WO2014115977A1 - Composition for preventing or treating cancer - Google Patents

Abstract

The present invention relates to a composition for preventing or treating cancer, containing fimasartan, a pharmaceutically acceptable salt thereof, a solvate of fimasartan and the salt or a hydrate of fimasartan or the salt, as an active ingredient, and it is possible to safely and effectively prevent or treat cancer.

Description

Cancer preventive or therapeutic composition

The present invention relates to a composition for preventing or treating cancer comprising fimasartan, a pharmaceutically acceptable salt thereof, a solvate thereof, or a hydrate thereof as an active ingredient, a method for preventing or treating cancer using the same, and a use thereof. .

Although the incidence of cancer increases with the development of civilization, the treatment of cancer patients relies on chemotherapy by surgical surgery, radiation therapy and chemotherapy with strong cytotoxicity. However, these therapies are usually limited to early cancer patients or certain cancers, and have various side effects. Therefore, the development of effective and low side effects anti-cancer drugs is required.

Meanwhile, fimasaltan is a compound having the chemical structure shown in FIG. 1 developed by the applicant of the present application (Boryeong Pharmaceuticals) {2-butyl-5-dimethylaminothiocarbonylmethyl-6-methyl-3-[[2 ']. -(1H-tetrazol-5-yl) biphenyl-4-yl] methyl] pyridin-4 (3H) -one} (Korean Patent No. 0354654). Fimasartan is a new Angiotensin II Receptor Blocker (ARB) used for hypertension and has a safety profile even after a fast oral dose of 20-480 mg [Chi YH., Lee H., Paik SH., Lee JH, Yoo BW, Kim JH, Tan HK, Kim SL., Am J Cardiovasc Drugs., 11 (5), 335-346 (2011). However, there is no known anticancer effect on fimasartan.

Therefore, the present inventors have completed the composition of the present invention that can be used as a safe and effective anti-cancer drug as a result of studies using cancer cell lines.

An object of the present invention is to provide a safe and effective composition for the prevention or treatment of cancer, a method for preventing or treating cancer using the same and its use.

In order to achieve the above object, the present invention provides a composition for the prevention and treatment of cancer comprising fimasartan, a pharmaceutically acceptable salt thereof, solvate thereof or a hydrate thereof as an active ingredient.

In the present invention, fimasaltan is a compound having the chemical structure shown in FIG. 1 {2-butyl-5-dimethylaminothiocarbonylmethyl-6-methyl-3-[[2 '-(1H-tetrazol-5-) Il) biphenyl-4-yl] methyl] pyridin-4 (3H) -one}.

In the present invention, fimasartan, pharmaceutically acceptable salts thereof, solvates thereof or hydrates thereof may be crystalline or amorphous, and the present invention includes their crystalline and / or amorphous forms.

In the present invention, pharmaceutically acceptable salts generally mean inorganic acid salts, organic acid salts, and metal salts used by pharmaceutical manufacturers to prepare medicines. As the "organic acid," hydrochloric acid, bromic acid, sulfuric acid, phosphoric acid, and the like may be used. "Organic acid" includes citric acid, acetic acid, lactic acid, tartaric acid, fumaric acid, formic acid, propionic acid, oxalic acid, trifluoroacetic acid, methanesulfonic acid, benzenesulfonic acid, maleic acid, benzoic acid, gluconic acid, glycolic acid, succinic acid, 4-morpholine Ethanesulfonic acid, camphorsulfonic acid, 4-nitrobenzenesulfonic acid, hydroxy-O-sulfonic acid, 4-toluenesulfonic acid, kalukturonic acid, emboic acid, glutamic acid, aspartic acid, adipate salt, camsylate salt or besylate salt And "metal" includes sodium, potassium, calcium, magnesium, and the like.

In the present invention, the pharmaceutically acceptable salt of fimasartan is preferably fimasartan potassium salt, hydrochloride, calcium salt, sulfate, adipate salt, camsylate salt or besylate salt, and the fimasaltan potassium salt is even more preferred. desirable.

In the present invention, as the hydrate of the pharmaceutically acceptable salt of fimasartan, the fimasartan trihydrate potassium salt is preferable.

In the present invention, the solvent in the "solvate" means a conventional organic solvent used to prepare an organic compound, for example, methanol, ethanol, 1-propanol, 2-propanol, 1-butanol, 2-butanol, 1 Acetates, acetone, acetic acid, anisole, tetrahydrofuran, methyl acetate, ethyl acetate, propyl acetate, isopropyl acetate, isobutyl acetate, n-butyl acetate, dimethyl sulfoxide, pentane, heptane, and the like. The solvates of the present invention are not limited.

In the present invention, "hydrate" and "solvate" may be contained in a 0.25 to 10 molar ratio with respect to 1 mole of fimasaltan, for example 0.5 mole, 1 mole, 1.5 mole, 2 mole, 2.5 mole, 3 mole 5 moles, etc., but the present invention is not limited thereto.

In the present invention, fimasartan, pharmaceutically acceptable salts thereof, solvates thereof or hydrates thereof can be purchased commercially and prepared by known methods (for example, Korean Patent Registration No. 0354654). And 05521980 and the like).

In the present invention, the cancer may be at least one selected from the group consisting of prostate cancer, colon cancer, stomach cancer, breast cancer, cervical cancer, skin cancer, kidney cancer, liver cancer, brain tumor, lung cancer, uterine cancer, colon cancer, bladder and pancreatic cancer. Preferably the cancer may be prostate cancer.

The composition of the present invention may be prepared by containing one or more pharmaceutically acceptable carriers in addition to the above components for administration. Pharmaceutically acceptable carriers may be used in combination with saline, sterile water, Ringer's solution, buffered saline, dextrose solution, maltodextrin solution, glycerol, ethanol and one or more of these components, if necessary, as antioxidants, buffers And other conventional additives such as bacteriostatic agents can be added.

Diluents, dispersants, surfactants, binders and lubricants may also be added in addition to formulate into injectable formulations, pills, capsules, granules or tablets such as aqueous solutions, suspensions, emulsions and the like. Furthermore, it may be preferably formulated according to each disease or component by a suitable method in the art or using the method disclosed in Remington's Pharmaceutical Science, Mack Publishing Company, Easton PA.

The composition of the present invention can be administered orally or parenterally (eg, applied intravenously, subcutaneously, intraperitoneally or topically) according to the desired method, and the dosage is based on the weight, age, sex, The range varies depending on the diet, the time of administration, the method of administration, the rate of excretion and the severity of the disease. The composition of the present invention may be administered once to several times a day. For example, the effective amount of fimasartan of the composition of the present invention is 150 mg to 900 mg based on a normal adult body weight of about 60 kg.

The compositions of the present invention may be injectable formulations such as aqueous solutions, suspensions, emulsions, and the like, pills, capsules, granules or tablets, or oral administration in the form of a kit, more preferably for oral administration, even more monolithic. desirable.

In addition, the present invention is to prevent or treat cancer by administering to the mammal, including humans in need thereof, a composition comprising fimasartan, a pharmaceutically acceptable salt thereof, solvate thereof or a hydrate thereof as an active ingredient. Provide a way to. Humans may be excluded from the method.

The present invention also provides the use of a composition comprising fimasartan, a pharmaceutically acceptable salt thereof, a solvate thereof or a hydrate thereof as an active ingredient for pharmaceutical preparation for preventing or treating cancer.

The composition according to the present invention can safely and effectively prevent or treat cancer.

1 is a diagram showing the chemical structure of fimasartan.

Figure 2 is a graph showing the anticancer effect of the treatment of angiotensin II receptor antagonists (pimasartan, losartan, valsartan and eprosaltan) in PC-3 cell line, a prostate cancer cell line.

Figure 3 is a graph showing the anticancer effect of the treatment of angiotensin II receptor antagonists (pimasartan, losartan, valsartan and eprosaltan) in the prostate cancer cell line DU-145.

Figure 4 is a graph showing the anticancer effect of the treatment of angiotensin II receptor antagonists (pimasaltan, losartan, valsartan and eprosaltan) to LNCap-LN3, a prostate cancer cell line.

Figure 5 is a Western blot showing the protein expression changes related to autophagy after fimasartan treatment in PC-3 cell line, a prostate cancer cell line.

Figure 6 is a graph showing the relative change in the ratio of LC3-II to LC3-I after fimasartan treatment in PC-3 cell line, a prostate cancer cell line.

FIG. 7 is a Western blot showing the changes in protein expression related to autophagy after treatment of losartan, valsartan and eprosaltan in PC-3 cell line, a prostate cancer cell line.

8 is a graph showing the relative change in the ratio of LC3-II to LC3-I after losartan treatment in PC-3 cell line, a prostate cancer cell line.

Figure 9 is a graph showing the relative change in ratio of LC3-II to LC3-I after valsartan treatment to PC-3 cell line, a prostate cancer cell line.

10 is a graph showing the relative change in the ratio of LC3-II to LC3-I after eprosaltan treatment in PC-3 cell line, a prostate cancer cell line.

11 is a diagram showing a confocal microscope observation result confirming whether autophagy by Pmasartan treatment of prostate cancer cell line PC-3 cell line using immunofluorescence.

FIG. 12 is a graph showing the number of LC3 spot forming cells relative to the total number of cells after pmasartan treatment of the prostate cancer cell line PC-3 cell line using immunofluorescence.

FIG. 13 is a diagram showing the anti-metastatic effect of fimasartan treatment on PC-3 cell line, a prostate cancer cell line.

Figure 14 is a graph showing the effect of the anti-metastasis effect when treated with Pimasaltan on PC-3 cell line, a prostate cancer cell line.

15 is a diagram showing the anti-metastasis effect of fimasartan treatment on DU145 cell line, a prostate cancer cell line.

Figure 16 is a graph showing the effect of the anti-transition effect when treated with pimasartan in DU145 cell line, a prostate cancer cell line.

Hereinafter, the present invention will be described in more detail with reference to the following examples. However, the following examples are not intended to limit the scope of the present invention, which will be construed as to aid the understanding of the present invention.

Example. Confirmation of the anticancer effect of fimasartan

1. Experimental Materials and Methods

A. Experimental Materials

DMEM, fetal bovine serum (FBS), penicillin, and streptomycin were obtained from Life Technologies Inc. (Grand Island, NY, USA). iNOS, p65, cJun, cFos, poly (ADP ribose) polymerase [PARP], β-actin monoclonal antibody, and peroxidase-conjugated secondary antibody are described in Santa Cruz Biotechnology, Inc. (SantaCruz, CA, USA). Optional oligonucleotide primers and M-MLV reverse transcriptase were purchased from Promega (Madison, WI, USA). SYBR green ex Taq is TaKaRa (Shiga, Japan). iNOS, β-actin oligonucleotide primers were purchased from Bioneer (Seoul, Korea). Fimasartan trihydrate potassium salt was obtained from Boryeong Pharmaceuticals (Seoul, Korea). MTT (3- (4,5-Dimethylthiazol-2-yl) -2,5-diphenyl tertazolium bromide), PMSF, DL-Dithiothreitol (DTT), NS-398, LPS ( Escherichia coli , serotype 0111: B4) and others All compounds were purchased from Sigma Chemical Co. (St. Louis, MO, USA).

B. Cell Culture

The following cell lines were obtained from ATCC and Korea Cell Line Bank (KCLB): PC-3, DU145 and LNCap-LN3

PC-3, DU145 and LNCap-LN3 cell lines contain 5% CO ₂ concentration, humid conditions, 37 ° C temperature in DMEM supplemented with 10% fetal bovine serum (FBS), 100 units / ml penicillin and 100 ug / ml streptomycin Incubated at.

C. WST-1 proliferation assay

After seeding the prostate cancer cell lines PC-3, DU-145 and LNCap-LN3 into 1 × 10 ³ cells / well in 96-well plates, Fimasartan, Losartan, Eposartan, and Valsartan (ARBs) were seeded into each prostate cancer cell line. The group treated with 200 and 400 uM and the control group without ARBs were divided into cells and cultured for 72 and 120 hours under the same conditions as the cell culture. Each well was treated with 10 μl of WST-1 (Roche Co., Catalog No. 11-644-807-001) solution and reacted for 3 hours in an incubator at 37 ° C. and 5% CO 2. Absorbance (A450 and A690) was measured at 450nm and 690nm, and the A450-A690 value was obtained by subtracting the reference value A690 from the A450 value. The percentage of viable cells was calculated from the relative A450-A690 values of the samples relative to the control group to compare the cell growth inhibition of each compound.

D. Western Blot Analysis

After prostate cancer cell line PC-3 was treated with 200 μM of fimasartan, the cells were harvested after 24 hours (h), 48 hours, 72 hours, and 96 hours at 0 h (control), followed by Western blot. .

PC-3 was collected by centrifugation and washed once with phosphate-buffered saline (PBS). The washed cell pellet was crushed by RIPA buffer solution (1% protein inhibitor) and protein was extracted from it. Protein concentrations were determined using Bradford assay reagents according to the manufacturer's instructions. Cell proteins from treated and untreated cell extracts were separated on 10-15% SDS-PAGE and then electrobloted on PVDF membranes. Immunoblots were incubated with blocking solution (5% skim milk) for 1 hour (h) followed by 2 hours with primary antibody. The blot was washed three times with Tween 20 / TBS (Tris-buffered saline) and was diluted 1: 1000 with horseradish peroxidase-conjugated secondary antibody for 1 hour 30 minutes at room temperature. Incubated. The blot was again washed three times with Tween 20 / TBS and then developed by enhanced chemiluminescence (ECL) [Amersham Life Science].

E. Confocal Microscopy

PC-3 cell line transfected with GFP-LC3 was treated with 200 μM of fimasartan, followed by 0 hours (control), 24 hours, 48 hours in Hanks' solution containing 0.1 μM bafilomycin A1 at 37 ° C. After 72 hours, cells were analyzed for GFP-LC3 spot formation, an autophagy biomarker by immunofluorescence confocal microscopy (Olympus, FV1000).

F. Cell Migration Assay

After seeding the prostate cancer cell lines PC-3 and DU-145 in 1 × 10 ⁴ / well in Transwell, divided into two groups treated with 200 μM of fimasartan and an untreated control for 24 hours in a 37 ° C. carbon dioxide incubator. Incubated for 6 hours. After incubation, after removing the medium of the transwell, it was fixed in 100% methanol for 15 minutes, and then washed twice with distilled water, and then reacted for 5 minutes in H & E staining solution. After the reaction was washed three times in distilled water, using a cotton swab to remove cells that did not pass through the transwell completely removed. After the transwells were completely dried, observations and photographs were taken at a magnification of 100 times to observe the cells that had passed through the transwells. For quantitative analysis, after the photographing, the absorbance was analyzed by immersing in a 10% acetic acid solution in transwell and measuring at 560nm wavelength.

G. Statistical Analysis

The results are expressed as mean ± standard deviation (S.D.) of three experiments. Statistically significant values were compared using Student's T test and p-values below 0.05 were considered statistically significant. (* P <0.05, ** P <0.01; *** P <0.001)

2. Results

A. WST-1 Cell Proliferation Assay

As shown in Figures 2 to 4, different at the same concentration treatment It was found that fimasartan showed antiproliferative effects of prostate cancer cell lines PC-3, DU-145 and LNCap-LN3 than angiotensin II receptor antagonists (rosaartan, valsartan and eprosaltan).

B. Confirmation of Apoptosis Mechanism by Autophagy of Fimasartan and Other Angiotensin II Receptor Antagonists (Rosartan, Valsartan and Eprosaltan)

Western blot confirmed the occurrence of apoptosis by autophagy of fimasartan and other angiotensin II receptor antagonists (rosaartan, valsartan and eprosaltan).

As shown in Figures 5 and 6, it can be seen that the treatment of PC-3, a prostate cancer cell line, with pimasartan increases the ratio of LC3-II / LC3-I, a marker of autophagy, over time.

In addition, as shown in Figures 7 to 10, even when treated with other angiotensin II receptor antagonists (rosaartan, valsartan and eprosaltan) to the cancer cell line prostate cancer cell line PC-3 is a marker of autophagy over time It can be seen that the ratio of LC3-II / LC3-I increases.

However, the ratio of LC3-II / LC3-I after 96 hours after treatment with Pmasartan to PC-3, a prostate cancer cell line, was about 23-fold, respectively, compared to other Angiotensin II receptor antagonists, Losartan, Valsartan and Eprosaltan. 35 times and 76 times higher was confirmed. In other words, it was confirmed that fimasartan can cause autophagy in cancer cells significantly more than other angiotensin II receptor antagonists.

C. Confirmation of the autophagy of fimasartan

Confocal microscopy confirmed the self-feeding effect of fimasartan.

As shown in FIGS. 11 and 12, GFP-LC3 showed a widespread staining pattern in PC-3, a prostate cancer cell line that was not treated with fimasartan, while PC-3, a prostate cancer cell line that was treated with fimasartan, showed Over time, an increase in cells showing GFP-LC3 spots was observed. This shows that fimasartan can potentiate autophagy in human prostate cancer cell line PC-3.

Therefore, confocal microscopy results indicate that fimasartan may have an effect of preventing and treating cancer by inducing cell death by inducing autophagy in cancer cells.

D. Determination of anti-metastatic ability of fimasartan cancer cells

Through cell migration analysis, promas cancer cell lines PC-3 and DU-145 were treated with 200 μM of fimasartan, and the anti-emergence effect was confirmed after 6 hours.

As shown in Figure 13 and 14, it was confirmed that after 6 hours after the prostate cancer cell line PC-3 treated with 200 μM of fimasartan, more than 12% of the cell migration is inhibited to inhibit the metastasis of cancer cells.

In addition, as shown in Figures 15 and 16, after the prostate cancer cell line DU-145 200 μM treatment with fimasartan 200 μM, it was confirmed that more than 13% of the cell migration is inhibited can inhibit the metastasis of cancer cells .

Therefore, the cell migration analysis results indicate that fimasartan not only induces death of cancer cells but also inhibits metastasis, thereby having a prophylactic and therapeutic effect of cancer.

The composition according to the invention can be used to prevent or treat cancer safely and effectively.

Claims (6)

1. Fimasaltan, a pharmaceutically acceptable salt thereof, solvate thereof or a hydrate thereof as an active ingredient, a composition for preventing and treating cancer.
2. The composition for preventing and treating cancer according to claim 1, comprising fimasartan trihydrate potassium salt as an active ingredient.
3. The cancer of claim 1, wherein the cancer is one or more selected from the group consisting of prostate cancer, colon cancer, gastric cancer, breast cancer, cervical cancer, skin cancer, kidney cancer, liver cancer, brain tumor, lung cancer, uterine cancer, colon cancer, bladder, and pancreatic cancer. Composition for the prevention and treatment of.
4. The composition for preventing and treating cancer of claim 1, wherein the cancer is prostate cancer.
5. A method for preventing or treating cancer by administering fimasartan, a pharmaceutically acceptable salt thereof, a solvate thereof or a hydrate thereof as an active ingredient to a mammal including a human in need thereof.
6. Use of a composition comprising fimasartan, a pharmaceutically acceptable salt thereof, a solvate thereof or a hydrate thereof as an active ingredient for the pharmaceutical preparation for preventing or treating cancer.

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