Application of berbamine in preparation of pharmaceutical composition for treating drug-resistant non-small cell lung cancer

Abstract

The invention relates to the field of biological medicine, in particular to application of berbamine in preparation of a medicine composition for treating drug-resistant non-small cell lung cancer. Berbamine can reverse osimertinib drug resistance of the non-small cell lung cancer, so that osimertinib which is ineffective or inefficient in treatment of drug-resistant non-small cell lung cancer originally becomes effective or efficient. Berbamine reverses the osimertinib drug resistance of the non-small cell lung cancer in vivo or in vitro, and the drug resistance reversion performance comprises the steps of remarkably inhibiting proliferation of drug-resistant cell strains of the non-small cell lung cancer, remarkably promoting apoptosis of the drug-resistant cell strains of the non-small cell lung cancer and remarkably inhibiting proliferation of drug-resistant cells of the non-small cell lung cancer in vivo.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and particularly to the application of berbamine (BBM) in the preparation of a drug combination for treating drug-resistant non-small cell lung cancer (NSCLC). Background Art

[0002] Research shows that the tumor driver genes of NSCLC patients have been discovered one by one, including epidermal growth factor receptor (EGFR), anaplastic lymphoma kinase (ALK), and receptor tyrosine kinase ROS proto-oncogene 1 (ROS 1) mutants. By targeting and regulating the signal pathways mediated by key genes that drive tumor growth and development, and using corresponding antibodies and inhibitors, the survival rate and quality of life of patients can be significantly improved. Among many targeted therapy strategies, tyrosine kinase inhibitors (EGFR-TKIs) targeting the EGFR signal pathway are the most widely used.

[0003] As a third-generation EGFR-TKI, osimertinib (OSI) effectively solves the problem of drug resistance of the first-generation and second-generation EGFR-TKIs caused by the T790M mutation. However, it has gradually been found in clinical applications that the problem of osimertinib drug resistance is still inevitable. Currently, the research on the drug resistance mechanism of osimertinib is not sufficient, and there is a lack of effective intervention means. Therefore, further research on the drug resistance mechanism of osimertinib and the development of new drugs and treatment methods that can overcome its drug resistance are of great significance for improving the prognosis of non-small cell lung cancer patients. Summary of the Invention

[0004] Based on this, the present invention provides the application of berbamine in the preparation of a drug combination for treating drug-resistant non-small cell lung cancer, which solves at least one problem in the prior art.

[0005] In a first aspect, the present invention provides the application of berbamine in the preparation of a drug combination for treating drug-resistant non-small cell lung cancer, wherein the drug-resistant non-small cell lung cancer is osimertinib-resistant non-small cell lung cancer.

[0006] Berbamine can reverse the osimertinib drug resistance of non-small cell lung cancer, making osimertinib, which was originally ineffective or less effective in treating drug-resistant non-small cell lung cancer, effective or more effective. Berbamine reverses osimertinib drug resistance in vivo or in vitro, and the manifestations of reversing drug resistance include significantly inhibiting the proliferation of non-small cell lung cancer drug-resistant cell lines, significantly promoting the apoptosis of non-small cell lung cancer drug-resistant cell lines, and significantly inhibiting the proliferation of non-small cell lung cancer drug-resistant cells in vivo.

[0007] In a second aspect, the present invention provides a drug combination for treating drug-resistant non-small cell lung cancer, which comprises berbamine and osimertinib.

[0008] Due to the adoption of the above technical solutions, the embodiments of the present invention have at least the following beneficial effects: Berbamine has the characteristics of low side effects and high safety, can effectively reverse the osimertinib resistance of non-small cell lung cancer, can improve the efficacy of osimertinib against osimertinib-resistant non-small cell lung cancer, and provides a new drug option for the treatment of osimertinib-resistant non-small cell lung cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0009] Figure 1 Shows the changes in cell viability of HCC827 OR cell line treated with OSI, BBM, and BBM combined with OSI respectively. Among them, NC represents cells without drug treatment, the concentration of OSI is 2.5 μM, and the concentrations of BBM are 0 μM, 5 μM, 10 μM, 15 μM, and 20 μM respectively.

[0010] Figure 2 Shows the changes in cell viability of PC9 OR cell line treated with OSI, BBM, and BBM combined with OSI respectively. Among them, NC represents cells without drug treatment, the concentration of OSI is 1 μM, and the concentrations of BBM are 0 μM, 5 μM, 10 μM, 15 μM, and 20 μM respectively.

[0011] Figure 3 Is the heat map of drug combination reaction of BBM combined with OSI in HCC827 OR cell line, where the blackened dotted line area represents the area of maximum synergy.

[0012] Figure 4 Is the heat map of drug combination reaction of BBM combined with OSI in PC9 OR cell line, where the blackened dotted line area represents the area of maximum synergy.

[0013] Figure 5 Shows the cell proliferation of HCC827 OR cell line treated with OSI, BBM, and BBM combined with OSI for 10 - 14 days. Among them, the concentrations of BBM are 0 μM, 5 μM, 10 μM, 15 μM, and 20 μM respectively, and the concentrations of OSI are 0 μM or 2.5 μM respectively.

[0014] Figure 6 Shows the cell proliferation of PC9 OR cell line treated with OSI, BBM, and BBM combined with OSI for 10 - 14 days. Among them, the concentrations of BBM are 0 μM, 5 μM, 10 μM, 15 μM, and 20 μM respectively, and the concentrations of OSI are 0 μM or 1 μM respectively.

[0015] Figure 7 Shows the statistical analysis results of the cell proliferation of HCC827 OR cell line treated with BBM and BBM combined with OSI for 10 - 14 days.

[0016] Figure 8 Shows the statistical analysis results of cell proliferation of PC9 OR cell line treated with BBM, BBM combined with OSI for 10 - 14 days respectively.

[0017] Figure 9 Shows the cell proliferation of HCC827 OR cell line detected by EdU kit after being treated with OSI, BBM, BBM combined with OSI for 48 hours respectively.

[0018] Figure 10 Shows the cell proliferation of PC9 OR cell line detected by EdU kit after being treated with OSI, BBM, BBM combined with OSI for 48 hours respectively.

[0019] Figure 11 Shows the statistical analysis results of cell proliferation of HCC827 OR cell line detected by EdU kit after being treated with OSI, BBM, BBM combined with OSI for 48 hours respectively.

[0020] Figure 12 Shows the statistical analysis results of cell proliferation of PC9 OR cell line detected by EdU kit after being treated with OSI, BBM, BBM combined with OSI for 48 hours respectively.

[0021] Figure 13 Shows the tumor volume change graphs of tumor - bearing mice treated with OSI, BBM, BBM combined with OSI respectively.

[0022] Figure 14 Shows the intuitive graphs of tumor volume of tumor - bearing mice treated with OSI, BBM, BBM combined with OSI respectively.

[0023] [[ID=)14]]Figure 15 Shows the statistical graphs of tumor weight of tumor - bearing mice treated with OSI, BBM, BBM combined with OSI respectively. Detailed implementation manners

[0024] The following will clearly and completely describe the concept and technical effects of the present invention to fully elaborate the purpose, scheme and effects of the present invention.

[0025] Berbamine (BBM) is a bis - benzylisoquinoline alkaloid, and its structural formula is: .

[0026] Berbamine, a natural small molecule drug, has been used to treat a variety of diseases, including autoimmune diseases, leukemia, common cancers, and inflammation. However, research on its potential to treat drug-resistant non-small cell lung cancer (NSCLC) remains limited. While conducting research on drug-resistant NSCLC, the inventors of the present application serendipitously discovered that berbamine could reverse Osimertinib resistance in NSCLC. Further research led to the present invention based on these findings.

[0027] In one aspect, the present invention provides use of berbamine in preparing a drug combination for treating drug-resistant non-small cell lung cancer, wherein the drug-resistant non-small cell lung cancer is osimertinib-resistant non-small cell lung cancer.

[0028] Osimertinib-resistant non-small cell lung cancer is non-small cell lung cancer that has developed resistance to osimertinib, and berbamine can reverse this resistance. Preferably, the present invention provides the use of berbamine in combination with osimertinib in the preparation of a drug combination for treating osimertinib-resistant non-small cell lung cancer.

[0029] In some optional embodiments, the drug-resistant non-small cell lung cancer cell line is HCC827 OR or PC9 OR.

[0030] Another aspect of the present invention provides a drug combination for treating drug-resistant non-small cell lung cancer, comprising an injection and an oral formulation, wherein the injection comprises berbamine and the oral formulation comprises osimertinib. The drug-resistant non-small cell lung cancer is osimertinib-resistant non-small cell lung cancer.

[0031] In some optional embodiments, the drug-resistant non-small cell lung cancer cell line is HCC827 OR or PC9 OR.

[0032] In some optional embodiments, the mass ratio of berbamine to osimertinib is 1-4: 1. Preferably, the mass ratio of berbamine to osimertinib is 4:1.

[0033] In some optional embodiments, the injection further includes at least one auxiliary material selected from water, physiological saline, and DMSO solution.

[0034] In some optional embodiments, the oral dosage form further comprises at least one auxiliary material selected from the group consisting of water, physiological saline, DMSO solution, gelatin, microcrystalline cellulose, starch, dextrin, silicon dioxide, and magnesium stearate.

[0035] In some optional embodiments, the mass concentration of the DMSO solution is 0.05%.

[0036] According to the embodiments of the present invention, a non-small cell lung cancer drug-resistant cell line was constructed by the concentration gradient method to verify the role of BBM in reversing the resistance of non-small cell lung cancer to OSI. The CCK8 experiment demonstrated the synergistic effect of BBM and OSI in NSCLC drug-resistant cells. The colony formation experiment proved that BBM combined with OSI significantly inhibited the proliferation of non-small cell lung cancer drug-resistant cell lines. The EdU kit was used to prove that BBM combined with OSI significantly inhibited the proliferation of NSCLC drug-resistant cell lines. The in vivo experiment on mice demonstrated that BBM combined with OSI could significantly inhibit the proliferation of non-small cell lung cancer drug-resistant cells in vivo.

[0037] In the verification experiment, the human EGFR-sensitive mutant NSCLC cell lines HCC827 (EGFR exon 19 mutation) and PC9 (EGFR exon 19 mutation) were purchased from Wuhan Procell Life Science Co., Ltd. (Procell), and the STR identification of the cells had been completed. Osimertinib (HY-15772, Osimertinib, OSI) and BBM (Berbamine, BBM) were both purchased from Shanghai MedChemExpress (MCE).

[0038] The following steps were taken to verify the synergistic effect of BBM and OSI in NSCLC drug-resistant cells: (1) HCC827 OR and PC9 OR cells in the logarithmic growth phase were digested and counted according to the cell culture method, and then inoculated into 96-well plates (at a density of 2000 cells / well), with a volume of 100 μL per well, and placed in an incubator at 37°C and 5% CO2 for incubation; (2) After 12 hours, the cell status and adhesion were observed. For HCC827 OR, 2.5 μM of OSI and / or BBM (0 μM, 5 μM, 10 μM, 15 μM, 20 μM) were added respectively, with 5 replicates for each concentration, and the cells were cultured for another 48 hours; for PC9 OR, 1 μM of OSI and / or BBM (0 μM, 5 μM, 10 μM, 15 μM, 20 μM) were added respectively, with 5 replicates for each concentration, and the cells were cultured for another 48 hours; (3) After the drug action time ended, a working solution of CCK8 reagent and complete medium was prepared at a ratio of 1:10. The original medium was discarded, and the CCK8 working solution was added to the 96-well plates at 100 μL / well, and then placed in an incubator at 37°C and 5% CO2 for continued incubation for 2 hours; (4) The plates were taken out, and the absorbance at 450 nm was measured with an enzyme-linked immunosorbent assay (ELISA) reader, and the cell viability and inhibition index were calculated (inhibition index = 100 - cell viability); (5)Using the online SynergyFinder software (https: / / synergy-finder.fimmm.fi), the response surface model and the zero interaction potency (ZIP) calculation method were adopted to calculate the synergy of OSI and BBM with the "inhibition index"; ZIP Synergy scores > 0 indicate that the two drugs have a synergistic effect (red area), ZIP Synergy score = 0 indicates an additive effect between the two drugs, ZIP Synergy score < 0 indicates an antagonistic effect between the two drugs, and ZIP Synergy score > 10 represents a strong synergistic effect between the two drugs; (6)The heat map of the combination of the two drugs was drawn by the online SynergyFinder software (https: / / synergy-finder.fimmm.fi).

[0039] The experimental results showed that although the single drug OSI or BBM could inhibit the proliferation of HCC827 OR and PC9 OR cells, the combination of the two drugs had the best effect (as Figure 1 and Figure 2 shown). Moreover, the average ZIP synergy score of BBM combined with OSI on HCC827 OR was 11.109, and the average ZIP synergy score of BBM combined with OSI on the PC9 OR cell line was 26.254, indicating that BBM combined with OSI had a strong synergistic effect in inhibiting the proliferation of NSCLC OSI-resistant cell lines (as Figure 3 and Figure 4 shown).

[0040] Verify the effects of BBM and OSI on the colony formation of NSCLC OSI-resistant cell lines according to the following steps: (1)Take HCC827 OR and PC9 OR cells in the logarithmic growth phase with good status. After digestion and counting according to the cell culture method, inoculate them into 6-well plates (500 cells / well) and place them in an incubator at 37°C and 5% CO2 for culture; (2)After 12 hours, observe the cell status and adhesion, and perform drug addition treatment, and continue to culture. Set 3 replicates for each concentration; among them, the OSI concentration is 0 μM (replaced with 0.05% DMSO solution), 2.5 μM or 1 μM, and the BBM concentrations are 0 μM (replaced with 0.05% DMSO solution), 5 μM, 10 μM, 15 μM, 20 μM; (3)Observe the growth of cells under the microscope every day, pay attention to observing the color of the culture medium and the cell growth status to ensure that the cells grow in the best state. When visible cell clone clusters appear, terminate the culture; (4) Discard the old culture medium, wash the cells 2 times with pre-cooled PBS, then add 1 mL of 4% paraformaldehyde to each well and fix at room temperature for 30 min; (5) Discard the fixing solution, add 1 mL of 0.1% crystal violet staining solution to each well and stain at room temperature for 5 min; (6) Aspirate the staining solution, then gently rinse the well plate with ddH2O, and take a photo after drying; (7) Use ImageJ software for colony counting and Graphpad software for statistical analysis.

[0041] As Figures 5 - 8 shown, in HCC827 OR cells, compared with the control group (DMSO) without drug treatment, both OSI alone and BBM alone could reduce the number of cell colonies (with statistical significance); after the combination of BBM and OSI, the number of cell colonies was further reduced. In PC9 OR cells, compared with the control group (DMSO), both OSI alone and BBM alone could reduce the number of cell colonies (with statistical significance), and the number of cell colonies was further reduced after the combination of BBM and OSI. The results showed that the combination of BBM and OSI could significantly inhibit the colony formation ability of OSI-resistant cell lines of NSCLC.

[0042] Detect the effect of BBM and OSI on the cell proliferation of OSI-resistant cell lines of NSCLC using the EdU kit according to the following steps: (1) Take cells in the logarithmic growth phase with good status, digest them according to the cell culture method, then inoculate them into a 96-well plate, with 10,000 cells in each well, set 5 replicates for each group, and place them in a 37 °C, 5% CO2 incubator for culture; (2) Observe the cell status and adhesion after 12 hours, and perform drug addition treatment. Control means no drug treatment of cells (replaced with 0.05% DMSO solution), O means adding 2.5 μM or 1 μM of OSI to the cell culture medium, B means adding 20 μM of BBM to the cell culture medium, O + B means adding OSI and BBM to the cell culture medium at the same time, and continue to culture for 48 hours; (3) After the culture is completed, aspirate 50 μL of the culture medium from the 96-well plate and add 50 μL of pre-warmed 20 μM EdU reaction solution, and continue to incubate in the incubator for 2 hours; (4) Aspirate and discard all the culture solution, add 100 μL of fixing solution, and fix at room temperature for 15 minutes; (5) Aspirate and discard the fixing solution, add 100 μL of washing solution, place the 96-well plate on a shaker and shake slowly for 10 minutes, repeat this operation 3 times, 5 minutes each time; (6) Aspirate and discard the washing solution, add 100 μL of permeabilization solution, and incubate at room temperature for 15 minutes; (7)Aspirate the permeabilization solution, add 100 μL of washing solution, place the 96-well plate on a shaker and shake slowly for 10 minutes. Repeat this operation 3 times, 5 minutes each time; (8)Aspirate the washing solution, add 50 μL of Click reaction solution (prepared freshly before use) to each well, gently shake to cover evenly, and incubate in the dark at room temperature for 30 minutes; (9)Aspirate the Click reaction solution, add 100 μL of washing solution, place the 96-well plate on a shaker and shake slowly for 10 minutes. Repeat this operation 3 times, 5 minutes each time; (10)Aspirate the washing solution, add 50 μL of Hoechst 33342 staining solution to each well, gently shake to cover evenly, and incubate in the dark at room temperature for 10 minutes; (11)Remove the Hoechst 33342 staining solution, add 100 μL of washing solution, place the 96-well plate on a shaker and shake slowly for 10 minutes. Repeat this operation 3 times, 5 minutes each time; (12)Observe the fluorescence intensity under a fluorescence microscope; (13)Count the fluorescent cells using ImageJ software; (14)Perform data analysis and graph plotting using Graphpad software.

[0043] As Figures 9 - 12 shown, in HCC827 OR, compared with the DMSO group, the single drug OSI could not inhibit cell proliferation, the single drug BBM could inhibit cell proliferation, and the combined treatment group of the two drugs had a more significant effect on inhibiting cell proliferation. In PC9 OR cells, the single drug OSI could not inhibit cell proliferation, the single drug BBM could inhibit cell proliferation, and the combined treatment group of the two drugs had a more significant effect on inhibiting cell proliferation.

[0044] Verify the effects of BBM and OSI on tumor formation in nude mice according to the following steps: (1)Male BALB / C nude mice (4 - 6 weeks old, 16 - 22 g) were purchased from Jiangsu Jicui Yakang Biotechnology Co., Ltd. (Animal experiment license number: SCXK (Su) 2023 - 0009) and were raised under the conditions of 20 - 25 °C temperature and 50 - 60% humidity; (2)Prepare a cell suspension (PC9 OR cells), adjust the cell concentration to 5×10 7 cells / mL according to cell counting, and perform subcutaneous injection on the back of nude mice at 5×10 6 cells / mouse; (3)Observe and record the growth of nude mice and the changes in tumors. Wait until the tumor volume reaches 100 mm 3At that time, the mice were randomly divided into 4 groups: the control group (Control group), the OSI group, the BBM group, and the BBM combined with OSI group, with 6 mice in each group, a total of 24 mice; (4)Measure the length and width of the tumor and the body weight of the mice every 2 days, and calculate the tumor volume = (tumor length × tumor width × tumor width) / 2; (5)After the drug treatment was completed, the mice were anesthetized, blood was taken from the eyeballs, centrifuged and stored at -80°C. Subsequently, the mice were sacrificed, and the tumor tissues were removed from the mice, weighed and photographed.

[0045] Table 1 Details of drug administration in each group of in vivo experiments on nude mouse tumorigenesis As Figures 13 - 15 It should be noted that there seems to be a small error in the original text where the ")" in "[[ID=)14]]Figure 15 " is likely a typo. It has been left as is in the translation to match the original as closely as possible. shown, in the xenograft subcutaneous tumorigenesis model, neither OSI monotherapy nor BBM monotherapy had a significant inhibitory effect on tumor growth, while the combination of BBM and OSI had a very significant inhibitory effect on tumor growth. The combination of BBM and OSI significantly inhibited the tumor growth rate and simultaneously significantly reduced the weight and volume of the tumor.

[0046] As described above, it is only a preferred embodiment of the present invention. The present invention is not limited to the above-mentioned embodiments. As long as it achieves the technical effects of the present invention by the same or equivalent means, it should fall within the protection scope of the present invention. Within the protection scope of the present invention, various different modifications and changes can be made to its technical solutions and / or implementation manners.

Claims

1. Use of berbamine in the preparation of a drug combination for treating drug-resistant non-small cell lung cancer, characterized in that, The drug-resistant non-small cell lung cancer is osimertinib-resistant non-small cell lung cancer.

2. The application according to claim 1, characterized in that, The application is the application of berbamine combined with osimertinib in the preparation of a drug combination for treating osimertinib-resistant non-small cell lung cancer.

3. The application according to claim 1, characterized in that, The cell line of the drug-resistant non-small cell lung cancer is HCC827OR or PC9OR.

4. A drug combination for treating drug-resistant non-small cell lung cancer, characterized in that, It includes an injection and an oral preparation. The injection includes berbamine, and the oral preparation includes osimertinib; wherein, the drug-resistant non-small cell lung cancer is osimertinib-resistant non-small cell lung cancer.

5. The pharmaceutical combination for treating drug-resistant non-small cell lung cancer according to claim 4, wherein The cell line of the drug-resistant non-small cell lung cancer is HCC827 OR or PC9 OR.

6. The pharmaceutical combination for treating drug-resistant non-small cell lung cancer according to claim 4, wherein, The mass ratio of berbamine to osimertinib is 1-4:

1.

7. The pharmaceutical combination for treating drug-resistant non-small cell lung cancer according to claim 6, wherein, The mass ratio of berbamine to osimertinib is 4:

1.

8. The pharmaceutical combination for treating drug-resistant non-small cell lung cancer according to claim 4, wherein The injection further includes at least one excipient selected from water, normal saline, and DMSO solution.

9. The pharmaceutical combination for treating drug-resistant non-small cell lung cancer according to claim 4, wherein, The oral preparation further includes at least one excipient selected from water, normal saline, DMSO solution, gelatin, microcrystalline cellulose, starch, dextrin, silicon dioxide, and magnesium stearate.

10. The pharmaceutical combination for treating drug-resistant non-small cell lung cancer according to claim 8 or 9, characterized in that, The mass concentration of the DMSO solution is 0.05%.

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