KR102595271B1 - Pharmaceutical composition comprising Niclosamide and Sunitinib for preventing or treating kidney cancer - Google Patents

Abstract

The present invention relates to a pharmaceutical composition for combined administration for the prevention or treatment of kidney cancer. Niclosamide not only has an anticancer effect against kidney cancer, but also has an anticancer effect when used in combination with sunitinib compared to the use of each drug alone. It was completed after confirming that there was a significant increase. Specifically, the present inventors confirmed that niclosamide exerts an anticancer effect alone by promoting apoptosis of kidney cancer cells while inhibiting cancer cell migration and invasion. As a result of analyzing the synergy effect of drug combinations, niclosamide and Suniti It was confirmed that the combination of nips achieved a synergistic effect in tumor suppression. In fact, as a result of confirming the combined effect of niclosamide and sunitinib in vitro and in vivo , it was confirmed that the tumor suppressive effect was more excellent compared to the single administration of each drug, and this synergistic effect was due to the DNA repair mechanism of niclosamide ( It was confirmed that it was due to the regulation of DNA repair pathway (DNA repair pathway) and cell cycle regulation mechanism. In other words, the combined use of niclosamide and sunitinib is expected to be used as an effective treatment strategy for kidney cancer as it can maximize the anticancer effect while reducing the side effects caused by the single use of each drug.

Description

Pharmaceutical composition comprising Niclosamide and Sunitinib for preventing or treating kidney cancer}

The present invention relates to a pharmaceutical composition for combined administration for preventing or treating kidney cancer.

Kidney cancer is a malignant tumor that occurs in the kidney. Depending on the location of occurrence, it is divided into renal pelvic cancer, which occurs in the renal pelvis, and renal cell carcinoma, which mainly occurs in the renal tubules. Renal cell cancer accounts for approximately 80% of all renal cancers, so it is generally classified as renal cancer. Kidney cancer refers to renal cell carcinoma. Kidney cancer is increasing in incidence worldwide every year, accounting for 2% of all new tumor cases.

Kidney cancer is often asymptomatic for a considerable period of time after the tumor develops, and symptoms do not appear until it has progressed to a significant degree. When treating kidney cancer, if there is no metastasis to other organs, the kidney and surrounding tissues are extensively resected. If the tumor size is relatively small, resection surgery is usually performed laparoscopically. Immunotherapy, chemotherapy, and radiation therapy are used as general anticancer treatments, but they are known to be largely ineffective in kidney cancer, and surgery is currently used as the main treatment method.

Sunitinib is a targeted anticancer treatment approved by the U.S. Food and Drug Administration (FDA) for the treatment of metastatic kidney cancer. Sunitinib exhibits anti-cancer effects by inhibiting the formation of new blood vessels, and shows a high response rate in clear renal cell carcinoma, the most common type of kidney cancer. However, sunitinib develops resistance within 6 to 15 months, which reduces its anticancer effect, and a recent study reported that sunitinib is less effective in preventing recurrence of kidney cancer after surgery compared to placebo. Therefore, recent research has been actively conducted on the development of new drugs and combination therapy with other drugs to overcome the resistance problem of sunitinib and improve its anticancer effect.

Meanwhile, niclosamide is an anthelmintic drug that has been used for a long time to prevent parasitic infections, and its safety has already been confirmed. Recently, it has been reported that niclosamide has an anticancer effect in some types of cancer, but the effectiveness of niclosamide in treating kidney cancer is not well known. In particular, there is insufficient research on using niclosamide as a sole anticancer agent.

Republic of Korea Patent No. 10-1456754

The present invention was conceived to solve the above problems, and was completed by confirming that niclosamide not only has an anticancer effect against kidney cancer, but also exhibits a significant anticancer effect when used in combination with sunitinib compared to the use of each drug alone. It has been done.

Accordingly, the object of the present invention is niclosamide or a pharmaceutically acceptable salt thereof; and sunitinib or a pharmaceutically acceptable salt thereof as an active ingredient to provide a pharmaceutical composition for combined administration for the prevention or treatment of kidney cancer.

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

The present invention relates to niclosamide or a pharmaceutically acceptable salt thereof; and sunitinib or a pharmaceutically acceptable salt thereof as an active ingredient. It provides a pharmaceutical composition for combined administration for the prevention or treatment of kidney cancer.

In one embodiment of the present invention, the pharmaceutical composition for combined administration includes the niclosamide or a pharmaceutically acceptable salt thereof; and sunitinib or a pharmaceutically acceptable salt thereof may be in the form of a mixture, but is not limited thereto.

In another embodiment of the present invention, the pharmaceutical composition for combined administration includes the niclosamide or a pharmaceutically acceptable salt thereof; and sunitinib or a pharmaceutically acceptable salt thereof may be formulated and administered simultaneously or sequentially, but is not limited thereto.

In another embodiment of the present invention, the niclosamide or a pharmaceutically acceptable salt thereof; and sunitinib or a pharmaceutically acceptable salt thereof may be mixed at a molar ratio of 1:0.1 to 25, but is not limited thereto.

In another embodiment of the present invention, the niclosamide or a pharmaceutically acceptable salt thereof; and sunitinib or a pharmaceutically acceptable salt thereof may be mixed at a weight ratio of 1:0.1 to 10, but is not limited thereto.

In another embodiment of the present invention, the composition may satisfy one or more characteristics selected from the group consisting of, but is not limited to:

(a) Inducing apoptosis of cancer cells;

(b) arresting cell cycle progression; and

(c) Regulating the repair mechanism of damaged DNA.

In another embodiment of the present invention, the composition may inhibit the expression of one or more genes selected from the group consisting of BRIP1, E2F2, FANCA, cyclin A, cyclin B, and CDK1, but is not limited thereto.

In another embodiment of the present invention, the composition may have a higher anticancer effect compared to the use of niclosamide alone or sunitinib alone, but the composition is not limited thereto.

In another embodiment of the present invention, the composition may be used for the treatment of an individual carrying a mutation in one or more genes selected from the group consisting of BRIP1, E2F2, FANCA, cyclin A, cyclin B, and CDK1. It is not limited.

Additionally, the present invention provides a kit for preventing or treating kidney cancer comprising the composition according to the present invention.

Additionally, the present invention provides a method for preventing or treating kidney cancer, comprising administering the pharmaceutical composition for combined administration according to the present invention to an individual in need thereof. That is, the present invention relates to niclosamide or a pharmaceutically acceptable salt thereof; and co-administering sunitinib or a pharmaceutically acceptable salt thereof to an individual in need thereof.

In addition, the present invention provides niclosamide or a pharmaceutically acceptable salt thereof for the manufacture of a drug for preventing or treating kidney cancer; And it provides a use of sunitinib or a pharmaceutically acceptable salt thereof. Preferably, the drug is a drug for combined administration.

Additionally, the present invention relates to niclosamide or a pharmaceutically acceptable salt thereof; And it provides a use of a composition containing sunitinib or a pharmaceutically acceptable salt thereof for preventing or treating kidney cancer.

The present invention relates to a pharmaceutical composition for combined administration for the prevention or treatment of kidney cancer. Niclosamide not only has an anticancer effect against kidney cancer, but also has an anticancer effect when used in combination with sunitinib compared to the use of each drug alone. It was completed after confirming that there was a significant increase. Specifically, the present inventors confirmed that niclosamide exerts an anticancer effect alone by promoting apoptosis of kidney cancer cells while inhibiting cancer cell migration and invasion. As a result of analyzing the synergy effect of drug combinations, niclosamide and Suniti It was confirmed that the combination of nips achieved a synergistic tumor suppressive effect. In fact, as a result of confirming the combined effect of niclosamide and sunitinib in vitro and in vivo , it was confirmed that the tumor suppressive effect was more excellent compared to the single administration of each drug, and this synergistic effect was due to the DNA repair mechanism of niclosamide. It was confirmed that it was due to regulation and cell cycle control mechanisms. In other words, the combined use of niclosamide and sunitinib is expected to be used as an effective treatment strategy for kidney cancer as it can maximize the anticancer effect while reducing the side effects caused by the single use of each drug.

Figure 1 shows the results of measuring cell viability (activity) by WST analysis after treating kidney cancer cells with sunitinib for 24 or 48 hours.
Figure 2 shows the results of measuring cell viability by WST analysis after treating kidney cancer cells with niclosamide for 24 or 48 hours.
Figure 3 shows the results of analyzing the degree of apoptosis of cancer cells using Annexin V-FITC PI after treating kidney cancer cells with niclosamide.
Figure 4 shows the results of treating kidney cancer cells with niclosamide, wounding the cell culture well with a pipette tip, and then photographing the cell culture well over time to confirm the degree of cancer cell movement (×50 magnification).
Figure 5 shows the results of treating kidney cancer cells with niclosamide and performing Matrigel-based transwell analysis. The degree of cancer cell invasion was confirmed by photographing cells that had infiltrated into the lower chamber (×200 magnification).
Figure 6 shows the results of measuring cell activity after treating kidney cancer cells with sunitinib and niclosamide at various concentrations for 48 hours, and comparing and analyzing the synergy effect of the two drugs using SynergyFinder 2.0. Drugs were treated alone and in combination at various concentrations, and the experiment was repeated at least three times for each condition.
Figure 7 is a synergy landscape (2D & 3D) based on the HSA model, showing the synergy score of drug response in color. From green to red, it means that the synergy score is higher. A synergy score > 0 was indicated as synergism, and a synergy score < 0 was indicated as antagonism.
Figure 8 shows the synergy scores of drug responses in color based on the HSA model. Synergy scores were categorized as synergy (>0, red), antagonism (<0, green), or additive (=0, white). Each synergy score was calculated by treating each drug alone or in combination at 7 different doses and repeating the experiment at least 3 times for each condition.
Figure 9 shows the results of measuring cell activity by performing WST analysis after a certain period of time after treating kidney cancer cells with 2.5 μM sunitinib and 1 μM niclosamide alone or in combination.
Figure 10 shows the results of analyzing the degree of apoptosis of cancer cells with Annexin V-FITC PI after treating kidney cancer cells with 2.5 μM sunitinib and 1 μM niclosamide for 48 hours (D, DMSO; S, sunitinib; N, niclosamide; C, combination).
Figure 11 shows control (PBS containing 1% DMSO and 1% Tween-80), sunitinib alone (20 mg/kg), niclosamide alone (20 mg/kg), and suniti in the ACHN xenograft mouse model. The results are shown by measuring the tumor volume every two weeks and calculating the average value while administering the combination of nip (20 mg/kg) and niclosamide (20 mg/kg) for 29 days (n=5, compared to the control group, * P < 0.05, ** P <0.01;^# P < 0.01 when comparing the single administration group and the combined administration group).
Figure 12 is a photograph of tumor tissue obtained by administering the drug as above to the ACHN xenograft mouse model for 29 days and then euthanizing the mouse.
Figure 13 shows the results of H&E staining and immunohistochemical staining on tumor tissue obtained after administering the drug as above to the ACHN xenograft mouse model for 29 days (scale bar, 100 μm). Immunohistochemical staining was performed with Ki-67 antibody and cleaved caspase-3 antibody.
Figure 14 shows the results of cross-analysis for identification of genes downregulated by single or combined treatment of sunitinib and niclosamide.
Figure 15 shows the results of KEGG pathway analysis of the down-regulated genes.
Figure 16 shows the results of confirming the γH2AX protein level by Western blot after treating kidney cancer cell lines with sunitinib alone (2.5 μM), niclosamide alone (1 μM), or a combination thereof for 24 hours.
Figure 17 shows the results of detecting the γH2AX locus in the nucleus by immunofluorescence staining after treating kidney cancer cell lines with sunitinib alone (2.5 μM), niclosamide alone (1 μM), or a combination thereof for 24 hours ( Green, γH2AX; blue, DAPI; scale bar = 20 μm). Fluorescence intensity was analyzed using ZEN 2012 Black Edition software (D, DMSO; S, sunitinib; N, niclosamide; C, combination).
Figure 18 shows the results of cell cycle analysis confirmed through flow cytometry, where each bar graph represents the cell distribution ratio (%) in G1, S, and G2/M phases (* P <0.05 compared to the control group, ^# P < 0.05 compared to the sunitinib treatment group alone).
Figure 19 shows the results of Western blot analysis of protein levels of Cyclin A, Cyclin B, CDK1, and β-actin after treatment of kidney cancer cell lines with sunitinib alone, niclosamide alone, or a combination thereof.
Figure 20 shows the results of comparing the gene expression levels of E2F2, TTK, BRIP1, FANCA, and DNA2 in tumor tissues and normal tissues based on the TCGA database-KIRC.
Figure 21 analyzes the mRNA expression levels of E2F2, TTK, BRIP1, FANCA, and DNA2 after treating kidney cancer cell lines with sunitinib alone (2.5 μM), niclosamide alone (1 μM), or a combination thereof for 48 hours. Shows one result.
Figure 22 shows the results of Western blot analysis of BRIP, E2F2, and FANCA protein levels after treating kidney cancer cell lines with sunitinib alone (2.5 μM), niclosamide alone (1 μM), or a combination thereof for 48 hours. (D, DMSO; S, sunitinib; N, niclosamide; C, combination).

The present invention relates to a pharmaceutical composition for combined administration for the prevention or treatment of kidney cancer. Niclosamide not only has an anticancer effect against kidney cancer, but also has a significant anticancer effect when used in combination with sunitinib compared to the use of each drug alone. It was completed after confirming that it was working.

Specifically, in one example of the present invention, as a result of confirming the anticancer effect of niclosamide alone, it was confirmed that niclosamide exerts an anticancer effect by increasing apoptosis of kidney cancer cells and inhibiting cell migration and invasion (Example 1 ).

In another embodiment of the present invention, the effect of the combination of niclosamide and sunitinib was confirmed using response analysis software according to the drug combination, and it was confirmed that the combination of the two drugs had a synergistic anticancer effect, and in vitro and in vivo experiments were performed. As a result of comparing the effects of single and combined use of the two drugs, it was confirmed that the cancer suppressing effect was significantly higher when treated in combination compared to treated with each drug alone (Example 2).

In another embodiment of the present invention, RNA sequencing and KEGG pathway analysis were performed to confirm the mechanism related to the anticancer effect of niclosamide. As a result, the anticancer effect of niclosamide was determined by regulation of the DNA repair pathway and cell cycle. It was confirmed that this was due to stopping (Example 3).

In another example of the present invention, as a result of analyzing genes whose expression changes are induced by niclosamide, it was confirmed that niclosamide exerts an anticancer effect by suppressing the expression of BRIP1, E2F2, and FANCA (Example 4) .

Hereinafter, the present invention will be described in detail.

The present invention relates to niclosamide or a pharmaceutically acceptable salt thereof; and sunitinib or a pharmaceutically acceptable salt thereof as an active ingredient. It provides a pharmaceutical composition for combined administration for the prevention or treatment of kidney cancer.

In the present invention, “Niclosamide” is a chlorinated salicylanilide that is effective in treating parasitic infections. When administered orally, it reacts with androgen receptor variant V7 through a proteasome-mediated pathway. It is a drug that inhibits the development of androgen receptor variants by inducing the decomposition of . Nicolasamide has been widely used as an anthelmintic around the world and is therefore guaranteed to be safe for the human body. Niclosamide according to the present invention may be represented by the following formula (1), but is not limited thereto.

[Formula 1]

In the present invention, “Sunitinib” is a tyrosine phosphorylation enzyme inhibitor used for the treatment of advanced renal cell carcinoma, advanced pancreatic endocrine tumor, and resistant gastrointestinal basal tumor, and was approved by the FDA as an anticancer treatment in 2006. received. Sunitinib inhibits angiogenesis and cancer cell proliferation by inhibiting the tyrosine kinase activities of vascular endothelial growth factor receptor 2 (VEGFR2), platelet-derived growth factor receptor b (PDGFRb), and c-kit. In the present invention, sunitinib may be represented by the following formula (2), but is not limited thereto.

[Formula 2]

The composition according to the present invention may include a pharmaceutically acceptable salt of niclosamide; And/or a pharmaceutically acceptable salt of sunitinib may be included 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, etc. Acid addition salts can be prepared by conventional methods, for example, by dissolving the compound in an excessive amount of aqueous 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 equimolar amounts of the compound and an acid or alcohol in water and then evaporating the mixture to dryness, or suction filtering 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 excessive amount of alkali metal hydroxide or alkaline earth metal hydroxide solution, filtering the undissolved compound salt, and then evaporating and drying the filtrate. At this time, it is particularly pharmaceutically suitable to produce sodium, potassium or calcium salts as metal salts, and the corresponding silver salts can be obtained by reacting an alkali metal or alkaline earth metal salt with an appropriate silver salt (eg, silver nitrate).

The niclosamide or a pharmaceutically acceptable salt thereof in the composition of the present invention; And the content of sunitinib or a pharmaceutically acceptable salt thereof can be appropriately adjusted depending on the symptoms of the disease, the degree of progression of the symptoms, the patient's condition, etc., for example, 0.0001 to 99.9% by weight, or 0.001 to 0.001% by weight, based on the total weight of the composition. It may be 50% by weight, but is not limited thereto. The content ratio is a value based on the dry amount with the solvent removed.

Preferably, in the present invention, the niclosamide or a pharmaceutically acceptable salt thereof; And sunitinib or a pharmaceutically acceptable salt thereof is 1:0.1 to 25, 1:0.1 to 20, 1:0.1 to 18, 1:0.1 to 16, 1:0.1 to 14, 1:0.1 to 12, 1 : 0.1 to 10, 1 : 0.1 to 8, 1 : 0.1 to 6, 1 : 0.1 to 5, 1 : 0.1 to 4, 1 : 0.1 to 3, 1 : 0.1 to 2, 1 : 0.1 to 1, 1 : 1 to 10, 1:1 to 8, 1:1 to 6, 1:1 to 4, 1:1 to 3, 1:1.5 to 5, 1:1.5 to 4.5, 1:1.5 to 4, 1:1.5 to 3.5 , 1:1.5 to 3, 1:2 to 5, 1:2 to 4.5, 1:2 to 4, 1:2 to 3.5, or 1:2 to 3, but is not limited thereto. .

Alternatively, in the present invention, niclosamide or a pharmaceutically acceptable salt thereof; And sunitinib or a pharmaceutically acceptable salt thereof is 1:0.1 to 10, 1:0.1 to 9, 1:0.1 to 8, 1:0.1 to 7, 1:0.1 to 6, 1:0.1 to 5, 1 : 0.1 to 4, 1 : 0.1 to 3, 1 : 0.1 to 2, 1 : 0.1 to 1.5, 1 : 0.5 to 5, 1 : 0.5 to 4.5, 1 : 0.5 to 4, 1 : 0.5 to 3.5, 1 : 0.5 to 3, 1:0.5 to 2.5, 1:0.5 to 2, 1:0.5 to 1.5, 1:0.7 to 3, 1:0.7 to 2.5, 1:0.7 to 2, 1:0.7 to 1.5, or 1:0.7 to It may be mixed at a weight ratio (w/w %) of 1.2, but is not limited thereto.

The pharmaceutical composition according to the present invention may further include appropriate carriers, excipients, and diluents commonly used in the preparation of pharmaceutical compositions. The excipient may be, for example, one or more selected from the group consisting of diluents, binders, disintegrants, lubricants, adsorbents, humectants, film-coating materials, and controlled-release additives.

The pharmaceutical composition according to the present invention can be prepared as powder, granules, sustained-release granules, enteric-coated granules, solutions, eye drops, ellipsis, emulsions, suspensions, spirits, troches, perfumes, and limonadese according to conventional methods. , tablets, sustained-release tablets, enteric-coated tablets, sublingual tablets, hard capsules, soft capsules, sustained-release capsules, enteric-coated capsules, pills, tinctures, soft extracts, dry extracts, liquid extracts, injections, capsules, perfusate, It can be formulated and used in the form of external preparations such as warning agents, lotions, pasta preparations, sprays, inhalants, patches, sterilized injection solutions, or aerosols, and the external preparations include creams, gels, patches, sprays, ointments, and warning agents. , it may have a dosage form such as 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, oligosaccharides, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum acacia, alginate, gelatin, and calcium. These include phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil.

When formulated, it is prepared using diluents or excipients such as commonly used fillers, extenders, binders, wetting agents, disintegrants, and surfactants.

Additives to tablets, powders, granules, capsules, pills, and troches according to the present invention include corn starch, potato starch, wheat starch, lactose, white sugar, glucose, fructose, di-mannitol, precipitated calcium carbonate, synthetic aluminum silicate, and phosphoric acid. Calcium monohydrogen, calcium sulfate, sodium chloride, sodium bicarbonate, purified lanolin, microcrystalline cellulose, dextrin, sodium alginate, methylcellulose, sodium carboxymethylcellulose, kaolin, urea, colloidal silica gel, hydroxypropyl starch, hydroxypropylmethyl. Excipients such as cellulose (HPMC) 1928, HPMC 2208, HPMC 2906, HPMC 2910, propylene glycol, casein, calcium lactate, and Primogel; Gelatin, gum arabic, ethanol, agar powder, cellulose acetate phthalate, carboxymethyl cellulose, calcium carboxymethyl cellulose, glucose, purified water, sodium caseinate, glycerin, stearic acid, sodium carboxymethyl cellulose, sodium methyl cellulose, methyl cellulose, microcrystalline cellulose, dextrin. , hydroxycellulose, hydroxypropyl starch, hydroxymethylcellulose, refined shellac, starch, hydroxypropylcellulose, hydroxypropylmethylcellulose, polyvinyl alcohol, polyvinylpyrrolidone, etc. binders can be used, Hydroxypropyl methyl cellulose, corn starch, agar powder, methyl cellulose, bentonite, hydroxypropyl starch, sodium carboxymethyl cellulose, sodium alginate, calcium carboxymethyl cellulose, calcium citrate, sodium lauryl sulfate, silicic acid anhydride, 1-hydroxy Propylcellulose, 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, white sugar, magnesium aluminum silicate, di-sorbitol solution, light anhydrous silicic acid; Calcium stearate, magnesium stearate, stearic acid, hydrogenated vegetable oil, talc, lycopodium, 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 acids, higher alcohol, silicone oil, paraffin oil, polyethylene glycol fatty acid ether, Lubricants such as starch, sodium chloride, sodium acetate, sodium oleate, dl-leucine, and light anhydrous silicic acid may be used.

Additives for the liquid according to the present invention include water, dilute hydrochloric acid, dilute sulfuric acid, sodium citrate, sucrose monostearate, polyoxyethylene sorbitol fatty acid esters (twin esters), 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.

A solution of white sugar, other sugars, or sweeteners, etc. may be used in the syrup according to the present invention, and if necessary, flavoring agents, colorants, preservatives, stabilizers, suspending agents, emulsifiers, thickening agents, etc. may be used.

Purified water can be used in the emulsion according to the present invention, and emulsifiers, preservatives, stabilizers, fragrances, etc. can be used as needed.

Suspensions 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. Topics may be used, and surfactants, preservatives, stabilizers, colorants, and fragrances may be used as needed.

Injections according to the present invention include distilled water for injection, 0.9% sodium chloride injection, IV solution, dextrose injection, dextrose + sodium chloride injection, PEG, lactated IV solution, ethanol, propylene glycol, non-volatile oil - sesame oil. solvents such as cottonseed oil, peanut oil, soybean oil, corn oil, ethyl oleate, isopropyl myristic acid, and benzene benzoate; Solubilizers such as sodium benzoate, sodium salicylate, sodium acetate, urea, urethane, monoethylacetamide, butazolidine, propylene glycol, Tween, nicotinic acid amide, hexamine, and dimethylacetamide; Weak acids and their salts (acetic acid and sodium acetate), weak bases and their salts (ammonia and ammonium acetate), organic compounds, proteins, albumin, peptone, and buffering agents such as gums; 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 ), and ethylenediaminetetraacetic acid; Sulfurizing agents such as sodium bisulfide 0.1%, sodium formaldehyde sulfoxylate, thiourea, disodium ethylenediaminetetraacetate, and acetone sodium bisulfite; Analgesics such as benzyl alcohol, chlorobutanol, procaine hydrochloride, glucose, and calcium gluconate; It may contain suspending agents such as CM sodium, sodium alginate, Tween 80, and aluminum monostearate.

Suppositories according to the present invention include cacao oil, lanolin, witepsol, polyethylene glycol, glycerogelatin, methylcellulose, carboxymethylcellulose, 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, Buytyrum Tego -G), Cebes Pharma 16, Hexalide Base 95, Cotomar, Hydrocote 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, Massaupol, Masupol-15, Neosupostal-N, Paramound-B, Suposiro (OSI, OSIX, A, B, C, D, H, L), suppositories type IV (AB, B, A, BC, BBG, E, BGF, C, D, 299), Supostal (N, Es), Wecobi (W, R, S, M, Fs), Tegestor triglyceride base (TG-95, MA, 57) and The same mechanism can be used.

Solid preparations for oral administration include tablets, pills, powders, granules, capsules, etc. These solid preparations include the extract with at least one excipient, such as starch, calcium carbonate, and sucrose. ) or prepared by mixing lactose, gelatin, etc. In addition to simple excipients, lubricants such as magnesium styrate talc are also used.

Liquid preparations for oral administration include suspensions, oral solutions, emulsions, and syrups. In addition to the commonly used simple diluents such as water and liquid paraffin, various excipients such as wetting agents, sweeteners, fragrances, and preservatives may be included. there is. Preparations for parenteral administration include sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, and suppositories. Non-aqueous solvents and suspensions include propylene glycol, polyethylene glycol, vegetable oil such as olive oil, and injectable ester such as ethyl oleate.

In the present invention, “kidney cancer” refers to any malignant tumor that occurs in the kidney. Specifically, in the present invention, renal cancer is renal cell carcinoma (RCC), and includes clear renal cell carcinoma, papillary renal cell carcinoma, chromophobe renal cell carcinoma, and collecting duct renal cell carcinoma. Preferably, the renal cancer in the present invention is clear renal cell carcinoma (clear cell RCC, ccRCC).

The present invention relates to niclosamide or a pharmaceutically acceptable salt thereof; and sunitinib or a pharmaceutically acceptable salt thereof, which maximizes the anticancer efficacy of each drug and minimizes side effects such as toxicity by reducing the dosage of each drug.

Therefore, the pharmaceutical composition for combined administration according to the present invention includes the niclosamide or a pharmaceutically acceptable salt thereof; and sunitinib or a pharmaceutically acceptable salt thereof, preferably in the form of a mixture for simultaneous administration of the two drugs.

Alternatively, the pharmaceutical composition for combined administration according to the present invention may include the niclosamide or a pharmaceutically acceptable salt thereof; and sunitinib or a pharmaceutically acceptable salt thereof may be formulated and administered simultaneously, or sequentially. In this case, the pharmaceutical composition for combined administration includes a first pharmaceutical composition containing a pharmaceutically effective amount of niclosamide or a pharmaceutically acceptable salt thereof; and a second pharmaceutical composition containing a pharmaceutically effective amount of sunitinib or a pharmaceutically acceptable salt thereof, and may be a pharmaceutical composition for combined administration for simultaneous or sequential administration. At this time, in the case of sequential administration, the order of administration is not limited, and the administration regimen can be appropriately adjusted depending on the patient's condition, etc.

That is, if the pharmaceutical composition is a pharmaceutical composition for combined administration for sequential administration, the composition is administered first, followed by niclosamide or a pharmaceutically acceptable salt thereof (“first ingredient”), followed by sunitinib or a pharmaceutically acceptable salt thereof. A pharmaceutically acceptable salt (“second component”) may be administered, or the reverse order is also possible.

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 the disease with a reasonable benefit/risk ratio applicable to medical treatment, and the effective dose level is determined by the type, severity, drug activity, and It can be determined based on factors including sensitivity to the drug, time of administration, route of administration and excretion rate, duration of treatment, drugs used simultaneously, and other factors well known in the medical field.

That is, in the composition according to the present invention, niclosamide or a pharmaceutically acceptable salt thereof; And there is no limitation to the pharmaceutically effective amount (i.e., administered dose) of sunitinib or a pharmaceutically acceptable salt thereof, and may vary depending on the patient's condition and severity of the disease, but is preferably 1 dose for each individual drug. The dosage per dose is 1 to 30 mg/kg, 1 to 28 mg/kg, 1 to 26 mg/kg, 1 to 24 mg/kg, 1 to 22 mg/kg, 1 to 20 mg/kg, and 1 to 18 mg/kg, respectively. mg/kg, 1 to 16 mg/kg, 1 to 14 mg/kg, 1 to 12 mg/kg, 1 to 10 mg/kg, 1 to 8 mg/kg, 1 to 6 mg/kg, 1 to 4 mg /kg, 1 to 2 mg/kg, 4 to 30 mg/kg, 6 to 28 mg/kg, 8 to 26 mg/kg, 10 to 24 mg/kg, 12 to 24 mg/kg, 14 to 24 mg/ kg, 16 to 24 mg/kg, 16 to 24 mg/kg, 18 to 22 mg/kg, or 19 to 22 mg/kg.

The present invention also provides a method for preventing or treating kidney cancer, comprising administering the pharmaceutical composition according to the present invention to an individual in need thereof. That is, the present invention relates to niclosamide or a pharmaceutically acceptable salt thereof; and co-administering sunitinib or a pharmaceutically acceptable salt thereof to an individual in need thereof.

The pharmaceutical composition according to the present invention may be administered as an individual therapeutic agent or in combination with other therapeutic agents, may be administered sequentially or simultaneously with conventional therapeutic agents, and may be administered singly or multiple times. Considering all of the above factors, it is important to administer an amount that can achieve the maximum effect with the minimum amount without side effects, and this can be easily determined by a person skilled in the art to which the present invention pertains.

The pharmaceutical composition of the present invention can be administered to an individual through various routes. All modes of administration are contemplated, including oral administration, subcutaneous injection, intraperitoneal administration, intravenous injection, intramuscular injection, paraspinal space (intrathecal) injection, sublingual administration, buccal administration, intrarectal injection, vaginal injection. It can be administered by internal insertion, ocular administration, ear administration, nasal administration, inhalation, spraying through the mouth or nose, dermal administration, transdermal administration, etc.

The pharmaceutical composition of the present invention is determined depending on the type of drug as the active ingredient along with various related factors such as the disease to be treated, the route of administration, the patient's age, gender, weight, and severity of the disease.

In the present invention, “individual” refers to a subject in need of treatment for a disease, and more specifically, human or non-human primates, mice, rats, dogs, cats, horses, cows, etc. refers to mammals of

In the present invention, “administration” means providing a given composition of the present invention to an individual by any appropriate method.

In the present invention, “prevention” refers to any action that suppresses or delays the onset of the desired disease, and “treatment” refers to the improvement or improvement of the desired disease and its associated metabolic abnormalities by administration of the pharmaceutical composition according to the present invention. It refers to all actions that are beneficially changed, and “improvement” refers to all actions that reduce parameters related to the target disease, such as the degree of symptoms, by administering the composition according to the present invention.

In summary, the composition or treatment method for combined administration according to the present invention is not limited to the method or order of administration of each drug, but includes niclosamide or a pharmaceutically acceptable salt thereof; and sunitinib or a pharmaceutically acceptable salt thereof, or all of them are administered, and the kidney cancer prevention, improvement, and/or treatment effect is higher (increased) compared to using each of the above ingredients alone. characterized by).

In the present invention, “increased” means that the level in the experimental group is at least 1%, 2%, 3%, 4%, 5%, 10% or more, for example, 5%, 10%, or more, compared to that in the control group. %, 20%, 30%, 40%, or 50%, 60%, 70%, 80%, 90% or higher, and/or 0.5 times, 1.1 times, 1.2 times, 1.4 times, 1.6 times, 1.8 times It means twice, 2 times, 2.2 times, 2.4 times, 2.6 times, 2.8 times, 3 times or more. Specifically, 1 to 10 times, 2 to 10 times, 2.5 to 10 times, 3 to 10 times, 3.5 to 10 times, 4 to 10 times, 4.5 to 10 times, 5 to 10 times, 5.5 to 10 times that of the control group. 10 times, 6 to 10 times, 6.5 to 10 times, 7 to 10 times, 7.5 to 10 times, 8 to 10 times, 1 to 1.5 times, 1.5 to 2 times, 2 to 2.5 times, 2.5 to 3 times, 3 to 3.5 times, 3.5 to 4 times, 4 to 4.5 times, 4.5 to 5 times, 5 to 5.5 times, 5.5 to 6 times, 6 to 6.5 times, 6.5 to 7 times, 7 to 7.5 times, 7.5 to 8 times, 8 to It may mean an increase of 8.5 times, 8.5 to 9 times, 9 to 9.5 times, 9.5 to 10 times, or more than 10 times, but is not limited thereto. The meaning of the opposite term can be understood by those skilled in the art as having the opposite meaning according to the above definition.

The composition according to the present invention is characterized by satisfying one or more characteristics selected from the group consisting of, but is not limited to: (a) inducing apoptosis of cancer cells; (b) cell cycle arrest; and (c) regulating the DNA repair pathway of damaged DNA.

In the present invention, “apoptosis” is a programmed cell death process, which is cell death regulated by the expression of a series of genes, and is morphologically characterized by cell shrinkage and blebbing of the cell membrane. ), nuclear fragmentation, and chromatin condensation (chromosomal condensation). In the present invention, niclosamide is characterized in that it exerts an anti-cancer effect by inducing apoptosis of cancer cells and causing them to die.

In the present invention, “cell cycle” refers to a series of processes from the occurrence of cell division until the next cell division occurs again, and “cell cycle arrest” refers to the cell cycle at some point. This means that DNA replication or cell division does not proceed because it stops at this point. In the present invention, niclosamide is characterized by inhibiting DNA replication and cell division of cancer cells by stopping the cell cycle progression of cancer cells. In the present invention, cell cycle arrest by niclosamide occurs in the G1 phase, S phase, G2 phase, or M phase, or in the G1/S transition phase, S/G2 transition phase, G2/M transition phase, or It may occur at the M/G1 transition stage, but is not limited to this.

In the present invention, “DNA repair pathway” refers to the overall process by which cells recognize and correct damage to DNA molecules. Every day, numerous DNA damage occurs due to the normal metabolic activities of cells in the body and environmental factors outside the body. The DNA repair mechanism recognizes this and repairs the DNA damage to prevent mutations or diseases caused by DNA damage. However, the DNA repair mechanism of cancer cells can be a factor that inhibits the efficacy of anticancer drugs. Various chemotherapy drugs induce cancer cell death by causing excessive damage to the DNA of cancer cells, and the DNA repair mechanism of cancer cells prevents cell death by repairing DNA damage caused by anticancer drugs. Therefore, the efficacy of anticancer drugs depends on the DNA repair ability of cancer cells, and in fact, preclinical cases have been confirmed in which DNA repair inhibitors enhance the efficacy of DNA damage-inducing anticancer drugs. For example, Olarparib, approved by the FDA, induces the death of cancer cells with BRCA1 and BRCA2 mutations through inhibition of poly ADP ribose polymerase, an enzyme involved in DNA repair. In the present invention, niclosamide is characterized by regulating the damaged DNA repair mechanism of cancer cells. Specifically, when DNA damage occurs in cancer cells, niclosamide regulates the expression of genes involved in the DNA damage repair mechanism of cancer cells. It is characterized by preventing the repair of damaged DNA by regulating its activity and ultimately inducing the death of cancer cells. Preferably, regulation of the DNA repair mechanism by niclosamide may be achieved through regulation of the expression or activity of BRIP1, E2F2, and/or FANCA.

In addition, the composition according to the present invention inhibits the expression of one or more genes selected from the group consisting of BRIP1 (BRCA1 interacting helicase 1), E2F2 (E2F transcription factor 2), FANCA (FA complementation group A), cyclin A, cyclin B, and CDK1. It is characterized by suppression. Therefore, the composition according to the present invention may be particularly suitable for preventing or treating kidney cancer in individuals carrying mutations in one or more genes selected from the group consisting of BRIP1, E2F2, FANCA, cyclin A, cyclin B, and CDK1. The mutation includes increased expression of the gene and/or increased protein activity.

BRIP1 is a gene involved in DNA damage repair and is known to be a treatment target for gastric cancer, and overexpression of BRIP1 is known to be a clinicopathological parameter of breast cancer. Specific information about BRIP1 can be found at NCBI (Gene ID: 83990).

E2F2 is a gene that regulates G1/S phase progression and the initiation of DNA replication. It was recently reported that E2F2 expression is increased in kidney cancer patients and can be used as a diagnostic biomarker for KIRC diagnosis (B Liang, et al.). Specific information about E2F2 can be found at NCBI (Gene ID: 1870).

FANCA is also a gene involved in DNA replication and DNA damage repair, and increased expression of FANCA is known to be associated with the progression and poor prognosis of chronic lymphocytic leukemia. Specific information about FANCA can be found at NCBI (Gene ID: 2175).

Additionally, the present invention provides a kit for preventing or treating kidney cancer comprising the composition according to the present invention.

In the present invention, “kit” refers to a combination of substances or devices that can be used to prevent or treat kidney cancer, and can be in a form that can be used or administered in combination with niclosamide and sunitinib, and can be used in a specific form. There is no limit to the shape. Therefore, the kit according to the present invention can include without limitation niclosamide and sunitinib, as well as any components known in the art as components of a kit for the treatment of a specific disease.

Throughout the specification of the present invention, when a part is said to “include” a certain component, this means that it may further include other components rather than excluding other components unless specifically stated to the contrary. The terms "about", "substantially", etc. used throughout the specification of the present invention are used to mean at or close to that value when manufacturing and material tolerances inherent in the stated meaning are presented, and the present invention Precise or absolute figures are used to aid understanding and to prevent unscrupulous infringers from taking unfair advantage of the disclosure.

Throughout the specification of the present invention, the term "combination thereof" included in the Markushi format expression means a mixture or combination of one or more selected from the group consisting of the components described in the Markushi format expression, It means containing one or more selected from the group consisting of constituent elements.

Below, preferred embodiments are presented to aid understanding of the present invention. However, the following examples are provided only to make the present invention easier to understand, and the content of the present invention is not limited by the following examples.

[Experimental materials and methods]

Sunitinib malate, niclosamide, and dimethyl sulfoxide (DMSO) were purchased from Sigma-Aldrich (MO, USA). Sunitinib and niclosamide were dissolved in DMSO and stored at -20°C. A-498, ACHN, and Caki-1 cells were obtained from Korea Cell Line Bank (Seoul, Korea). A-498 and ACHN cells were incubated with 10% fetal bovine serum (FBS; Gibco; Thermo Fisher Scientific, Inc.; MA, USA), 100 units/mL penicillin, and 0.1 mg/mL streptomycin (Gibco). Cultured in Dulbecco's modified Eagles' medium (DMEM) (Gibco). Caki-1 cells were cultured in RPMI1640 medium (Gibco) containing 10% FBS, 100 units/mL of penicillin, and 0.1 mg/mL of streptomycin. All cell cultures were grown at a temperature of 37°C in humidified air containing 5% CO ₂ .

Cell viability assay

Cells were seeded in a 96-well plate and the experiment was performed 24 hours later. Cells were treated with sunitinib or niclosamide alone at various doses, and similarly, cells were treated with a combination of sunitinib and niclosamide at various doses. Cell activity was assessed using EZ-CYTOX (Daeil Lab Service; Seoul, Korea) according to the manufacturer's instructions.

Wound healing assay

Cells were seeded in a 6-well plate and the experiment was performed 24 hours later. After creating a wound with a 200 μL pipette tip, cells were treated with DMSO or niclosamide at various concentrations (0.5 to 5 μM). The wound area was observed under a light microscope (Carl Zeiss Inc.; Jena, Germany).

Cell invasion assay

Cell invasion assays were performed in 24-well transwell chambers equipped with 8 μm pore polycarbonate membrane inserts coated with 25 μg/mL Matrigel (Corning Inc.; NY, USA). Serum-free medium containing DMSO or various concentrations of niclosamide (0.5 to 5 μM) was used to plate in the upper well of the chamber. The lower chamber was filled with culture medium containing 10% FBS as a chemoattractant. After 48 hours of incubation, cells on the bottom of the membrane were fixed, stained using 0.1% crystal violet solution (Sigma-Aldrich), and observed under a light microscope (Carl Zeiss Inc.). The membrane was dissolved in 20% acetic acid and its color was measured at 570 nm.

Apoptosis assay

Cells were harvested 48 hours after drug treatment and washed three times with phosphate-buffered saline (PBS). Cells were stained with Annexin V and propidium iodide (PI) using the Annexin-FITC/PI staining kit (BD Biosciences; CA, USA) according to the manufacturer's instructions and FACS Canto II system (BD Biosciences). analyzed.

Drug interaction analysis

Cell activity was measured after treatment with single or combined drugs at various concentrations. To identify synergistic combinations, synergy scores were evaluated based on the Highest single agent (HSA) reference model using SynergyFider 2.0 software. Overall level synergy/antagonism was summarized against the overall dose-response matrix using delta scores to quantify the combination effect. HSA synergy scores were visualized as inhibition heatmaps and synergy landscapes (2D & 3D).

in vivo research

All experimental procedures were approved by the Catholic University Animal Care Committee (CUMC-2018-0313-01). Five-week-old male BALB/c nude mice (body weight 20-25 g) were purchased from Orient Bio (Gyeonggi, Korea). Mice (n=5) were injected into the flanks with 1×10 ⁷ ACHN cells dispersed in 100 μL of PBS containing 100 μL of Matrigel (Corning). When the tumor volume reached approximately 250 mm ³ , mice were randomly divided into 4 groups based on similar average size, and each group was administered vehicle, sunitinib (20 mg/kg) alone, or niclosamide (20 mg) daily. /kg) alone or in combination with niclosamide and sunitinib (niclosamide 20 mg/kg and sunitinib 20 mg/kg) were orally gavaged. Body weight and tumor size were measured every 3 days during the entire experiment period, and tumor volume was calculated as (horizontal length) ² × (vertical length) × 0.5. After 4 weeks of drug treatment, mice were euthanized and tumor tissues were obtained for further analysis.

Histological analysis

For histological analysis, tumor tissue was cut at 4 μM thickness and paraffin sections were stained with hematoxylin and eosin (H&E) staining. Paraffin sections were immunostained with the following primary antibodies: rabbit Ki-67 (1:300) (Abcam; Cambridge, UK), cleaved caspase-3 (1:200) (Cell Signaling Technology). . HRP-conjugated secondary antibody was used. The color development reaction of HRP-conjugated secondary antibody was performed with 3,3'-diaminobenzidine tetrahydrochloride (DAB) (Vector laboratories Inc., Burlingame; USA, CA). Counterstaining was performed with hematoxylin. Tissue fragments were observed under a light microscope (Carl Zeiss Inc.).

mRNA sequencing and data analysis

RNA was extracted from ACHN cells treated with 2.5 μM sunitinib and 1 μM niclosamide alone or in combination. A library was derived from 2 μg of total RNA using the SMARTer Stranded RNA-Seq Kit (Clontech Laboratories Inc.; USA). Isolation of mRNA was performed using the Poly(A) RNA Selection Kit (LEXOGEN Inc.; Austria). The isolated mRNA was used for cDNA synthesis and shearing. Indexing was performed using Illumina indexes 1-12. The enrichment step was performed using PCR. Afterwards, the library was checked using an Agilent 2100 bioanalyzer (DNA High Sensitivity Kit) and the average fragment size was measured. Quantification was performed with a library quantification kit using the StepOne Real-Time PCR System (Life Technologies Inc.; USA). High-throughout sequencing was performed using HiSeq 2500 (Illumina, Inc.; USA) with paired-end 100 sequencing. Alignment files were obtained by mapping mRNA-Seq reads using TopHat software tools. Differentially expressed genes (DEGs) were determined based on these counts from unique multiple alignments using coverage in Bedtool. Read count data were processed based on the Quantile normalization method using EdgeR within R using Bioconductor. Alignment files were used to collect transcripts and measure their abundance, and were also used to measure differential expression of genes or isoforms using cufflinks. Additionally, FPKM (fragments per kilobase of exon per million fragments) was used as a method to determine the expression level of a gene region.

TCGA dataset

All RNA sequencing (RNA-Seq) data and clinical data were downloaded from the Genomic Data Commons (GDC) data portal on January 20, 2021 using the TCGA Biolink package. These databases (kidney renal clear cell carcinoma (KIRC)), created using the Illumina HiSeq RNASeq platform, contain 533 kidney cancer tissues (primary solid tumors; tumors) and 72 adjacent non-neoplastic kidney tissues ( Solid tissue normal (normal) samples are included.

Data processing and DEG screening

Gene expression data from ccRCC RNA-Seq includes 19,947 official genes labeled in NCBI ( https://www.ncbi.nlm.nih.gov/ ) or GENCODE database ( http://www.gencodegenes.org ). there is. All data were normalized and processed with the TCGA Biolink pipeline. The principle of TCGA biolinking of differentially expressed genes (DEGs) follows the following formula: (1) convert the count matrix to an edgeR object, (2) assign the same variance estimate to each gene, (3) Perform pair-wise test for differential expression between two groups, (4) use False Discovery Rate (FDR) correction to obtain output and return top DEGs. The cutoff criteria are FDR<0.01 and |Log ₂ FC|>2.0.

Quantitative reverse transcription-PCR (qRT-PCR)

Total RNA from cells was extracted using TRIzol ^® reagent (Invitrogen Life Technologies; Thermo Fisher Scientific, Inc.; MA, USA) according to the manufacturer's protocol. A total of 1 μg of total RNA was then reverse transcribed into cDNA using the PrimeScript ^TM RT reagent kit (Takara Bio Inc.; Shiga, Japan). PCR was performed with TB Green™ Premix Ex Taq™ II (Takara Bio Inc.). Relative gene expression levels were determined by normalizing to β-actin using the 2- ^ΔΔCT method. Primers used in the experiment are shown in Table 1 below.

gene Forward sequence (5′ to 3′) Reverse sequence (5′ to 3′) BRIP1 GGAAGAAGCAGGGGAAAGCAG GAGGCACTATTCTCTGATCGACC E2F2 GACTCGGTATTGACACTTCGCTG CGTTGGTGATGTCATAGATGCG TTK ATGATGATGGCAAACAACCC CGCTTGACTGTAACGACCA FANCA GGTGGGTATTCTCTCAGCCG CACAGGGTGACTGGTCTCCG DNA2 GTGTTCTGTTCCAGTAGAGCCA CCTAAAAGTTTCACTCAGGACAGC GAPDH GCTACAGCAACAGGGTGGTG GGTCTACATGGCAACTGTGAGG

Western blot analysis

After harvesting the cells, they were lysed using RIPA lysis buffer (Cell Signaling Technology; MA, USA). Proteins were separated by SDS-PAGE and then transferred to a nitrocellulose membrane. After blocking the membrane for 1 hour, BRIP1 (1:2000, Cell Signaling Technology), E2F1 (1:1000, Santa Cruz Biotechnology, Inc.), FANCA (1:500, Cell Signaling Technology), γH2AX (1:5000; Cell Signaling Technology), Cyclin A (1:1000, Santa Cruz Biotechnology, Inc.), Cyclin B (1:1000, Santa Cruz Biotechnology, Inc.), CDK1 (1:1000, Santa Cruz Biotechnology, Inc.), β -actin (1:2500, Santa Cruz Biotechnology, Inc.), and GAPDH (1:10000, Santa Cruz Biotechnology, Inc.) were treated and incubated overnight at 4 °C. The membrane was then incubated with horseradish peroxidase-conjugated anti-rabbit IgG or anti-mouse IgG (GenDEPOT; TX, USA). Protein bands were visualized using an ECL kit (DoGenBio; Seoul, Korea) and detected using X-ray film.

immunocytochemistry

Cells were cultured on coverslips and treated with 2.5 μM sunitinib and 1 μM niclosamide alone or in combination. After 24 hours, cells were fixed with 4% formaldehyde for 15 minutes and permeabilized with 0.1% Triton-100 for 10 minutes. Samples were blocked with 10% bovine serum albumin for 1 hour and incubated overnight with primary rabbit anti-phospho-H2AX (Ser139; γH2AX; 1:500; Cell Signaling Technology). After washing with PBS, the slides were incubated with secondary goat anti-rabbit-FITC antibody at room temperature for 1 hour, and DNA was stained with 4', 6-diamidino-2-phenylindole (DAPI). Confocal images were acquired using an LSM510 meta confocal laser scanning microscope (Carl Zeiss Inc.).

Cell cycle analysis

Cell cycle arrest analysis was performed 48 hours after cells were treated with 2.5 μM sunitinib and 1 μM niclosamide alone or in combination. Cells were harvested, washed twice with PBS, and fixed in 70% ethanol. Fixed cells were washed twice with PBS and then incubated with 100 μg/ml RNase A and 50 μg/ml PI. Cell cycle distribution was analyzed using the FACS Canto II system (BD Biosciences).

statistical analysis

In vitro test results are expressed as mean ± SD (standard deviation), and in vivo test results are expressed as SE (standard error). Differences between values were analyzed using the Student t test using IBM SPSS version 24.0 software (SPSS, Inc.; IL, USA). It was considered statistically significant when the p-value was <0.05. Throughout the specification, * P < 0.05, ** P < 0.01, *** P < 0.001, ^# P < 0.01.

[Example]

Example 1. Confirmation of anticancer effect of niclosamide

Before confirming the combined effect of niclosamide and sunitinib, the anticancer effect of each drug in kidney cancer was confirmed. As shown in Figure 1, sunitinib inhibited the activity of renal cancer cells in a dose-dependent manner, and niclosamide was also shown to inhibit the cellular activity of renal cancer cell lines in a dose- and time-dependent manner ( Figure 2 ). In addition, the apoptosis analysis results showed that niclosamide dose-dependently increased apoptosis of cancer cells (Figure 3), and the wound healing analysis and Matrigel penetration assay results showed that niclosamide dose-dependently increased the migration of kidney cancer cells (cell It was confirmed that both migration and invasion were inhibited (Figures 4 and 5).

The above results show that niclosamide has the effect of inhibiting the growth of kidney cancer cells, inducing apoptosis, and inhibiting invasion in proportion to the treatment concentration.

Example 2. Confirmation of synergistic effect by combined use of sunitinib and niclosamide

The present inventors confirmed whether the combined use of niclosamide and sunitinib could achieve a synergistic anticancer effect. Specifically, the activity of cancer cells was measured after treating sunitinib and niclosamide alone or in combination with ACHN cell lines at various concentrations. Synergy scores for drug interactions were calculated using SynergyFinder 2.0 software, based on the Highest Single Agent (HSA) reference model (Figure 6). As a result, it was confirmed that the combination of niclosamide and sunitinib exhibited a synergistic effect with a positive synergy score of 5.663 (FIG. 7). Among these combinations, above the appropriate concentration, a more red (synergy) area was shown on the 2D/3D plot, indicating a relatively higher synergy score, and the ACHN cell line showed the highest score of 28.826 (FIG. 8). The combination of niclosamide (1 μM) and sunitinib (2.5 μM) showed a high synergy score of 9.46 at the lowest dose (Figure 8). Therefore, subsequent experiments were conducted using combinations of the above ratios.

Next, to evaluate the effect of drug combination on other kidney cancer cell lines, cell activity analysis and flow cytometry were performed. As shown in Figure 9, the combination of niclosamide-sunitinib significantly reduced the proliferation of cancer cells compared to the other groups. In addition, compared to the case of treatment with niclosamide or sunitinib alone, when the two drugs were treated in combination, the number of kidney cancer cells that caused apoptosis was found to increase further (FIG. 10).

Next, to confirm the therapeutic effect of the niclosamide-sunitinib combination in vivo , an ACHN-xenograft model was created and experiments were conducted. Mice were orally administered vehicle, sunitinib alone, niclosamide alone, or niclosamide-sunitinib combination. There was no difference in mouse body weight between groups. Rapid tumor growth was observed in the control group compared to other drug administration groups. On the other hand, in the group treated with sunitinib or niclosamide alone, tumor growth was inhibited by 46% and 32%, respectively, and in the group treated with niclosamide-sunitinib together, tumor growth was inhibited by 65%. (Figures 11 and 12). In other words, this shows that the combination treatment of niclosamide-sunitinib can effectively inhibit cancer growth, and in particular, the tumor growth inhibition effect is superior compared to the group administered the drug alone. In addition, tumor tissues were obtained from mice and the expression levels of Ki-67 and cleaved caspase-3 were confirmed. As a result, the sunitinib alone, niclosamide alone, and niclosamide-sunitinib combination groups had Ki-67 higher than the control group. The level of was significantly decreased, and cleaved caspase-3 was confirmed to be increased (Figure 13). The above changes were particularly noticeable in the combination treatment group.

The above results show that the combination of sunitinib-niclosamide exerts a synergistic anticancer effect.

Example 3. Confirmation of the effect of niclosamide on controlling the DNA repair mechanism and stopping the cell cycle.

In clear renal cell carcinoma, sunitinib is known to exert an anticancer effect by inhibiting the formation of new blood vessels. The present inventors predicted that niclosamide would exert anticancer effects through a different biological mechanism from sunitinib, and to identify genes related to the mechanism of each drug, niclosamide alone, sunitinib alone, or a combination of the two drugs were used. RNA sequencing was performed after processing. Both single and combined treatments of each drug were shown to significantly reduce the expression of various genes in kidney cancer cells. Among these, the present inventors found that treatment with sunitinib alone had no effect, but treatment with niclosamide alone; Alternatively, 729 genes that were downregulated during the combined treatment of niclosamide and sunitinib were selected (|fold difference|>1.5, p-value<0.05; Figure 14). These genes were analyzed by KEGG pathway analysis (Kyoto Encyclopedia of Genes and Genomes pathway analysis) (p-value<0.001).

As a result, as shown in Figure 15 and Table 2, the genes were found to be highly related to the Fanconi anethia pathway (gene count; 11), DNA replication (gene count; 12), and cell cycle (gene count; 24). . These results suggest that the main mechanism of the anticancer effect of niclosamide is related to the regulation of the DNA repair pathway and the cell cycle.

Terms count p-value gene hsa04110:Cell cycle 24 1.26E-10 CDC7, E2F2, CDK1, YWHAZ, RBL1, SKP2, YWHAB, TTK, CHEK1, CDK6, CHEK2, MCM2, SMC3, WEE1, CDC25B, CCNE2, YWHAH, YWHAQ, PCNA, ORC5, ORC6,
ABL1, SMC1A, TFDP1 hsa03030:DNA replication 12 4.36E-08 POLD3, RPA1, PRIM1, DNA2, RFC3, RFC4, POLE, PRIM2, PCNA, POLA1, MCM2, RNASEH2A hsa03460:Fanconi anemia pathway 11 2.17E-05 RPA1, RAD51C, BLM, FANCD2, FANCI, FAAP24, BRIP1, FANCA, RMI1, UBE2T, BRCA1 hsa03430:Mismatch repair 6 0.001378 POLD3, RPA1, RFC3, RFC4, MSH2, PCNA hsa03040:Spliceosome 14 0.001379 SRSF10, TRA2B, LSM6, RBMX, HNRNPU, HNRNPA3, HNRNPK, HSPA2, SRSF7, DHX15, SNRNP40, SNRPE, HSPA8, RBM17 hsa05203:Viral carcinogenesis 18 0.001708 CDK1, YWHAZ, ACTN4, RBL1, SKP2, YWHAB, ACTN1, CHEK1, CDK6, SRF, PKM, CCNE2, CDC42, NRAS, CASP3, YWHAH, HNRNPK, YWHAQ hsa05130:Pathogenic Escherichia coli infection 8 0.002712 ACTB, CDC42, EZR, TUBB6, ARPC4, ABL1, TUBA1C, CTNNB1 hsa00240:Pyrimidine metabolism 11 0.004404 POLD3, PRIM1, TYMS, UMPS, POLE, PRIM2, DHODH, DCK, POLA1, POLR2D, DUT hsa03013:RNA transport 15 0.005061 UPF1, NUP85, NUP155, PNN, NDC1, SUMO3, EIF4G2, NUP62, EIF1AX, NUP50, RPP30, THOC7, NUP107, TGS1, GEMIN5 hsa05100:Bacterial invasion of epithelial cells 9 0.008507 ACTB, CDC42, CAV1, ARHGEF26, BCAR1, ARPC4, SEPT11, CTNNB1, VCL hsa04810:Regulation of actin cytoskeleton 16 0.012206 ACTB, FGFR4, PDGFB, ACTN4, LIMK1, BCAR1, IQGAP3, ACTN1,ARPC4, VCL, CDC42, NRAS, EZR, CFL1, PIP4K2A, F2R hsa04115:p53 signaling pathway 8 0.012276 CCNE2, CDK1, CASP3, SERPINE1, CHEK1, CDK6, CHEK2, THBS1 hsa04520:Adherens junction 8 0.016555 ACTB, PTPRJ, CDC42, ACTN4, ACTN1, PTPN1, CTNNB1, VCL hsa03440:Homologous recombination 5 0.021947 POLD3, RPA1, RAD51C, XRCC2, BLM hsa04510:Focal adhesion 15 0.022617 ACTB, CAV1, PDGFB, ACTN4, BCAR1, ACTN1, CAPN2, VASP, VCL, CTNNB1, CDC42, COL6A3, LAMC1, ZYX, THBS1 hsa04114:Oocyte meiosis 10 0.023104 CCNE2, CDK1, YWHAZ, YWHAH, PPP2R5D, YWHAB, YWHAQ, SMC1A, SMC3, CALM2 hsa04390:Hippo signaling pathway 12 0.026119 ACTB, YWHAZ, YWHAH, TP53BP2, SERPINE1, YWHAB, YWHAQ, TEAD2, LIMD1, GLI2, WWTR1, CTNNB1 hsa04670:Leukocyte transendothelial migration 10 0.028289 ACTB, CDC42, EZR, ACTN4, BCAR1, ACTN1, CLDN2, VASP, CTNNB1, VCL hsa03420:Nucleotide excision repair 6 0.030341 POLD3, RPA1, RFC3, RFC4, POLE, PCNA hsa04530:Tight junction 8 0.044133 ACTB, CDC42, ACTN4, CGN, ACTN1, CLDN2, AMOTL1, MYH9 hsa05161:Hepatitis B 11 0.045447 CCNE2, NRAS, E2F2, CASP3, YWHAZ, PCNA, YWHAB, YWHAQ, FADD, CDK6, NFATC2 hsa04360:Axon guidance 10 0.048634 CDC42, NRAS, NRP1, LIMK1, CFL1, DPYSL5, SEMA4D, NFATC2, ABL1, EPHB2 hsa04015:Rap1 signaling pathway 14 0.051695 ACTB, FGFR4, PDGFB, BCAR1, KITLG, APBB1IP, VASP, CTNNB1, CDC42, NRAS, RAPGEF5, THBS1, CALM2, F2R hsa03050:Proteasome 5 0.081512 PSMC6, PSMA4, PSMA3, PSMD1, PSME3 hsa04151:PI3K-Akt signaling pathway 19 0.096590 SGK1, YWHAZ, FGFR4, PHLPP2, PDGFB, PPP2R5D, YWHAB, KITLG, CDK6, BRCA1, CCNE2, NRAS, YWHAH, COL6A3, YWHAQ, LAMC1, THBS1, PPP2R3C, F2R hsa05222:Small cell lung cancer 7 0.098805 CCNE2, CKS1B, E2F2, CKS2, SKP2, CDK6, LAMC1

As shown in Figures 16 and 17, the expression and nuclear foci of γ-H2AX increased in all drug treatment groups compared to the DMSO treatment group. Additionally, compared to sunitinib treatment, γ-H2AX expression increased further during niclosamide treatment, and when niclosamide and sunitinib were combined, γ-H2AX expression increased more dramatically.

Next, we observed how niclosamide treatment affected cell cycle progression. When treated with sunitinib alone, the difference in cell cycle was less variable than when treated with niclosamide alone or when the two drugs were combined. On the other hand, when treated with niclosamide alone or in combination with the two drugs, the proportion of ACHN and A498 cells arrested in the G0/G1 phase was found to increase (FIG. 18). G0/G1 arrest is mediated by various proteins such as cyclin A, cyclin B, and CDK1, and changes in the levels of these proteins according to drug treatment were confirmed. As shown in Figure 19, cyclin A, cyclin B, and CDK1 proteins were significantly decreased in the niclosamide treatment group alone and in the combination treatment group compared to the DMSO or sunitinib treatment group alone.

The above results show that niclosamide exerts an anticancer effect by regulating the DNA repair mechanism of cancer cells and suppressing excessive cell cycle progression.

Example 4. Confirmation of the function of niclosamide to regulate the DNA repair mechanism and expression of genes involved in the cell cycle.

Significant genes identified through KEGG pathway analysis are shown in Table 2 above. Forty-four regulated genes, including three overlapping genes affected by niclosamide, were selected for further validation.

In order to select genes that are clinically significant and can be used as therapeutic targets among the 44 genes, transcriptome data from the TCGA database-KIRC (Log FC>2.0, FDR<0.05) was used to determine the gene expression in tumor tissue compared to normal tissue. Candidate genes with significantly increased expression were screened. As a result, the following five genes were selected whose expression was significantly increased in primary tissues compared to solid normal tissues: E2F2, TTK, BRIP1, FANCA and DNA2 (FIG. 20). The other 39 genes had no significant expression differences between tumor tissues and normal tissues. Next, it was confirmed whether the above five genes were affected by niclosamide in kidney cancer cell lines. qRT-PCR results showed that the mRNA expression levels of BRIP1, E2F2, and FANCA in A-498 and ACHN cells were significantly reduced when treated with niclosamide alone or in combination with the two drugs compared to when treated with DMSO or sunitinib alone. It was found that (Figure 21). This result was also found to be the same when protein levels were analyzed by Western blot (FIG. 22).

The above results suggest that niclosamide regulates DNA repair mechanisms and mediates cell cycle arrest by targeting BRIP1, E2F2, and FANCA genes, and niclosamide achieves a synergistic anticancer effect through a different mechanism from sunitinib. It shows that it does.

As discussed above, the present inventors confirmed that niclosamide exerts an anticancer effect against kidney cancer, and in particular, the combined use of niclosamide and sunitinib can achieve a synergistic anticancer effect compared to the use of each drug alone. It was confirmed that it exists. In addition, it was confirmed that this synergistic effect was due to the regulation of DNA repair mechanism and cell cycle arrest, which are the regulatory mechanisms of niclosamide, which are different from those of sunitinib. In other words, the combined use of niclosamide and sunitinib is expected to be used as an effective treatment strategy for kidney cancer as it can maximize the anti-cancer effect while reducing the side effects caused by the single use of high doses of each drug.

The description of the present invention described above is for illustrative purposes, and those skilled in the art will understand that the present invention can be easily modified into other specific forms without changing the technical idea or essential features of the present invention. will be. Therefore, the embodiments described above should be understood as illustrative in all respects and not restrictive.

<110> THE CATHOLIC UNIVERSITY OF KOREA INDUSTRY-ACADEMIC COOPERATION FOUNDATION <120> Pharmaceutical composition comprising Niclosamide and Sunitinib for preventing or treating kidney cancer <130> MP21-127 <160> 12 <170> KoPatentIn 3.0 <210> 1 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> BRIP1_primer_FW <400> 1 ggaagaagca gggaaagcag 20 <210> 2 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> BRIP1_primer_RV <400> 2 gaggcactat tctctgatga cc 22 <210> 3 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> E2F2_primer_FW <400> 3 gactcggtat gacacttcgc tg 22 <210> 4 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> E2F2_primer_RV <400> 4 cgttggtgat gtcatagatg cg 22 <210> 5 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> TTK_primer_FW <400> 5 atgatgatgg caaacaaccc 20 <210> 6 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> TTK_primer_RV <400> 6 cgcttgactg taacgacca 19 <210> 7 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> FANCA_primer_FW <400> 7 ggtgggtatt ctctcagccg 20 <210> 8 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> FANCA_primer_RV <400> 8 cacagggtga ctggtctccg 20 <210> 9 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> DNA2_primer_FW <400> 9 gtgttctgtt ccagtagagc ca 22 <210> 10 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> DNA2_primer_RV <400> 10 cctaaaagtt tcactcagga cagc 24 <210> 11 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> GAPDH_primer_FW <400> 11 gctacagcaa cagggtggtg 20 <210> 12 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> GAPDH_primer_RV <400> 12 ggtctacatg gcaactgtga gg 22

Claims (10)

Niclosamide or a pharmaceutically acceptable salt thereof; and a pharmaceutical composition for combined administration for preventing or treating kidney cancer, comprising sunitinib or a pharmaceutically acceptable salt thereof as an active ingredient.
According to paragraph 1,
The pharmaceutical composition for combined administration includes the niclosamide or a pharmaceutically acceptable salt thereof; A pharmaceutical composition for combined administration, characterized in that it is in the form of a mixture of sunitinib or a pharmaceutically acceptable salt thereof.
According to paragraph 1,
The pharmaceutical composition for combined administration includes the niclosamide or a pharmaceutically acceptable salt thereof; and sunitinib or a pharmaceutically acceptable salt thereof are each formulated and administered simultaneously or sequentially.
According to paragraph 1,
The niclosamide or a pharmaceutically acceptable salt thereof; and sunitinib or a pharmaceutically acceptable salt thereof are mixed at a molar ratio of 1:0.1 to 25.
According to paragraph 1,
The niclosamide or a pharmaceutically acceptable salt thereof; and sunitinib or a pharmaceutically acceptable salt thereof are mixed at a weight ratio of 1:0.1 to 10. A pharmaceutical composition for combined administration.
According to paragraph 1,
The composition is a pharmaceutical composition for combined administration, characterized in that it satisfies one or more characteristics selected from the group consisting of:
(a) Inducing apoptosis of cancer cells;
(b) arresting cell cycle progression; and
(c) Regulating the repair mechanism of damaged DNA.
According to paragraph 1,
The composition is a pharmaceutical composition for combined administration, characterized in that it inhibits the expression of one or more genes selected from the group consisting of BRIP1, E2F2, FANCA, cyclin A, cyclin B, and CDK1.
According to paragraph 1,
A pharmaceutical composition for combined administration, characterized in that the composition has a higher anticancer effect compared to the use of niclosamide alone or sunitinib alone.
According to paragraph 1,
The composition is a pharmaceutical composition for combined administration for use in the treatment of an individual carrying a mutation in one or more genes selected from the group consisting of BRIP1, E2F2, FANCA, cyclin A, cyclin B, and CDK1.
A kit for preventing or treating kidney cancer, comprising the pharmaceutical composition for combined administration of any one of claims 1 to 9.