Application of berbamine in preparing drug combination for treating drug-resistant non-small cell lung cancer

Abstract

The present invention relates to the field of biomedicine, and more specifically to the use of berbamine in the preparation of a drug combination for treating drug-resistant non-small cell lung cancer. Berbamine can reverse osimertinib resistance in non-small cell lung cancer, rendering osimertinib, which was previously ineffective or ineffective in treating drug-resistant non-small cell lung cancer, effective or highly effective. Berbamine reverses osimertinib resistance in non-small cell lung cancer in vivo or in vitro, with manifestations of reversal of resistance including significant inhibition of proliferation of drug-resistant non-small cell lung cancer cell lines, significant promotion of apoptosis of drug-resistant non-small cell lung cancer cell lines, and significant inhibition of proliferation of drug-resistant non-small cell lung cancer cells in vivo.

Description

Technical Field

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

[0002] Research has shown that tumor-driving genes in NSCLC patients have been identified one by one, including epidermal growth factor receptor (EGFR), stromal lymphoma kinase (ALK), and receptor tyrosine kinase ROS proto-oncogene 1 (ROS-1) mutants. By targeting signaling pathways mediated by key genes that regulate tumor growth and progression, and using corresponding antibodies and inhibitors, patients' survival rates and quality of life can be significantly improved. Among the many targeted therapy strategies, tyrosine kinase inhibitors targeting the EGFR signaling pathway (EGFR-TKIs) are the most widely used.

[0003] Osimertinib (OSI), a third-generation EGFR-TKI, effectively addresses the resistance to first- and second-generation EGFR-TKIs caused by the T790M mutation. However, clinical application has gradually revealed that osimertinib resistance is still inevitable. Currently, research on the mechanisms of osimertinib resistance is inadequate, and effective interventions are lacking. Therefore, further investigation of the mechanisms of osimertinib resistance and the development of novel drugs and treatments that can overcome this resistance are crucial for improving the prognosis of patients with non-small cell lung cancer. Summary of the Invention

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

[0005] In a first 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.

[0006] Berbamine can reverse osimertinib resistance in non-small cell lung cancer, rendering osimertinib, previously ineffective or ineffective, effective or highly effective. Berbamine reverses osimertinib resistance in non-small cell lung cancer both in vitro and in vivo. Significance of resistance reversal includes significant inhibition of proliferation of drug-resistant non-small cell lung cancer cell lines, significant promotion of apoptosis of drug-resistant non-small cell lung cancer cell lines, and significant inhibition of proliferation of drug-resistant non-small cell lung cancer cells in vivo.

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

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

[0009] Figure 1 The figure shows the changes in cell viability of HCC827 OR cell lines treated with OSI, BBM, and BBM combined with OSI, respectively. NC means cells were not treated with drugs. The OSI concentration was 2.5 μM, and the BBM concentrations were 0 μM, 5 μM, 10 μM, 15 μM, and 20 μM, respectively.

[0010] Figure 2 The figure shows the changes in cell viability of PC9 OR cell lines treated with OSI, BBM, and BBM combined with OSI, respectively. NC means cells were not treated with drugs, the OSI concentration was 1 μM, and the BBM concentrations were 0 μM, 5 μM, 10 μM, 15 μM, and 20 μM, respectively.

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

[0012] Figure 4 Figure 3 is a heat map of the drug combination reaction of BBM combined with OSI in PC9 OR cell line, where the black dotted area represents the area of ​​maximum synergy.

[0013] Figure 5 The cell proliferation of HCC827 OR cell lines treated with OSI, BBM, and BBM combined with OSI for 10-14 days is shown, where the BBM concentrations were 0 μM, 5 μM, 10 μM, 15 μM, and 20 μM, and the OSI concentrations were 0 μM or 2.5 μM.

[0014] Figure 6 The cell proliferation of PC9 OR cells treated with OSI, BBM, and BBM combined with OSI for 10-14 days is shown, where the BBM concentrations were 0 μM, 5 μM, 10 μM, 15 μM, and 20 μM, and the OSI concentrations were 0 μM or 1 μM.

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

[0016] Figure 8 Shown are the statistical analysis results of cell proliferation of PC9 OR cell lines treated with BBM and BBM combined with OSI for 10-14 days.

[0017] Figure 9 The figure shows the cell proliferation of HCC827 OR cell lines detected by EdU kit after 48 hours of treatment with OSI, BBM, and BBM combined with OSI.

[0018] Figure 10 The cell proliferation of PC9 OR cell lines treated with OSI, BBM, and BBM combined with OSI for 48 hours was detected using an EdU kit.

[0019] Figure 11 The statistical analysis results of cell proliferation of HCC827 OR cell lines treated with OSI, BBM, and BBM combined with OSI for 48 hours are shown using the EdU kit.

[0020] Figure 12 The statistical analysis results of cell proliferation of PC9 OR cell lines treated with OSI, BBM, and BBM combined with OSI for 48 hours are shown using the EdU kit.

[0021] Figure 13 The graphs showing the changes in tumor volume of tumor-bearing mice treated with OSI, BBM, and BBM combined with OSI, respectively.

[0022] Figure 14 Shown are intuitive graphs of tumor volumes in mice bearing tumors treated with OSI, BBM, and BBM combined with OSI, respectively.

[0023] Figure 15 Statistical graphs of tumor weights of tumor-bearing mice treated with OSI, BBM, and BBM combined with OSI are shown. DETAILED DESCRIPTION

[0024] The following is a clear and complete description of the concept of the present invention and the technical effects produced, so as to fully explain the purpose, scheme and effects of the present invention.

[0025] Berbamine (BBM) is a bisbenzylisoquinoline alkaloid with the structural formula:

[0026] .

[0027] 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.

[0028] 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.

[0029] 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.

[0030] In some optional embodiments, the drug-resistant non-small cell lung cancer cell line is HCC827 OR or PC9 OR.

[0031] 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.

[0032] In some optional embodiments, the drug-resistant non-small cell lung cancer cell line is HCC827 OR or PC9 OR.

[0033] 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.

[0034] In some optional embodiments, the injection further includes at least one auxiliary material selected from water, physiological saline, and DMSO solution.

[0035] 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.

[0036] In some optional embodiments, the mass concentration of the DMSO solution is 0.05%.

[0037] According to an embodiment of the present invention, a non-small cell lung cancer resistant cell line was constructed using a concentration gradient method to verify the role of BBM in reversing the OSI resistance of non-small cell lung cancer. CCK8 experiments demonstrated that BBM and OSI have a synergistic effect in NSCLC resistant cells. Clonogenic experiments demonstrated that BBM combined with OSI significantly inhibited the proliferation of non-small cell lung cancer resistant cell lines. Using an EdU kit, it was demonstrated that BBM combined with OSI significantly inhibited the proliferation of NSCLC resistant cell lines. In vivo experiments in mice demonstrated that BBM combined with OSI can significantly inhibit the proliferation of non-small cell lung cancer resistant cells in vivo.

[0038] In the validation experiments, human EGFR-sensitive mutation NSCLC cell lines HCC827 (EGFR exon 19 mutation) and PC9 (EGFR exon 19 mutation) were purchased from Wuhan Procell Life Science Co., Ltd. (Procell). STR analysis was performed on all cells. Osimertinib (HY-15772, OSI) and berbamine (BBM) were purchased from Shanghai MedChenExpress (MCE).

[0039] Follow the steps below to verify the synergistic effect of BBM and OSI in drug-resistant NSCLC cells:

[0040] (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 (density of 2000 cells / well) with a volume of 100 μL per well and incubated in a 37°C, 5% CO2 incubator;

[0041] (2) After 12 hours, the cell status and adhesion were observed. For HCC827 OR, 2.5 μM OSI and / or BBM (0 μM, 5 μM, 10 μM, 15 μM, 20 μM) were added, with 5 replicates per concentration, and the culture was continued for 48 hours. For PC9 OR, 1 μM OSI and / or BBM (0 μM, 5 μM, 10 μM, 15 μM, 20 μM) were added, with 5 replicates per concentration, and the culture was continued for 48 hours.

[0042] (3) After the drug action time is over, the CCK8 reagent and complete culture medium are prepared into a working solution at a ratio of 1:10. The original culture medium is discarded, and the CCK8 working solution is added to the 96-well plate at 100 μL / well. The plate is placed in a 37°C, 5% CO2 incubator and incubated for 2 hours.

[0043] (4) Remove the well plate and measure the absorbance at 450 nm using a microplate reader to calculate the cell viability and inhibition index (inhibition index = 100 - cell viability);

[0044] (5) The synergistic effect of OSI and BBM was calculated using the response surface model and the zero interaction potency (ZIP) calculation method using the online SynergyFinder software (https: / / synergy-finder.fimmm.fi). The “inhibition index” was used to calculate the synergistic effect of OSI and BBM. A ZIP synergy score (ZIP Synergy score) > 0 indicates that the two drugs have a synergistic effect (red area), a ZIP synergy score = 0 indicates that there is an additive effect between the two drugs, a ZIP synergy score < 0 indicates that there is an antagonistic effect between the two drugs, and a ZIP synergy score > 10 indicates that the two drugs have a strong synergistic effect.

[0045] (6) Draw a heat map of the two-drug combination using the online SynergyFinder software (https: / / synergy-finder.fimmm.fi).

[0046] The experimental results showed that although OSI or BBM alone could inhibit the proliferation of HCC827 OR and PC9 OR cells, the combination of the two drugs had the best effect (e.g. Figure 1 and Figure 2 Moreover, the average ZIP synergy score of BBM combined with OSI in HCC827 OR was 11.109, and the average ZIP synergy score of BBM combined with OSI in PC9 OR cell line was 26.254, indicating that BBM combined with OSI has a strong synergistic effect in inhibiting the proliferation of NSCLC OSI-resistant cell lines (as shown in Figure 2). Figure 3 and Figure 4 shown).

[0047] The following steps were performed to verify the effects of BBM and OSI on the clonogenicity of OSI-resistant NSCLC cell lines:

[0048] (1) HCC827 OR and PC9 OR cells in good condition in the logarithmic growth phase were obtained, digested and counted according to the cell culture method, and then inoculated into 6-well plates (500 cells / well) and cultured in a 37°C, 5% CO2 incubator;

[0049] (2) After 12 hours, the cell status and adhesion were observed, and drug treatment was performed. The cells were cultured continuously, and three replicate wells were set for each concentration. Among them, the OSI concentration was 0 μM (replaced by 0.05% DMSO solution), 2.5 μM, or 1 μM, and the BBM concentration was 0 μM (replaced by 0.05% DMSO solution), 5 μM, 10 μM, 15 μM, and 20 μM.

[0050] (3) Observe the cell growth under a microscope every day, pay attention to the color of the culture medium and the cell growth status, ensure that the cells grow in the best condition, and terminate the culture when visible cell clones appear;

[0051] (4) Discard the old culture medium, wash the cells twice with pre-cooled PBS, add 1 mL of 4% paraformaldehyde to each well, and fix at room temperature for 30 minutes;

[0052] (5) Discard the fixative solution and add 1 mL of 0.1% crystal violet staining solution to each well. Stain at room temperature for 5 minutes.

[0053] (6) Remove the staining solution by aspiration, then slowly rinse the plate with ddH2O, dry it, and take a photo;

[0054] (7) Use ImageJ software to count clones and Graphpad software for statistical analysis.

[0055] like Figure 5-Figure 8 As shown, in HCC827 OR cells, both OSI and BBM alone reduced the number of cell clones compared to the untreated control (DMSO) (statistically significant), and the combination of BBM and OSI further reduced the number of cell clones. In PC9 OR cells, both OSI and BBM alone reduced the number of cell clones compared to the control (DMSO) (statistically significant), and the combination of BBM and OSI further reduced the number of cell clones. These results demonstrate that the combination of BBM and OSI significantly inhibits the clonogenicity of OSI-resistant NSCLC cell lines.

[0056] Follow the steps below to use the EdU kit to detect the effects of BBM and OSI on the proliferation of OSI-resistant NSCLC cell lines:

[0057] (1) Take cells in the logarithmic growth phase and in good condition, digest them according to the cell culture method, and inoculate them into 96-well plates, with 10,000 cells per well. Set up 5 replicate wells for each group and culture them in a 37°C, 5% CO2 incubator;

[0058] (2) After 12 hours, the cell status and adhesion were observed and drug treatment was performed. Control means that the cells were not treated with drugs (replaced with 0.05% DMSO solution), O means that 2.5 μM or 1 μM OSI was added to the cell culture medium, B means that 20 μM BBM was added to the cell culture medium, and O+B means that OSI and BBM were added to the cell culture medium at the same time and cultured for another 48 hours.

[0059] (3) After the incubation period, aspirate 50 μL of culture medium from the 96-well plate and add 50 μL of preheated 20 μM EdU reaction solution, and continue to incubate in the incubator for 2 hours;

[0060] (4) Aspirate all the culture medium, add 100 μL of fixative solution, and fix at room temperature for 15 minutes;

[0061] (5) Aspirate the fixative, add 100 μL of washing solution, and place the 96-well plate on a shaker and shake slowly for 10 minutes. Repeat this operation 3 times, 5 minutes each time.

[0062] (6) Aspirate the wash solution, add 100 μL of permeabilization solution, and incubate at room temperature for 15 minutes;

[0063] (7) Aspirate the permeabilization solution, add 100 μL of washing solution, and place the 96-well plate on a shaker and shake slowly for 10 minutes. Repeat this operation 3 times, 5 minutes each time.

[0064] (8) Aspirate the wash solution and add 50 μl of Click reaction solution (freshly prepared) to each well. Shake gently to cover evenly and incubate at room temperature in the dark for 30 minutes.

[0065] (9) Aspirate the Click reaction solution, add 100 μL of washing solution, and place the 96-well plate on a shaker and shake slowly for 10 minutes. Repeat this operation 3 times, 5 minutes each time.

[0066] (10) Aspirate the wash solution and add 50 μl of Hoechst 33342 dye to each well. Shake gently to cover evenly and incubate at room temperature for 10 minutes in the dark.

[0067] (11) Remove the Hoechst 33342 staining solution, add 100 μL of washing solution, and place the 96-well plate on a shaker and shake slowly for 10 minutes. Repeat this operation 3 times, 5 minutes each time;

[0068] (12) Observe the fluorescence intensity under a fluorescence microscope;

[0069] (13) Count fluorescent cells using ImageJ software;

[0070] (14) Graphpad software was used for data analysis and graphing.

[0071] like Figures 9-12 As shown in the figure, in HCC827 OR cells, compared with the DMSO group, OSI alone failed to inhibit cell proliferation, while BBM alone did, and the inhibitory effect of the combination of the two drugs was more significant. In PC9 OR cells, OSI alone failed to inhibit cell proliferation, while BBM alone did, and the inhibitory effect of the combination of the two drugs was more significant.

[0072] Follow the steps below to verify the effects of BBM and OSI on tumor formation in nude mice:

[0073] (1) Male BALB / C nude mice (4-6 weeks old, 16-22 g) were purchased from Jiangsu Jicui Yaokang Biotechnology Co., Ltd. (Animal Experiment License No.: SCXK (Su) 2023-0009) and maintained at a temperature of 20-25°C and a humidity of 50-60%.

[0074] (2) Prepare cell suspension (PC9 OR cells) and adjust the cell concentration to 5×10 7 cells / mL, and 5×10 6 cells / subcutaneous injection only;

[0075] (3) Observe and record the growth of nude mice and changes in tumors. 3 At the same time, the mice were randomly divided into 4 groups: control group, OSI group, BBM group and BBM combined with OSI group, with 6 mice in each group, for a total of 24 mice;

[0076] (4) Measure the length and width of the tumor and the weight of the mouse every 2 days, and calculate the tumor volume = (tumor length × tumor width × tumor width) / 2;

[0077] (5) After drug treatment, the mice were anesthetized, blood was collected from the eyeballs, centrifuged and stored at -80°C. The mice were then killed, and the tumor tissues were removed from the mice, weighed, and photographed.

[0078] Table 1 Details of drug administration in each group in the in vivo nude mouse tumor formation experiment

[0079]

[0080] like Figure 13-15 As shown in the results, in a xenograft subcutaneous tumor model, neither OSI nor BBM alone had a significant inhibitory effect on tumor growth, but BBM combined with OSI significantly inhibited tumor growth. The combination of BBM and OSI significantly inhibited tumor growth rate and reduced tumor weight and volume.

[0081] The above description is merely a preferred embodiment of the present invention. The present invention is not limited to the above-described embodiments. Any method that achieves the technical effects of the present invention by the same or equivalent means shall fall within the scope of protection of the present invention. Within the scope of protection of the present invention, various modifications and variations of the technical solutions and / or implementation methods are possible.

Claims

1. Use of berbamine combined with osimertinib 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, and the mass ratio of berbamine to osimertinib is 4:

1.

2. A drug combination for treating drug-resistant non-small cell lung cancer, characterized in that: The invention comprises an injection and an oral preparation, wherein the injection comprises berbamine and the oral preparation comprises osimertinib; wherein the drug-resistant non-small cell lung cancer is osimertinib-resistant non-small cell lung cancer; and the mass ratio of berbamine to osimertinib is 4:

1.

3. The drug combination for treating drug-resistant non-small cell lung cancer according to claim 2, characterized in that: The injection further comprises at least one auxiliary material selected from water, physiological saline, and DMSO solution.

4. The drug combination for treating drug-resistant non-small cell lung cancer according to claim 2, characterized in that: 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.

5. The drug combination for treating drug-resistant non-small cell lung cancer according to claim 3 or 4, characterized in that: The mass concentration of the DMSO solution is 0.05%.

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