KR101916283B1 - Pharmaceutical composition for enhancing the radiotherapy of cancer - Google Patents

Abstract

The present invention relates to a pharmaceutical composition capable of enhancing the radiotherapeutic effect on cancer, and more particularly, to a pharmaceutical composition comprising a histone deacetylation inhibitor (HDAC inhibitor) and a kinase inhibitor as an active ingredient And to a pharmaceutical composition for promoting radiation therapy for cancer.

Description

[0001] The present invention relates to a pharmaceutical composition for enhancing the radiotherapy of cancer,

The present invention relates to a pharmaceutical composition capable of enhancing the radiation treatment effect on cancer.

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.

Currently, 35% of the cancer patients in Korea and 50% in the US are receiving radiation therapy, and the proportion of cancer patients receiving radiation therapy is steadily increasing in Korea, and the importance of radiation therapy in cancer treatment is also increasing. Radiation therapy is currently considered to be an indispensable treatment for various types of cancer, but the radiation resistance of cancer cells and the presence of hypoxic cells are important factors that cause local failure in conventional cancer radiation therapy (Int. J. Radiation oncology Biol. Phys. , 1988, 14, 831-833). In addition, there has been a problem in that a side effect of radiation therapy such as destruction of normal tissue in human body appears in high doses of radiation treatment with increased radiation dose due to the failure of such radiotherapy.

Therefore, studies on radiation therapy enhancers and radiation sensitizers that can increase the efficiency of radiation therapy have been continuing (Oncogene, 2004, 23, 1599-1607, Int. J. Radiation Oncology Biol. Phys. 2006, 64 (5), 1466-1474; Clin, Cancer Res, 2007,13 (16), 4891-4899).

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.

It is an object of the present invention to provide a pharmaceutical composition capable of enhancing the radiation treatment effect on cancer.

Another object of the present invention is to provide a method of radiotherapy of cancer which can enhance the radiation treatment effect by administering the composition as a radiation therapy enhancer.

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 promoting radiation therapy for 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)

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.

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 in combination with radiation therapy for cancer by synergistic effect, The growth of cancer stem cells known to cause recurrence of cancer cells can also be effectively inhibited and ultimately prevent or treat cancer, and further, can prevent resistance to an anticancer drug, cancer metastasis, or cancer recurrence.

The type of cancer to be treated in the present invention is not particularly limited, and examples thereof include cancer of the thyroid, breast cancer, bile duct cancer, gallbladder cancer, pancreatic cancer, colon cancer, uterine cancer, esophageal cancer, gastric cancer, Renal cancer, ovarian cancer, prostate cancer, uterine cancer, head and neck cancer, skin cancer, blood cancer, and liver cancer, and preferably may be a thyroid cancer.

In the present invention, "treatment" may include, without limitation, any act of irradiating radiation to improve or ameliorate the symptoms of 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.

The pharmaceutical composition of the present invention can be administered in combination with other anticancer agents, thereby further enhancing the effect of radiation therapy on cancer.

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, calmophor, 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.

According to another embodiment of the present invention, there is provided a pharmaceutical composition for enhancing radiation therapy according to the present invention; And irradiating the cancer with radiation.

The radiation therapy after the administration of the pharmaceutical composition for enhancing radiation therapy provided in the present invention can significantly enhance the effect of radiation therapy according to the synergy effect when irradiated with radiation. Specifically, the radiation therapy effect can be improved not only in cancer cells, It is possible to effectively inhibit the growth of cancer stem cells known to cause recurrence and ultimately to prevent or treat cancer, and further to prevent resistance to an anticancer drug, cancer metastasis, or cancer recurrence.

In addition, in the present invention, the animal to be subjected to radiation therapy may be any mammal including humans, livestock, and pets, but is not limited thereto.

In the present invention, the above-mentioned radiation irradiation may be applied to any radiation method used for radiation therapy of a conventional cancer, or to a later developed radiation method for a cancer.

The type of cancer to be treated in the present invention is not particularly limited, and examples thereof include cancer of the thyroid, breast cancer, bile duct cancer, gallbladder cancer, pancreatic cancer, colon cancer, uterine cancer, esophageal cancer, gastric cancer, Renal cancer, ovarian cancer, prostate cancer, uterine cancer, head and neck cancer, skin cancer, blood cancer, and liver cancer, and preferably may be a thyroid cancer.

When the pharmaceutical composition according to the present invention is administered and then treated with radiation, the effect of radiation therapy is remarkably enhanced, and the growth of cancer stem cells, which is known to cause resistance to cancer drugs and cancer recurrence, And can finally prevent or treat cancer, and further, can prevent resistance to an anticancer drug, cancer metastasis, or cancer recurrence.

FIG. 1 (a) is a graph showing changes in cell survival rate of 8505C according to drug treatment concentration in Experimental Example 1. FIG.
FIG. 1 (b) is a graph showing changes in KSA1 cell survival rate according to drug treatment concentration in Experimental Example 1. FIG.
2 (a) is a graph showing changes in the number of star 8505C cells according to drug treatment time in Experimental Example 2. FIG.
FIG. 2 (b) 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 8505C cells in Experimental Example 3. FIG.
FIG. 3 (b) 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 the weight of tumor measured after inoculation of 8505C cells in Experimental Example 3 and 40 days after treatment of each drug.
FIG. 4 (b) 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 of 8505C cells in Experimental Example 4 and time after treatment of each drug. FIG.
FIG. 5 (b) is a graph showing changes in the weight of the mice according to time after inoculation of KSA1 cells and treatment of each drug in Experimental Example 4. 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 who were biochemically and histologically diagnosed with 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 8505C cells

Patient-derived malignant thyroid tumor cell line 8505C was purchased from ATCC (American Type Culture Colleciton) and cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum.

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

HNHA and sorafenib treatment, the changes in cell viability were measured using MTT assay. Specifically, 8505C and KSA1 cells were each plated on a 96-well plate at 5 × 10 ³ cells per well, and then cultured overnight at 70% confluency. A 1: 1 concentration mixture of HNHA, sorafenib, and HNHA and sorapenib was treated at a concentration of 0 to 64 μM, and no treatment was done as a negative control. Cells were then irradiated with Faxitron X-rays (Faxitro Bioptics, AZ, USA). After 48 hours from irradiation, MTT was added to the cells, followed by further incubation. After that, the absorbance at 490 nm was measured and the cell survival rate of each cell line was shown in FIG. 1 (a) and (b). 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 Sorapanib HNHA + RT Sorapenip + RT HNHA + sorafenib + RT 8505C Undifferentiated thyroid cancer human 16.19
(± 0.6) 10.17
(± 0.6) 6.71
(± 0.1) 7.12
(± 0.9) 2.74
(± 0.6) KSA1 Undifferentiated thyroid cancer human 18.16
(± 0.4) 12.14
(± 0.5) 8.14
(± 1.3) 10.33
(± 0.7) 4.08
(± 0.2)

As shown in Table 1, when irradiated with HNHA and sorafenib, irradiation was more effective than IC _{50 in the} case where only HNHA or sorapanib was treated alone, or each of them was irradiated with radiation, And the cell inhibition effect of 50% can be confirmed even at a very low concentration.

In addition, as shown in FIG. 1, HNHA or HNHA was detected in 8505C cells (Endocrine Journal 2014, 61 (5), 481-490), which are known as thyroid cancer stem cells and resistant to anticancer drugs, and KSA1 cells derived from ATC patients The survival rate of HNHA and sorafenib in combination with sorafenib was significantly lower than that in the case of sorafenib treated with radiation alone.

[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, each of the 8505C and KSA1 cells was dispensed into a 6-well plate, and a mixture of HNHA, sorapenib, and HNHA and sorapenib was treated at the concentrations shown in the following Table 2 and transferred to a Faxitron X-ray (Faxitro Bioptics, AZ , USA). After incubation for 24 hours, the medium was removed, and 0.04% trypan blue was added. The cells were further cultured for 7 days, and the number of living cells was measured daily using a heamocytometer, The results are shown in Figs. 2 (a) and 2 (b).

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

As shown in FIG. 2, in 8505C cells, which are known as thyroid cancer stem cells, and 8505C cells that are resistant to anticancer drugs and KSA1 cells derived from ATC patients, only radiation alone, HNHA or sorapenib alone, In comparison with the case of irradiation, the effect of irradiation with HNHA and sorafenib in combination with radiation was remarkably superior to the inhibition of cell proliferation.

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

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 were grouped, and a mixture of 25 mg / kg HNHA, 80 mg / kg sorapenib, and 6.5 mg / kg HNHA and 25 mg / kg sorapenib was injected every other day for 10 to 12 Respectively. After that, the Small Animal Radiation Research Platform (SARRP) [High-resolution, small animal radiation research platform with x-ray tomographic guidance capabilities / PMID; 18640502]. In other words, a circular beam with a diameter of 1 cm was irradiated to the tumor site with three consecutive daily doses of 3Gy. The volume of the tumor was measured daily using calipers for 40 days. 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 FIG. 3 and FIG. 4, compared with the case where only radiation was irradiated in both 8505C and KSA1 cells, or when HNHA and sorafenib were separately treated and then irradiated, the combination of HNHA and sorafenib was irradiated The tumor growth inhibitory effect is remarkably excellent.

[Experimental Example 4] In-vivo toxicity assay [

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 a mixture of 25 mg / kg HNHA, 80 mg / kg sorafenib, and 6.5 mg / kg HNHA and 25 mg / kg sorapenib was orally orally administered to each group. Small Animal Radiation Research Platform (SARRP) [High-resolution, small animal radiation research platform with x-ray tomographic guidance capabilities / PMID; 18640502]. In other words, a circular beam with a diameter of 1 cm was irradiated to the tumor site with three consecutive daily doses of 3Gy. 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 were measured for 40 days, and the results were shown graphically in FIG. 5 (a) and (b) for each cell line injected.

Generally, weight change due to hepatotoxicity occurs largely when a side effect of a drug occurs. However, as shown in FIGS. 5 (a) to 5 (b), the change in body weight is considerable even when radiation is irradiated after combination treatment with HNHA and sorafenib It can be seen that there is no side effect.

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 (12)

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:
[Chemical Formula 1]

delete The method according to claim 1,
Wherein the kinase inhibitor is a compound represented by the following formula 2:
(2)

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. The method according to claim 1,
Wherein said cancer comprises cancer stem cells. The method according to claim 1,
The cancer is selected from the group consisting of thyroid cancer, breast cancer, biliary cancer, gallbladder cancer, pancreatic cancer, colon cancer, uterine cancer, esophageal cancer, stomach cancer, brain cancer, rectal cancer, lung cancer, bladder cancer, kidney cancer, ovarian cancer, prostate cancer, Liver cancer, and liver cancer. The method according to claim 6,
Wherein said cancer is thyroid cancer. Administering a pharmaceutical composition for enhancing radiation therapy to cancer comprising as an active ingredient a histone deacetylation inhibitor (HDAC inhibitor) and a kinase inhibitor to an animal other than a human; And
Irradiating the substrate with radiation,
Wherein the histone deacetylation inhibitor is a compound represented by the following formula 1:
[Chemical Formula 1]

delete 9. The method of claim 8,
Wherein the kinase inhibitor is a compound represented by the following formula 2:
(2)

9. The method of claim 8,
Wherein said cancer comprises cancer stem cells. 9. The method of claim 8,
The cancer is selected from the group consisting of thyroid cancer, breast cancer, biliary cancer, gallbladder cancer, pancreatic cancer, colon cancer, uterine cancer, esophageal cancer, stomach cancer, brain cancer, rectal cancer, lung cancer, bladder cancer, kidney cancer, ovarian cancer, prostate cancer, Hepatocellular carcinoma, and liver cancer.

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