Application of combined pharmaceutical composition in the preparation of a drug for preventing and / or treating liver cancer

Abstract

The present invention provides the use of a combined pharmaceutical composition in the preparation of a drug for preventing and / or treating liver cancer. The present invention first discovered that Cpd-63 can inhibit the proliferation, migration and invasion activity of liver cancer cells, and further combined sorafenib and Cpd-63 to produce a synergistic inhibitory effect on the proliferation of liver cancer cells. Therefore, it can be used as a combined pharmaceutical composition in the preparation of a drug for preventing and / or treating liver cancer, and has clinical application prospects.

Description

Technical Field

[0001] The present invention relates to the technical field of molecular biology, and in particular to the use of a combined pharmaceutical composition in the preparation of a drug for preventing and / or treating liver cancer. Background Art

[0002] Primary liver cancer (PLC) is one of the most commonly diagnosed cancers worldwide and the third leading cause of cancer-related death. Hepatocellular carcinoma (HCC) accounts for approximately 75-85% of all PLC cases. Its pathogenesis is extremely complex and can have multiple causes, including chronic hepatitis B virus or hepatitis C virus infection, exposure to aflatoxin B1, alcoholic and non-alcoholic fatty diseases, etc. Although recent studies have discovered many genes and pathways that affect the development of HCC, treatment and patient prognosis remain unsatisfactory.

[0003] Although many trials have been conducted to treat patients with advanced HCC, systemic therapy, including sorafenib, lenvatinib, or atezolizumab plus bevacizumab, is the main treatment option. The overall response rate of sorafenib is only 2%, and the overall median survival of the sorafenib group is only 10.7 months. The efficacy of lenvatinib and atezolizumab combined with bevacizumab is still far from satisfactory, although it is slightly better than sorafenib. This situation highlights the need for more therapeutic approaches to improve the clinical benefit of HCC treatment.

[0004] Therefore, it is necessary to develop a new drug for preventing and / or treating liver cancer. Summary of the invention

[0005] The purpose of the present invention is to overcome the defects of the prior art and provide the use of a combination pharmaceutical composition in the preparation of a drug for preventing and / or treating liver cancer. The present application found that the combination of sorafenib and Cpd-63 can achieve a synergistic effect in preventing and / or treating liver cancer.

[0006] The present invention is achieved in that:

[0007] In a first aspect of the present invention, a combination pharmaceutical composition is provided for use in the preparation of a medicament for preventing and / or treating liver cancer, wherein the combination pharmaceutical composition contains sorafenib and Cpd-63, and the structural formula of Cpd-63 is:

[0008]

[0009] Furthermore, the drug for preventing and / or treating liver cancer inhibits the proliferation, invasion and metastasis of liver cancer cells.

[0010] Further, the mass ratio of sorafenib to Cpd-63 is 1:1-4. Preferably, the mass ratio of sorafenib to Cpd-63 is 1:1.

[0011] As a specific embodiment, the dosage of sorafenib and Cpd-63 is 10-50 mg / kg, preferably 30 mg / kg.

[0012] Furthermore, the application concentration of sorafenib is 1 μM-50 μM, and the application concentration of Cpd-63 is 1 μM-50 μM.

[0013] In a second aspect of the present invention, a drug for preventing and / or treating liver cancer is provided, wherein the drug for preventing and / or treating liver cancer contains sorafenib and Cpd-63, and the structural formula of Cpd-63 is:

[0014]

[0015] Furthermore, the drug for preventing and / or treating liver cancer contains an effective amount of sorafenib and an effective amount of Cpd-63.

[0016] Furthermore, the drug for preventing and / or treating liver cancer contains a pharmaceutically acceptable carrier and / or excipient. The excipient is selected from one of a filler, a disintegrant, a binder, an excipient, a diluent, a lubricant, a sweetener or a colorant.

[0017] Furthermore, the dosage form of the drug for preventing and / or treating liver cancer is selected from one of granules, tablets, pills, capsules, injections and dispersants.

[0018] In the third aspect of the present invention, provided is the use of Cpd-63 in the preparation of a medicament for preventing and / or treating liver cancer.

[0019] One or more technical solutions in the embodiments of the present invention have at least the following technical effects or advantages:

[0020] The present invention proves through a large amount of experimental data that Cpd-63 can inhibit the proliferation activity of liver cancer. Furthermore, by combining sorafenib and Cpd-63, a synergistic inhibitory effect can be produced on liver cancer cells, that is, the inhibitory effect of the two drugs on liver cancer cells when used in combination is stronger than the sum of the inhibitory effects exerted when the two drugs are used alone. Therefore, it can be used as a combined pharmaceutical composition in the preparation of a drug for preventing and / or treating liver cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 The results of the CCK8 test of single drug Cpd-52 and Cpd-63 on cells of different liver cancer cell lines in Example 1; wherein, Figure 1 A is the structural formula of Cpd-52, Figure 1B is the CCK8 test result of Cpd-52 on cells of different liver cancer cell lines; Figure 1 C is the structural formula of Cpd-63, Figure 1 D is the CCK8 test result of Cpd-63 on cells of different liver cancer cell lines;

[0022] Figure 2 The experimental results of the effects of single-agent Cpd-52 and single-agent Cpd-63 on the migration and invasion of liver cancer cells in Example 2; wherein, Figure 2 A is the scratch test result, Figure 2 B is the result of Transwell experiment.

[0023] Figure 3 The results of the in vivo treatment experiment in nude mice in Example 3; wherein, Figure 3 A is the experimental process of nude mouse subcutaneous tumor and treatment. Figure 3 B is the tumor size, Figure 3 C Tumor volume and weight, Figure 3 D is Ki67 proliferation index immunohistochemical staining, Figure 3 E is the nude mouse tail vein injection liver cancer lung metastasis model and treatment flow chart, Figure 3 F is the result of nude mouse in vivo imaging. Figure 3 G shows the HE staining results of nude mouse lung tissue and the count and size statistics of metastatic foci.

[0024] Figure 4 The results of the cell growth test of Cpd-63, sorafenib and their combination in Example 4;

[0025] Figure 5 The results of CCK8 tests of Cpd-63, sorafenib and their combination on cells of different liver cancer cell lines in Example 4;

[0026] Figure 6 The combined index analysis results in Example 4;

[0027] Figure 7 The results of the experiment on the effects of single-agent Cpd-63, sorafenib and their combination on the migration and invasion of liver cancer cells in Example 5 are shown in FIG. Figure 7 A is the scratch test result, Figure 7 B is the result of Transwell experiment. DETAILED DESCRIPTION

[0028] The present invention will be described in detail below in conjunction with specific implementations and examples, and the advantages and various effects of the present invention will be more clearly presented. It should be understood by those skilled in the art that these specific implementations and examples are used to illustrate the present invention, rather than to limit the present invention.

[0029] Throughout the specification, unless otherwise specifically stated, the terms used herein should be understood as meanings commonly used in the art. Therefore, unless otherwise defined, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art to which the present invention belongs. In the event of a conflict, the present specification takes precedence.

[0030] Unless otherwise specified, various raw materials, reagents, instruments and equipment used in the present invention can be purchased from the market or obtained by existing methods.

[0031] In order to solve the technical problem of the present invention, the overall idea of ​​the present invention is as follows:

[0032] Cpd-63, the structural formula of the Cpd-63 is:

[0033]

[0034] Cpd-63 is a derivative of N-[5-(phenoxymethyl)-1,3,4-thiadiazol-2-yl]acetamide. The literature "Identification of N-(5-(phenoxymethyl)-1,3,4-thiadiazol-2-yl)acetamidederivatives as novel protein tyrosine phosphatase epsilon inhibitors exhibiting anti-osteoclastic activity" found that Cpd-63 has anti-osteoclastic activity.

[0035] The present application discovered through experiments that Cpd-63 can inhibit the proliferation, invasion and metastasis of liver cancer cells.

[0036] Furthermore, the combination of sorafenib and Cpd-63 was found to have a synergistic inhibitory effect on the proliferation of liver cancer cells. Cpd-63 can enhance the in vitro response of sorafenib.

[0037] These results suggest that Cpd-63 may be a potential therapeutic approach for HCC treatment.

[0038] The application of the combined pharmaceutical composition of the present application in the preparation of a drug for preventing and / or treating liver cancer will be described in detail below in conjunction with examples and experimental data.

[0039] The term "combination" or "combination therapy group" as used herein means that two or more active substances can be administered to a subject simultaneously as a single preparation, or sequentially in any order as a single preparation.

[0040] "Effective amount" as described herein refers to an amount sufficient to prevent or treat the symptoms or diseases of a medical condition. After use in a specific patient or medical subject, the following changes may occur: the condition to be treated, the overall health of the patient / subject is improved. An effective amount may also be a dosage regimen below the maximum dose to avoid significant side effects or toxic effects.

[0041] The Cpd-63 and Cpd-52 of the examples of the present invention were synthesized by WuXi AppTec.

[0042] Example 1: Effects of single-agent Cpd-52 and single-agent Cpd-63 on proliferation of liver cancer cells

[0043] 1. The CCK8 method was used to detect the effects of different concentrations of single-agent Cpd-52 (1μM, 5μM, 10μM, 25μM, 50μM) and single-agent Cpd-63 (1μM, 5μM, 10μM, 25μM, 50μM) on the proliferation of different liver cancer cell lines (HepG2, Huh7, PLC / PRF / 5, HLF, MHCC-97H, HCC-LM3). The specific steps are as follows:

[0044] The cells in the exponential growth phase were digested with trypsin, blown into a single cell suspension, counted, and inoculated into a 96-well culture plate (3000 cells / well). After 24 hours of culture in the incubator, the above-mentioned different concentrations of Cpd-52 and Cpd-63 were added separately after the cells adhered to the wall. After 72 hours of action, the culture was terminated, 10 μl of CCK8 (5 mg / mL) was added to each well, and incubated at 37°C in the dark for 1 hour. The cell optical density value was measured at a wavelength of 450nm on an ELISA reader, and the cell survival rate was calculated according to the following formula:

[0045] Survival rate % = [(experimental group value - blank group value) ÷ (control group value - blank group value)] × 100%.

[0046] The results are as follows Figure 1 As shown, in human liver cancer cell lines, both Cpd-52 and Cpd-63 showed a certain effect of inhibiting the proliferation activity of liver cancer cells, and with the increase of drug concentration, the cell activity of the above liver cancer cell lines gradually decreased, and the effect of Cpd-63 alone was better than that of Cpd-52 alone.

[0047] Example 2: Effects of single-agent Cpd-52 and single-agent Cpd-63 on migration and invasion of liver cancer cells

[0048] 1. Scratch healing experiment

[0049] The migration ability of tumor cells was evaluated by artificially creating a blank area on the confluent monolayer cells and then observing the "healing" of the scratch at different times. The specific steps were as follows: MHCC-97H, a human hepatoma cell line in the logarithmic growth phase, was cultured at 2.0×10 5 The cells were inoculated into a 6-well plate and cultured in a 37°C, 5% CO2 incubator; when the cell density reached 80%, a 1 cm scratch was made from top to bottom in the center of each well with a gun tip, the culture medium was discarded, and the plates were washed three times with PBS, and photographed under an inverted microscope; after the PBS was discarded, the human liver cancer cell line MHCC-97H was treated with single drug Cpd-52 (10 μM) and single drug Cpd-63 (10 μM), respectively, and cultured in an incubator for 24 hours; after the supernatant was discarded, the plates were washed twice with PBS, and photographed again under an inverted microscope, and the experiment was repeated three times. The results are shown in Figure 2. Figure 2 As shown in A.

[0050] 2. Transwell experiment:

[0051] MHCC-97H cells were treated with 0.01% DMSO, Cpd-52 alone, and Cpd-63 alone for 24 hours, and then subjected to cell migration and invasion assays.

[0052] Add 800 μL of medium containing 10% fetal bovine serum to the lower chamber and add 5x10 cells in 100 μL of serum-free medium. 4 Cells were inoculated into the upper chamber in equal parts. After incubation at 37°C for 24 hours, the cell membrane was scraped from the upper surface with a cotton swab to remove cells that did not migrate or invade. Cells on the lower surface of the filter were fixed with 4% paraformaldehyde for 15 minutes at room temperature and stained with 0.1% crystal violet for 1 hour at room temperature. The number of cells was counted under an optical microscope. Each experiment was repeated at least three times.

[0053] The results are as follows Figure 2 As shown, it can be seen that single-agent Cpd-52 and single-agent Cpd-63, especially Cpd-63, can significantly inhibit the migration and invasion of MHCC-97H cells.

[0054] Example 3: Cpd-63 alone inhibits subcutaneous tumor growth and lung metastasis in nude mice with hepatocellular carcinoma

[0055] 1. Establishment of a nude mouse subcutaneous liver cancer model

[0056] A total of 20 male BALB / c nude mice (5 weeks old) were used, and 1 million HCC-LM3 cells were injected subcutaneously into each nude mouse. Two weeks later, the mice were given control solvent, Cpd-52 (30 mg / kg / day), Cpd-63 (30 mg / kg / day), and sorafenib (30 mg / kg / day) for four weeks of intragastric administration. After two weeks of drug withdrawal, the nude mice were killed, and the tumors were completely removed and photographed. The weight of the nude mice, tumor mass and volume were also measured.

[0057] The results are as follows Figure 3 As shown in, Figure 3 B shows the size of subcutaneous tumors in nude mice after treatment with Cpd-52 alone, Cpd-63 alone, and sorafenib alone. It can be seen that sorafenib, Cpd-63 alone, and Cpd-52 can significantly inhibit the growth of subcutaneous tumors; compared with the control group, the tumor volume and weight of the Cpd-52 treatment group and the Cpd-63 treatment group were significantly reduced. At the same dose (30 mg / kg / day), the effects of Cpd-52 or Cpd-63 are not inferior to sorafenib.

[0058] 2. Establishment of a nude mouse tail vein injection model of liver cancer lung metastasis

[0059] A total of 15 male BALB / c nude mice (5 weeks old) were used. Each nude mouse was injected with 1 million HCC-LM3-Luc cells through the tail vein. Two weeks later, they were given control solvent, Cpd-63 (30 mg / kg / day), and sorafenib (30 mg / kg / day) by gavage for 4 weeks. Two weeks after stopping the drug, in vivo imaging was performed to detect lung metastasis. The mice were then killed to isolate and fix the lung tissues, and the number and size of lung metastatic nodules were measured.

[0060] like Figure 3 As shown in F, the analysis of the fluorescence signal of in vivo imaging showed that the fluorescence intensity of the Cpd-63-treated group was significantly lower than that of the control group. There was no significant difference in bioluminescence between the Cpd-63-treated group and the sorafenib-treated group. Figure 3 As shown in G, the number and size of lung nodules in the Cpd-63-treated group were significantly smaller than those in the control group.

[0061] Taken together, these results demonstrate that Cpd-63 can effectively inhibit the growth and metastasis of HCC in vivo.

[0062] Example 4: Effect of Cpd-63 combined with sorafenib on the proliferation of liver cancer cells

[0063] 1. Cell cloning experiments were used to detect the effects of sorafenib, Cpd-63 and their combination on the growth of different liver cancer cells

[0064] The MHCC-97H in the exponential growth phase was digested with trypsin, blown into a single cell suspension, counted, and inoculated into a 6-well culture plate. 1500 cells were inoculated in each well of the single-drug group and 2500 cells were inoculated in each well of the combination group. After culturing in the incubator for 24 hours, 10μM Cpd-63 alone, 10μM sorafenib alone and the combination group were added, and the culture was terminated after 14 days. The supernatant culture medium was discarded, rinsed once with PBS, and 0.25% crystal violet solution was added. After standing for 15 minutes, it was rinsed with running water, photographed after drying, and then clone counting analysis was performed.

[0065] Results Figure 4 It can be seen that the combination of Cpd-63 and sorafenib can more effectively inhibit the colony formation of MHCC-97H cells. The combination of sorafenib and Cpd-63 drugs can synergistically inhibit the growth of liver cancer cells.

[0066] 2. The CCK8 method was used to detect the effects of Cpd-63 alone (concentration of 10 μM), sorafenib alone (concentration of 10 μM), and the combination of Cpd-63 and sorafenib (Cpd-63 and sorafenib concentrations were both 10 μM) on the proliferation of liver cancer cell lines MHCC-97H and HCC-LM3.

[0067] The results are as follows Figure 5 As shown, the combination of sorafenib and Cpd-63 for 48 hours can synergistically inhibit the proliferation of liver cancer cells, and Cpd-63 significantly promoted the inhibitory effect of sorafenib on cell activity.

[0068] 3. At the same time, the above test results were analyzed by combination index, where the combination index (CI) is the sum of the fractional inhibitory concentrations of the two drugs, and its calculation formula is CI = C 50A / IC 50A + C 50B / IC 50B;

[0069] Wherein, C50A and C50B refer to the drug concentrations when the combination of drugs A and B achieves 50% inhibition, respectively; IC50A and IC50B refer to the drug concentrations when a single drug achieves 50% inhibition, respectively.

[0070] When CI is less than 1, the combination of the two drugs has a synergistic effect; when CI is equal to 1, the combination of the two drugs has an additive effect; when CI is greater than 1, the combination of the two drugs has an antagonistic effect.

[0071] The combined index analysis results of the Cpd-63 and sorafenib combined treatment group after 48 hours were as follows Figure 6 As shown, it can be seen that after Cpd-63 and sorafenib were used in combination, their CIs were all <1, indicating that Cpd-63 and sorafenib had obvious synergistic effects after being used in combination, and could better inhibit the proliferation of liver cancer cells.

[0072] Example 5: Effect of Cpd-63 combined with sorafenib on migration and invasion of liver cancer cells

[0073] We performed Transwell assay and wound healing assay on MHCC-97H cells to study the anti-migratory effect of combined therapy. The experimental steps were the same as in Example 2, and the doses of Cpd-63 monotherapy, sorafenib monotherapy, and combined therapy groups were all 10 μM. Figure 7 As shown, the inhibitory effect of the combined treatment group on the migration and invasion of MHCC-97H cells was significantly stronger than that of the Cpd-63 or sorafenib treatment group.

[0074] Finally, it should be noted that the terms "comprises," "includes," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that includes a series of elements includes not only those elements, but also other elements not explicitly listed, or also includes elements that are inherent to such process, method, article, or apparatus.

[0075] Although the preferred embodiments of the present invention have been described, those skilled in the art may make other changes and modifications to these embodiments once they have learned the basic creative concept. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments and all changes and modifications that fall within the scope of the present invention.

[0076] Obviously, those skilled in the art can make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include these modifications and variations.

Claims

1. Use of a combined pharmaceutical composition in the preparation of a drug for preventing and / or treating liver cancer, characterized in that: The combined pharmaceutical composition contains sorafenib and Cpd-63, and the structural formula of Cpd-63 is: The drug for preventing and / or treating liver cancer inhibits the proliferation, invasion and metastasis of liver cancer cells. The mass ratio of sorafenib to Cpd-63 is 1:1-4. The application concentration of sorafenib is 1μM-50μM, and the application concentration of Cpd-63 is 1μM-50μM.

2. A drug for preventing and / or treating liver cancer, characterized in that: The drug for preventing and / or treating liver cancer contains sorafenib and Cpd-63, and the structural formula of Cpd-63 is: 。 3. The drug for preventing and / or treating liver cancer according to claim 2, characterized in that: The medicine for preventing and / or treating liver cancer contains an effective amount of sorafenib and an effective amount of Cpd-63.

4. The drug for preventing and / or treating liver cancer according to claim 2, characterized in that: The medicine for preventing and / or treating liver cancer contains a pharmaceutically acceptable carrier and / or auxiliary material.

5. The drug for preventing and / or treating liver cancer according to claim 2, characterized in that: The dosage form of the drug for preventing and / or treating liver cancer is selected from one of granules, tablets, pills, capsules, injections and dispersants.

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