High-efficiency low-toxicity anticancer combined medicine and pharmaceutical composition

Abstract

The invention belongs to the technical field of medicines, and particularly relates to a high-efficiency low-toxicity anticancer combined medicine and a pharmaceutical composition. The invention provides application of guanidine compounds combined with piperlongumine in preparation of anti-cancer drugs. Wherein the guanidine compound can be selected from metformin. Compared with a single-dose drug, the combined drug provided by the invention has better anticancer activity. Compared with platinum chemotherapeutic drugs, the drug combination scheme has the same tumor killing efficacy, and has no obvious influence on mouse weight, immune system, liver function, kidney function and the like, which indicates that the toxic and side effects of the drug combination are obviously weaker than those of the platinum chemotherapeutic drugs. Therefore, the drug combination scheme provided by the invention has a very good application prospect.

Description

[0001] This application is a divisional application. The original application has the patent application number "2023106172903", the application date is "May 29, 2023", and the invention title is "A highly effective and low-toxicity anticancer combination drug and drug composition". Technical Field

[0002] This invention belongs to the field of pharmaceutical technology, specifically relating to a highly effective and low-toxicity anticancer combination drug and pharmaceutical composition. Background Technology

[0003] Cancer is a major malignant disease threatening human life and health. In recent years, cancer has shown a trend of affecting younger people, with incidence and mortality rates rising year by year. Currently, chemotherapy drugs remain the primary treatment for cancer. Statistics show that chemotherapy drugs account for 73% of the domestic oncology drug market, targeted therapies account for 23%, and immunotherapy drugs account for approximately 4%. However, while chemotherapy is effective, its toxic side effects are significant, a classic case of "killing a thousand enemies while injuring eight hundred of your own." These side effects severely reduce patients' quality of life and can even become a direct cause of death for cancer patients, representing a major bottleneck in oncology chemotherapy. Therefore, developing novel, highly effective, and low-toxicity chemotherapy drugs is of great importance to improving the quality of life for cancer patients.

[0004] Metformin is a first-line drug for treating type 2 diabetes. Numerous clinical and basic studies have shown that metformin not only has low toxicity but also possesses anticancer activity. Research indicates that metformin can selectively kill various types of tumor cells without significantly affecting normal cells. These findings provide a solid theoretical basis for developing metformin as an effective and low-toxicity novel anticancer drug. However, clinical trials have shown that metformin alone is not significantly effective in treating cancer; therefore, developing metformin adjuvants is an important topic in this field. Currently, some studies have attempted to enhance the anticancer activity of metformin through combination therapy. However, the current combination therapy regimens do not yet provide an ideal level of enhancement to the anticancer activity of metformin, failing to make its anticancer effect comparable to conventional chemotherapy drugs (such as platinum-based chemotherapy drugs).

[0005] Piperazine is an alkaloid with various activities including anti-inflammatory, antibacterial, anti-angiogenic, antioxidant, anticancer, and antidiabetic effects. However, the anticancer effect of piperazine alone is still not significant. Furthermore, no literature has yet reported its anticancer efficacy when used in combination with metformin.

[0006] In conclusion, with the aim of enhancing anticancer activity while reducing drug toxicity, it is still necessary in this field to develop more combination therapy regimens that enhance the anticancer activity of metformin. Summary of the Invention

[0007] To address the problems of existing technologies, this invention provides a highly effective and low-toxicity anticancer combination drug and drug composition, with the aim of enhancing the anticancer activity of guanidine compounds such as metformin through combination drug use.

[0008] The use of guanidine compounds in combination with long pepperamide in the preparation of anticancer drugs, wherein the ratio of the guanidine compound to long pepperamide is within one of the following ranges:

[0009] The molar ratio ranges from greater than 250:5 to less than or equal to 500:5.

[0010] The mass ratio is taken as greater than or equal to 50:1 and less than 100:1.

[0011] The mass ratio is taken in the range of greater than 100:1 and less than or equal to 150:1.

[0012] The present invention also provides the use of guanidine compounds in combination with long pepperamide in the preparation of anticancer drugs, wherein the molar ratio of the guanidine compound to long pepperamide is 250:5-500:5 or the mass ratio is 50:1-150:1, and the anticancer drugs are used to treat cancers other than lung cancer, breast cancer or colorectal cancer.

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

[0014] Preferably, the anticancer drug is used to treat lung cancer, breast cancer, colorectal cancer, or head and neck cancer.

[0015] The present invention also provides a combination drug for anticancer treatment, wherein the combination drug is a guanidine compound and long pepperamide administered separately or simultaneously, and the ratio of the guanidine compound to long pepperamide is one of the following ranges:

[0016] The molar ratio ranges from greater than 250:5 to less than or equal to 500:5.

[0017] The mass ratio is taken as greater than or equal to 50:1 and less than 100:1.

[0018] The mass ratio is taken in the range of greater than 100:1 and less than or equal to 150:1.

[0019] The present invention also provides a combination drug for anticancer treatment, wherein the combination drug is a guanidine compound and long pepperamide administered separately or simultaneously, wherein the molar ratio of the guanidine compound to long pepperamide is 250:5-500:5 or the mass ratio is 50:1-150:1, and the anticancer drug is used to treat cancers other than lung cancer, breast cancer or colorectal cancer.

[0020] Preferably, the guanidine compound is selected from metformin.

[0021] Preferably, the combined medication is used to treat lung cancer, breast cancer, colorectal cancer, or head and neck cancer.

[0022] The present invention also provides a pharmaceutical composition comprising a guanidine compound and long pepperamide as active ingredients, and the addition of pharmaceutically acceptable excipients or auxiliary ingredients, wherein the ratio of the guanidine compound to long pepperamide is one of the following ranges:

[0023] The molar ratio ranges from greater than 250:5 to less than or equal to 500:5.

[0024] The mass ratio is taken as greater than or equal to 50:1 and less than 100:1.

[0025] The mass ratio is taken in the range of greater than 100:1 and less than or equal to 150:1.

[0026] Preferably, the guanidine compound is selected from metformin.

[0027] This invention combines long peppermintamide with guanidine compounds (e.g., metformin) to significantly enhance the tumor-specific killing activity of guanidine compounds. This efficacy and specificity have been repeatedly validated in various tumor models, including a patient-derived xenograft (PDX) model of lung / breast / colorectal cancer and a spontaneous mouse model of breast cancer induced by MMTV-PyMT. Importantly, compared to platinum-based chemotherapy drugs, this combination therapy exhibits the same tumor-killing efficacy, but has no significant impact on mouse body weight, immune system, liver function, or kidney function, indicating that its toxic side effects are significantly weaker than those of platinum-based chemotherapy drugs. Therefore, the combination therapy method of this invention has excellent application prospects.

[0028] It should be noted that the experimental examples of this invention include in vitro cell experiments, showing that the co-treatment of tumor cells with long pepperamide and guanidine compounds exhibits a good selective killing effect on tumor cells. Therefore, the long pepperamide and guanidine compounds of this invention, when used to treat cancer, can be used in combination with existing pharmaceutical preparations, or they can be formulated into a new formulation for administration.

[0029] Obviously, based on the above description of the present invention, and according to common technical knowledge and conventional methods in the field, various other modifications, substitutions or alterations can be made without departing from the basic technical concept of the present invention.

[0030] The following detailed embodiments further illustrate the above-described content of the present invention. However, this should not be construed as limiting the scope of the present invention to the following examples. All technologies implemented based on the above-described content of the present invention fall within the scope of the present invention. Attached Figure Description

[0031] Figure 1 Experimental results show that reactive oxygen species play a key role in the selective killing of tumor cells by metformin. Cells were treated with 250 μM metformin alone or in combination with 5 μM piperlongumine (piper) for 48 hours, and cell viability was then assessed using trypan blue staining (A) or PI (B).

[0032] Figure 2 This study demonstrates the significant inhibition of MMTV-PyMT-induced spontaneous mammary tumor growth in mice by the combined use of metformin and long peppermint. (A) Tumor growth in female MMTV-PyMT mice at 95 days of age. (B) Dosing regimen diagram. Following the dosing regimen in B, 49-day-old female MMTV-PyMT-positive mice (n=4 per group) were treated intraperitoneally with 100 mg / kg metformin and 1 mg / kg long peppermint, either alone or in combination, for 46 consecutive days. At 95 days of age, tumor size (C), tumor number (D), and tumor weight (E) were observed.

[0033] Figure 3 This study investigated the experimental results showing that the combination of metformin and long peppermint significantly inhibited the growth of xenograft tumors of breast and colorectal cancer in mice and prolonged their survival. 1*10 5 4 T-1 cells (A and B) or 5*10 5 Two CT-26 cells (C and D) were subcutaneously injected into the right neck of 6-week-old female Balb / c mice. On the third day after cell inoculation, mice were intraperitoneally injected daily with 100 mg / kg metformin and 1 mg / kg piperazine (MP), while the control group received an equal volume of PBS (Ctrl). Mice in one group received the treatment for 18 consecutive days, and tumors were removed and their weight measured (A and C). Mice in the other group continued treatment until the tumor volume reached 1500 mm². 3 The mice were identified as having died ethically, and survival curves (C and D) were plotted.

[0034] Figure 4 This study presents experimental results demonstrating the significant inhibition of PDX (Patient-derived xenograft) tumor growth in lung cancer, breast cancer, and colorectal cancer by the combined use of metformin and long peppermint. (The tumors were 1-3 mm in size.) 3PDX 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 mice were injected intraperitoneally with MP (100 mg / kg metformin and 1 mg / kg long peppermint, daily), while 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 monitored using calipers. When the tumors reached day 24, they were removed, photographed, and weighed.

[0035] Figure 5 This is an experimental result demonstrating that the combination of metformin and long peppermint has the same tumor-killing effect as platinum-based chemotherapy drugs. (A) Dosing diagram. 1*10 5 4 T-1 cells (B and C) or 5*10 5 Two LLC cells (D and E) were subcutaneously injected into the right neck of female Balb / c (4T-1) or C57BL / 6 (LLC) mice. On day 3 post-inoculation, the mice were randomly divided into four groups (ctrl control group, MP group, Cis group, and Car group). The MP group received intraperitoneal injections of 100 mg / kg metformin and 1 mg / kg piperazine daily; the Cis group received intraperitoneal injections of 5 mg / kg cisplatin every 5 days; the Car group received intraperitoneal injections of 50 mg / kg carboplatin every 5 days; and the ctrl control group received an equal volume of PBS daily. On day 15, tumors were removed and tumor weight (BE) was measured.

[0036] Figure 6 The results show that the combined use of metformin and long peppermint had no significant effect on mouse body weight, immune system, or liver and kidney function. Figure 6 The effects of metformin, cisplatin, and carboplatin on body weight, spleen weight, and peripheral blood leukocyte count in mice were investigated using the following administration methods (AB). Female FVB mice were treated with 100 mg / kg metformin and 1 mg / kg long peppermint for 35 days (intraperitoneal injection daily), while the control group received an equal volume of PBS (Ctrl). On day 20 of administration, the levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), creatinine, and cystatin C (Cys-C) in the peripheral blood of mice were measured (C and D).

[0037] Figure 7The results show that platinum-based drugs indiscriminately kill normal and tumor cells, but the combination of metformin and long peppermint can selectively kill tumor cells. (AH) Cells were treated with cisplatin or carboplatin at the concentrations shown in the figure, and cell viability was then assessed using trypan blue staining. (IJ) Cells were treated with 250 μM metformin alone or in combination with 5 μM long peppermint, and cell viability was then assessed using trypan blue staining. *** indicates p < 0.0001.

[0038] Figure 8 The results of cell inhibition experiments on MDA-MB-231 breast cancer cells treated with 250 μM metformin in combination with different concentrations of long pepperamide are presented.

[0039] Figure 9 The results show the cell inhibition effects of treating MDA-MB-231 breast cancer cells, H292 lung cancer cells, and FaDu head and neck cancer cells with 5 μM piperazine in combination with different concentrations of metformin.

[0040] Figure 10 This represents the experimental results validating the combination of metformin (Met) and piperazine (Piper) in esophageal cancer KYSE450 cells and gastric cancer SGC-7901 cells. *** indicates p<0.0001. Detailed Implementation

[0041] In the following examples and experimental cases, all reagents and raw materials used were commercially available products.

[0042] Example 1: Combination drugs for anticancer treatment

[0043] This embodiment provides a combination anticancer drug, which is an injection containing 250 μM metformin and 5 μM piperazine.

[0044] Example 2: Combination drugs for anticancer treatment

[0045] This embodiment provides a combination anticancer drug, which is an injection containing 500 μM metformin and 5 μM piperazine.

[0046] Example 3: Combination drugs for anticancer treatment

[0047] This embodiment provides a combination anticancer drug, which is a tablet containing 100 mg metformin and 1 mg long pepperamide.

[0048] Example 4: Composition for anticancer purposes

[0049] This embodiment provides an anticancer composition, which is an injection containing 250 μM metformin and 5 μM piperazine.

[0050] Example 5: Composition for anticancer purposes

[0051] This embodiment provides an anticancer composition, which is an injection containing 500 μM metformin and 5 μM piperazine.

[0052] Example 6: Composition for anticancer purposes

[0053] This embodiment provides a combination anticancer drug, which is a tablet containing 80 mg metformin and 1 mg of piperazine.

[0054] Example 7: Composition for anticancer purposes

[0055] This embodiment provides a combination anticancer drug, which is a tablet containing 120 mg metformin and 1 mg long pepperamide.

[0056] The technical solution of the present invention will be further illustrated by the following experiments. In the following experimental examples, the combined dosing regimen of metformin and piperlongumine is referred to as MP.

[0057] Experimental Example 1: Anticancer activity of metformin and its combination with piperazine

[0058] I. Experimental Methods

[0059] 1. Cell viability detection

[0060] Cultured tumor cells were digested with trypsin and collected; then washed twice with PBS, and counted to achieve a cell concentration of 1×10⁻⁶. 5 100 Take 2×10 5 Tumor cells (using lung cancer cell line H292, breast cancer cell line MDA-MB-231, and head and neck cancer cell line FaDu, respectively) were placed in 12-well plates and cultured overnight at 37°C. The cells were treated with 250 μM metformin alone or in combination with 5 μM piperlongumine (piper) for 48 hours. All cells (including suspension cells) were collected, and dead cells were stained with trypan blue or PI (propidium iodide). The cell death rate was then detected using a LUNA-FLTM fully automated cell counter.

[0061] 2. Detection of the anticancer activity of MP using an MMTV-PyMT-induced spontaneous breast cancer mouse model.

[0062] Female MMTV-PyMT-positive mice aged 49 days were randomly divided into two groups (ctrl control group and MP group), with 4 mice in each group. The experimental group mice were injected intraperitoneally with MP (100 mg / kg metformin and 1 mg / kg piperazine, daily), while the control group was injected with an equal volume of PBS (Ctrl). On day 46 after injection, tumors were removed, photographed, and the number and weight of tumors were recorded.

[0063] 3. Investigate the anticancer activity of MP using the CDX (Cell-derived xenograft) model.

[0064] 1*10 5 4 T-1 cells or 5*10 5 One CT-26 cell was subcutaneously injected into the right neck of 6-week-old female Balb / c mice. On the third day after cell inoculation, mice were intraperitoneally injected daily with 100 mg / kg metformin and 1 mg / kg piperazine (MP), while the control group received an equal volume of PBS (Ctrl). One group of mice received the treatment for 18 consecutive days, after which tumors were removed and their weight measured. The other group of mice continued the treatment until their tumors reached 1500 mm². 3 The mice were identified as having died ethically, and survival curves were plotted.

[0065] 4. Detecting the anticancer activity of MP using a patient-derived xenograft (PDX) model for lung / breast / colorectal cancer.

[0066] Fresh human lung / breast / colorectal tumor tissue was cut into 1-3 mm³ pieces (F0 generation) and subcutaneously injected into the right neck of female severely immunodeficient mice (NSG mice) (F1 generation); the tumors were allowed to grow to 400-600 mm³. 3 Remove the tumor and re-cut it into 1-3mm pieces. 3 Small pieces were then re-inoculated subcutaneously into the right neck of NSG mice (F2 generation); the tumor volume was then allowed to grow to 400-600 mm. 3 Tumor tissue was stored in liquid nitrogen or injected subcutaneously into the right neck of new NSG mice (F3-F6 generations) for drug efficacy evaluation. Tumor tissue was retained in each generation to identify the molecular and histological consistency of tumor samples from each generation.

[0067] Sixteen 6-week-old female NSG mice were prepared and randomly divided into a control group and an experimental group (n=8 per group); the PDX tumors of each F3 generation were cut into 1-3 mm sections. 3 Small pieces were inoculated subcutaneously into the right neck of NSG mice; the PDX tumors were allowed to grow to 15-20 mm. 3Mice in the experimental group were injected intraperitoneally with MP (100 mg / kg metformin and 1 mg / kg piperazine, daily), while mice in the control group were injected with an equal volume of PBS (Ctrl). Tumor growth was dynamically monitored using calipers on days 0, 4, 8, 12, 16, 18, and 24 after injection. When the tumor reached day 24, it was removed, photographed, and weighed.

[0068] 5. Using the CDX model to compare the tumor-killing effects of MP and platinum-based chemotherapy drugs.

[0069] 1*10 5 4 T-1 cells or 5*10 5 LLC cells were subcutaneously inoculated into the right neck of female Balb / c (4T-1) or C57BL / 6 (LLC) mice. On the third day after cell inoculation, the mice were randomly divided into four groups (ctrl control group, MP group, Cis group, and Car group, with 6 mice in each group). The MP group received intraperitoneal injections of 100 mg / kg metformin and 1 mg / kg piperazine daily; the Cis group received intraperitoneal injections of 5 mg / kg cisplatin every 5 days; the Car group received intraperitoneal injections of 50 mg / kg carboplatin every 5 days; and the ctrl control group received an equal volume of PBS daily. On day 15 of drug administration, tumors were removed and their weight was measured.

[0070] II. Experimental Results

[0071] 1. Anticancer activity of metformin alone or in combination with piperazine

[0072] First, the effect of pipelongumine (piper) on the anticancer activity of low-dose metformin (250 μM) was examined. Figure 1 As shown in A and B, compared with the use of low-dose metformin or long pepper alone, the combined use of metformin and long pepper significantly inhibited the survival of various tumor cells (including lung cancer cell line H292, breast cancer cell line MDA-MB-231, and head and neck cancer cell line FaDu).

[0073] 2. The combination of metformin and long peppermint significantly inhibited the growth of spontaneous mammary tumors induced by MMTV-PyMT in mice.

[0074] The above studies indicate that long pepperamide plays a crucial role in the anticancer activity of metformin. Therefore, the combination of metformin and long pepperamide may be a novel cancer treatment strategy. To verify the in vivo anticancer activity of the combined metformin and long pepperamide, spontaneous breast cancer in MMTV-PyMT mice was first selected as the research model (in this model, polyoma virus middle T antigen / PyMT can be specifically expressed in mammary cells, leading to abnormal proliferation of mammary cells; mice develop primary mammary tumors at week 5; and significant lung metastases at week 12). Figure 2 As shown in Figure A, MMTV-PyMT mice developed significant tumors around the mammary glands at 95 days of age. Based on this, in this experiment, mice were intraperitoneally injected with 100 mg / kg metformin and 1 mg / kg long peppermint at 49 days of age, either alone or in combination, for 46 consecutive days. When the mice reached 95 days of age, the size and number of tumors were observed. Figure 2 B). For example Figure 2 In mice, metformin or long peppermint alone had no significant effect on tumor growth, but the combined use of metformin and long peppermint significantly inhibited mammary tumor growth. These results suggest that the combination of metformin and long peppermint represents a potential novel strategy for cancer treatment.

[0075] 3. The combination of metformin and long pepper significantly inhibited the growth of xenograft tumors of breast cancer and colorectal cancer in mice and prolonged the survival of mice.

[0076] Next, the anticancer activity of the combination of metformin and long peppermint was tested in mouse xenograft tumor models mediated by mouse breast cancer cells 4T-1 and mouse colorectal cancer cells CT-26. Figure 3 As shown, MP significantly inhibited tumor growth in mice and significantly prolonged the survival of tumor-bearing mice.

[0077] 4. The combination of metformin and long peppermint significantly inhibited tumor growth in PDX mouse models of lung cancer / breast cancer / colorectal cancer.

[0078] Furthermore, this experimental example investigated the inhibitory effects of metformin and long peppermint on tumor growth in a patient-derived xenograft (PDX) model. This experimental example successfully established human PDX models for colorectal cancer, lung cancer, and triple-negative breast cancer (TNBC). Figure 4 As shown, compared with the control group (Ctrl), MP significantly inhibited colorectal cancer in the PDX model. Figure 4 AB), lung cancer ( Figure 4 CD) and triple-negative breast cancer ( Figure 4 EF) tumor growth.

[0079] 5. The combination of metformin and long pepper has the same tumor-killing effect as platinum-based chemotherapy drugs.

[0080] Currently, chemotherapy drugs remain the primary treatment for cancer. Platinum-based chemotherapy drugs are one of the main types of cancer chemotherapy drugs. Next, this experiment compared the inhibitory effects of monosodium glutamate (MP) and platinum-based drugs (cisplatin and carboplatin) on tumor growth in mouse 4T-1 breast cancer cells and LLC-mediated xenograft tumor models. Figure 5 As shown, MP exhibited the same tumor-suppressive effect as cisplatin or carboplatin in both tumor models.

[0081] 6. The combined use of metformin and long pepper dichlorvos had no significant effect on body weight, immune system, or liver and kidney function in mice.

[0082] Next, this experiment compared the effects of MP and platinum-based drugs (cisplatin and carboplatin) on normal physiological indicators in mice using xenograft models mediated by mouse breast cancer cells 4T-1 and mouse lung cancer cells LLC. Figure 6 As shown in AB, cisplatin and carboplatin significantly inhibited mouse body weight, spleen weight, and peripheral blood leukocyte count; however, MP had no significant effect on these indicators. Furthermore, MP had no significant effect on the levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), creatinine, and cystatin C (Cys-C) in mouse peripheral blood, indicating that MP has no toxic side effects on mouse liver and kidney function.

[0083] 7. Platinum-based drugs indiscriminately kill both normal and tumor cells, but when combined with metformin and long peppermint, they can selectively kill tumor cells.

[0084] Experiments showed that 40 μM cisplatin and 320 μM carboplatin could kill 80% of tumor cells. Figure 7 AF), but it also indiscriminately inhibited the survival of 80% of normal cells. Figure 7 GH). The combination of 250 μM metformin and 5 μM piperazine can also kill 80% of tumor cells. Figure 7 I), but it has no significant effect on normal cells. Figure 7 (I) The results showed that, while MP and platinum-based drugs were equivalent in killing tumor cells, platinum-based drugs indiscriminately inhibited the survival of normal cells, while MP had no significant effect on the survival of normal cells. This result provides a cellular basis for the high efficiency and low toxicity of MP.

[0085] Experimental Example 2: Screening of the dosage ratio of metformin and long peppermint.

[0086] I. Experimental Methods

[0087] MDA-MB-231, H292, or FaDu cells were seeded at a density of 5,000–8,000 cells per well in 96-well plates and incubated overnight at 37°C in a 5% CO2 incubator. Subsequently, different concentrations of the drug were added for treatment for 48–72 hours according to the experimental groups. After treatment, 20 μL of MTS reagent (Promega, CellTiter) was added to each well. The AQueous OneSolution Cell Proliferation Assay was transferred to wells containing 100 μL of culture medium and incubated for 1–4 hours in the dark. After the reaction, the absorbance (OD490) of each well was read at 490 nm using a microplate reader. Blank wells (cell-free but containing MTS) served as background controls. Cell viability was calculated using the following formula:

[0088] Cell viability (%) = (OD490 of experimental group - OD490 of blank group) / (OD490 of control group - OD490 of blank group) × 100%.

[0089] II. Experimental Results

[0090] To investigate the optimal ratio of metformin to long pepperamide, we first treated MDA-MB-231 breast cancer cells with 250 μM metformin combined with different concentrations of long pepperamide. Figure 8 As shown, when metformin was used in combination with 1.25 μM long pepperamide, it inhibited cell viability by approximately 20%; while when used in combination with 20 μM long pepperamide, the inhibition rate increased to approximately 80%. Therefore, under the condition that metformin concentration is fixed at 250 μM, long pepperamide can enhance its anticancer activity within the range of 1.25–20 μM, and the effective molar ratio of the two is 250:1.25–250:20.

[0091] Furthermore, we treated MDA-MB-231 breast cancer cells, H292 lung cancer cells, and FaDu head and neck cancer cells with 5 μM piperazine in combination with different concentrations of metformin. Figure 9 As shown, metformin at 250–2000 μM and long pepperamide at 5 μM both exhibited significant anticancer activity, indicating that metformin has the ability to synergistically inhibit tumor cell growth in the range of 250–2000 μM under the condition of 5 μM long pepperamide, with corresponding ratios of 250:5–2000:5 (i.e., 50:1 to 400:1).

[0092] In summary, the results from the two parts indicate that metformin and long pepperamide can both produce good anticancer effects when the ratio is between 250:20 and 2000:5.

[0093] Experimental Example 3: The effect of metformin combined with long peppermint on the inhibition of gastric and esophageal cancer.

[0094] I. Experimental Methods

[0095] Cultured tumor cells were digested with trypsin and collected; then washed twice with PBS, and counted to achieve a cell concentration of 1×10⁻⁶. 5 100 μL; take 2 × 10 5 Tumor cells (using the esophageal cancer cell line KYSE450 and the gastric cancer cell line SGC-7901, respectively) were placed in 12-well plates and cultured overnight at 37°C. The cells were treated with 250 μM metformin alone or in combination with 5 μM piperlongumine (piper) for 48 hours. All cells (including suspension cells) were collected, and dead cells were stained with trypan blue or PI (propidium iodide). The cell death rate was then detected using the LUNA-FLTM fully automated cell counter.

[0096] II. Experimental Results

[0097] To further verify the broad-spectrum anticancer activity of the combination of metformin (Met) and piperazine (Piper), we conducted validation studies in esophageal cancer KYSE450 cells and gastric cancer SGC-7901 cells. Figure 10 As shown, 250 μM metformin combined with 5 μM long pepper significantly inhibited the survival rate of both cell types, and the effect was significantly better than that of the experimental group using either drug alone. This result indicates that the two drugs produced synergistic anticancer activity.

[0098] Based on the aforementioned experimental results, this combined regimen demonstrated good anti-cancer activity in various tumor cells, including lung cancer, breast cancer, colorectal cancer, gastric cancer, head and neck cancer, and esophageal cancer.

[0099] In summary, the experimental results of this example demonstrate that, compared to using metformin or long pepperamide alone, the combined use of metformin and long pepperamide significantly enhances anticancer activity, with MP's anticancer activity comparable to platinum-based chemotherapy drugs. Furthermore, compared to platinum-based chemotherapy drugs, MP exhibits no toxic side effects and selectively kills tumor cells while maintaining comparable anticancer activity. Therefore, the combined drug regimen of this invention has excellent application prospects.

Claims

1. The use of guanidine compounds in combination with piperazine in the preparation of anticancer drugs, characterized in that: The ratio of the guanidine compound to piperazine is one of the following ranges: The molar ratio ranges from greater than 250:5 to less than or equal to 500:

5. The mass ratio is taken as greater than or equal to 50:1 and less than 100:

1. The mass ratio is taken in the range of greater than 100:1 and less than or equal to 150:

1.

2. The use of guanidine compounds in combination with piperazine in the preparation of anticancer drugs, characterized in that: The ratio of the guanidine compound to piperazine is 250:5-500:5 in molar ratio or 50:1-150:1 in mass ratio. The anticancer drug is used to treat cancers other than lung cancer, breast cancer, or colorectal cancer.

3. The use according to claim 1 or 2, characterized in that: The guanidine compound is selected from metformin.

4. The use according to claim 1, characterized in that: The anticancer drug is used to treat lung cancer, breast cancer, colorectal cancer, or head and neck cancer.

5. A combination drug for anticancer use, characterized in that: The combined medication is a guanidine compound and long pepperamide administered separately or simultaneously, wherein the ratio of the guanidine compound to long pepperamide is one of the following ranges: The molar ratio ranges from greater than 250:5 to less than or equal to 500:5; The mass ratio is taken as greater than or equal to 50:1 and less than 100:

1. The mass ratio is taken in the range of greater than 100:1 and less than or equal to 150:

1.

6. A combination drug for anticancer use, characterized in that: The combined drug is a guanidine compound and long pepperamide administered separately or simultaneously, wherein the ratio of the guanidine compound to long pepperamide is a molar ratio of 250:5-500:5 or a mass ratio of 50:1-150:1, and the anticancer drug is used to treat cancers other than lung cancer, breast cancer, or colorectal cancer.

7. The combination drug according to claim 5 or 6, characterized in that: The guanidine compound is selected from metformin.

8. The combination drug according to claim 5, characterized in that: The combined medications are used to treat lung cancer, breast cancer, colorectal cancer, or head and neck cancer.

9. A pharmaceutical composition, characterized in that: It is made with guanidine compounds and long pepperamide as active ingredients, and with the addition of pharmaceutically acceptable excipients or auxiliary ingredients, wherein the ratio of the guanidine compounds to long pepperamide is one of the following ranges: The molar ratio ranges from greater than 250:5 to less than or equal to 500:

5. The mass ratio is taken as greater than or equal to 50:1 and less than 100:

1. The mass ratio is taken in the range of greater than 100:1 and less than or equal to 150:

1.

10. The pharmaceutical composition according to claim 9, characterized in that: The guanidine compound is selected from metformin.

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