Application of pamidronate in preparation of medicine for preventing, relieving and / or treating non-small cell lung cancer

Abstract

The invention relates to application of pamidronate in preparation of a medicine for preventing, relieving and / or treating non-small cell lung cancer. According to the application, new application of the pamidate is found, that is, the pamidate can inhibit the growth of the non-small cell lung cancer resistant to the tyrosine kinase inhibitor EGFR-TKIs, which indicates that the pamidate can be applied to the preparation of the medicine for preventing, relieving and / or treating the non-small cell lung cancer. As an inhibitor of the deubiquitinating enzyme USP20, the compound can target the deubiquitinating enzyme USP20, reverses the drug resistance of non-small cell lung cancer to a tyrosine kinase inhibitor, and plays a synergistic anti-tumor role with the tyrosine kinase inhibitor.

Description

Technical Field

[0001] This application relates to the field of biomedical technology, and in particular to the use of pamidronate in the preparation of a medicament for the prevention, relief and / or treatment of non-small cell lung cancer. Background Technology

[0002] Lung cancer is the malignant tumor with the highest incidence and mortality rate in my country, and it places the heaviest burden on the medical system. Among them, lung adenocarcinoma has become the most common pathological subtype of lung cancer. In the Asian lung adenocarcinoma patient population, the mutation rate of epidermal growth factor receptor (EGFR) is 40%-55%. Currently, tyrosine kinase inhibitors (EGFR-TKIs), targeted drugs developed for EGFR mutations, have become the first-line treatment for advanced lung adenocarcinoma.

[0003] However, many patients inevitably develop resistance during EGFR-TKI treatment, eventually leading to tumor progression. Therefore, innovative resistance targets and drugs targeting these targets urgently need to be identified and developed. Summary of the Invention

[0004] This application provides the use of pamidronate in the preparation of a medicament for the prevention, relief and / or treatment of non-small cell lung cancer, which can inhibit the growth of non-small cell lung cancer resistant to tyrosine kinase inhibitors EGFR-TKIs, and can target the deubiquitinase USP20 to reverse the resistance of non-small cell lung cancer to tyrosine kinase inhibitors.

[0005] In one aspect, embodiments of this application provide the use of pamidronate in the preparation of a medicament for the prevention, relief, and / or treatment of non-small cell lung cancer.

[0006] In conjunction with the first aspect, in one embodiment, the pamidronate comprises pamidronate monosodium C3H 10 NO7P2·Na and / or disodium pamidronate C3H9NO7P2·Na2.

[0007] In conjunction with the first aspect, in one embodiment, the non-small cell lung cancer includes lung adenocarcinoma.

[0008] In conjunction with the first aspect, in one embodiment, the lung adenocarcinoma is a lung adenocarcinoma resistant to the tyrosine kinase inhibitor EGFR-TKIs.

[0009] Secondly, embodiments of this application provide a medicament for the prevention, relief, and / or treatment of non-small cell lung cancer, the medicament comprising an active substance, the active substance being pamidronate.

[0010] In conjunction with the second aspect, in one embodiment, the pamidronate comprises pamidronate monosodium C3H. 10 NO7P2·Na and / or disodium pamidronate C3H9NO7P2·Na2.

[0011] In conjunction with the second aspect, in one embodiment, the non-small cell lung cancer includes lung adenocarcinoma.

[0012] In conjunction with the second aspect, in one embodiment, the lung adenocarcinoma is a lung adenocarcinoma resistant to the tyrosine kinase inhibitor EGFR-TKIs.

[0013] Thirdly, embodiments of this application provide a pharmaceutical composition for the prevention, relief, and / or treatment of non-small cell lung cancer, comprising two or more drugs for the prevention, relief, and / or treatment of non-small cell lung cancer, wherein one drug is pamidronate as the active substance.

[0014] In conjunction with the third aspect, in one embodiment, another drug is a tyrosine kinase inhibitor EGFR-TKIs; And / or, the pamidronate comprises C3H 10 NO7P2·Na and / or pamidronate disodium C3H9NO7P2·Na2; And / or, the non-small cell lung cancer includes lung adenocarcinoma.

[0015] The beneficial effects of the technical solution provided in this application include: (1) This application discovers a novel use of pamidronate, namely that pamidronate can inhibit the growth of non-small cell lung cancer resistant to tyrosine kinase inhibitors EGFR-TKIs, indicating that it can be used in the preparation of drugs for the prevention, relief and / or treatment of non-small cell lung cancer.

[0016] (2) As an inhibitor of deubiquitinase USP20, it can target deubiquitinase USP20, reverse the drug resistance of non-small cell lung cancer to tyrosine kinase inhibitors, and exert a synergistic anti-tumor effect with tyrosine kinase inhibitors. Attached Figure Description

[0017] To more clearly illustrate the technical solutions in the embodiments of this application, the accompanying drawings used in the description of the embodiments will be briefly introduced below. Obviously, the accompanying drawings described below are only some embodiments of this application. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0018] Figure 1 Comparison of immunohistochemical staining results of USP20 in lung adenocarcinoma tissues before and after osimertinib resistance, as provided in Example 1 of this application; Figure 2 Comparison of USP20 immunohistochemical staining results of lung adenocarcinoma tissues before and after drug resistance in 17 osimertinib-resistant patients provided in Example 1 of this application (*P<0.05). Figure 3 For 30 patients with lung adenocarcinoma who received osimertinib adjuvant therapy after surgery, as provided in Example 1 of this application, the staining results of high and low USP20 expression and the difference in recurrence rate were compared (***P<0.001). Figure 4 This is a comparison of the differences in disease-free survival between high and low expression of USP20 provided in Example 1 of this application; Figure 5 The cell proliferation results of PC9 lung cancer cells overexpressing USP20 WT and USP20 CS provided in Example 2 of this application after treatment with osimertinib for 72 hours; Figure 6 The cell proliferation results of NCI-H1975 lung cancer cells overexpressing USP20 WT and USP20 CS provided in Example 2 of this application after treatment with osimertinib for 72 hours; Figure 7 This is a comparison of the final size of subcutaneous tumors in nude mice after different interventions, as provided in Example 2 of this application. Figure 8 The comparison results of the subcutaneous tumor growth rate in nude mice after different interventions provided in Example 2 of this application are as follows: (The subcutaneous tumor growth rate of the "osimertinib + USP20 wild-type" group was significantly greater than that of the "osimertinib + empty lentivirus" group, ***P<0.001; the subcutaneous tumor growth rate of the "osimertinib + USP20 enzyme functional domain inactivated" group was significantly smaller than that of the "osimertinib + USP20 wild-type" group, ***P<0.001, and there was no statistical difference with the "osimertinib + empty lentivirus" group). Figure 9The image shows a comparison of immunohistochemical staining results of USP20 and Ki67 subcutaneous tumors in nude mice after different interventions, along with immunohistochemical scores and Ki67+ cell count / per field of view results (Regarding the USP20 immunohistochemical staining score, the scores of the "osimertinib + USP20 wild-type" group (***P<0.001) and the "osimertinib + USP20 enzyme functional domain inactivated" group (**P<0.01) were significantly higher than those of the "osimertinib + USP20 wild-type" group). In the "osimertinib + USP20 wild-type" group, it was demonstrated that USP20 was successfully transfected and expressed in subcutaneous tumor cells of nude mice. Regarding the Ki67+ cell count per field of view, the number of Ki67+ cells in the "osimertinib + USP20 wild-type" group was significantly greater than that in the "osimertinib + empty lentivirus" group (***P<0.001), while there was no statistically significant difference in the number of Ki67+ cells between the "osimertinib + USP20 enzyme functional domain inactivated" group and the "osimertinib + empty lentivirus" group (ns P>0.05). This demonstrates that USP20 overexpression in lung cancer cells can induce osimertinib resistance. Figure 10 The cell proliferation results of USP20 knocked-down PC9OR lung cancer cells provided in Example 3 of this application after 72 hours of osimertinib treatment; Figure 11 The cell proliferation results of USP20 knocked-down NCI-H1975OR lung cancer cells provided in Example 3 of this application after 72 hours of osimertinib treatment; Figure 12 This is a comparison of the final size of subcutaneous tumors in nude mice after different interventions, as provided in Example 3 of this application. Figure 13 The comparison results of the subcutaneous tumor growth rate in nude mice after different interventions provided in Example 3 of this application (in NCI-H1975OR cells, the subcutaneous tumor growth rate of the "osimertinib + USP20 gene knockdown lentivirus" group was significantly smaller than that of the "osimertinib + empty lentivirus" group, ***P<0.001). Figure 14The image shows a comparison of immunohistochemical staining results of USP20 and Ki67 in nude mouse subcutaneous tumors after different interventions, along with immunohistochemical scores and Ki67+ cell counts per field of view, provided in Example 3 of this application. (Regarding the USP20 immunohistochemical staining score, the score of the "osimertinib + USP20 gene knockdown lentivirus" group was significantly lower than that of the "osimertinib + empty lentivirus" group (***P<0.001), and the score of the "solvent + USP20 gene knockdown lentivirus" group was also significantly lower than that of the "solvent + empty lentivirus" group, demonstrating that shUSP20 was successfully infected and expressed in nude mouse subcutaneous tumor cells; regarding the Ki67+ cell count per field of view, the number of Ki67+ cells in the "osimertinib + USP20 gene knockdown lentivirus" group was significantly smaller than that in the "osimertinib + empty lentivirus" group (***P<0.001), demonstrating that knocking down USP20 in osimertinib-resistant lung cancer cells can reverse osimertinib resistance). Figure 15 The computer-simulated molecular docking model of compound T33873 and USP20 provided in Example 4 of this application; Figure 16 The conformational stability level between the protein complex and T33873 during a simulation period of 100 nanoseconds, as provided in Example 4 of this application; Figure 17 The interaction analysis between T33873 and USP20 provided in Example 4 of this application shows that green and blue represent hydrogen bonds and water clusters, respectively. Figure 18 The binding model obtained from molecular dynamics simulations provided in Example 4 of this application; Figure 19 In the osimertinib-resistant PC9OR cell line provided in Example 5 of this application, after treatment with osimertinib or compound T33873 at increasing concentrations for 72 hours, the cell proliferation results were detected by CCK-8 assay. Figure 20 In the osimertinib-resistant H1975OR cell line provided in Example 5 of this application, after treatment with osimertinib or compound T33873 at increasing concentrations for 72 hours, the cell proliferation results were detected by CCK-8 assay. Figure 21 This is a comparison of the final size of subcutaneous tumors in each group of nude mice provided in Example 5 of this application; Figure 22 The comparison results of the subcutaneous tumor growth rate of each group of nude mice provided in Example 5 of this application (the subcutaneous tumor growth rate of the "T33873+ osimertinib" group was significantly lower than that of the "T33873" group (**P<0.01) and the "osimertinib" group (***P<0.001)). Figure 23The immunohistochemical staining results and Ki67+ cell count / per field of view of each group of nude mice provided in Example 5 of this application are compared. (Regarding the Ki67+ cell count / per field of view, the Ki67+ cell count in the "T33873+osimertinib" group was significantly smaller than that in the "T33873" group (***P<0.001) and the "osimertinib" group (***P<0.001), demonstrating that T33873 combined with osimertinib can synergistically inhibit the survival of osimertinib-resistant nude mouse subcutaneous tumors).

[0019] Figure 24 The H&E staining results of nude mouse heart, liver, spleen, lung and kidney tissues after different treatments with solvent, osimertinib, compound T33873 and osimertinib combined with compound T33873 provided in Example 6 of this application. Detailed Implementation

[0020] To make the objectives, technical solutions, and advantages of the embodiments of this application clearer, the technical solutions of the embodiments of this application will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of this application, not all embodiments. Based on the embodiments of this application, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this application.

[0021] Ubiquitination is an important post-translational modification pathway in cells, regulating key cellular processes such as protein homeostasis, proliferation signaling, and the cell cycle. Dysregulation of this pathway is closely related to tumorigenesis and development, and represents a potential target for anti-tumor therapy. USP20, as a deubiquitinase, regulates protein stability and activity by removing ubiquitination modifications. Currently, the role of USP20 in regulating resistance to tyrosine kinase inhibitors such as EGFR-TKIs (osimertinib) in non-small cell lung cancer (NSCLC) remains unclear.

[0022] Pamidronate monosodium C3H 10 NO7P2·Na and pamidronate disodium C3H9NO7P2·Na2 (CAS No. 109552-15-0) can both treat bone-related diseases.

[0023] CAS number 89131-02-2, molecular formula C3H 10The NO7P2·Na compound T33873 has been shown in earlier studies to inhibit bone loss, alleviating the progression of osteoarthritis and osteoporosis by suppressing subchondral bone loss. Its action is related to the inhibition of the Wnt / β-catenin signaling pathway, and it has therefore been used in research related to osteosarcoma. However, the mechanism by which this compound exerts its antitumor effect through the regulation of ubiquitination modification remains unclear. This application reveals for the first time that compound T33873 can target the deubiquitinase USP20, reversing resistance to osimertinib in lung adenocarcinoma, providing a new target and therapeutic strategy for overcoming targeted drug resistance in clinical practice.

[0024] The compound T33873 described in this application can effectively inhibit the USP20-CYS154 site, thereby inhibiting its deubiquitinating enzyme activity, and exerts a synergistic anti-tumor effect with osimertinib, reversing the resistance of lung adenocarcinoma to osimertinib. T33873 can be called pamidronate monosodium, and its structural formula is as follows:

[0025] Based on this, embodiments of this application provide the use of pamidronate in the preparation of a medicament for the prevention, relief and / or treatment of non-small cell lung cancer.

[0026] The pamidronate includes pamidronate monosodium C3H. 10 NO7P2·Na and / or disodium pamidronate C3H9NO7P2·Na2.

[0027] In some embodiments, the non-small cell lung cancer includes lung adenocarcinoma.

[0028] In some embodiments, the lung adenocarcinoma is lung adenocarcinoma resistant to the tyrosine kinase inhibitor EGFR-TKIs.

[0029] In some embodiments, the tyrosine kinase inhibitor EGFR-TKIs include one or more of gefitinib, erlotinib, afatinib, and osimertinib. Preferably, the tyrosine kinase inhibitor EGFR-TKI is osimertinib.

[0030] This application provides a medicament for the prevention, alleviation, and / or treatment of non-small cell lung cancer, the medicament comprising an active substance, wherein the active substance is pamidronate. The medicament may also include pharmaceutically acceptable excipients or auxiliary ingredients.

[0031] In some embodiments, the pamidronate includes pamidronate monosodium C3H. 10 NO7P2·Na and / or disodium pamidronate C3H9NO7P2·Na2.

[0032] In some embodiments, the non-small cell lung cancer includes lung adenocarcinoma.

[0033] In some embodiments, the lung adenocarcinoma is lung adenocarcinoma resistant to the tyrosine kinase inhibitor EGFR-TKIs.

[0034] In some embodiments, the tyrosine kinase inhibitor EGFR-TKIs include one or more of gefitinib, erlotinib, afatinib, and osimertinib. Preferably, the tyrosine kinase inhibitor EGFR-TKI is osimertinib.

[0035] This application provides a pharmaceutical composition for the prevention, alleviation, and / or treatment of non-small cell lung cancer, comprising two or more drugs for the prevention, alleviation, and / or treatment of non-small cell lung cancer, wherein one drug is pamidronate as the active substance. The drug may also include pharmaceutically acceptable excipients or auxiliary ingredients.

[0036] In some embodiments, another drug is a tyrosine kinase inhibitor, EGFR-TKIs.

[0037] In some embodiments, the tyrosine kinase inhibitor EGFR-TKIs include one or more of gefitinib, erlotinib, afatinib, and osimertinib. Preferably, the tyrosine kinase inhibitor EGFR-TKI is osimertinib.

[0038] In some embodiments, the pamidronate includes pamidronate monosodium C3H. 10 NO7P2·Na and / or disodium pamidronate C3H9NO7P2·Na2.

[0039] In some embodiments, the non-small cell lung cancer includes lung adenocarcinoma.

[0040] This application proposes compound T33873 as a novel deubiquitinase inhibitor, elucidating the specific mechanism by which compound T33873 reverses osimertinib resistance in lung adenocarcinoma at multiple levels, including molecular, cellular, and animal models. It can effectively reverse osimertinib resistance in lung adenocarcinoma. Mechanistically, compound T33873 inhibits the deubiquitinase activity by specifically binding to the CYS154 site of USP20. The combination of compound T33873 and osimertinib exhibits a synergistic antitumor effect, effectively inhibiting the growth of osimertinib-resistant lung adenocarcinoma in both cell and animal models, without significant toxicity to major organs in mice. This application provides a novel approach for the clinical treatment of EGFR-TKI-resistant lung adenocarcinoma.

[0041] To enable those skilled in the art to better understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below with reference to specific embodiments.

[0042] Example 1: USP20 is highly expressed in osimertinib-resistant lung adenocarcinoma and is associated with worse patient survival. This study investigated lung adenocarcinoma tissue samples from 17 osimertinib-resistant patients before and after resistance, and surgical tissue samples from 30 lung adenocarcinoma patients. All samples were obtained from patients treated at Union Hospital, Tongji Medical College, Huazhong University of Science and Technology. This study was conducted entirely in accordance with the ethical guidelines established by the Declaration of Helsinki, and the research protocol has been formally approved by the Institutional Review Committee of Union Hospital, Tongji Medical College, Huazhong University of Science and Technology.

[0043] Lung adenocarcinoma tissue samples underwent tissue fixation, dehydration and embedding, sectioning, baking, dewaxing, hydration, endogenous peroxidase blockade, antigen retrieval, blocking, incubation with USP20 primary antibody, incubation with secondary antibody, staining and counterstaining, and mounting. The USP20 staining results were then observed under a microscope. Representative results are shown in […]. Figure 1 and Figure 3 .

[0044] The t-test analysis of USP20 protein levels in 17 lung adenocarcinoma tissue samples before and after drug resistance was performed using GraphPad Prism 9 software. The results are shown in [Figure 1]. Figure 2 Significant differences were found in the USP20 protein levels in lung adenocarcinoma tissue samples before and after drug resistance.

[0045] Thirty lung adenocarcinoma surgical tissue samples were divided into two groups: one with high USP20 expression and the other with low USP20 expression. The recurrence rate after osimertinib treatment in both groups was analyzed using a chi-square test with GraphPadPrism 9 software. The results are shown below. Figure 3 Patients with high USP20 expression had a higher relapse rate. Log-rank test analysis of disease-free survival in both groups was performed using GraphPad Prism 9 software; results are shown below. Figure 4 Individuals with high USP20 expression have a worse disease-free survival rate.

[0046] Figures 1-4 This indicates that high expression of USP20 in lung adenocarcinoma patients' tissues is associated with osimertinib resistance and shortened survival.

[0047] Example 2: Overexpression of USP20 significantly enhances the resistance of lung adenocarcinoma cells to osimertinib. (1) Overexpression of USP20 can enhance the resistance of PC9 and NCI-H1975 cells to osimertinib.

[0048] Log-phase PC9 and NCI-H1975 cells (purchased from American Type Culture Collection; PC9 and NCI-H1975 are both lung adenocarcinoma cell lines) were prepared into 5 × 10⁶ cells using complete medium (Gibco RPMI-1640 or DMEM medium (Thermo Fisher Scientific, USA), supplemented with 10% fetal bovine serum (Pronosai, China), 100 μg / mL streptomycin, and penicillin). 4 Cell suspension of cells / ml was seeded into 6-well plates, 2 ml per well. After incubation at 37°C for 24 h, the medium was changed, and 1 ml of Opti-MEM medium (Thermo Fisher Scientific, USA) was added to each well.

[0049] For plasmid transfection experiments, vector (empty vector plasmid), USP20WT (USP20 wild-type plasmid), and USP20 CS (USP20 enzyme functional domain inactivation plasmid) were purchased from Wuhan Qinda Biotechnology Co., Ltd. X-tremeGENETM HP DNA transfection reagent (Roche, Switzerland; concentration: transfection reagent (μL): DNA (μg) = 3:1) was used for plasmid transfection. 1 μg of each of the three different plasmids was co-cultured with PC9 and NCI-H1975 lung cancer cells at 37℃ for 24 h, and then replaced with complete culture medium.

[0050] PC9 and NCI-H1975 cells transfected with vector, USP20 WT, and USP20 CS in logarithmic growth phase were seeded in 96-well plates at a density of 5000 cells per well. After 12 hours, osimertinib (100 nM) was added, and after 72 hours, a CCK8 assay (Dojin Chemical Research Institute, Japan) was performed to detect changes in cell viability. The CCK8 reagent was diluted 1:10 using fresh culture medium, and 100 μL of the diluted reagent was added to each well of the 96-well plate. The plates were then incubated at 37°C in the dark for 2 hours. After incubation, the OD values ​​of each well were measured using a multi-mode microplate reader, and cell viability and half-inhibitory concentration (IC50) under different interventions were further analyzed using Graphapd Prism 6.0 software. Figure 5 , Figure 6 As shown, overexpression of USP20 WT instead of USP20 CS enhances the resistance of PC9 and NCI-H1975 cells to osimertinib.

[0051] (2) Overexpression of USP20 in a nude mouse subcutaneous tumor model can enhance the resistance of lung adenocarcinoma cells to osimertinib.

[0052] For lentiviral infection experiments, empty vector lentivirus, wild-type USP20 lentivirus, and USP20 enzyme functional domain-inactivated lentivirus were purchased from Wuhan Qinda Biotechnology Co., Ltd., and co-cultured with PC9 and NCI-H1975 lung cancer cells at 37℃ for 16 h, respectively, before being replaced with complete culture medium. Stable strains were selected 72 h post-infection using 3 μg / ml puromycin (MCE, USA).

[0053] Twenty 4-week-old female BALB / c nude mice (Jicui Pharmaceutical, China) were randomly divided into four groups of five mice each: "solvent + empty lentivirus" group, "osimertinib + empty lentivirus" group, "osimertinib + USP20 wild-type" group, and "osimertinib + USP20 enzyme functional structure inactivated type" group. 5 × 10⁵ mmol / L was subcutaneously injected into the dorsal junction of the right hind limb of each mouse. 6 NCI-H1975 cells stably transfected with lentivirus were used to construct subcutaneous xenograft tumor models. The "solvent + empty lentivirus" and "osimertinib + empty lentivirus" groups used NCI-H1975 cells stably transfected with empty lentivirus. The "osimertinib + USP20 wild-type" and "osimertinib + USP20 enzyme functional domain inactivated lentivirus" groups used NCI-H1975 cells stably transfected with USP20 wild-type lentivirus and stably transfected with USP20 enzyme functional domain inactivated lentivirus, respectively. Subcutaneous tumors were induced to reach approximately 50 mm² in size. 3 According to the group settings, treatment was administered every 48 hours. The "solvent + empty lentivirus" group was administered the solvent (10% DMSO + 90% corn oil, volume fraction, the same below, by gavage), while the other three groups were administered osimertinib (5 mg / kg, calculated based on mouse body weight, the same below, dissolved in 10% DMSO + 90% corn oil, by gavage). During this period, the length and width of the subcutaneous tumors in nude mice were measured with calipers (the longest axis is the length, and the width is measured at the point where the length is uniformly perpendicular to the length), and the tumor volume was calculated as V = 1 / 2 × length × width. 2 The nude mice were weighed, and measurements were taken every two days. The experiment ended 18 days after drug administration (or when the tumor volume exceeded 2000 mmHg). 3 Or, if the mice lose more than 20% of their body weight, the experiment is terminated. Nude mice are euthanized by cervical dislocation, and subcutaneous tumors are collected, along with images of the tumors. Figures 7-8 ,in Figure 7 The image shows the tumor volume in the nude mouse at the end of the study. Figure 8The graph shows the change in tumor volume in nude mice. These results demonstrate that overexpression of USP20 WT instead of USP20 CS enhances the resistance of lung adenocarcinoma cells to osimertinib. Subcutaneous tumors in nude mice were then fixed, dehydrated and embedded, sectioned, baked, dewaxed, hydrated, subjected to endogenous peroxidase blockade, antigen retrieval, blocking, incubation with USP20 and Ki67 primary antibodies, incubation with secondary antibodies, staining and counterstaining, and then observed under a microscope. Figure 9 Overexpression of USP20 WT instead of USP20 CS can inhibit the decrease in Ki67 positivity rate caused by osimertinib. Figures 5-9 This indicates that overexpression of USP20 can significantly enhance the resistance of lung adenocarcinoma cells to osimertinib.

[0054] Example 3: Inhibiting USP20 expression levels can restore the sensitivity of lung adenocarcinoma cell lines to osimertinib. (1) Knockdown of USP20 can enhance the sensitivity of PC9OR and NCI-H1975OR cells to osimertinib.

[0055] Wild-type PC9 and wild-type NCI-H1975 cells were induced with a 10 nM-5 μM concentration gradient of osimertinib (MCE, USA) for six months, successfully establishing PC9OR and NCI-H1975OR resistant cell lines. Logarithmic-phase PC9OR and NCI-H1975OR cells (PC9OR cells represent osimertinib-resistant PC9 cells, and NCI-H1975OR cells represent osimertinib-resistant NCI-H1975 cells) were prepared into 5 × 10⁶ cells / years using complete culture medium (Pronosur, China). 4 Cell suspensions of 10 cells / ml were seeded into 6-well plates, 2 ml per well. After incubation at 37°C for 24 h, the medium was changed, and 1 ml of Opti-MEM medium (Thermo Fisher Scientific, USA) was added to each well. For lentiviral infection experiments, shNC (empty vector lentivirus) and shUSP20 (USP20 gene knockdown lentivirus) were purchased from Wuhan Qinda Biotechnology Co., Ltd., and co-cultured with lung cancer cells at 37°C for 16 h, followed by replacement with complete medium. 72 h post-infection, stable strains were selected using 3 μg / ml puromycin (MCE, USA).

[0056] Log-phase PC9OR and NCI-H1975OR cells stably transfected with shNC and shUSP20 were seeded into 96-well plates at a density of 5000 cells per well. After 12 hours, osimertinib (100 nM) was added, and after 72 hours, a CCK8 assay (Dojin Chemical Research Institute, Japan) was performed to detect changes in cell viability. The CCK8 reagent was diluted 1:10 using fresh culture medium, and 100 μL of the diluted reagent was added to each well of the 96-well plate. The plates were then incubated at 37°C in the dark for 2 hours. After cell incubation, the OD values ​​of each well were measured using a multi-mode microplate reader, and cell viability and half-inhibitory concentration (IC50) under different interventions were further analyzed using Graphapd Prism 6.0 software. Figure 10 , Figure 11 As shown, USP20 knockdown can enhance the sensitivity of PC9OR and NCI-H1975OR cells to osimertinib. Two sensitivity experiments were conducted using shUSP20: shUSP20#1 (USP20 gene knockdown lentivirus #1) and shUSP20#2 (USP20 gene knockdown lentivirus #2).

[0057] (2) Knockdown of USP20 in a nude mouse subcutaneous tumor model can reverse the resistance of lung adenocarcinoma cells to osimertinib.

[0058] Twenty 4-week-old female BALB / c nude mice (from Jicui Pharmaceutical, China) were randomly divided into four groups of five mice each: "solvent + empty vector lentivirus" group, "osimertinib + empty vector lentivirus" group, "solvent + USP20 gene knockdown lentivirus" group, and "osimertinib + USP20 gene knockdown lentivirus" group. 5 × 10⁵ mmol / L saturates were subcutaneously injected into the dorsal junction of the right hind limb of each mouse. 6 NCI-H1975OR cells stably transfected with lentivirus were used to construct subcutaneous xenograft tumor models. The "solvent + empty lentivirus" and "osimertinib + empty lentivirus" groups used NCI-H1975OR cells stably transfected with empty lentivirus, while the "solvent + USP20 knockdown lentivirus" and "osimertinib + USP20 knockdown lentivirus" groups used NCI-H1975OR cells stably transfected with USP20 knockdown lentivirus. Subcutaneous tumors were constructed when their volume reached approximately 50 mm². 3According to the group settings, drug treatment was administered every 48 hours. The "solvent + empty lentivirus" group and the "solvent + USP20 gene knockdown lentivirus" group were administered the solvent (10% DMSO + 90% corn oil, by gavage). The "osimertinib + empty lentivirus" group and the "osimertinib + USP20 gene knockdown lentivirus" group were administered osimertinib (5 mg / kg, calculated based on mouse body weight, dissolved in 10% DMSO + 90% corn oil, by gavage). During this period, the length and width of subcutaneous tumors in nude mice were measured using calipers (length was the longest axis, and width was measured at the points perpendicular to the length). Tumor volume was calculated as V = 1 / 2 × length × width. 2 The nude mice were weighed, and measurements were taken every two days. The experiment ended 18 days after drug administration (or when the tumor volume exceeded 2000 mmHg). 3 Or, if the mice lose more than 20% of their body weight, the experiment is terminated. Nude mice are euthanized by cervical dislocation, and subcutaneous tumors are collected, along with images of the tumors. Figures 12-13 ,in Figure 12 The image shows the tumor volume in the nude mouse at the end of the study. Figure 13 The graph shows the change in tumor volume in nude mice. These results demonstrate that USP20 knockdown can reverse the resistance of lung adenocarcinoma cells to osimertinib. Subcutaneous tumors in nude mice were then fixed, dehydrated and embedded, sectioned, baked, dewaxed, hydrated, subjected to endogenous peroxidase blockade, antigen retrieval, blocking, incubated with USP20 and Ki67 primary antibodies, incubated with secondary antibodies, stained and counterstained, and mounted. Microscopic observation was then performed. Figure 14 Knockdown of USP20 can promote an increase in Ki67 positivity rate induced by osimertinib.

[0059] Figures 10-14 This indicates that inhibiting USP20 can reverse the resistance of lung adenocarcinoma cells to osimertinib.

[0060] Example 4: Molecular docking and molecular dynamics simulations resolve the stable conformation of the T33873-USP20 complex. (1) Construction of molecular docking model: The small molecule library used for screening was provided by TOPSCIENCE Co., Ltd. (Shanghai, China). All small molecules were protonated at pH 7.4 before molecular docking, and their three-dimensional structures were generated using Open Babel. Protein and ligand pretreatment was performed using AutoDock Tools (ADT3). In virtual screening, the binding pocket containing the catalytic residue CYS154 was defined as the target region, and docking grid boxes were generated around this site using the AutoGrid program. Initial high-throughput virtual screening was performed using Quick Vina 2, and the top 3% of compounds with the highest scores were selected for more precise evaluation using smina. To ensure drug-likeness, we applied quantitative drug similarity indicators, retaining only compounds with a QED score greater than 0.3; simultaneously, all broad-spectrum interfering compounds were systematically excluded to eliminate potential false positives. Protein-ligand interaction patterns were visualized and analyzed using PyMOL. Figure 15 As shown, the computer simulation of the molecular docking model of the T33873-USP20 complex shows a binding energy of -6.39 kcal / mol, indicating strong binding potential.

[0061] (2) Molecular dynamics simulation: Molecular dynamics simulations were performed using Desmond / Maestro software (non-commercial version 2022.1). A TIP3P water molecule model was added to the system, and the charge was neutralized with 0.15 M NaCl solution. After energy minimization and equilibrium treatment, isothermal and isobaric ensemble simulations were performed at 300 K and 1 bar pressure for 100 ns, with trajectory coordinates recorded every 100 ps. The results were analyzed using the Simulation Interaction Diagram module in Desmond. Figure 16 Molecular dynamics simulations show that the system rapidly reaches equilibrium within 100 ns of simulation time. The USP20 protein complex (blue-gray curve) converges to 7.0-9.0 Å, and the RMSD (Root Mean Square Deviation) of conformational stability between it and compound T33873 (dark red curve) stabilizes at 2.0 Å, demonstrating good conformational stability. Figure 17 For protein-ligand interaction analysis, green and blue represent hydrogen bonds and water clusters, respectively. The results suggest that USP20-CYS154 and T33873 can form stable hydrogen bonds (occupancy rate of 100%). Figure 18 The binding model is derived from molecular dynamics simulations.

[0062] Figures 15-18 The results of molecular docking and kinetic simulations demonstrate that compound T33873 can be stably bound to USP20.

[0063] Example 5: Compound T33873 has the potential to modulate the reversal of osimertinib resistance in NSCLC in cell and animal experiments.

[0064] (1) Compound T33873 showed a significant effect in reversing resistance in lung adenocarcinoma cell lines resistant to osimertinib.

[0065] Log-phase PC9OR and NCI-H1975OR cells were seeded in 96-well plates at a density of 5000 cells per well. After 12 h, 0, 0.625, 1.25, 2.5, 5, and 10 μM osimertinib or 0, 10, and 15 μM compound T33873 were added, with each group repeated three times. After 72 h, a CCK8 assay (Dojin Chemical Research Institute, Japan) was performed to detect changes in cell viability. The CCK8 reagent was diluted 1:10 using fresh culture medium, and 100 μL of the diluted CCK8 reagent was added to each well of the 96-well plate. The 96-well plates were then incubated at 37°C in the dark for 2 h. After cell incubation, the OD value of each well in the 96-well plate was measured using a multi-mode microplate reader. Graphapd Prism 6.0 software was used to further analyze cell viability and half-inhibitory concentration (IC50) under different interventions. Figure 19 , Figure 20 As shown, the combination of compound T33873 and osimertinib can significantly inhibit the proliferation of PC9OR and NCI-H1975OR cell lines resistant to osimertinib.

[0066] (2) Compound T33873 showed a significant effect in reversing osimertinib resistance in NSCLC in a nude mouse subcutaneous xenograft model.

[0067] Twenty 4-week-old female BALB / c nude mice (Jicui Pharmaceutical, China) were randomly divided into four groups of five mice each: solvent group, osimertinib group, T33873 group, and osimertinib + T33873 group. 5 × 10⁵ mice were subcutaneously injected into the dorsal junction of the right hind limb. 6 NCI-H1975OR cells were used at a density of 100 μL / 100 μL to construct a subcutaneous xenograft model. The subcutaneous tumor volume was expected to be approximately 50 mm². 3According to the group settings, the drug treatment was administered every 48 hours. The solvent group was given the solvent (10% DMSO + 90% corn oil, by gavage), the osimertinib group was given osimertinib (5 mg / kg, dissolved in 10% DMSO + 90% corn oil, by gavage), the T33873 group was given T33873 (10 mg / kg, dissolved in 10% DMSO + 90% corn oil, by intraperitoneal injection), and the osimertinib + T33873 group was given osimertinib and T33873 (osimertinib 5 mg / kg, dissolved in 10% DMSO + 90% corn oil, by gavage, compound T33873 10 mg / kg, dissolved in 10% DMSO + 90% corn oil, by intraperitoneal injection). During the process, the length and width of the subcutaneous tumors in nude mice were measured using vernier calipers (length was measured along the longest axis, and width was measured along the uniformly perpendicular part of the length). The tumor volume was calculated as V = 1 / 2 × length × width. 2 The nude mice were weighed, and measurements were taken every two days. The experiment ended 18 days after drug administration (or when the tumor volume exceeded 2000 mmHg). 3 (The experiment was terminated if the mice lost more than 20% of their body weight). Nude mice were euthanized by cervical dislocation, and subcutaneous tumors were collected and images of the tumors were preserved. Figure 21 The image shows the tumor volume of nude mice after administration and sacrifice. Figure 22 The graph shows the change in tumor volume in nude mice. These results demonstrate that T33873 can inhibit the growth of NCI-H1975OR subcutaneous tumors resistant to osimertinib, and the inhibitory effect is even stronger when used in combination with osimertinib. Subcutaneous tumors in nude mice were then fixed, dehydrated and embedded, sectioned, baked, dewaxed, hydrated, subjected to endogenous peroxidase blockade, antigen retrieval, blocking, incubation with Ki67 primary antibody, incubation with secondary antibody, staining and counterstaining, and then the staining results were observed under a microscope. Figure 23 The compound T33873 showed that it could significantly reduce the positive rate of Ki67 in NCI-H1975OR subcutaneous tumors, and the effect was stronger when used in combination with osimertinib.

[0068] Figures 19-23 This indicates that compound T33873 has the regulatory potential to reverse osimertinib resistance in NSCLC in cell and animal experiments.

[0069] Example 6: Compound T33873 showed no significant toxic side effects on vital organs of nude mice at effective doses. (1) Collect the heart, liver, spleen, lung and kidney of nude mice in Example 5, perform tissue fixation, dehydration and embedding, sectioning, baking, dewaxing, hydration, hematoxylin staining, differentiation blue staining, eosin staining, dehydration and clearing and mounting, and then observe the staining results under a microscope. Figure 24The results showed that neither compound T33873 nor compound T33873 in combination with osimertinib caused significant tissue changes in the heart, liver, spleen, lungs and kidneys of nude mice, proving that compound T33873 is safe for normal body tissues at effective doses.

[0070] The above description is merely a specific embodiment of this application, enabling those skilled in the art to understand or implement this application. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of this application. Therefore, this application is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features claimed herein.

Claims

1. The use of a pamidronate in the preparation of a medicament for the prevention, relief and / or treatment of non-small cell lung cancer.

2. The application as described in claim 1, characterized in that: The pamidronate includes pamidronate monosodium C3H 10 NO7P2·Na and / or disodium pamidronate C3H9NO7P2·Na2.

3. The application as described in claim 1, characterized in that: The non-small cell lung cancer includes lung adenocarcinoma.

4. The application as described in claim 3, characterized in that: The lung adenocarcinoma mentioned is lung adenocarcinoma resistant to the tyrosine kinase inhibitor EGFR-TKIs.

5. A medicament for the prevention, alleviation, and / or treatment of non-small cell lung cancer, characterized in that: The drug includes an active substance, which is pamidronate.

6. The drug as described in claim 5, characterized in that: The pamidronate includes pamidronate monosodium C3H 10 NO7P2·Na and / or disodium pamidronate C3H9NO7P2·Na2.

7. The drug as described in claim 5, characterized in that: The non-small cell lung cancer includes lung adenocarcinoma.

8. The drug as described in claim 7, characterized in that: The lung adenocarcinoma mentioned is lung adenocarcinoma resistant to the tyrosine kinase inhibitor EGFR-TKIs.

9. A pharmaceutical composition for the prevention, alleviation, and / or treatment of non-small cell lung cancer, characterized in that: It includes two or more drugs used to prevent, alleviate and / or treat non-small cell lung cancer, one of which uses pamidronate as the active ingredient.

10. The pharmaceutical composition according to claim 9, characterized in that: Another type of drug is the tyrosine kinase inhibitor EGFR-TKIs; And / or, the pamidronate comprises C3H 10 NO7P2·Na and / or pamidronate disodium C3H9NO7P2·Na2; And / or, the non-small cell lung cancer includes lung adenocarcinoma.

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