KR20200031219A - Pharmaceutical Composition For Treating Cancer Comprising Tyrosine Kinase Inhibitor - Google Patents

Abstract

The present invention relates to a pharmaceutical composition for treating cancer containing a tyrosine kinase activity inhibitor as an active component. The composition of the present invention has a cancer cell killing effect, increases the expression of sodium iodine co-transporter (NIS), and induces re-differentiation of cancer cells, so that when administered in combination with radioactive iodine (RAI), cancer cells can be effectively killed, thereby being able to be used for cancer treatment. In addition, the composition can be effectively utilized in the treatment of cancer with resistance to radioactive iodine treatment, thereby being able to be used as an anticancer adjuvant for treating cancer diseases.

Description

Pharmaceutical Composition For Treating Cancer Comprising Tyrosine Kinase Inhibitor}

The present invention relates to a pharmaceutical composition for treating cancer and an anticancer adjuvant comprising a tyrosine kinase inhibitor as an active ingredient. Specifically, the composition comprising the tyrosine kinase inhibitor of the present invention has a cancer cell killing effect and increases the expression (NIS) of the sodium iodine cotransporter, thereby promoting the absorption of radioactive substances, thereby increasing the radiation treatment effect.

Thyroid cancer is the most common endocrine cancer, and its incidence has increased significantly in recent years. In particular, in women, the recent incidence rate has increased rapidly, making thyroid cancer the most common cancer among women. According to the pathological distinction established by the WHO, thyroid cancer is divided into 5 types: papillary carcinoma, follicular carcinoma, Hurtle cell neoplasm, anaplastic carcinoma, undifferentiated thyroid cancer. ), Medullary thyroid carcinoma, and the like.

Differentiated cancers such as papillary and follicular cancers are the main treatments with surgical treatment and radioactive iodine treatment, and the 10-year survival rate is more than 90%, with a good prognosis. It is characterized by rapid progression, poor prognosis and high mortality. Undifferentiated thyroid cancer, unlike differentiated thyroid cancer, does not exhibit the characteristics of thyroid follicular cells such as iodine uptake and thyroglobulin synthesis, and radioactive iodine (radioactive iodine) RAI) It is resistant to treatment. Therefore, treatment of undifferentiated thyroid cancer alone or in combination with surgery, radiation therapy, and chemotherapy does not have an effect on patient survival, and thus, the development of new treatment methods is urgently needed.

The sodium iodine cotransporter (NIS) protein is a cell membrane transporter and has 13 domains, and two sodium cations (Na ²⁺ ) and one iodine are caused by differences in the concentration of sodium ions generated inside and outside the cell membrane by sodium-potassium ATPase. It carries a ^- anion (I). With the recent development of molecular biology, several properties of NIS proteins have been revealed, and cloning of NIS genes has become possible, and the molecular biological mechanism of iodine intake has been revealed, and various studies have been conducted to implement basic experiments using this into clinical medicine. Is becoming. Sodium iodine co-transporter (NIS) is a transporter that introduces iodine necessary for synthesizing thyroid hormone into the thyroid follicle.If radioactive iodine is administered, it enters into the thyroid cells through NIS and destroys the thyroid cells, so radioactive iodine therapy is a thyroid cancer patient It is mainly used as a treatment method. However, in recent years, it is not limited to the treatment of thyroid cancer, and new fields of application such as radionuclide gene therapy using NIS and molecular genetic imaging have been developed.

On the other hand, protein kinase is an enzyme that catalyzes the phosphorylation of hydroxy groups located in tyrosine, serine and threonine residues of proteins, and plays an important role in signal transmission of growth factors that cause cell growth, differentiation and proliferation. In order to maintain the homeostasis of the living body, the signal transmission system in the living body needs to balance on and off smoothly. However, mutation or overexpression of a specific protein kinase disrupts the normal intracellular signal transduction system, causing various diseases such as cancer, inflammation, metabolic disease, and brain disease. Recently, among several protein kinases, research is being conducted to develop anticancer drugs through the development of compounds having selective inhibitory activity against specific kinases.

Therefore, the inventors of the present application have the effect that a compound having a tyrosine kinase inhibitory activity kills cancer cells, increases the sodium iodine cotransporter (NIS) expression in cancer cells, promotes radioactive iodine absorption, and induces re-differentiation of cancer cells to induce radioactive iodine. The present invention was completed by confirming that the effect of treatment can be increased.

Republic of Korea Registered Patent No. 10-1348550

An object of the present invention is to provide a pharmaceutical composition for preventing or treating cancer, which contains a tyrosine kinase inhibitor or a pharmaceutically acceptable salt thereof as an active ingredient.

Another object of the present invention is to provide an anti-cancer adjuvant for use in the treatment of cancer diseases comprising a tyrosine kinase inhibitor or a pharmaceutically acceptable salt thereof as an active ingredient.

One aspect of the present invention provides a composition for preventing or treating cancer comprising the compound of Formula 1 or a pharmaceutically acceptable salt thereof as an active ingredient.

[Formula 1]

Compound 1 is "5- (5- {4H, 5H, 6H-cyclopenta [b] thiophen-2-yl} -1,3,4-oxadiazol-2-yl) -1-methyl- 1,2-dihydropyridin-2-one ", which exhibits tyrosine kinase inhibitory activity, and thus can be used as a pharmaceutical composition for the prevention or treatment of diseases caused by abnormal cell growth, such as cancer, and in vitro ( in It was confirmed through an in vitro or in vivo experiment that the composition of the present invention has an effect of killing cancer cells.

According to an embodiment of the present invention, the compound of Formula 1 may be obtained by extraction and separation from natural products, or may be prepared by a conventional organic synthesis method, but is not limited thereto.

In the present invention, "prevention" may include, without limitation, any action to block or suppress or delay cancer symptoms using the pharmaceutical composition of the present invention.

"Treatment" in the present invention may include, without limitation, any action that improves or benefits cancer symptoms using the pharmaceutical composition of the present invention.

"Pharmaceutically acceptable salt" in the present invention means a salt in a form that can be used pharmacologically among salts, which are materials in which cations and anions are bound by electrostatic attraction, typically salts with metal salts and organic bases. , Salts with inorganic acids, salts with organic acids, salts with basic or acidic amino acids, and the like. For example, the metal salt may be an alkali metal salt (sodium salt, potassium salt, etc.), an alkaline earth metal salt (calcium salt, magnesium salt, barium salt, etc.), aluminum salt, or the like; Salts with organic bases include triethylamine, pyridine, picoline, 2,6-lutidine, ethanolamine, diethanolamine, triethanolamine, cyclohexylamine, dicyclohexylamine, N, N-dibenzylethylenediamine Salt with the back; Salts with inorganic acids can be salts with hydrochloric acid, hydrobromic acid, nitric acid, sulfuric acid, phosphoric acid, etc .; Salts with organic acids can be salts with formic acid, acetic acid, trifluoroacetic acid, phthalic acid, fumaric acid, oxalic acid, tartaric acid, maleic acid, citric acid, succinic acid, methanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, etc .; Salts with basic amino acids can be salts with arginine, lysine, ornithine, etc .; Salts with acidic amino acids can be salts with aspartic acid, glutamic acid, and the like.

Cancers that can be prevented or treated with the composition of the present invention include uterine cancer, fallopian tube cancer, ovarian cancer, stomach cancer, brain cancer, rectal cancer, colon cancer, small intestine cancer, rectal cancer, esophageal cancer, lymph node cancer, gallbladder cancer, lung cancer, skin cancer, kidney cancer, bladder cancer, Blood cancer, pancreatic cancer, prostate cancer, endocrine cancer, oral cancer, and liver cancer may be selected from the group consisting of, and in one embodiment of the present invention, the cancer may be thyroid cancer (thyroid cancer) or breast cancer (breast cancer).

For the purposes of the present invention, thyroid cancer that can be prevented or treated with the composition is included without limitation, and in one embodiment of the present invention, the thyroid cancer may be undifferentiated thyroid cancer (anaplastic thyroid cancer) or differentiated thyroid cancer (differentiated thyroid cancer). .

In one embodiment of the present invention, the composition may be to increase the expression of a sodium iodide symporter (NIS).

In the present invention, "sodium iodide symporter (NIS)" is a cell membrane protein having 13 transmembrane domains, and two sodium ions due to the difference in concentration inside and outside the cell membrane of sodium ions generated by sodium-potassium ATPase. And one iodine ion to move into the thyroid cells.

The pharmaceutical composition of the present invention may further include a pharmaceutically acceptable additive, wherein the pharmaceutically acceptable additive is starch, gelatinized starch, microcrystalline cellulose, lactose, povidone, colloidal silicon dioxide, calcium hydrogen phosphate , Lactose, mannitol, syrup, gum arabic, pregelatinized starch, corn starch, powdered cellulose, hydroxypropyl cellulose, opadry, sodium starch glycolate, carnauba lead, synthetic aluminum silicate, stearic acid, magnesium stearate, aluminum stearate, Calcium stearate, white sugar, dextrose, sorbitol and talc can be used.

The composition of the present invention may be administered in various formulations, oral and parenteral, in actual clinical administration. When formulated, diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrants, surfactants, etc., which are usually used, are used. Can be prepared. Solid preparations for oral administration include tablets, pills, powders, granules, capsules, etc. These solid preparations include at least one excipient, for example, starch, calcium, in the herbal composition with increased fat-soluble polyphenol component of the present invention. It can be prepared by mixing calcium carbonate, sucrose, lactose, or gelatin. In addition, lubricants such as magnesium stearate talc may be used in addition to simple excipients.

The composition of the present invention may be administered orally or parenterally depending on the desired method, and when parenterally administered, an intraperitoneal injection, rectal injection, subcutaneous injection, intravenous injection, intramuscular injection or intrathoracic injection is selected. It is preferred. The dosage range varies according to the patient's weight, age, sex, health status, diet, administration time, administration method, excretion rate and disease severity.

The composition of the present invention is administered in a pharmaceutically effective amount. In the present invention, "a pharmaceutically effective amount" means an amount sufficient to treat the disease at a ratio of rational benefit / risk applicable to medical treatment, and the effective dose level is the type of patient's disease, severity, drug activity, drug Sensitivity to, time of administration, route of administration and rate of discharge, duration of treatment, factors including co-drugs, and other factors well known in the medical field. The composition of the present invention may be administered as an individual therapeutic agent or in combination with other therapeutic agents, and may be administered sequentially or simultaneously with a conventional therapeutic agent, and may be administered single or multiple. Considering all of the above factors, it is important to administer an amount that can achieve the maximum effect in a minimal amount without side effects, which can be easily determined by those skilled in the art.

Specifically, the effective amount of the composition according to the present invention may vary depending on the patient's age, sex, and body weight, and in general, 1 mg to 200 mg per kg of body weight, preferably 10 mg to 200 mg daily or every other day Alternatively, it may be administered in 1 to 3 times a day. However, since the dosage may be increased or decreased depending on the route of administration, the severity of the disease, sex, weight, and age, the dosage does not limit the scope of the present invention in any way.

One aspect of the present invention provides an anti-cancer adjuvant for use in the treatment of cancer diseases comprising the compound of Formula 1 or a salt thereof as an active ingredient.

The inventors of the present application have discovered that the compound of Formula 1, which is a tyrosine kinase inhibitor, increases the expression of sodium iodine cotransporter (NIS), and the anticancer drug is tested in vitro or in vivo . When co-administration of adjuvant and radioactive iodine, it was confirmed that it promotes absorption of radioactive iodine and is effective in killing cancer cells.

In the present invention, "anti-cancer adjuvant" means an agent that can improve, improve or enhance the anti-cancer effect of anti-cancer agents. For example, when a concentration-dependent agent exhibiting anti-cancer activity is used in combination with an anti-cancer agent at a level that does not exhibit anti-cancer activity itself, it can be used as an anti-cancer adjuvant that can improve, improve or enhance the anti-cancer effect of the anti-cancer agent.

In one embodiment of the present invention, the anti-cancer adjuvant may be to increase the expression of sodium iodine co-transporter (NIS).

Cancer objects that can be treated with the anti-cancer adjuvant for the purposes of the present invention include without limitation, in one embodiment of the present invention, the cancer disease may be thyroid cancer or breast cancer, and the thyroid cancer may be undifferentiated thyroid cancer (anaplastic thyroid cancer) or differentiated thyroid cancer (differentiated thyroid cancer).

Undifferentiated thyroid cancer does not respond well to radioactive iodine treatment and is resistant to radioactive iodine treatment.NIS, thyroglobulin (Tg), thyroid peroxidase (TPO), and thyroid stimulating hormone receptor (thyroid-) are also present in differentiated thyroid cancer. Through a series of dedifferentiation processes in which thyroid-specific gene expression such as stimulating hormone receptor (TSHR) decreases, it may become resistant to radioactive iodine treatment. In one embodiment of the present invention, the anti-cancer adjuvant may be one that enhances the sensitivity of radioactive iodine treatment by inducing re-differentiation of thyroid cancer with resistance to radioactive iodine treatment due to loss of radioactive iodine intake.

The route of administration of the anti-cancer adjuvant can be administered through any general route as long as it can reach the target tissue. The anticancer adjuvant of the present invention may be administered intraperitoneally, intravenously, intramuscularly, subcutaneously, intradermally, orally as desired, but is not limited thereto. In addition, the anti-cancer adjuvant can be administered by any device capable of transporting the active agent to the target cell.

In addition, the anti-cancer adjuvant may be administered alone before or after administration of the anti-cancer agent, and may be administered together as an adjuvant for the treatment of cancer at the same time as the anti-cancer agent. When the anti-cancer adjuvant of the present invention is administered together with the anti-cancer agent, it may be administered together with the anti-cancer agent in an appropriate ratio depending on the patient's condition, the dose of the anti-cancer agent, the administration period of the anti-cancer agent, and specifically, 0.01 to 10 compared to the total weight of the anti-cancer agent It may be administered by pear.

The anti-cancer adjuvant of the present invention may be used in combination with other anti-cancer agents such as other anti-cancer agents, surgical surgery treatment, anti-cancer chemotherapy, or radiation treatment.

Sodium iodine co-transporter (NIS) is a transporter that introduces iodine necessary for synthesizing thyroid hormone into the thyroid follicle. When radioactive iodine is administered, radioactive iodine enters the thyroid cells through NIS and destroys thyroid cells. Using this mechanism, radioactive iodine treatment is used as a treatment method for thyroid cancer patients. Recently, attempts to apply radioactive iodine treatment to cancer cells in which NIS is present as well as thyroid cancer are active, but there are cases in which radioactive iodine treatment is resistant due to decreased expression and positional change of NIS.

The anti-cancer adjuvant of the present invention can increase the intake of radioactive iodine by increasing the expression of NIS and induce re-differentiation of cancer cells. Therefore, when administered in combination with radioactive iodine, radioactive iodine is ingested into the cells and destroys cancer cells to treat cancer diseases. As it shows the effect, in one embodiment of the present invention, the anti-cancer adjuvant may be used individually, sequentially or simultaneously with radioactive iodine treatment.

The pharmaceutical composition according to an embodiment of the present invention has a cancer cell killing effect and may be used for cancer treatment. The anticancer adjuvant according to an embodiment of the present invention expresses sodium iodine cotransporter (NIS) in cancer cells. Since it increases and re-differentiates cancer cells, it can be effectively used to treat cancer diseases because it can effectively kill cancer cells by administering it in combination with radioactive iodine. In particular, it can be effectively used to treat cancer diseases that are resistant to radioactive iodine treatment.

1A to 1C show the results of screening TKI to increase NIS promoter activity. Specifically, FIG. 1A shows the activity of Rluc, which means the viability of cells, FIG. 1B, shows the activity of Fluc, which means NIS promoter activity, and FIG. 1C, the Fluc, normalized to Rluc activity in cells and vehicles administered with selected TKI It shows activity.
2A to 2C show the TKI concentration and treatment time effectively showing NIS promoter activity. Specifically, FIG. 2A shows a pattern of Fluc activity according to TKI concentration and treatment time, FIG. 2B shows a pattern of Rluc activity according to TKI concentration and treatment time, and FIG. 2C shows normalized Rluc activity according to TKI concentration and treatment time. Fluc activity.
Figure 3 shows the relative mRNA expression of thyroid specific genes (NIS, TPO, Tg and TSHR) and thyroid transcription factors (TTF-1 and Pax-8) by administration of TKI.
4A to 4C show the expression level and expression position of the NIS protein in 8505C cells. Specifically, FIG. 4A shows Western blot results for NIS proteins in cells among total proteins, and FIGS. 4B and 4C show Western blot results for cell membrane NIS proteins and cytoplasmic NIS proteins, respectively.
5A and 5B show the expression level of NIS protein in breast cancer cells. 5A and 5B show Western blot results for NIS protein in MDA-MB-231 cells and MCF-7 cells, respectively.
6 shows the results of observing NIS expression in cells through immunofluorescence imaging.
7A and 7B show the results of observing changes in thyroid iodine metabolic protein when TKI is administered. Specifically, FIG. 7A shows Western blot results for thyroid specific proteins (Tg, TPO, NIS, and TSHR) when TKI is administered, and FIG. 7B shows Western thyroid transcription factors (Pax-8 and TTF-1) when TKI is administered. Blot results are shown.
8 shows Western blot results of genes associated with MAPK and PI3K-AKT signaling pathways.
9A to 9C show the results of evaluating the absorption of radioactive iodine by TKI administration in 8505C cells. Specifically, Figure 9a shows the result of a significant increase in the absorption capacity of radioactive iodine concentration-dependently by the administration of TKI, Figure 9b and Figure 9c are radioactive iodine by administration of 12.5μM and 25μM TKI and KClO ₄ respectively It shows absorption capacity.
10A and 10B show the survival rate of 8505C cells when administered alone or in combination with ¹³¹ I and TKI, specifically, FIG. 10A shows the survival rate of 8505C cells when administered alone or in combination with ¹³¹ I and 12.5 μM TKI, and FIG. 10B Shows the survival rate of 8505C cells when administered with TKI alone or in combination of ¹³¹ I and 25 μM.
11A and 11B show the degree of DNA damage when administered alone or in combination with ¹³¹ I and TKI. Specifically, Figure 11a shows the results of γH2A.X foci formation assay when administered alone or in combination with ¹³¹ I and TKI, Figure 11b shows the Western blot results for γH2A.X when administered alone or in combination with ¹³¹ I and TKI Shows.
12A to 12D show the result of an increase in NIS promoter activity when TKI is administered by an in vivo experiment. Specifically, FIG. 12A shows Fluc activity in vehicle and TKI administration groups in a tumor xenograft mouse model. 12B shows the results of quantitative analysis of Fluc activity in the vehicle and TKI administration groups in a tumor xenograft mouse model. 12C shows Rluc activity in vehicle and TKI administration groups in a tumor xenograft mouse model. 12D shows the results of quantitative analysis of NIS promoter activity normalized to Rluc activity in the vehicle and TKI administration groups in a tumor xenograft mouse model.
13A and 13B show the result of an increase in the amount of NIS expression in tumor tissue. Specifically, FIG. 13A shows Western blot results for NIS proteins in tumor tissues of tumor xenograft mice, and FIG. 13B shows the results of immunohistochemical staining of NIS in tumor tissues of tumor xenograft mice.
14A to 14C, 15A, 15B, and 16 show cancer treatment effects mediated by ¹³¹ I when TKI is administered by in vivo experiments.
Figure 14a is ¹³¹ I and the TKI represents the Rluc activity in tumor xenograft mouse models administered alone or in combination, Figure 14b shows the tumor volume for administration of ¹³¹ I and TKI alone or in combination tumor xenograft mouse. 14C shows the weight change of tumor xenograft mice during in vivo experiments.
15A shows tumor tissue isolated from tumor xenograft mice administered ¹³¹ I and TKI alone or in combination, and FIG. 15B shows tumor tissue isolated from tumor xenograft mice administered ¹³¹ I and TKI alone or in combination. It represents the average weight.
16 shows the results of immunohistochemical staining of γH2A.X in tumor tissue isolated from tumor xenograft mice.

Hereinafter, the present invention will be described in more detail through examples. However, these examples are for illustrative purposes only, and the scope of the present invention is not limited to these examples.

Example 1. Selection of substances that increase NIS expression

<1.1> Cell lines and chemicals

The human undifferentiated thyroid cancer (anaplastic thyroid cancer, ATC) cell line 8505C was purchased from Deutsche Sammlung von Mikroorganismen und Zellkulturen (DSMZ), and the breast cancer cell lines MDA-MB-231 and MCF-7 were purchased from the American Type Culture Collection (ATCC). 8505C is Roswell Park Memorial Institute (RPMI) -1640 medium (HyClone, Logan, UT) supplemented with 10% fetal bovine serum (FBS, Gibco, Grand Island, NY, USA) and 1% penicillin-streptomycin. , USA) (HyClone) was maintained in a humidified incubator at 37 ° C. and 5% CO ₂ conditions. MDA-MB-231 and MCF-7 cells were DMEM (Dulbecco's modified eagle medium) supplemented with 10% fetal bovine serum (FBS, Gibco, Grand Island, NY, USA) and 1% penicillin-streptomycin. The medium (HyClone, Logan, UT, USA) (HyClone) was maintained in a humidified incubator at 37 ° C. and 5% CO ₂ conditions.

The TKI library used for the experiment was provided by the Korea Research Institute of Chemical Technology (Korea) from the Korea Compound Bank (http://www.chembank.org/).

Tyrosine kinase inhibitor (TKI), "5- (5- {4H, 5H, 6H-cyclopenta [b] thiophen-2-yl} -1,3,4-oxadiazol-2-yl) -1- Methyl-1,2-dihydropyridin-2-one "was purchased from Enamine (NJ, USA) and 50 mM of stock solution was dissolved in dimethyl sulfoxide (DMSO) and stored at -20 ° C.

<1.2>. Construction of pNIS-Fluc-TurboFP635-pCMV-Rluc recombinant vector

The pNIS-Fluc-TurboFP635-pCMV-Rluc recombinant vector expressing a reporter gene operated by a double promoter was prepared using the pNIS-Fluc-TurboFP635 vector and pcDNA3.1 / Hygro (+) vector. Ltd. (Korea). Specifically, in order to screen a substance that increases the activity of the sodium iodide symporter (NIS) promoter, Firefly-luciferase2 (Fluc) and TurboFP635 were included in the lower portion (clockwise) of the NIS promoter. The other side of the NIS promoter (counterclockwise) included the CMV promoter and Renilla-luciferase (Rluc) to confirm cell viability. The pNIS-Fluc-TurboFP635 vector is Provided by Abhijit De (ACTREC, India), pcDNA3.1 / Hygro (+) vector was purchased from Invitrogen (USA).

<1.3> Stable cell line production

The pNIS-Fluc-TurboFP635-pCMV-Rluc recombinant vector was transfected into 8505C cell line with Fu-GENE HD solution according to the manufacturer's (Promega) Fu-GENE HD protocol. The ratio of the recombinant vector and the Fu-GENE HD solution was 1: 4, and transfected cells were selected using geneticin (AG Scientific) at a concentration of 600 μg / ml. The selected cell line stably expressing the dual reporter gene system was designated as '8505C-PNIS-PCMV'.

<1.4> Bioluminescence to measure promoter activity

Lucifer to expose 8505C-PNIS-PCMV cells to a tyrosine kinase inhibitor (TKI) library and to enhance NIS promoter activity and observe cell viability using IVIS Lumina III (Perkin-Elmer, Wellesley, MA, USA) Lase activity was measured.

The detailed protocol is as follows. NIS promoter activity was measured with IVIS Lumina III (Perkin-Elmer, Wellesley, MA, USA) by adding D-luciferin (150 μg / ml) to the cells. To normalize NIS promoter activity for cell number, Rluc activity was measured by adding h-coelenterazine at a concentration of 10 μg / ml, and Fluc activity was measured for Rluc activity. Divided by Fluc activity was normalized to Rluc activity .

<1.5> Tyrosine kinase inhibitor (TKI) screening that increases NIS promoter activity

In order to select a tyrosine kinase inhibitor (TKI) that enhances the activity of a sodium iodide symporter (NIS) promoter, a quantitative analysis of bioluminescence imaging (hereinafter referred to as BLI) activity was performed by the method of <1.4>. Rluc activity indicating cell viability and Fluc activity meaning NIS promoter activity were observed by BLI. Figure 1a shows the activity of Rluc, Figure 1b shows the activity of Fluc. Compounds showing a tendency to increase Fluc activity (compounds located at B4 in FIGS. 1A and 1B) compared to vehicles were selected, and the name of the selected compounds was "5- (5- {4H, 5H, 6H-cyclo Penta [b] thiophen-2-yl} -1,3,4-oxadiazol-2-yl) -1-methyl-1,2-dihydropyridin-2-one ". 1C shows Fluc activity normalized to Rluc activity in cells and vehicles administered with the selected TKI. The Fluc activity normalized to Rluc activity in the cells administered with the selected TKI was 2.67 times higher than the vehicle. The selected TKI has the structure of Formula 1 below and detailed information about it is shown in Table 1.

[Formula 1]

Hereinafter, "TKI" used in the following experiment is "5- (5- {4H, 5H, 6H-cyclopenta [b] thiophen-2-yl} -1,3,4-oxadiazole-2- 1) -1-methyl-1,2-dihydropyridin-2-one.

<1.6> Selection of TKI concentration and treatment time in which NIS promoter activity is active upon administration of TKI

To observe the activity of the NIS promoter, 8505C-PNIS-PCMV cells were cultured with TKI at various times and concentrations. Figure 2a shows the pattern of Fluc activity according to TKI concentration and treatment time. The Fluc activity, which means the activity of the NIS promoter, was increased in the 72 hour TKI administration group compared to the other time TKI administration group. As the TKI concentration increased, the activity of Fluc increased up to 25 μM, and particularly, the strongest Fluc activity at 6.25 μM, 12.5 μM, and 25 μM concentrations in the 72-hour TKI administration group. However, at 50 μM concentration, Fluc activity was significantly reduced.

Figure 2b shows the pattern of Rluc activity according to TKI concentration and administration time. Rluc activity, which means cell viability, gradually decreased as TKI concentration increased.

In order to normalize NIS promoter activity with respect to cell number, Fluc activity was divided by Rluc activity. 2c shows Fluc activity normalized to Rluc activity according to TKI concentration and treatment time. The 50 μM concentration of the TKI administration group appeared to exhibit the highest NIS promoter activity, but this was because relatively low Rluc activity was reflected. Therefore, except for 50 μM, the highest NIS promoter activity was shown in the TKI administration group at the concentrations of 12.5 μM and 25 μM for 72 hours, so that 12.5 μM and 25 μM TKI was administered for 72 hours to conduct subsequent experiments.

Example 2. Increased mRNA expression of thyroid-specific genes and thyroid transcription factors by TKI administration

<2.1> RNA extraction and real-time quantitative PCR analysis

8505C cells were exposed to 12.5 μM and 25 μM TKI for 72 hours. After exposure, total RNA was isolated from cells using the TRIzol reagent (Invitrogen, San Diego, CA, USA) according to the manufacturer's instructions. According to the manufacturer's instructions, 1 μg of total RNA was converted to cDNA using a High Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Foster City, CA, USA). The CFX96 Touch Real-Time PCR detection system (Bio-rad Laboratories, Inc., Hercules, CA, USA) using the SsoAdvanced Universal SYBR Green Supermix (Bio-Rad Laboratories, Inc., Hercules, USA) was used according to the manufacturer's instructions. Real-time quantitative PCR analysis was performed. Sodium iodide symporter (NIS), thyroid peroxidase (TPO), thyroglobulin (Tg) and thyroid-stimulating hormone receptor (TSHR) primers (see Table 2) were used to analyze changes in mRNA levels of thyroid-specific genes after TKI administration at 8505C. , TTF-1 (thyroid transcription factor-1) and Pax-8 (paired box gene-8) primers (see Table 2) were used to analyze changes in mRNA levels of thyroid transcription factors after TKI administration to 8505C. The r18s primer (see Table 2) was used as an internal control for real-time quantitative PCR. For real-time quantitative PCR analysis, the relative expression of the target gene was calculated using the 2 ^{-Δ △ Ct} method.

<2.2>  Increased mRNA expression of thyroid-specific genes and thyroid transcription factors by administration of TKI in 8505C cells

Since the thyroid-specific gene and thyroid transcription factor including NIS are important for collecting and maintaining radioactive iodine (RAI), we examined the effect of TKI on the expression of these genes in 8505C cells. Figure 3 shows the relative mRNA expression of thyroid specific genes (NIS, TPO, Tg and TSHR) and thyroid transcription factors (TTF-1 and Pax-8) by administration of TKI (12.5 μM and 25 μM).

NIS mRNA expression increased significantly compared to vehicle when administered with TKI, especially 13.11 times increased compared to vehicle when administered with 25 μM TKI, and increased 2.02 times compared to vehicle when administered with 12.5 μM of TKI. Moreover, the amount of gene expression of TPO, Tg and TSHR as thyroid specific genes increased by 10.08 ± 3.15, 19.08 ± 2.75 and 12.39 ± 1.99 times, respectively, compared to the vehicle by administration of TKI at 25 μM. In addition, mRNA expression of TTF-1 and Pax-8 as thyroid transcription factors increased by 2.59 ± 0.24 and 12.1 ± 4.10 times, respectively, compared to the vehicle by administration of 25 μM TKI.

Therefore, it was confirmed that the re-differentiation of cancer cells was induced by TKI by confirming that the amount of mRNA expression of thyroid-specific genes including NIS and thyroid transcription factors was increased by TKI administration.

Example 3. Increased expression and localization of endogenous NIS protein in cells upon administration of TKI

<3.1> Protein extraction

8505C, MDA-MB-231 and MCF-7 cells were incubated for 72 hours with appropriate concentrations (12.5 μM and 25 μM) of TKI. After 72 hours of TKI administration, cell pellets are collected to isolate total protein and buffered with radioimmunoprecipitation assay (RIPA) containing protease and phosphatase inhibitor cocktail kits (Thermo Fisher Scientific, Rockford, IL, USA) Solution (Thermo Fisher Scientific, Rockford, IL, USA). The lysed cells were gently vortexed 3 times at a time interval and centrifuged at 13,000 Хg at 4 ° C.

Membrane protein and cytoplasmic protein were extracted from a soluble protein fraction according to the manufacturer's instructions using a Mem-PER ™ Plus kit (Thermo scientific, Rockford, IL, USA). . Briefly, the cell pellet was washed with a cooled cell wash solution and centrifuged twice at 300 xg for 5 minutes. After discarding the supernatant, the cell pellet was administered with a permeabilization buffer containing protease and phosphatase inhibitor cocktail kits. In order to obtain a homogeneous cell suspension, the cell pellet was vortexed and incubated by continuously mixing at 4 ° C. for 10 minutes. After incubation, the cell pellet was centrifuged and the supernatant containing cytoplasmic protein was isolated. The remaining pellets were administered with continuous mixing at 4 ° C. for 10 minutes with a solubilization buffer containing protease and phosphatase inhibitor cocktail kits. After centrifugation at 300 xg for 5 minutes, the membrane protein was transferred to a new tube. Protein samples were quantified using a bicinchoninic acid (BCA) protein analysis kit (Thermo Fisher Scientific, Rockford, IL, USA).

For Western blot analysis from tumor tissue, T-PER tissue protein extraction reagent (Thermo Scientific) was used to polish frozen tumor tissue and isolate proteins according to the manufacturer's instructions.

<3.2> Western Blot

The same amount of protein extracted by the method of <3.1> was loaded onto 10% SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis) and transferred to a PVDF membrane (Millipore, Billerica, MA, USA). The membrane was blocked with Tween-20 containing TBS (TBS-T) for 3 hours with 3% Bovine serum albumin (BSA), and contacted at 1 ° C. with 1% BSA at 4 ° C. overnight. After washing three times with TBS-T, the membrane was contacted with a horseradish peroxidase (HRP) -binding secondary antibody for 1 hour at room temperature. The membrane was washed 3 times with TBS-T and the signal was visualized using enhanced chemiluminescence (ECL) detection reagent (GE Healthcare Life Sciences, Pittsburgh, PA, USA). The band was detected using a Fusion FX chemiluminescence analyzer system (Vilber lourmat, Marne-la-Vallιe, France) according to the manufacturer's instructions. Table 1 lists the primary antibodies used in the experiment. HRP-binding secondary antibody was used as anti-mouse (Cell Signaling) and anti-rabbit (Cell Signaling).

<3.3> Increase and localization of endogenous NIS protein expression in 8505C cells

It was confirmed by Western blot by the method of <3.2> whether the expression level of the NIS protein (endogenous NIS protein) in 8505C cells and the position thereof changed by TKI administration.

4A shows Western blot results for NIS proteins in cells among total proteins. As shown in FIG. 4A, it can be seen that when the TKI administration of 12.5 μM and 25 μM was increased, the expression level of NIS protein in the cells increased by 1.21 ± 1.54 times and 1.52 ± 0.09 times compared to the vehicle, respectively.

Since NIS expression of the cell membrane plays an important role in iodine intake, it was confirmed whether the location where the NIS protein is expressed is the cell membrane or cytoplasm. 4B and 4C show Western blot results of the cell membrane NIS protein and cytoplasmic NIS protein, respectively. As shown in Figures 4b and 4c, the NIS expression level shows an increasing pattern in both the cell membrane and the cytoplasm, and in particular, unlike the cytosolic NIS protein, the expression level of the cell membrane NIS protein increases significantly up to 1.96 times when administered with 25 μM TKI.

<3.4> Increased expression of endogenous NIS protein in breast cancer cells

Whether breast cancer cells MDA-MB-231 and MCF-7 expressed changes in the expression level of endogenous NIS protein by TKI administration was measured by Western blot as in <3.2>.

5A and 5B show Western blot results for NIS protein in MDA-MB-231 cells and MCF-7 cells, respectively. As shown in Figures 5a and 5b, it was confirmed that the amount of NIS protein increased in a concentration-dependent manner by TKI administration in both MDA-MB-231 cells and MCF-7 cells. As a result of Western blot quantitative analysis, the expression of NIS protein in MDA-MB-231 cells increased by 1.17 fold in 12.5 μM and 1.73 fold in 25 μM concentration treatment group compared to vehicle group. In addition, in MCF-7 cells, a pattern that promotes up to 1.56 times in the 12.5 μM concentration treatment group and 2.67 times in the 25 μM treatment group was confirmed compared to the vehicle group.

Example 4. Immunofluorescence imaging for NIS expression analysis

<4.1> Immunofluorescence imaging

8505C cells were incubated (2.5x10 ⁴ ) for 24 hours in a 4-well chamber slide in a humidified atmosphere of 37 ° C., 5% CO ₂ . For NIS immunofluorescence imaging, 8505C cells were exposed to 25 μM TKI for an additional 72 hours. Thereafter, cells were fixed with methanol for 10 minutes at a temperature of -20 ° C and washed 3 times with PBS for 10 minutes. The cells were then incubated with 0.3% Triton X-100 for 90 seconds to make them permeabilized and then washed 3 times with PBS for 10 minutes. Cells were blocked with 3% BSA in PBS for 1 hour and incubated overnight at 4 ° C. with anti-NIS (Abcam, Cambridge, UK) primary antibody (1:50). After the primary antibody incubation, cells were washed 3 times with PBS for 10 minutes and contacted with Alexa-Fluor 488-conjugated secondary antibody (Thermo Fisher Scientific) at a dilution ratio of 100: 1 for 1 hour. Cells were mounted on cover slips using a Vecta mounting medium containing DAPI (4 ′, 6-diamidino-2-phenylindole; Vector Laboratories, Burlingame, CA, USA), and confocal laser microscopy (Zeiss , LSM 5 exciter, Germany).

<4.2> NIS expression observation through immunofluorescence imaging

6 shows the results of observing NIS expression in cells through immunofluorescence imaging. In the group where 25 μM TKI was administered to 8505C cells, active expression of NIS protein in the cell was observed in the cytoplasm or cell membrane compared to the vehicle, and this result indicates that TKI administration induces NIS expression in the cell again.

Moreover, when synthesizing the results of the Western blot of <3.3> and the results of the above-mentioned immunofluorescence imaging, it can be seen that, by TKI administration, the degree of increase in expression of the cell membrane NIS protein is greater than that of the cytoplasmic NIS protein.

Example 5. Increased protein expression of thyroid specific proteins and thyroid transcription factors when TKI is administered

As described above in <2.2>, in the case of 8505C cells, mRNA expression levels of both thyroid specific genes and thyroid transcription factors were significantly increased. Next, Western blot was performed by administering TKI to 8505C cells for 72 hours (12.5 μM and 25 μM) by the method of <3.2> in order to measure the change in the expression level of the important protein handling iodine uptake in 8505C cells. 7A shows Western blot results for Tg, TPO, NIS and TSHR as thyroid specific proteins upon TKI administration. 7B also shows Western blot results for Pax-8 and TTF-1 proteins as thyroid transcription factors upon TKI administration.

As shown in FIGS. 7A and 7B, since thyroid-specific protein and thyroid transcription factors increased expression at the protein level depending on TKI concentration, it was confirmed that TKI induced re-differentiation of cancer cells.

Example 6. Investigation of signaling pathways leading to NIS expression.

Signaling pathways that increase NIS expression in cells were identified. Previous studies have demonstrated that intracellular NIS expression and thyroid cancer tumor formation are associated with mitogen-activated protein kinase (MAPK) and PI3K-AKT signaling pathways. Therefore, in order to define the signaling pathway by focusing on both the MAPK and PI3K-AKT signaling pathways, Western blot was performed by the method of <3.2>. 8 shows Western blot results of genes associated with MAPK and PI3K-AKT signaling pathways. As shown in FIG. 8, TKI administration was found to affect both signaling pathways downstream. However, it was confirmed that phosphorylation of Erk is more inhibited than phosphorylation of Akt. Phosphorylation of BRAF was also tested because the 8505C cell line had a BRAF ^v600E mutation. Since phosphorylation of BRAF was also reduced by TKI administration, it can be seen that, as a result, re-induction of NIS expression in cells by TKI administration was more related to the MAPK signaling pathway than PI3K-AKT.

Example 7. Measurement of accumulation of radioactive iodine (hereinafter referred to as RAI) by NIS after TKI administration

<7.1> In cells ¹²⁵ I absorption capacity analysis ( In vitro ¹²⁵ I uptake assay)

Dispense 8505C cells into 24-well plates (5 Х 10 ⁴ ) and treat TKI at a concentration of 12.5 μM or 25 μM at 37 ° C. and 5% CO ₂ under RPMI (Roswell Park Memorial Institute) -1640 medium (HyClone, Logan , UT, USA) (HyClone) for 72 hours. After incubation, the medium was removed and 8505C cells were washed with bHBSS (warm Hank's balanced salt solution containing 0.5% BSA). Cells in a wet incubator were 500 μl bHBSS, 3.7 kBq carrier-free ¹²⁵ I (Perkin-Elmer, Waltham, MA, USA) and 10 μM / L sodium iodide (NaI, specific activity of 740 MBq / mM) for 30 min at 37 ° C. Cultured for a while. Before the addition of ¹²⁵ I, 50 μM of potassium perchlorate (KClO ₄ ) as a competitive inhibitor of iodine transporter was administered to 8505C cells for 30 minutes. After incubation, cells were washed twice with cooled bHBSS and lysed with 500 μl of RIPA buffer. Radioactivity was measured with a Cobra II gamma-counter (Canberra Packard, Canada), and the ¹²⁵ I absorption capacity was normalized against the total protein amount measured with a BCA protein analysis kit, and the results were expressed in cpm / µg.

<7.2> Measurement of accumulation of radioactive iodine (RAI) by NIS after TKI administration

To test the recovery of radioactive iodine (RAI) accumulation by NIS after TKI administration, the ¹²⁵ I uptake capacity was analyzed by the method of <7.1> above in 8505C cells. Figure 9a shows the result of significantly increased RAI accumulation capacity in a concentration-dependent manner by TKI administration. TKI administration of 12.5 μM and 25 μM increased 1.72 and 2.54 fold RAI intake, respectively, compared to vehicle. We also tried to reveal the correlation between RAI intake and functional NIS expression using potassium chlorate (KClO ₄ ), a competitive inhibitor of iodine transport. 9B and 9C show the absorption capacity of RAI by administration of 12.5 μM and 25 μM of TKI and KClO ₄ , respectively. Administration of TKI alone at concentrations of 12.5 μM and 25 μM in 8505C cells increased RAI accumulation, but iodine uptake was blocked by administration of KCIO ₄ .

As shown in the above results, TKI administration improves RAI accumulation and KCIO ₄ interferes with RAI intake to completely block RAI accumulation, so it was confirmed that TKI directly affects RAI increase through NIS.

Example 8. TKI administration ¹³¹ Improvement of I mediated cytotoxicity effect

<8.1> In vitro ¹³¹ I colony formation analysis ( In vitro ¹³¹ I Clonogenic assay)

8505C cells were dispensed (1 Х 10 ⁵ ) into 6-well plates and treated with 12.5 μM or 25 μM concentration of TKI in RPMI (Roswell Park Memorial Institute) -1640 medium (HyClone, Logan, UT, USA) (HyClone) 72 Incubated for hours. Thereafter, the medium was removed and the cells were washed twice with bHBSS (warm Hank's balanced salt solution containing 0.5% BSA), followed by the presence of 50 μCi / ml ¹³¹ I (KIRAMS, Seoul, Korea) containing 30 μM NaI or Cells were cultured at 37 ° C. for 7 hours in the absence. After incubation, cells were washed twice with bHBSS, trypsin was administered, and redistributed to a new 6-well plate at a density of 1,000 cells / well. Cells were incubated for 10 days at 37 ° C. in 5% CO ₂ to allow colony formation. On day 10, medium was removed and cells were washed twice with PBS. Each colony of the 6-well plate was then fixed with a fixed buffer containing acetic acid: methanol in a 1: 7 volume ratio. The fixed colonies were stained with 0.05% crystal violet for 1 hour and immersed in tap water to wash the crystal violet. By counting colonies with 50 or more cells, colony forming efficiency and survival fraction (%) were calculated using the following equation.

[Equation 1]

Colony efficiency (Plating efficiency: PE,%) = (number of colonies counted / number of cells dispensed) x 100

[Equation 2]

Surviving fraction (SF,%) = (PE of treated cells / PE in vehicle) x 100

<8.2> TKI administration in 8505C cells ¹³¹ Improvement of I mediated cytotoxic effects

Whether the avidity of radioactive iodine (RAI) is improved through intracellular NIS expression and the therapeutic efficacy of undifferentiated thyroid cancer cells (8505C cells) when administered in combination with ¹³¹ I and TKI are evaluated. 10A shows the survival rate of 8505C cells when administered alone or in combination with ¹³¹ I and 12.5 μM TKI, and FIG. 10B shows the survival rate of 8505C cells when administered alone or in combination with TKI at ¹³¹ I and 25 μM. Survival Fraction (%) of the groups administered with vehicle and ¹³¹ I, 12.5 μM TKI, 25 μM TKI alone were 100 ± 13.08%, 71.98 ± 3.68%, 77.94 ± 8.17% and 77.60 ± 8.77%, respectively. In addition, when the combination of ¹³¹ I and 12.5 μM of TKI was administered, the survival rate of cells was significantly reduced, resulting in approximately 52.52 ± 3.94% (FIG. 10A). In addition, when a combination of ¹³¹ I and 25 μM TKI is administered, a cell survival rate of 32.95 ± 2.27% is shown (FIG. 10B).

Therefore, through these results, it was confirmed that the administration of ¹³¹ I or TKI alone has the effect of killing cancer cells, and the administration of ¹³¹ I and TKI in combination improves NIS re-induction and intake of RAI and is effective in treating thyroid cancer. .

Example 9. ¹³¹ DNA damage measurement when I and TKI are administered

The TKI and ¹³¹ I to 8505C cells through γH2A.X foci formation assay was observed damage to the DNA when administered alone or in combination to identify the cancer cell killing effect of the TKI and ¹³¹ I.

<9.1> Immunofluorescence imaging for γH2A.X foci formation analysis

8505C cells were incubated (2.5x10 ⁴ ) for 24 hours in a 4-well chamber slide in a humidified atmosphere of 37 ° C., 5% CO ₂ . γH2A.X for immunofluorescence imaging for analysis of foci formation, was administered to 8505C cells with 25μM and TKI ¹³¹ I, alone or in combination, were administered during the administration of the combination, and TKI ¹³¹ I is administered after 72 hours. After incubation, cells were fixed with methanol for 10 minutes at a temperature of -20 ° C and washed 3 times with PBS for 10 minutes. Then, the cells were incubated with 0.3% Triton X-100 for 90 seconds to make them permeabilized, and then washed three times with PBS for 10 minutes. Cells were blocked with 3% BSA in PBS for 1 hour and incubated overnight at 4 ° C. with anti-NIS (Abcam, Cambridge, UK) primary antibody (1:50). After the primary antibody incubation, the cells were rinsed three times with PBS for 10 minutes and contacted with a Alexa-Fluor 488-conjugated secondary antibody (Thermo Fisher Scientific) at a dilution ratio of 100: 1 for 1 hour.

For γH2A.X foci, incubation was performed at room temperature for 4 hours using anti-γH2A.X conjugated with Dylight 488 (Abcam, Cambridge, UK). Cells were mounted on cover slips using a Vecta mounting medium containing DAPI (4 ′, 6-diamidino-2-phenylindole; Vector Laboratories, Burlingame, CA, USA), and confocal laser microscopy (Zeiss , LSM 5 exciter, Germany).

<9.2> In 8505C cells ¹³¹ DNA damage measurement when I and TKI are administered

Analysis of γH2A.X foci formation was observed through immunofluorescence imaging of the method of <9.1>, and DNA damage was analyzed when administered alone or in combination with ¹³¹ I and TKI. 11A shows the results of γH2A.X foci formation assay when administered with ¹³¹ I and TKI alone or in combination. As shown in FIG. 11A, the formation of γH2A.X foci was increased in the TKI and ¹³¹ I alone groups compared to vehicle. Also it increased the number of significantly more than the administration γH2A.X foci in the group treated with a combination of ¹³¹ I and ¹³¹ I as TKI and TKI alone group.

11B shows Western blot results for γH2A.X when administered alone or in combination with ¹³¹ I and TKI. Same as γH2A.X foci formation analysis by the immune fluorescence imaging as ¹³¹ I and see that in a group administered with a combination of TKI ¹³¹ I and TKI the protein expression level of γH2A.X higher than the group administered alone Could.

From the above results, ¹³¹ I or It can be seen that administration of TKI alone showed the effect of cancer cell death, and administration of ¹³¹ I and TKI in combination showed a synergistic effect on cancer cell death.

Example 10. Increase of NIS expression in cells by TKI administration in vivo

<10.1> Construction of tumor xenograft mice model

Female 5-week Balb / c nude mice were purchased from Hamamatsu (Shizuoka, Japan). Mice were kept under pathogen-free conditions for one week to adapt to the experimental conditions, and animals were kept at room temperature (20-25 ° C) and 40-70% relative humidity. To create a tumor xenograft mouse model, 8505C-PNIS-PCMV cells (5Х10 ⁶ ) were mixed with Matrigel (Corning, Bedford, MA, USA) at a volume ratio of 1: 1 and injected subcutaneously in the right flank region of the mouse. In vivo experiments began when the average tumor size of the mice reached 50 mm ³ .

<10.2> Bioluminescence imaging in vivo ( In vivo  bioluminescence imaging (hereinafter referred to as BLI)

It was observed whether the activity of the NIS promoter was increased for mice in the vehicle group and the 100 mg / kg TKI administration group (n = 5 for each group). 100 mg / kg TKI is administered daily to the mice daily for 7 days by intraperitoneal injection, and -1 (before administration), 3, during TKI administration on the basis of the first TKI administration day (day 0), 3, Observations were made on days 5 and 7. To observe Rluc activity, 15 μg / ml of h-coelenterazine was administered by intravenous (i.v.) injection, and images were taken using IVIS Lumina III. Similar to Rluc activity, Fluc activity was performed by administering 150 μg / ml d-luciferin by intraperitoneal (i.p) injection and was observed by IVIS Lumina III.

<10.3> Immunohistochemistry staining

For immunohistochemical staining, tumors were fixed overnight with 4% formalin. Then, the specimen was embedded in paraffin, sectioned to a thickness of 4 μm, and mounted on a slide. Paraffin was removed from the specimen section slides and stained with hematoxylin and eosin (H & E). NIS (Thermo Fisher Scientific; 1: 200 dilution) and γH2A.X (Abcam; 1: 200 dilution) antibodies were used.

<10.4> Increase of NIS promoter activity by TKI administration in vivo

To observe the change in NIS promoter activity after TKI administration, BLI images for Fluc activity were collected on Days -1, 3, 5 and 7 by the method of <10.2> above. 12A shows BLI images for Fluc activity in vehicle and TKI administration groups in a tumor xenograft mouse model. As shown in FIG. 12A, the TKI administration group actively increased Fluc activity. Figure 12b shows the results of quantitative analysis for Fluc activity in the vehicle and TKI administration group in a tumor xenograft mouse model. Quantitative analysis confirmed that the FLuc activity was significantly increased in the TKI administration group. TIS dosing group showed NIS promoter activity of 1.00 ± 0.00, 3.95 ± 2.76, 4.92 ± 2.16 and 8.83 ± 5.05 on day -1, 3, 5, and 7, respectively, and -1, 3, 5, and 7 in vehicle group It shows relative NIS promoter activity of 1.00 ± 0.00, 1.55 ± 1.27, 2.28 ± 1.29 and 2.11 ± 1.15, respectively, on the first day. The TKI administration group increased the NIS promoter activity 8-fold at 2 and 7 days compared to the vehicle group on days 5 and 7.

However, since the tumor size was not reflected in the quantitative analysis, the tumor size was normalized after TKI administration by observing Rluc activity. 12C shows BLI images for Rluc activity in vehicle and TKI administration groups in a tumor xenograft mouse model. As shown in Figure 12c, both vehicle and TKI administration groups showed an increase in Rluc activity. 12D shows the results of quantitative analysis by normalizing Fluc activity with Rluc activity in the vehicle and TKI administration groups in a tumor xenograft mouse model. Although the activity of the NIS promoter was increased in the vehicle between Days -1, 3, 5 and 7, there was no statistically significant increase. Compared to the vehicle group, the TKI administration group showed a significant increase in NIS promoter activity. The vehicle group on day 7 with the highest Fluc activity showed an increase of 1.56 ± 0.88 times NIS promoter activity compared to day -1, but the TKI administration group had 3.22 ± 0.97 times NIS promoter activity on day 7 compared to day -1. It was confirmed that it increased.

<10.5> Increase of NIS expression in cells by TKI administration

Through Western blot analysis according to the method of <3.2>, the expression of NIS protein in cells was confirmed in tumor tissue derived from mice sacrificed on the 7th day after TKI administration. 13A shows Western blot results for NIS protein in tumor tissue derived from tumor xenograft mice. As shown in FIG. 13A, it was confirmed that NIS expression in cells was re-induced by TKI administration in the tumor tissue of the mouse.

Immunohistochemical staining was performed using the tumor tissue of the mouse in the method of <10.3>. Figure 13b shows the results of immunohistochemical staining of NIS in the tumor tissue of a tumor xenograft mouse. As shown in FIG. 13A, it was confirmed that the amount of NIS expression was higher in the tumor tissue of the mice administered with 100 mg / kg TKI than in the vehicle group.

Example 11. For mice ¹³¹ Treatment effect by administration of I and TKI

<11.1> Bioluminescence imaging for Rluc activity in a tumor xenograft mouse model

To evaluate the therapeutic effect of TKI and ¹³¹ I alone or in combination through serial bioluminescence imaging, tumor xenograft mice were treated with ① vehicle group (solvent not containing TKI daily from day 0 to day 7) Dosing), ② ¹³¹ I dosing group (daily administration of solvents that do not contain TKI from day 0 to day 7 and 1 mCi Na- ¹³¹ I administration by intravenous injection on day 7), ③ TKI dosing group (day 0 to day 7) During daily administration of TKI at a concentration of 100 mg / kg by intraperitoneal injection) and ④ in combination groups of ¹³¹ I and TKI (at day 7 from day 0 to day 7 after daily administration of TKI at a concentration of 100 mg / kg by intraperitoneal injection daily) 1mCi Na- ¹³¹ I was administered by intravenous injection). Rluc activity was 15 μg / ml h-coelenterazine in mice on days 0, 3 and 6 after administration of ¹³¹ I or TKI (day 0, 3, and 6 after administration of ¹³¹ I in the group of combination administration). Was observed by injection and imaged with IVIS Lumina III. Mice were anesthetized with Isofluorane (Forane, ChongWae Co., Seoul, Korea) and placed in the imaging chamber with continuous 2.5% isofluorane administration through a nasal cone.

14A shows Rluc activity for vehicle, ¹³¹ I and TKI administration in a tumor xenograft mouse model. The vehicle, ¹³¹ I and TKI alone dosing group showed a continuous increase in Rluc activity by Day 6, but the combination administration group of ¹³¹ I and TKI significantly inhibited the increase in Rluc activity.

<11.2> Tumor volume, mouse weight and tumor weight measurement

Tumor xenograft mice were ① vehicle group (solvent administration without TKI daily for days 0 to 7), ② ¹³¹ I administration group (solvent administration without TKI daily for days 0 to 7 and intravenous on day 7 1mCi Na- ¹³¹ I administration by intravenous injection), ③ TKI administration group (TKI administration at 100 mg / kg concentration by intraperitoneal injection daily from day 0 to day 7) and ④ ¹³¹ I and TKI combination administration group (day 0 Tumor volume, mouse weight and tumor weight were measured by dividing into 1 mCi Na- ¹³¹ I by intravenous injection on day 7 after TKI administration at a concentration of 100 mg / kg by intraperitoneal injection daily for 7 days from day .

The length and width of the tumor were measured with a caliper at -1 (before TKI or solvent administration), on days 6, 10, and 14 on the basis of the first day of administration of TKI (or solvent without TKI) (day 0) Tumor volume (mm ³ ) was calculated using Equation 3.

[Equation 3]

Volume (V) = 0.5 x length x width ²

FIG. 14B shows tumor volumes measured on day -1 (before TKI or solvent administration), Days 6, 10, and 14 based on the first day of administration of TKI (or TKI-free solvent). As shown in Fig. 14B, it was shown that the combination administration group of ¹³¹ I and TKI significantly inhibited tumor growth compared to other groups.

14C shows the weight change of tumor xenograft mice during in vivo experiments. Although there may be side effects during the in vivo experiment, as shown in FIG. 14C, it can be seen that there was no side effect of ¹³¹ I and TKI administration in all groups, as the weight of mice did not change between groups.

 Mice were sacrificed by cervical vertebrae dislocation at the end of the mouse in vivo experiment. On day 14 after TKI administration, tumors were collected, imaged, and tumor weights were measured.

15A shows tumor tissue isolated from tumor xenograft mice. As shown in FIG. 15A, the tumor size of the ¹³¹ I and TKI combination administration group was significantly smaller than that of the other groups. 15B shows the average weight for tumor tissue. From the average tumor weight shown in Fig. 15B, it was confirmed that the tumors of the ¹³¹ I and TKI combination administration group inhibited the growth of 2 times or more. From these results, it was found that tumor growth was significantly inhibited when ¹³¹ I and TKI were administered in combination.

<11.3> Immunohistochemical staining of γH2A.X in tumor tissue

① Vehicle group (solvent administration without TKI daily for days 0 to 7), ② ¹³¹ I administration group (solvent administration without TKI daily for days 0 to 7 and 1 mCi by intravenous injection on day 7 Na- ¹³¹ I administration), ③ TKI administration group (TKI administration daily for 7 days at a concentration of 100 mg / kg by intraperitoneal injection), and ④ above <10.3 in tumor tissues isolated from mice of the ¹³¹ I and TKI combination administration groups. Immunohistochemical staining of γH2A.X was performed by the method of>. 16 shows the results of immunohistochemical staining of γH2A.X in tumor tissue isolated from tumor xenograft mice. As shown in FIG. 16, it can be seen that the degree of DNA damage was significantly increased in the ¹³¹ I and TKI combination administration group, so it can be seen that tumor growth is significantly inhibited when the ¹³¹ I and TKI combination administration is administered.

Statistical analysis

All experimental results were expressed as mean ± standard deviation (means ± SD), and the results of the two groups were statistically analyzed by Student's t-test method using GraphPad Prism 5 software version 5.01 (GraphPad Software, Inc. USA). When the p value was less than 0.05, it was judged to be statistically significant, and the error bars represent standard deviation (SD).

<110> Kyungpook National University Industry-Academic Cooperation Foundation <120> Pharmaceutical Composition For Treating Cancer Comprising          Tyrosine Kinase Inhibitor <130> PN180163 <160> 14 <170> KoPatentIn 3.0 <210> 1 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> NIS_Forward <400> 1 ctgccccact ccagtacatg cc 22 <210> 2 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> NIS_Reverse <400> 2 tgacggtgaa ggaaccctga ag 22 <210> 3 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> TPO_Forward <400> 3 ggagtctcgt tgctctagcg t 21 <210> 4 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> TPO_Reverse <400> 4 ctctgcactg tggcgtacat 20 <210> 5 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Tg_Forward <400> 5 aagaatacgt cgtcttctcc ac 22 <210> 6 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Tg_Reverse <400> 6 caggcataac atctccctcc g 21 <210> 7 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> TSHR_Forward <400> 7 ggaatggggt tgtcgtctcc 20 <210> 8 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> TSHR_Reverse <400> 8 gcgttgaatt accttgcagg t 21 <210> 9 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> TTF-1_Forward <400> 9 agcacacgac tccgtcttc 19 <210> 10 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> TTF-1_Reverse <400> 10 gcccacttct ttgtagcttt cc 22 <210> 11 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> PAX-8_Forward <400> 11 atccggcctg ggatgatagg 20 <210> 12 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> PAX-8_Reverse <400> 12 tggcgttgtt agtccccaat c 21 <210> 13 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> r18s_Forward <400> 13 gaataatgga ataggaccgc gg 22 <210> 14 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> r18s_Reverse <400> 14 ggaactacga cggtatctga tc 22

Claims (9)

A pharmaceutical composition for preventing or treating cancer comprising the compound of Formula 1 or a pharmaceutically acceptable salt thereof as an active ingredient.
[Formula 1]

The composition according to claim 1, wherein the cancer is thyroid cancer or breast cancer.
The method according to claim 2, wherein the thyroid cancer is an undifferentiated thyroid cancer (anaplastic thyroid cancer) or a differentiated thyroid cancer (differentiated thyroid cancer).
The composition of claim 1, wherein the composition increases expression of a sodium iodide symporter (NIS).
An anticancer adjuvant for use in the treatment of cancer diseases comprising the compound of Formula 1 or a pharmaceutically acceptable salt thereof as an active ingredient.
[Formula 1]

The anti-cancer adjuvant of claim 5, wherein the anti-cancer adjuvant increases the expression of a sodium iodide symporter (NIS).
The anticancer adjuvant according to claim 5, wherein the cancer disease is thyroid cancer or breast cancer.
The anti-cancer adjuvant of claim 7, wherein the thyroid cancer is an undifferentiated thyroid cancer or a differentiated thyroid cancer.
The anti-cancer adjuvant of claim 5, wherein the anti-cancer adjuvant is used individually, sequentially or simultaneously with radioactive iodine treatment.

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