show increase or decrease in protein expression.

That is, through the above results, it was confirmed that the change in TG2 was insignificant in terms of protein expression, but the expression of PD-L1 was increased.

Example 4. Confirmation of protein expression according to streptonigrin and IFN-γ combination treatment

When IFN-γ, known as a factor that activates immune T cells, was treated, the effect of streptonigrin on PD-L1, STAT1 and IRF1 protein expression was confirmed.

Specifically, HT29 cells, a colorectal cancer cell line, were treated with streptonigrin (0, 250, 500 nM) and IFN-γ (10 IU/mL). was heated for 5 minutes. After electrophoresis on 12% SDS-polyacrylamide gel for 2 hours, the protein was transferred to a nitrocellulose membrane. Then, 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-β -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.

As a result, as shown in FIGS. 4 and 5 , strong expression of PD-L1 was confirmed even when IFN-γ was treated. This is due to the stimulation of IFN-γ, immune T cells are activated and the increase in PD-L1 is induced.

In addition, looking at the mechanism of operation of the immune cells and cancer cells in FIG. 6 , it was confirmed that the receptor (IFNγR) increased by the treated IFN-γ during cell culture, and the mechanism for increasing the transcription factor into the nucleus works. .

That is, the STAT1 factor is activated in cancer cells, and through the activation and elevation of IRF1, it is bound to the promoter portion of PD-L1, expresses the corresponding PD-L1 gene, and is translated into protein to induce an increase in PD-L1. can Therefore, when streptonigrin is co-treated with IFN-γ, an in vivo-like immune environment is created and STAT1 is activated in colorectal cancer cells, thereby increasing IRF1 and PD-L1.

Example 5. Confirmation of the effect of streptonigrin on apoptosis-related proteins

It is a widely known fact that cancer can be treated by regulating proteins involved in apoptosis in cancer cells. Accordingly, the expression of 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).

Specifically, 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. The protein was photosensitized to determine the expression level of apoptosis-related protein. Anti-caspase 3 antibody (Cell Signaling Technology), anti-cleaved caspase 3 antibody (Cell Signaling Technology), anti-PARP antibody (Cell Signaling Technology), anti-cleaved PARP antibody (Cell Signaling) Technology), anti-cytochrome C antibody (Abcam), and β-actin (Sigma-Aldrich) primary antibody were used.

As shown in FIG. 7 , the expression levels of apoptosis-related proteins cleaved Caspase 3, cleaved PARP, and cytochrome C were significantly increased in a concentration-dependent manner with the treatment of streptonigrin.

Example 6. Confirmation of the effect of streptonigrin on PD-L1 and MHC-I protein expression

For flow cytometry, 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 μl PBS.

As a result, as shown in FIG. 8 , it was found that there was no significant difference in the expression of MHC-I when streptonigrin was treated, but the expression of PD-L1 increased in a concentration-dependent manner.

Example 7. Confirmation of protein expression related to antigen presentation mechanism according to combined treatment with streptonigrin and IFN-γ

When IFN-γ, known as a factor that activates immune T cells, was treated, the effect of streptonigrin on the expression of antigen-presenting mechanism-related proteins (TAP1, TAP2, PSMB8, PSMB9, PSMB10) was confirmed.

Specifically, HT29 cells, a colorectal cancer cell line, were treated with streptonigrin (0, 250, 500 nM) and IFN-γ (10 IU/mL). was heated for 5 minutes. After electrophoresis on 12% SDS-polyacrylamide gel for 2 hours, the protein was transferred to a nitrocellulose membrane. Then, 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.

As a result, as shown in FIG. 9 , it was confirmed that the expression level of antigen presentation mechanism related proteins (TAP1, TAP2, PSMB8, PSMB9, PSMB10) was significantly reduced even when IFN-γ was treated.

Example 8. Confirmation of the effect of streptonigrin on NK activation ligand protein

Activation of NK cells is enhanced by the binding of NKG2D to other activating receptors such as 2B4. 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. Various types of NKG2D ligands (MICA, MICB, ULBP1, ULBP2, ULBP3) exist, and their expression is induced by stress, and various expression patterns are shown in various types of carcinoma. In the case of MHC class I-related chain A and B (MICA/B), expression is induced by stress such as heat shock, radiation, oxidative stress and viral infection, and UL-16 In the case of binding proteins (ULBPs), it is known that the expression is induced by viral infection. Accordingly, 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.

As shown in FIG. 10 , it was confirmed that the expression levels of ULBP1 and MIC A/B significantly increased according to the treatment with streptonigrin compared to the untreated control group.

Example 9. Confirmation of colon cancer cell line killing effect according to combined treatment with streptonigrin and anti-PD-1 antibody

First, through the above examples, when streptonigrin was treated at a concentration of 250 nM or higher in colorectal cancer cell lines, the mechanism capable of increasing the anticancer drug sensitivity in cancer cells in the group treated with IFN-γ 10 IU/mL was p- It was confirmed by confirming the protein expression of STAT1 and IRF1. That is, it was confirmed that only a single treatment of streptonigrin had a significant anticancer effect.

Based on this, it was confirmed whether the anticancer effect could be increased when streptonigrin and anti-PD-1 antibody were co-treated. Specifically, 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. During co-culture, streptonigrin (0, 50, 100 nM) and 10 μg/mL of an anti-Igg isotype control antibody (BioLegend) or 10 μg/mL of an 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).

As a result, as shown in FIG. 11 , it was confirmed that the level of cancer cell death significantly increased in colorectal cancer cells treated in combination with streptonigrin and an anti-PD-1 antibody. Compared to the control group treated with streptonigrin alone, when the anti-PD-1 antibody and streptonigrin were treated in combination, the cell viability was significantly reduced in colorectal cancer cell lines, and the experimental group in which the anti-PD-1 antibody and streptonigrin were administered alone It was confirmed that the cell death effect was significantly increased compared to that. These results showed the most significant effect when streptonigrin and anti-PD-1 antibody were treated at a concentration ratio of 1:200 (w/v%).

Summarizing the above examples, the present inventors confirmed that 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.

Therefore, it is expected that the streptonigrin of the present invention and an immune checkpoint inhibitor can be used in combination to prevent or treat colorectal cancer.

The above description of the present invention is for illustration, and those of ordinary skill in the art to which the present invention pertains can understand that it can be easily modified into 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 illustrative in all respects and not restrictive.

Claims (9)

streptonigrin or a pharmaceutically acceptable salt thereof; And a pharmaceutical composition for preventing or treating colon cancer comprising an immune checkpoint inhibitor as an active ingredient.
According to claim 1,
The streptonigrin is a pharmaceutical composition, characterized in that represented by the following formula (1).
[Formula 1]

According to claim 1,
The streptonigrin and the immune checkpoint inhibitor are 1:0.01 to 500 concentration ratio (w / v%) characterized in that mixed, the pharmaceutical composition.
According to claim 1,
The immune checkpoint inhibitor is a PD-L1 inhibitor or a PD-1 inhibitor, characterized in that the pharmaceutical composition.
5. The method of claim 4, wherein the PD-L1 inhibitor is atezolizumab, avelumab, duvalumab, envafolimab, cosibelimab, AUNP12, CA-170 and BMS-986189, a pharmaceutical composition selected from the group consisting of.
5. The method of claim 4, wherein the PD-1 inhibitor is nivolumab, pembrolizumab, semiplimab, spartalizumab, camrelizumab, scintilimab ( sintilimab), tislelizumab, toripalimab, dostarlimab, INCMGA00012, AMP-224 and AMP-514, characterized in that selected from the group consisting of, a pharmaceutical composition.
According to claim 1,
The streptonigrin and the immune checkpoint inhibitor are characterized in that administered at the same time, the pharmaceutical composition.
According to claim 1,
The streptonigrin and the immune checkpoint inhibitor are characterized in that sequentially administered, the pharmaceutical composition.
According to claim 1,
The composition is characterized in that it increases the expression of one or more proteins selected from the group consisting of PD-L1, STAT1 and IRF1, a pharmaceutical composition.

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