WO2024244607A1 - Efficient and low-toxicity anti-cancer combined drug and pharmaceutical composition - Google Patents

Abstract

The present invention belongs to the technical field of medicine, and in particular relates to an efficient and low-toxicity anti-cancer combined drug and a pharmaceutical composition. Provided is the use of a guanidine compound combined with piperlongumine in the preparation of an anti-cancer drug. The guanidine compound can be selected from metformin. Compared with a single drug, the combined drug has a better anti-cancer activity. Furthermore, compared with platinum-based chemotherapeutic drugs, the combined drug regimen has the same tumor killing efficacy, but has no significant effect on the body weight, immune system, liver function, kidney function, etc., of mice, indicating that the toxicity and side effects of the combined drug regimen are significantly weaker than those of the platinum-based chemotherapeutic drugs. Therefore, the combined drug scheme has good application prospects.

Description

A highly effective and low-toxic anticancer combined drug and drug composition Technical Field

The present invention belongs to the field of medical technology, and in particular relates to a highly effective and low-toxic anticancer combined drug and a drug composition.

Background Art

Cancer is a major malignant disease that threatens human life and health. In recent years, cancer has become younger, and the incidence and mortality rates have increased year by year. At present, cancer treatment drugs are still mainly chemotherapy drugs. According to statistics, chemotherapy drugs account for 73% of the domestic tumor drug market, targeted drugs account for 23%, and immunotherapy drugs account for about 4%. However, although chemotherapy is effective, its toxic side effects are very obvious, that is, the so-called killing one thousand enemies and injuring eight hundred of oneself. These toxic 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 to the quality of life of cancer patients.

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 toxicity and side effects, but also has anti-cancer activity. Studies have 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, so the development of metformin enhancers (adjuvants) is an important topic in this field. At present, there have been related studies attempting to enhance the anti-cancer activity of metformin by combining drugs, however, the current combination drug regimens are still not ideal for enhancing the anti-cancer activity of metformin, and cannot make the anti-cancer effect of metformin comparable to conventional chemotherapy drugs (such as platinum chemotherapy drugs).

Piper longum amide is an alkaloid with multiple activities such as anti-inflammatory, antibacterial, anti-angiogenic, antioxidant, anti-cancer and anti-diabetic. However, the anti-cancer effect of piperon longum amide alone is still not significant enough. There is no literature report on the anti-cancer effect of its combination with metformin.

In conclusion, for the purpose of enhancing anti-cancer activity while reducing drug toxicity, it is still necessary in the art to develop more combined drug regimens that enhance the anti-cancer activity of metformin.

Summary of the invention

In view of the problems of the prior art, the present invention provides a highly effective and low-toxic anticancer combination drug and a pharmaceutical composition, the purpose of which is to enhance the anticancer activity of guanidine compounds such as metformin through combined use.

Use of guanidine compounds combined with piperlongum amide in the preparation of anticancer drugs.

Preferably, the molar ratio of the guanidine compound to the piper longum amide is 250:5-500:5 or the mass ratio is 50:1-150:1. Preferably, the molar ratio of the guanidine compound to the piper longum amide is 250:5 or the mass ratio is 100:1.

Preferably, the guanidine compound is selected from metformin.

Preferably, the anticancer drug is used to treat lung cancer, breast cancer or colorectal cancer.

The present invention also provides a combined drug for anticancer, wherein the combined drug is a guanidine compound and piperlongum amide which are administered separately or simultaneously.

Preferably, the molar ratio of the guanidine compound to the piper longum amide is 250:5-500:5 or the mass ratio is 50:1-150:1. Preferably, the molar ratio of the guanidine compound to the piper longum amide is 250:5 or the mass ratio is 100:1.

Preferably, the guanidine compound is selected from metformin.

Preferably, the combination drug is used to treat lung cancer, breast cancer or colorectal cancer.

The present invention also provides a pharmaceutical composition, which is prepared by using guanidine compounds and piperlongum amide as active ingredients and adding pharmaceutically acceptable excipients or auxiliary ingredients.

Preferably, the molar ratio of the guanidine compound to the piper longum amide is 250:5-500:5 or the mass ratio is 50:1-150:1. Preferably, the molar ratio of the guanidine compound to the piper longum amide is 250:5 or the mass ratio is 100:1.

Preferably, the guanidine compound is selected from metformin.

Preferably, the pharmaceutical composition is used to treat lung cancer, breast cancer or colorectal cancer.

The present invention combines longanamide with guanidine compounds (such as metformin) to 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 models) and MMTV-PyMT-induced spontaneous breast cancer mouse models. More importantly, compared with platinum-based chemotherapy drugs, this combination therapy has the same tumor killing efficacy, but it has no significant effect on mouse body weight, immune system, liver function, kidney function, etc., indicating that its toxic side effects are significantly weaker than platinum-based chemotherapy drugs. Therefore, the combined drug therapy method of the present invention has a good application prospect.

It should be noted that the experimental examples of the present invention include in vitro cell experiments, and the use of piperlongumamide and guanidine compounds to treat tumor cells together showed a good selective killing effect on tumor cells. Therefore, when the piperlongumamide and guanidine compounds of the present invention are used to treat cancer, they can be used in combination with existing pharmaceutical preparations, or they can be used together in the form of a combination to prepare a new preparation for administration.

Obviously, according to the above content of the present invention, according to the common technical knowledge and customary means in this field, Without departing from the above basic technical concept of the present invention, other various forms of modification, replacement or change can be made.

The above contents of the present invention are further described in detail below through specific implementation methods in the form of embodiments. However, this should not be understood as the scope of the above subject matter of the present invention being limited to the following examples. All technologies realized based on the above contents of the present invention belong to the scope of the present invention.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 shows the experimental results that reactive oxygen species play a key role in the selective killing of tumor cells by metformin. The 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 staining (B).

Figure 2 shows the experimental results that the combination of metformin and piperlongumide 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. (B) Dosage pattern diagram. According to the dosing pattern in B, 100 mg/kg of metformin and 1 mg/kg of piperlongumide were used intraperitoneally alone or in combination to treat female MMTV-PyMT-positive mice (4 mice in each group) at the age of 49 days. The treatment lasted for 46 days. When the mice grew to 95 days, the tumor size of the mice was observed (C), the number of tumors (D) and the tumor weight (E) were measured.

Figure 3 shows the experimental results that the combination of metformin and piperlongumide significantly inhibited the growth of breast cancer and colorectal cancer xenografts in mice and prolonged the survival of mice. 1*10 ⁵ 4T-1 cells (A and B) or 5*10 ⁵ CT-26 cells (C and D) were inoculated subcutaneously in 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 piperlongumide (MP) were intraperitoneally injected 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 weight was measured (A and C); the other group of mice continued to be administered, and the ethical death of the mice was determined when the tumor volume grew to 1500 mm ³ , and the survival curve was drawn (C and D).

Figure 4 shows the experimental results that the combination of metformin and piperlongumide significantly inhibited the growth of lung cancer, breast cancer and colorectal cancer PDX (Patient-derived xenograft) tumors. 1-3mm ³ PDX tumors were cut into small pieces and inoculated subcutaneously in the right neck of NSG mice; when the PDX tumors grew to 15-20mm, the experimental group mice were intraperitoneally injected with MP (100mg/kg of metformin and 1mg/kg of piperlongumide, every day), 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 tumor grew to day 24, the tumor was removed, photographed and weighed.

Figure 5 shows the experimental results that the combination of metformin and piperidine has the same tumor killing effect as platinum chemotherapy drugs. (A) Dosage pattern diagram. 1*10 ⁵ 4T-1 cells (B and C) or 5*10 ⁵ LLC 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 cell inoculation, the mice were evenly divided into four groups (ctrl control group, MP group, Cis group and Car group). The MP group was intraperitoneally injected with 100 mg/kg of metformin and 1 mg/kg of piperidine every day; the Cis group was intraperitoneally injected with 5 mg/kg of cisplatin once every 5 days; the Car group was intraperitoneally injected with 50 mg/kg of carboplatin once every 5 days; and the ctrl control group was injected with an equal volume of PBS every day. On the 15th day of administration, the tumors were removed and the tumor weight (BE) was measured.

Figure 6 shows the experimental results that the combination of metformin and piperlongumide has no significant effect on the body weight, immune system, liver and kidney function of mice. According to the dosing mode of Figure 6, the effects of MP, cisplatin and carboplatin on the body weight, spleen weight and peripheral blood leukocyte count of mice were detected (A-B). Female FVB mice were treated with 100 mg/kg of metformin and 1 mg/kg of piperlongumide for 35 days (intraperitoneal injection every day), and the control group was injected with an equal amount of PBS (Ctrl). On the 20th day of administration, the levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), creatinine (Creatinine) and cystatin C (Cys-C) in the peripheral blood of mice were detected (C and D).

Figure 7 shows the experimental results that platinum drugs indiscriminately kill normal and tumor cells, but can selectively kill tumor cells when combined with metformin and piperlongumide. (A-H) Each cell was treated with cisplatin or carboplatin at the concentration shown in the figure, and then the survival of each cell was detected by trypan blue staining. (I-J) The indicated cells were treated with 250μm metformin alone or in combination with 5μm piperlongumide, and then the survival of each cell was detected by trypan blue staining. *** represents p<0.0001.

DETAILED DESCRIPTION

In the following examples and experimental examples, the reagents and raw materials used are all commercially available.

Example 1 Combination drug for anticancer

This embodiment provides an anticancer combination drug, which is an injection containing 250 μM metformin and 5 μM piperidine.

Example 2 Combination Drugs for Anticancer

This embodiment provides an anticancer combination drug, which is an injection containing 500 μm metformin and 5 μm piperlongum amide.

Example 3 Combination drugs for anticancer

This embodiment provides an anticancer combined drug, which is a tablet containing 100 mg of metformin and 1 mg of piperonil.

Example 4 Composition for Anticancer

This embodiment provides an anticancer composition, which is an injection containing 250 μm metformin and 5 μm piperlongum amide.

Example 5 Composition for Anticancer

This embodiment provides an anticancer composition, which is an injection containing 500 μm metformin and 5 μm piperlongum amide.

Example 6 Composition for Anticancer

This embodiment provides an anticancer combined drug, which is a tablet containing 80 mg of metformin and 1 mg of piperonil.

Example 7 Composition for Anticancer

This embodiment provides an anticancer combined drug, which is a tablet containing 120 mg of metformin and 1 mg of piperonil.

The technical solution of the present invention is further described below through experiments. In the following experimental examples, the medication scheme of combining metformin and piperlongumine is referred to as MP.

Experimental Example 1 Anticancer activity of metformin and its combination with piperlongum amide

1. Experimental Methods

1. Cell activity detection

The cultured tumor cells were digested with trypsin and collected; the cells were washed twice with PBS and then counted to a cell concentration of 1×10 ⁵ /100L; 2×10 ⁵ tumor cells were placed in a 12-well plate and cultured in a 37°C constant temperature incubator overnight; the 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, dead cells were stained with trypan blue or PI (Propidium Iodide), and then the cell death rate was detected using a LUNA-FLTM fully automatic cell counter.

2. Detection of the anticancer activity of MP using the spontaneous breast cancer model induced by MMTV-PyMT in mice

MMTV-PyMT-positive female mice at 49 days of age were evenly divided into two groups (ctrl control group, MP group), with 4 mice in each group. The experimental group mice were intraperitoneally injected with MP (100 mg/kg of metformin and 1 mg/kg of piperlongum amide, every day), 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 tumors were counted.

3. Using the CDX (Cell-derived xenograft) model to explore the anticancer activity of MP

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 of metformin and 1 mg/kg of piperidine (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; the other group of mice continued to be administered, and the ethical death of the mice was determined when the tumor volume grew to 1500 mm ³ , and the survival curve was drawn.

4. Detect the anticancer activity of MP using lung cancer/breast cancer/colorectal cancer PDX (Patient-derived xenograft) models

Fresh human lung/breast/colorectal tumor tissues were cut into 1-3mm3 small pieces (F0 generation) and inoculated into the right neck subcutaneous of female severely immunodeficient mice (NSG mice) (F1 generation); when the tumor volume grew to ^400-600mm3 , the tumor was removed and re-cut into ^1-3mm3 small pieces and re-inoculated into the right neck subcutaneous of NSG mice (F2 generation); when the tumor volume grew to ^400-600mm3 , the tumor tissue was stored in liquid nitrogen or inoculated into the right neck subcutaneous of new NSG mice (F3-F6 generations) for drug efficacy evaluation. Tumor tissues need to be retained for each generation to identify the molecular and tissue morphological consistency of each generation of tumor samples.

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); each F3 generation PDX tumor was cut into 1-3 mm ³ small pieces and inoculated into the right neck subcutaneous of NSG mice; when the PDX tumor grew to 15-20 mm ³ , the experimental group mice were intraperitoneally injected with MP (100 mg/kg of metformin and 1 mg/kg of piperlongum amide, every day), 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 tumor grew to day 24, the tumor was removed, photographed, and weighed.

5. Comparison of the tumor-killing effects of MP and platinum chemotherapy drugs using the CDX model

1*10 ⁵ 4T-1 cells or 5*10 ⁵ LLC cells were inoculated 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 of metformin and 1 mg/kg of piperidine every day, the Cis group was intraperitoneally injected with 5 mg/kg of cisplatin every 5 days; the Car group was intraperitoneally injected with 50 mg/kg of carboplatin every 5 days, and the ctrl control group was injected with an equal volume of PBS every day. On the 15th day of administration, the tumors were removed and the tumor weight was measured.

2. Experimental Results

1. Anticancer activity of metformin alone or in combination with piperlongumide

First, the effect of piperlongumine (piper) on low-dose metformin (250 μM) was tested. Effects on anticancer activity. As shown in Figures 1A and B, the combined use of metformin and piperlongumide can more significantly inhibit the survival of multiple tumor cells than the use of low-dose metformin or piperlongumide alone.

2. The combination of metformin and piperlongum amide significantly inhibited the growth of spontaneous breast tumors in mice induced by MMTV-PyMT

The above studies show that piperlongumide plays a key role in the anticancer activity of metformin. Therefore, the combination of metformin and piperlongumide may be a new tumor treatment strategy. In order to verify the anti-tumor activity of the combination of metformin and piperlongumide in vivo, spontaneous breast cancer in MMTV-PyMT mice was first selected as a research model (in this model, Polyoma virus middle T antigen/PyMT can be specifically expressed in breast cells, leading to abnormal proliferation of breast cells; mice develop primary breast tumors in the 5th week; obvious lung metastasis appears in the 12th week). As shown in Figure 2A, MMTV-PyMT mice had obvious tumors around the mammary gland at 95 days. Based on this, in this experimental case, 100 mg/kg of metformin and 1 mg/kg of piperlongumide were injected intraperitoneally alone or in combination at 49 days of age, and the treatment was continued for 46 days. When the mice grew to 95 days old, the size and number of tumors in the mice were observed (Figure 2B). As shown in Figure 2C-E, the use of metformin or piperlongamide alone had no significant effect on mouse tumor growth, but the combined use of metformin and piperlongamide significantly inhibited the growth of mouse breast tumors. The results show that the combination of metformin and piperlongamide is a potential new strategy for tumor treatment.

3. The combination of metformin and piperlongum amide significantly inhibited the growth of breast cancer and colorectal cancer xenografts in mice and prolonged the survival of mice

Next, the anticancer activity of the combined metformin and piperlongum amide was tested in mouse xenograft models mediated by mouse breast cancer cell 4T-1 and mouse colorectal cancer cell CT-26. As shown in Figure 3, MP significantly inhibited tumor growth in mice and significantly prolonged the survival of tumor-bearing mice.

4. Combination of metformin and piperlongumide significantly inhibits tumor growth in lung cancer/breast cancer/colorectal cancer PDX mouse models

Further, this experimental example detected the inhibitory effect of metformin and piperlongum on tumor growth in a patient-derived xenograft model. This experimental example successfully established human colorectal cancer (Colon cancer), lung cancer (Lung Cancer) and triple-negative breast cancer (TNBC) PDX models. As shown in Figure 4, compared with the control group (Ctrl), MP can significantly inhibit the growth of colorectal cancer (Figure 4A-B), lung cancer (Figure 4C-D) and triple-negative breast cancer (Figure 4E-F) tumors in the PDX model.

5. Combination of metformin and piperidine has the same tumor killing effect as platinum chemotherapy drugs

At present, the main cancer treatment drugs are still chemotherapy drugs. Platinum is one of the main types of cancer chemotherapy drugs. Next, this experiment was conducted on mouse breast cancer cells 4T-1 and mouse lung cancer cells LLC. The inhibitory effects of MP and platinum drugs (cisplatin and carboplatin) on tumor growth were compared in a xenograft model induced by PD-1. As shown in Figure 5, MP showed the same tumor inhibitory effect as cisplatin or carboplatin in both tumor models.

6. The combination of metformin and piperlongumide had no significant effect on the body weight, immune system, liver and kidney function of mice

Next, this experimental example used mouse breast cancer cell 4T-1 and mouse lung cancer cell LLC mediated xenograft tumor models to compare the effects of MP and platinum drugs (cisplatin and carboplatin) on normal physiological indicators of mice. As shown in Figure 6A-B, cisplatin and carboplatin significantly inhibited the weight of mice, spleen weight and peripheral blood leukocyte count, but MP had no significant effect on these indicators. In addition, MP had no significant effect on the levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), creatinine (Creatinine) and cystatin C (Cys-C) in the peripheral blood of mice, indicating that MP has no toxic side effects on the liver and kidney function of mice.

7. Platinum drugs indiscriminately kill normal and tumor cells, but combined with metformin and piperidine can selectively kill tumor cells

The experiment found that 40μM cisplatin and 320μM carboplatin can kill 80% of tumor cells (Figure 7A-F), but they also indifferently inhibited the survival of 80% of normal cells (Figure 7G-H). The combination of 250μM metformin and 5μM piperidine can also kill 80% of tumor cells (Figure 7I), but has no significant effect on normal cells (Figure 7I). The results show that on the basis of the equivalent killing of tumor cells by MP and platinum drugs, platinum drugs indifferently inhibit the survival of normal cells, while MP has no significant effect on the survival of normal cells. This result provides a cellular basis for the high efficiency and low toxicity of MP.

In summary, the experimental results of this experimental example show that compared with the use of metformin or piperlongamide alone, the combined use of metformin and piperlongamide can significantly enhance the anticancer activity, and the anticancer activity of MP is comparable to that of platinum chemotherapy drugs. At the same time, compared with platinum chemotherapy drugs, under the condition of comparable anticancer activity, MP has no toxic side effects and can selectively kill tumor cells.

Therefore, the combined drug regimen of the present invention has a good application prospect.

Claims (12)

Use of guanidine compounds combined with piperlongum amide in the preparation of anticancer drugs. The use according to claim 1 is characterized in that the usage ratio of the guanidine compound to piperlongum amide is: a molar ratio of 250:5-500:5 or a mass ratio of 50:1-150:1. The use according to claim 1, characterized in that the guanidine compound is selected from metformin. The use according to claim 1 is characterized in that the anticancer drug is used to treat lung cancer, breast cancer or colorectal cancer. A combined drug for anticancer, characterized in that the combined drug is a guanidine compound and piperlongum amide administered separately or simultaneously. The combined drug according to claim 5, characterized in that the usage ratio of the guanidine compound to piperlongum amide is: a molar ratio of 250:5-500:5 or a mass ratio of 50:1-150:1. The combined drug according to claim 5, characterized in that the guanidine compound is selected from metformin. The combined drug according to claim 5 is characterized in that the combined drug is used to treat lung cancer, breast cancer or colorectal cancer. A pharmaceutical composition, characterized in that it is prepared by using guanidine compounds and piperlongum amide as active ingredients and adding pharmaceutically acceptable excipients or auxiliary ingredients. The pharmaceutical composition according to claim 9, characterized in that the usage ratio of the guanidine compound to piperlongum amide is: a molar ratio of 250:5-500:5 or a mass ratio of 50:1-150:1. The pharmaceutical composition according to claim 9, characterized in that the guanidine compound is selected from metformin. The pharmaceutical composition according to claim 9 is characterized in that the pharmaceutical composition is used to treat lung cancer, breast cancer or colorectal cancer.