A highly effective and low-toxicity anti-cancer combination drug and pharmaceutical composition

Abstract

The present invention belongs to the field of pharmaceutical technology, and particularly relates to a highly effective and low-toxic anti-cancer combination drug and a pharmaceutical composition. The present invention provides the use of a guanidine compound in combination with piperlongumine in the preparation of an anti-cancer drug. Among them, the guanidine compound can be selected from metformin. The combination drug of the present invention has better anti-cancer activity compared with single-agent drugs. Moreover, compared with platinum-based chemotherapy drugs, this combination drug regimen has the same tumor-killing efficacy, and has no significant impact on the body weight, immune system, liver function, kidney function, etc. of mice, indicating that its toxic and side effects are significantly weaker than those of platinum-based chemotherapy drugs. Therefore, the combination drug regimen of the present invention has good application prospects.

Description

Technical Field

[0001] The present invention belongs to the field of pharmaceutical technology, and particularly relates to a highly effective and low-toxic anti-cancer combination drug and a pharmaceutical composition. Background Art

[0002] Cancer is a major malignant disease threatening human life and health. In recent years, cancer has shown a trend of getting younger, and the incidence and mortality rates have been increasing year by year. Currently, cancer treatment drugs are still mainly chemotherapy drugs. According to statistics, the proportion of chemotherapy drugs in the domestic oncology drug market is 73%, the proportion of targeted drugs is 23%, and the proportion of immunotherapy drugs is about 4%. However, although chemotherapy is effective, its toxic and side effects are very obvious, that is, the so-called "killing one thousand enemies and hurting eight hundred oneself". These toxic and side effects lead to a serious decline in the quality of life of patients, and even become the direct cause of death of cancer patients, becoming the main bottleneck of tumor chemotherapy. Therefore, the development of new chemotherapy drugs with high efficiency and low toxicity is of great significance for the quality of life of cancer patients.

[0003] Metformin is a first-line drug for the treatment of type 2 diabetes. A large number of clinical and basic studies have shown that metformin not only has low toxic and side effects, but also has anti-cancer activity. Research has shown that metformin can selectively kill various types of tumor cells, but has no significant effect on normal cells. These findings provide a solid theoretical basis for the development of metformin as an effective anti-cancer and low-toxic new drug. However, clinical experiments have shown that the anti-cancer effect of metformin alone is not significant. Therefore, the development of metformin synergists (adjuvants) is an important topic in this field. Currently, relevant studies have tried to enhance the anti-cancer activity of metformin by combining drugs. However, the current combination drug regimens still do not enhance the anti-cancer activity of metformin to an ideal extent, and cannot make the anti-cancer effect of metformin comparable to that of conventional chemotherapy drugs (such as platinum-based chemotherapy drugs).

[0004] Piperlongumine is an alkaloid with various activities such as anti-inflammatory, antibacterial, anti-angiogenic, antioxidant, anti-cancer and anti-diabetic. However, the anti-cancer effect of using piperlongumine alone is still not significant enough. And there is no literature report on the anti-cancer effect of its combination with metformin.

[0005] In summary, for the purpose of enhancing anti-cancer activity while reducing drug toxicity, it is still necessary to develop more combination drug regimens to enhance the anti-cancer activity of metformin in this field. Summary of the Invention

[0006] In view of the problems of the prior art, the present invention provides a highly effective and low-toxic anti-cancer combination drug and a pharmaceutical composition, aiming to enhance the anti-cancer activity of guanidine compounds such as metformin by combining drugs.

[0007] Use of a guanidine compound in combination with piperlongumine for the preparation of an anti-cancer drug.

[0008] Preferably, the dosage ratio of the guanidine compound to piperlongumine is: molar ratio of 250:5 - 500:5 or mass ratio of 50:1 - 150:1. Preferably, the dosage molar ratio of the guanidine compound to piperlongumine is 250:5 or the mass ratio is 100:1.

[0009] Preferably, the guanidine compound is selected from metformin.

[0010] Preferably, the anticancer drug is used for treating lung cancer, breast cancer or colorectal cancer.

[0011] The present invention also provides a combined drug for anticancer, and the combined drug is a guanidine compound and piperlongumine administered separately or simultaneously.

[0012] Preferably, the dosage ratio of the guanidine compound to piperlongumine is: molar ratio of 250:5 - 500:5 or mass ratio of 50:1 - 150:1. Preferably, the dosage molar ratio of the guanidine compound to piperlongumine is 250:5 or the mass ratio is 100:1.

[0013] Preferably, the guanidine compound is selected from metformin.

[0014] Preferably, the combined drug is used for treating lung cancer, breast cancer or colorectal cancer.

[0015] The present invention also provides a pharmaceutical composition, which is prepared by using a guanidine compound and piperlongumine as active ingredients and adding pharmaceutically acceptable excipients or auxiliary ingredients.

[0016] Preferably, the dosage ratio of the guanidine compound to piperlongumine is: molar ratio of 250:5 - 500:5 or mass ratio of 50:1 - 150:1. Preferably, the dosage molar ratio of the guanidine compound to piperlongumine is 250:5 or the mass ratio is 100:1.

[0017] Preferably, the guanidine compound is selected from metformin.

[0018] Preferably, the pharmaceutical composition is used for treating lung cancer, breast cancer or colorectal cancer.

[0019] The present invention combines piperlonguminine with guanidine compounds (such as metformin), which can significantly enhance the tumor-specific killing activity of guanidine compounds. This effectiveness and specificity have been repeatedly verified in a variety of tumor models, including lung cancer / breast cancer / colorectal cancer PDX models (Patient-derived xenograft model) and MMTV-PyMT-induced spontaneous breast cancer mouse models. Particularly importantly, compared with platinum-based chemotherapeutic drugs, this combined drug treatment regimen has the same tumor-killing efficacy, but has no significant impact on mouse body weight, immune system, liver function, kidney function, etc., indicating that its toxic and side effects are significantly weaker than those of platinum-based chemotherapeutic drugs. Therefore, the combined drug treatment method of the present invention has good application prospects.

[0020] It should be specifically noted that the experimental examples of the present invention include in vitro cell experiments. After treating tumor cells with piperlonguminine and guanidine compounds together, a good selective killing effect on tumor cells is shown. Therefore, when piperlonguminine and guanidine compounds of the present invention are used for treating cancer, they can either be combined with existing pharmaceutical preparations for drug combination, or be made into a new preparation in the form of a composition for administration together.

[0021] Obviously, based on the above content of the present invention, other various forms of modifications, substitutions or changes can be made without departing from the above basic technical idea of the present invention according to the common general technical knowledge and customary means in the art.

[0022] The following is a further detailed description of the above content of the present invention through specific embodiments in the form of examples. However, this should not be construed as limiting the scope of the above subject matter of the present invention to the following examples. All technologies implemented based on the above content of the present invention belong to the scope of the present invention. Brief Description of the Drawings

[0023] Figure 1 The experimental results show that reactive oxygen species play a key role in the process of metformin selectively killing tumor cells. The indicated cells were treated with 250 μM metformin alone or in combination with 5 μM piperlongumine (piper) for 48 hours, and then the cell viability was detected by trypan blue staining (A) or PI (B) staining.

[0024] Figure 2Results of the experiment that metformin combined with piperlongumine significantly inhibited the growth of spontaneous mammary tumors induced by MMTV-PyMT in mice. (A) Tumor growth in female MMTV-PyMT mice at 95 days of age. (B) Schematic diagram of the dosing pattern. According to the dosing pattern in B, female MMTV-PyMT positive mice (4 mice per group) at 49 days of age were treated intraperitoneally with 100 mg / kg of metformin alone or in combination with 1 mg / kg of piperlongumine for 46 consecutive days. When the mice reached 95 days of age, the tumor size (C), the number of tumors (D), and the tumor weight (E) were observed.

[0025] Figure 3 Results of the experiment that metformin combined with piperlongumine significantly inhibited the growth of xenografted tumors of breast cancer and colorectal cancer in mice and prolonged the survival time of mice. 1×10 5 4T-1 cells (A and B) or 5×10 5 CT-26 cells (C and D) were inoculated subcutaneously into the right neck of 6-week-old female Balb / c mice. On the third day after cell inoculation, 100 mg / kg of metformin and 1 mg / kg of piperlongumine (MP) were injected intraperitoneally every day, and the control group was injected with an equal volume of PBS (Ctrl); one group of mice was continuously administered for 18 days, and the tumors were removed and the tumor weights were measured (A and C); another group of mice was continuously administered until the tumor volume reached 1500 mm 3 which was defined as the ethical death of the mice, and the survival curves were plotted (C and D).

[0026] Figure 4 Results of the experiment that metformin combined with piperlongumine significantly inhibited the growth of PDX (Patient-derived xenograft) tumors of lung cancer, breast cancer, and colorectal cancer. 1-3 mm 3 PDX tumors were cut into small pieces and inoculated subcutaneously into the right neck of NSG mice; when the PDX tumors grew to 15-20 mm, the experimental group of mice was injected intraperitoneally with MP (100 mg / kg of metformin and 1 mg / kg of piperlongumine, daily), and the control group was injected with an equal volume of PBS (Ctrl); on days 0, 4, 8, 12, 16, 18, and 24 after injection, the tumor growth was dynamically detected with a vernier caliper; when the tumors reached day 24, the tumors were removed, photographed, and weighed.

[0027] Figure 5 Results of the experiment that metformin combined with piperlongumine had the same tumor-killing effect as platinum-based chemotherapeutic drugs. (A) Schematic diagram of the dosing pattern. 1×10 5 4T-1 cells (B and C) or 5×10 5LLC cells (D and E) were inoculated subcutaneously into the right neck of female Balb / c mice (4T-1) or C57BL / 6 mice (LLC). On the third day after inoculating the cells, the mice were evenly divided into four groups (ctrl control group, MP group, Cis group, and Car group). In the MP group, 100 mg / kg of metformin and 1 mg / kg of piperlongumine were intraperitoneally injected daily; in the Cis group, 5 mg / kg of cisplatin was intraperitoneally injected once every 5 days; in the Car group, 50 mg / kg of carboplatin was intraperitoneally injected once every 5 days; the ctrl control group was injected with an equal volume of PBS daily. On the 15th day of administration, the tumors were removed and the tumor weights were measured (B-E).

[0028] Figure 6 The experimental results showed that metformin combined with piperlongumine had no significant effect on the body weight, immune system, liver and kidney functions of mice. According to the Figure 6 administration mode, the effects of MP, cisplatin, and carboplatin on the body weight, spleen weight, and peripheral blood white blood cell count of mice were detected (A-B). Female FVB mice treated with 100 mg / kg of metformin and 1 mg / kg of piperlongumine by intraperitoneal injection daily for 35 days were used. The control group was injected with an equal amount of PBS (Ctrl). On the 20th day of administration, the contents of alanine aminotransferase (ALT), aspartate aminotransferase (AST), creatinine, and cystatin C (Cys-C) in the peripheral blood of the mice were detected (C and D).

[0029] Figure 7 The experimental results showed that platinum drugs killed normal and tumor cells without discrimination, but metformin combined with piperlongumine could selectively kill tumor cells. (A-H) Cells were treated with cisplatin or carboplatin at the indicated concentrations as shown in the figure, and then the viability of each cell was detected by trypan blue staining. (I-J) Cells were treated with 250 μM metformin alone or in combination with 5 μM piperlongumine, and then the viability of each cell was detected by trypan blue staining. *** represents p < 0.0001. Detailed implementation mode

[0030] In the following examples and experimental examples, the reagents and raw materials used were all commercially available products.

[0031] Example 1 Combination drug for anti-cancer

[0032] This example provides a combination drug for anti-cancer, which is an injection containing 250 μM metformin and 5 μM piperlongumine.

[0033] Example 2 Combination drug for anti-cancer

[0034] This embodiment provides a combined drug for anti-cancer, which is an injection containing 500 μM metformin and 5 μM piperlongumine.

[0035] Example 3 Combined drug for anti-cancer

[0036] This embodiment provides a combined drug for anti-cancer, which is a tablet containing 100 mg metformin and 1 mg piperlongumine.

[0037] Example 4 Composition for anti-cancer

[0038] This embodiment provides a composition for anti-cancer, which is an injection containing 250 μM metformin and 5 μM piperlongumine.

[0039] Example 5 Composition for anti-cancer

[0040] This embodiment provides a composition for anti-cancer, which is an injection containing 500 μM metformin and 5 μM piperlongumine.

[0041] Example 6 Composition for anti-cancer

[0042] This embodiment provides a combined drug for anti-cancer, which is a tablet containing 80 mg metformin and 1 mg piperlongumine.

[0043] Example 7 Composition for anti-cancer

[0044] This embodiment provides a combined drug for anti-cancer, which is a tablet containing 120 mg metformin and 1 mg piperlongumine.

[0045] The technical solution of the present invention is further illustrated by experiments below. In the following experimental examples, the medication regimen combining metformin and piperlongumine is abbreviated as MP.

[0046] Experimental Example 1 Anti-cancer activity of metformin and its combination with piperlongumine

[0047] I. Experimental method

[0048] 1. Cell viability detection

[0049] The cultured tumor cells were digested with trypsin and collected; then the cells were washed twice with PBS and counted to make the cell concentration 1×10 5 cells / 100 μL; 2×10 5Tumor cells were placed in a 12-well plate and cultured overnight in an incubator at 37°C. Cells were treated with 250 μM metformin alone or in combination with 5 μM piperlongumine (piper) for 48 hours. All cells (including suspended cells) were collected, and dead cells were stained with trypan blue or PI (Propidium Iodide), and then the cell death rate was detected using a LUNA-FLTM automatic cell counter.

[0050] 2. Detect the anti-cancer activity of MP using a mouse spontaneous breast cancer model induced by MMTV-PyMT

[0051] Forty-nine-day-old female MMTV-PyMT-positive mice were evenly divided into two groups (ctrl control group, MP group), with 4 mice in each group. The experimental group of mice was intraperitoneally injected with MP (100 mg / kg metformin and 1 mg / kg piperlongumine, daily), and the control group was injected with an equal volume of PBS (Ctrl). On the 46th day after injection, the tumors were removed, photographed, and the number and weight of the tumors were counted.

[0052] 3. Explore the anti-cancer activity of MP using a CDX (Cell-derived xenograft) model

[0053] 1×10 5 4T-1 cells or 5×10 5 CT-26 cells were inoculated subcutaneously into the right neck of 6-week-old female Balb / c mice. On the third day after cell inoculation, 100 mg / kg metformin and 1 mg / kg piperlongumine (MP) were intraperitoneally injected daily, and the control group was injected with an equal volume of PBS (Ctrl). One group of mice was continuously administered for 18 days, and the tumors were removed and the tumor weight was measured. Another group of mice was continuously administered until the tumor volume reached 1500 mm 3 and was considered to have died ethically, and the survival curve was plotted.

[0054] 4. Detect the anti-cancer activity of MP using a lung cancer / breast cancer / colorectal cancer PDX (Patient-derived xenograft) model

[0055] Fresh human lung / breast / colorectal tumor tissues were cut into 1-3 mm3 small pieces (F0 generation) and inoculated subcutaneously into the right neck of female severely immunodeficient mice (NSG mice) (F1 generation). When the tumor volume reached 400-600 mm 3 , the tumors were removed, and the tumors were re-cut into 1-3 mm 3 small pieces and re-inoculated subcutaneously into the right neck of NSG mice (F2 generation). When the tumor volume reached 400-600 mm 3, the tumor tissues were stored in liquid nitrogen or inoculated subcutaneously into the right neck of new NSG mice (F3 - F6 generations) for efficacy evaluation. Tumor tissues were retained for each generation to identify the molecular and histological consistency of tumor samples in each generation.

[0056] Sixteen 6 - week - old female NSG mice were prepared and evenly divided into a control group and an experimental group (8 mice in each group); the PDX tumors of the F3 generation were cut into small pieces of 1 - 3 mm 3 and inoculated subcutaneously into the right neck of NSG mice; when the PDX tumors grew to 15 - 20 mm 3 , the mice in the experimental group were intraperitoneally injected with MP (100 mg / kg metformin and 1 mg / kg piperlongumine amide, daily), and the control group was injected with an equal volume of PBS (Ctrl); on days 0, 4, 8, 12, 16, 18, and 24 after injection, the tumor growth was dynamically detected using a vernier caliper; when the tumors grew to day 24, the tumors were removed, photographed, and weighed.

[0057] 5. Use the CDX model to compare the antitumor effects of MP and platinum - based chemotherapeutic drugs

[0058] 1×10 5 4T - 1 cells or 5×10 5 LLC cells were inoculated subcutaneously into the right neck of female Balb / c mice (4T - 1) or C57BL / 6 mice (LLC). On the third day after cell inoculation, the mice were evenly divided into four groups (ctrl control group, MP group, Cis group, and Car group, 6 mice in each group). The MP group was intraperitoneally injected with 100 mg / kg metformin and 1 mg / kg piperlongumine amide daily, the Cis group was intraperitoneally injected with 5 mg / kg cisplatin every 5 days; the Car group was intraperitoneally injected with 50 mg / kg carboplatin every 5 days, and the ctrl control group was injected with an equal volume of PBS daily. On the 15th day of drug administration, the tumors were removed and the tumor weights were measured.

[0059] II. Experimental Results

[0060] 1. Anticancer activity of metformin alone or in combination with piperlongumine amide

[0061] First, the effect of piperlongumine (piper) on the anticancer activity of low - dose metformin (250 μM) was detected. As Figure 1 shown in A and B, compared with the use of low - dose metformin or piperlongumine alone, the combination of metformin and piperlongumine could more significantly inhibit the survival of various tumor cells.

[0062] 2. The combination of metformin and piperlongumine significantly inhibits the growth of MMTV - PyMT - induced spontaneous mammary tumors in mice

[0063] The above research indicates that piperlongumine plays a crucial role in the anti-cancer activity of metformin. Therefore, the combination of metformin and piperlongumine may be a novel tumor treatment strategy. To verify the anti-cancer activity of the combination of metformin and piperlongumine in vivo, the spontaneous breast cancer of MMTV-PyMT mice was first selected as the research model (in this model, Polyoma virus middle T antigen / PyMT can be specifically expressed in breast cells, leading to abnormal hyperplasia of breast cells; primary breast tumors appear in mice at the 5th week; obvious lung metastases appear at the 12th week). As Figure 2 shown in A, obvious tumors appeared around the mammary glands of MMTV-PyMT mice at 95 days. Based on this, in this experimental example, at 49 days of the mice, metformin at 100 mg / kg and piperlongumine at 1 mg / kg were intraperitoneally injected alone or in combination for 46 consecutive days. When the mice grew to 95 days, the size and number of tumors in the mice were observed ( Figure 2 B). As Figure 2 shown in C-E, the use of metformin or piperlongumine alone had no significant effect on the tumor growth of mice, but the combination of metformin and piperlongumine significantly inhibited the growth of mammary tumors in mice. The results indicate that the combination of metformin and piperlongumine is a potential new strategy for tumor treatment.

[0064] 3. The combination of metformin and piperlongumine significantly inhibits the growth of orthotopic tumors of breast cancer and colorectal cancer in mice and prolongs the survival period of mice

[0065] Next, the anti-cancer activity of the combination of metformin and piperlongumine was detected in a mouse orthotopic tumor model mediated by mouse breast cancer cells 4T-1 and mouse colorectal cancer cells CT-26. As Figure 3 shown, MP significantly inhibited the tumor growth in mice and significantly prolonged the survival period of tumor-bearing mice at the same time.

[0066] 4. The combination of metformin and piperlongumine significantly inhibits the tumor growth in the PDX mouse models of lung cancer / breast cancer / colorectal cancer

[0067] Furthermore, in this experimental example, the inhibitory effect of metformin and piperlongumine on tumor growth was detected in a human-derived PDX model (Patient-derived xenograft). In this experimental example, PDX models of human colorectal cancer, lung cancer, and triple-negative breast cancer were successfully established. As Figure 4 shown, compared with the control group (Ctrl), MP could significantly inhibit the tumor growth of colorectal cancer ( Figure 4 A-B), lung cancer ( Figure 4 C-D), and triple-negative breast cancer ( Figure 4 E-F) in the PDX model.

[0068] 5. Combining metformin and piperlongumine has the same tumor-killing effect as platinum-based chemotherapeutic drugs

[0069] Currently, chemotherapy drugs are still the mainstay of tumor treatment. Platinum-based drugs are one of the main types of tumor chemotherapy drugs. Next, in this experimental example, the inhibitory effects of MP and platinum-based drugs (cisplatin and carboplatin) on tumor growth were compared in a xenograft tumor model mediated by mouse breast cancer cells 4T-1 and mouse lung cancer cells LLC. As Figure 5 shown, in both tumor models, MP exhibited the same tumor inhibitory effect as cisplatin or carboplatin.

[0070] 6. Combining metformin and piperlongumine has no significant effect on the body weight, immune system, liver and kidney functions of mice

[0071] Next, in this experimental example, the effects of MP and platinum-based drugs (cisplatin and carboplatin) on the normal physiological indexes of mice were compared in a xenograft tumor model mediated by mouse breast cancer cells 4T-1 and mouse lung cancer cells LLC. As Figure 6 shown in A - B, cisplatin and carboplatin significantly inhibited the body weight, spleen weight and the number of peripheral blood leukocytes of mice. However, MP had no significant effect on these indexes. In addition, MP also had no significant effect on the contents of alanine aminotransferase (ALT), aspartate aminotransferase (AST), creatinine and cystatin C (Cys-C) in the peripheral blood of mice, indicating that MP has no toxic and side effects on the liver and kidney functions of mice.

[0072] 7. Platinum-based drugs kill normal and tumor cells without discrimination, but combining metformin and piperlongumine can selectively kill tumor cells

[0073] The experiment found that 40 μM cisplatin and 320 μM carboplatin could kill 80% of tumor cells ( Figure 7 A - F), but they also inhibited the survival of 80% of normal cells without discrimination ( Figure 7 G - H). Combining 250 μM metformin and 5 μM piperlongumine could also kill 80% of tumor cells ( Figure 7 I), but had no significant effect on normal cells ( Figure 7 I). The results showed that on the basis of MP and platinum-based drugs having equivalent tumor cell killing effects, platinum-based drugs inhibited the survival of normal cells without difference, while MP had no significant effect on the survival of normal cells. This result provided a cellular basis for the high efficiency and low toxicity of MP.

[0074] In summary, according to the experimental results of this experimental example, compared with the single use of metformin or piperlongumine, the combined use of metformin and piperlongumine can significantly enhance the anti-cancer activity, and the anti-cancer activity of MP can be comparable to that of platinum-based chemotherapeutic drugs. At the same time, compared with platinum-based chemotherapeutic drugs, in the case of comparable anti-cancer activity, MP has no toxic and side effects and can selectively kill tumor cells.

[0075] Therefore, the combined drug use scheme of the present invention has good application prospects.

Claims

1. Use of a composition composed of metformin and piperlongumine in the preparation of a drug for treating lung cancer, breast cancer or colorectal cancer, characterized in that: The dosage ratio of metformin to piperlongumine amide is as follows: the molar ratio is 250:5 or the mass ratio is 100:

1.

2. A drug for treating lung cancer, breast cancer or colorectal cancer, characterized in that: The drug consists of metformin and piperlongumine amide, and the dosage ratio of metformin to piperlongumine amide is as follows: the molar ratio is 250:5 or the mass ratio is 100:

1.

3. A pharmaceutical composition for treating lung cancer, breast cancer or colorectal cancer, characterized in that: It is prepared by using metformin and piperlongumine amide as active ingredients and adding pharmaceutically acceptable excipients or auxiliary components. The dosage ratio of metformin to piperlongumine amide is as follows: the molar ratio is 250:5 or the mass ratio is 100:1.

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