CN102688493B - Pharmaceutical composition containing resveratrol, resveratrol derivatives and Bc1-2 inhibitor and application thereof - Google Patents

Abstract

The invention relates to a pharmaceutical composition containing resveratrol, resveratrol derivatives and a Bcl-2 inhibitor and application thereof in preparing medicaments for treating colon cancer, liver cancer, lung cancer, kidney cancer, stomach cancer, brain tumor, sarcoma, glioma, pancreatic cancer, ovarian cancer, breast cancer or prostate cancer.

Description

Pharmaceutical composition containing resveratrol, resveratrol derivatives and Bc1-2 inhibitor and application thereof

technical field

The invention relates to a pharmaceutical composition and its application in the preparation of medicines for treating cancer, in particular to a pharmaceutical composition containing resveratrol, resveratrol derivatives and Bcl-2 inhibitors and its application in the preparation and treatment of cancer. Use in medicine for colon, liver, lung, kidney, stomach, brain, sarcoma, glioma, pancreas, ovary, breast or prostate cancer. the

Background technique

The survey report of the World Health Organization shows that the global cancer situation is getting worse. In the next 20 years, the number of new patients will increase from the current 10 million to 15 million per year, and the number of deaths from cancer will also increase from 6 million to 10 million per year. Primary liver cancer is a cancer that occurs in liver cells and intrahepatic bile duct epithelial cells, and is one of the most common malignant tumors in humans; the incidence of colon cancer is related to the environment, living habits, especially diet. Insufficient cellulose is the main cause of the disease. With the improvement of living standards and changes in dietary structure, the incidence of colon cancer is increasing year by year; glioma is a tumor that originates from glial cells and occurs in the neuroectoderm Its main features are diffuse infiltrating growth of tumor cells, no clear boundaries, unlimited proliferation and high invasiveness. Despite the continuous improvement of neurosurgery skills, precise positioning of radiotherapy and continuous development of chemotherapy drugs in recent years, the prognosis of glioma patients is still unsatisfactory, so it is imminent to study drugs for glioma treatment. the

Currently, there are many anti-tumor drugs on the market, such as alkylating agent drugs, antimetabolite drugs, anti-tumor antibiotics, immunomodulators, etc., but most of the drugs are not tolerated by patients due to their high toxicity. A large number of clinical practices have proved that traditional Chinese medicine or the combination of traditional Chinese and Western medicine can effectively treat malignant tumors, and at the same time reduce the toxic and side effects of radiotherapy and chemotherapy. Using modern medical methods, it is found that some active natural products can effectively inhibit the growth of tumor cells and induce apoptosis. Many antibiotics and antineoplastic drugs currently used are either directly derived from natural products or obtained through structural modification. Therefore, active natural products with high safety will have broad prospects for clinical application in the treatment of cancer. With the research on the molecular mechanism of tumor occurrence and development becoming more and more clear, molecular targeted therapy for various malignant tumors has received extensive attention and high attention. Molecular targeted drugs are highly selective, broad-spectrum effective, and their safety is superior to cytotoxic chemotherapy drugs. They are currently a new development direction in the field of tumor treatment. the

Resveratrol (Resveratrol) is a polyphenol compound widely present in grapes, knotweed, peanuts and other plants. It is a natural phytoalexin and a green anticancer drug for preventing and treating tumors. Experiments in vitro and in vivo have shown that resveratrol can inhibit or even reverse the three main stages of most tumors, namely initiation, proliferation and development. Its anti-tumor mechanism can be through anti-oxidation, blocking cell cycle, promoting tumor cell apoptosis, inducing tumor cell differentiation, inhibiting the activity of cyclooxygenase and cytochrome P450, interfering with related signal transduction pathways, and inhibiting tumor angiogenesis Wait for it to work. the

Apoptosis (programmed cell death) is the body's natural way of getting rid of abnormal or unwanted cells, which if affected can lead to the development of various diseases such as cancer. Bcl-2 family proteins are important regulators of apoptosis, among which Bcl-2 and Bcl-xL are overexpressed in various types of tumors, which are considered to be related to the occurrence, development and drug resistance of tumors, so targeting Bcl -2 and Bcl-xL anti-apoptotic protein drug development has become a research hotspot in anti-tumor therapy in recent years. ABT-263 and ABT-737 are small molecule Bcl-2 inhibitors developed by Abbott Pharmaceuticals in the United States. They have significant effects on a variety of tumors and can be used orally, with good application prospects. the

With the progress of tumor molecular biology research, tumor molecular targeted therapy has become a hot spot in tumor research and plays an important role in the treatment of various tumors. However, the biological behavior of most tumors is not dominated by a single signal transduction pathway, but multiple signal transduction pathways work together. Traditional Chinese medicine is attracting increasing attention due to its advantages of multi-gene and multi-target effects. Therefore, a reasonable combination of drugs has incomparable advantages over single drugs. Targeted therapy for multiple targets in combination will not only reduce or delay the emergence of drug resistance and reduce toxicity, but also treat cancer cells with multiple drugs. The synergistic effect of killing can achieve better curative effect. the

Contents of the invention

In view of the above technical defects, the present invention provides a pharmaceutical composition and its application in the preparation of drugs for treating cancer, specifically a pharmaceutical composition containing resveratrol, resveratrol derivatives and Bcl-2 inhibitors The method and its application in the preparation of medicines for treating colon cancer, liver cancer, lung cancer, kidney cancer, stomach cancer, brain tumor, sarcoma, glioma, pancreas cancer, ovarian cancer, breast cancer or prostate cancer. the

In the pharmaceutical composition containing resveratrol, resveratrol derivatives and Bcl-2 inhibitors of the present invention, the resveratrol and resveratrol derivatives are resveratrol; Bcl-2 The inhibitor can be ABT-263 or ABT-737, or their corresponding structural analogs and derivatives. the

The resveratrol and resveratrol derivatives in the pharmaceutical composition of the present invention are preferably resveratrol, and its corresponding structural formula is shown in formula I. the

In the pharmaceutical composition of the present invention, the components are not limited to resveratrol itself, but may also be pharmaceutically acceptable salts, hydrates or derivatives thereof. the

In the present invention, the Bcl-2 inhibitor can be any structural type of Bcl-2 inhibitor drug, preferably ABT-263 or ABT-737. Wherein ABT-263 is the compound shown in formula II recorded in US2007027135:

Wherein ABT-737 is the compound shown in the formula III recorded in WO2005049594 and WO2005049593:

In the pharmaceutical composition of the present invention, the components are not limited to the above-mentioned ABT-263 and ABT-737 themselves, but may also be their hydrates, analogs, derivatives and other organic or inorganic salts. the

In the pharmaceutical composition containing resveratrol, resveratrol derivatives and Bcl-2 inhibitors of the present invention, the molar ratio of resveratrol, resveratrol derivatives and Bcl-2 inhibitors is 15.0 -200.0:0.5-4.0; more preferably, the molar ratio of resveratrol and resveratrol derivatives to Bcl-2 inhibitor is 30.0-100.0:1.0-2.0. the

The pharmaceutical composition containing resveratrol, resveratrol derivatives and Bcl-2 inhibitors of the present invention can be used to treat various tumors, and said tumors include but not limited to colon cancer, liver cancer, lung cancer, kidney cancer , gastric cancer, brain tumor, sarcoma, glioma, pancreatic cancer, ovarian cancer, breast cancer, or prostate cancer. the

The present invention preferably uses the pharmaceutical composition of resveratrol, resveratrol derivatives and Bcl-2 inhibitors in the preparation of medicines for treating colon cancer, liver cancer and neuroglioma. the

In the application of the pharmaceutical composition of the present invention in the preparation of medicines for treating colon cancer, liver cancer and glioma, the molar ratio of resveratrol and resveratrol derivatives to Bcl-2 inhibitor is 30.0-100.0: 1.0-2.0; preferably the molar ratio of resveratrol and resveratrol derivatives to Bcl-2 inhibitor is 50.0-100.0:1.5-2.0; more preferably resveratrol and resveratrol derivatives The molar ratio with Bcl-2 inhibitor is 100.0:2.0. the

Compositions containing resveratrol, resveratrol derivatives and Bcl-2 inhibitors are used in the preparation and treatment of colon cancer, liver cancer, lung cancer, kidney cancer, gastric cancer, brain tumor, sarcoma, glioma, pancreatic cancer, ovarian cancer In the application of drugs for cancer, breast cancer or prostate cancer, in the scheme of making the composition of the present invention into a drug for simultaneous administration, resveratrol and resveratrol derivatives and Bcl-2 inhibitors may contain In the same pharmaceutical preparation such as tablets or capsules, resveratrol and resveratrol derivatives and Bcl-2 inhibitors can also be made into preparations, such as tablets or capsules respectively, and the They are packaged or combined together in a conventional way in the field, and the patient then takes them at the same time according to the instructions of the drug instructions; Bcl-2 derivatives and Bcl-2 inhibitors are made into different preparations, and they are packaged or combined in a conventional way in the art, and patients then take them in the order indicated in the drug instructions, or combine the ingredients in the above compositions Two components are made into a controlled-release preparation, one component in the composition is released first, and then another component in the composition is released, and the patient only needs to take the controlled-release composition preparation; In the plan of preparing medicines for cross-administration, resveratrol and resveratrol derivatives and Bcl-2 inhibitors can be made into different preparations respectively, and they are packaged or packaged in a conventional way in the art Combining them together, patients then take them according to the cross sequence indicated by the drug instructions, or the pharmaceutical composition is prepared as a controlled release preparation for cross release of resveratrol and resveratrol derivatives and Bcl-2 inhibitors. the

Resveratrol, resveratrol derivatives and Bcl-2 inhibitor composition are used in the preparation and treatment of colon cancer, liver cancer, lung cancer, kidney cancer, gastric cancer, brain tumor, sarcoma, glioma, pancreatic cancer, ovarian cancer , breast cancer or prostate cancer medicine, the resveratrol and resveratrol derivatives and Bcl-2 inhibitors in the composition can be used simultaneously or in any sequential order, such as can Resveratrol and resveratrol derivatives and Bcl-2 inhibitors are administered to patients at the same time; resveratrol and resveratrol derivatives can also be administered to patients first, and then Bcl-2 inhibitors can be administered to patients. Inhibitors, or take Bcl-2 inhibitors first, and then take resveratrol and resveratrol derivatives. There is no special requirement for the time interval between the two drugs, but it is preferred that the time interval between the two drugs should not exceed one day; or alternate administration of the two drugs. the

In the present invention, resveratrol, resveratrol derivatives and Bcl-2 inhibitors of the present invention can be prepared into drugs suitable for gastrointestinal administration or parenteral administration by using conventional methods in the art Preparations, the present invention preferably makes resveratrol, resveratrol derivatives and Bcl-2 inhibitors into pharmaceutical preparations for gastrointestinal administration, and its preparation form can be conventional tablets or capsules, or controlled release , Sustained-release preparations. In the pharmaceutical preparation of resveratrol, resveratrol derivatives and Bcl-2 inhibitor composition of the present invention, according to different preparation forms and preparation specifications, the content of the composition in the preparation can be It is 1-99%, preferably 10%-90%; the adjuvant used in the preparation can adopt the conventional adjuvant in this field, on the premise of not reacting with the composition of the present invention or not affecting the curative effect of the medicament of the present invention; The preparation method can be prepared by using conventional preparation methods in the art. the

In the present invention, the preparation method of the composition is not particularly limited, resveratrol and resveratrol derivatives and Bcl-2 inhibitors can be directly mixed and then made into preparations, or separately and/or corresponding auxiliary materials They are mixed separately to make preparations, and then packaged together in a conventional manner in the art, or mixed with corresponding auxiliary materials separately and then mixed to make preparations. the

The dosage of the pharmaceutical composition in the present invention can be appropriately changed according to the difference in the administration object, the route of administration or the preparation form of the medicine, but to ensure that the pharmaceutical composition can reach an effective blood drug concentration of premise. the

In the present invention, resveratrol, resveratrol derivatives and Bcl-2 inhibitors are combined to kill DLD1 (colon cancer cell line), HUH-7 (liver cancer cell line) and U251 (glioma cell line). strain) test, the results show that the combination of resveratrol and resveratrol derivatives and Bcl-2 inhibitors of the present invention has a significant synergistic effect in the treatment of colon cancer, liver cancer and glioma, and improves the curative effect of the drug , reducing the dosage and reducing the occurrence of side effects. the

Detailed ways

The present invention is further described in conjunction with the following examples, but the present invention is not limited thereto. the

Example

Reagents and methods:

Cells: DLD1 (colon cancer cell line), HUH-7 (hepatoma cell line) and U251 (glioma cell line) were purchased from American Type Culture Collection (ATCC), Rockville, MD, USA. the

Medicines: the pharmaceutical compositions used in the following examples are all prepared according to the following method 1 or method 2; The synthesis references of ABT-263 and ABT-737 are: Synthesis, 15, 2398-2404, WO2005049594, WO2005049593 and US2007027135. the

Method 1: Accurately weigh the components of the corresponding pharmaceutical composition, dissolve them separately in dimethyl sulfoxide, and make 10mM stock solutions respectively, store them at -20°C, and dilute them with fresh medium when used. concentration, and then take 1 microliter of the solutions of each component and mix them together for later use. In all experiments, the final concentration of dimethyl sulfoxide should be ≤5g/L so as not to affect the viability of the cells. the

All the cells were cultured in RPMI 1640 medium containing 10% calf serum, 100kU/L penicillin, and 100mg/L streptomycin at 37°C and 5% CO ₂ humidity conditions. Cells were inoculated at 2×10 ⁵ /well on a six-well plate, and then the pharmaceutical composition solution prepared by the above method was added to the cells to make each component reach its working concentration, see 1-6 in Table 1 for details.

After drug treatment, cell death was determined by Trypan Blue and cells were trypsinized with trypsin sodium/EDTA for 10 min at 37°C. As dead cells dislodged from the incubator into the medium, all cells were collected by centrifugation at 1200 rpm, and the pellet was resuspended in medium and mixed with trypan blue dye. After staining, counts were performed using a light microscope and a hemocytometer. Dead cells were counted as those stained blue by the dye. 500 cells were randomly selected for counting, and the dead cells were expressed as a percentage of the total counted cells. the

Method 2: Accurately weigh each component of the corresponding pharmaceutical composition, dissolve them in dimethyl sulfoxide, make 10 mM stock solutions, and store them at -20°C. When used, dilute to an appropriate concentration with fresh culture medium, and then take 1 microliter of each component solution for future use. In all experiments, the final concentration of dimethyl sulfoxide should be ≤5g/L so as not to affect the viability of the cells. the

All the cells were cultured in RPMI 1640 medium containing 10% calf serum, 100kU/L penicillin, and 100mg/L streptomycin at 37°C and 5% CO ₂ humidity conditions. Cells were inoculated at 2×10 ⁵ /well on a six-well plate, and then the components of the pharmaceutical composition prepared by the above method were added to the cells in any order, so that each component reached its working concentration, see Table 1 for details Nos. 7-12 in the middle.

After drug treatment, cell death was determined by Trypan Blue and cells were trypsinized with trypsin sodium/EDTA for 10 min at 37°C. As dead cells dislodged from the incubator into the medium, all cells were collected by centrifugation at 1200 rpm, and the pellet was resuspended in medium and mixed with trypan blue dye. After staining, counts were performed using a light microscope and a hemocytometer. Dead cells were counted as those stained blue by the dye. 500 cells were randomly selected for counting, and the dead cells were expressed as a percentage of the total counted cells. the

Among the drug combinations shown in Table 1 below, combinations No. 1-6 were prepared according to Method 1, and combinations No. 7-12 were prepared according to Method 2. the

Table 1

Example 1 The combination of different ratios of resveratrol and ABT-263 synergistically promotes the death of DLD1 cells, see Table 2. the

Table 2

In the experiment investigating the cell death of colon cancer cell line DLD1 caused by related compounds, it was found that when 2.0 μM ABT-263 or lower concentration was used alone, there was almost no cell death, even when 100.0 μM resveratrol was used alone, only about 20% cell death; and when the two were combined at a lower concentration (50.0 μM resveratrol + 1.5 μM ABT-263), there was an obvious synergistic effect, resulting in the death of 48% of cancer cells; When the ratio of veratrol + 2.0μM ABT-263 was combined, a more significant synergistic effect was produced, resulting in the death of 68% of cancer cells. the

Example 2 The combination of different ratios of resveratrol and ABT-737 synergistically promotes the death of DLD1 cells, see Table 3. the

table 3

In the experiment investigating the cell death of colon cancer cell line DLD1 caused by related compounds, it was found that only about 15-20% of the cells died when 2.0 μM ABT-737 or 100.0 μM resveratrol were used alone; When used together at lower concentrations (50.0 μM resveratrol + 1.5 μM ABT-737), a more obvious synergistic effect was produced, resulting in the death of 31% of cancer cells; When used in combination, a more significant synergistic effect was produced, resulting in the death of 55% of cancer cells. the

Example 3 The combination of different ratios of resveratrol and ABT-263 synergistically promotes the death of HUH-7 cells, see Table 4. the

Table 4

In the experiment of investigating the cell death of the liver cancer cell line HUH-7 caused by related compounds, it was found that only about 10% of the cells died when 2.0 μM ABT-263 or lower concentrations were used alone, and almost none when 50.0 μM resveratrol was used alone Cell death, even when 100.0μM resveratrol was used alone, only about 25% of the cells died; when the two were combined at a lower concentration (50.0μM resveratrol + 1.5μM ABT-263), it was more obvious The synergistic effect of ABT-263 resulted in the death of 31% of cancer cells; when the two were combined at the ratio of 100.0 μM resveratrol + 2.0 μM ABT-263, a more significant synergistic effect was produced, resulting in the death of 86% of cancer cells. the

Example 4 The combination of different ratios of resveratrol and ABT-263 synergistically promotes the death of U251 cells, see Table 5. the

table 5

In the experiment of investigating the cell death of glioma cell line U251 caused by related compounds, it was found that only about 10% of the cells died when 1.5 μM ABT-263 or 50.0 μM resveratrol was used alone; 2.0 μM ABT-263 alone or 100.0μM resveratrol, about 20-35% of the cells died; and when the two were used together at a lower concentration (50.0μM resveratrol + 1.5μM ABT-263), there was an obvious synergistic effect, resulting in 44% of cancer cells died; when the two were combined at the ratio of 100.0 μM resveratrol + 2.0 μM ABT-263, a more significant synergistic effect was produced, resulting in the death of 90% of cancer cells. the

Although the above-mentioned embodiments have described the technical solution of the present invention in detail, the technical solution of the present invention is not limited to the above embodiments. Any modification will fall within the scope defined by the claims of the present invention. the

Claims (3)

1. a pharmaceutical composition that is used for the treatment of cancer, is characterized in that, described pharmaceutical composition contains resveratrol and Bcl-2 inhibitor; Wherein, the mol ratio of described resveratrol and Bcl-2 inhibitor is 30.0-100.0:1.0-2.0; Described Bcl-2 inhibitor is ABT-263 or ABT-737; Described cancer is colon cancer, hepatocarcinoma or glioma.

2. pharmaceutical composition according to claim 1, is characterized in that, in being used for the treatment of colon cancer, hepatocarcinoma and gliomatous pharmaceutical composition, the mol ratio of described resveratrol and Bcl-2 inhibitor is 30.0-100.0:1.0-2.0.

3. pharmaceutical composition according to claim 2, is characterized in that, the mol ratio of described resveratrol and Bcl-2 inhibitor is 50.0-100.0:1.5-2.0.