JP2019199477A - Cancer treatment agent - Google Patents

Abstract

To provide a cancer treatment agent and a cancer treatment method having stronger therapeutic effects on various types of cancer than those of conventional chemotherapy.SOLUTION: The present inventors have conducted intensive studies to search for an agent that can enhance therapeutic effects of an anticancer agent by combination with an anticancer agent and, as a result, find out that a combination of caffeine citrate and an anticancer agent synergistically improves therapeutic effects of the anticancer agent.SELECTED DRAWING: None

Description

The present invention relates to a cancer therapeutic agent and a cancer treatment method characterized by combining caffeine citrate as an active ingredient with an anticancer agent as an active ingredient.
This application claims the priority of Japanese Patent Application No. 2016-203793 and Japanese Patent Application No. 2017-061056, which are incorporated herein by reference.

(Osteosarcoma)
Osteosarcoma is the most common primary malignant bone tumor that occurs mainly in young people in their 10s, but the cause is unknown. The incidence in Japan is said to be 1 per 1 million (130 to 260 per year). In the days when amputation was the only treatment option, osteosarcoma had a 2 year survival rate of 15-20%, a very poor prognosis.
However, with the introduction / advancement of chemotherapy and the development of diagnostic imaging and surgical therapy, the 5-year survival rate has improved to 70% or more (Non-patent Document 1). The inventors have disclosed “caffeine combination chemotherapy”. This therapy dramatically improved the 5-year survival rate of 89% in patients with no metastasis at the first visit, but the improvement in prognosis is still insufficient. (Non-patent document 2). In patients who have relapsed or metastasized since the first visit, the 5-year survival rate is 10-30%, which has not improved significantly over the past 40 years. I think that the improvement of an effect is required (nonpatent literature 3).

(Soft tissue sarcoma)
Soft tissue sarcomas have various classifications such as leiomyosarcoma, fibrosarcoma, liposarcoma, rhabdomyosarcoma, and the like. Each has a different nature, and the rarity is very high. In non-small round cell sarcomas excluding extraosseous Ewing sarcoma and rhabdomyosarcoma, the 10-year survival rate of surgery alone has been reported to be around 35% when the extremity is limited to stage III. Reference 4). Furthermore, regarding the effect of chemotherapy for advanced malignant soft tissue sarcoma, the response rate has been reported to be 30-40%, and soft tissue sarcoma is a disease that is more difficult to improve the prognosis than osteosarcoma. (Non-patent documents 5 to 7).
Current standard treatments for soft tissue sarcoma are based on a combination of preoperative chemotherapy, surgery (broad resection), and postoperative chemotherapy, depending on the grade, type of tumor and patient condition. The above-mentioned chemotherapy is a multidrug combination chemotherapy using cisplatin, doxorubicin and the like.
As described above, this treatment method cannot be said to have a sufficient therapeutic effect, but has a problem that development of a new drug is difficult to proceed due to the high scarcity of soft tissue sarcoma.

  As described above, a new therapeutic agent that exerts an effect on various cancer types and has a stronger therapeutic effect than conventional chemotherapy has been desired.

N Engl J Med 1986; 314: 1600-6. Anticancer Res 1999; 18 (1B): 657-66. Cancer Treat Rev 2014; 40: 523-32. Semin Radiat Oncol 1999; 9: 349-51. Am J Clin Oncol 1988; 11: 39-45. Eur J Cancer Clin Oncol 1987; 23: 311-21. Hematol Oncol Clin North Am 1995; 9: 765-85.

  An object of the present invention is to provide a cancer therapeutic agent and a cancer treatment method having a stronger therapeutic effect than conventional chemotherapy in various cancer types.

  In order to solve the above problems, the present inventors have intensively studied a drug that can enhance the therapeutic effect of an anticancer agent by combining it with an anticancer agent, and as a result, “the combination of caffeine citrate and an anticancer agent has a therapeutic effect of the anticancer agent. The present invention has been completed by finding “enhancing synergistically”.

That is, this invention consists of the following.
1. A cancer therapeutic agent comprising a combination of an active ingredient caffeine citrate and an anticancer agent as an active ingredient.
2. 2. The cancer therapeutic agent according to 1 above, wherein the cancer is primary malignant bone tumor, soft tissue sarcoma, lung cancer, stomach cancer, breast cancer, uterine cancer, colon cancer, skin cancer, ovarian cancer, pancreatic cancer, bile duct cancer or hepatocellular carcinoma. .
3. The cancer therapeutic agent according to 1 or 2 above, wherein the anticancer agent is cisplatin, gemcitabine or adriamycin.
4). 4. The cancer therapeutic agent according to any one of items 1 to 3, wherein the cancer is osteosarcoma.
5. 4. The cancer therapeutic agent according to any one of items 1 to 3, wherein the cancer is soft tissue sarcoma.
6). 4. The cancer therapeutic agent according to any one of items 1 to 3, wherein the cancer is lung cancer.
7. 4. The cancer therapeutic agent according to any one of items 1 to 3, wherein the cancer is pancreatic cancer.
8). 4. The cancer therapeutic agent according to any one of items 1 to 3, wherein the cancer is gastric cancer.
9. 4. The cancer therapeutic agent according to any one of items 1 to 3, wherein the cancer is hepatocellular carcinoma.
10. 4. The cancer therapeutic agent according to any one of items 1 to 3, wherein the cancer is uterine cancer.
11. 4. The cancer therapeutic agent according to any one of items 1 to 3, wherein the cancer is breast cancer.
12 4. The cancer therapeutic agent according to any one of items 1 to 3, wherein the cancer is ovarian cancer.
13. 4. The cancer therapeutic agent according to any one of items 1 to 3, wherein the cancer is skin cancer.
14 4. The cancer therapeutic agent according to any one of items 1 to 3, wherein the cancer is cholangiocarcinoma.
15. A method for treating cancer, comprising administering a caffeine citrate as an active ingredient and an anticancer agent as an active ingredient to a cancer patient.
16. The cancer according to item 15 above, wherein the cancer patient is a primary malignant bone tumor, soft tissue sarcoma, lung cancer, stomach cancer, breast cancer, uterine cancer, colon cancer, skin cancer, ovarian cancer, pancreatic cancer, bile duct cancer or hepatocellular carcinoma patient. Method of treatment.
17. The cancer treatment method according to 15 or 16 above, wherein the anticancer agent is cisplatin, adriamycin, or gemcitabine.
18. A therapeutic agent for soft tissue sarcoma characterized by combining caffeine citrate, which is an active ingredient, with gemcitabine, which is an anticancer agent, which is an active ingredient.
19. A therapeutic agent for gastric cancer, wherein caffeine citrate as an active ingredient is combined with gemcitabine as an anticancer agent as an active ingredient.
20. A therapeutic agent for skin cancer, characterized in that caffeine citrate, which is an active ingredient, is combined with gemcitabine, which is an anticancer agent, which is an active ingredient.
21. A therapeutic agent for pancreatic cancer, characterized in that caffeine citrate, which is an active ingredient, is combined with gemcitabine, which is an anticancer agent, which is an active ingredient.
22. A therapeutic agent for gastric cancer, characterized in that caffeine citrate, which is an active ingredient, is combined with adriamycin, which is an anticancer agent, which is an active ingredient.
23. A therapeutic agent for uterine cancer, comprising a combination of caffeine citrate, which is an active ingredient, with adriamycin, which is an anticancer agent, which is an active ingredient.
24. A lung cancer therapeutic agent characterized by combining caffeine citrate, which is an active ingredient, with cisplatin, which is an anticancer agent, which is an active ingredient.
25. A therapeutic agent for breast cancer, comprising a combination of caffeine citrate, which is an active ingredient, with cisplatin, which is an anticancer agent, which is an active ingredient.
26. A therapeutic agent for uterine cancer, characterized in that caffeine citrate, which is an active ingredient, is combined with cisplatin, which is an anticancer agent, which is an active ingredient.
27. A skin cancer therapeutic agent characterized by combining caffeine citrate as an active ingredient with cisplatin as an anticancer agent as an active ingredient.
28. A therapeutic agent for ovarian cancer characterized by combining caffeine citrate as an active ingredient with cisplatin as an anticancer agent as an active ingredient.
29. A therapeutic agent for cholangiocarcinoma characterized by combining caffeine citrate as an active ingredient with cisplatin as an anticancer agent as an active ingredient.

  The cancer therapeutic agent of the present invention has a synergistic effect capable of synergistically enhancing the therapeutic effect of the anticancer agent by combining at least caffeine citrate with the anticancer agent.

The result of the WST-8 assay using the osteosarcoma cell line (HOS) in Example 1. The vertical axis represents cell viability, and the horizontal axis represents cisplatin concentration (CDDP concentration). Diamond (♦) is cisplatin (CDDP) alone, black square (■) is cisplatin + anhydrous caffeine (CDDP + caf), gray square is cisplatin + citric acid (CDDP + CA), circle is cisplatin + caffeine citrate (CDDP + cafCA) ). The CDDP + cafCA group was significantly different from all other groups at each cisplatin concentration (p <0.01). The result of the WST-8 assay using the soft tissue sarcoma cell line (HT1080) in Example 1. The vertical axis represents cell viability, and the horizontal axis represents cisplatin concentration. A diamond (♦) indicates CDDP alone, a black square (■) indicates CDDP + caf, a gray square indicates CDDP + CA, and a circle indicates CDDP + cafCA. The CDDP + cafCA group was significantly different from all other groups at each cisplatin concentration (p <0.01). The result of the WST-8 assay using the lung cancer cell line (RERF-LC-AI) in Example 1. The vertical axis represents cell viability, and the horizontal axis represents cisplatin concentration. A diamond (♦) indicates CDDP alone, a black square (■) indicates CDDP + caf, a gray square indicates CDDP + CA, and a circle indicates CDDP + cafCA. The CDDP + cafCA group was significantly different from all other groups at each cisplatin concentration (p <0.01). The result of the WST-8 assay using the gastric cancer cell line (MKN45) in Example 1. The vertical axis represents cell viability, and the horizontal axis represents cisplatin concentration. A diamond (♦) indicates CDDP alone, a black square (■) indicates CDDP + caf, a gray square indicates CDDP + CA, and a circle indicates CDDP + cafCA. The CDDP + cafCA group was significantly different from all other groups at each cisplatin concentration (p <0.01). The result of having analyzed the result of the WST-8 assay using the osteosarcoma cell line (HOS) in Example 1 by the Isobologram method. In the graph in the figure, the vertical axis represents the CDDP concentration of CDDP + caf (μM), and the horizontal axis represents the CDDP concentration of CDDP + CA (μM). X represents Effective Dose 50 (ED50), + represents Effective Dose 75 (ED75), and ● represents Effective Dose 90 (ED90). X, +, and ● on the vertical axis represent each effective dose of CDDP + caf, and x, +, and ● on the horizontal axis represent each effective dose of CDDP + CA, and x, +, and ● surrounded by a circular broken line are Each effective dose of CDDP + cafCA is shown. A synergistic effect is observed when the circular broken line (Effective Dose of CDDP + cafCA) is located on the lower left side of the curve connecting CDDP + caf and Effective Dose of CDDP + CA. In the table in the figure, CI (Combination Index), which is an index of synergistic effect, is expressed using CDDP + caf Effective Dose, CDDP + CA Effective Dose and CDDP + caf Effective Dose shown in the above graph, and CDDP + caf Effective Dose fCDDP + a Was calculated as CDDP + cafCA. A synergistic effect is observed when CI <1.0. The result of having analyzed the result of the WST-8 assay using the soft tissue sarcoma cell line (HT1080) in Example 1 by the Isobologram method. In the graph in the figure, the vertical axis represents the CDDP concentration of CDDP + caf (μM), and the horizontal axis represents the CDDP concentration of CDDP + CA (μM). X represents ED50, + represents ED75, and ● represents ED90. X, +, and ● on the vertical axis represent each effective dose of CDDP + caf, and x, +, and ● on the horizontal axis represent each effective dose of CDDP + CA, and x, +, and ● surrounded by a circular broken line are Each effective dose of CDDP + cafCA is shown. A synergistic effect is observed when the circular broken line (Effective Dose of CDDP + cafCA) is located at the lower left of the curve connecting the Effective Dose. In the table in the figure, CI uses CDDP + ca and CDDP + CA as a combination of CDDP + caf and CDDP + CA by using each effective dose of CDDP + caf, each effective dose of CDDP + CA, and each effective dose of CDDP + cafCA shown in the above graph. A synergistic effect is observed when CI <1.0. The result of having analyzed the result of the WST-8 assay using the lung cancer cell line (RERF-LC-AI) in Example 1 by the Isobologram method. In the graph in the figure, the vertical axis represents the CDDP concentration of CDDP + caf (μM), and the horizontal axis represents the CDDP concentration of CDDP + CA (μM). X represents ED50, + represents ED75, and ● represents ED90. X, +, and ● on the vertical axis represent each effective dose of CDDP + caf, and x, +, and ● on the horizontal axis represent each effective dose of CDDP + CA, and x, +, and ● surrounded by a circular broken line are Each effective dose of CDDP + cafCA is shown. A synergistic effect is observed when the circular broken line (Effective Dose of CDDP + cafCA) is located at the lower left of the curve connecting the Effective Dose. In the table in the figure, CI uses CDDP + ca and CDDP + CA as a combination of CDDP + caf and CDDP + CA by using each effective dose of CDDP + caf, each effective dose of CDDP + CA, and each effective dose of CDDP + cafCA shown in the above graph. A synergistic effect is observed when CI <1.0. The result of having analyzed the result of the WST-8 assay using the gastric cancer cell line (MKN45) in Example 1 by the Isobologram method. In the graph in the figure, the vertical axis represents the CDDP concentration of CDDP + caf (μM), and the horizontal axis represents the CDDP concentration of CDDP + CA (μM). X represents ED50, + represents ED75, and ● represents ED90. X, +, and ● on the vertical axis represent each effective dose of CDDP + caf, and x, +, and ● on the horizontal axis represent each effective dose of CDDP + CA, and x, +, and ● surrounded by a circular broken line are Each effective dose of CDDP + cafCA is shown. A synergistic effect is observed when the circular broken line (Effective Dose of CDDP + cafCA) is located at the lower left of the curve connecting the Effective Dose. In the table in the figure, CI uses CDDP + ca and CDDP + CA as a combination of CDDP + caf and CDDP + CA by using each effective dose of CDDP + caf, each effective dose of CDDP + CA, and each effective dose of CDDP + cafCA shown in the above graph. A synergistic effect is observed when CI <1.0. The results of the WST-8 assay for the osteosarcoma cell line (HOS), soft tissue sarcoma cell line (HT1080), lung cancer cell line (RERF-LC-AI) and gastric cancer cell line (MKN45) in Example 1 were compared with the median effect method and Isobologram. The figure which summarized the result analyzed by the method. As shown in the figure, when CI (Combination Index) <1.0, it indicates a synergistic effect, when CI = 1.0, an additive effect, and when CI> 1.0, it indicates an antagonistic effect. In each figure, in each series, the bars are shown in order of HOS, HT1080, RERF-LC-AI, and MKN45 from the left. The result of the WST-8 assay using the hepatocellular carcinoma cell line (HepG2) in Example 2. The way of viewing the graph is the same as in FIG. The analysis result of the synergistic effect using the hepatoma cell line (HepG2) by the Median Effect method in Example 2. FIG. The result of having analyzed the result of the WST-8 assay using the hepatocellular carcinoma cell line (HepG2) in Example 2 by the Isobologram method. The way of viewing the graph and table is the same as in FIG. The result of the WST-8 assay using the uterine cancer cell line (Hela) in Example 2. The way of viewing the graph is the same as in FIG. The analysis result of the synergistic effect using the uterine cancer cell line (HepG2) by the Median Effect method in Example 2. FIG. The result of having analyzed the result of the WST-8 assay using the uterine cancer cell line (HepG2) in Example 2 by the Isobologram method. The way of viewing the graph and table is the same as in FIG. The result of the WST-8 assay using the breast cancer cell line (MCF7) in Example 2. The way of viewing the graph is the same as in FIG. The analysis result of the synergistic effect using the breast cancer cell line (HepG2) by the Median Effect method in Example 2. FIG. The result of having analyzed the result of the WST-8 assay using the breast cancer cell line (MCF7) in Example 2 by the Isobologram method. The way of viewing the graph and table is the same as in FIG. The result of the WST-8 assay using the ovarian cancer cell line (OVCAR-3) in Example 2. The way of viewing the graph is the same as in FIG. The analysis result of the synergistic effect using the ovarian cancer cell line (HepG2) by the Median Effect method in Example 2. FIG. The result of having analyzed the result of the WST-8 assay using the ovarian cancer cell line (OVCAR-3) in Example 2 by the Isobologram method. The way of viewing the graph and table is the same as in FIG. The result of the WST-8 assay using the osteosarcoma cell line (HOS) in Example 3. The vertical axis represents cell viability, and the horizontal axis represents adriamycin concentration (ADM concentration). Diamonds (♦) are adriamycin (ADM) alone, black squares (■) are adriamycin + anhydrous caffeine (ADM + caf), gray squares are adriamycin + citric acid (ADM + CA), circles are adriamycin + caffeine citrate (ADM + cafCA) ). The ADM + cafCA group was significantly different from all other groups at each adriamycin concentration (p <0.01). The analysis result of the synergistic effect using the osteosarcoma cell line (HOS) by the Median Effect method in Example 3. FIG. The result of having analyzed the result of the WST-8 assay using the osteosarcoma cell line (HOS) in Example 3 by the Isobologram method. In the graph in the figure, the vertical axis represents the ADM + caf ADM concentration (nM), and the horizontal axis represents the ADM + CA ADM concentration (nM). X represents Effective Dose 50 (ED50), + represents Effective Dose 75 (ED75), and ● represents Effective Dose 90 (ED90). X, +, and ● on the vertical axis indicate each effective dose of ADM + caf, and x, +, and ● on the horizontal axis indicate each effective dose of ADM + CA, and X, +, and ● surrounded by a circular broken line are Each effective dose of ADM + cafCA is shown. A synergistic effect is observed when the circular broken line (Effective Dose of ADM + cafCA) is located on the lower left side of the curve connecting ADM + caf and Effective Dose of ADM + CA. In the table in the figure, CI (Combination Index), which is an index of synergistic effect, is expressed by using ADM + cf Effective Dose, ADM + CA Effective Dose, and ADM + caf Effective Dose shown in the above graph, ADM + c + Was calculated as ADM + cafCA. A synergistic effect is observed when CI <1.0. The result of the WST-8 assay using the soft tissue sarcoma cell line (HT1080) in Example 3. The way of viewing the graph is the same as in FIG. The analysis result of the synergistic effect using the soft tissue sarcoma cell line (HT1080) by the Median Effect method in Example 3. FIG. The result of having analyzed the result of the WST-8 assay using the osteosarcoma cell line (HOS) in Example 3 by the Isobologram method. The way of viewing the graph and the table is the same as in FIG. The result of the WST-8 assay using the gastric cancer cell line (MKN45) in Example 3. The way of viewing the graph is the same as in FIG. The analysis result of the synergistic effect using the gastric cancer cell line (MKN45) by the Median Effect method in Example 3. The result of having analyzed the result of the WST-8 assay using the gastric cancer cell line (MKN45) in Example 3 by Isobologram method. The way of viewing the graph and the table is the same as in FIG. The result of the WST-8 assay using the uterine cancer cell line (Hela) in Example 3. The way of viewing the graph is the same as in FIG. The analysis result of the synergistic effect using the uterine cancer cell line (Hela) by the Median Effect method in Example 3. FIG. The result of having analyzed the result of the WST-8 assay using the uterine cancer cell line (Hela) in Example 3 by the Isobologram method. The way of viewing the graph and the table is the same as in FIG. The result of the WST-8 assay using the osteosarcoma cell line (HOS) in Example 4. The vertical axis represents cell viability, and the horizontal axis represents gemcitabine concentration (GEM concentration). Diamond (♦) is gemcitabine (GEM) alone, black square (■) is gemcitabine + anhydrous caffeine (GEM + caf), gray square is gemcitabine + citric acid (GEM + CA), circle is gemcitabine + caffeine citrate (GEM + cafCA) ). The GEM + cafCA group was significantly different from all other groups at each gemcitabine concentration (p <0.01). The analysis result of the synergistic effect using the osteosarcoma cell line (HOS) by the Median Effect method in Example 4. The result of having analyzed the result of the WST-8 assay using the osteosarcoma cell line (HOS) in Example 4 by the Isobologram method. In the graph in the figure, the vertical axis indicates the GEM + caf GEM concentration (nM), and the horizontal axis indicates the GEM + CA GEM concentration (nM). X represents Effective Dose 50 (ED50), + represents Effective Dose 75 (ED75), and ● represents Effective Dose 90 (ED90). X, +, and ● on the vertical axis indicate GEM + caf Effective Dose, and x, +, and ● on the horizontal axis indicate GEM + CA Effective Dose, and X, +, and ● surrounded by a circular broken line are Each effective dose of GEM + cafCA is shown. A synergistic effect is recognized when the circular broken line (Effective Dose of GEM + cafCA) is located on the lower left side of the curve connecting GEM + caf and Effective Dose of GEM + CA. In the table in the figure, CI (Combination Index), which is an index of synergistic effect, uses GEM + caf Effective Dose, GEM + CA Effective Dose and GEM + caf Effective Dose shown in the graph above, GEM + cf, GEM + c + GEM + cf Was calculated as GEM + cafCA. A synergistic effect is observed when CI <1.0. The result of the WST-8 assay using the gastric cancer cell line (MKN45) in Example 4. The way of viewing the graph is the same as in FIG. The analysis result of the synergistic effect using the gastric cancer cell line (MKN45) by the Median Effect method in Example 4. The result of having analyzed the result of the WST-8 assay using the gastric cancer cell line (MKN45) in Example 4 by the Isobologram method. The way of viewing the graph and table is the same as in FIG. The result of the WST-8 assay using the ovarian cancer cell line (OVCAR-3) in Example 4. The way of viewing the graph is the same as in FIG. The analysis result of the synergistic effect using the ovarian cancer cell line (OVCAR-3) by the Median Effect method in Example 4. The result of having analyzed the result of the WST-8 assay using the ovarian cancer cell line (OVCAR-3) in Example 4 by the Isobologram method. The way of reading the table is the same as in FIG. Results of cell proliferation (EdU) assay using osteosarcoma cell line (HOS) in Example 5. The result of taking a photograph for a representative fixed point visual field of each group is shown. Hoechst is a photographed result of Hoechst detection, Hoechst + EdU is an image obtained by combining the photographed results of Hoechst detection and EdU detection, and EdU is a photographed result of EdU detection. Results of cell proliferation (EdU) assay using the soft tissue sarcoma cell line (HT1080) in Example 5. The result of taking a photograph for a representative fixed point visual field of each group is shown. Hoechst is a photographed result of Hoechst detection, Hoechst + EdU is an image obtained by combining the photographed results of Hoechst detection and EdU detection, and EdU is a photographed result of EdU detection. The analysis result of the significant difference of the EdU coloring rate in the cell proliferation (EdU) assay using the osteosarcoma cell line (HOS) in Example 5, and a soft tissue sarcoma cell line (HT1080). * Indicates p <0.05, and ** indicates p <0.01. The result of the mitochondrial membrane potential change (TMRE) assay using the osteosarcoma cell line (HOS) in Example 6. The result of taking a photograph for a representative fixed point visual field of each group is shown. Hoechst is a photographed result of Hoechst detection, Hoechst + TMRE is an image obtained by combining the photographed results of Hoechst detection and TMRE detection, and TMRE is a photographed result of TMRE detection. The result of a mitochondrial membrane potential change (TMRE) assay using the soft tissue sarcoma cell line (HT1080) in Example 6. The result of taking a photograph for a representative fixed point visual field of each group is shown. Hoechst is a photographed result of Hoechst detection, Hoechst + TMRE is an image obtained by combining the photographed results of Hoechst detection and TMRE detection, and TMRE is a photographed result of TMRE detection. The analysis result of the significant difference of the TMRE brightness | luminance per cell in the mitochondrial membrane potential change (TMRE) assay using the osteosarcoma cell line (HOS) in Example 6, and a soft tissue sarcoma cell line (HT1080). The vertical axis shows the ratio of TMRE luminance per cell. * Indicates p <0.05, and ** indicates p <0.01. The result of the WST-8 assay using the skin cancer cell line (A375) in Example 7. The vertical axis represents cell viability, and the horizontal axis represents cisplatin concentration (CDDP concentration). Diamonds (♦) indicate cisplatin (CDDP) alone, triangles (▲) indicate cisplatin + anhydrous caffeine (CDDP + caf), squares indicate cisplatin + citric acid (CDDP + CA), and circles indicate cisplatin + caffeine citrate (CDDP + cafCA). The CDDP + cafCA group was significantly different from all other groups at each cisplatin concentration (p <0.01). The result of the WST-8 assay using the pancreatic cancer cell line (PANC-1) in Example 7. The vertical axis represents cell viability, and the horizontal axis represents cisplatin concentration (CDDP concentration). Diamonds (♦) indicate cisplatin (CDDP) alone, triangles (▲) indicate cisplatin + anhydrous caffeine (CDDP + caf), squares indicate cisplatin + citric acid (CDDP + CA), and circles indicate cisplatin + caffeine citrate (CDDP + cafCA). The CDDP + cafCA group was significantly different from all other groups at each cisplatin concentration (p <0.01). The result of the WST-8 assay using the cancer-bearing cell line (TFK-1) in Example 7. The vertical axis represents cell viability, and the horizontal axis represents cisplatin concentration (CDDP concentration). Diamonds (♦) indicate cisplatin (CDDP) alone, triangles (▲) indicate cisplatin + anhydrous caffeine (CDDP + caf), squares indicate cisplatin + citric acid (CDDP + CA), and circles indicate cisplatin + caffeine citrate (CDDP + cafCA). The CDDP + cafCA group was significantly different from all other groups at each cisplatin concentration (p <0.01). The result of the WST-8 assay using the fibrosarcoma cell line (HT1080) in Example 8. The vertical axis represents cell viability, and the horizontal axis represents gemcitabine concentration (GEM concentration). Diamonds (♦) indicate gemcitabine (GEM) alone, triangles (▲) indicate gemcitabine + anhydrous caffeine (GEM + caf), squares indicate gemcitabine + citric acid (GEM + CA), and circles indicate gemcitabine + caffeine citrate (GEM + cafCA). The GEM + cafCA group was significantly different from all other groups at each gemcitabine concentration (p <0.01). The result of the WST-8 assay using the skin cancer cell line (A375) in Example 8. The vertical axis represents cell viability, and the horizontal axis represents gemcitabine concentration (GEM concentration). Diamonds (♦) indicate gemcitabine (GEM) alone, triangles (▲) indicate gemcitabine + anhydrous caffeine (GEM + caf), squares indicate gemcitabine + citric acid (GEM + CA), and circles indicate gemcitabine + caffeine citrate (GEM + cafCA). The result of the WST-8 assay using the pancreatic cancer cell line (PANC-1) in Example 8. The vertical axis represents cell viability, and the horizontal axis represents gemcitabine concentration (GEM concentration). Diamonds (♦) indicate gemcitabine (GEM) alone, triangles (▲) indicate gemcitabine + anhydrous caffeine (GEM + caf), squares indicate gemcitabine + citric acid (GEM + CA), and circles indicate gemcitabine + caffeine citrate (GEM + cafCA). The GEM + cafCA group was significantly different from all other groups at each gemcitabine concentration (p <0.01). The result of the WST-8 assay using the cancer-bearing cell line (TFK-1) in Example 8. The vertical axis represents cell viability, and the horizontal axis represents gemcitabine concentration (GEM concentration). Diamonds (♦) indicate gemcitabine (GEM) alone, triangles (▲) indicate gemcitabine + anhydrous caffeine (GEM + caf), squares indicate gemcitabine + citric acid (GEM + CA), and circles indicate gemcitabine + caffeine citrate (GEM + cafCA). The GEM + cafCA group was significantly different from all other groups at each gemcitabine concentration (p <0.01). Administration schedule in Example 9. The change of the tumor volume of the nude mouse which transplanted LM8 cell which is a mouse | mouth osteosarcoma cell line in Example 9 subcutaneously on the back. The tumor weight of the 15th day of the nude mouse which transplanted LM8 cell derived from the mouse | mouth in Example 9 subcutaneously in the back part. Transition of tumor volume of nude mice transplanted with human-derived HT1080 cells subcutaneously in the back in Example 9. The tumor weight of the 15th day of the nude mouse which transplanted the human-derived HT1080 cell in Example 9 to the back subcutaneously. The transition of the tumor volume of the nude mouse which transplanted the LM8 cell derived from the mouse | mouth characterized by the low dose of caffeine citrate in Example 9 to the back subcutaneously. The tumor weight of the 15th day of the nude mouse | mouth which transplanted the LM8 cell derived from the mouse | mouth characterized by the low dose of caffeine citrate in Example 9 to the back subcutaneously. The change of the tumor volume of the nude mouse which transplanted the LM8 cell derived from the mouse | mouth characterized by the low dosage of the anticancer agent in Example 9 to the back subcutaneously. The tumor weight of the 15th day of the nude mouse which transplanted the LM8 cell derived from the mouse | mouth characterized by the low dosage of the anticancer agent in Example 9 to the back subcutaneously. Change in body weight of nude mice (not cancer-bearing mice) in Example 10. The measurement date of the upper left graph is the first day (before drug administration), and the measurement date of the upper right graph is the 22nd day (after the end of the third course). The upper row shows the results of using CDDP as an anticancer agent, and the lower row shows the results of using ADM as an anticancer agent.

  The present invention relates to "a cancer therapeutic agent characterized by combining caffeine citrate as an active ingredient with an anticancer agent as an active ingredient" and "an anticancer agent as an active ingredient and caffeine citrate as an active ingredient" Is a method for treating cancer, comprising administering to a cancer patient.

(Caffeine citrate)
In the present invention, caffeine citrate (caffeine citrate) is an anhydrous caffeine represented by the following formula (1) and citric acid (2) and / or citric acid (2 ′). Containing citric acid hydrate). Here, citric acid represented by formula (2) and formula (2 ′) means a mixture of citric acid represented by formula (2) and citric acid represented by formula (2 ′). .
The ratio of anhydrous caffeine and citric acid in caffeine citrate is a molar ratio: anhydrous caffeine: citric acid = 0.1: 9.9 to 9.9: 0.1, preferably 1: 9 to 9 : 1, more preferably 1: 1.
As the caffeine citrate, a commercially available caffeine citrate may be used. For example, the trade name “Respia (registered trademark)” is marketed by Nobel Pharma Co., Ltd.
In addition, as a compound similar to anhydrous caffeine (anhydrous caffeine analog), caffeine hydrate, sodium benzoate caffeine (so-called Annaka) and the like can be used, and since caffeine is a xanthine derivative, xanthines Xanthine-based nerve center stimulants (phenethylin, proventferrin), xanthine-based vasodilators (pentoxyferrin, naxiferin, xanthinol nicotinate, pentifylline, lorophilin, tonapoferrin), xanthine diuretics (theobromine, pamaprom), etc. Can also be used.
As a compound similar to citric acid (citric acid analog), potassium citrate, lithium citrate, copper citrate, ammonium iron citrate, and hydrates thereof can also be used.
Therefore, a combination of a compound similar to anhydrous caffeine and a compound similar to citric acid can be used as a drug similar to caffeine citrate.

Although caffeine citrate has not been reported on effects on cancer types and combined use with anticancer agents, it is used as a therapeutic agent for premature infant apnea attacks. That is, caffeine citrate is a therapeutic agent that is already used in other diseases and its safety is well established.
Thus, caffeine citrate is an agent that has already been prescribed in insurance practice, and is therefore advantageous in early clinical applications compared to new agents.

(Anticancer agent)
The anticancer agent as an active ingredient of the cancer therapeutic agent of the present invention is preferably cisplatin, adriamycin (doxorubicin), ifosfamide or gemcitabine.

(Cisplatin)
Cisplatin is an anticancer agent that inhibits DNA replication by binding to the N-7 position of guanine and adenine in DNA and forming a bridge in the DNA strand.
As cisplatin, a commercially available product can be used. For example, it is marketed by Nippon Kayaku Co., Ltd. under the trade name “IA Coal”.
(Adriamycin)
Adriamycin is an anticancer agent that suppresses both DNA and RNA biosynthesis by intercalating between DNA base pairs of tumor cells and inhibiting DNA synthase and RNA synthase.
Commercially available adriamycin can be used. For example, it is commercially available from Kyowa Hakko Kirin Co., Ltd. under the trade name “Adriacin”.
(Ifosfamide)
Ifosfamide is an anticancer agent that inhibits DNA replication by alkylating guanine in DNA and forming a bridge between DNA double strands.
A commercially available product can be used as ifosfamide. For example, it is marketed by Shionogi Pharmaceutical Co., Ltd. under the trade name “Ihomide”.
(Gemcitabine)
Gemcitabine is an anticancer agent that induces apoptosis in DNA synthesis of tumor cells by incorporating it into the DNA strand in the same manner as cytidine having a similar structure and stopping DNA strand elongation.
A commercially available gemcitabine can be used. For example, it is marketed by Eli Lilly Japan Co., Ltd. under the trade name “Gemzar”.

Cisplatin is a platinum preparation, and the same platinum preparation such as carboplatin, oxaliplatin, nedaplatin and the like is expected to have the same effect.
Doxorubicin is an anticancer antibiotic, and the same anticancer antibiotics such as actinomycin D, pirarubicin, daunorubicin and the like are expected to have the same effect.
Ifosfamide is an alkylating agent, and the same alkylating agent such as cyclophosphamide, busulfan, dacarbazine and the like is expected to have the same effect.
Gemcitabine is an antimetabolite and the same antimetabolite is expected to have the same effect.
In addition to the above, similar effects are expected in all types of drugs that inhibit DNA synthesis including plant alkaloids such as vincristine and etoposide.

(Cancer type)
The target cancer types of the cancer therapeutic agent and cancer treatment method of the present invention are primary malignant bone tumor, soft tissue sarcoma, lung cancer, stomach cancer, breast cancer, uterine cancer, colon cancer, skin cancer, ovarian cancer, pancreatic cancer, bile duct cancer or liver Examples include cell cancer.
The primary malignant bone tumor is not particularly limited, and examples thereof include osteosarcoma, chondrosarcoma, Ewing sarcoma, and bone undifferentiated pleomorphic sarcoma.
The soft tissue sarcoma is not particularly limited, and examples thereof include fibrosarcoma, synovial sarcoma, leiomyosarcoma, liposarcoma, rhabdomyosarcoma, undifferentiated pleomorphic sarcoma, extraosseous osteosarcoma, extraosseous Ewing sarcoma, and the like. Can be mentioned.
Although it does not specifically limit as lung cancer, For example, non-small cell lung cancer, such as adenocarcinoma, squamous cell carcinoma, and large cell cancer, small cell lung cancer, etc. are mentioned.
Uterine cancer is not particularly limited, and examples thereof include cervical cancer and endometrial cancer.
Although it does not specifically limit as skin cancer, For example, malignant melanoma, basal cell carcinoma, squamous cell carcinoma etc. are mentioned.
In addition, the cancer types mentioned in this specification also include all squamous cell carcinomas in organs and organs in which those cancers occur. For example, gastric cancer includes squamous cell carcinoma in the stomach.

(Administration method or cancer treatment method of the cancer therapeutic agent of the present invention)
The administration method of the cancer therapeutic agent of the present invention or the dosage and administration schedule in the cancer treatment method can be adjusted depending on the required amount for each individual treatment target, the treatment method, the disease or the degree of necessity. Specifically, the dosage can be determined according to age, weight, general health condition, sex, diet, administration time, administration method, excretion rate, combination of drugs, patient condition, etc. It may be determined in consideration of these factors.
Although the following can be illustrated, it is not specifically limited.
(1) One dose Caffeine citrate (total of administration for 3 days): 0.1 to 10,000 mg / m ² body surface area, preferably 1 to 10000 mg / m ² body surface area, more preferably 50 to 9000 mg / ^{m 2} body surface area anticancer: cisplatin (administration 1 day) is, 0.1 to 150 mg / ^{m 2} body surface area, preferably 10 to 150 mg / ^{m 2} body surface area, more preferably 60 to 120 mg / m ^Two- body surface area. Doxorubicin (total for 2 days administration) is 0.1 to 120 mg / m ² body surface area, preferably 10 to 90 mg / m ² body surface area, more preferably 30 to 60 mg / m ² body surface area. Ifosfamide (total for 3 to 5 days administration) is 0.1 to 30 mg / m ² body surface area, preferably 1 to 20 mg / m ² body surface area, more preferably 4.5 to 15 mg / m ² body surface area. . Gemcitabine (administered one day) it is, 0.1 to 1000 mg / m @ 2 of body surface area, preferably 10 to 900 mg / ^{m 2} body surface area, more preferably 675~900 mg / ^{m 2} body surface area.
(2) Administration interval Caffeine citrate: Administered 1 to 70 times for 10 weeks, preferably administered 1 to 2 times for 2 days, more preferably administered once a day. Specifically, it is administered 1 to 10 times a day for 1 to 30 consecutive days as 1 course for 1 to 10 weeks, preferably 1 to 3 times daily for 1 to 15 days taking 1 course as 1 course, More preferably, once a day for 3 to 7 consecutive days with 1 to 2 weeks as one course.
Anticancer agent: administration once per 10 to 10 weeks, preferably once per 2 to 8 weeks, more preferably once per 3 to 6 weeks.
(3) Concomitant administration Caffeine citrate and anticancer agent are administered simultaneously, administered several hours apart, administered one day apart, administered several days apart, several weeks apart Either administration after an interval or administration after an interval of several months may be used.
Further, the caffeine citrate and the anticancer agent may be mixed with a physiologically acceptable solvent, carrier, excipient, binder, etc. to form a pharmaceutical composition, and then the composition may be administered to the patient. . In addition, the caffeine citrate and / or the anticancer agent in the present invention also includes prodrugs or pharmacologically acceptable salts thereof.
Although it does not specifically limit as a solvent, For example, water, alcohol, physiological saline, etc. are mentioned.
Although it does not specifically limit as a support | carrier, For example, an acid, a base, a buffer solution, a stabilizer, an isotonizing agent, a preservative, etc. are mentioned.
Although it does not specifically limit as an excipient | filler, For example, a filler, a coagulant, a slipping agent, a disintegrating agent, a pigment | dye, a sweetener, a fragrance | flavor, a coating agent etc. are mentioned.
The binder is not particularly limited. For example, water, ethanol, propanol, simple syrup, glucose solution, starch solution, gelatin solution, carboxymethyl cellulose, hydroxypropyl cellulose, hydroxypropyl starch, methyl cellulose, ethyl cellulose, shellac, calcium phosphate, polyvinyl Examples include pyrrolidone and the like.
Further, when the caffeine citrate and the anticancer agent are separately formulated, the separately formulated product may be mixed with a diluent at the time of use to make a mixture, and then the mixture may be administered to the patient. Good.
In the method for administering a cancer therapeutic agent or the method for treating cancer according to the present invention, it can be administered orally or parenterally. For oral administration, known dosage forms for administration such as tablets, capsules, coated tablets, troches, solutions such as solutions or suspensions can be used. Parenteral administration includes intravenous, intramuscular, or subcutaneous administration by injection, transmucosal administration such as nasal cavity or oral cavity using spray or aerosol, rectal administration using suppositories, patches or liniments. And transdermal administration using gel and gel. Preferred examples include oral administration, nasal administration, and intravenous administration by injection.

  The present invention will be specifically described with reference to the following examples, but the present invention is not limited thereto.

[Evaluation of enhanced cisplatin effect of caffeine citrate in cultured cells of osteosarcoma, soft tissue sarcoma, lung cancer and gastric cancer]
The following methods were used to evaluate the enhancement of anticancer drug effects of caffeine citrate against osteosarcoma, soft tissue sarcoma, lung cancer, and gastric cancer.

<Material>
-Cell line Human-derived HOS cells (obtained from the American Type Culture Collection (ATCC)) were used as osteosarcoma.
As the soft tissue sarcoma, human-derived HT1080 cells (obtained from ATCC) were used.
As lung cancer, human-derived RERF-LC-AI cells (obtained from the National Institute for Science and Technology Bioresource Center (RIKEN BRC) via the National Bioresource Project of the Ministry of Education, Culture, Sports, Science and Technology) were used.
As gastric cancer, human-derived MKN45 cells (obtained from RIKEN BRC through the Ministry of Education, Culture, Sports, Science and Technology's National BioResource Project) were used.
-Anticancer agent Cisplatin (obtained from Wako Pure Chemical Industries, Ltd., hereinafter sometimes referred to as CDDP) was used.
Concomitant drugs Caffeine citrate (Caffeine citrate, Nobel Pharma Co., Ltd., trade name: Respia (registered trademark), hereinafter may be referred to as cafCA), anhydrous caffeine (Wako Pure Chemical Industries, Ltd., hereinafter) , Or caf) or citric acid (Wako Pure Chemical Industries, Ltd., hereinafter may be referred to as CA) was used.

<WST-8 assay>
(1) For each of the above cancer type cultured cells, HOS cells, RERF-LC-AI cells, and MKN45 cells are Roswell Park Memorial Institute (RPMI) 1640 medium, and HT1080 cells are Dulbecco's modified Eagle medium (DMEM). The cells were cultured in a 96-well culture plate for 24 hours at 37 ° C. and 5% CO ₂ at a concentration of 3000 to 5000 cells / 100 μL.
(2) After culturing, each cancer type cultured cell was divided into four groups, and 100 μL of drug (CDDP, CDDP + caf, CDDP + CA, CDDP + cafCA) was added. The CDDP concentration of the drug added to each group is 0.125, 0.00 by dissolving a CDDP powder in water (sterilized and purified water) to prepare a high concentration, and then diluting with a culture solution according to the cells. Prepared to 25, 0.5, 1.0, 2.0 μM. The concentrations of caf and CA, which are concomitant drugs in each group, were uniformly adjusted to 0.5 mM by dissolving powders in water and preparing high concentrations, respectively, and then diluting with a culture solution according to the cells. Since the product was a liquid, the concentration of cafCA was diluted as it was with a culture solution according to the cells and adjusted to 0.5 mM, which was the same as for caf and CA. As a control, no drug was added to some wells (CDDP concentration 0 μM).
(3) 72 hours after drug administration, color was developed with Cell Counting Kit-8 (CCK-8) (Dojindo), and absorbance at 450 nm was measured 2 to 4 hours later. The cell viability was determined from the absorbance ratio, with the absorbance of the control being 1.
(4) The results of (3) were subjected to analysis of variance with Statcel 3 (OMS Publishing), and the significant difference in cell viability between the four groups at each CDDP concentration was analyzed to examine the effect of CDDP + cafCA.
(5) CalcuSyn ver. 2 (BIOSOFT) was analyzed for the synergistic effect of the anticancer agent enhancing effect caused by cafCA and the anticancer agent enhancing effect caused by caf / CA. For the analysis, the median effect method and the isobologram method were used (see: Ting-Chao Chou, Cancer Res 2010; 70: 440-6 .; Tallarida RJ, et al., Life Sci 1989; 45: 947-61 .; Roering SC et al., J Pharmacol Exp Ther 1988; 247: 603-8 .; Tallarida RJ, Chapman & Hall / CRC, Florida, USA, 2000.).
For each CDDP concentration of 0.125, 0.25, 0.5, 1.0, and 2.0 μM, the combination of the caf concentration in CDDP + caf and the CA concentration in CDDP + CA is defined as a combination index (cfCA concentration in CDDP + cafCA). CI) was determined.
Here, CDDP has the same amount and the same concentration before and after the combination. For example, when the CDDP concentration is 1.0 μM, the combination of CDDP (1.0 μM) + caf (0.5 μM) and CDDP (1.0 μM) + CA (0.5 μM) is CDDP (1.0 μM). μM) + cafCA (0.5 μM).
The CI by the median effect method is based on the “CDDP concentration that is considered necessary for 50% cell survival (ED50)” derived from the cell viability at each concentration, and the concentrations of caf, CA, and cafCA. It is a numerical value calculated for the cell viability at that time.
The CI by the Isobologram method is a numerical value calculated using “Effective doses (ED50, ED75, ED90)” of CDDP + caf, CDDP + CA and CDDP + cafCA derived from the cell viability by each CDDP concentration.
When CI <1, the synergistic effect was determined. When CI = 1, it is determined as an additive effect, and when CI> 1, it is determined as an antagonistic effect.

<Results of osteosarcoma>
1. Cell Viability FIG. 1 shows the cell viability based on the 450 nm absorbance of four groups in HOS cells. As is clear from FIG. 1, in HOS cells (osteosarcoma), the CDDP + cafCA group had the lowest cell viability compared to the other groups at all CDDP concentrations.
As a result of analysis of variance, the CDDP + cafCA group was significantly different from all other groups at each cisplatin concentration (p <0.01).
Therefore, it was confirmed that caffeine citrate enhances the anticancer drug effect of cisplatin when added in combination with cisplatin at all cisplatin concentrations of 0.125 to 2.0 μM.

2. Median Effect Method Next, Table 1 shows the analysis result of the synergistic effect in HOS cells by the Median Effect method. CI, which is an index of synergistic effect, was calculated based on the cell viability for each CDDP concentration in FIG. 1 as a combination of CDDP + caf and CDDP + CA as CDDP + cafCA. A synergistic effect is observed when CI <1.0.
As is clear from Table 1, the CDDP + cafCA group had a CI <1.0 at all cisplatin concentrations of 0.125 to 2.0 μM, and a synergistic effect was observed for CDDP + caf and CDDP + CA.

3. Isobologram Method FIG. 5 shows the analysis result of the synergistic effect in HOS cells by the Isobologram method.
In the graphs of the analysis results of the Isobologram method in FIGS. 5 to 8, if a circular broken line indicating each Effective Dose of CDDP + cafCA is on a curve connecting each Effective Dose of CDDP + caf and each Effective Dose of CDDP + CA An additive effect is shown, and if it is in the lower left of the curve, it shows a synergistic effect, and if it is in the upper right of the curve, it shows an antagonistic effect. For example, if the circular broken line indicating the ED50 of CDDP + cafCA is on a curve connecting the ED50 of CDDP + caf and the ED50 of CDDP + CA, an additive effect is shown, and if it is on the lower left of the curve, a synergistic effect is shown. If it is in the upper right, an antagonistic effect is shown. The same applies to ED75 and ED90.
“Effective Dose” in the present analysis indicates the CDDP concentration necessary for reducing each carcinoma cell culture, and is derived from the cell viability for each CDDP concentration. For example, ED50 means the concentration of CDDP required to reduce cells by 50% from the base (to make cell viability 50%), and ED75 reduces cells by 75% (cell viability). ED90 means the CDDP concentration required to reduce the cells by 90% (to bring the cell viability to 10%).
As is clear from the graph of FIG. 5, since the circular broken lines indicating the effective doses of CDDP + cafCA are located at the lower left of the curves connecting the effective doses of CDDP + caf and CDDP + CA, a synergistic effect was recognized. .
As is apparent from the table of FIG. 5, the CDDP + cafCA group had a CI <1.0 at all effective doses of 50 to 90%, and a synergistic effect was observed for CDDP + caf and CDDP + CA.

<Results of soft tissue sarcoma>
1. Cell viability Cell viability based on 450 nm absorbance of four groups in HT1080 cells is shown in FIG. As is clear from FIG. 2, in HT1080 cells (soft tissue sarcoma), the CDDP + cafCA group had the lowest cell viability compared to the other groups at all CDDP concentrations.
As a result of analysis of variance, the CDDP + cafCA group was significantly different from all other groups at each cisplatin concentration (p <0.01).
Therefore, it was confirmed that caffeine citrate enhances the anticancer drug effect of cisplatin when added in combination with cisplatin at all cisplatin concentrations of 0.125 to 2.0 μM.

2. Median Effect Method Table 1 shows the analysis result of the synergistic effect in HT1080 cells by the Median Effect method. CI, which is an index of the synergistic effect, was calculated as a combination of CDDP + caf and CDDP + CA as CDDP + cafCA based on the cell viability for each CDDP concentration in FIG.
As is clear from Table 1, the CDDP + cafCA group had a CI <1.0 at all cisplatin concentrations of 0.125 to 2.0 μM, and a synergistic effect was observed for CDDP + caf and CDDP + CA.

3. Isobologram Method FIG. 6 shows the analysis result of the synergistic effect in HT1080 cells by the Isobologram method.
As is clear from the graph of FIG. 6, since the circular broken lines indicating the effective doses of CDDP + cafCA are located at the lower left of the curves connecting the effective doses of CDDP + caf and CDDP + CA, a synergistic effect was recognized. .
As is apparent from the table of FIG. 6, the CDDP + cafCA group had CI <1 at all effective doses of 50 to 90%, and a synergistic effect was observed for CDDP + caf and CDDP + CA.

<Results of lung cancer>
1. Cell viability Cell viability based on 450 nm absorbance of four groups in RERF-LC-AI cells is shown in FIG. As is clear from FIG. 3, in RERF-LC-AI cells (lung cancer), the CDDP + cafCA group had the lowest cell viability compared to the other groups at all CDDP concentrations.
As a result of analysis of variance, the CDDP + cafCA group was significantly different from all other groups at each cisplatin concentration (p <0.01).
Therefore, it was confirmed that caffeine citrate enhances the anticancer drug effect of cisplatin when added in combination with cisplatin at all cisplatin concentrations of 0.125 to 2.0 μM.

2. Median Effect Method Table 1 shows the results of analysis of the synergistic effect in RERF-LC-AI cells by the Median Effect method. CI, which is an index of synergistic effect, was calculated based on the cell viability for each CDDP concentration in FIG. 3 as a combination of CDDP + caf and CDDP + CA as CDDP + cafCA.
As is clear from Table 1, the CDDP + cafCA group had a CI <1.0 at all cisplatin concentrations of 0.125 to 2.0 μM, and a synergistic effect was observed for CDDP + caf and CDDP + CA.

3. Isobologram Method FIG. 7 shows the analysis result of the synergistic effect in RERF-LC-AI cells by the Isobologram method.
As is clear from the graph of FIG. 7, since the circular broken lines indicating the effective doses of CDDP + cafCA are located at the lower left of the curves connecting the effective doses of CDDP + caf and CDDP + CA, a synergistic effect was recognized. .
As is clear from the table of FIG. 7, the CDDP + cafCA group had CI <1 at all effective doses of 50 to 90%, and a synergistic effect was observed for CDDP + caf and CDDP + CA.

<Results of stomach cancer>
1. Cell viability Cell viability based on 450 nm absorbance of four groups in MKN45 cells is shown in FIG. As is clear from FIG. 4, in MKN45 cells (gastric cancer), the CDDP + cafCA group had the lowest cell viability compared with the other groups at all CDDP concentrations.
As a result of analysis of variance, the CDDP + cafCA group was significantly different from all other groups at each cisplatin concentration (p <0.01).
Therefore, it was confirmed that caffeine citrate enhances the anticancer drug effect of cisplatin when added in combination with cisplatin at all cisplatin concentrations of 0.125 to 2.0 μM.

2. Median Effect Method Table 1 shows the analysis results of the synergistic effect in MKN45 cells by the Median Effect method. CI, which is an index of synergistic effect, was calculated based on the cell viability for each CDDP concentration in FIG. 4 as the combination of CDDP + caf and CDDP + CA as CDDP + cafCA.
As is clear from Table 1, the CDDP + cafCA group had a CI <1.0 at all cisplatin concentrations of 0.125 to 2.0 μM, and a synergistic effect was observed for CDDP + caf and CDDP + CA.

3. Isobologram Method FIG. 8 shows the analysis result of the synergistic effect in MKN45 cells by the Isobologram method.
As is clear from the graph of FIG. 8, a circular broken line indicating each effective dose of CDDP + cafCA is located at the lower left of the curve connecting each effective dose of CDDP + caf and CDDP + CA, and thus a synergistic effect was recognized. .
As is apparent from the table of FIG. 8, the CDDP + cafCA group had CI <1 at all effective doses of 50 to 90%, and a synergistic effect was observed for CDDP + caf and CDDP + CA.

<Comprehensive evaluation>
The experimental results of the Median Effect method and Isobologram method in the above four cell lines are summarized as shown in FIG.
As is clear from FIG. 9, the cancer therapeutic agent of the present invention has a synergistic effect in all the cancer types tested, in which the CDDP + cafCA group has a CI <1.0 by the analysis of both the Median Effect method and the Isobologram method. It was confirmed to have.

  From the above results, in the cancer therapeutic agent of the present invention, the combination of cisplatin and caffeine citrate is significantly more significant than cisplatin alone, cisplatin and anhydrous caffeine, and cisplatin and citric acid. Further enhancing the anticancer agent effect of cisplatin, the anticancer agent enhancing effect of caffeine citrate is not just an additive effect of the anticancer agent enhancing effect of anhydrous caffeine and the anticancer agent enhancing effect of citric acid, It became clear that it was a synergistic effect.

[Evaluation of enhancement of cisplatin effect of caffeine citrate in cultured cells of hepatocellular carcinoma, uterine cancer, breast cancer and ovarian cancer]
The enhancement of the anticancer agent (cisplatin) effect of caffeine citrate against hepatocellular carcinoma, uterine cancer, breast cancer and ovarian cancer was evaluated. The test was performed using the same materials and methods as in Example 1 except that the following cell lines and media were used.

<Material>
-Cell line As hepatocellular carcinoma, human-derived HepG2 cells (obtained from RIKEN BRC through the Ministry of Education, Culture, Sports, Science and Technology's National BioResource Project) were used.
As uterine cancer, human-derived Hela cells (obtained from RIKEN BRC through the Ministry of Education, Culture, Sports, Science and Technology's National BioResource Project) were used.
As breast cancer, human-derived MCF7 cells (obtained from RIKEN BRC through the Ministry of Education, Culture, Sports, Science and Technology's National BioResource Project) were used.
As the ovarian cancer, human-derived OVCAR-3 cells (obtained from RIKEN BRC through the Ministry of Education, Culture, Sports, Science and Technology National BioResource Project) were used.
-Medium As for each of the above cancer type cultured cells, RPMI1640 medium was used for HepG2 cells, Hela cells, MCF7 cells, and OVCAR-3 cells.

<Result>
1. Cell viability Figures 10, 13, 16 and 19 are based on absorbance at 450 nm of four groups (CDDP, CDDP + caf, CDDP + CA, CDDP + cafCA) in HepG2 cells, Hela cells, MCF7 cells and OVCAR-3 cells, respectively. Cell viability is shown. As is clear from FIGS. 10, 13, 16, and 19, in the HepG2 cell (hepatocellular carcinoma), the Hela cell (uterine cancer), the MCF7 cell (breast cancer), and the OVCAR-3 cell (ovarian cancer), the CDDP + cafCA group Had the lowest cell viability compared to the other groups at all CDDP concentrations.
As a result of analysis of variance, the CDDP + cafCA group was significantly different from all other groups at each cisplatin concentration (p <0.01).
Therefore, it was confirmed that caffeine citrate enhances the anticancer drug effect of cisplatin when added in combination with cisplatin at all cisplatin concentrations of 0.125 to 2.0 μM.

2. Median Effect Method Next, the analysis results of the synergistic effect in HepG2 cells, Hela cells, MCF7 cells and OVCAR-3 cells by the Median Effect method are shown in FIGS. 11, 14, 17 and 20. FIG. CI, which is an index of synergistic effect, was calculated as a combination of CDDP + caf and CDDP + CA as CDDP + cafCA based on the cell viability for each CDDP concentration in FIG. 11, FIG. 14, FIG. 17 and FIG. A synergistic effect is observed when CI <1.0.
As is clear from FIGS. 11, 14, 17 and 20, the CDDP + cafCA group has a CI <1.0 at all cisplatin concentrations of 0.125 to 2.0 μM, and for CDDP + caf and CDDP + CA, A synergistic effect was observed.

3. Isobologram method The results of analysis of the synergistic effect in HepG2 cells, Hela cells, MCF7 cells and OVCAR-3 cells by the Isobologram method are shown in FIG. 12, FIG. 15, FIG. 18 and FIG.
As is clear from the graphs of FIGS. 12, 15, 18 and 21, the circular broken lines indicating the effective doses of CDDP + cafCA are located on the lower left of the curves connecting the effective doses of CDDP + caf and CDDP + CA, respectively. Therefore, a synergistic effect was recognized.
As is apparent from the tables of FIGS. 12, 15, 18 and 21, the CDDP + cafCA group has a CI <1.0 at 50 to 90% of all effective doses, and has a synergistic effect on CDDP + caf and CDDP + CA. Was recognized.

[Evaluation of enhanced adriamycin effect of caffeine citrate in cultured cells of osteosarcoma, soft tissue sarcoma, gastric cancer and uterine cancer]
The enhancement of anticancer agent (adriamycin) effect of caffeine catenate on osteosarcoma, soft tissue sarcoma, gastric cancer and uterine cancer was evaluated. The test was performed using the same materials as in Example 1 except that the following cell lines, anticancer agents and medium were used.

<Material>
Cell line Human-derived HOS cells (obtained from ATCC) were used as osteosarcoma.
As the soft tissue sarcoma, human-derived HT1080 cells (obtained from ATCC) were used.
As gastric cancer, human-derived MKN45 cells (obtained from RIKEN BRC through the Ministry of Education, Culture, Sports, Science and Technology's National BioResource Project) were used.
As uterine cancer, human-derived Hela cells (obtained from RIKEN BRC through the Ministry of Education, Culture, Sports, Science and Technology's National BioResource Project) were used.
Anticancer agent Adriamycin (obtained from Wako Pure Chemical Industries, Ltd., hereinafter sometimes referred to as ADM) was used.
-Medium About each said cancer type cultured cell, RPMI1640 medium was used for HOS cell, MKN45 cell, and Hela cell. For HT1080 cells, DMEM was used.

<WST-8 assay>
A test was performed according to the method of Example 1 except that the step (2) was changed as follows.
(2) After culturing, each cancer type cultured cell was divided into four groups, and 100 μL of a drug (ADM, ADM + caf, ADM + CA, ADM + cafCA) was added. The ADM concentration of the drug added to each group is 12.5, 25, by dissolving the powder of ADM in water (sterilized purified water) to make a high concentration, and then diluting with a culture solution according to the cells. Prepared to 50, 100, 200 nM. The concentrations of caf and CA, which are concomitant drugs in each group, were uniformly adjusted to 0.5 mM by dissolving powders in water and preparing high concentrations, respectively, and then diluting with a culture solution according to the cells. Since the product was a liquid, the concentration of cafCA was diluted as it was with a culture solution according to the cells and adjusted to 0.5 mM, which was the same as for caf and CA. As a control, no drug was added to some wells (ADM concentration 0 μM).

<Result>
1. Cell viability FIGS. 22, 25, 28 and 31 show cell survival based on absorbance at 450 nm of four groups (ADM, ADM + caf, ADM + CA, ADM + cafCA) in HOS cells, HT1080 cells, MKN45 cells and Hela cells, respectively. Indicates the rate. As is clear from FIG. 22, FIG. 25, FIG. 28 and FIG. 31, in the HOS cell (osteosarcoma), HT1080 cell (soft tissue sarcoma), MKN45 cell (gastric cancer) and Hela cell (uterine cancer), the ADM + cafCA group is all The cell viability was lowest at the ADM concentration compared to the other groups.
As a result of analysis of variance, the ADM + cafCA group was significantly different from all other groups in each adriamycin concentration (p <0.01).
Therefore, it was confirmed that caffeine citrate enhances the anticancer drug effect of adriamycin by adding it in combination with adriamycin at all adriamycin concentrations of 12.5 to 200 nM.

2. Median Effect Method Next, FIG. 23, FIG. 26, FIG. 29, and FIG. 32 show the analysis results of the synergistic effect in HOS cells, HT1080 cells, MKN45 cells, and Hela cells by the Median Effect method. CI, which is an index of synergistic effect, was calculated as a combination of ADM + caf and ADM + CA as ADM + cafCA based on the cell viability for each ADM concentration in FIGS. 23, 26, 29, and 32. A synergistic effect is observed when CI <1.0.
As is apparent from FIGS. 23, 26, 29 and 32, the ADM + cafCA group has a CI <1.0 at all adriamycin concentrations from 12.5 to 200 nM, and has a synergistic effect on ADM + caf and ADM + CA. Was recognized.

3. Isobologram method FIGS. 24, 27, 30 and 33 show analysis results of synergistic effects in HOS cells, HT1080 cells, MKN45 cells and Hela cells by the Isobologram method.
As is clear from the graphs of FIGS. 24, 27, 30 and 33, the circular broken lines indicating the effective doses of ADM + cafCA are located on the lower left side of the curves connecting the effective doses of ADM + caf and ADM + CA, respectively. Therefore, a synergistic effect was recognized.
As is apparent from the tables of FIGS. 24, 27, 30 and 33, the ADM + cafCA group has a CI <1.0 at all effective doses of 50 to 90%, and has a synergistic effect on ADM + caf and ADM + CA. Was recognized.

  From the above results, in the cancer therapeutic agent of the present invention, the combination of adriamycin and caffeine citrate is significantly more significant than the combination of adriamycin alone, the combination of adriamycin and anhydrous caffeine, and the combination of adriamycin and citric acid. Further enhancing the anticancer drug effect of adriamycin, the anticancer drug potentiating effect of caffeine citrate is not just an additive effect of the anticancer drug potentiating effect of anhydrous caffeine and the anticancer drug potentiating effect of citric acid, It became clear that it was a synergistic effect.

[Evaluation of enhancement of gemcitabine effect of caffeine citrate in cultured cells of osteosarcoma, gastric cancer and ovarian cancer]
The enhancement of anticancer agent (gemcitabine) effect of caffeine citrate against osteosarcoma, gastric cancer and ovarian cancer was evaluated. The test was performed using the same materials as in Example 1 except that the following cell lines, anticancer agents and medium were used.

<Material>
Cell line Human-derived HOS cells (obtained from ATCC) were used as osteosarcoma.
As gastric cancer, human-derived MKN45 cells (obtained from RIKEN BRC through the Ministry of Education, Culture, Sports, Science and Technology's National BioResource Project) were used.
As the ovarian cancer, human-derived OVCAR-3 cells (obtained from RIKEN BRC through the Ministry of Education, Culture, Sports, Science and Technology National BioResource Project) were used.
Anticancer agent Gemcitabine (obtained from Wako Pure Chemical Industries, Ltd., hereinafter sometimes referred to as GEM) was used.
-Medium About each said cancer type cultured cell, RPMI1640 medium was used for HOS cell, MKN45 cell, and OVCAR-3 cell.

<WST-8 assay>
A test was performed according to the method of Example 1 except that the step (2) was changed as follows.
(2) After the culture, each cancer type cultured cell was divided into four groups, and 100 μL of a drug (GEM, GEM + caf, GEM + CA, GEM + cafCA) was added. The GEM concentration of the drug added to each group is 0.5, 1 by dissolving the GEM powder in water (sterilized purified water) to make a high concentration, and then diluting with a culture solution according to the cells. Prepared to 2, 4, 8 nM. The concentrations of caf and CA, which are concomitant drugs in each group, were uniformly adjusted to 0.5 mM by dissolving powders in water and preparing high concentrations, respectively, and then diluting with a culture solution according to the cells. Since the product was a liquid, the concentration of cafCA was diluted as it was with a culture solution according to the cells and adjusted to 0.5 mM, which was the same as for caf and CA. As a control, no drug was added to some wells (GEM concentration 0 μM).

<Result>
1. Cell Viability FIG. 34, FIG. 37 and FIG. 40 show the cell viability based on the absorbance at 450 nm of four groups (GEM, GEM + caf, GEM + CA, GEM + cafCA) in HOS cells, MKN45 cells and OVCAR-3 cells, respectively. As is apparent from FIGS. 34, 37 and 40, in HOS cells (osteosarcoma), MKN45 cells (gastric cancer) and OVCAR-3 cells (ovarian cancer), the GEM + cafCA group is the other group at all GEM concentrations. The cell viability was the lowest compared with.
As a result of analysis of variance, the GEM + cafCA group was significantly different from all other groups in each gemcitabine concentration (p <0.01).
Therefore, it was confirmed that caffeine citrate enhances the anticancer agent effect of gemcitabine by adding it in combination with gemcitabine at all gemcitabine concentrations of 0.5 to 8.0 nM.

2. Median Effect Method Next, FIG. 35, FIG. 38, and FIG. 41 show the analysis results of synergistic effects in HOS cells, MKN45 cells, and OVCAR-3 by the Median Effect method. CI, which is an index of the synergistic effect, was calculated as a combination of GEM + caf and GEM + CA as GEM + cafCA based on the cell viability for each GEM concentration in FIG. 35, FIG. 38 and FIG. A synergistic effect is observed when CI <1.0.
As is apparent from FIGS. 35, 38 and 41, the GEM + cafCA group has a CI <1.0 at all gemcitabine concentrations from 0.5 to 8.0 nM, and has a synergistic effect on GEM + caf and GEM + CA. Admitted.

3. Isobologram method The results of analysis of the synergistic effect in HOS cells, MKN45 cells and OVCAR-3 by the Isobologram method are shown in FIGS. 36, 39 and 42.
As is apparent from the graphs of FIGS. 36 and 39, since the circular broken lines indicating the effective doses of GEM + cafCA are located on the lower left side of the curves connecting the effective doses of GEM + caf and GEM + CA, respectively, there is a synergistic effect. Admitted.
As is clear from the tables of FIGS. 36, 39 and 42, the GEM + cafCA group has a CI <1.0 at all effective doses of 50 to 90%, and a synergistic effect is observed with respect to GEM + caf and GEM + CA. It was.

  From the above results, in the cancer therapeutic agent of the present invention, the combination of gemcitabine and caffeine citrate is significantly more significant than the combination of gemcitabine alone, the combination of gemcitabine and anhydrous caffeine, or the combination of gemcitabine and citric acid. To enhance the anticancer agent effect of gemcitabine, the anticancer agent enhancing effect of caffeine citrate is not just an additive effect of the anticancer agent enhancing effect of anhydrous caffeine and the anticancer agent enhancing effect of citric acid, It became clear that it was a synergistic effect.

[Evaluation of enhancement of cell growth inhibitory effect of caffeine citrate by anticancer agent in cultured cells of osteosarcoma and soft tissue sarcoma]
By the following method, the enhancement of the cell growth inhibitory effect of caffeine catenate anticancer agent against osteosarcoma and soft tissue sarcoma was evaluated.

<Material>
Cell line Human-derived HOS cells (obtained from ATCC) were used as osteosarcoma.
As the soft tissue sarcoma, human-derived HT1080 cells (obtained from ATCC) were used.
-The anticancer agent cisplatin (available from Wako Pure Chemical Industries, Ltd.) was used.
-Concomitant drugs Caffeine citrate (cafCA, citrate caffeine, obtained from Nobel Pharma Co., Ltd.), anhydrous caffeine (caf, obtained from Wako Pure Chemical Industries, Ltd.) or citric acid (CA, Wako Pure Chemical Industries Ltd.) Obtained from the company).
-Medium HMI cells used RPMI1640 medium, and HT1080 cells used DMEM.

<Cell proliferation (EdU) assay>
EdU (5-ethynyl-2′-deoxyuridine) is a nucleoside analog (analog) of thymidine that is incorporated into DNA during active DNA synthesis. That is, by detecting EdU, proliferating cells in which active DNA synthesis is occurring can be detected.
Therefore, in this experiment, by detecting EdU according to the following procedure, the change in cell growth ability due to drug administration was confirmed, and the enhancement of the cell growth inhibitory effect by CDDP + cafCA was examined.
(1) For each cancer type cultured cells described above, at a concentration of 3000 to 5000 pieces / 100 [mu] L each, above the medium, 37 ° C., with _CO 2 5% of the conditions, 24 hours, chamber slides (Lab-Tec (registered (Trademark), Thermo Scientific Fisher).
(2) After the culture, each cancer type cultured cell was divided into five groups (Non-drug, CDDP, CDDP + caf, CDDP + CA, CDDP + cafCA), and the culture solution was replaced. Non-drug group added 0.5 mL of culture solution, and drug-administered group added 0.5 mL of culture solution adjusted so that CDDP was 0.25 μM, caf, CA, and cafCA were each 0.5 mM. did. The CDDP concentration of the drug to be added to each group is adjusted to 0.25 μM by dissolving CDDP powder in water (sterilized purified water) to make a high concentration, and then diluting with a culture solution suitable for the cells. did. The concentrations of caf and CA, which are concomitant drugs in each group, were uniformly adjusted to 0.5 mM by dissolving powders in water and preparing high concentrations, respectively, and then diluting with a culture solution according to the cells. Since the product was a liquid, the concentration of cafCA was diluted as it was with a culture solution according to the cells and adjusted to 0.5 mM, which was the same as for caf and CA. As a control, no drug was added to some wells (Non-drug: 0 μM).
(3) 24 hours after drug addition, Click-iT (registered trademark) Plus EdU Alexa Fluor (registered trademark) 555 Imaging Kit (Thermo Fisher Scientific), and NucBlue (registered trademark) Live Cell Stent Reagent trademark Using probes by life technologies (Thermo Fisher Scientific), the nuclei of cells undergoing DNA synthesis were stained according to the manufacturer's manual (Hoechst (Hoechst 33342): nuclei detected, EdU detection reagent (Alexa Fluor (registered Alexa Fluor (registered Alexa Fluor))) (Trademark) 555): detection of nuclei during DNA synthesis). With a fluorescence microscope (BIOREVO: BZ-9000, KEYENCE), the filter for illuminating Hoechst is DAPI (excitation wavelength: 360 ± 40 nm, dichroic mirror wavelength: 400 nm, absorption wavelength: 460 ± 50 nm), EdU TRITC (excitation wavelength: 540 ± 25 nm, dichroic mirror wavelength: 565 nm, absorption wavelength: 605 ± 55 nm) was used as a filter for illuminating, and five fixed points of each field were photographed. Hoechst and EdU were detected. It can be said that as the EdU detection signal is smaller (weaker) compared to non-drug, cell proliferation is suppressed.
(4) Cells in the screen of the photo in (3) were counted. The EdU color development rate was calculated as follows. EdU color development rate = DNA synthetic cell number (EdU detected cell number) / cell total number (Hoechst detected cell number). The average value of the EdU color development rate of each group of 5 visual fields is calculated, and from the ratio where the average value of Non-drug is 1, dispersion of the significant difference in the EdU color development rate of each group using Statcel 3 (OMS Publishing) analyzed.

<Result>
The results in HOS cells and HT1080 cells are shown in FIGS. 43 and 44, respectively.
As is clear from FIG. 43, in the HOS cells (osteosarcoma), the CDDP + cafCA group had the smallest ratio of the EdU detected cells to the Hoechst detected cells compared to the other groups. That is, in the CDDP + cafCA group, cell growth of HOS cells (osteosarcoma) was most suppressed as compared with other groups.
As is clear from FIG. 44, in the HT1080 cells (soft tissue sarcoma), the CDDP + cafCA group had the lowest ratio of the EdU detection signal to the Hoechst detection signal compared to the other groups. That is, the CDDP + cafCA group most suppressed the cell growth of HT1080 cells (soft tissue sarcoma) as compared with the other groups.
The analysis result of the significant difference of the EdU coloring rate of each group in HOS cell and HT1080 cell is shown in FIG.
As is clear from FIG. 45, the CDDP + cafCA group was significantly different from all other groups in HOS cells and HT1080 cells (p <0.01). In HOS cells, the EdU color development rate of the CDDP + cafCA group is about 51% compared to the non-drug group, about 58% compared to the CDDP group, about 66% compared to the CDDP + caf group, and compared to the CDDP + CA group. About 61%. In HT1080 cells, the EdU color development rate of the CDDP + cafCA group is about 51% compared to the Non-Drug group, about 56% compared to the CDDP group, about 72% compared to the CDDP + caf group, and compared to the CDDP + CA group. It was about 57%, which was greatly reduced.
Therefore, it was confirmed that caffeine citrate enhances the cell growth inhibitory effect of cisplatin when added in combination with cisplatin in osteosarcoma and soft tissue sarcoma.

  From the above results, in the cancer therapeutic agent of the present invention, the combination of cisplatin and caffeine citrate is cisplatin alone, cisplatin and anhydrous caffeine, cisplatin and citric acid in osteosarcoma and soft tissue sarcoma. Compared with the combination, it was found that the cell growth inhibitory effect of cisplatin was significantly enhanced.

[Evaluation of enhancement of apoptosis (cell death) inducing effect of caffeine citrate by anticancer agent in cultured cells of osteosarcoma and soft tissue sarcoma]
By the following method, the enhancement of apoptosis-inducing effect of caffeine citrate anticancer agent against osteosarcoma and soft tissue sarcoma was evaluated.

<Material>
Cell line Human-derived HOS cells (obtained from ATCC) were used as osteosarcoma.
As the soft tissue sarcoma, human-derived HT1080 cells (obtained from ATCC) were used.
-The anticancer agent cisplatin (available from Wako Pure Chemical Industries, Ltd.) was used.
-Concomitant drugs Caffeine citrate (cafCA, citrate caffeine, obtained from Nobel Pharma Co., Ltd.), anhydrous caffeine (caf, obtained from Wako Pure Chemical Industries, Ltd.) or citric acid (CA, Wako Pure Chemical Industries Ltd.) Obtained from the company).
-Medium HMI cells used RPMI1640 medium, and HT1080 cells used DMEM.

<Mitochondrial membrane potential change (TMRE) assay>
TMRE (tetramethylrhodamine, ethyl ester) is a pigment that accumulates in mitochondria where the membrane potential is maintained. When the mitochondrial membrane potential disappears, TMRE is dispersed and the detection signal intensity is weakened. In apoptosis, as an early stage, the outer mitochondrial membrane is destroyed and the membrane potential disappears, so that TMRE accumulation is reduced and the TMRE detection signal intensity is weakened.
Therefore, in this experiment, TMRE was detected by the following procedure to confirm the change in membrane potential due to drug administration, and the enhancement of mitochondrial membrane potential attenuation / disappearance (apoptosis induction effect) by CDDP + cafCA was examined.
(1) For each cancer type cultured cells described above, at a concentration of 3000 to 5000 pieces / 100 [mu] L each, above the medium, 37 ° C., with _CO 2 5% of the conditions, 24 hours, chamber slides (Lab-Tec (registered (Trademark), Thermo Scientific Fisher).
(2) After the culture, each cancer type cultured cell was divided into five groups (Non-drug, CDDP, CDDP + caf, CDDP + CA, CDDP + cafCA), and the culture solution was replaced. Non-drug group added 0.5 mL of culture solution, and drug administration group added 0.5 mL of culture solution adjusted so that CDDP was 0.25 μM, caf, CA and cafCA were 0.5 mM each. did. The CDDP concentration of the drug to be added to each group is adjusted to 0.25 μM by dissolving CDDP powder in water (sterilized purified water) to make a high concentration, and then diluting with a culture solution suitable for the cells. did. The concentrations of caf and CA, which are concomitant drugs in each group, were uniformly adjusted to 0.5 mM by dissolving powders in water and preparing high concentrations, respectively, and then diluting with a culture solution according to the cells. Since the product was a liquid, the concentration of cafCA was diluted as it was with a culture solution according to the cells and adjusted to 0.5 mM, which was the same as for caf and CA. As a control, no drug was added to some wells (Non-drug: 0 μM).
(3) Twenty-four hours after the addition of the drug, the nucleus of the cell undergoing DNA synthesis was stained according to the manufacturer's manual using TMRE-Mitochondral Membrane Potential Assay Kit (Abcam) (Hoechst (Hoechst 33342): Nucleus Detection, TMRE: Detect mitochondria with membrane potential maintained). A filter for illuminating Hoechst with a fluorescence microscope (BIOREVO: BZ-9000, KEYENCE) emits TMRE with DAPI (excitation wavelength: 360 ± 40 nm, dichroic mirror wavelength: 400 nm, absorption wavelength: 460 ± 50 nm). As a filter for this purpose, TRITC (excitation wavelength: 540 ± 25 nm, dichroic mirror wavelength: 565 nm, absorption wavelength: 605 ± 55 nm) was used, and five fixed points of each field were photographed. Detection of Hoechst and TMRE was performed. It can be said that apoptosis is more induced as the TMRE detection signal becomes weaker than non-drug.
(4) Cells in the screen of the photo in (3) were counted. The TMRE luminance per cell was calculated as follows. TMRE luminance per cell = TMRE luminance (detection signal intensity) sum / cell total number (Hoechst detection cell number). The average value of TMRE luminance per cell in each group of 5 visual fields was calculated, and the TMRE per cell of each group was calculated using Statcel 3 (OMS Publishing) from the ratio where the average value of Non-drug was 1. Analysis of variance was performed for significant differences in luminance.

<Result>
The results in HOS cells and HT1080 cells are shown in FIGS. 46 and 47, respectively.
As is clear from FIG. 46, in the HOS cells (osteosarcoma), the CDDP + cafCA group had the weakest TMRE detection signal intensity with respect to the number of Hoechst detected cells as compared with the other groups. That is, the CDDP + cafCA group was confirmed to be most likely to induce apoptosis of HOS cells (osteosarcoma) compared to the other groups.
As is clear from FIG. 47, in HT1080 cells (soft tissue sarcoma), the CDDP + cafCA group had the weakest TMRE detection signal intensity with respect to the number of Hoechst detected cells as compared with the other groups. That is, the CDDP + cafCA group was confirmed to be most likely to induce apoptosis of HT1080 cells (soft tissue sarcoma) compared to other groups.
The analysis result of the significant difference of TMRE brightness | luminance per cell of each group in HOS cell and HT1080 cell is shown in FIG.
As is clear from FIG. 48, the CDDP + cafCA group was significantly different from all other groups in HOS cells and HT1080 cells (p <0.05 or p <0.01).
In addition, in HOS cells, the TMRE brightness per cell in the CDDP + cafCA group is about 51% compared to the Non-Drug group, about 65% compared to the CDDP group, about 77% compared to the CDDP + caf group, CDDP + CA Compared with the group, it was about 71%, which was greatly reduced. In HT1080 cells, the TMRE brightness per cell of the CDDP + cafCA group is about 35% compared to the Non-Drug group, about 38% compared to the CDDP group, about 57% compared to the CDDP + caf group, and the CDDP + CA group Compared to about 52%, it was greatly reduced.
Therefore, it was confirmed that caffeine citrate was added in combination with cisplatin in osteosarcoma and soft tissue sarcoma, whereby the mitochondrial membrane potential was attenuated / disappeared and the apoptosis-inducing effect of cisplatin could be enhanced.

  From the above results, in the cancer therapeutic agent of the present invention, the combination of cisplatin and caffeine citrate is cisplatin alone, cisplatin and anhydrous caffeine, cisplatin and citric acid in osteosarcoma and soft tissue sarcoma. Compared with the combination, the possibility of significantly enhancing the apoptosis-inducing effect of cisplatin was confirmed.

[Evaluation of enhancement of cisplatin effect of caffeine citrate in skin, pancreatic and cholangiocarcinoma cells]
The enhancement of anticancer agent (cisplatin) effect of caffeine citrate against skin cancer, pancreatic cancer and bile duct cancer was evaluated. The test was performed using the same materials and methods as in Example 1 except that the following cell lines and media were used.

<Material>
-Cell line Human skin A375 cells (obtained from DS Pharma Biomedical Co., Ltd.) were used as skin cancer.
As pancreatic cancer, human-derived PANC-1 cells (obtained from RIKEN BRC through the Ministry of Education, Culture, Sports, Science and Technology's National BioResource Project) were used.
As bile duct cancer, TFK-1 cells derived from humans (obtained from RIKEN BRC through the Ministry of Education, Culture, Sports, Science and Technology's National BioResource Project) were used.
-Medium About RPMA1640 culture medium, as for each A375 cell, PANC-1 cell, and TFK-1 cell about each said cancer type cultured cell.

<Result>
1. Cell Viability FIG. 49, FIG. 50 and FIG. 51 show the cell viability based on the absorbance at 450 nm of four groups (CDDP, CDDP + caf, CDDP + CA, CDDP + cafCA) in A375 cells, PANC-1 cells and TFK-1 cells, respectively. Show. As is apparent from FIGS. 49, 50 and 51, in A375 cells (skin cancer), PANC-1 cells (pancreatic cancer) and TFK-1 cells (bile duct cancer), the CDDP + cafCA group is the other at all CDDP concentrations. The cell viability was the lowest as compared with the group.
As a result of analysis of variance, the CDDP + cafCA group was significantly different from all other groups at each cisplatin concentration (p <0.01).
Therefore, it was confirmed that caffeine citrate enhances the anticancer drug effect of cisplatin when added in combination with cisplatin at all cisplatin concentrations of 0.125 to 2.0 μM.

2. Median Effect Method A synergistic effect on A375 cells, PANC-1 cells and TFK-1 cells was analyzed by the Median Effect method (not shown). CI, which is an index of synergistic effect, was calculated as CDDP + cafCA as a combination of CDDP + caf and CDDP + CA based on the cell viability for each CDDP concentration, as in Example 1. A synergistic effect is observed when CI <1.0.
In the CDDP + cafCA group, CI <1.0 at all cisplatin concentrations of 0.125 to 2.0 μM, and a synergistic effect was observed for CDDP + caf and CDDP + CA.

3. Isobologram method The synergistic effect in A375 cells, PANC-1 cells, and TFK-1 cells was analyzed by the Isobologram method (not shown).
Similar to Example 1, since the circular broken lines indicating the effective doses of CDDP + cafCA are located at the lower left of the curves connecting the effective doses of CDDP + caf and CDDP + CA, a synergistic effect was recognized.
In the CDDP + cafCA group, CI <1.0 at all effective doses of 50 to 90%, and a synergistic effect was observed for CDDP + caf and CDDP + CA.

[Evaluation of enhancement of gemcitabine effect of caffeine citrate in cultured soft tissue sarcoma, skin cancer, pancreatic cancer and cholangiocarcinoma cells]
The enhancement of the anticancer agent (gemcitabine) effect of caffeine citrate against soft tissue sarcoma, skin cancer, pancreatic cancer and bile duct cancer was evaluated. The test was conducted using the same materials and methods as in Example 4 except that the following cell lines and media were used.

<Material>
-Cell line As soft tissue sarcoma, human-derived HT1080 cells (obtained from ATCC) were used.
As skin cancer, human-derived A375 cells (obtained from DS Pharma Biomedical Co., Ltd.) were used.
As pancreatic cancer, human-derived PANC-1 cells (obtained from RIKEN BRC through the Ministry of Education, Culture, Sports, Science and Technology's National BioResource Project) were used.
As bile duct cancer, TFK-1 cells derived from humans (obtained from RIKEN BRC through the Ministry of Education, Culture, Sports, Science and Technology's National BioResource Project) were used.
-Medium About each said cancer type cultured cell, HT1080 cell used DMEM, and A375 cell, PANC-1 cell, and TFK-1 cell all used RPMI1640 medium.

<Result>
1. Cell viability FIGS. 52, 53, 54 and 55 show the absorbance at 450 nm of four groups (GEM, GEM + caf, GEM + CA, GEM + cafCA) in HT1080 cells, A375 cells, PANC-1 cells and TFK-1 cells, respectively. The cell viability based on is shown. As is apparent from FIGS. 52, 53, 54 and 55, HT1080 cells (soft tissue sarcoma (fibrosarcoma)), A375 cells (skin cancer), PANC-1 cells (pancreatic cancer) and TFK-1 cells (bile duct cancer) ), The GEM + cafCA group had the lowest cell viability compared to the other groups at all gemcitabine concentrations.
As a result of analysis of variance, the GEM + cafCA group was significantly different from all other groups (p <0.01).
Therefore, it was confirmed that caffeine citrate enhances the anticancer drug effect of gemcitabine when added in combination with gemcitabine.

2. Median Effect Method The synergistic effect in HT1080 cells, A375 cells, PANC-1 cells and TFK-1 cells was analyzed by the Median Effect method (not shown). CI, which is an index of the synergistic effect, was calculated as GEM + cafCA based on the cell viability for each GEM concentration as in Example 1, based on the combination of GEM + caf and GEM + CA. A synergistic effect is observed when CI <1.0.
The GEM + cafCA group had a CI <1.0 at all gemcitabine concentrations from 0.125 to 2.0 μM, and a synergistic effect was observed for GEM + caf and GEM + CA.

3. Isobologram method The synergistic effect in HT1080 cells, A375 cells, PANC-1 cells, and TFK-1 cells was analyzed by the Isobologram method (not shown).
As in Example 1, a circular broken line indicating each GEM + cafCA Effective Dose is located at the lower left of the curve connecting each GEM + caf and GEM + CA Effective Dose, and thus a synergistic effect was recognized.
In the GEM + cafCA group, CI <1.0 at all effective doses of 50 to 90%, and a synergistic effect was observed for GEM + caf and GEM + CA.

[Evaluation of enhancement of anticancer effects of caffeine citrate in mice bearing cancer cells]
The enhancement of anticancer effect of caffeine citrate in mice carrying cancer cells was evaluated by the following method. Further, the enhancement of anticancer agent effect of caffeine citrate was evaluated by changing the concentration of caffeine citrate and anticancer agent.

<Material>
-As the mouse, BALB / c-nu (obtained from Charles River, Japan) was used.
-Cell line As the osteosarcoma, mouse-derived LM8 cells (obtained from RIKEN BRC through the Ministry of Education, Culture, Sports, Science and Technology's National BioResource Project) were used.
As the soft tissue sarcoma, human-derived HT1080 cells (obtained from ATCC) were used.
-The anticancer agent cisplatin (available from Wako Pure Chemical Industries, Ltd.) was used.
-Concomitant drugs Caffeine citrate (cafCA, citrate caffeine, obtained from Nobel Pharma Co., Ltd.), anhydrous caffeine (caf, obtained from Wako Pure Chemical Industries, Ltd.) or citric acid (CA, Wako Pure Chemical Industries Ltd.) Obtained from the company).
-Medium LM8 cells used RPMI1640 medium, and HT1080 cells used DMEM.

<Evaluation method>
(1) Evaluation system in nude mice transplanted subcutaneously on the back of mice with LM8 cells (5 × 10 ⁵ cells) derived from mice According to the schedule shown in Table 2 and FIG. The anticancer drug efficacy enhancement of was evaluated.
(2) Evaluation system in nude mice transplanted with human-derived HT1080 cells (5 × 10 ⁵ cells) subcutaneously on the back caffeine citrate in mice carrying cancer cells according to the schedule shown in Table 2 below and FIG. The anticancer drug efficacy enhancement of was evaluated.
(3) Evaluation system in nude mice in which LM8 cells (5 × 10 ⁵ cells) derived from mice characterized by low doses of caffeine citrate were subcutaneously implanted in the back, according to the schedule in Table 3 below and FIG. The enhancement of anticancer drug effect of caffeine citrate was evaluated in mice bearing cancer cells.
(4) Evaluation system in nude mice in which LM8 cells (5 × 10 ⁵ cells) derived from mice characterized by a low dose of anticancer agent are transplanted subcutaneously on the back, carrying cancer cells according to the schedule of Table 4 below and FIG. The anticancer effect enhancement of caffeine citrate was evaluated in mice.

<Evaluation results>
57 and 58 show the results of an evaluation system using nude mice transplanted with mouse-derived LM8 cells subcutaneously on the back.
The results of the evaluation system in nude mice transplanted with human-derived HT1080 cells subcutaneously in the back are shown in FIG. 59 and FIG.
61 and 62 show the results of the evaluation system in nude mice in which LM8 cells derived from mice characterized by low doses of caffeine citrate are transplanted subcutaneously in the back.
FIG. 63 and FIG. 64 show the results of the evaluation system in nude mice in which LM8 cells derived from mice characterized by low doses of anticancer agents were transplanted subcutaneously in the back.
As is apparent from FIGS. 57 to 64, the CDDP + cafCA group suppressed tumor growth (volume and weight) very strongly as compared with the other groups. In addition, even when the concentrations of caffeine citrate and anticancer agent were changed, the CDDP + cafCA group suppressed tumor growth (volume and weight) very strongly compared to the other groups.
From the above results, it was confirmed that the anticancer agent (caffeine citrate) and the treatment method of the present invention have an anticancer agent enhancing effect even in an in vitro evaluation system.

[Evaluation of safety]
The safety of the cancer therapeutic agent of the present invention was evaluated by the following method.

<Safety evaluation method>
According to the following Table 5, the weight transition of nude mice (not cancer-bearing mice) was evaluated.
Outsourced, the nude mice were bled and organs removed, and blood and histopathological examinations were performed.

The evaluation of weight transition is shown in FIG. There was no significant difference in weight gain in each county.
Blood tests and histopathology revealed no adverse events.
From the above, it was confirmed that the cancer therapeutic agent and cancer treatment method of the present invention have no problem in safety.

[Evaluation of myocardial damage by ADM]
ADM can cause adverse events of myocardial injury. Therefore, the damage of cardiomyocytes caused by the cancer therapeutic agent containing the ADM of the present invention was evaluated by an in vitro WST-8 assay. In this Example, the decrease in the number of viable cells of cardiomyocytes was equivalent to ADM + caf, ADM + CA, and ADM + cafCA, respectively, with ADM alone.

<Overview>
From the above results, it was revealed that the cancer therapeutic agent and cancer treatment method of the present invention have the following advantageous effects.
(1) The combination of cisplatin and caffeine citrate significantly enhances the anticancer drug effect of cisplatin compared to cisplatin alone, cisplatin and anhydrous caffeine, or cisplatin and citric acid. . The anticancer agent enhancement effect of caffeine citrate is not a mere additive effect of the anticancer agent enhancement effect of anhydrous caffeine and the anticancer agent enhancement effect of citric acid, but a synergistic effect.
(2) The combination of adriamycin and caffeine citrate significantly enhances the anticancer drug effect of adriamycin, compared to adriamycin alone, a combination of adriamycin and anhydrous caffeine, and a combination of adriamycin and citric acid. . The anticancer agent enhancement effect of caffeine citrate is not a mere additive effect of the anticancer agent enhancement effect of anhydrous caffeine and the anticancer agent enhancement effect of citric acid, but a synergistic effect.
(3) The combination of gemcitabine and caffeine citrate significantly enhances the anticancer drug effect of gemcitabine compared to gemcitabine alone, a combination of gemcitabine and anhydrous caffeine, or a combination of gemcitabine and citric acid . The anticancer agent enhancement effect of caffeine citrate is not a mere additive effect of the anticancer agent enhancement effect of anhydrous caffeine and the anticancer agent enhancement effect of citric acid, but a synergistic effect.
(4) The combination of cisplatin and caffeine citrate is significantly more effective in osteosarcoma and soft tissue sarcoma than cisplatin alone, cisplatin and anhydrous caffeine, and cisplatin and citric acid. To further enhance the cell growth inhibitory effect.
(5) The combination of cisplatin and caffeine citrate is significantly more effective in osteosarcoma and soft tissue sarcoma than cisplatin alone, cisplatin and anhydrous caffeine, and cisplatin and citric acid. There is a possibility of further enhancing the apoptosis-inducing effect.
(6) There is no problem in safety.

  Provided are a cancer therapeutic agent and a cancer treatment method having a stronger therapeutic effect than conventional chemotherapy.

Claims (12)

A therapeutic agent for soft tissue sarcoma characterized by combining caffeine citrate, which is an active ingredient, with gemcitabine, which is an anticancer agent, which is an active ingredient.
A therapeutic agent for gastric cancer, wherein caffeine citrate as an active ingredient is combined with gemcitabine as an anticancer agent as an active ingredient.
A skin cancer therapeutic agent comprising a combination of caffeine citrate, which is an active ingredient, with gemcitabine, which is an anticancer agent, which is an active ingredient.
A therapeutic agent for pancreatic cancer, characterized in that caffeine citrate, which is an active ingredient, is combined with gemcitabine, which is an anticancer agent, which is an active ingredient.
A therapeutic agent for gastric cancer, characterized in that caffeine citrate, which is an active ingredient, is combined with adriamycin, which is an anticancer agent, which is an active ingredient.
A therapeutic agent for uterine cancer, comprising a combination of caffeine citrate, which is an active ingredient, with adriamycin, which is an anticancer agent, which is an active ingredient.
A lung cancer therapeutic agent characterized by combining caffeine citrate, which is an active ingredient, with cisplatin, which is an anticancer agent, which is an active ingredient.
A therapeutic agent for breast cancer, comprising a combination of caffeine citrate, which is an active ingredient, with cisplatin, which is an anticancer agent, which is an active ingredient.
A therapeutic agent for uterine cancer, characterized in that caffeine citrate, which is an active ingredient, is combined with cisplatin, which is an anticancer agent, which is an active ingredient.
A skin cancer therapeutic agent characterized by combining caffeine citrate as an active ingredient with cisplatin as an anticancer agent as an active ingredient.
A therapeutic agent for ovarian cancer, characterized by combining caffeine citrate as an active ingredient with cisplatin as an anticancer agent as an active ingredient.
  A therapeutic agent for cholangiocarcinoma characterized by combining caffeine citrate as an active ingredient with cisplatin as an anticancer agent as an active ingredient.