COMPOSITION FOR PREVENTING, ALLEVIATING OR TREATING CANCER

Abstract

A compound represented by Formula 1 according to the present disclosure, when used in combination with an anticancer drug, may significantly improve the anticancer effect of the anticancer drug, and may induce the same anticancer effect even when the anticancer drug is used in a significantly smaller amount than the conventionally used amount, thereby reducing the side effects caused by administration of the anticancer drug. Furthermore, the compound represented by Formula 1 makes it possible to effectively prevent, alleviate or treat either anticancer-resistant cancer or cancer which recurs or metastasizes after anticancer drug treatment.

Claims

1. A method for treating a subject having cancer or for enhancing sensitivity of a subject to an anticancer drug or for overcoming or inhibiting anticancer drug resistance of a subject, or for preventing cancer in a subject, comprising providing a pharmaceutical composition containing, as an active ingredient, hydroflumethiazide represented by the following Formula 1 or a pharmaceutically acceptable salt thereof, in a carrier, ##STR00002## wherein the cancer is metastatic cancer selected from the group consisting of colorectal cancer, breast cancer, uterine cancer, fallopian tube cancer, ovarian cancer, stomach cancer, brain cancer, rectal cancer, small intestine cancer, esophageal cancer, lymph gland cancer, gallbladder cancer, lung cancer, skin cancer, kidney cancer, bladder cancer, blood cancer, pancreatic cancer, prostate cancer, thyroid cancer, endocrine adenocarcinoma, oral cancer, and liver cancer, and wherein the cancer is resistant to at least one anticancer drug selected from the group consisting of nitrogen mustard, imatinib, rituximab, panitumumab, erlotinib, neratinib, lapatinib, gefitinib, vandetanib, nirotinib, semaxanib, bosutinib, axitinib, cediranib, lestaurtinib, sorafenib, lenvatinib, trastuzumab, gefitinib, bortezomib, sunitinib, carboplatin, 5-fluorouracil (5-FU), bevacizumab, cisplatin, cetuximab, aflibercept, regorafenib, viscumalbum, asparaginase, tretinoin, hydroxycarbamide, dasatinib, estramustine, gemtuzumab ozogamicin, ibritumomab tiuxetan, heptaplatin, methylaminolevulinic acid, amsacrine, alemtuzumab, procarbazine, alprostadil, holmium nitrate chitosan, gemcitabine, doxifluridine, pemetrexed, tegafur, capecitabine, gimeracil, oteracil, azacitidine, methotrexate, uracil, cytarabine, fluorouracil, fludarabine, enocitabine, flutamide, decitabine, mercaptopurine, thioguanine, cladribine, leucovorine, carmofur, raltitrexed, interferon alpha-2a, docetaxel, paclitaxel, irinotecan, belotecan, topotecan, vinorelbine, etoposide, vincristine, vinblastine, teniposide, doxorubicin, idarubicin, epirubicin, mitoxantrone, mitomycin, bleomycin, daunorubicin, dactinomycin, pirarubicin, aclarubicin, pepromycin, temsirolimus, temozolomide, busulfan, ifosfamide, cyclophosphamide, melphalan, altretamine, dacabazine, thiotepa, nimustine, chlorambucil, mitolactol, leucovorin, tretonin, exemestane, aminoglutethimide, anagrelide, navelbine, fadrazole, tamoxifen, toremifene, testolactone, anastrozole, letrozole, vorozole, bicalutamide, lomustine, and carmustine, and administering an effective amount of the pharmaceutical composition to the individual.

2. The method of claim 1, wherein the anticancer drug is at least one selected from the group consisting of cetuximab, panitumumab, irinotecan, vinorelbine, capecitabine, leucovorin, cisplatin, carboplatin, sorafenib, 5-fluorouracil (5-FU), bevacizumab, aflibercept, and regorafenib.

3. The method of claim 1, wherein the cancer is any one or more cancers selected from the group consisting of colorectal cancer, breast cancer, uterine cancer, fallopian tube cancer, ovarian cancer, stomach cancer, brain cancer, rectal cancer, small intestine cancer, esophageal cancer, lymph gland cancer, gallbladder cancer, lung cancer, skin cancer, kidney cancer, bladder cancer, blood cancer, pancreatic cancer, prostate cancer, thyroid cancer, endocrine adenocarcinoma, oral cancer, and liver cancer.

4. The method of claim 1, wherein the active ingredient is administered in a dosage amount of 0.0001 to 50 mg/kg/day or 0.001 to 50 mg/kg/day.

5. The method of claim 1, wherein the pharmaceutical composition is administered once to several times per day.

6. The method of claim 1, wherein the active ingredient and anticancer drug is administered at a ratio of 1:0.001 to 1:1,000.

7. The method of claim 1, wherein the active ingredient is administered as a solid formulation, a liquid formulation or an aerosol formulation for inhalation thereof.

8. The method of claim 1, wherein the composition is administered to a subject via oral administration, intravenous administration, intramuscular administration, intra-arterial administration, intramedullary administration, intradural administration, intracardiac administration, transdermal administration, subcutaneous administration, intraperitoneal administration, intranasal administration, gastrointestinal administration, topical administration, sublingual administration or intrarectal administration routes.

9. The method of claim 1, wherein the active ingredient and anticancer drug is administered at a ratio of 1:0.01 to 1:100.

10. The method of claim 1, wherein the active ingredient and anticancer drug is administered at a ratio of 1:0.1 to 1:10.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0078] The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

[0079] FIG. 1 shows the results of microarray analysis performed to analyze gene expression changes in YUMC-C1, YUMC-H2 and YUMC-P1 cell lines according to one example of the present disclosure.

[0080] FIG. 2 graphically shows the results of measuring the changes in cell viability after treating YUMC-C1, YUMC-H2 and YUMC-P1 cell lines with drugs according to one example of the present disclosure.

[0081] FIG. 3 graphically shows the results of measuring the changes in tumor size after administering drugs to mouse models xenografted with YUMC-C1, YUMC-H2 and YUMC-P1 cell lines, respectively, according to one example of the present disclosure.

[0082] FIG. 4 graphically shows the results of measuring the changes in net tumor weight after administering drugs to mouse models xenografted with YUMC-C1, YUMC-H2 and YUMC-P1 cell lines, respectively, according to one example of the present disclosure.

[0083] FIG. 5 graphically shows the results of measuring the changes in mouse body weight after administering drugs to mouse models xenografted with YUMC-C1, YUMC-H2 and YUMC-P1 cell lines, respectively, according to one example of the present disclosure.

DETAILED DESCRIPTION

[0084] Hereinafter, the present disclosure will be described in detail with reference to the following examples. However, the following examples are only illustrative of the present disclosure, and the scope of the present disclosure is not limited by the following examples.

Examples

[Example 1] Isolation of Cancer Cells from Cancer Patients Who have Relapsed after Anticancer Drug Treatment

[0085] Cancer cells were isolated from patients with colorectal cancer, liver cancer, and thyroid cancer as described in Table 1 below, who have relapsed after anticancer drug treatment.

TABLE-US-00001 TABLE 1 Patient 2 Patient 3 Patient 1 (YUMC- (YUMC- (YUMC-P1 C1 cell line) H2 cell line) cell line) Age 71 71 57 Sex Female Male Male Location of primary Colon Liver Thyroid disease Stage IVc IVc IVc Primary pathology Colorectal cancer Liver cancer Thyroid (recurred and (recurred after papillary metastasized after sorafenib) cancer FOLFOX*) (recurred after sorafenib) Classification of Fresh tumor Fresh tumor Fresh tumor samples used in culture Source Severance Hospital Severance Severance (Seoul, Korea) Hospital Hospital (Seoul, Korea) (Seoul, Korea) *FOLFOX: Triple co-administration of Folinic acid, 5-Fluorouracil (5-FU) and Oxaliplatin in colorectal cancer

[Example 2] Analysis of Gene Expression by Microarray

[0086] Using the microarray analysis method, gene expression was compared between the YUMC-C1 cell line, YUMC-H2 cell line and YUMC-P1 cell line of Example 1, and a control group which is the thyroid cancer cell line (YUMC-M1) isolated from a thyroid cancer patient who had successfully undergone anticancer therapy by administration of an anticancer drug for standard anticancer therapy.

[0087] Specifically, RNA purity and integrity were measured using an ND-1000 spectrophotometer (NanoDrop) and an Agilent 2100 Bioanalyzer (Agilent Technologies). RNA labeling and hybridization was performed using an Agilent One-Color Microarray-Based Gene Expression Analysis protocol (Agilent Technology, V 6.5, 2010). 100 ng of total RNA from each sample was amplified linearly and labeled with Cy3-dCTP, and the labeled cRNA was purified using an RNeasy Mini Kit (Qiagen). The concentration and specific activity of the labeled cRNA (pmol Cy3/g cRNA) were measured using NanoDrop ND-1000. Then, 600 ng of each labeled cRNA was fragmented by adding 5 l of 10 blocking agent and 1 l of 25 fragmentation buffer, followed by heating at 60 C. for 30 minutes. Finally, 25 l of 2GE hybridization buffer was added to dilute the labeled cRNA. 40 l of hybridization solution was dispensed into a gasket slide and assembled to the Agilent SurePrint G3 Human GE 860K V3 Microarrays (Agilent). Raw data were extracted using Agilent Feature Extraction Software (v11.0.1.1), and then the raw data for each gene were summarized in the Agilent feature extraction protocol to generate a raw data text file, thus providing expression information for each gene. Gene-enrichment and functional annotation analysis for the significant probe list was performed using gene ontology and Kyoto Encyclopedia for Genes and Genomes (KEGG) analyses. All data analysis and visualization of differentially expressed genes were conducted using R 3.1.2. FIG. 1 shows the results of analyzing the differentially expressed genes.

[0088] As shown in FIG. 1, it could be confirmed that the expression levels of genes corresponding to cancer stem cell markers significantly increased in the YUMC-C1, YUMC-H2 and YUMC-P1 cell lines that are anticancer drug-resistant cancer cells, compared to the gene expression levels in the YUMC-M1 cell line which is a non-anticancer drug-resistant cancer cell line.

[0089] Through these results, it can be seen that the YUMC-C1, YUMC-H2 and YUMC-P1 cell lines of the present disclosure are cancer stem cells having anticancer drug resistance.

[Example 3] Results of Evaluating the Effect of Killing Anticancer Drug-Resistant Cancer Cells Lines

[0090] Each of the YUMC-C1, YUMC-H2 and YUMC-P1 cell lines of Example 1 was inoculated into 96-well plates at a density of 610.sup.3 cells/well and then cultured overnight to a confluence of 90%. After hydroflumethiazide (Candidate 23) represented by Formula 1 and oxaliplatin or sorafenib were added to final concentrations of 0 to 200 M, the cells were cultured. However, when hydroflumethiazide was used in combination with oxaliplatin or sorafenib, the treatment concentration of each of oxaliplatin and sorafenib was 30 M. Thereafter, cell viability was measured using the MTT reagent. The absorbance was measured at 550 nm, and viable cells were counted using trypan blue staining. FIG. 2 graphically shows the change in cell viability after each treatment, and Table 2 below shows the IC.sub.50 value when treated with hydroflumethiazide in combination with oxaliplatin or sorafenib.

TABLE-US-00002 TABLE 2 IC.sub.50 (M) YUMC-H2 cell YUMC-C1 cell line line YUMC-P1 cell line Candidate 23 + 80 oxaliplatin Candidate 23 + 80 60 sorafenib

[0091] As shown in FIG. 2, it could be confirmed that, even when the YUMC-C1 cell line was treated with oxaliplatin at a high concentration of 200 M, more than 80% of the cells survived, and even when the treatment concentration of sorafenib for the YUMC-H2 and YUMC-P1 cell lines was increased to 200 M, more than 80% of the cells also survived, suggesting that the cell lines showed high resistance to these anticancer drugs. However, it could be confirmed that, when each of the above-described cell lines was treated with hydroflumethiazide in combination with oxaliplatin or sorafenib, the death rate of the cancer cells significantly increased. In addition, as shown in Table 2 above, the IC.sub.50 concentrations of hydroflumethiazide (Candidate 23) plus standard anticancer drug (oxaliplatin or sorafenib) for the anticancer drug-resistant YUMC-C1, YUMC-H2 and YUMC-P1 cell lines were low at 80 M, 80 M and 60 M, respectively.

[Example 4] Results of Evaluating Therapeutic Effect in Mouse Models Xenografted with Anticancer Drug-Resistant Cancer Cell Lines

[0092] Each of the YUMC-C1, YUMC-H2 and YUMC-P1 cell lines (4.510.sup.6 cells/mouse) of Example 1 was cultured in test tubes and then was subcutaneously injected into the upper left flank regions of 6-week-old female BALB/c nude, NOD/Shi-scid, IL-2R KOJic (NOG) mice. 15 days after injection of each of the cell lines, when the tumor volume reached about 100 to 200 mm.sup.3, mice having a tumor with the above volume were randomly grouped (n=10/group), and then each drug was administered to the mice 13 times for a total of 41 days under the conditions shown in Table 3 below. Then, the changes in tumor volume during this administration were measured, and the results of the measurement are shown in FIG. 3. In addition, the tumor was extracted and the net weight of the tumor was measured, and the results of the measurement are shown in FIG. 4. In addition, changes in the body weights of the mice were measured, and the results of the measurement are shown in FIG. 5. Here, nothing was administered to the control group.

TABLE-US-00003 TABLE 3 Administration conditions Group 1 Group 2 Oxaliplatin (17 mg/kg) alone, oral administration Group 3 Sorafenib (80 mg/kg) alone, oral administration Group 4 Hydroflumethiazide (25 mg/kg, Candidate 23) represented by Formula 1 alone, oral administration Group 5 Oxaliplatin (8.5 mg/kg), oral administration Hydroflumethiazide (25 mg/kg, Candidate 23), oral administration Group 6 Sorafenib (25 mg/kg), oral administration Hydroflumethiazide (25 mg/kg, Candidate 23), oral administration

[0093] As shown in FIGS. 3 and 4, when oxaliplatin was administered to the mouse model xenografted with the YUMC-C1 cell line, the mouse model showed anticancer drug resistance compared to the control group, and thus the volume and weight of the tumor did not significantly decrease. In addition, even when sorafenib was administered to the mouse models xenografted with the YUMC-H2 and YUMC-P1 cell lines, no significant changes in the tumor volume and weight were observed. However, it could be confirmed that, when hydroflumethiazide (Candidate 23) represented by Formula 1, together with the anticancer drug oxaliplatin or sorafenib, was administered to these mouse models, the tumor volume and weight significantly decreased.

[0094] Meanwhile, as shown in FIG. 5, it could be confirmed that, when hydroflumethiazide represented by Formula 1 was administered alone or in combination with the anticancer drug oxaliplatin or sorafenib to the mouse models xenografted with the YUMC-C1, YUMC-H2 and YUMC-P1 cell lines, there was no changes in the body weights of all the mice, suggesting that there was no toxicity problem.

[0095] Through the above results, it can be seen that, when the hydroflumethiazide compound represented by Formula 1 according to the present disclosure is used in combination with an anticancer drug against various kinds of cancers showing resistance to the anticancer drug, it can not only significantly improve the anticancer effect of the anticancer drug, but also can induce the same anticancer effect even when the anticancer drug is used in a significantly smaller amount than the conventionally used amount, thereby reducing the side effects caused by administration of the anticancer drug.

[0096] As described above, the compound represented by Formula 1, when used in combination with an anticancer drug, can significantly improve the anticancer effect of the anticancer drug, and can induce the same anticancer effect even when the anticancer drug is used in a significantly smaller amount than the conventionally used amount, thereby reducing the side effects caused by administration of the anticancer drug. Furthermore, the compound represented by Formula 1 can also effectively prevent, alleviate or treat either anticancer-resistant cancer or cancer which recurs or metastasizes after anticancer drug treatment.