Method for treating cancer with dihydropyridine calcium antagonist

Abstract

The present invention discloses a method for treating a cancer with a dihydropyridine calcium antagonist, which comprises administering, to a cancer patient, a dihydropyridine calcium antagonist in an amount effective in inhibiting the cancer metastasis and reducing the proliferation of cancer cells, so as to achieve the effect of increasing the survival rate of patients with cancers.

Claims

1. A method for treating a cancer with a dihydropyridine calcium antagonist, comprising administering an effective amount of a dihydropyridine calcium antagonist to a cancer patient, wherein the dihydropyridine calcium antagonist is used at a dosage from 100 mg/90 day to 120 mg/30 day, wherein the administration of the dihydropyridine calcium antagonist to the cancer patient is to inhibit cancer metastasis.

2. The method for treating a cancer with a dihydropyridine calcium antagonist according to claim 1, wherein the dihydropyridine calcium antagonist is Amlodipine.

3. The method for treating a cancer with a dihydropyridine calcium antagonist according to claim 2, wherein Amlodipine is used at an average dosage of 1 mg/day to 4 mg/day.

4. The method for treating a cancer with a dihydropyridine calcium antagonist according to claim 1, wherein the cancer is selected from the group consisting of gastric cancer, liver cancer, lung cancer, breast cancer, colorectal cancer, pancreatic cancer, bladder cancer, and cervical cancer.

5. The method for treating a cancer with a dihydropyridine calcium antagonist according to claim 1, wherein the cancer is advanced cancer.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1 shows the result of statistical analysis at various times after patients having gastric cancer receive or receive no treatment with Amlodipine.

(2) FIG. 2 shows the result of statistical analysis at various times after patients having colorectal cancer receive or receive no treatment with Amlodipine.

(3) FIG. 3 shows the result of statistical analysis at various times after patients having liver cancer receive or receive no treatment with Amlodipine.

(4) FIG. 4 shows the result of statistical analysis at various times after patients having pancreatic cancer receive or receive no treatment with Amlodipine.

(5) FIG. 5 shows the result of statistical analysis at various times after patients having lung cancer receive or receive no treatment with Amlodipine.

(6) FIG. 6 shows the result of statistical analysis at various times after patients having breast cancer receive or receive no treatment with Amlodipine.

(7) FIG. 7 shows the result of statistical analysis at various times after patients having cervical cancer receive or receive no treatment with Amlodipine.

(8) FIG. 8 shows the result of statistical analysis at various times after patients having bladder cancer receive or receive no treatment with Amlodipine.

(9) FIG. 9 shows the result of cell viability of HepG2 cells detected after treatment with various concentrations of Amlodipine for 72 hrs.

(10) FIG. 10 shows the result of cell viability of Hep3B cells detected after treatment with various concentrations of Amlodipine for 72 hrs.

(11) FIG. 11A shows the result of cell viability by flow cytometry of HepG2 cells receiving no treatment with Amlodipine.

(12) FIG. 11B shows the result of cell viability by flow cytometry of HepG2 cells after treatment with 1.5625 M Amlodipine for 48 hrs.

(13) FIG. 11C shows the result of cell viability by flow cytometry of HepG2 cells after treatment with 3.125 M Amlodipine for 48 hrs.

(14) FIG. 11D shows the result of cell viability by flow cytometry of HepG2 cells after treatment with 6.25 M Amlodipine for 48 hrs.

(15) FIG. 11E shows the result of cell viability by flow cytometry of HepG2 cells after treatment with 12.5 M Amlodipine for 48 hrs.

(16) FIG. 11F shows the result of cell viability by flow cytometry of HepG2 cells after treatment with 25 M Amlodipine for 48 hrs.

(17) FIG. 12 shows the result of cell viability of HepG2 cells detected after treatment with various concentrations of Amlodipine for 48 hrs.

(18) FIG. 13A shows the result of cell viability by flow cytometry of Hep3B cells receiving no treatment with Amlodipine (Batch 1).

(19) FIG. 13B shows the result of cell viability by flow cytometry of Hep3B cells (Batch 1) after treatment with 1.5625 M Amlodipine for 48 hrs.

(20) FIG. 13C shows the result of cell viability by flow cytometry of Hep3B cells (Batch 1) after treatment with 3.125 M Amlodipine for 48 hrs.

(21) FIG. 13D shows the result of cell viability by flow cytometry of Hep3B cells (Batch 1) after treatment with 6.25 M Amlodipine for 48 hrs.

(22) FIG. 13E shows the result of cell viability by flow cytometry of Hep3B cells (Batch 1) after treatment with 12.5 M Amlodipine for 48 hrs.

(23) FIG. 13F shows the result of cell viability by flow cytometry of Hep3B cells (Batch 1) after treatment with 25 M Amlodipine for 48 hrs.

(24) FIG. 14A shows the result of cell viability by flow cytometry of Hep3B cells receiving no treatment with Amlodipine (Batch 2).

(25) FIG. 14B shows the result of cell viability by flow cytometry of Hep3B cells (Batch 2) after treatment with 1.5625 M Amlodipine for 48 hrs.

(26) FIG. 14C shows the result of cell viability by flow cytometry of Hep3B cells (Batch 2) after treatment with 3.125 M Amlodipine for 48 hrs.

(27) FIG. 14D shows the result of cell viability by flow cytometry of Hep3B cells (Batch 2) after treatment with 6.25 M Amlodipine for 48 hrs.

(28) FIG. 14E shows the result of cell viability by flow cytometry of Hep3B cells (Batch 2) after treatment with 12.5 M Amlodipine for 48 hrs.

(29) FIG. 14F shows the result of cell viability by flow cytometry of Hep3B cells (Batch 2) after treatment with 25 M Amlodipine for 48 hrs.

(30) FIG. 15 shows the result of cell viability of Hep3B cells detected after treatment with various concentrations of Amlodipine for 48 hrs.

(31) FIG. 16A shows the detection result of cell cycle of HepG2 cells receiving no treatment with Amlodipine.

(32) FIG. 16B shows the detection result of cell cycle of HepG2 cells after treatment with 1.5625 M Amlodipine.

(33) FIG. 16C shows the detection result of cell cycle of HepG2 cells after treatment with 3.125 M Amlodipine.

(34) FIG. 16D shows the detection result of cell cycle of HepG2 cells after treatment with 6.25 M Amlodipine.

(35) FIG. 16E shows the detection result of cell cycle of HepG2 cells after treatment with 12.5 M Amlodipine.

(36) FIG. 16F shows the detection result of cell cycle of HepG2 cells after treatment with 25 M Amlodipine.

(37) FIG. 17 shows the result of cell cycle by flow cytometry of HepG2 cells after treatment with various concentrations of Amlodipine.

(38) FIG. 18A shows the detection result of cell cycle of Hep3B cells receiving no treatment with Amlodipine.

(39) FIG. 18B shows the detection result of cell cycle of Hep3B cells after treatment with 1.5625 M Amlodipine.

(40) FIG. 18C shows the detection result of cell cycle of Hep3B cells after treatment with 3.125 M Amlodipine.

(41) FIG. 18D shows the detection result of cell cycle of Hep3B cells after treatment with 6.25 M Amlodipine.

(42) FIG. 18E shows the detection result of cell cycle of Hep3B cells after treatment with 12.5 M Amlodipine.

(43) FIG. 18F shows the detection result of cell cycle of Hep3B cells after treatment with 25 M Amlodipine.

(44) FIG. 19 shows the result of cell cycle by flow cytometry of Hep3B cells after treatment with various concentrations of Amlodipine.

(45) FIG. 20A shows the result of cell cycle by flow cytometry of HepG2 cells receiving no treatment with Amlodipine.

(46) FIG. 20B shows the result of cell cycle by flow cytometry of HepG2 cells after treatment with 12.5 M Amlodipine for 12 hrs.

(47) FIG. 20C shows the result of cell cycle by flow cytometry of HepG2 cells after treatment with 12.5 M Amlodipine for 24 hrs.

(48) FIG. 20D shows the result of cell cycle by flow cytometry of HepG2 cells after treatment with 12.5 M Amlodipine for 36 hrs.

(49) FIG. 20E shows the result of cell cycle by flow cytometry of HepG2 cells after treatment with 12.5 M Amlodipine for 48 hrs.

(50) FIG. 21 shows the result of cell cycle by flow cytometry of HepG2 cells after treatment with Amlodipine for various times.

(51) FIG. 22A shows the result of cell cycle by flow cytometry of Hep3B cells receiving no treatment with Amlodipine.

(52) FIG. 22B shows the result of cell cycle by flow cytometry of Hep3B cells after treatment with 25 M Amlodipine for 12 hrs.

(53) FIG. 22C shows the result of cell cycle by flow cytometry of Hep3B cells after treatment with 25 M Amlodipine for 24 hrs.

(54) FIG. 22D shows the result of cell cycle by flow cytometry of Hep3B cells after treatment with 25 M Amlodipine for 36 hrs.

(55) FIG. 22E shows the result of cell cycle by flow cytometry of Hep3B cells after treatment with 25 M Amlodipine for 48 hrs.

(56) FIG. 23 shows the result of cell cycle by flow cytometry of Hep3B cells after treatment with Amlodipine for various times.

(57) FIG. 24 shows the result (I) of protein expressions in HepG2 cells detected after treatment with various concentrations of Amlodipine.

(58) FIG. 25 shows the result (I) of protein expressions in Hep3B cells detected after treatment with various concentrations of Amlodipine.

(59) FIG. 26 shows the result (II) of protein expressions in HepG2 cells detected after treatment with various concentrations of Amlodipine.

(60) FIG. 27 shows the result (II) of protein expressions in Hep3B cells detected after treatment with various concentrations of Amlodipine.

(61) FIG. 28A shows the result of fluorescence intensity from HepG2 cells detected after treatment with various concentrations of Amlodipine for 48 hrs.

(62) FIG. 28B shows the result of cell count of HepG2 cells detected after treatment with various concentrations of Amlodipine for 48 hrs.

(63) FIG. 29 shows the result of cell migration of HepG2 cells observed after treatment with various concentrations of Amlodipine for 48 hrs.

(64) FIG. 30A shows the result of fluorescence intensity from Hep3B cells detected after treatment with various concentrations of Amlodipine for 72 hrs.

(65) FIG. 30B shows the result of cell count of Hep3B cells detected after treatment with various concentrations of Amlodipine for 72 hrs.

(66) FIG. 31 shows the result of cell migration of Hep3B cells observed after treatment with various concentrations of Amlodipine for 72 hrs.

(67) FIG. 32A shows the result of fluorescence intensity from HepG2 cells detected after treatment with various concentrations of Amlodipine for 72 hrs.

(68) FIG. 32B shows the result of cell count of HepG2 cells detected after treatment with various concentrations of Amlodipine for 72 hrs.

(69) FIG. 33 shows the result of cell migration of HepG2 cells observed after treatment with various concentrations of Amlodipine for 72 hrs.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

(70) The present invention will be further described below by way of examples in connection with the drawings.

Example 1: Statistical Analysis (I)

(71) In this example, statistical analysis was performed on the statistics of the effects of Amlodipine in the treatment of cancers, using data from the National Health Insurance Research Database (NHIRD) covering detailed healthcare data for the population in Taiwan. Then, the diagnostic data at the time of admission in the database and the database of patients with registered major injuries were used, and statistical analysis was performed using data from patients with gastric cancer. The results are as shown in Table 1 below, in which 24/30 represents treatment with Amlodipine for at least 24 days within 30 days after cancer diagnosis, 48/60 represents treatment with Amlodipine for at least 48 days within 60 days after cancer diagnosis, and 72/90 represents treatment with Amlodipine for at least 72 days within 90 days after cancer diagnosis.

(72) TABLE-US-00001 TABLE 1 Effect of Amlodipine in the treatment of cancers Day of treatment Number of Cases of with patients death Crude HR Cancer type Amlodipine With/without Amlodipine HRs (95% C.I.) P value Gastric 24/30 1577/6308 727/3644 0.43 (0.39-0.47) <.0001 cancer 48/60 1324/5296 621/3097 0.43 (0.39-0.48) <.0001 72/90 1185/4740 549/2715 0.42 (0.38-0.47) <.0001 Colorectal 24/30 6673/26692 2713/11833 0.46 (0.44-0.49) <.0001 cancer 48/60 5705/22820 2283/10176 0.44 (0.42-0.47) <.0001 72/9 5044/20176 1999/8911 0.44 (0.42-0.47) <.0001 Liver 24/30 3084/12336 2292/8197 0.55 (0.53-0.59) <.0001 cancer 48/60 2598/10392 1928/6918 0.54 (0.51-0.57) <.0001 72/90 2297/9188 1704/5967 0.55 (0.52-0.58) <.0001 Pancreatic 24/30 235/940 192/729 0.62 (0.51-0.75) <.0001 cancer 48/60 196/784 161/618 0.63 (0.51-0.77) <.0001 72/90 171/684 144/503 0.71 (0.57-0.88) 0.002 Lung cancer 24/30 2312/9248 1757/6375 0.66 (0.62-0.70) <.0001 48/60 1959/7836 1469/5358 0.62 (0.58-0.67) <.0001 72/9 1720/6880 1285/4725 0.62 (0.58-0.66) <.0001 Breast 24/30 4005/16020 1025/3825 0.54 (0.50-0.59) <.0001 cancer 48/60 3489/13956 885/3360 0.53 (0.48-0.57) <.0001 72/90 3145/12580 786/3085 0.50 (0.46-0.55) <.0001 Cervical 24/30 2769/11076 815/3868 0.50 (0.46-0.55) <.0001 cancer 48/60 2320/9280 670/3180 0.51 (0.46-0.56) <.0001 72/90 2041/8164 589/2889 0.46 (0.42-0.51) <.0001 Bladder 24/30 1930/7720 889/3237 0.54 (0.50-0.59) <.0001 cancer 48/60 1634/6536 754/2725 0.55 (0.50-0.60) <.0001 72/90 1421/5684 648/2360 0.55 (0.50-0.61) <.0001

(73) It can be seen from Table 1 that administration of Amlodipine to cancer patients can effectively reduce the mortality of cancer patients, and, in particular, it can be known from Table 1 that the longer the duration of the administration time is, the lower the mortality rate is.

Example 2: Statistical Analysis (II)

(74) The dose of Amlodipine used in Example 1 was at least 120 mg/30 days. Therefore, in order to obtain the minimum effective dose, the survival rate of patients treated with Amlodipine for days in 90 days after cancer diagnosis was further statistically analyzed. The results are shown in Table 2 below.

(75) TABLE-US-00002 TABLE 2 Analysis of survival rate after treatment with Amlodipine for 20 days within 90 days after diagnosis With Without Cancer Amlodipine/ Amlodipine/ Crude HR type death death HRs (95% C.I.) p-value Gastric cancer 1147/625 4588/2571 0.73 (0.66-0.80) <.0001 Colorectal 4645/2158 18580/8241 0.76 (0.72-0.80) <.0001 cancer Liver cancer 2865/2189 11460/7490 0.73 (0.69-0.77) <.0001 Pancreatic 227/192 908/658 0.77 (0.65-0.93) 0.0059 cancer Lung cancer 2221/1771 8884/6013 0.85 (0.80-0.90) <.0001 Breast cancer 2532/733 10128/2601 0.91 (0.83-0.99) 0.0339 Cervical cancer 1343/465 5372/1693 1.00 (0.89-1.11) 0.9418 Bladder cancer 1396/678 5584/2375 0.82 (0.75-0.90) <.0001

(76) The survival rate was analyzed for each cancer. The results are shown in FIGS. 1-8, respectively.

(77) The results of this example show that administration of Amlodipine does increase the survival rate of cancer patients compared with cancer patients receiving no treatment with Amlodipine, and can effectively reduce the mortality of cancer patients as shown by the long-term follow-up results (3 years). In addition, from the results of this example, it can be seen that the use of 100 mg of Amlodipine in 90 days can achieve the effect of increasing the survival rate of cancer patients.

Example 3: Cell Assay

(78) Two liver cancer cell lines HepG2 and Hep3B were cultured. The medium used for the culture of the cells was DMEM medium containing 10% fetal bovine serum, 10,000 U/ml penicillin and 10,000 g/ml streptomycin. The culture occurred in an environment at 37 C. with 5% CO.sub.2, and the medium was changed every 2-3 days during the culture.

(79) After the cells were cultured to about 70% confluence, they were treated with various concentrations of Amlodipine: 0, 1.5625, 3.125, 6.25, 12.5, 25, and 50 M for various times: 48, and 72 hrs. After the experiment, the viability of each liver cancer cell line was observed and the cell viability and cell cycle were analyzed.

Example 4: Cell Viability of Liver Cancer Cell Lines

(80) The HepG2 and Hep3B cells treated with various concentrations of Amlodipine for 72 hrs were added to WST-1 reagent. After 330 minutes of reaction, the absorbance at a wavelength of 420-480 nm was measured. The results are shown in FIGS. 9 and 10.

(81) The HepG2 and Hep3B cells treated with various concentrations of Amlodipine for 48 hrs were scraped off with 0.05% Trypsin-EDTA, and then added to 2 mg/ml PI solution. The cell viability was analyzed by flow cytometry. The results are shown in FIGS. 11 to 15.

(82) The results from FIGS. 9 to 15 show that the cell viability of liver cancer cells treated with Amlodipine is decreased, and as the concentration administered increases, the cell viability of liver cancer cells decreases accordingly. When the Amlodipine concentration is greater than or equal to 12.5 M, the best inhibitory effect on the growth of liver cancer cells is achieved.

Example 5: Analysis of the Cell Cycle

(83) The cell cycle of each liver cancer cell line treated under different conditions was analyzed by flow cytometry. The results are shown in FIGS. 16 to 23.

(84) Referring to FIGS. 16 to 19, when each liver cancer cell line was treated with Amlodipine at a concentration of 12.5 M or more for 48 hrs, most cells were unable to undergo cell division, specifically as shown in Tables 3 and 4 below.

(85) TABLE-US-00003 TABLE 3 Cell cycle distribution of liver cancer cells HepG2 treated with various concentrations of Amlodipine for 48 hrs Amlodipine concentration Proportion of cell distribution in cell cycle (%) (M) M1: M2: M3: M4: 0 1.95 68.79 10.58 18.84 1.5625 1.58 70.28 8.89 19.34 3.125 1.36 69.60 8.91 20.23 6.25 1.91 66.92 10.28 21.01 12.5 81.27 12.49 3.31 2.95 25 99.75 0.22 0.00 0.94

(86) TABLE-US-00004 TABLE 4 Cell cycle distribution of liver cancer cells Hep3B treated with various concentrations of Amlodipine for 48 hrs Amlodipine concentration Proportion of cell distribution in cell cycle (%) (M) M1: M2: M3: M4: 0 5.15 72.09 8.38 14.55 1.5625 5.34 74.88 7.06 12.82 3.125 6.51 73.83 6.63 13.17 6.25 6.48 72.91 6.74 13.93 12.5 16.96 67.77 6.58 8.83 25 57.32 28.75 8.02 6.10

(87) Still further, each liver cancer cell line was treated with Amlodipine at a concentration of 12.5 M for 0, 12, 24, 36 and 48 hrs, and the cell cycle was analyzed by flow cytometry. The results are shown in FIGS. 20 to 23. The proportion of cell distribution at each phase of the cell cycle is shown in Tables 5 and 6 below. The results show that as the time of treatment with Amlodipine increases, more liver cancer cells are inhibited from undergoing cell division.

(88) TABLE-US-00005 TABLE 5 Cell cycle distribution of liver cancer cells HepG2 treated with Amlodipine for various times Treatment Proportion of cell distribution in cell cycle (%) time (hr) M1: M2: M3: M4: 0 1.52 66.25 11.49 20.94 12 5.01 76.16 6.10 12.87 24 16.20 65.01 6.24 12.69 36 65.46 22.64 4.81 7.22 48 83.26 11.13 1.73 3.93

(89) TABLE-US-00006 TABLE 6 Cell cycle distribution of liver cancer cells Hep3B treated with Amlodipine for various times Treatment Proportion of cell distribution in cell cycle (%) time (hr) M1: M2: M3: M4: 0 9.25 71.49 7.63 11.87 12 24.89 62.89 4.22 8.08 24 30.77 56.25 5.46 7.55 36 39.90 47.73 5.03 7.47 48 56.61 29.59 7.08 6.82

Example 6: Protein Expression

(90) The expression of apoptosis-related genes in each liver cancer cell line treated with various concentrations of Amlodipine for 24 hours were detected by Western blotting. The results are shown in FIGS. 24 to 27.

(91) From the results shown in FIGS. 24 to 27, it can be seen that treatment of the liver cancer cells with Amlodipine results in the intracellular expression of apoptotic proteins, indicating that Amlodipine promotes apoptosis and is effective in treating cancers.

Example 7: Cell Metastasis

(92) The HepG2 and Hep3B cells were separately cultured in an insert of the Transwell plate, then added with 10 M Amlodipine and cultured for 48 or 72 hrs. The insert was placed in a serum-free DMEM medium containing 8 mM Calcien AM. Fluorescent staining was carried out in a culture environment at 37 C. After about 45-60 min, the cells were scraped off with 0.05% Trypsin-EDTA, and the fluorescence intensity at a wavelength of 490 to 520 nm was measured. The results are shown in FIGS. 28 to 31.

(93) From the results of this example, the status of cancer cell metastasis gets known. In other words, the fluorescence intensity from the liver cancer cells treated with Amlodipine is lower than that from the liver cancer cells not treated with Amlodipine, thus indicating that Amlodipine can improve the status of cancer metastasis.

Example 8: Cell Invasion

(94) The HepG2 and Hep3B cells were separately cultured in the insert of the Transwell plate, then added with 10 M Amlodipine and cultured for 48 or 72 hrs. The cells on the inner surface of the insert were taken out, and the insert was placed in methanol and stained with a crystal violet solution. The results photographed under a microscope are shown in FIGS. 32 and 33. From the results, it is found that the fluorescence intensity from the liver cancer cells treated with Amlodipine for 72 hrs is lower than that from the liver cancer cells not treated with Amlodipine. That is, Amlodipine is effective in slowing the progression of cancers, thereby achieving a therapeutic effect in the treatment of cancers.

(95) It can be seen from the above embodiments and examples that the dihydropyridine calcium antagonist disclosed in the present invention has the effect of inhibiting the division and promoting the apoptosis of cancer cells, that is, the dihydropyridine calcium antagonist disclosed in the present invention is useful in a pharmaceutical composition for treating cancers. Furthermore, as the dose of the dihydropyridine calcium antagonist administered is increased or as the duration of administration is increased, the effect of inhibiting the growth of cancer cells is better.