Disclosure of Invention
The application aims to overcome the defects of the prior art and provide application of a combined drug composition in preparing drugs for preventing and/or treating liver cancer, and the application discovers that the drug effect for cooperatively preventing and/or treating liver cancer can be realized through the combined use of sorafenib and Cpd-63 drugs.
The invention is realized in the following way:
in a first aspect of the invention, there is provided the use of a combination pharmaceutical composition comprising sorafenib and Cpd-63 in the manufacture of a medicament for the prevention and/or treatment of liver cancer, said Cpd-63 having the structural formula:
further, the medicament for preventing and/or treating liver cancer inhibits proliferation, invasion and metastasis of liver cancer cells.
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.
As a specific embodiment, the dosage of both said sorafenib and said Cpd-63 is from 10 to 50mg/kg, preferably 30mg/kg.
Further, the sorafenib is used at a concentration of 1. Mu.M to 50. Mu.M and the Cpd-63 is used at a concentration of 1. Mu.M to 50. Mu.M.
In a second aspect of the present invention, there is provided a medicament for the prophylaxis and/or treatment of liver cancer, the medicament for the prophylaxis and/or treatment of liver cancer comprising sorafenib and Cpd-63, the Cpd-63 having the formula:
further, the medicine for preventing and/or treating liver cancer contains an effective amount of sorafenib and an effective amount of Cpd-63.
Further, the medicine for preventing and/or treating liver cancer contains pharmaceutically acceptable carriers and/or auxiliary materials. The auxiliary material is selected from one of filler, disintegrating agent, adhesive, excipient, diluent, lubricant, sweetener or colorant.
Further, the dosage form of the medicament for preventing and/or treating liver cancer is selected from one of granules, tablets, pills, capsules, injections and dispersing agents.
In a third aspect of the present invention there is provided the use of Cpd-63 in the manufacture of a medicament for the prevention and/or treatment of liver cancer.
One or more technical solutions in the embodiments of the present invention at least have the following technical effects or advantages:
According to the invention, a large amount of experimental data prove that Cpd-63 can inhibit liver cancer proliferation activity. And further, by combining sorafenib and Cpd-63, a synergistic inhibition effect can be generated on liver cancer cells, that is, the inhibition effect on liver cancer cells when the two medicines are combined is stronger than the sum of inhibition effects exerted when the two medicines are used independently. Therefore, the compound can be used as a combined drug composition to prepare drugs for preventing and/or treating liver cancer.
Detailed Description
The advantages and various effects of the present invention will be more clearly apparent from the following detailed description and examples. It will be understood by those skilled in the art that these specific embodiments and examples are intended to illustrate the invention, not to limit the invention.
Throughout the specification, unless specifically indicated otherwise, the terms used herein should be understood as meaning as commonly used in the art. Accordingly, unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In case of conflict, the present specification will control.
Unless specifically indicated otherwise, the various raw materials, reagents, instruments, equipment, etc., used in the present invention are commercially available or may be obtained by existing methods.
In order to solve the technical problem of the invention, the general idea of the invention is as follows:
Cpd-63, wherein the structural formula of Cpd-63 is:
Cpd-63 is an N- [5- (phenoxymethyl) -1,3, 4-thiadiazol-2-yl ] acetamide derivative and Cpd-63 was found to have anti-osteoclast activity in literature Identification of N-(5-(phenoxymethyl)-1,3,4-thiadiazol-2-yl)acetamide derivatives as novel protein tyrosine phosphatase epsilon inhibitors exhibiting anti-osteoclastic activity.
Experiments show that Cpd-63 can inhibit proliferation, invasion and metastasis of liver cancer cells.
Further, the combination of sorafenib and Cpd-63 was found to produce a synergistic inhibitory effect on liver cancer cell proliferation. Cpd-63 may enhance the in vitro reaction of sorafenib.
These results indicate that Cpd-63 may be a potential therapeutic approach for the treatment of HCC.
The use of the combination pharmaceutical composition of the present application for preparing a medicament for preventing and/or treating liver cancer will be described in detail below with reference to examples and experimental data.
As used herein, "combination" or "combination therapy" and the like means that two or more active agents can each be administered to a subject simultaneously as a single formulation, or sequentially in any order, each as a single formulation.
As used herein, an "effective amount" refers to an amount that comprises symptoms or diseases sufficient to prevent or treat a medical condition. After use in a particular patient or medical subject, a change may be made in the condition to be treated, the overall health of the patient/subject being improved. The effective amount may also be a dosage regimen that is below the maximum dose that avoids significant side effects or toxic effects.
Cpd-63, cpd-52 of the examples of the present invention were synthesized by the Ming Kangde company.
Example 1 Effect of Cpd-52 and Cpd-63 alone on proliferation of liver cancer cells
1. The effect of the different concentrations of the single drug Cpd-52 (1. Mu.M, 5. Mu.M, 10. Mu.M, 25. Mu.M, 50. Mu.M) and the single drug Cpd-63 (1. Mu.M, 5. Mu.M, 10. Mu.M, 25. Mu.M, 50. Mu.M) on proliferation of different liver cancer cell lines (HepG 2, huh7, PLC/PRF/5, HLF, MHCC-97H, HCC-LM 3) was examined by the CCK8 method, and the specific steps are:
Cells in the exponentially growing phase were digested with pancreatin, blown off into single cell suspensions, counted and plated into 96-well plates (3000 cells/well). Culturing in an incubator for 24 hours, after the cells are attached, independently adding Cpd-52 and Cpd-63 with different concentrations, respectively, stopping culturing after 72 hours, adding 10 μl of CCK8 (5 mg/mL) into each hole, incubating for 1 hour at 37 ℃ in a dark place, measuring the optical density value of the cells at the wavelength of 450nm of an enzyme-labeled instrument, and calculating the survival rate of the cells according to the following formula:
Percent survival = (experimental group number-blank group number)/(control group number-blank group number) ×100%.
As shown in FIG. 1, in the human liver cancer cell line, cpd-52 single drug and Cpd-63 single drug both show a certain effect of inhibiting proliferation activity of liver cancer cells, and with the increase of drug concentration, the cell activity of the liver cancer cell line gradually decreases, and the effect of Cpd-63 single drug is better than that of Cpd-52 single drug.
Example 2 Effect of Cpd-52 and Cpd-63 alone on migration and invasion of liver cancer cells
1. Scratch healing experiments
Tumor cell migration ability was evaluated by artificially creating a blank area on the fused monolayer cells, and then observing the condition of scratch healing at different times, specifically comprising the steps of inoculating a human liver cancer cell line MHCC-97H in a logarithmic growth phase to a 6-well plate in a number of 2.0X105 cells, culturing in a 37 ℃ and 5% CO2 incubator, after the cell density reaches 80%, scraping a scratch of about 1cm from top to bottom at the center of each well with a gun head, discarding the culture solution, washing 3 times with PBS, photographing with an inverted microscope, treating the human liver cancer cell line MHCC-97H with a single drug Cpd-52 (10 mu M) and a single drug Cpd-63 (10 mu M) respectively after discarding the PBS, culturing in the incubator for 24 hours, washing 2 times with PBS, and photographing again with an inverted microscope, wherein the experiment is repeated three times. The results are shown in FIG. 2A.
2. Transwell experiments:
MHCC-97H cells were treated with 0.01% DMSO, cpd-52 single drug and Cpd-63 single drug, respectively, for 24 hours, followed by cell migration and invasion assays.
Mu.L of medium containing 10% fetal bovine serum was added to the lower chamber and 100. Mu.L of 5X104 cells in serum-free medium were aliquoted into the upper chamber. After incubation at 37 ℃ for 24 hours, the cell membrane was scraped off the upper surface with a cotton swab to remove non-migrated or non-invaded cells. Cells on the lower surface of the filter were fixed with 4% paraformaldehyde for 15min at room temperature and stained with 0.1% crystal violet for 1 hour at room temperature. The cell number was counted under an optical microscope. Each experiment was repeated at least three times.
As shown in FIG. 2, it is clear that the single agent Cpd-52 and the single agent Cpd-63, and in particular Cpd-63, significantly inhibited the migration and invasion of MHCC-97H cells.
EXAMPLE 3 inhibition of subcutaneous tumor growth and pulmonary metastasis in liver cancer nude mice by Cpd-63 Single drug
1. Establishing a nude mouse liver cancer subcutaneous tumor model
A total of 20 male BALB/c nude mice (5 weeks old) were used, each injected subcutaneously with 100 ten thousand HCC-LM3 cells. After two weeks, control solvents, cpd-52 (30 mg/kg/day), cpd-63 (30 mg/kg/day), sorafenib (30 mg/kg/day) were administered separately for 4 weeks of gastric lavage treatment, and the nude mice were sacrificed after two weeks of drug withdrawal, dissected and the tumors were removed completely and photographed while weighing the weight, tumor mass and volume of the nude mice.
The results are shown in FIG. 3, where the size of the subcutaneous tumor in nude mice after administration of Cpd-52 single drug, cpd-63 single drug, or sorafenib single drug is shown in FIG. 3B, and it can be seen that both sorafenib and Cpd-63 single drug, cpd-52 significantly inhibited the growth of subcutaneous tumors, and that Cpd-52 treated groups and Cpd-63 treated groups have significantly reduced tumor volume and weight compared to control groups. At the same dose (30 mg/kg/day), neither Cpd-52 nor Cpd-63 was less effective than sorafenib.
2. Establishing a model of liver cancer lung metastasis by intravenous injection of naked tail
A total of 15 male BALB/c nude mice (5 weeks old) were used, 100 ten thousand HCC-LM3-Luc cells were intravenously injected into each of the nude mice, and after two weeks, a control solvent, cpd-63 (30 mg/kg/day), sorafenib (30 mg/kg/day) were administered for intragastric treatment for 4 weeks, and after two weeks of drug withdrawal, in vivo imaging was performed to detect lung metastasis, and after the mice were sacrificed, lung tissues were isolated and fixed, and the number and size of lung metastasis nodules were measured.
As shown in FIG. 3F, analysis of in vivo imaging fluorescence signals showed that the fluorescence intensity of Cpd-63 treated groups was significantly lower than that of control groups. There was no significant difference in bioluminescence between the Cpd-63 treated group and the sorafenib treated group. As shown in FIG. 3G, the number and size of lung nodules in the Cpd-63 treated group were significantly smaller than in the control group.
Taken together, these results demonstrate that Cpd-63 is effective in inhibiting HCC growth and metastasis in vivo.
Example 4 Effect of Cpd-63 in combination with sorafenib on liver cancer cell proliferation
1. Cell clone experiment is adopted to detect influence of sorafenib, cpd-63 and combined group thereof on growth of different liver cancer cells
And (3) carrying out pancreatin digestion on MHCC-97H in an exponential growth phase, blowing off to form single cell suspension, counting, inoculating into a 6-hole culture plate, inoculating 1500 cells per hole of a single drug group and 2500 cells per hole of a combined group, culturing for 24 hours in an incubator, respectively adding 10 mu M Cpd-63 single drug, 10 mu M sorafenib single drug and the combined group, stopping culturing after 14 days, discarding supernatant culture solution, washing once by PBS, adding 0.25% crystal violet solution, standing for 15 minutes, washing by running water, taking a photo after drying, and carrying out clone counting analysis.
As a result, and as seen in FIG. 4, cpd-63 and sorafenib in combination are more effective in inhibiting the colony formation of MHCC-97H cells. The combination of sorafenib and Cpd-63 can synergistically inhibit liver cancer cell growth.
2. The effect of Cpd-63 alone (at a concentration of 10. Mu.M), sorafenib alone (at a concentration of 10. Mu.M), cpd-63 in combination with Sorafenib (Cpd-63, sorafenib at a concentration of 10. Mu.M) on proliferation of hepatoma cell lines MHCC-97H and HCC-LM3 was examined using the CCK8 method.
As shown in FIG. 5, the combination of sorafenib and Cpd-63 for 48 hours can synergistically inhibit the proliferation of liver cancer cells, and Cpd-63 significantly promotes the inhibition of the cell activity by sorafenib.
3. Meanwhile, the above test results were subjected to a combination index analysis, wherein the Combination Index (CI), which is the sum of the partial formulas of the inhibitory concentrations of the two drugs, was calculated as ci=c50a/IC 50a+c50b/IC 50B;
wherein, C50A and C50B respectively refer to the drug concentration when A, B two drugs are combined to achieve 50% inhibition, and IC 50A and IC 50B respectively refer to the drug concentration when a certain drug alone achieves 50% inhibition.
When CI <1, the two drug combinations have a synergistic effect, when ci=1, the two drug combinations have a additive effect, and when CI >1, the two drug combinations have an antagonistic effect.
The results of the combined index analysis of Cpd-63 and sorafenib combined treatment group for 48 hours are shown in FIG. 6, and it can be seen that CI of Cpd-63 and sorafenib combined treatment group is <1, which indicates that Cpd-63 and sorafenib combined treatment group has obvious synergistic effect and can better inhibit proliferation of liver cancer cells.
Example 5 influence of Cpd-63 in combination with sorafenib on liver cancer cell migration and invasion
We performed a Transwell test and scratch healing test on mhc c-97H cells to investigate the anti-migration effect of the combination therapy. Experimental procedure As in example 2, cpd-63 single drug, sorafenib single drug and combination treatment group doses were all 10. Mu.M. The results are shown in FIG. 7, where the inhibition of migration and invasion of MHCC-97H cells was significantly greater in the combination treatment group than in the Cpd-63 or sorafenib treatment groups.
Finally, it is also noted that the terms "comprises," "comprising," or any other variation thereof, are intended to cover a non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements does not include only those elements but may include other elements not expressly listed or inherent to such process, method, article, or apparatus.
While preferred embodiments of the present invention have been described, additional variations and modifications in those embodiments may occur to those skilled in the art once they learn of the basic inventive concepts. It is therefore intended that the following claims be interpreted as including the preferred embodiments and all such alterations and modifications as fall within the scope of the invention.
It will be apparent to those skilled in the art that various modifications and variations can be made to the present invention without departing from the spirit or scope of the invention. Thus, it is intended that the present invention also include such modifications and alterations insofar as they come within the scope of the appended claims or the equivalents thereof.