CN117414367A - Pharmaceutical composition for reversing drug resistance of lenvatinib and application thereof - Google Patents
Pharmaceutical composition for reversing drug resistance of lenvatinib and application thereof Download PDFInfo
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Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/33—Heterocyclic compounds
- A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
- A61K31/535—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with at least one nitrogen and one oxygen as the ring hetero atoms, e.g. 1,2-oxazines
- A61K31/5375—1,4-Oxazines, e.g. morpholine
- A61K31/5377—1,4-Oxazines, e.g. morpholine not condensed and containing further heterocyclic rings, e.g. timolol
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/33—Heterocyclic compounds
- A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
- A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
- A61K31/47—Quinolines; Isoquinolines
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
- A61P1/16—Drugs for disorders of the alimentary tract or the digestive system for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
Abstract
The invention relates to the field of biological medicine, in particular to a pharmaceutical composition for reversing the drug resistance of lenvatinib and application thereof. It is an object of the present invention to provide a pharmaceutical composition comprising domperidone and lenvatinib. Through the synergistic effect of the coupannix and the lenvatinib, the inhibition effect on the drug-resistant cells of the liver cancer, namely the lenvatinib, is obviously improved, meanwhile, the formation of colonies of the drug-resistant cells of the lenvatinib can be obviously inhibited, the apoptosis of the drug-resistant cells of the lenvatinib is promoted, and a better tumor inhibition effect is obtained in a liver cancer tumor-bearing mouse experiment.
Description
Technical Field
The invention relates to the field of biological medicine, in particular to a pharmaceutical composition for reversing the drug resistance of lenvatinib and application thereof.
Background
At present, liver cancer is the fourth most frequently seen in cancer incidence and the second most frequently seen in death rate in China, so that the health of the vast people in China is seriously threatened. Whereas, the incidence of liver cancer is the sixth leading of common malignant tumors at the world level. Although hepatitis B patients receive increasing attention on a global scale, early diagnosis of liver cancer is still not perfect due to different economic conditions and areas. Especially in rural and remote mountainous areas, where economy is behind, the patient does not begin to visit until clinical symptoms appear, at which time the cancer has mostly progressed to mid-to late stages. The present treatment of middle and late liver cancer is always a difficult problem faced by human beings. Although there are a number of treatments available, long-term survival of patients is difficult to achieve. The advent of each new drug has caused a tremendous adverse effect on the medical community, with many problems that must be addressed. Most critical is that resistance occurs with almost every long-term use of a chemotherapeutic agent.
Lenvatinib (trade name Le Weima) is the active ingredient of the lenvatinib mesylate, and the molecular formula is: c (C) 21 H 19 ClN 4 O 4 ·CH 4 O 3 S, molecular weight: 522.96 a plurality of tyrosine kinase receptors, including Fibroblast Growth Factor Receptor (FGFR) 1-4, vascular Endothelial Growth Factor Receptor (VEGFR) 1-4, platelet-derived growth factor receptor (PDGR) alpha, proto-oncogene RET and c-KIT, can be inhibited. And the lenvatinib can inhibit tumor angiogenesis by blocking VEGF receptor, and can inhibit tumor cell proliferation by blocking FGFR, PDFR alpha, RET and c-KIT, and finally the lenvatinib shows a strong anti-tumor effect. The main expression is that the lenvatinib can prolong the overall survival time and the progression-free survival time of patients with advanced liver cancer, and can convert a part of unresectable liver cancer into resectable liver cancer. Currently, lenvatinib has been FDA approved for targeted therapeutic first-line administration to patients with advanced liver cancer. However, in clinical work, some liver cancer patients were found to respond poorly to lenvatinib or tumor progression after treatment. The reasons for this can be broadly divided into two categories: one reason is that tumors are naturally resistant to lenvatinib; another reason is that tumors produce an adaptive modulation of lenvatinib, allowing tumor cells to survive high concentrations of lenvatinib. However, there is still a scientific problem to be solved regarding drug resistance mechanism of liver cancer and remedial therapeutic measures after drug resistance.
Disclosure of Invention
The present invention aims to solve at least one of the technical problems in the related art to some extent. Therefore, an object of the invention is to provide a pharmaceutical composition and application thereof in reversing drug resistance of liver cancer and lenvatinib, which can obviously improve inhibition effect on liver cancer and lenvatinib drug-resistant cells through synergistic effect of the coupannix and the lenvatinib, and can obviously inhibit formation of liver cancer drug-resistant cell colonies, promote apoptosis of liver cancer drug-resistant cells, and obtain better tumor inhibition effect in liver cancer tumor-bearing mice experiments.
The present invention provides in a first aspect a pharmaceutical composition. According to an embodiment of the invention, the pharmaceutical composition contains domperidone and lenvatinib.
The drug composition provided by the invention has the advantages that the contained coupanone can reverse the drug resistance of the lenvatinib, obviously inhibit the proliferation and colony formation of the lenvatinib-resistant liver cancer cells, promote the apoptosis of the lenvatinib-resistant liver cancer cells and effectively reverse the drug resistance of the liver cancer. Aiming at the drug-resistant liver cancer cells of the lenvatinib, compared with the independent drug of the coupannixin and the lenvatinib, the combined drug of the coupannixin and the lenvatinib has higher anticancer activity.
According to an embodiment of the present invention, in the pharmaceutical composition, the molar ratio of the domperidone and the lenvatinib is (1 to 20): 10. therefore, the proliferation and colony formation of the drug-resistant cells of the lenvatinib can be further inhibited, and the apoptosis of the drug-resistant cells can be promoted.
In a second aspect the invention provides the use of domperidone for the preparation of a reverse drug resistance agent for lenvatinib.
In a third aspect the invention provides the use of domperidone for reversing resistance to lenvatinib.
The coupannixie can not only reverse the drug resistance of the lenvatinib, but also obviously reduce the concentration of the drug and improve the anti-tumor curative effect when being used together.
In a fourth aspect, the present invention provides the use of coumarone in inhibiting proliferation of a drug-resistant liver cancer cell of lenvatinib and cell colony formation.
In a fifth aspect, the invention provides the use of domperidone for promoting apoptosis of a drug resistant liver cancer cell of lenvatinib.
In a sixth aspect, the present invention provides use of the pharmaceutical composition of the first aspect in the preparation of a medicament for treating liver cancer.
The pharmaceutical composition provided by the invention is especially aimed at tissues or individuals with drug resistance to the lenvatinib, and the drug combination can obviously inhibit proliferation and colony formation of drug-resistant cells and promote apoptosis of the drug-resistant cells. Therefore, the pharmaceutical composition can be used for preparing medicines for treating liver cancer.
Additional aspects and advantages of the invention will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of the invention.
Drawings
The foregoing and/or additional aspects and advantages of the invention will become apparent and may be better understood from the following description of embodiments taken in conjunction with the accompanying drawings in which:
FIG. 1 shows Huh7LR cell screening process and morphological changes; graph A shows that the concentration change and the time axis of the lenvatinib in the process of obtaining the Huh7LR cells by inducing the Huh7LR cells by multi-round lenvatinib; panel B shows the morphological differences between Huh 7P cells and Huh7LR cells;
FIG. 2 shows the proliferation assay IC of Huh 7P cells and Huh7LR cells 50 Detecting a result;
FIG. 3 shows the comparison of the proliferation rates of Huh 7P cells and Huh7LR cells and the results of cell colony formation experiments, wherein, A graph shows the comparison of the proliferation rates of Huh 7P cells and Huh7LR cells, B graph shows the results of colony formation of Huh 7P and Huh7LR cells after treatment with lenvatinib, and C graph shows quantitative comparison of colony formation by Image J software, wherein P is less than 0.001;
FIG. 4 shows the effect of using flow cytometry to detect apoptosis of Huh 7P and Huh7LR cells by Renzitinib, wherein, A shows apoptosis detected by flow cytometry after 48 hours of treatment of Huh 7P and Huh7LR cells by Renzitinib, B shows statistical analysis results of apoptosis of different groups, P <0.001;
fig. 5 shows the effect of different concentrations of lenvatinib (denoted Len), elclidade (denoted Ela) and combinations thereof on Huh7LR cell proliferation, wherein panel a shows the effect of MTT detection of different concentrations of two drugs alone and in combination on drug-resistant cell proliferation activity, and panel B shows Plot and CI values plotted based on quantitative methods of Chou-Talalay drug combination effect, P <0.001;
fig. 6 shows the effect of lenvatinib (10 μm), elclidamide (2.5 μm) alone and in combination on Huh7LR cell colony formation, panel a shows different groups of cell colonies, panel B shows statistical analysis of cell colonies after different groups of treatments, ×p <0.001;
FIG. 7 shows the effect of lenvatinib (20. Mu.M), icridazole (10. Mu.M) alone and in combination on Huh7LR apoptosis, wherein Panel A shows the morphology of Huh7LR cells after 72 hours of treatment with different drugs; panel B shows the detection of apoptosis in different drug treatment groups by flow cytometry, with early apoptotic cells in the lower right quadrant and late apoptotic cells in the upper right quadrant; panel C shows the results of statistical analysis of apoptosis rate, P <0.001;
fig. 8 shows a schematic of drug action targets targeting EGFR/PI3K pathway, wherein Gefitinib (Gefitinib) targets EGFR and Copantinib (Copantinib) targets PI3K.
FIG. 9 shows the effect of various concentrations of lenvatinib, gefitinib, coumaperib alone and as well as of the combination of lenvatinib + gefitinib, of the combination of lenvatinib + coumaperib on the proliferation of Huh7LR cells, wherein panel A shows the effect of MTT detection on the proliferation activity of drug-resistant cells Huh7LR cells in different drug combinations and concentration gradients, five columns in the bar chart being sequentially from left to right, of the group consisting of lenvatinib, gefitinib, lenvatinib + gefitinib, coumaperib, and lenvatinib + coumaperib; panel B shows Plot and CI values plotted based on a quantitative method of Chou-Talalay drug combination, P <0.001;
FIG. 10 shows the effect of lenvatinib, gefitinib, coupaninib alone and combination of lenvatinib + gefitinib, combination of lenvatinib + coupannib on apoptosis of Huh7LR, wherein Panel A shows the morphology of Huh7LR cells after 72 hours of treatment with different drugs; panel B shows the detection of apoptosis ratios of different drug treatment groups by flow cytometry, with early apoptotic cells in the lower right quadrant of the figure and late apoptotic cells in the upper right quadrant of the figure; panel C shows the results of statistical analysis of apoptosis rate, P <0.001;
fig. 11 shows the evaluation of the therapeutic effect of the solvent group, gefitinib single drug group, escitalopram single drug group, lenvatinib single drug group, lenvatinib+gefitinib group, and lenvatinib+escitalopram group on the treatment effect of lenvatinib resistant BALB/C mice, wherein graph a shows the tumor volume change of different drug groups, graph B shows the change of the body weight of the mice with time, P <0.001, and Veh: solvent group (5%o sodium carboxymethylcellulose); gef: gefitinib group (80 mg/kg/d); ela: iclidade group (80 mg/kg/d); len: lenvatinib group (5 mg/kg/d); len+ Gef: the group of the lamvastatin and the gefitinib (5 mg/kg/d of the lamvastatin and 80mg/kg/d of the gefitinib); len+ela: lenvatinib + irinotecan group (5 mg/kg/d of lenvatinib, 80mg/kg/d of irinotecan);
fig. 12 shows photographs showing the therapeutic effect of the solvent group, gefitinib single drug group, eketanide single drug group, lenvatinib single drug group, lenvatinib+gefitinib group, and lenvatinib+eketanide group on the lenvatinib-resistant BALB/C mice and tumor weight detection, a graph a shows the size and morphology of tumors of different drug groups, and B graph B shows tumor weight, P <0.001.
Detailed Description
The invention will now be described with reference to specific examples, which are intended to be illustrative only and not limiting in any way.
The reagents used in the experiments of the examples, unless otherwise specified, are all commercially available.
Furthermore, the terms "first," "second," and the like, are used for descriptive purposes only and are not to be construed as indicating or implying a relative importance or implicitly indicating the number of technical features indicated. Thus, a feature defining "a first" or "a second" may explicitly or implicitly include at least one such feature. In the description of the present disclosure, the meaning of "a plurality" is at least two, such as two, three, etc., unless explicitly specified otherwise.
According to a specific embodiment of the present invention, the present invention provides a pharmaceutical composition comprising copanide and lenvatinib. Compared with the single use of the medicines, the combined use of the medicines can obviously inhibit the proliferation of liver cancer tumor cells and reverse the drug resistance of the tumors.
Iclidada (Elacridar): is an inhibitor of the dual pumps of MDR1 and BCRP. In vitro, preclinical and clinical studies have shown that combined use of exendin can reverse MDR1 and/or BCRP mediated drug resistance of chemotherapeutic drugs, while exendin can increase the blood concentration of antitumor drugs by inhibiting drug efflux pumps.
Gefitinib (Gefitinib): selectively inhibiting EGFR receptor, the medicine is firstly applied to other non-small cell lung cancer patients who fail to be treated by the medicine. Thereafter, use as a single agent or in combination with other agents is also useful in the treatment of other tumors. Regarding the application of gefitinib in liver cancer, gefitinib can inhibit the growth of liver cancer cells, promote the apoptosis of the liver cancer cells and cause cell cycle arrest, and meanwhile, gefitinib can play a role in inhibiting the formation of liver cancer nodules on a rat model.
Copanlisib (Copanlisib): is an inhibitor of PI3K and was first used for recurrent follicular lymphoma, after which the drug was also used for the treatment of other advanced and recurrent solid tumors. In vitro studies on their role in liver cancer have shown that the combined use of domperidone and sorafenib can promote apoptosis of liver cancer cells.
Gefitinib and domperidone are FDA approved drugs.
According to a specific embodiment of the present invention, the present invention provides a pharmaceutical composition comprising domperidone and lenvatinib.
According to a specific embodiment of the present invention, the pharmaceutical composition may contain, in addition to the coupannix and lenvatinib as active ingredients, other excipients which can be added to the drugs known in the art.
According to a specific embodiment of the present invention, the pharmaceutical composition of the present invention comprises a combination of copan and lenvatinib to form a combination drug, and also comprises the independent formation of copan and lenvatinib to form two drugs, only at the time of administration.
According to a specific embodiment of the present invention, the pharmaceutical composition comprises domperidone and lenvatinib, wherein the molar ratio of the domperidone to the lenvatinib is (1-20): 10.
according to a specific embodiment of the invention, in the pharmaceutical composition, for example, the concentration of coupannixin is 3 μm, the concentration of lenvatinib is 30 μm, or the concentration of coupannixin is 12.5 μm, the concentration of lenvatinib is 10 μm, or the concentration of coupannixin is 25 μm, and the concentration of lenvatinib is 20 μm.
In the present invention, "Huh 7LR cells" means Huh7 Lenvatinib resistance (Huh 7 LR), namely, lenvatinib-resistant cells.
The aspects of the present disclosure will be explained below with reference to examples. Those skilled in the art will appreciate that the following examples are illustrative of the present disclosure and should not be construed as limiting the scope of the present disclosure. The examples are not to be construed as limiting the specific techniques or conditions described in the literature in this field or as per the specifications of the product. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention.
EXAMPLE 1 preparation of Lun-lvatinib resistant liver cancer cell line Huh7LR
1. Huh7LR liver cancer cell line for inducing and obtaining lenvatinib drug resistance
Over a 10 month induction period with increasing drug doses of lenvatinib, huh7 lenvatinib resistant (Huh 7 lenvatinib resistance, huh7 LR) cells can tolerate 20. Mu.M concentrations of lenvatinib, after which Huh7LR cells are maintained in 20. Mu.M of lenvatinib-containing medium. The Huh7LR cell screening procedure and drug dosing procedure are shown in figure 1, panel a. Compared with the original cell (Huh 7P), the morphology of the Huh7LR cell is changed, the appearance is reduced, the cell grows in an aggregation way, and the cell morphology is shown as a B diagram in FIG. 1.
2. Detection of drug resistance in Huh7LR cells
(1) Drug resistance of Huh7LR cells was tested by cell proliferation assay (MTT), 96-hour MTT assay showed that Huh 7P cells had been tested for lenvatinib IC 50 The value was 5.34.+ -. 1.07. Mu.M, whereas Huh7LR cells had a value of lenvatinib IC 50 The value is greater than 20. Mu.M. Two IC 50 The difference was significant (P < 0.05) (FIG. 2). Semi-inhibitory concentration of drug (Half Maximal Inhibitory Concentration, IC) 50 ) Refers to a drug concentration that reduces the activity of the cells to 50%.
The cell proliferation assay procedure was as follows:
taking cells in logarithmic growth phase, digesting by trypsin, centrifuging after stopping digestion, and collecting the cells to prepare single cell suspension. After counting by using a cell counting plate, the cell density was adjusted to 4X 10 4 And each ml. Mixing the above cell suspension, adding 100 μl/well of 96-well plate, i.e. 4000 cells/well, placing in cell incubator (37deg.C, 5% CO) 2 ) Is cultured overnight. In the process, cells are not added to the edge holes of the 96-well plate, PBS is added, and the influence of liquid evaporation on cell proliferation is reduced. The next day, the culture medium is discarded, and after medicines with different concentration gradients are respectively added, the culture is continued for 96 hours. After 96 hours, the medium was discarded, and MTT working solution (5 mg/ml final working concentration) was added thereto, followed by further incubation for 4 hours. After 4 hours, 100. Mu.l DMSO was added to each well, and after incubation at 37℃for 10 minutes, the bottom crystalline material was sufficiently dissolved, and then the mixture was detected on a multifunctional microplate detector at a wavelength of 490nm to measure OD. In the whole process, a blank control group (without adding cells) is subjected to the same operation, and more than or equal to 3 gradient repeat holes are formed. Cell activity (%) = (experimental group OD value-blank group OD value)/(non-dosing group OD value-blank group OD value) was calculated.
(2) Evaluation of colony Forming ability of Huh7LR cells
The real-time proliferation rate of drug-resistant cells was measured using a real-time cell activity counter (RTCA), and the results showed that the proliferation rate of Huh7LR cells in the drug-containing medium of lenvatinib (20 μm) was faster than that of Huh 7P cells (panel a in fig. 3). In addition, colony forming experiments were used to compare the colony forming ability of primordial cells with drug resistant cells. The results showed that the colony formation number of Huh7LR cells was significantly greater than that of Huh7 cells (B, C plot in FIG. 3) in the drug medium containing lenvatinib (20. Mu.M) under the same culture conditions with the same number of cells inoculated, and the difference was statistically significant (P < 0.001). Colony formation experimental results show that Huh7LR cells have significantly stronger colony formation ability than Huh 7P cells.
The colony formation assay was as follows:
taking cells in logarithmic growth phase, adopting trypsin for digestion, and re-suspending the cells into cell suspension after centrifugation. To compare the colony forming ability of drug-resistant cells with that of primary cells, 1000 cells were inoculated per well using a 6-well plate, and a drug medium containing lenvatinib (20. Mu.M concentration of lenvatinib) was added. When comparing the cell colony formation ability after different drug combinations, using 6-well plate, adding 2000 cells into each well, placing in cell incubator (37deg.C, 5% CO) 2 ) Culturing overnight; after the culture medium is discarded, the culture medium is replaced by a culture medium containing medicines with different concentrations, and the culture is continued for 72 hours; after 72 hours to 2 weeks, the medium was changed to a medium without drug at intervals of 3 days/time. At 2 weeks of the above two treatments, colonies were visible to the naked eye, after 2 washes with medium and PBS, fixation was performed for 20 minutes with methanol, staining was performed for 10 minutes with 0.1% crystal violet, and after photographing with a camera, colonies were counted with Image J software.
(3) Flow cytometry evaluation of anti-apoptotic capacity of Huh7LR cells
And detecting the apoptosis resistance of the cells by using flow cytometry. Huh7 cells and Huh7LR cells were treated with lenvatinib (20. Mu.M) respectively. The apoptosis rate of Huh7 cells after 48 hours was 11.74%, while that of Huh7LR cells was 3.27%; both were statistically analyzed and the differences were significant (P < 0.001) (panels A and B in FIG. 4).
Flow-through apoptosis assay was as follows:
taking cells in the logarithmic growth phase, discarding the culture medium, washing with PBS, then using trypsin to digest, stopping and preparing cell suspension, and counting; cells were seeded in 6-well plates at a cell density of 200,000 cells/well and cultured overnight. The next day, the medium was changed to one containing a different drug concentration and incubation was continued for 72 hours. The following steps are as follows:
(1) after the culture is finished, the cells are washed by PBS, and collected cells are digested by pancreatin without EDTA in a flow sample tube for 5 minutes at 1500 g/min; washing once by using PBS, and repeating centrifugation once continuously;
(2) the supernatant was discarded and 195. Mu.l of Annexin V-FITC conjugate was added to lightly resuspend the cells;
(3) add 5. Mu.l Annexin V FITC and mix gently;
(4) adding 10 μl of Propidium Iodide (PI) staining solution, and gently mixing;
(5) incubation was performed at room temperature for 15 minutes in the dark for flow detection, and at least 50,000 cells were analyzed per sample.
Example 2 synergistic inhibition of Huh7LR cell proliferation by Iclidas and lenvatinib
1. Cell proliferation assay
Taking Huh7LR cells in logarithmic growth phase, digesting by trypsin, centrifuging after stopping digestion, and collecting cells to prepare single cell suspension. After counting by using a cell counting plate, the cell density was adjusted to 4X 10 4 And each ml. Mixing the above cell suspension, adding 100 μl/well of 96-well plate, i.e. 4000 cells/well, placing in cell incubator (37deg.C, 5% CO) 2 ) Is cultured overnight. In the process, cells are not added to the edge holes of the 96-well plate, PBS is added, and the influence of liquid evaporation on cell proliferation is reduced. The next day, the culture medium is discarded, and after medicines with different concentration gradients are respectively added, the culture is continued for 96 hours. After 96 hours, the medium was discarded, and MTT working solution (5 mg/ml final working concentration) was added thereto, followed by further incubation for 4 hours. After 4 hours, 100. Mu.l DMSO was added to each well, and after incubation at 37℃for 10 minutes, the bottom crystalline material was sufficiently dissolved, and then the mixture was detected on a multifunctional microplate detector at a wavelength of 490nm to measure OD. In the whole processThe same procedure was performed for the blank group (no cell addition group), and the number of wells was not less than 3 per gradient repeat. Cell activity (%) = (experimental group OD value-blank group OD value)/(non-dosing group OD value-blank group OD value) was calculated.
2. Experimental grouping design and results
The MTT assay was used to examine the proliferation activity of the cells of the different treatment groups as shown in FIG. 5, and the results showed that: (1) the cell activity was hardly affected when lenvatinib (10 μm, indicated by Len) alone or elclidine (2.5 μm, indicated by Ela) alone, whereas the cell activity was reduced to 71.70% when lenvatinib (10 μm) and elclidine (2.5 μm) were used in combination; (2) the cell activity was 76.23% when lenvatinib (20 μm) alone and 92.49% when elvan (5 μm) alone was used, whereas the cell activity was reduced to 39.67% when lenvatinib (20 μm) and elvan (5 μm) were used in combination (fig. 5, panel a). Based on the cell proliferation rate under the two groups of concentration gradients, a quantitative method of the combined action of Chou-Talalay medicaments is further applied to calculate a Combined Index (CI): combination of 10 μm of lenvatinib with 2.5 μm of escitalopram, ci=0.57; lenvatinib 20 μm in combination with 5.0 μm of escitalopram, ci=0.720. Given that the above CI values were all less than 1, it was shown that lenvatinib and ekindac could synergistically inhibit Huh7LR cell proliferation (panel B in fig. 5).
Example 3 Effect of Icleidad in combination with Lunlukatinib on Huh7LR colony Forming Capacity
1. Colony formation experimental method
Taking Huh7LR cells in logarithmic growth phase, using trypsin to digest, and re-suspending the cells into cell suspension after centrifugation. To compare the colony forming ability of drug-resistant cells with that of primary cells, 1000 cells were inoculated per well using a 6-well plate, and a drug medium containing lenvatinib (20. Mu.M concentration of lenvatinib) was added. When comparing the cell colony formation ability after different drug combinations, using 6-well plate, adding 2000 cells into each well, placing in cell incubator (37deg.C, 5% CO) 2 ) Culturing overnight; after the culture medium is discarded, the culture medium is replaced by a culture medium containing medicines with different concentrations, and the culture is continued for 72 hours; changing into non-medicated culture medium within a period of 72 hr to 2 weeks at intervals of3 days/time. At 2 weeks of the above two treatments, colonies were visible to the naked eye, after 2 washes with medium and PBS, fixation was performed for 20 minutes with methanol, staining was performed for 10 minutes with 0.1% crystal violet, and after photographing with a camera, colonies were counted with Image J software.
2. Experimental grouping and results
The tumor stem-like cells have the potential of abnormal differentiation and self-renewal of stem cells and are related to the drug resistance of chemotherapeutics of various tumors. Colony formation experiments were performed to evaluate the effect of drugs on tumor cell stem proliferation, and were divided into 4 groups: control, lenvatinib (10. Mu.M), icaridada (2.5. Mu.M), and lenvatinib (10. Mu.M) +Icaridada (2.5. Mu.M). The results of the study are shown in fig. 6, which shows that single drug of lenvatinib can inhibit the formation of Huh7LR cell colonies, and that the combination of lenvatinib and elklida can further inhibit the formation of Huh7LR cell colonies (fig. 6, panel a); further statistical analysis was performed, the differences were significant (P < 0.001) (panel B in fig. 6).
Example 4 Effect of Icleidad in combination with lenvatinib on apoptosis of Huh7LR cells
1. Flow type apoptosis detection experimental design
Taking Huh7LR cells in the logarithmic growth phase, discarding the culture medium, washing with PBS, then using trypsin to digest, stopping and preparing cell suspension, and counting; cells were seeded in 6-well plates at a cell density of 200,000 cells/well and cultured overnight.
The next day, the medium was changed to one containing a different drug concentration and incubation was continued for 72 hours. The following steps are as follows:
(1) after the culture is finished, the cells are washed by PBS, and collected cells are digested by pancreatin without EDTA in a flow sample tube for 5 minutes at 1500 g/min; washing once by using PBS, and repeating centrifugation once continuously;
(2) the supernatant was discarded and 195. Mu.l of Annexin V-FITC conjugate was added to lightly resuspend the cells;
(3) add 5. Mu.l Annexin V FITC and mix gently;
(4) adding 10 μl of Propidium Iodide (PI) staining solution, and gently mixing;
(5) incubation was performed at room temperature for 15 minutes in the dark for flow detection, and at least 50,000 cells were analyzed per sample.
2. Experimental grouping and results
Huh7LR cells were treated with different drugs (duration of action 72 hours): control, lenvatinib (20 μm), escitalopram (10 μm), lenvatinib (20 μm) +escitalopram (10 μm) group (panel a in fig. 7). Then, the ratio of apoptosis (early apoptosis+late apoptosis) of the different treatment groups was examined by flow cytometry using an Annexin V-FITC/PI staining method (panel B in fig. 7), and further statistical analysis was performed (panel C in fig. 7). The results show that: compared with single administration, the combination of the Icleidad and the lenvatinib can obviously promote the apoptosis of Huh7LR cells, and the difference has statistical significance (P is less than 0.001).
Example 5EGFR/PI3K inhibitor and Lunlukatinib synergistically inhibit Huh7LR cell proliferation
EGFR signaling pathway and its downstream PI3K/AKT pathway activation play an important role in mediating malignant tumor resistance. Whereas the tyrosine kinase receptors that work on cell membrane surfaces by lenvatinib include VEGFR, PDGFR, FGFR, c-KIT and RET, but do not include EGFR. Based on the above, the inventors speculate that EGFR pathway activation may be involved in mediating resistance to lenvatinib. Here, follow-up studies were performed with FDA approved drugs gefitinib (targeting EGFR) and cooperant (targeting PI 3K), and fig. 8 shows a schematic of drug action targets targeting EGFR/PI3K pathways.
Cell proliferation activity was examined using MTT in the same manner as in example 2. As shown in a graph a in fig. 9, the results indicate that: (1) 10. Mu.M of lenvatinib (represented by Len) and 20. Mu.M of gefitinib (represented by Gef) were used alone, their cell activities were 98.01% and 76.30% respectively, 5. Mu.M of gefitinib (represented by Gef) and 10. Mu.M of gefitinib were 86.93% and 77.23% respectively, 12.5nM of coupannix (represented by Cop) and 25nM of coupannix were used alone, and their cell activities were 64.77% and 58.20% respectively; (2) the cell activities of lenvatinib (10. Mu.M) +gefitinib (5. Mu.M), lenvatinib (20. Mu.M) +gefitinib (10. Mu.M) were 60.04% and 48.20%, respectively, and of lenvatinib (10. Mu.M) +coumonixib (12.5 nM) and of lenvatinib (20. Mu.M) +gefitinib (25 nM) were 46.74% and 31.8%, respectively. The Combination Index (CI) was calculated using the quantitative method of Chou-Talay drug combination: combination of 10 μm of lenvatinib with 5.0 μm of gefitinib, ci=0.611; combination of 20 μm of lenvatinib with 10.0 μm of gefitinib, ci=0.967; combination of 10 μm of lenvatinib with 12.5nM of coupannix, ci=0.542; lenvatinib 20 μm in combination with 25.0nM of coupannix, ci=0.700. Given that the above CI values are less than 1, it is shown that lenvatinib and gefitinib (or copanib) act synergistically in inhibiting Huh7LR cell proliferation (panel B in fig. 9).
Example 6EGFR/PI3K inhibitor in combination with lenvatinib promotes apoptosis of Huh7LR cells
To further evaluate the effect of EGFR/PI3K pathway inhibitors in combination with lenvatinib on apoptosis, flow cytometry was further used to detect apoptosis. The same flow assay design as in example 4 was used to detect apoptosis.
First, huh7LR cells were treated with different drugs (duration of action 72 hours): control group, lenvatinib group (30 μm), gefitinib group (30 μm), domperidone group (3 μm), lenvatinib (30 μm) +gefitinib (30 μm), lenvatinib (30 μm) +domperidone (3 μm) (panel a in fig. 10). Then, the ratio of apoptosis (early apoptosis+late apoptosis) of the different treatment groups was examined by flow cytometry using an Annexin V-FITC/PI staining method (panel B in fig. 10), and further statistical analysis was performed (panel C in fig. 10). The results show that: compared with the single drug of the lenvatinib, the combination group of the 'lenvatinib and the gefitinib' or the 'lenvatinib and the coupanone' can obviously promote the apoptosis of Huh7LR cells (P < 0.001).
Example 7 inhibition of apoptosis
1. BALB/c nude mice tumor-bearing experiment
And constructing a lenvatinib drug resistance model by using BALB/c nude mice. The steps and the flow are as follows:
(1) mice were inoculated subcutaneously 1X 10 on the right dorsal side 7 Huh7 lenvatinib resistant cells (suspension cell fluid and Matrigel glue are mixed according to a ratio of 1:1, and total 200 μl);
(2) animals were randomly divided into 6 groups when tumor diameter was about 6 mm: the solvent group (5%o sodium carboxymethyl cellulose solution), gefitinib group (80 mg/kg/d), irinotecan group (80 mg/kg/d), lenvatinib group (5 mg/kg/d), lenvatinib (5 mg/kg/d) +gefitinib (80 mg/kg/d), and lenvatinib (5 mg/kg/d) +irinotecan (80 mg/kg/d) were administered by intragastric administration (5 times/week). Wherein the combination of the two components is administered 2 hours before the administration of the two components;
(3) in this procedure, tumor volume (mm) and mouse body weight were measured every 2-3 days 3 ) =0.5×tumor longest meridian×tumor shortest meridian 2 。
2. In vivo combined drug for inhibiting tumor growth
A tumor-bearing model is established on a BALB/C nude mouse by using Huh7 lenvatinib drug-resistant cells so as to evaluate the in-vivo anti-tumor effect of the combined drug. After 1 week of subcutaneous inoculation of drug-resistant cells, the tumor diameters were approximately 6mm, and the animals were randomly divided into the above 6 groups, a solvent group (denoted by Veh in fig. 11), a gefitinib single drug group, an irinotecan single drug group, a lenvartinib single drug group, a lenvartinib+gefitinib group, and a lenvartinib+irinotecan group, and administration was started according to the method recorded above. Tumor volume and mouse weight were monitored, animals were euthanized 2 weeks after dosing, tumors were obtained, photographed and weighed. As shown in a diagram a in fig. 11: gefitinib or ibrida alone does not inhibit tumor growth compared to the solvent group, but the lenvatinib alone combination group can significantly inhibit tumor growth. The inhibition of tumor growth was more pronounced in the combination group of lenvatinib (lenvatinib + gefitinib, lenvatinib + escitalopram) compared to the single group of lenvatinib, with the most pronounced inhibition of tumor growth in the group of lenvatinib + escitalopram (fig. 11, panel a). Meanwhile, the body weight of the mice in different groups does not fluctuate greatly during the administration process, which indicates that the combination administration does not produce obvious side effects (B diagram in FIG. 11).
The tumor appearance was observed and tumor weights were measured for the different drug-treated groups as shown in panels a and B of fig. 12. The discovery is as follows: (1) the tumors of the solvent group, gefitinib single drug group, and the ekindac single drug group were red blood-rich tumors, while the tumors of the lenvatinib single drug group, lenvatinib+gefitinib group, and lenvatinib+ekindac group were (light pink-white) blood-rich tumors (fig. 12, panel a); (2) further tumor weighing was performed with the tumor weight trend consistent with its tumor volume (i.e., the application of solvent set, gefitinib single drug set and ekeridade single drug set with the greatest tumor weight, the lenvatinib single drug set with the centered tumor weight, the lenvatinib+gefitinib set with smaller tumor weight, and the lenvatinib+ekeridade set with the smallest tumor weight) (fig. 12, panel B).
In the description of the present specification, the descriptions of the terms "one embodiment," "some embodiments," "examples," "particular examples," "some embodiments," or "some examples," etc., mean that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the present invention. In this specification, schematic representations of the above terms are not necessarily directed to the same embodiment or example. Furthermore, the particular features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples. Furthermore, the different embodiments or examples described in this specification and the features of the different embodiments or examples may be combined and combined by those skilled in the art without contradiction.
While embodiments of the present invention have been shown and described above, it will be understood that the above embodiments are illustrative and not to be construed as limiting the invention, and that variations, modifications, alternatives and variations may be made to the above embodiments by one of ordinary skill in the art within the scope of the invention.
Claims (7)
1. A pharmaceutical composition comprising domperidone and lenvatinib.
2. The pharmaceutical composition according to claim 1, wherein the molar ratio of domperidone to lenvatinib is (1-20): 10.
3. use of copanide in the preparation of a reverse drug resistance agent of lenvatinib.
4. Use of copanide for reversing resistance to lenvatinib.
5. Use of coupannix for inhibiting proliferation of a cut-resistant hepatoma cell and colony formation.
6. Use of coupannix in promoting apoptosis of a drug resistant liver cancer cell of lenvatinib.
7. Use of the pharmaceutical composition of claim 1 or 2 in the manufacture of a medicament for treating liver cancer.
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