JP6820567B2 - Cancer treatment - Google Patents

Description

The present invention relates to a cancer therapeutic agent and a cancer treating method, which are characterized in that caffeine citrate, which is an active ingredient, is combined with an anticancer agent, which is 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 teens, but the cause is unknown. The frequency of occurrence in Japan is said to be 1 in 1 million (130 to 260 per year). In the days when amputation was once the only treatment option, the 2-year survival rate for osteosarcoma was 15-20%, a very poor prognosis.
However, with the introduction and progress of chemotherapy and the development of diagnostic imaging and surgical therapy, the 5-year survival rate has now improved to 70% or more (Non-Patent Document 1). The present inventors disclose "chemotherapy with caffeine". This therapy dramatically improved the 5-year survival rate of 89% in patients who completed treatment without metastasis at the first visit, but the prognosis is still insufficient, so further improvement in therapeutic effect is required. (Non-Patent Document 2). In addition, the 5-year survival rate is 10 to 30% in patients with recurrence or metastasis from the first visit, which has not been significantly improved in the past 40 years, and further treatment in this respect as well. It is considered necessary to improve the effect (Non-Patent Document 3).

(Soft tissue sarcoma)
Soft tissue sarcoma is classified into various categories such as leiomyosarcoma, fibrosarcoma, liposarcoma, and rhabdomyosarcoma. Each has different properties and is extremely rare. In non-small round cell sarcoma excluding extraosseous Ewing sarcoma and rhabdomyosarcoma, it has been reported that the 10-year survival rate by surgery alone is about 35% when limited to stage III in limb development (non-patent). Document 4). Furthermore, the effect of chemotherapy on advanced cases of high-grade soft tissue sarcoma has been reported to be 30-40% in response rate, 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 (extensive resection), and postoperative chemotherapy, depending on malignancy, tumor type, and patient condition. The above-mentioned chemotherapy is a multidrug combination chemotherapy using cisplatin, doxorubicin and the like.
As described above, the therapeutic method does not have a sufficient therapeutic effect, but there is a problem that the development of a new drug is difficult to proceed due to the high rarity of soft tissue sarcoma.

As described above, a new therapeutic agent that is effective against 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 diligently studied a drug that can enhance the therapeutic effect of the anticancer drug by combining with the anticancer drug. As a result, "the combination of caffeine citrate and the anticancer drug has the therapeutic effect of the anticancer drug. The present invention was completed with the finding of "synergistic enhancement".

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

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

Results of WST-8 assay using osteosarcoma cell line (HOS) in Example 1. The vertical axis shows cell viability, and the horizontal axis shows cisplatin concentration (CDDP concentration). The diamond (◆) is cisplatin (CDDP) alone, the black square (■) is cisplatin + anhydrous caffeine (CDDP + caf), the gray square is cisplatin + citric acid (CDDP + CA), and the circle is cisplatin + caffeine citrate (CDDP + cafCA). ) Is shown. The CDDP + cafCA group was significantly different from all other groups at each cisplatin concentration (p <0.01). Results of WST-8 assay using soft tissue sarcoma cell line (HT1080) in Example 1. The vertical axis shows cell viability and the horizontal axis shows cisplatin concentration. The diamond (◆) indicates CDDP alone, the black square (■) indicates CDDP + caf, the gray square indicates CDDP + CA, and the circle indicates CDDP + cafCA. The CDDP + cafCA group was significantly different from all other groups at each cisplatin concentration (p <0.01). Results of WST-8 assay using lung cancer cell line (RERF-LC-AI) in Example 1. The vertical axis shows cell viability and the horizontal axis shows cisplatin concentration. The diamond (◆) indicates CDDP alone, the black square (■) indicates CDDP + caf, the gray square indicates CDDP + CA, and the circle indicates CDDP + cafCA. The CDDP + cafCA group was significantly different from all other groups at each cisplatin concentration (p <0.01). Results of WST-8 assay using gastric cancer cell line (MKN45) in Example 1. The vertical axis shows cell viability and the horizontal axis shows cisplatin concentration. The diamond (◆) indicates CDDP alone, the black square (■) indicates CDDP + caf, the gray square indicates CDDP + CA, and the circle indicates CDDP + cafCA. The CDDP + cafCA group was significantly different from all other groups at each cisplatin concentration (p <0.01). Results of analysis of the results 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 shows the CDDP concentration (μM) of CDDP + caf, and the horizontal axis shows the CDDP concentration (μM) of CDDP + CA. X indicates Effective Dose 50 (ED50), + indicates Effective Dose 75 (ED75), and ● indicates Effective Dose 90 (ED90). X, + and ● on the vertical axis indicate each Effective Dose of CDDP + caf, ×, + and ● on the horizontal axis indicate each Effective Dose of CDDP + CA, and ×, + and ● surrounded by a circular broken line are. , CDDP + cafCA, each Effective Dose is shown. A synergistic effect is observed when the circular broken line (CDDP + cafCA Effective Dose) is located at the lower left of the curve connecting the CDDP + caf and the CDDP + CA Effective Dose. In the table in the figure, the CI (Combination Index), which is an index of the synergistic effect, is CDDP + ca using each Effective Dose of CDDP + caf, each Effective Dose of CDDP + CA, and each Effective Dose of CDDP + caf CA and CDDP + c. The combination of was calculated as CDDP + cafCA. When CI <1.0, a synergistic effect is observed. Results of analysis of the results 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 shows the CDDP concentration (μM) of CDDP + caf, and the horizontal axis shows the CDDP concentration (μM) of CDDP + CA. X indicates ED50, + indicates ED75, and ● indicates ED90. X, + and ● on the vertical axis indicate each Effective Dose of CDDP + caf, ×, + and ● on the horizontal axis indicate each Effective Dose of CDDP + CA, and ×, + and ● surrounded by a circular broken line are. , CDDP + cafCA, each Effective Dose is shown. A synergistic effect is observed when the circular broken line (each Effective Dose of CDDP + cafCA) is located at the lower left of the curve connecting the respective Effective Dose. In the table in the figure, CI is a combination of CDDP + caf and CDDP + CA calculated as CDDP + caf 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. When CI <1.0, a synergistic effect is observed. Results of analysis of the results 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 shows the CDDP concentration (μM) of CDDP + caf, and the horizontal axis shows the CDDP concentration (μM) of CDDP + CA. X indicates ED50, + indicates ED75, and ● indicates ED90. X, + and ● on the vertical axis indicate each Effective Dose of CDDP + caf, ×, + and ● on the horizontal axis indicate each Effective Dose of CDDP + CA, and ×, + and ● surrounded by a circular broken line are. , CDDP + cafCA, each Effective Dose is shown. A synergistic effect is observed when the circular broken line (each Effective Dose of CDDP + cafCA) is located at the lower left of the curve connecting the respective Effective Dose. In the table in the figure, CI is a combination of CDDP + caf and CDDP + CA calculated as CDDP + caf 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. When CI <1.0, a synergistic effect is observed. Results of analysis of the results 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 shows the CDDP concentration (μM) of CDDP + caf, and the horizontal axis shows the CDDP concentration (μM) of CDDP + CA. X indicates ED50, + indicates ED75, and ● indicates ED90. X, + and ● on the vertical axis indicate each Effective Dose of CDDP + caf, ×, + and ● on the horizontal axis indicate each Effective Dose of CDDP + CA, and ×, + and ● surrounded by a circular broken line are. , CDDP + cafCA, each Effective Dose is shown. A synergistic effect is observed when the circular broken line (each Effective Dose of CDDP + cafCA) is located at the lower left of the curve connecting the respective Effective Dose. In the table in the figure, CI is a combination of CDDP + caf and CDDP + CA calculated as CDDP + caf 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. When CI <1.0, a synergistic effect is observed. The results of the WST-8 assay of 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 are shown in 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 is a synergistic effect, when CI = 1.0, it is an additive effect, and when CI> 1.0, it is an antagonistic effect. In the figure, in each series, the bars are shown in the order of HOS, HT1080, RERF-LC-AI, and MKN45 from the left. Results of WST-8 assay using hepatocellular carcinoma cell line (HepG2) in Example 2. The way to read the graph is the same as in FIG. Analysis result of synergistic effect using hepatocellular carcinoma cell line (HepG2) by the Median Effect method in Example 2. Results of analysis of the results of the WST-8 assay using the hepatocellular carcinoma cell line (HepG2) in Example 2 by the Isobologram method. How to read the graph and the table is the same as in FIG. Results of WST-8 assay using uterine cancer cell line (Hela) in Example 2. The way to read the graph is the same as in FIG. Analysis result of synergistic effect using uterine cancer cell line (HepG2) by Median Effect method in Example 2. The result of analyzing the result of the WST-8 assay using the uterine cancer cell line (HepG2) in Example 2 by the Isobologram method. How to read the graph and the table is the same as in FIG. Results of WST-8 assay using breast cancer cell line (MCF7) in Example 2. The way to read the graph is the same as in FIG. Analysis result of synergistic effect using a breast cancer cell line (HepG2) by the Median Effect method in Example 2. The result of analyzing the result of the WST-8 assay using the breast cancer cell line (MCF7) in Example 2 by the Isobologram method. How to read the graph and the table is the same as in FIG. Results of WST-8 assay using ovarian cancer cell line (OVCAR-3) in Example 2. The way to read the graph is the same as in FIG. Analysis result of synergistic effect using ovarian cancer cell line (HepG2) by the Median Effect method in Example 2. Results of analysis of the results of the WST-8 assay using the ovarian cancer cell line (OVCAR-3) in Example 2 by the Isobologram method. How to read the graph and the table is the same as in FIG. Results of WST-8 assay using osteosarcoma cell line (HOS) in Example 3. The vertical axis shows cell viability, and the horizontal axis shows adriamycin concentration (ADM concentration). Rhombus (◆) is adriamycin (ADM) alone, black square (■) is adriamycin + anhydrous caffeine (ADM + caf), gray square is adriamycin + citric acid (ADM + CA), circle is adriamycin + caffeine citrate (ADM + cafCA) ) Is shown. The ADM + cafCA group was significantly different from all other groups at each adriamycin concentration (p <0.01). Analysis result of synergistic effect using osteosarcoma cell line (HOS) by Median Effect method in Example 3. Results of analysis of the results 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 shows the ADM concentration (nM) of ADM + caf, and the horizontal axis shows the ADM concentration (nM) of ADM + CA. X indicates Effective Dose 50 (ED50), + indicates Effective Dose 75 (ED75), and ● indicates Effective Dose 90 (ED90). X, + and ● on the vertical axis indicate each Effective Dose of ADM + caf, ×, + and ● on the horizontal axis indicate each Effective Dose of ADM + CA, and ×, + and ● surrounded by a circular broken line are. , ADM + cafCA Each Effective Dose is shown. A synergistic effect is observed when the circular broken line (Effective Dose of ADM + cafCA) is located at the lower left of the curve connecting each Effective Dose of ADM + caf and ADM + CA. In the table in the figure, CI (Combination Index), which is an index of synergistic effect, is determined by using each Effective Dose of ADM + caf, each Effective Dose of ADM + CA, and each Effective Dose of ADM + cafCA shown in the above graph, and ADM + ca. The combination of ADM + cafCA was calculated. When CI <1.0, a synergistic effect is observed. Results of WST-8 assay using soft tissue sarcoma cell line (HT1080) in Example 3. The way to read the graph is the same as in FIG. Analysis result of synergistic effect using soft tissue sarcoma cell line (HT1080) by the Median Effect method in Example 3. Results of analysis of the results of the WST-8 assay using the osteosarcoma cell line (HOS) in Example 3 by the Isobologram method. How to read the graph and the table is the same as in FIG. 24. Results of WST-8 assay using gastric cancer cell line (MKN45) in Example 3. The way to read the graph is the same as in FIG. Analysis result of synergistic effect using gastric cancer cell line (MKN45) by Median Effect method in Example 3. Results of analysis of the results of the WST-8 assay using the gastric cancer cell line (MKN45) in Example 3 by the Isobologram method. How to read the graph and the table is the same as in FIG. 24. Results of WST-8 assay using uterine cancer cell line (Hela) in Example 3. The way to read the graph is the same as in FIG. Analysis result of synergistic effect using uterine cancer cell line (Hela) by Median Effect method in Example 3. Results of analysis of the results of the WST-8 assay using the uterine cancer cell line (Hela) in Example 3 by the Isobologram method. How to read the graph and the table is the same as in FIG. 24. Results of WST-8 assay using osteosarcoma cell line (HOS) in Example 4. The vertical axis shows cell viability, and the horizontal axis shows gemcitabine concentration (GEM concentration). Rhombus (◆) 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) ) Is shown. The GEM + cafCA group was significantly different from all other groups at each gemcitabine concentration (p <0.01). Analysis result of synergistic effect using osteosarcoma cell line (HOS) by Median Effect method in Example 4. Results of analysis of the results 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 shows the GEM concentration (nM) of GEM + caf, and the horizontal axis shows the GEM concentration (nM) of GEM + CA. X indicates Effective Dose 50 (ED50), + indicates Effective Dose 75 (ED75), and ● indicates Effective Dose 90 (ED90). X, + and ● on the vertical axis indicate each Effective Dose of GEM + caf, ×, + and ● on the horizontal axis indicate each Effective Dose of GEM + CA, and ×, + and ● surrounded by a circular broken line are. , GEM + cafCA, each Effective Dose is shown. A synergistic effect is observed when the circular broken line (Effective Dose of GEM + cafCA) is located at the lower left of the curve connecting the Effective Dose of GEM + caf and GEM + CA. In the table in the figure, CI (Combination Index), which is an index of synergistic effect, is GEM + ca using each Effective Dose of GEM + caf, each Effective Dose of GEM + CA, and each Effective Dose of GEM + caf CA shown in the above graph. The combination of was calculated as GEM + cafCA. When CI <1.0, a synergistic effect is observed. Results of WST-8 assay using gastric cancer cell line (MKN45) in Example 4. The way to read the graph is the same as in FIG. 34. Analysis result of synergistic effect using gastric cancer cell line (MKN45) by Median Effect method in Example 4. Results of analysis of the results of the WST-8 assay using the gastric cancer cell line (MKN45) in Example 4 by the Isobologram method. How to read the graph and the table is the same as in FIG. Results of WST-8 assay using ovarian cancer cell line (OVCAR-3) in Example 4. The way to read the graph is the same as in FIG. 34. Analysis result of synergistic effect using ovarian cancer cell line (OVCAR-3) by Median Effect method in Example 4. Results of analysis of the results 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 results of taking photographs of one typical fixed point visual field of each group are shown. Hoechst indicates the photographed result of Hoechst detection, Hoechst + EdU indicates the combined image of the photographed result of Hoechst detection and EdU detection, and EdU indicates the photographed result of EdU detection. Results of Cell Proliferation (EdU) Assay Using Soft Tissue Sarcoma Cell Line (HT1080) in Example 5. The results of taking photographs of one typical fixed point visual field of each group are shown. Hoechst indicates the photographed result of Hoechst detection, Hoechst + EdU indicates the combined image of the photographed result of Hoechst detection and EdU detection, and EdU indicates the photographed result of EdU detection. Analysis result of significant difference in EdU color development rate in cell proliferation (EdU) assay using osteosarcoma cell line (HOS) and soft tissue sarcoma cell line (HT1080) in Example 5. * Indicates p <0.05, ** indicates p <0.01. Results of mitochondrial membrane potential change (TMRE) assay using osteosarcoma cell line (HOS) in Example 6. The results of taking photographs of one typical fixed point visual field of each group are shown. Hoechst indicates the photographed result of Hoechst detection, Hoechst + TMRE indicates the combined image of the photographed result of Hoechst detection and TMRE detection, and TMRE indicates the photographed result of TMRE detection. Results of mitochondrial membrane potential change (TMRE) assay using soft tissue sarcoma cell line (HT1080) in Example 6. The results of taking photographs of one typical fixed point visual field of each group are shown. Hoechst indicates the photographed result of Hoechst detection, Hoechst + TMRE indicates the combined image of the photographed result of Hoechst detection and TMRE detection, and TMRE indicates the photographed result of TMRE detection. Analysis result of significant difference in TMRE brightness per cell in mitochondrial membrane potential change (TMRE) assay using osteosarcoma cell line (HOS) and soft tissue sarcoma cell line (HT1080) in Example 6. The vertical axis shows the ratio of TMRE brightness per cell. * Indicates p <0.05, ** indicates p <0.01. Results of WST-8 assay using skin cancer cell line (A375) in Example 7. The vertical axis shows cell viability, and the horizontal axis shows cisplatin concentration (CDDP concentration). A diamond (◆) indicates cisplatin (CDDP) alone, a triangle (▲) indicates cisplatin + anhydrous caffeine (CDDP + caf), a square indicates cisplatin + citric acid (CDDP + CA), and a circle indicates cisplatin + caffeine citrate (CDDP + cafCA). The CDDP + cafCA group was significantly different from all other groups at each cisplatin concentration (p <0.01). Results of WST-8 assay using pancreatic cancer cell line (PANC-1) in Example 7. The vertical axis shows cell viability, and the horizontal axis shows cisplatin concentration (CDDP concentration). A diamond (◆) indicates cisplatin (CDDP) alone, a triangle (▲) indicates cisplatin + anhydrous caffeine (CDDP + caf), a square indicates cisplatin + citric acid (CDDP + CA), and a circle indicates cisplatin + caffeine citrate (CDDP + cafCA). The CDDP + cafCA group was significantly different from all other groups at each cisplatin concentration (p <0.01). Results of WST-8 assay using cancer-bearing cell line (TFK-1) in Example 7. The vertical axis shows cell viability, and the horizontal axis shows cisplatin concentration (CDDP concentration). A diamond (◆) indicates cisplatin (CDDP) alone, a triangle (▲) indicates cisplatin + anhydrous caffeine (CDDP + caf), a square indicates cisplatin + citric acid (CDDP + CA), and a circle indicates cisplatin + caffeine citrate (CDDP + cafCA). The CDDP + cafCA group was significantly different from all other groups at each cisplatin concentration (p <0.01). Results of WST-8 assay using fibrosarcoma cell line (HT1080) in Example 8. The vertical axis shows cell viability, and the horizontal axis shows 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). Results of WST-8 assay using skin cancer cell line (A375) in Example 8. The vertical axis shows cell viability, and the horizontal axis shows 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). Results of WST-8 assay using pancreatic cancer cell line (PANC-1) in Example 8. The vertical axis shows cell viability, and the horizontal axis shows 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). Results of WST-8 assay using cancer-bearing cell line (TFK-1) in Example 8. The vertical axis shows cell viability, and the horizontal axis shows 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. Changes in tumor volume of nude mice transplanted with LM8 cells, which is a mouse osteosarcoma cell line in Example 9, subcutaneously in the back. The weight of the tumor on day 15 of a nude mouse in which LM8 cells derived from mice in Example 9 were transplanted subcutaneously on the back. Changes in tumor volume of nude mice transplanted with human-derived HT1080 cells subcutaneously in the back in Example 9. Tumor weight on day 15 of nude mice transplanted with human-derived HT1080 cells subcutaneously in the back in Example 9. Changes in tumor volume of nude mice in which LM8 cells derived from mice characterized by a low dose of caffeine citrate in Example 9 were transplanted subcutaneously on the back. Tumor weight on day 15 of nude mice transplanted subcutaneously into the back of mouse-derived LM8 cells characterized by low doses of caffeine citrate in Example 9. Changes in tumor volume of nude mice in which LM8 cells derived from mice characterized by low dose of anticancer drug in Example 9 were transplanted subcutaneously on the back. Tumor weight on day 15 of nude mice transplanted subcutaneously in the back of mouse-derived LM8 cells characterized by low doses of anti-cancer agent in Example 9. Body weight transition of nude mice (not cancer-bearing mice) in Example 10. The measurement date in the upper and lower left graphs is the first day (before drug administration), and the measurement date in the upper and lower right graphs is the 22nd day (after the completion of the three courses). The upper row shows the result of using CDDP as an anti-cancer agent, and the lower row shows the result of using ADM as an anti-cancer agent.

The present invention is "a cancer therapeutic agent characterized by combining caffeine citrate as an active ingredient with an anticancer agent as an active ingredient" and "a caffeine citrate as an active ingredient and an anticancer agent as an active ingredient". Is a cancer treatment method characterized by 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 represented by the formulas (2) and / or the formula (2'). It is a drug containing (including citric acid hydrate). Here, the citric acid represented by the formula (2) and the formula (2') means a mixture of the citric acid represented by the formula (2) and the citric acid represented by the 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 commercially available by Nobel Pharma Co., Ltd.
Further, as a compound similar to anhydrous caffeine (anhydrous caffeine-like compound), caffeine hydrate, sodium benzoate caffeine (so-called Annaka) and the like can be used, and since caffeine is a xanthine derivative, xanthines are used. Xanthine-based nerve center stimulants (phenetylin, proventferin), xanthine-based vasodilators (pentoxyferin, naxisferin, xanthinol nicotinate, pentiphyllin, lorophyllin, tonapoferin), xanthine-based diuretics (theobromine, pamaprom), etc. Can also be used.
As a compound similar to citric acid (citric acid-like compound), potassium citrate, lithium citrate, copper citrate, ammonium iron citrate, hydrates thereof and the like can also be used.
Therefore, as a drug similar to caffeine citrate, a combination of the above-mentioned compound similar to anhydrous caffeine and a compound similar to citric acid can also be used.

Caffeine citrate has not been reported to be effective against cancer types or used in combination with anticancer agents, but it is used as a therapeutic agent for apnea of prematurity in premature infants. That is, caffeine citrate is a therapeutic agent that has already been used in other diseases and its safety has been well established.
As described above, since caffeine citrate is a drug that has already been prescribed in medical insurance, it is advantageous in early clinical application as compared with a new drug.

(Anti-cancer drug)
As the anticancer agent as an active ingredient of the cancer therapeutic agent of the present invention, cisplatin, adriamycin (doxorubicin), ifosfamide or gemcitabine is preferable.

(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 crosslink in the DNA strand.
Commercially available cisplatin can be used. For example, it is marketed by Nippon Kayaku Co., Ltd. under the trade name "IACOL".
(Adriamycin)
Adriamycin is an anticancer agent that suppresses the biosynthesis of both DNA and RNA by inserting it between the base pairs of DNA in 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 crosslink between DNA double strands.
Commercially available ifosfamide can be used. For example, it is marketed by Shionogi Pharmaceutical Co., Ltd. under the trade name "Ihomide".
(Gemcitabine)
Gemcitabine is an anticancer drug that induces apoptosis in DNA synthesis of tumor cells by being incorporated into the DNA strand in the same manner as cytidine having a similar structure and stopping the elongation of the DNA strand.
Commercially available gemcitabine can be used. For example, it is marketed by Eli Lilly Japan K.K. under the trade name "Gemzar".

Cisplatin is a platinum preparation, and the same platinum preparation, for example, carboplatin, oxaliplatin, nedaplatin, etc. is expected to have the same effect.
Doxorubicin is an anticancer antibiotic, and the same anticancer antibiotics such as actinomycin D, pirarubicin, and daunorubicin are expected to have similar effects.
Ifosfamide is an alkylating agent, and the same alkylating agent, for example, 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 for 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 the cancer treatment method of the present invention are primary malignant bone tumor, soft tissue sarcoma, lung cancer, gastric 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's sarcoma, and undifferentiated polymorphic bone tumor.
The soft tissue sarcoma is not particularly limited, and examples thereof include fibrosarcoma, synovial sarcoma, leiomyosarcoma, liposarcoma, rhabdomyosarcoma, undifferentiated polymorphic sarcoma, extraosseous osteosarcoma, and extraosseous Ewing sarcoma. Can be mentioned.
The lung cancer is not particularly limited, and examples thereof include non-small cell lung cancer such as adenocarcinoma, squamous cell carcinoma, and large cell carcinoma, and small cell lung cancer.
The uterine cancer is not particularly limited, and examples thereof include cervical cancer and endometrial cancer.
The skin cancer is not particularly limited, and examples thereof include malignant melanoma, basal cell carcinoma, and spinous cell carcinoma.
In addition, the cancer types listed in the present specification also include all squamous cell carcinomas in the organs in which those cancers occur. For example, gastric cancer includes squamous cell carcinoma of the stomach.

(A method of administering a cancer therapeutic agent of the present invention or a method of treating cancer)
The dose and administration plan in the administration method or the cancer treatment method of the cancer therapeutic agent of the present invention can be adjusted depending on the required amount, the treatment method, the degree of disease or necessity for each individual treatment target, and the like. The dose can be specifically determined according to age, body weight, general health, gender, diet, administration time, administration method, excretion rate, drug combination, patient's medical condition, etc. It may be decided in consideration of the factors of.
The following can be exemplified, but the present invention is not particularly limited.
(1) Single dose Caffeine citrate (total of 3 days administration): 0.1 to 10000 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 areas. Doxorubicin (total of 2-day administration) has a surface area of 0.1 to 120 mg / m ² body, preferably 10 to 90 mg / m ² body surface area, more preferably 30 to 60 mg / m ² body surface area. Ifosfamide (total of 3-5 day doses) 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 from 675 to 900 mg / ^{m 2} body surface area.
(2) Administration interval Caffeine citrate: Administered 1 to 70 times for 10 weeks, preferably 1 to 2 times a day, more preferably once a day. Specifically, 1 to 10 weeks as one course, 1 to 10 times a day for 1 to 30 consecutive days, preferably 1 to 6 weeks as 1 course, 1 to 15 days in a row, 1 to 3 times a day. More preferably, it is administered once a day for 3 to 7 consecutive days with 1 course for 2 to 3 weeks.
Anti-cancer agent: Administer once for 10 to 10 weeks, preferably once for 2 to 8 weeks, more preferably once for 3 to 6 weeks.
(3) Combined administration Caffeine citrate and anticancer drug are administered simultaneously, at intervals of several hours, at intervals of one day, at intervals of several days, and at intervals of several weeks. It may be administered at intervals of several months or at intervals of several months.
In addition, caffeine citrate and an 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 a patient. .. The caffeine citrate and / or anticancer agent in the present invention also includes prodrugs or pharmacologically acceptable salts thereof.
The solvent is not particularly limited, and examples thereof include water, alcohol, and physiological saline.
The carrier is not particularly limited, and examples thereof include acids, bases, buffers, stabilizers, tonicity agents, preservatives and the like.
Excipients are not particularly limited, and examples thereof include fillers, coagulants, slip agents, disintegrants, pigments, sweeteners, flavors, and coating agents.
The binder is not particularly limited, but is, for example, water, ethanol, propanol, simple syrup, glucose solution, starch solution, gelatin solution, carboxymethyl cellulose, hydroxypropyl cellulose, hydroxypropyl starch, methyl cellulose, ethyl cellulose, shelac, calcium phosphate, polyvinyl. Examples include pyrrolidone.
Furthermore, when caffeine citrate and an anticancer agent are separately formulated, the separately formulated ones may be mixed with a diluent or the like at the time of use to form a mixture, and then the mixture may be administered to the patient. Good.
In the method for administering the therapeutic agent for cancer or the method for treating cancer of the present invention, it can be administered orally or parenterally. For oral administration, known dosage forms 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 and oral cavity using spray or aerosol, rectal administration using suppository, patch or liniment. And percutaneous administration using gel or the like can be mentioned. Preferably, oral administration, nasal administration, or intravenous administration by injection can be mentioned.

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 enhancement of the anticancer drug effect of caffeine citrate on osteosarcoma, soft tissue sarcoma, lung cancer, and gastric cancer was evaluated by the following method.

<Material>
-As the cell line osteosarcoma, human-derived HOS cells (obtained from the American Type Culture Collection (ATCC)) were used.
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 BioResource Center (RIKEN BRC), RIKEN 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 via the National BioResource Project of the Ministry of Education, Culture, Sports, Science and Technology) were used.
-The anticancer drug cisplatin (obtained from Wako Pure Chemical Industries, Ltd., hereinafter sometimes referred to as CDDP) was used.
・ Concomitant drug caffeine citrate (caffeine citrate, Nobelpharma Co., Ltd., trade name: Respia (registered trademark), hereinafter sometimes referred to as cafCA), anhydrous caffeine (Wako Pure Chemical Industries, Ltd., hereinafter , Caf) or citric acid (Wako Pure Chemical Industries, Ltd., hereinafter sometimes referred to as CA) was used.

<WST-8 Assay>
(1) For each of the above-mentioned cultured cells of cancer type, HOS cells, RERF-LC-AI cells, and MKN45 cells were prepared with Roswell Park Memorial Institute (RPMI) 1640 medium, and HT1080 cells were used with Dalveco modified Eagle's medium (DMEM). used at a concentration of 3000 to 5000 pieces / 100 [mu] L, respectively, 37 ° C., with _CO 2 5% of the conditions, 24 hours and cultured in a culture plate 96 well.
(2) After culturing, each cancer type cultured cell was divided into four groups, and 100 μL of a drug (CDDP, CDDP + caf, CDDP + CA, CDDP + cafCA) was added. The CDDP concentration of the drug added to each group is 0.125, 0. By dissolving CDDP powder in water (sterile purified water) to prepare a high concentration, and then diluting with a culture solution suitable for 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 powder in water to prepare high concentrations of each, and then diluting with a culture solution suitable for the cells. Since the product is a liquid, the concentration of cafCA was diluted with a culture solution suitable for the cells as it was, 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 administration of the drug, color was developed with Cell Counting Kit-8 (CCK-8) (Dojindo), and the 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 analyzed by analysis of variance with Statcel 3 (OMS Publishing), the significant difference in cell viability between the four groups at each CDDP concentration was analyzed, and the effect of CDDP + cafCA was examined.
(5) CalcuSyn ver. In 2 (BIOSOFT), the synergistic effect of the anti-cancer agent enhancing effect brought about by cafCA and the anti-cancer agent enhancing effect brought about by caf / CA was analyzed. The median effect method and the Isobolog method were used for the analysis (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.).
At each CDDP concentration of 0.125, 0.25, 0.5, 1.0, and 2.0 μM, the combination of the caf concentration at CDDP + caf and the CA concentration at CDDP + CA is defined as the cafCA concentration at CDDP + cafCA, and the combination index ( CI) was sought.
Here, CDDP has the same amount and 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 (ED50) considered necessary for cells to survive 50%" derived from the cell viability at each concentration, and the respective concentrations of caf, CA, and cafCA. It is a numerical value calculated for the cell viability at that time.
The CI by the Cisplatin 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 according to each CDDP concentration.
When CI <1, it was judged to be a synergistic effect. When CI = 1, it is determined to be an additive effect, and when CI> 1, it is determined to be an antagonistic effect.

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

2. 2. Median Effect method Next, the analysis results of the synergistic effect in HOS cells by the Median Effect method are shown in Table 1. The CI, which is an index of synergistic effect, was calculated by calculating the combination of CDDP + caf and CDDP + CA as CDDP + cafCA based on the cell viability for each CDDP concentration in FIG. When CI <1.0, a synergistic effect is observed.
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. 3. Isobologram method The analysis result of the synergistic effect in HOS cells by the Isobologram method is shown in FIG.
In the graph of the analysis result of the Isobologram method of FIGS. 5 to 8, if the circular broken line indicating each Effective Dose of CDDP + cafCA is on the curve connecting each Effective Dose of CDDP + caf and each Effective Dose of CDDP + CA, respectively. , An additive effect is shown, a synergistic effect is shown if it is in the lower left of the curve, and an antagonistic effect is shown if it is in the upper right of the curve. For example, if the circular broken line indicating the ED50 of CDDP + cafCA is on the curve connecting the ED50 of CDDP + caf and the ED50 of CDDP + CA, it indicates an additive effect, and if it is on the lower left of the curve, it indicates a synergistic effect. If it is in the upper right, it shows an antagonistic effect. The same applies to ED75 and ED90.
“Effective Dose” in this analysis indicates the CDDP concentration required to reduce each carcinoma cultured cell, and is derived from the cell viability for each CDDP concentration. For example, ED50 means the CDDP concentration required to reduce cells by 50% from the base (cell viability to 50%), and ED75 means to reduce cells by 75% (cell viability). The CDDP concentration required to reduce the number of cells to 25%, and ED90 means the CDDP concentration required to reduce cells by 90% (to increase the cell viability to 10%).
As is clear from the graph of FIG. 5, the circular dashed 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, respectively, so that a synergistic effect was recognized. ..
As is clear from the table of FIG. 5, the CDDP + cafCA group had a CI <1.0 in all Effective Dose of 50 to 90%, and a synergistic effect was observed for CDDP + caf and CDDP + CA.

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

2. 2. Median Effect Method Table 1 shows the analysis results of the synergistic effect in HT1080 cells by the Median Effect method. The CI, which is an index of the synergistic effect, was calculated by calculating the 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. 3. Isobologram method The analysis result of the synergistic effect in HT1080 cells by the Isobologram method is shown in FIG.
As is clear from the graph of FIG. 6, the 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, respectively, so that a synergistic effect was recognized. ..
As is clear from the table of FIG. 6, the CDDP + cafCA group had a CI <1 in all Effective Dose of 50 to 90%, and a synergistic effect was observed for CDDP + caf and CDDP + CA.

<Results of lung cancer>
1. 1. Cell viability The cell viability based on the absorbance at 450 nm of the 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 at all CDDP concentrations as compared with the other groups.
In addition, as a result of analysis of variance, the CDDP + cafCA group was significantly different from all other groups in each cisplatin concentration (p <0.01).
Therefore, it was confirmed that caffeine citrate enhances the anticancer agent effect of cisplatin when added in combination with cisplatin at all cisplatin concentrations of 0.125 to 2.0 μM.

2. 2. Median Effect Method Table 1 shows the analysis results of the synergistic effect in RERF-LC-AI cells by the Median Effect method. The CI, which is an index of the synergistic effect, was calculated by calculating the 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. 3. Isobologram method The analysis result of the synergistic effect in RERF-LC-AI cells by the Isobologram method is shown in FIG.
As is clear from the graph of FIG. 7, the 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, respectively, so that a synergistic effect was recognized. ..
As is clear from the table in FIG. 7, the CDDP + cafCA group had a CI <1 in all Effective Dose of 50 to 90%, and a synergistic effect was observed for CDDP + caf and CDDP + CA.

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

2. 2. Median Effect Method Table 1 shows the analysis results of the synergistic effect in MKN45 cells by the Median Effect method. The CI, which is an index of the synergistic effect, was calculated by calculating the 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. 3. Isobologram method The analysis result of the synergistic effect on MKN45 cells by the Isobologram method is shown in FIG.
As is clear from the graph of FIG. 8, the 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, respectively, so that a synergistic effect was recognized. ..
As is clear from the table in FIG. 8, the CDDP + cafCA group had a CI <1 in all Effective Dose of 50 to 90%, and a synergistic effect was observed for CDDP + caf and CDDP + CA.

<Comprehensive evaluation>
FIG. 9 summarizes the experimental results of the Median Effect method and the Isobologram method in the above four types of cell lines.
As is clear from FIG. 9, the cancer therapeutic agent of the present invention has a synergistic effect in all the cancer types tested, with the CDDP + cafCA group having a CI <1.0 by 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 significant as compared with 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 a mere 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 enhanced 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 on hepatocellular carcinoma, uterine cancer, breast cancer and ovarian cancer was evaluated. The test was carried out 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 via the National BioResource Project of the Ministry of Education, Culture, Sports, Science and Technology) were used.
As uterine cancer, human-derived Hela cells (obtained from RIKEN BRC via the National BioResource Project of the Ministry of Education, Culture, Sports, Science and Technology) were used.
As breast cancer, human-derived MCF7 cells (obtained from RIKEN BRC via the National BioResource Project of the Ministry of Education, Culture, Sports, Science and Technology) were used.
As ovarian cancer, human-derived OVCAR-3 cells (obtained from RIKEN BRC via the National BioResource Project of the Ministry of Education, Culture, Sports, Science and Technology) were used.
-Medium For each of the above-mentioned cancer type cultured cells, RPMI1640 medium was used for HepG2 cells, Hela cells, MCF7 cells and OVCAR-3 cells.

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

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

3. 3. Isobologram method The analysis results of the synergistic effect on HepG2 cells, Hela cells, MCF7 cells and OVCAR-3 cells by the Isobologram method are shown in FIGS. 12, 15, 18 and 21.
As is clear from the graphs of FIGS. 12, 15, 18 and 21, the circular broken line indicating each Effective Dose of CDDP + cafCA is located at the lower left of the curve connecting the Effective Dose of CDDP + caf and CDDP + CA, respectively. Therefore, a synergistic effect was recognized.
As is clear from the tables of FIGS. 12, 15, 18 and 21, the CDDP + cafCA group had a CI <1.0 at all Effective Dose of 50-90% and had 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 the anticancer agent (adriamycin) effect of caffeine citrate on osteosarcoma, soft tissue sarcoma, gastric cancer and uterine cancer was evaluated. The test was carried out using the same materials as in Example 1 except that the following cell lines, anticancer agents and media were used.

<Material>
-As the cell line osteosarcoma, human-derived HOS cells (obtained from ATCC) were used.
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 via the National BioResource Project of the Ministry of Education, Culture, Sports, Science and Technology) were used.
As uterine cancer, human-derived Hela cells (obtained from RIKEN BRC via the National BioResource Project of the Ministry of Education, Culture, Sports, Science and Technology) were used.
-The anticancer drug adriamycin (obtained from Wako Pure Chemical Industries, Ltd., hereinafter sometimes referred to as ADM) was used.
-Medium For the above-mentioned cultured cells of each cancer type, RPMI1640 medium was used as HOS cells, MKN45 cells and Hela cells. DMEM was used for HT1080 cells.

<WST-8 Assay>
The test was carried out 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 ADM powder in water (sterile purified water) to prepare a high concentration, and then diluting with a culture solution suitable for 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 powder in water to prepare high concentrations of each, and then diluting with a culture solution suitable for the cells. Since the product is a liquid, the concentration of cafCA was diluted with a culture solution suitable for the cells as it was, 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. 1. Cell viability Figure 22, FIG. 25, FIG. 28 and FIG. 31 show cell survival based on the 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. Shows the rate. As is clear from FIGS. 22, 25, 28 and 31, in HOS cells (osteosarcoma), HT1080 cells (soft tissue sarcoma), MKN45 cells (stomach cancer) and Hela cells (uterine cancer), the ADM + cafCA group is all At the ADM concentration of, the cell viability was the lowest compared with the other groups.
In addition, as a result of analysis of variance, the ADM + cafCA group was significantly different from all the other groups in each adriamycin concentration (p <0.01).
Therefore, it was confirmed that caffeine citrate enhances the anticancer agent effect of adriamycin when added in combination with adriamycin at all adriamycin concentrations of 12.5 to 200 nM.

2. 2. Median Effect Method Next, the analysis results of the synergistic effect on HOS cells, HT1080 cells, MKN45 cells and Hela cells by the Median Effect method are shown in FIGS. 23, 26, 29 and 32. The CI, which is an index of the synergistic effect, was calculated by calculating the 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. When CI <1.0, a synergistic effect is observed.
As is clear from FIGS. 23, 26, 29 and 32, the ADM + cafCA group had a CI <1.0 at all adriamycin concentrations of 12.5 to 200 nM, a synergistic effect on ADM + caf and ADM + CA. Was recognized.

3. 3. Isobologram method The analysis results of the synergistic effect on HOS cells, HT1080 cells, MKN45 cells and Hela cells by the Isobologram method are shown in FIGS. 24, 27, 30 and 33.
As is clear from the graphs of FIGS. 24, 27, 30 and 33, the circular broken line indicating each Effective Dose of ADM + cafCA is located at the lower left of the curve connecting each Effective Dose of ADM + caf and ADM + CA, respectively. Therefore, a synergistic effect was recognized.
As is clear from the tables of FIGS. 24, 27, 30 and 33, the ADM + cafCA group had a CI <1.0 at all Effective Dose of 50-90% and had 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 significant as compared with the combination of adriamycin alone, adriamycin and anhydrous caffeine, and adriamycin and citric acid. In addition, the anticancer agent enhancing effect of adriamycin and the anticancer agent enhancing effect of caffeine citrate are not merely additive effects 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 enhanced gemcitabine effect of caffeine citrate in cultured cells of osteosarcoma, gastric cancer and ovarian cancer]
The enhancement of the anticancer drug (gemcitabine) effect of caffeine citrate on osteosarcoma, gastric cancer and ovarian cancer was evaluated. The test was carried out using the same materials as in Example 1 except that the following cell lines, anticancer agents and media were used.

<Material>
-As the cell line osteosarcoma, human-derived HOS cells (obtained from ATCC) were used.
As gastric cancer, human-derived MKN45 cells (obtained from RIKEN BRC via the National BioResource Project of the Ministry of Education, Culture, Sports, Science and Technology) were used.
As ovarian cancer, human-derived OVCAR-3 cells (obtained from RIKEN BRC via the National BioResource Project of the Ministry of Education, Culture, Sports, Science and Technology) were used.
-An anticancer drug gemcitabine (obtained from Wako Pure Chemical Industries, Ltd., hereinafter sometimes referred to as GEM) was used.
-Medium For each of the above-mentioned cultured cells of cancer type, RPMI1640 medium was used as HOS cells, MKN45 cells and OVCAR-3 cells.

<WST-8 Assay>
The test was carried out 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 (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 GEM powder in water (sterile purified water) to prepare a high concentration, and then diluting with a culture solution suitable for 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 powder in water to prepare high concentrations of each, and then diluting with a culture solution suitable for the cells. Since the product is a liquid, the concentration of cafCA was diluted with a culture solution suitable for the cells as it was, 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. 1. Cell viability FIGS. 34, 37 and 40 show cell viability based on the absorbance at 450 nm of the four groups (GEM, GEM + caf, GEM + CA, GEM + cafCA) in HOS cells, MKN45 cells and OVCAR-3 cells, respectively. As is clear 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.
In addition, 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 when caffeine citrate was added in combination with gemcitabine, the anticancer agent effect of gemcitabine was enhanced at all gemcitabine concentrations of 0.5 to 8.0 nM.

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

3. 3. Isobologram method The analysis results of the synergistic effect on HOS cells, MKN45 cells and OVCAR-3 by the Isobologram method are shown in FIGS. 36, 39 and 42.
As is clear from the graphs of FIGS. 36 and 39, the circular broken line indicating each Effective Dose of GEM + cafCA is located at the lower left of the curve connecting each Effective Dose of GEM + caf and GEM + CA, respectively, so that the synergistic effect is obtained. Admitted.
As is clear from the tables of FIGS. 36, 39 and 42, the GEM + cafCA group had a CI <1.0 in all 50-90% of Effective Dose, and a synergistic effect was observed for 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 significant as compared with gemcitabine alone, gemcitabine and anhydrous caffeine, and gemcitabine and citric acid. To further enhance the anticancer agent effect of gemcitabine, the anticancer agent enhancing effect of caffeine citrate is not a mere 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 drug in osteosarcoma and soft tissue sarcoma cultured cells]
The enhancement of the cell growth inhibitory effect of caffeine citrate on anticancer agents on osteosarcoma and soft tissue sarcoma was evaluated by the following method.

<Material>
-As the cell line osteosarcoma, human-derived HOS cells (obtained from ATCC) were used.
As the soft tissue sarcoma, human-derived HT1080 cells (obtained from ATCC) were used.
-The anticancer drug cisplatin (obtained from Wako Pure Chemical Industries, Ltd.) was used.
-Concomitant drug caffeine citrate (cafCA, caffeine citrate, obtained from Nobelpharma 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) was used.
-Medium HOS cells used RPMI1640 medium, and HT1080 cells used DMEM.

<Cell proliferation (EdU) assay>
EdU (5-ethynyl-2'-deoxyuridine) is a nucleoside 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 by the following procedure, the change in cell proliferation ability due to drug administration was confirmed, and the enhancement of the cell proliferation inhibitory effect by CDDP + cafCA was investigated.
(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) was cultured.
(2) After culturing, each cancer type cultured cell was divided into 5 groups (Non-drug, CDDP, CDDP + caf, CDDP + CA, CDDP + cafCA), and the culture medium was replaced. The Non-drug group added 0.5 mL of the culture solution, and the drug-administered group added 0.5 mL of the culture solution adjusted so that the CDDP was 0.25 μM and the CDDP was 0.5 mM for each of caf, CA and cafCA. did. The CDDP concentration of the drug added to each group was adjusted to 0.25 μM by dissolving CDDP powder in water (sterile purified water) to prepare 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 powder in water to prepare high concentrations of each, and then diluting with a culture solution suitable for the cells. Since the product is a liquid, the concentration of cafCA was diluted with a culture solution suitable for the cells as it was, 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, Click-iT (registered trademark) Plus EdU Alexa Fluor (registered trademark) 555 Imaging Kit (Thermo Fisher Scientific), and NucBlue (trademark) Live Cell Reagent (trademark) Live Cell Reagent 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 (AlexaFlu) Trademark) 555): Detects 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), and EdU. A TRITC (excitation wavelength: 540 ± 25 nm, dichroic mirror wavelength: 565 nm, absorption wavelength: 605 ± 55 nm) was used as a filter for illuminating, and 5 fixed-point visual fields of each group were photographed. Hoechst and EdU were detected. It can be said that the smaller (weaker) the EdU detection signal is, the more the cell proliferation is suppressed as compared with Non-drug.
(4) The cells in the screen of the photograph of (3) were counted. The EdU color development rate was calculated as follows. EdU color development rate = number of DNA-synthesizing cells (number of EdU-detected cells) / total number of cells (number of Hoechst-detected cells). The average value of the EdU color development rate of the 5 fields of view in each group was calculated, and from the ratio with the average value of Non-drug as 1, using Statcel 3 (OMS Publishing), the significant difference in the EdU color development rate of each group was dispersed. 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 HOS cells (osteosarcoma), the ratio of the number of EdU-detected cells to the number of Hoechst-detected cells was the smallest in the CDDP + cafCA group as compared with the other groups. That is, the CDDP + cafCA group suppressed the cell proliferation of HOS cells (osteosarcoma) most as compared with the other groups.
As is clear from FIG. 44, in HT1080 cells (soft tissue sarcoma), the CDDP + cafCA group had the lowest ratio of the EdU detection signal to the Hoechst detection signal as compared with the other groups. That is, the CDDP + cafCA group suppressed the cell proliferation of HT1080 cells (soft tissue sarcoma) most as compared with the other groups.
The analysis result of the significant difference in the EdU color development rate of each group in HOS cells and HT1080 cells 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 was about 51% as compared with the Non-Drug group, about 58% as compared with the CDDP group, about 66% as compared with the CDDP + caf group, and compared with the CDDP + CA group. It was about 61%, which was a large decrease. In HT1080 cells, the EdU color development rate of the CDDP + cafCA group was 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 a large decrease.
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 the combination of cisplatin alone, cisplatin and anhydrous caffeine, and cisplatin and citric acid in osteosarcoma and soft sarcoma. It was revealed that the effect of cisplatin on suppressing cell proliferation was significantly enhanced as compared with the combination.

[Evaluation of enhancement of apoptosis (cell death) -inducing effect of caffeine citrate by anticancer drug in osteosarcoma and soft tissue sarcoma cultured cells]
The enhancement of the apoptosis-inducing effect of caffeine citrate on anticancer agents on osteosarcoma and soft tissue sarcoma was evaluated by the following method.

<Material>
-As the cell line osteosarcoma, human-derived HOS cells (obtained from ATCC) were used.
As the soft tissue sarcoma, human-derived HT1080 cells (obtained from ATCC) were used.
-The anticancer drug cisplatin (obtained from Wako Pure Chemical Industries, Ltd.) was used.
-Concomitant drug caffeine citrate (cafCA, caffeine citrate, obtained from Nobelpharma 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) was used.
-Medium HOS cells used RPMI1640 medium, and HT1080 cells used DMEM.

<Mitochondrial Membrane Potential Change (TMRE) Assay>
TMRE (tetramethylrhodamine, ester) is a dye 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 is lost, so that TMRE accumulation is reduced and the TMRE detection signal intensity is weakened.
Therefore, in this experiment, the change in the membrane potential due to drug administration was confirmed by detecting TMRE by the following procedure, and the attenuation / disappearance (apoptosis-inducing effect) of the mitochondrial membrane potential by CDDP + cafCA was investigated.
(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) was cultured.
(2) After culturing, each cancer type cultured cell was divided into 5 groups (Non-drug, CDDP, CDDP + caf, CDDP + CA, CDDP + cafCA), and the culture medium was replaced. The Non-drug group added 0.5 mL of the culture solution, and the drug-administered group added 0.5 mL of the culture solution adjusted so that the CDDP was 0.25 μM and the CDDP was 0.5 mM for each of caf, CA and cafCA. did. The CDDP concentration of the drug added to each group was adjusted to 0.25 μM by dissolving CDDP powder in water (sterile purified water) to prepare 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 powder in water to prepare high concentrations of each, and then diluting with a culture solution suitable for the cells. Since the product is a liquid, the concentration of cafCA was diluted with a culture solution suitable for the cells as it was, 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 nuclei of cells undergoing DNA synthesis were stained using TMRE-Mitochondrial Membrane Potential Assay Kit (Abcam) according to the manufacturer's manual (Hoechst (Hoechst 33342): nuclei Detection, TMRE: Detects mitochondria whose membrane potential is maintained). The filter for illuminating Hoechst with a fluorescence microscope (BIOREVO: BZ-9000, KEYENCE) uses DAPI (excitation wavelength: 360 ± 40 nm, dichroic mirror wavelength: 400 nm, absorption wavelength: 460 ± 50 nm) to illuminate TMRE. A TRITC (excitation wavelength: 540 ± 25 nm, dichroic mirror wavelength: 565 nm, absorption wavelength: 605 ± 55 nm) was used as a filter for this purpose, and 5 fixed-point visual fields of each group were photographed. Hoechst and TMRE were detected. It can be said that the weaker the TMRE detection signal is, the more apoptosis is induced as compared with Non-drug.
(4) The cells in the screen of the photograph of (3) were counted. The TMRE brightness per cell was calculated as follows. TMRE brightness per cell = TMRE brightness (detection signal intensity) total / total number of cells (Hoechst detection cell number). The average value of TMRE brightness per cell in each group of 5 visual fields was calculated, and from the ratio with the average value of Non-drug as 1, using Statcel 3 (OMS Publishing), TMRE per cell in each group was used. Analysis of variance was performed on the significant difference in brightness.

<Result>
The results in HOS cells and HT1080 cells are shown in FIGS. 46 and 47, respectively.
As is clear from FIG. 46, in HOS cells (osteosarcoma), the CDDP + cafCA group had the weakest TMRE detection signal intensity with respect to the Hoechst detection cell number as compared with the other groups. That is, it was confirmed that the CDDP + cafCA group has the highest possibility of inducing apoptosis of HOS cells (osteosarcoma) as compared with 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 Hoechst detection cell number as compared with the other groups. That is, it was confirmed that the CDDP + cafCA group has the highest possibility of inducing apoptosis of HT1080 cells (soft tissue sarcoma) as compared with other groups.
FIG. 48 shows the analysis results of the significant difference in TMRE brightness per cell in each group of HOS cells and HT1080 cells.
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 HOS cells, the TMRE brightness per cell in the CDDP + cafCA group was about 51% as compared with the Non-Drug group, about 65% as compared with the CDDP group, about 77% as compared with the CDDP + caf group, and CDDP + CA. It was about 71% compared to the group, which was a large decrease. In HT1080 cells, the TMRE brightness per cell in the CDDP + cafCA group was 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. It was about 52% in comparison, which was a large decrease.
Therefore, it was confirmed that caffeine citrate may attenuate or eliminate the mitochondrial membrane potential and enhance the apoptosis-inducing 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 the combination of cisplatin alone, cisplatin and anhydrous caffeine, and cisplatin and citric acid in osteosarcoma and soft sarcoma. It was confirmed that cisplatin may significantly enhance the apoptosis-inducing effect as compared with the combination.

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

<Material>
-Cell line 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 via the National BioResource Project of the Ministry of Education, Culture, Sports, Science and Technology) were used.
As bile duct cancer, human-derived TFK-1 cells (obtained from RIKEN BRC via the National BioResource Project of the Ministry of Education, Culture, Sports, Science and Technology) were used.
-Medium For each of the above-mentioned cultured cells of cancer type, RPMI1640 medium was used for all of A375 cells, PANC-1 cells and TFK-1 cells.

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

2. 2. Median Effect method The synergistic effect on A375 cells, PANC-1 cells and TFK-1 cells was analyzed by the Median Effect method (not shown). For CI, which is an index of synergistic effect, the combination of CDDP + caf and CDDP + CA was calculated as CDDP + cafCA based on the cell viability for each CDDP concentration, as in Example 1. When CI <1.0, a synergistic effect is observed.
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 on CDDP + caf and CDDP + CA.

3. 3. Isobologram method The synergistic effect on A375 cells, PANC-1 cells and TFK-1 cells by the Isobologram method was analyzed (not shown).
Similar to Example 1, the 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, respectively, so that a synergistic effect was recognized.
In the CDDP + cafCA group, CI <1.0 in all Effective Dose of 50 to 90%, and a synergistic effect was observed for CDDP + caf and CDDP + CA.

[Evaluation of enhanced gemcitabine effect of caffeine citrate in cultured cells of soft tissue sarcoma, skin cancer, pancreatic cancer and cholangiocarcinoma]
The enhancement of the anticancer agent (gemcitabine) effect of caffeine citrate on soft tissue sarcoma, skin cancer, pancreatic cancer and cholangiocarcinoma was evaluated. The test was carried out 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 the 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 via the National BioResource Project of the Ministry of Education, Culture, Sports, Science and Technology) were used.
As bile duct cancer, human-derived TFK-1 cells (obtained from RIKEN BRC via the National BioResource Project of the Ministry of Education, Culture, Sports, Science and Technology) were used.
-Medium For each of the above-mentioned cultured cells of cancer type, DMEM was used for HT1080 cells, and RPMI1640 medium was used for A375 cells, PANC-1 cells and TFK-1 cells.

<Result>
1. 1. Cell viability In FIGS. 52, 53, 54 and 55, 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 clear 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 at all gemcitabine concentrations as compared with the other groups.
In addition, 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 agent effect of gemcitabine by adding it in combination with gemcitabine.

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

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

[Evaluation of enhancement of anti-cancer drug effect of caffeine citrate in mice carrying cancer cells]
The enhancement of the anticancer drug effect of caffeine citrate in mice carrying cancer cells was evaluated by the following method. Furthermore, the enhancement of the anticancer agent effect of caffeine citrate was evaluated by changing the concentrations of caffeine citrate and the anticancer agent.

<Material>
-As the mouse, BALB / c-nu (obtained from Charles River Laboratories, Japan) was used.
-As the cell line osteosarcoma, mouse-derived LM8 cells (obtained from RIKEN BRC via the National BioResource Project of the Ministry of Education, Culture, Sports, Science and Technology) were used.
As the soft tissue sarcoma, human-derived HT1080 cells (obtained from ATCC) were used.
-The anticancer drug cisplatin (obtained from Wako Pure Chemical Industries, Ltd.) was used.
-Concomitant drug caffeine citrate (cafCA, caffeine citrate, obtained from Nobelpharma 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) was used.
-Medium For LM8 cells, RPMI1640 medium was used, and for HT1080 cells, DMEM was used.

<Evaluation method>
(1) Evaluation system in nude mice in which mouse-derived LM8 cells (5 × 10 ⁵ ) were transplanted subcutaneously in the back Caffeine citrate in mice carrying cancer cells according to the schedule shown in Table 2 and FIG. 56 below. The enhancement of the anticancer drug effect was evaluated.
(2) according to the schedule in Table 2 and Figure 56 evaluation system of the following human-derived HT1080 cells (5 × 10 ⁵ cells) in nude mice implanted subcutaneously on the back, caffeine citrate in mice carrying cancer cells The enhancement of the anticancer drug effect was evaluated.
(3) Evaluation system in nude mice in which mouse-derived LM8 cells (5 × 10 ⁵ cells) characterized by a low dose of caffeine citrate were transplanted subcutaneously on the back, according to the schedules in Table 3 and FIG. 56 below. The enhancement of the anticancer drug effect of caffeine citrate in mice carrying cancer cells was evaluated.
(4) Evaluation system in nude mice in which LM8 cells (5 × 10 ⁵ ) derived from mice characterized by a low dose of anticancer drug were transplanted subcutaneously on the back, cancer cells were supported according to the schedules in Table 4 and FIG. 56 below. The enhancement of the anticancer drug effect of caffeine citrate was evaluated in the mice.

<Evaluation result>
The results of the evaluation system in nude mice in which mouse-derived LM8 cells were transplanted subcutaneously on the back are shown in FIGS. 57 and 58.
The results of the evaluation system in nude mice transplanted with human-derived HT1080 cells subcutaneously on the back are shown in FIGS. 59 and 60.
The results of the evaluation system in nude mice in which LM8 cells derived from mice characterized by a low dose of caffeine citrate were transplanted subcutaneously on the back are shown in FIGS. 61 and 62.
The results of the evaluation system in nude mice in which LM8 cells derived from mice characterized by a low dose of anticancer drug were transplanted subcutaneously on the back are shown in FIGS. 63 and 64.
As is clear from FIGS. 57-64, 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 as compared with the other groups.
From the above results, it was confirmed that the anticancer agent (caffeine citrate) and the therapeutic method of the present invention also have an anticancer agent enhancing effect in the in vitro evaluation system.

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

<Safety evaluation method>
According to Table 5 below, the body weight transition of nude mice (not cancer-bearing mice) was evaluated.
The nude mice were outsourced, blood was collected, organs were removed, and blood tests and histopathological tests were performed.

The evaluation of the body weight transition is shown in FIG. There was no significant difference in weight gain in each county.
Blood tests and histopathological examination showed no adverse events.
From the above, it was confirmed that the cancer therapeutic agent and the 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 damage. Therefore, in vitro WST-8 assay was used to evaluate cardiomyocyte damage caused by the ADM-containing cancer therapeutic agent of the present invention. In this example, the decrease in the number of surviving cardiomyocytes was equivalent to ADM + caf, ADM + CA, and ADM + cafCA, respectively, as compared to ADM alone.

<General>
From the above results, it was clarified that the cancer therapeutic agent and the cancer treatment method of the present invention have the following advantageous effects.
(1) The combination of cisplatin and caffeine citrate significantly enhances the anticancer agent effect of cisplatin as compared with the combination of cisplatin alone, the combination of cisplatin and anhydrous caffeine, and the combination of cisplatin and citric acid. .. The anti-cancer agent enhancing effect of caffeine citrate is not a mere additive effect but a synergistic effect of the anti-cancer agent enhancing effect of anhydrous caffeine and the anti-cancer agent enhancing effect of citric acid.
(2) The combination of adriamycin and caffeine citrate significantly enhances the anticancer drug effect of adriamycin as compared with adriamycin alone, adriamycin and anhydrous caffeine, and adriamycin and citric acid. .. The anti-cancer agent enhancing effect of caffeine citrate is not a mere additive effect but a synergistic effect of the anti-cancer agent enhancing effect of anhydrous caffeine and the anti-cancer agent enhancing effect of citric acid.
(3) The combination of gemcitabine and caffeine citrate significantly enhances the anticancer agent effect of gemcitabine as compared with gemcitabine alone, gemcitabine and anhydrous caffeine, and gemcitabine and citric acid. .. The anti-cancer agent enhancing effect of caffeine citrate is not a mere additive effect but a synergistic effect of the anti-cancer agent enhancing effect of anhydrous caffeine and the anti-cancer agent enhancing effect of citric acid.
(4) The combination of cisplatin and caffeine citrate is significantly more cisplatin in osteosarcoma and soft tissue sarcoma than cisplatin alone, cisplatin plus anhydrous caffeine, or cisplatin plus citric acid. To further enhance the cell growth inhibitory effect.
(5) The combination of cisplatin and caffeine citrate is significantly more cisplatin in osteosarcoma and soft sarcoma than cisplatin alone, cisplatin plus anhydrous caffeine, or cisplatin plus citric acid. It may enhance the effect of inducing apoptosis.
(6) There is no problem with 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, which is characterized by combining caffeine citrate, which is an active ingredient, with gemcitabine, which is an anticancer agent, which is an active ingredient.
A gastric cancer therapeutic agent characterized by combining caffeine citrate, which is an active ingredient, with gemcitabine, which is an anticancer agent, which is an active ingredient.
A skin cancer therapeutic agent characterized by combining caffeine citrate, which is an active ingredient, with gemcitabine, which is an anticancer agent, which is an active ingredient.
A pancreatic cancer therapeutic agent characterized by combining caffeine citrate, which is an active ingredient, with gemcitabine, which is an anticancer agent, which is an active ingredient.
A gastric cancer therapeutic agent characterized by combining caffeine citrate, which is an active ingredient, with adriamycin, which is an anticancer agent, which is an active ingredient.
A uterine cancer therapeutic agent characterized by combining 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 breast 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 uterine 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 skin 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.
An ovarian 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 cholangiocarcinoma characterized by combining caffeine citrate, which is an active ingredient, with cisplatin, which is an anticancer agent, which is an active ingredient.