Application of telocinobufagin in preparation of anti-hepatoma drugs

Abstract

The invention provides an application of telocinobufagin in preparation of an anti-hepatoma drug and further provides a pharmaceutical composition, the pharmaceutical composition comprises telocinobufagin and 5-fluorouracil, telocinobufagin can remarkably inhibit proliferation of hepatoma cells and can be applied to preparation of the anti-hepatoma drug, and when the telocinobufagin and 5-fluorouracil are used in a combined mode, the telocinobufagin and 5-fluorouracil can be used for preparing the anti-hepatoma drug. Under a specific concentration, the compound has a remarkable synergistic effect in the aspect of liver cancer resistance.

Description

Technical Field

[0001] This invention patent relates to the field of pharmaceutical technology, and particularly to the application of telocinobufagin in the preparation of anti-hepatocellular carcinoma drugs. Background Art

[0002] Venenum Bufonis is the dried secretion of the toad Bufo bufo gargarizans Cantor or Bufo melanostictus Schneider of the family Bufonidae. As a traditional Chinese medicine, it has effects such as analgesia, anti-inflammation, anesthesia, and anti-tumor. The main components of Venenum Bufonis include bufadienolide, indole alkaloids, steroids, etc. Among them, bufadienolides and indole alkaloids are two of the most important bioactive substances. Among them, telocinobufagin is a kind of bufadienolide and has not been applied to the research and treatment of liver cancer at present.

[0003] Telocinobufagin has the effects of detoxifying and detumescence, opening orifices and stopping pain, strengthening the heart and inducing diuresis, etc. Existing research has confirmed that telocinobufagin can inhibit the growth and metastasis of osteosarcoma by inhibiting the JAK2 / STAT3 signaling pathway and has the effect of enhancing immunity. In addition, telocinobufagin has currently been confirmed to be able to inhibit the proliferation of a variety of tumor cells, including breast cancer 4T1 cells, non-small cell lung cancer A549 cells, and head and neck squamous cell carcinoma HNSCC cells, etc. It has extremely broad application and development prospects in anti-cancer, but the research on the effect of telocinobufagin on liver cancer is still shallow at present.

[0004] Liver cancer is a malignant tumor of the liver and is the most common primary liver cancer, which can be divided into two major categories: primary and secondary. Due to the high recurrence rate and metastasis rate of liver cancer, the prognosis is poor. Liver transplantation is currently the most effective method for treating liver cancer, but due to reasons such as tumor burden and poor liver function, more than 70% of advanced patients are not suitable for liver transplantation and need to rely on chemotherapy, but chemotherapy has certain toxic and side effects.

[0005] Therefore, it is of great significance to develop new drugs for the treatment of liver cancer. Summary of the Invention

[0006] The molecular formula of telocinobufagin: C 24 H 34 O 5 , molecular weight: 402.52. The structural formula is shown in Formula 1:

[0007]

[0008] The present invention aims to solve at least one of the technical problems existing in the prior art to some extent. Thus, in the first aspect of the present invention, the present invention provides an application of telocinobufagin in the preparation of an anti-hepatocellular carcinoma drug.

[0009] In the second aspect of the present invention, the present invention provides an application of a pharmaceutical preparation in the preparation of an anti-hepatocellular carcinoma drug, wherein the concentration of telocinobufagin in the pharmaceutical preparation is 25 - 500 nM.

[0010] In the third aspect of the present invention, the present invention provides a pharmaceutical composition, which comprises telocinobufagin and 5-fluorouracil.

[0011] Preferably, the concentration of telocinobufagin is 0.25 - 1 nM, and the concentration of 5-fluorouracil is 0.25 - 0.5 μg / mL.

[0012] More preferably, the concentration of telocinobufagin is 0.5 - 1 nM, and the concentration of 5-fluorouracil is 0.25 μg / mL.

[0013] More preferably, the concentration of telocinobufagin is 0.5 - 1 nM, and the concentration of 5-fluorouracil is 0.5 μg / mL.

[0014] More preferably, the concentration of telocinobufagin is 1 nM, and the concentration of 5-fluorouracil is 0.5 μg / mL.

[0015] More preferably, the pharmaceutical composition further comprises pharmaceutically acceptable excipients and carriers.

[0016] More preferably, the dosage form of the pharmaceutical composition is selected from one of granules, tablets, pills, capsules and injections.

[0017] In the fourth aspect of the present invention, the present invention provides an application of the above-mentioned pharmaceutical composition in the preparation of an anti-hepatocellular carcinoma drug.

[0018] The beneficial effects of the present invention are as follows:

[0019] 1. The present application provides an application of telocinobufagin in the preparation of an anti-hepatocellular carcinoma drug. Experiments in the present application have proved that telocinobufagin has a significant growth inhibitory effect on hepatocellular carcinoma cells, significantly inhibits the invasion of hepatocellular carcinoma cells and significantly inhibits the proliferation of hepatocellular carcinoma cells. And experiments have proved the therapeutic effect of telocinobufagin on a hepatocellular carcinoma animal model. Experiments have proved that telocinobufagin can significantly inhibit the volume and weight of tumors in H22-bearing hepatocellular carcinoma mice, and has a significant effect on inhibiting hepatocellular carcinoma tumors. It can be used in the preparation of drugs for treating hepatocellular carcinoma, increasing the new uses of telocinobufagin and expanding the selection of drugs for treating hepatocellular carcinoma.

[0020] 2. This application provides a pharmaceutical composition comprising telocinobufagin and 5-fluorouracil. Experimental results of this application demonstrate that 5-fluorouracil and telocinobufagin have a synergistic inhibitory effect on the cell proliferation of human hepatocellular carcinoma cell line SK-HEP-1 at specific concentrations. When the concentration of 5-fluorouracil is 0.25 μg / mL, the CI values are 0.796 and 0.599 when the concentrations of telocinobufagin are 0.5 nM and 1 nM respectively; when the concentration of 5-fluorouracil is 0.5 μg / mL, the CI value is 0.779 when the concentration of telocinobufagin is 1 nM, indicating that telocinobufagin combined with 5-fluorouracil has a synergistic effect against liver cancer at this concentration. When the concentration of 5-fluorouracil is 0.25 μg / mL and the concentration of telocinobufagin is 1 nM, the CI value is 0.599, proving that the two have a significant synergistic effect at this concentration.

[0021] 3. Telocinobufagin designed in this application is one of the main active ingredients of toad venom. Nowadays, the purity of this drug can reach ≥98%, meeting the internationally recognized standard for monomeric compounds, solving serious problems such as complex components, poor quality controllability, and large batch-to-batch quality differences in the extracts of toad venom in the past, which may lead to safety risks, providing an important material basis for its safe clinical use, and also providing a guarantee for clarifying the action targets. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 It is a graph showing the detection of cell viability of telocinobufagin on human hepatocellular carcinoma SK-HEP-1 cells;

[0023] Figure 2 It is the tumor volume proliferation curve of each group (control group, low-dose group (Telocinobufagin (0.5 mg / kg)), high-dose group (Telocinobufagin (1 mg / kg)), positive drug group (5-FU (30 mg / kg))) in the H22-bearing liver cancer mouse model;

[0024] Figure 3 It is the result graph of tumor weight of each treatment group (control group, low-dose group (TEL (0.5 mg / kg)), high-dose group (TEL (1 mg / kg)), positive drug group (5-FU (30 mg / kg))) at the end point of the experiment in the H22-bearing liver cancer mouse model;

[0025] Figure 4 It is the mouse body weight proliferation curve of each group (control group, low-dose group (Telocinobufagin (0.5 mg / kg)), high-dose group (Telocinobufagin (1 mg / kg)), positive drug group (5-FU (30 mg / kg))) in the H22-bearing liver cancer mouse model;

[0026] Figure 5 Cell viability detection chart of yuanhuacin combined with 5-fluorouracil on human hepatocarcinoma cell line SK-HEP-1 Detailed implementation manners

[0027] The present invention will be further described below in conjunction with specific embodiments. However, the following embodiments are only used to illustrate the present invention and should not be regarded as limiting the scope of the present invention. For those not specified in the following embodiments, they are carried out according to conventional conditions or conditions recommended by the manufacturer. The methods used are all conventional methods well-known in the art unless otherwise specified, and the consumables and reagents used are all commercially available unless otherwise specified. Unless otherwise stated, the professional and scientific terms used herein have the same meaning as those familiar to those skilled in the art. In addition, any method or material similar or equivalent to the described content can also be applied to the present invention.

[0028] Cell line: Human hepatocarcinoma SK-HEP-1 cell line was purchased from Beina Biotechnology Co., Ltd.; CCK8 reagent (product number: B4261GG1) was purchased from Nanjing Novizan Biotech Co., Ltd.; Yuanhuacin (product number: BP1370) was purchased from Chengdu PuriFa Technology Co., Ltd.; 5-fluorouracil (product number: S17073) was purchased from Shanghai Yuanye Bio-Technology Co., Ltd.

[0029] Example 1: Effect of yuanhuacin on the proliferation of human hepatocarcinoma cells

[0030] Treatment method: CCK-8 was used to evaluate the growth inhibitory effect of yuanhuacin on human hepatocarcinoma cells: Human hepatocarcinoma SK-HEP-1 cells were inoculated into 96-well plates, with 3500 cells per well. After being placed in the incubator for 24 h, different concentrations of drugs were added, with 5 replicates in each group, and they were treated for 24 or 48 h respectively. After the corresponding time ended, the cell culture medium was aspirated, 100 μL of 10% CCK8 was added to each well, and it was placed in the incubator and incubated for 1 h. The absorbance of each well, that is, the OD value, was detected with an enzyme-linked immunosorbent assay (ELISA) reader at a wavelength of 450 nm. The calculation formula for cell proliferation rate: Cell proliferation rate (%) = (OD value of the drug treatment group / average OD value of the control group) × 100%.

[0031] The results are as Figure 1 shown. Yuanhuacin has a proliferation inhibitory effect on human hepatocarcinoma cell line SK-HEP-1 cells, and it has time- and dose-dependence.

[0032] Example 2: Effect of yuanhuacin on tumor growth in a murine model of hepatocarcinoma H22 tumor-bearing mice

[0033] Experimental animals: SPF-grade Kunming mice, male, 4 weeks old, with a body weight of about 20 g, purchased from Jinan Pengyue Experimental Animal Breeding Co., Ltd.

[0034] Experimental method: Mouse liver cancer H22 cells were cultured in DMEM medium containing 10% fetal bovine serum. H22 cells in the exponential growth phase were harvested, resuspended in PBS, and the cell suspension was used for subcutaneous tumor inoculation. Each mouse was inoculated with 200 μL (containing 2×10 6 H22 cells) of the cell suspension under the right axilla, and then randomly divided into groups of 10 each. Administration was by intraperitoneal injection, and the specific dosing regimen is shown in Table 1. The tumor length and width were measured every other day with a vernier caliper. Tumor volume calculation formula: Tumor volume (mm 3 ) = 1 / 2×(a×b 2 ) (where a represents the major axis and b represents the minor axis).

[0035] Table 1 Dosing regimen

[0036] Group Dosing dose Dosing dose Dosing cycle Control group PBS 200 μL Once a day for two consecutive weeks Low-dose group Telocinobufagin 0.5 mg / kg Once a day for two consecutive weeks High-dose group Telocinobufagin 1 mg / kg Once a day for two consecutive weeks Positive drug group 5-Fluorouracil 30 mg / kg Once every two days for two consecutive weeks

[0037] Experimental results: The experiment was terminated on the 14th day after dosing for each group. The results are as shown in Figure 2 . It can be seen from the results that the average tumor volume of the control group was 11.32 mm 3 , the average tumor volume of the low-dose group of telocinobufagin was 7.28 mm 3 , the average tumor volume of the high-dose group of telocinobufagin was 5.48 mm 3 . 5-Fluorouracil (5-FU) is a positive drug for the treatment of liver cancer, and the average tumor volume after its treatment was 4.28 mm 3 . Thus, it can be seen that 5-FU has the best tumor inhibitory effect. In addition, tumor samples were collected at the end of the experiment and the tumor weights were measured. The results are as shown in Figure 2 . It can be seen from the results that compared with the PBS control group, telocinobufagin treatment could significantly reduce the tumor weight. 5-FU had the best tumor inhibitory effect, and the results were consistent with those of the tumor volume measurement. The results indicate that telocinobufagin can inhibit the growth of tumors in H22 liver cancer-bearing mice.

[0038] Meanwhile, the body weight changes of the mice were monitored regularly during the dosing period, once a day. The results are as shown in Figure 4 . It can be seen from the results that the body weights of the control group, the low- and high-dose groups of telocinobufagin, and the 5-FU group all increased steadily and continuously. The results indicate that the mice grew normally after telocinobufagin administration, without obvious drug toxic and side reactions, which also illustrates the safety of telocinobufagin from the side.

[0039] Example 3: Effect of telocinobufagin combined with 5-fluorouracil on the proliferation of human liver cancer SK-HEP-1 cells

[0040] Treatment method: Use CCK-8 to evaluate the growth inhibitory effect of telocinobufagin combined with 5-fluorouracil on human liver cancer cells: Inoculate human liver cancer SK-HEP-1 cells in a 96-well plate, with 3,500 cells per well. After placing them in an incubator for 24 hours, administer 5-fluorouracil at 0, 0.25, 0.5 μg / mL and telocinobufagin at 0, 10, 25, 50 nM in combination. There are 5 replicate wells in each group, and treat for 48 hours. After the corresponding time ends, aspirate the cell culture medium, add 100 μL of 10% CCK8 to each well, place it in an incubator, and incubate for 1 hour. Detect the absorbance of each well, that is, the OD value, with an enzyme-linked immunosorbent assay (ELISA) reader at a wavelength of 450 nm. The formula for calculating the cell proliferation rate: Cell proliferation rate (%) = (OD value of the drug-administered group / average OD value of the control group) * 100%. Calculate the combination index CI of the two drugs based on the obtained cell proliferation rate, calculate the relationship between the CI value and 1. According to the judgment method of Soriano et al., 0.9 ≤ CI ≤ 1.1 is additive effect, 0.8 ≤ CI < 0.9 is low-degree synergistic effect, 0.6 ≤ CI < 0.8 is moderate-degree synergistic effect, 0.4 ≤ CI < 0.6 is high-degree synergistic effect, 0.2 ≤ CI < 0.4 is strong synergistic effect.

[0041] The results are as Figure 5 shown. The results show that when 5-fluorouracil is 0.25 μg / mL, the CI values when the concentrations of telocinobufagin are 0.25, 0.5, 1 nM are 0.997, 0.796, 0.599 respectively; when 5-fluorouracil is 0.5 μg / mL, the CI values when the concentrations of telocinobufagin are 0.25, 0.5, 1 nM are 0.994, 0.918, 0.779 respectively. The CI values are all < 1, proving that the two have a synergistic effect at this concentration. It can be seen from the results that when 5-fluorouracil and telocinobufagin are at specific concentrations, they have a significant synergistic inhibitory effect on the cell proliferation of human liver cancer cells SK-HEP-1.

[0042] Although the embodiments of the present invention have been shown and described above, it can be understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those of ordinary skill in the art can make changes, modifications, substitutions, and variations to the above embodiments within the scope of the present invention, and all should be included within the protection scope of the present invention.

Claims

1. A use of telocinobufagin in the preparation of anti-liver cancer drugs.

2. Use of a pharmaceutical preparation in the preparation of an anti-liver cancer drug, characterized in that: The concentration of telocinobufagin in the pharmaceutical preparation is 25-500 nM.

3. A pharmaceutical composition, characterized in that The pharmaceutical composition comprises telocinobufagin and 5-fluorouracil.

4. The pharmaceutical composition according to claim 3, characterized in that The concentration of telocinobufagin is 0.25-1 nM, and the concentration of 5-fluorouracil is 0.25-0.5 μg / mL.

5. The pharmaceutical composition according to claim 4, characterized in that The concentration of telocinobufagin is 0.5-1 nM, and the concentration of 5-fluorouracil is 0.25 μg / mL.

6. The pharmaceutical composition according to claim 4, characterized in that The concentration of telocinobufagin is 0.5-1 nM, and the concentration of 5-fluorouracil is 0.5 μg / mL.

7. The pharmaceutical composition according to claim 4, characterized in that The concentration of telocinobufagin was 1 nM, and the concentration of 5-fluorouracil was 0.5 μg / mL.

8. The pharmaceutical composition according to any one of claims 3 to 7, characterized in that The pharmaceutical composition further comprises pharmaceutically acceptable excipients and carriers.

9. The pharmaceutical composition according to any one of claims 3 to 7, characterized in that The dosage form of the pharmaceutical composition is selected from one of granules, tablets, pills, capsules and injections.

10. Use of the pharmaceutical composition according to any one of claims 3 to 9 in the preparation of anti-liver cancer drugs.

Images