WO2014047782A1

WO2014047782A1 - Pharmaceutical composition containing resveratrol and resveratrol derivative and bcl-2 inhibitor and use thereof

Info

Publication number: WO2014047782A1
Application number: PCT/CN2012/081926
Authority: WO
Inventors: 赵镭
Original assignee: 鼎泓国际投资（香港）有限公司
Priority date: 2012-09-25
Filing date: 2012-09-25
Publication date: 2014-04-03

Abstract

Provided in the present invention are a pharmaceutical composition containing resveratrol and resveratrol derivative and the use thereof in the preparation of medicine for treating colon cancer, liver cancer, lung cancer, kidney cancer, gastric cancer, brain tumours, sarcoma, glioma, pancreatic cancer, ovarian cancer, breast cancer or prostate cancer.

Description

Containing resveratrol and resveratrol derivatives and Bc l-2 inhibitors

Pharmaceutical composition and application thereof

The present invention relates to a pharmaceutical composition and its use in the preparation of a medicament for treating cancer, in particular to a pharmaceutical composition comprising resveratrol and a resveratrol derivative and a Bcl-2 inhibitor and preparation thereof Use in the treatment of drugs for colon cancer, liver cancer, lung cancer, kidney cancer, gastric cancer, brain tumor, sarcoma, glioma, pancreatic cancer, ovarian cancer, breast cancer or prostate cancer. Background technique

The World Health Organization survey shows that the global cancer situation is getting worse. The number of new patients will increase from the current 10 million to 15 million in the next 20 years, and the number of deaths due to cancer will increase from 6 million to 10 million per year. Primary liver cancer is a cancer that occurs in hepatocytes and intrahepatic bile duct epithelial cells. It is one of the most common malignant tumors in humans. The incidence of colon cancer is related to the environment, living habits, especially the way of eating. It is generally considered to be a high-fat diet and Insufficient cellulose is the main cause of the disease. With the improvement of living standards and the changes in diet structure, the incidence of colon cancer is increasing year by year. Glioma is a tumor originating from glial cells and occurring in the neuroectodermal layer. Its main features are diffuse infiltration and growth of tumor cells, no clear boundaries, infinite proliferation and high invasiveness. Despite the continuous improvement of neurosurgery skills, the precise positioning of radiotherapy and the continuous development of chemotherapy drugs in recent years, the recovery of glioma patients is still unsatisfactory, so the treatment of glioma is imminent.

At present, there are many anti-tumor drugs listed, such as alkylating agents, anti-metabolites, anti-tumor antibiotics, immunomodulators, etc., but most of the drugs are intolerant due to their toxicity. A large number of clinical practices have proved that Chinese medicine or combination of traditional Chinese and Western medicine can effectively treat malignant tumors, and at the same time can alleviate the side effects of radiotherapy and chemotherapy. Using modern medical methods, it was found that some active natural products can effectively inhibit the growth of tumor cells and induce apoptosis. Many of the antibiotics and anti-tumor drugs currently in use are either directly derived from natural products or have been structurally modified. Therefore, the use of highly safe active natural products for clinical treatment of cancer will have broad prospects. With the development of molecular mechanisms for the development of tumors, molecular targeted therapy for a variety of evils Sexual tumors have received extensive attention and high attention. Molecular targeting drugs have high selectivity and wide language, and their safety is superior to cytotoxic chemotherapy drugs, which is a new direction in the field of cancer therapy.

Resveratrol (Resveratrol) is a polyphenolic compound widely found in various plants such as grapes, Polygonum cuspidatum, and peanut. It is a natural phytoalexin and a green anticancer drug for cancer prevention and treatment. In vivo and in vivo, the resveratrol showed inhibition and even reversal of the three major stages of initiation, proliferation and development of most tumors. Its anti-tumor mechanism can inhibit tumor cell angiogenesis, inhibit cell cycle, promote tumor cell apoptosis, induce tumor cell differentiation, inhibit epoxidase and cytochrome P450 activity, interfere with related signal transduction pathways, and inhibit tumor angiogenesis. And so on.

Apoptosis (programmed cell death) is a natural pathway by which the body removes abnormal or unwanted cells, which, if affected, can lead to various diseases such as cancer. Bc l-2 family proteins are important regulators of apoptosis, and Bc l-2 and Bc l-xL are overexpressed in various types of tumors, which are thought to be related to tumorigenesis, development and drug resistance. Therefore, the development of anti-apoptotic proteins against Bc l-2 and Bc l-xL has become a research hotspot in anti-tumor therapy in recent years. ABT-263 and ABT-737 are small molecule Bc l-2 inhibitors developed by Abbott Pharmaceuticals. They have a remarkable effect on a variety of tumors and can be used orally. They have good application prospects.

With the advancement of tumor molecular biology, tumor molecular targeted therapy has become a hot spot in cancer research and has played an important role in the treatment of various tumors. However, the biological behavior of most tumors is not dominated by a single signaling pathway, but multiple signaling pathways work together. Traditional Chinese medicine is receiving increasing attention due to its multi-gene multi-target advantage. Therefore, a reasonable combination of drugs, the incomparable superiority of single drugs, combined therapy for multi-target targeted therapy will not only reduce or delay the emergence of drug resistance, reduce toxicity, but also through multiple drugs against cancer cells The synergistic effect of killing achieves better results. Summary of the invention

In view of the above technical deficiencies, the present invention provides a pharmaceutical composition and application thereof in the preparation of a medicament for treating cancer, specifically a pharmaceutical composition comprising resveratrol and a resveratrol derivative and a Bc l-2 inhibitor And its preparation in the treatment of colon cancer, liver cancer, lung cancer, kidney cancer, stomach cancer, Use in drugs for brain tumors, sarcomas, gliomas, pancreatic cancer, ovarian cancer, breast cancer or prostate cancer.

The pharmaceutical composition containing the resveratrol and the resveratrol derivative and the Bc l-2 inhibitor, wherein the resveratrol and the resveratrol derivative are resveratrol; Bc l The -2 inhibitor can be ABT-263 or ABT-737, or a corresponding structural analog of the two, ^ [stained organism.

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

I

In the pharmaceutical composition of the present invention, the component is not limited to the resveratrol drug itself, but may be a pharmaceutically acceptable salt, hydrate or derivative thereof.

In the present invention, the Bc l-2 inhibitor may be a drug of any structural type of Bc l-2 inhibitor, preferably ABT-263 or ABT-737. Wherein ABT-263 is a compound of formula II as described in US2007027135:

II

Wherein ABT-737 is a compound of formula III as described in WO2005049594 and WO2005049593:

III

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

The molar ratio of resveratrol and resveratrol derivatives to Bc l-2 inhibitor in the pharmaceutical composition containing resveratrol and resveratrol derivatives and Bc l-2 inhibitor 0至100. 0: 0. The ratio of the molar ratio of the resveratrol and the resveratrol derivative to the Bc l-2 inhibitor is 30. 0-100. 0: 1. 0-2. 0.

The pharmaceutical composition of the present invention comprising resveratrol and a resveratrol derivative and a Bcl-2 inhibitor can be used for treating various tumors including, 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.

In the present invention, a pharmaceutical composition of resveratrol and a resveratrol derivative and a Bc l-2 inhibitor is preferably used for the preparation of a medicament for treating colon cancer, liver cancer and glioma.

The molar ratio of the resveratrol and the resveratrol derivative to the Bc l-2 inhibitor is 30. 0. The pharmaceutical composition of the present invention is used in the preparation of a medicament for treating colon cancer, liver cancer and glioma. The ratio of the molar ratio of the resveratrol and the resveratrol derivative to the Bc l-2 inhibitor is 50. 0-100. 0: 1. 5-2 0: 2. 0。 The molar ratio of the resveratrol and resveratrol derivatives to the Bc l-2 inhibitor is 100. 0: 2. 0.

A composition comprising resveratrol and a resveratrol derivative and a Bcl-2 inhibitor for the treatment of colon cancer, liver cancer, lung cancer, kidney cancer, gastric cancer, brain tumor, sarcoma, glioma, pancreatic cancer, In the application of a medicament for ovarian cancer, breast cancer or prostate cancer, in the composition of the present invention In the case of a drug to be administered simultaneously, the resveratrol and the resveratrol derivative and the Bc l-2 inhibitor may be contained in the same pharmaceutical preparation such as a tablet or a capsule, or may be a white gourd. The alcohol and the resveratrol derivative and the Bc l-2 inhibitor are respectively formulated into a tablet, or a capsule, respectively, and the instructions of the instructions are taken at the same time; the composition of the present invention is formulated into a drug for sequential administration. In the solution, the resveratrol and the resveratrol derivative and the Bc l-2 inhibitor may be separately prepared according to the order indicated in the instructions of the drug, or the two components in the above composition may be made. In a controlled release formulation, one component of the composition is first released, and then the other component of the composition is released, the patient only needs to take the controlled release composition formulation; the composition of the invention is prepared to cross In the regimen of the administered medicament, the resveratrol and the resveratrol derivative and the Bcl-2 inhibitor may be separately formulated into different preparations and packaged or combined together in a manner conventional in the art. , the patient then follows the instructions for the drug CROSS taking the indicated order, or the pharmaceutical composition is a controlled release formulation of resveratrol and resveratrol derivatives and Bc l-2 inhibitor to prepare a cross-release.

Resveratrol and resveratrol derivatives and Bcl-2 inhibitor compositions are prepared for the treatment of colon cancer, liver cancer, lung cancer, kidney cancer, gastric cancer, brain tumor, sarcoma, glioma, pancreatic cancer, ovary In the use of a medicament for cancer, breast cancer or prostate cancer, the resveratrol and resveratrol derivatives and the Bcl-2 inhibitor in the composition may be used simultaneously or in any order. For example, resveratrol and resveratrol derivatives and Bc l-2 inhibitors can be administered to patients at the same time; resveratrol and resveratrol derivatives can be administered to patients first, and then taken Bc l-2 inhibitor, or taking Bc l-2 inhibitor first, then taking resveratrol and resveratrol derivative drugs, there is no special requirement for the time interval between the two, but it is preferred to take two drugs The time interval is no more than one day; or two drugs are administered alternately.

In the present invention, the resveratrol and resveratrol derivatives of the present invention and the Beb 2 inhibitor can be prepared into a medicament suitable for gastrointestinal administration or parenteral administration by a method conventional in the art. Formulation, the present invention preferably comprises a resveratrol and a resveratrol derivative and a Be 1 -2 inhibitor as a pharmaceutical preparation for gastrointestinal administration, and the preparation form may be a conventional tablet or capsule, or a control Release, sustained release preparation. In the pharmaceutical preparation of the resveratrol and resveratrol derivatives and the Bcl-2 inhibitor composition of the present invention, the composition is contained in the preparation according to different preparation forms and formulation specifications. The amount may be from 1 to 99% by mass, preferably from 10% to 90%; the excipient used in the formulation may be a conventional excipient in the art, and may not react with the composition of the present invention or affect the invention of the present invention. The preparation method of the composition is not particularly limited, and both the resveratrol and the resveratrol derivative and the Beb 2 inhibitor may be directly mixed and then formulated, or separately and/or correspondingly excipients are separately mixed. Formulations are prepared and then packaged together in a manner conventional in the art, or separately mixed with the corresponding adjuvants and then mixed to form a formulation.

The dosage of the pharmaceutical composition of the present invention can be appropriately changed depending on the administration target, the administration route or the preparation form of the drug, but to ensure that the pharmaceutical composition can achieve an effective blood concentration in the mammal. As a prerequisite.

The present invention separately combines resveratrol and resveratrol derivatives and Bc l-2 inhibitors to kill DLD1 (colon cancer cell line), HUH-7 (hepatoma cell line) and U251 (glioma). The test results of the cell strain showed that the combination of resveratrol and resveratrol derivatives and Be 1-2 inhibitor of the present invention has significant synergistic effects on colon cancer, liver cancer and glioma, and improved. The efficacy of the drug reduces the amount of the drug and reduces the occurrence of side effects. detailed description

The invention is further illustrated by the following examples, but the invention is not limited thereto. Example

Reagents and methods:

Cells: DLD1 (colon cancer cell line), HUH-7 (hepatoma cell line) and U251 (glioma cell line) were purchased from Amer i can Type Technology (ATCC), Rockaway, Maryland, USA Seoul.

Drugs: The pharmaceutical compositions used in the following examples were prepared as described in Method 1 or Method 2 below; Resveratrol was purchased from Nanjing Institute of Traditional Chinese Medicine; Bc l-2 inhibitors were synthesized according to the literature. , ABT-263 and ABT-737 synthetic references are: Synthes is, 15, 2398-2404, W02005049594, WO2005049593 and US2007027135.

Method 1: Accurately weigh the components of the corresponding pharmaceutical composition, dissolve them separately with dimethyl sulfoxide, prepare 10 mM stock solutions, store at -20 ° C, and use fresh medium when using. Dilute to the appropriate concentration, then take 1 μl of each component solution and mix for use. In all tests, the final concentration of dimethyl sulfoxide should be < 5g/L so as not to affect the activity of the cells.

All cells were cultured in RPMI 1640 medium containing 10% calf serum, 100 kU/L penicillin, 100 mg/L streptomycin, and humidity at 37 ° C, 5 % C0 ₂ before dosing. One day, cells were seeded with 2 x 107 wells on a six-well plate, and then the pharmaceutical composition solution prepared as described above was added to the cells to bring the components to their working concentrations, as shown in Tables 1 to 6 in Table 1.

After drug treatment, cell death was measured by Trypan Blue, and the cells were trypsinized with trypsin sodium/EDTA for 10 minutes at 37 °C. Since the dead cells were detached from the incubator into the medium, all the cells were collected by centrifugation at 1200 rpm, and then the precipitate was resuspended in the medium, and mixed with the trypan blue dye. After staining, counting was performed using an optical microscope and a hemocytometer. The dyed blue color is counted as a dead cell. 500 cells were randomly selected for counting, and the dead cells were expressed as a percentage of the total counted cells.

Method 2: Each component of the corresponding pharmaceutical composition was accurately weighed, dissolved separately with dimethyl sulfoxide, and each was formulated into a 10 mM stock solution, and stored at - 20 °C. Dilute to a suitable concentration with fresh medium, and then take 1 μl of each component solution for use. In all tests, the final concentration of dimethyl sulfoxide should be < 5g/L so as not to affect the activity of the cells.

All cells were cultured in RPMI 1640 medium containing 10% calf serum, 100 kU/L penicillin, 100 mg/L streptomycin, and humidity at 37 ° C, 5 % C0 ₂ before dosing. One day, cells were seeded with 2×107 wells on a six-well plate, and then the components of the pharmaceutical composition prepared as described above were added to the cells in any order to bring the components to their working concentrations, as shown in Table 1. In the seventh 7-12.

After drug treatment, cell death was measured by Trypan Blue, and the cells were trypsinized with trypsin sodium/EDTA for 10 minutes at 37 °C. Since the dead cells were detached from the incubator into the medium, all the cells were collected by centrifugation at 1200 rpm, and then the precipitate was resuspended in the medium, and mixed with the trypan blue dye. After staining, counting was performed using an optical microscope and a hemocytometer. Dyed in dyed blue Cell. 500 cells were randomly selected for counting, and dead cells were expressed as a percentage of the total counted cells.

In the pharmaceutical combination shown in Table 1 below, the combination of the first to 1-6 is prepared according to the method 1, and the combination of the seventh to the fourth is prepared according to the method 1.

Table 1

Example 1 The synergistic effect of different ratios of resveratrol and ABT-263 on promoting DLD1 cell death test is shown in Table 2.

Table 2

Group Usage (μΜ) Cellular mortality (%) Control group 2.3 ±0.9 Low dose Resveratrol 30.0 4.8±1.0

ABT-263 1.0 3.3±1.1 Resveratrol+ABT-263 30.0 + 1.0 13.4 ±2.3 Medium dose resveratrol 50. 0 18. 6 ± 2. 4

ABT-263 1. 5 3. 6 ± 0. 9

Resveratrol +ABT-263 50. 0 + 1. 5 47. 6 ± 3. 4 High dose Resveratrol 100. 0 22. 3 ± 3. 4

ABT-263 2. 0 4. 6 ± 1. 2

Resveratrol + ABT-263 100. 0 + 2. 0 68. 3 ± 4. 2 In the test face of the relevant compound causing death of colon cancer cell line DLD1, it was found that when using 2.0 μM ABT-263 alone or There is almost no cell death at lower concentrations, even if 100% ΟμΜ resveratrol alone, only about 20% of the cells die; and when the two are combined at a lower concentration (50. ΟμΜ resveratrol + 1. 5μΜ ABT-263) produced a significant synergistic effect, resulting in 48% cancer cell death; when the two were combined in a ratio of 100. ΟμΜ resveratrol + 2. ΟμΜ ABT-263, a more significant synergistic effect was produced. , causing 68% of cancer cells to die.

Example 2 The synergistic effect of different ratios of resveratrol and ABT-737 on promoting DLD1 cell death test is shown in Table 3.

table 3

In examining the test results of the compound-induced death of colon cancer cell line DLD1, it was found that only about 15-20% of the cells died when using 1. ΟμΜ ABT-737 or 100. ΟμΜ resveratrol alone; When used at lower concentrations (50. ΟμΜ resveratrol + 1. 5 μΜ ABT-737) It produces a more pronounced synergy, resulting in 31% of cancer cell death; when the two are combined in a ratio of 100. ΟμΜ resveratrol + 2. 0μΜ ΑΒΤ-737, a more significant synergistic effect is produced, resulting in 55 % Cancer cells die.

Example 3 The synergistic effect of different ratios of resveratrol and strontium-263 promoted the death test of HUH-7 cells, see Table 4.

Table 4

In the test to examine the death of the liver cancer cell line HUH-7 by the relevant compound, it was found that when only 1.10 μM of ABT-263 or lower was used alone, only about 10% of the cells died, and when 50. ΜμΜ resveratrol was used alone. There is almost no cell death, even if 100. ΟμΜ resveratrol alone, only about 25% of the cells die; and when the two are combined at a lower concentration (50. ΟμΜ resveratrol + 1. 5μΜ ABT-263 ), which produced a more pronounced synergy, resulting in 31% of cancer cell death; when the two were combined in a ratio of 100. ΟμΜ resveratrol + 2. 0μΜ ABT-263, a more significant synergistic effect was produced, resulting in 86 % of cancer cells die.

Example 4 The synergistic effect of different ratios of resveratrol and ABT-263 on the promotion of U251 cell death test is shown in Table 5.

table 5

Group use (μΜ) Cell death rate (%) Control group 1. 3 ± 0. 6 Low dose resveratrol 30.0 3.6±1.1

ABT-263 1.0 3.1 ±1.0

Resveratrol +ABT-263 30.0 + 1.0 15.6±2.1 medium dose resveratrol 50.0 12.3±2.3

ABT-263 1.5 12.5 ± 1.6 resveratrol +ABT-263 50.0 + 1.5 44.3± 3.6 high dose resveratrol 100.0 34.3± 3.5

ABT-263 2.0 21.3±2.6 Resveratrol+ABT-263 100.0 + 2.0 90.3± 3.5 In the test of the death of glioma cell line U251 by related compounds, it was found that when using 1.5μΜ ABT-263 alone or 50. Only about 10% of cells die when ΟμΜ resveratrol; about 20-35% of cell death when using ΟμΜ ABT-263 or 100. ΟμΜ resveratrol; and when the two are at lower concentrations When combined (50. ΜμΜ resveratrol + 1.5 μΜ ABT-263 ), there is a significant synergistic effect, resulting in 44% of cancer cell death; when both are ΙΟΟ. ΟμΜ resveratrol + 2. ΟμΜ ABT- When the ratio of 263 is combined, a more significant synergy occurs, resulting in 90% of cancer cells dying. Although the above embodiments have been described in detail with reference to the technical solutions of the present invention, the technical solutions of the present invention are not limited to the above embodiments, and the technical solutions of the present invention are made without departing from the spirit and scope of the present invention. Any modifications are intended to fall within the scope of the appended claims.

Claims

claims

1. A pharmaceutical composition, characterized in that the pharmaceutical composition contains resveratrol and resveratrol derivatives and a Bcl-2 inhibitor.

2. The pharmaceutical composition according to claim 1, characterized in that the molar ratio of the resveratrol and resveratrol derivatives to the Bcl-2 inhibitor is 15.0-200.0: 0. 5-4. 0.

3. The pharmaceutical composition according to claim 2, characterized in that the molar ratio of the resveratrol and resveratrol derivatives to the Bcl-2 inhibitor is 30.0-100.0: 1. 0-2. 0.

4. The pharmaceutical composition according to any one of claims 1 to 3, characterized in that, the resveratrol and resveratrol derivatives are resveratrol; the Bcl-2 inhibitor for ABT-263 or ABT-737.

5. Application of the pharmaceutical composition according to any one of claims 1 to 4 in the preparation of drugs for treating cancer.

6. The application according to claim 5, wherein the cancer is colon cancer, liver cancer, lung cancer, kidney cancer, gastric cancer, brain tumor, sarcoma, glioma, pancreatic cancer, ovarian cancer, breast cancer or Prostate cancer.

7. Application according to claim 6, characterized in that, in the preparation of drugs for the treatment of colon cancer, liver cancer and glioma, the resveratrol and resveratrol derivatives are combined with Bc The molar ratio of l-2 inhibitor is 30. 0-100. 0: 1. 0-2. 0.

8. The application according to claim 7, characterized in that the molar ratio of the resveratrol and resveratrol derivatives to the Bcl-2 inhibitor is 50.0-100.0:1. 5-2. 0.

9. The application according to claim 8, characterized in that: the resveratrol and resveratrol-type contaminants are resveratrol; the Bcl-2 inhibitor is ABT-263 or ABT -737.

10. The application according to any one of claims 5 to 9, characterized in that, resveratrol and resveratrols in the pharmaceutical composition are used simultaneously with fouling organisms and Bcl-2 inhibitors. or in any order.