KR101800129B1 - Pharmaceutical composition for preventing and treating cancer - Google Patents

Abstract

The present invention relates to a pharmaceutical composition for preventing or treating cancer and, more specifically, to a pharmaceutical composition capable of effectively preventing or treating thyroid cancer by effectively inhibiting growth of cancer cells or cancer stem cells. The pharmaceutical composition includes a histone deacetylation inhibitor (HDAC inhibitor) and a kinase inhibitor as active ingredients, wherein the histone deacetylation inhibitor is a compound represented by chemical formula 1, and the kinase inhibitor is a compound represented by chemical formula 2.

Description

[0001] The present invention relates to a pharmaceutical composition for preventing or treating cancer,

The present invention relates to a pharmaceutical composition for preventing or treating cancer, and more particularly, to a pharmaceutical composition capable of effectively preventing or treating cancer by effectively inhibiting the growth of cancer cells or cancer stem cells.

Cancer is one of the incurable diseases that humanity needs to solve. In the world, huge capital is invested in development to heal it globally. In Korea, it is the first disease among the causes of death, It is diagnosed and more than 60,000 people are dying.

Smoking, ultraviolet rays, chemicals, food, and other environmental factors are among the carcinogens that cause cancer, but the development of therapeutic agents is difficult due to the various causes, and the effects of the therapeutic agents are also different depending on the site where they occur. Currently, substances used as therapeutic agents have considerable toxicity. Since they can not selectively remove only cancer cells, it is urgently necessary to develop a less toxic and effective anticancer drug to prevent the cancer from occurring after cancer development .

On the other hand, thyroid cancer is a cancer that is more prevalent among women than men, and the cancer incidence rate of National Cancer Information Center is 7.4% of total female cancer in 2002. According to recent reports from the Korean Thyroid Disease Society, it ranked fourth in the incidence of cancer in Korea as of 2006, and women are ranked first.

These thyroid cancers are the most common malignant tumors of the endocrine system, including papillary carcinoma, follicular carcinoma, hurthle cell neoplasm, anaplastic carcinoma, undifferentiated thyroid cancer, (Medullary thyroid carcinoma).

The cause of thyroid cancer is known as radiation exposure, sex hormone, etc. Although the incidence of thyroid cancer is higher than that of other cancers, the early diagnosis and early survival rates are high. However, there is a problem in the already advanced cancer, especially when it does not respond to radioactive iodine (RAI) treatment.

Therefore, recent studies on methods for treating thyroid cancer more effectively have been actively conducted.

The present invention aims at providing a pharmaceutical composition capable of preventing and / or treating cancer in the end, by effectively inhibiting the growth of cancer cells or cancer stem cells and suppressing proliferation, invasion and metastasis of cancer cells.

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

According to one embodiment of the present invention, there is provided a pharmaceutical composition for preventing or treating cancer comprising, as an active ingredient, a histone deacetylation inhibitor (HDAC inhibitor) and a kinase inhibitor.

In the present invention, the above-mentioned histone deacetylation inhibitor inhibits the activity of histone deacetylase (HDAC), which is known to play an important role in the gene expression regulating mechanism, and it can inhibit the hypoacetylation of histone and Induced changes in gene expression. Wherein the histone deacetylation inhibitor is selected from the group consisting of a) a domain capable of binding a metal (especially zinc), b) a linker capable of filling the channels of the histone deacetylase, and c) a histone deacetylase (J. Med. Chem., 2003, 46 (24), 5097-5116), which can interact with structures at the edges of the active region. In the present invention, the above-mentioned histone deacetylation inhibitor is preferably (N-hydroxy-7- (2-naphthylthio) heptanomide (N-hydroxy- )): ≪ / RTI >

[Chemical Formula 1]

Also, in the present invention, the kinase inhibitor may be a multi-kinase inhibitor targeting VEGFR, PDGFR and Raf, and preferably sorafenib (trade name: Nexavar, Nexavar). In the present invention, the sorapenib is represented by the following general formula (2), and 4- (4- (3- (4-chloro-3- (trifluoromethyl) phenyl) ureido) phenoxy) (4- (4- (3- (4-Chloro-3- (trifluoromethyl) phenyl) ureido) phenoxy) -N-methylpicolinamide)

(2)

Among the inhibitors of histone deacetylation in the present invention, particularly (N-hydroxy-7- (2-naphthylthio) heptanumide) and 4- (4- (3- (4- When 3- (trifluoromethyl) phenyl) ureido) phenoxy) -N-methylpicolinamide is administered in combination, cancer cell proliferation can be effectively inhibited and cancer can be prevented or treated.

In the present invention, the histone deacetylation inhibitor and the kinase inhibitor may be used in a weight ratio of 1: 1 to 100, preferably 1: 2 to 20.

Examples of the cancer to be prevented or treated in the present invention include, but are not limited to, thyroid cancer, breast cancer, biliary cancer, gallbladder cancer, pancreatic cancer, colon cancer, uterine cancer, esophageal cancer, stomach cancer, brain cancer, , Renal cancer, ovarian cancer, prostate cancer, uterine cancer, head and neck cancer, skin cancer, blood cancer, and liver cancer, preferably thyroid cancer.

In the present invention, "prevention" may include, without limitation, any act that blocks cancer symptoms using the pharmaceutical composition of the present invention, or inhibits or delays cancer symptoms.

Further, in the present invention, "treatment" may include, without limitation, any action of improving or ameliorating cancer symptoms using the pharmaceutical composition of the present invention.

According to another embodiment of the present invention, there is provided a pharmaceutical composition for inhibiting the growth of cancer stem cells comprising a histone deacetylation inhibitor and a kinase inhibitor as an active ingredient.

In the present invention, the histone deacetylation inhibitor may be (N-hydroxy-7- (2-naphthylthio) heptanumide) represented by the above formula (1).

In addition, in the present invention, the kinase inhibitor may be selected from the group consisting of 4- (4- (3- (4-chloro-3- (trifluoromethyl) phenyl) ureido) phenoxy) It may be nemide.

Among the inhibitors of histone deacetylation in the present invention, particularly (N-hydroxy-7- (2-naphthylthio) heptanumide) and 4- (4- (3- (4- - (trifluoromethyl) phenyl) ureido) phenoxy) -N-methylpicolinamide is administered in combination, the growth of cancer stem cells is effectively inhibited, and furthermore, Cancer recurrence or metastasis can be prevented or treated.

In the present invention, the histone deacetylation inhibitor and the kinase inhibitor may be used in a weight ratio of 1: 1 to 100, preferably 1: 2 to 20.

The types of cancer stem cells to be subject to growth inhibition in the present invention are not particularly limited, and examples thereof include thyroid cancer, breast cancer, biliary cancer, gallbladder cancer, pancreatic cancer, colon cancer, uterine cancer, esophageal cancer, gastric cancer, The present invention may be a stem cell of at least one cancer selected from the group consisting of bladder cancer, kidney cancer, ovarian cancer, prostate cancer, uterine cancer, head and neck cancer, skin cancer, blood cancer and liver cancer, and preferably a thyroid cancer stem cell.

In the present invention, the term "cancer stem cell" refers to a cancer cell having a comprehensive meaning that has the ability to regenerate or differentiate into a stem cell-specific ability. The normal tumor growth condition of the cancer stem cell (the "normal tumor growth condition" refers to a state in which the nutrients (glucose) necessary for cell growth are sufficient and the condition of growth of the tumor microenvironment is abundant and cell stress is absent) The expression of PGC-1a and other transcription factors may be different from that of normal tumor cells. For example, the expression of PGC-1a and the like may be different from that of normal tumor cells. And the function of major metabolic control substances may be different compared to normal cancer cells. This collectively refers to cells that acquire resistance to apoptosis in the nutritional deficiency state and control invasion and / or metastasis through the modulation of these different metabolic regulatory capacities and the cell signaling pathways associated therewith. However, the present invention is not limited to the cells that can differentiate into common cancer cells.

In the present invention, " inhibition of growth of cancer stem cells " may include the inhibition of cancer stem cell maintenance (mainstance), inhibition of cancer stem cell malignancy, and inhibition of cancer stem cell invasion .

According to another embodiment of the present invention, there is provided a pharmaceutical composition for overcoming tolerance to endometrial cancer, which comprises a histone deacetylation inhibitor and a kinase inhibitor as an active ingredient.

In the present invention, the histone deacetylation inhibitor may be (N-hydroxy-7- (2-naphthylthio) heptanumide) represented by the above formula (1).

In addition, in the present invention, the kinase inhibitor may be selected from the group consisting of 4- (4- (3- (4-chloro-3- (trifluoromethyl) phenyl) ureido) phenoxy) It may be nemide.

Among the inhibitors of histone deacetylation in the present invention, particularly (N-hydroxy-7- (2-naphthylthio) heptanumide) and 4- (4- (3- (4- - (trifluoromethyl) phenyl) ureido) phenoxy) -N-methylpicolinamide is administered in combination, it is possible to overcome the tolerance of drug resistance by increasing the sensitivity to the drug for cancer treatment, Further inhibiting growth and inducing apoptosis.

In the present invention, the histone deacetylation inhibitor and the kinase inhibitor may be used in a weight ratio of 1: 1 to 100, preferably 1: 2 to 20.

The type of endometrial cancer to be overcome by the present invention is not particularly limited, and examples thereof include thyroid cancer, breast cancer, biliary cancer, gallbladder cancer, pancreatic cancer, colon cancer, uterine cancer, And may be at least one type of cancer selected from the group consisting of gastric cancer, brain cancer, rectal cancer, lung cancer, bladder cancer, kidney cancer, ovarian cancer, prostate cancer, uterine cancer, head and neck cancer, skin cancer, have.

In the present invention, "drug for treating cancer" is a drug for treating cancer, which may be an anticancer agent, and the kind thereof is not particularly limited, but may be preferably a drug for the treatment of biliary cancer or pancreatic cancer. Specific examples include, but are not limited to, Nitrogen mustard, Imatinib, Oxaliplatin, Rituximab, Erotinib, Neratib, Lapatinib, Zetitib, Bandetanib, Nilotinib, Semathanib, , Corticosteroids, cisplatin, cetuximab, bismuth alum, asparaginase, tretinoin, hydroxycarbamate, terephthalic acid, tretinoin, Amlodipine, amlodipine, amlodipine, amlodipine, amlodipine, amide, amlodipine, amlodipine, amlodipine, amlodipine, But are not limited to, chitosan, gemcitabine, doxifluridine, femetrexed, tegafur, capecitabine, gimeracin, oteracil, azacytidine, methotrexate, uracil, cytarabine, fluorouracil, Tabin, Plutamid, De But are not limited to, cholesterol, cholesterol, cholesterol, cholesterol, cholesterol, cholesterol, cholesterol, cholesterol, cholesterol, But are not limited to, atorvastatin, tinifoside, doxorubicin, dirubicin, epirubicin, mitoxantrone, mitomycin, blormomycin, daunorubicin, dactinomycin, pyrabicin, aclarubicin, But are not limited to, polysaccharides, polysaccharides, polysaccharides, polysaccharides, polysaccharides, polysaccharides, polysaccharides, Aminoglutethimide, anagrelide, navelin, pradrazol, tamoxifen, toremifene, testolactone, anastrozole, letrozole, borozol, bicalutamide, romestin, borinostat, , 5FU, and carmustine, Preferably one or more selected from the group Gemcitabine, Cisplatin, 5FU, Capecitabine, Olsaliplatine, Vorinistat, and Entinostat. And more preferably selected from the group consisting of gemcitabine, cisplatin, 5-FU, Capecitabine, Olsaliplatine, Vorinistat and Entinostat. And the like, but is not limited thereto.

In the present invention, the term " endometrial cancer for a drug for cancer treatment "means an extremely low sensitivity to a drug for cancer treatment, particularly, an anticancer drug, and the treatment of the cancer, Means cancer that does not exhibit symptoms of treatment. The cancer drug for cancer treatment may be resistant to a specific anticancer drug from the beginning and may not be resistant to the same therapeutic agent due to gene mutation in cancer cells due to a long time drug treatment, . In the cancer-resistant cells against the drug, a) reduction in the action of the drug due to cell membrane changes, b) promotion of drug release by multidrug resistance (MRP), which is a transporter gene of p-glycoprotein and ABC , c) a decrease in activity of a drug-activating enzyme such as P-450, and d) an increase in DNA repair activity (topoisomerase).

In the present invention, the term "overcoming tolerance" means an action of increasing sensitivity to a drug for treatment of a cancer cell that has acquired resistance to a specific drug. For the purpose of the present invention, the specific drug means a drug for treating cancer, in particular, an anti-cancer agent. The increase in the sensitivity is more likely to be achieved when the concentrations exhibiting the effects of inhibiting the growth of cancer cells and acquiring resistance to cell death are equal to or higher than those exhibiting effects such as growth inhibition and the like for cancer cells having no resistance . As a synonym for overcoming resistance, there are "tolerance inhibition "," resistance release ","resistance release ",and" sensitivity enhancement ".

The pharmaceutical composition of the present invention can be administered in combination with other anticancer drugs, and the therapeutic effect of cancer stem cells as well as cancer cells can be further improved.

Examples of the anticancer agent include nitrogene mustard, imatinib, oxaliplatin, rituximab, elotinib, neratinib, lapatinib, zetitib, bandetanib, nilotinib, semathanib, conservinib, acitinib, Corticosteroids, cisplatin, cetuximab, bismuth alum, asparaginase, tretinoin, hydroxycarbamides, corticosteroids, rheumatoid arthritis, rheumatoid arthritis, rheumatoid arthritis, Amlodipine, amitriptyline, amitriptyline, amitriptyline, amlodipine, amlodipine, amlodipine, amlodipine, amlodipine, amlodipine, But are not limited to, but are not limited to, tamavir, tamifluuridine, femetrexed, tegafur, capecitabine, gimeracin, oteracil, azacytidine, methotrexate, uracil, cytarabine, fluorouracil, Tamide, decitabine, Wherein the compound is selected from the group consisting of mercaptopurine, thioguanine, cladribine, calmopar, ralitriptycde, docetaxel, paclitaxel, irinotecan, velotecan, topotecan, vinorelbine, etoposide, vincristine, vinblastine, teniposide, doxorubicin, But are not limited to, rubicin, epirubicin, mitoxantrone, mitomycin, blaromycin, daunorubicin, dactinomycin, pyrabicin, aclarubicin, pepromycin, temsirolimus, temozolomide, But are not limited to, corticosteroids, corticosteroids, cyclophosphamide, cyclophosphamide, melphalan, altretamine, dacarbazine, thiotepa, nimustine, chlorambucil, mitolactol, leucovorin, At least one selected from the group consisting of anagrelide, navelbine, pradrazol, tamoxifen, toremifene, testolactone, anastrozole, letrozole, borozol, bicalutamide, However, But is not limited to.

In the present invention, the pharmaceutical composition may be in the form of a capsule, a tablet, a granule, an injection, an ointment, a powder or a drink, and the pharmaceutical composition may be a human.

The pharmaceutical composition of the present invention may be formulated in the form of oral preparations such as powders, granules, capsules, tablets, aqueous suspensions, etc., external preparations, suppositories and sterilized injection solutions according to a conventional method, . The pharmaceutical composition of the present invention may include a pharmaceutically acceptable carrier. The pharmaceutically acceptable carrier may be a binder, a lubricant, a disintegrant, an excipient, a solubilizing agent, a dispersing agent, a stabilizer, a suspending agent, a coloring matter, a perfume or the like in the case of oral administration. A solubilizing agent, an isotonic agent, a stabilizer and the like may be mixed and used. In the case of topical administration, a base, an excipient, a lubricant, a preservative and the like may be used. Formulations of the pharmaceutical compositions of the present invention may be prepared in a variety of ways by mixing with pharmaceutically acceptable carriers as described above. For example, oral administration may be in the form of tablets, troches, capsules, elixirs, suspensions, syrups, wafers, etc. In the case of injections, they may be formulated in unit dosage ampoules or in multiple dosage forms have. Other, solutions, suspensions, tablets, capsules, sustained-release preparations and the like.

Examples of suitable carriers, excipients and diluents for formulation include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltoditol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, Cellulose, methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate or mineral oil. Further, it may further include a filler, an anticoagulant, a lubricant, a wetting agent, a flavoring agent, an emulsifying agent, an antiseptic, and the like.

The route of administration of the pharmaceutical composition according to the present invention may be, but is not limited to, oral, intravenous, intramuscular, intraarterial, intramedullary, intrathecal, intracardiac, transdermal, subcutaneous, intraperitoneal, intranasal, , Sublingual or rectal. Oral or parenteral administration is preferred.

In the present invention, "parenteral" includes subcutaneous, intradermal, intravenous, intramuscular, intraarticular, intrasynovial, intrasternal, intrathecal, intralesional and intracranial injection or infusion techniques. The pharmaceutical compositions of the present invention may also be administered in the form of suppositories for rectal administration.

The pharmaceutical composition of the present invention may be administered orally or parenterally depending on various factors including the activity of the specific compound used, age, weight, general health, sex, diet, time of administration, route of administration, rate of excretion, drug combination, And the dose of the pharmaceutical composition may be appropriately selected by a person skilled in the art depending on the condition of the patient, the body weight, the degree of disease, the mode of administration, the route of administration and the period of time, and is preferably from 0.0001 to 50 mg / kg or 0.001 to 50 mg / kg. The administration may be carried out once a day or divided into several times. The dose is not intended to limit the scope of the invention in any way. The pharmaceutical composition according to the present invention can be formulated into pills, dragees, capsules, solutions, gels, syrups, slurries, suspensions.

The pharmaceutical composition according to the present invention effectively inhibits the proliferation of cancer cells or cancer stem cells by using a histone deacetylation inhibitor and a kinase inhibitor in combination to prevent or treat cancer, It is also possible to prevent the transition or recurrence of the disease.

FIG. 1 (a) is a graph showing changes in SNU-80 cell survival rate according to drug treatment concentration in Experimental Example 1. FIG.
FIG. 1 (b) is a graph showing changes in cell survival rate of 8505C according to drug treatment concentration in Experimental Example 1. FIG.
FIG. 1 (c) is a graph showing changes in KSA1 cell survival rate according to drug treatment concentration in Experimental Example 1. FIG.
FIG. 2 (a) is a graph showing changes in the number of SNU-80 cells per starter according to the drug treatment time in Experimental Example 2. FIG.
FIG. 2 (b) is a graph showing changes in the number of star 8505C cells according to drug treatment time in Experimental Example 2. FIG.
FIG. 2 (c) is a graph showing changes in the number of KSA1 cells per treatment time according to the treatment time in Experimental Example 2. FIG.
FIG. 3 (a) is a graph showing changes in tumor volume with time after treatment of each drug with SNU-80 cells in Experimental Example 3. FIG.
FIG. 3 (b) is a graph showing changes in tumor volume over time after inoculation of 8505C cells in Experimental Example 3 and treatment of each drug.
FIG. 3 (c) is a graph showing changes in tumor volume over time after inoculation of KSA1 cells in Experimental Example 3 and treatment of each drug.
FIG. 4 (a) is a graph showing tumor weights measured after inoculation of SNU-80 cells in Experimental Example 3 and 40 days after treatment of each drug.
FIG. 4 (b) is a graph showing the weight of the tumor after 40 days from the inoculation of 8505C cells in Experimental Example 3 and treatment of each drug.
4 (c) is a graph showing the weights of the tumors measured after inoculation of KSA1 cells in Experimental Example 3 and 40 days after the treatment of each drug.
FIG. 5 (a) is a graph showing changes in the body weight of mice after inoculation with SNU-80 cells in Experimental Example 4 and time after treatment with each drug.
FIG. 5 (b) is a graph showing changes in the weight of mice after inoculation of 8505C cells in Experimental Example 4 with time after treatment of each drug.
FIG. 5 (c) is a graph showing changes in the body weight of mice after inoculation with KSA1 cells in Experimental Example 4 and time after treatment with each drug.
6 (a) shows the results of immunohistochemical staining of tumor sections after inoculation with SNU-80 cells in Experimental Example 5 and treatment of each drug.
FIG. 6 (b) is a graph showing the immunostaining intensity of tumor sections after inoculation of SNU-80 cells and treatment of each drug in Experimental Example 5. FIG.
Fig. 7 (a) shows the results of immunohistochemical staining of tumor sections after inoculation of 8505C cells in Experimental Example 5 and treatment of each drug.
FIG. 7 (b) is a graph showing the immunohistochemical staining intensity of tumor sections after inoculation of 8505C cells in Experimental Example 5 and treatment of each drug.
8 (a) shows the results of immunostaining of tumor sections after inoculation of KSA1 cells in Experimental Example 5 and treatment of each drug.
FIG. 8 (b) is a graph showing the immunostaining intensity of tumor sections after inoculation of KSA1 cells and treatment of each drug in Experimental Example 5. FIG.

Hereinafter, the present invention will be described in more detail with reference to Examples. It will be apparent to those skilled in the art that these embodiments are only for describing the present invention in more detail and that the scope of the present invention is not limited by these embodiments in accordance with the gist of the present invention .

Example

[Preparation Example 1] Preparation of KSA1 cells

Tumor tissues were obtained from patients diagnosed biochemically and histologically as anaplastic thyroid cancer (ATC) at Yonsei University College of Medicine, Gangnam Severance Hospital (Seoul, Korea). Specifically, a tumor mass was obtained by surgical resection of the tumor from the initial position and the metastatic position of the ATC, and then stored in saline containing antifungal agent and antibiotic after 1 day. Normal tissue and fat were then removed from the tumor mass, and only the tumor tissue was washed with 1X HBSS. Tumor tissues were milled in dissociation medium. At this time, DMEM / F12 supplemented with 20% FBS supplemented with 1 mg / ml collagenase type IV (Sigma, USA) was used as the dissociation medium. The tumor cells suspended and suspended were suspended in 50 ml 1X HBSS And then centrifuged at 1,400 rpm for 5 minutes. Then, the cells were centrifuged at 4,000 rpm for 5 minutes. Then, the obtained cells were suspended in RPMI-1640 (Hyclone, South Logan, UT, USA) supplemented with 10% fetal bovine serum (Hyclone) and 2% penicillin / streptomycin solution (Gibco, Grand Island, NY, USA) Lt; / RTI >

[Preparation Example 2] Preparation of SNU-80 and 8505C cells

Patient-derived malignant thyroid tumor cell lines SNU-80 and 8505C were purchased from Korean Cell Line Bank (Seoul National University, Seoul, Korea) and ATCC (American Type Culture Colleciton). They were cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum.

[Experimental Example 1] Evaluation of cell viability (Cell viability assay)

Changes in cell viability by treatment with HNHA and sorapenib were measured using MTT assay. Specifically, SNU-80, 8505C and KSA1 cells were each plated in a 96-well plate at 5 × 10 ³ cells per well and then cultured overnight at 70% confluency. A 1: 1 concentration ratio mixture of HNHA, sorafenib, HNHA and sorapenib and a 1: 1 concentration ratio mixture of SAHA and sorapenib as a positive control were treated at a concentration of 0-100 μM, and no treatment was performed as a negative control. After 48 hours from the treatment, MTT was added to further cultivate the cells. Then, the absorbance at 490 nm was measured, and the cell survival rate of each cell line was shown in FIG. 1 (a) to (c). In addition, the cell sensitivity to the drug treatment is represented by the drug concentration (IC ₅₀ ) at which 50% of the cell inhibition is exhibited, and is shown in Table 1 below. All experiments were performed three times. Data are expressed as a percentage of the signal observed in the vehicle-treated cell, expressed as the mean ± standard deviation of the mean (SEM) value of the triplicate experiments.

Cell line Histopathology
(Histopathology) origin Cell sensitivity IC ₅₀ ([mu] M) HNHA + Sorafanib HNHA Sorapanib SNU-80 Undifferentiated thyroid cancer human 0.87 (+/- 1.1) 2.28 (+/- 0.4) 5.14 (+/- 1.1) 8505C Undifferentiated thyroid cancer human 3.82 (+/- 0.5) 17.42 (+/- 0.6) 10.21 (+/- 0.9) KSA1 Undifferentiated thyroid cancer human 8.72 (+/- 0.5) 20.14 (+/- 0.5) 23.45 (+/- 0.6)

As shown in the above Table 1, IC ₅₀ is remarkably decreased when HNHA and sorapenib are used alone, and 50% of the cell inhibitory effect can be confirmed even at very low concentrations.

As shown in FIG. 1, SNU-80 and 8505C cells (Endocrine Journal 2014, 61 (5), 481-490), which are known as thyroid cancer stem cells and resistant to anticancer agents, and KSA1 cells derived from ATC patients Compared with the case of using HNHA and sorafenib alone, the survival rate of HNHA and sorafenib was significantly decreased in proportion to the concentration, and the cell survival rate was significantly lower than that of the combination of SAHA and sorafenib Can be seen.

[Experimental Example 2] Evaluation of cell proliferation

Changes in cell proliferative capacity following treatment with HNHA and sorapenib were measured using the trypan blue dye exclusion method. Specifically, SNU-80, 8505C and KSA1 cells were each divided into 6-well plates and then a mixture of HNHA, sorapenib, HNHA and sorapenib, and a mixture of SAHA and sorapenib were treated at the concentrations shown in Table 2 below . Each cell was incubated for 24 hours, and the medium was removed. After 0.04% trypan blue was added, the cells were cultured for 7 days, and the number of viable cells was measured daily using a heamocytometer, The results are shown in Figs. 2 (a) to 2 (c).

division HNHA (μM) Sorapanib (μM) SAHA (μM) Comparative Example 1 - - - Comparative Example 2 2.28 - - Comparative Example 3 - 5.14 - Example 1 0.87 0.87 - Comparative Example 4 - 1.98 1.98

As shown in FIG. 2, HNHA and sorapenib were found to be more effective against both SNU-80 and 8505C cells, which are resistant to anticancer agents, and KSA1 cells derived from ATC patients, It can be seen that the effect of inhibiting cell proliferation is remarkably excellent when phenob is used together with the cell proliferation effect is superior to that of the combination of SAHA and sorafenib.

[Experimental Example 3] Animal transplantation model of human thyroid cancer cell

SNU-80, 8505C and KSA1 cells were cultured in in-vitro, and cells cultured subcutaneously in the upper left flank of BALB / c nude female mice were injected at 2.0 × 10 ⁷ cells / mouse. Seven days later, groups of 10 animals were grouped and then treated with a mixture of 25 mg / kg HNHA, 80 mg / kg sorapenib, 6.5 mg / kg HNHA and 25 mg / kg sorafenib, 6.5 mg / kg SAHA and 25 mg / kg sorafenib The mixture was injected once every two days and injected 10 to 12 times in total. The volume of the tumor was measured daily for 40 days using calipers. Here, the volume of the tumor was evaluated using the following formula. After 40 days, the mouse was dissected and the tumor was weighed. The experiments were performed under specific sterile conditions (SPF). FIG. 3 shows changes in tumor volume by each cell line, and FIG. 4 shows the weight of tumors measured after 40 days.

The volume of tumor = LXS ^2/2

(Where L is the longest diameter and S is the shortest diameter)

As shown in FIGS. 3 and 4, HNHA and sorapenib were found to be more effective in treating SNU-80 and 8505C cells, which are resistant to anticancer agents, and KSA1 cells derived from ATC patients, And sorafenib, the tumor growth inhibitory effect is remarkably excellent, and the tumor growth inhibitory effect is superior to that of the combination of SAHA and sorafenib.

[Experimental Example 4] In-vivo toxicity assay [

SNU-80, 8505C and KSA1 cells were cultured in in-vitro, and cells cultured subcutaneously in the upper left flank of BALB / c nude female mice were injected at 2.0 × 10 ⁷ cells / mouse. Seven days later, groups of 10 animals were grouped and then treated with a mixture of 25 mg / kg HNHA, 80 mg / kg sorapenib, 6.5 mg / kg HNHA and 25 mg / kg sorafenib, 6.5 mg / kg SAHA and 25 mg / kg sorafenib The mixture was administered intraperitoneally or orally. Mice were monitored periodically to identify toxic or lethal external signals. Five mice were added to each cage and the light was adjusted at 22 ° C and 40-60% humidity for 12 hours. The changes in the body weight of the mice for 40 days were measured and the results were shown graphically in FIG. 5 (a) to (c) for each cell line injected.

Generally, when a side effect of a drug occurs, the body weight changes largely due to hepatotoxicity. However, as shown in Figs. 5 (a) to 5 (c), when the combination of HNHA and sorafenib is used, .

[Experimental Example 5] Immunohistochemistry < RTI ID = 0.0 >

After 40 days in Experimental Example 3, the tumor was dissected and the obtained tumor tissue was fixed in 10% neutral-buffered formalin and fixed in paraffin wax. After removal of the wax from tissue sections of 5 mu m thickness, antigen-retrieval was performed in citrate buffer for 5 minutes at 120 DEG C using an electric pressure cooker set. Tissue sections were incubated in 3% hydrogen peroxide for 5 minutes to quench the peroxidase. All tissue sections were contrast dyed with hematoxylin and then water was removed. Photographs and staining intensity after immunostaining for each injected cell are shown in FIGS. 6 to 8, respectively.

6 (a), 7 (a) and 8 (a), the brown dyed portion is the survival protein Bcl-2. When the drug is administered, In particular, the combination of HNHA and sorafenib showed significantly less brown areas than when they were administered alone or in combination with SAHA and sorafenib.

6 (b), 7 (b), and 8 (b) show that the combination of HNHA and sorafenib results in a higher dyeing intensity than when sorafenib is administered alone or when SAHA is combined with sorapanib It can be seen that it is remarkably low.

Statistical analysis was performed using GraphPad Prism software (GraphPad Software Inc., La Jolla, CA, USA). Immunohistochemical results were obtained by one-way ANOVA and Bonferroni post hoc tests. The values were expressed as mean ± SEM, and when the P value was less than 0.05, it was judged to be a significant result.

While the present invention has been particularly shown and described with reference to exemplary embodiments thereof, it is to be understood that the invention is not limited to the disclosed exemplary embodiments, but, on the contrary, It will be obvious to those of ordinary skill in the art.

Claims (11)

A histone deacetylation inhibitor (HDAC inhibitor) and a kinase inhibitor as an active ingredient,
Wherein the histone deacetylation inhibitor is a compound represented by the following formula 1 and the kinase inhibitor is a compound represented by the following formula 2:
[Chemical Formula 1]

(2)

delete delete The method according to claim 1,
Wherein the histone deacetylation inhibitor and the kinase inhibitor are contained in a weight ratio of 1: 1 to 100. delete A histone deacetylation inhibitor (HDAC inhibitor) and a kinase inhibitor as an active ingredient,
Wherein the histone deacetylation inhibitor is a compound represented by the following formula 1 and the kinase inhibitor is a compound represented by the following formula 2:
[Chemical Formula 1]

(2)

delete delete The method according to claim 6,
Wherein the histone deacetylation inhibitor and the kinase inhibitor are contained in a weight ratio of 1: 1 to 100. delete A histone deacetylation inhibitor (HDAC inhibitor) and a kinase inhibitor as an active ingredient,
Wherein the histone deacetylation inhibitor is a compound represented by the following formula 1 and the kinase inhibitor is a compound represented by the following formula 2:
[Chemical Formula 1]

(2)

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