Combined medicine for treating lung cancer

Abstract

The invention discloses a combined medicine for treating lung cancer. It is found for the first time that lomitapide has a remarkable anti-cancer effect on NSCLC patients and osimertinib drug-resistant NSCLC patients when used independently or used in combination with other drugs, and in addition, it is found that lomitapide is a potential autophagy inducer and can activate cell autophagy. The application provides a novel clinical treatment method for NSCLC patients and osimertinib drug-resistant NSCLC patients, and has a wide application prospect.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine, and in particular relates to a combined drug for treating lung cancer. Background Art

[0002] Lung cancer is a highly prevalent tumor worldwide and ranks first among malignant tumors in my country. Non-small cell lung cancer (NSCLC) is the most common histological type of lung cancer, accounting for about 85% of lung cancer patients. Gene mutations, especially L858R and T790M mutations of epidermal growth factor receptor (EGFR), are the main causes of NSCLC patients.

[0003] Osimertinib is an irreversible third-generation EGFR-TKI. Compared with the first- and second-generation EGFR-TKIs, osimertinib has a stronger inhibitory effect on EGFR and can overcome the acquired resistance of the first- and second-generation drugs caused by the T790M mutation. However, acquired resistance to osimertinib is also inevitable and remains the biggest dilemma and obstacle for osimertinib to achieve long-term efficacy.

[0004] Therefore, finding new and more effective treatments for lung cancer is of special importance and urgency. Summary of the invention

[0005] To make up for the deficiencies of the prior art, the present invention provides a combined drug for treating lung cancer.

[0006] To achieve the above purpose, the present invention adopts the following technical solution.

[0007] A first aspect of the present invention provides a pharmaceutical composition for treating lung cancer / reversing lung cancer drug resistance, the pharmaceutical composition comprising lomitripadip and / or osimertinib.

[0008] Furthermore, the drug resistance is osimertinib resistance.

[0009] Furthermore, the lung cancer is selected from non-small cell lung cancer.

[0010] Furthermore, the non-small cell lung cancer is non-small cell lung cancer with EGFR mutation.

[0011] Furthermore, the EGFR mutation is EGFR-L858R / T790M mutation.

[0012] Furthermore, the pharmaceutical composition also includes pharmaceutically acceptable excipients.

[0013] The second aspect of the present invention provides a pharmaceutical preparation for treating lung cancer / reversing lung cancer drug resistance, wherein the pharmaceutical preparation comprises the pharmaceutical composition according to the first aspect of the present invention.

[0014] Furthermore, the dosage form of the pharmaceutical preparation includes a dosage form for enteral administration and a dosage form for parenteral administration.

[0015] Furthermore, the dosage forms for administration through the gastrointestinal tract include tablets, granules, capsules, solutions, dry suspensions, powders, sustained-release agents, effervescent agents, emulsions, suspensions, syrups, drops, and chewable agents.

[0016] Furthermore, the non-intestinal administration dosage forms include injection dosage forms, respiratory tract administration dosage forms, cavity administration dosage forms, mucosal administration dosage forms, and skin administration dosage forms.

[0017] The third aspect of the present invention provides the use of lomitropium and / or osimertinib in the preparation of a pharmaceutical composition for treating lung cancer / reversing lung cancer drug resistance.

[0018] Furthermore, the drug resistance is osimertinib resistance.

[0019] Furthermore, the lung cancer is selected from non-small cell lung cancer.

[0020] Furthermore, the non-small cell lung cancer is non-small cell lung cancer with EGFR mutation.

[0021] Furthermore, the EGFR mutation is EGFR-L858R / T790M mutation.

[0022] The fourth aspect of the present invention provides the use of lomitropium and / or osimertinib in the preparation of a pharmaceutical preparation for treating lung cancer / reversing lung cancer drug resistance.

[0023] Furthermore, the drug resistance is osimertinib resistance.

[0024] Furthermore, the lung cancer is selected from non-small cell lung cancer.

[0025] Furthermore, the non-small cell lung cancer is non-small cell lung cancer with EGFR mutation.

[0026] Furthermore, the EGFR mutation is EGFR-L858R / T790M mutation.

[0027] A fifth aspect of the present invention provides a method for regulating cellular autophagy in vitro, the method comprising administering lomitapide.

[0028] Furthermore, the method is a non-therapeutic method.

[0029] The sixth aspect of the present invention provides the use of lomitapide in the preparation of a medicament for regulating cellular autophagy.

[0030] The seventh aspect of the present invention provides a method for regulating apoptosis / metastasis of lung cancer cells / lung cancer resistant cells in vitro, the method comprising administering lomitripadip and / or osimertinib.

[0031] Furthermore, the method is a non-therapeutic method.

[0032] The eighth aspect of the present invention provides the use of lomistatin and / or osimertinib in the preparation of a medicament for regulating apoptosis / metastasis of lung cancer cells / lung cancer resistant cells.

[0033] Advantages and beneficial effects of the present invention: This application first discovered that lomitapide, when used alone or in combination with other drugs, has a significant anti-cancer effect on NSCLC patients and osimertinib-resistant NSCLC patients. In addition, it was found that lomitapide is a potential autophagy inducer that can activate cellular autophagy. This application provides a new clinical treatment method for NSCLC patients and osimertinib-resistant NSCLC patients, which has broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 The figure shows the establishment and identification of osimertinib-resistant cells, where 1A is a schematic diagram of the establishment of H1975 / OR cells, 1B is a cell viability graph, 1C is a cell apoptosis flow cytometry graph and statistical graph, 1D is a cell clone formation ability graph, 1E is a cell 3D sphere formation ability graph, and 1F is a cell invasion ability graph; Figure 2 2A is a graph showing the reversal of NSCLC resistance by lomitaxime and osimertinib in vitro, where 2A is a graph showing cell apoptosis after the addition of lomitaxime and osimertinib, and 2B is a graph showing the 3D spheroidization ability of cells after the addition of lomitaxime and osimertinib; Figure 3 Figure 3 is a graph showing that lomitapide reverses osimertinib resistance through autophagy, where 3A is a graph showing cell viability after lomitapide addition, 3B is a graph showing pathways that are significantly enriched in cells after lomitapide treatment compared with H1975 / OR cells through KEGG analysis, 3C is a graph showing LC3 (green) fluorescence imaging of cells after treatment with designated drugs, 3D is a graph showing immunoblotting and expression statistics of autophagy-related proteins after treatment with designated drugs, 3E is a representative transmission electron micrograph of cells treated with or without lomitapide, and 3F is a graph showing immunoblotting and expression statistics of autophagy-related proteins induced by lomitapide under the action of CQ (chloroquine, an autophagy inhibitor); Figure 4 4A is a graph showing the effect of CQ on lomitapide treatment, wherein 4A is a graph showing the drug sensitivity of H1975 / OR cells to osimertinib, and 4B is a graph showing the effect of CQ on cell apoptosis in lomitapide treatment; Figure 55A is a graph showing the reversal of osimertinib resistance by lomitripapine and osimertinib in vivo, wherein 5A is a graph showing the tumor volume of transplanted tumor mice, 5B is a graph showing the tumor volume of transplanted tumor mice, 5C is a graph showing the tumor weight of transplanted tumor mice, 5D is a graph showing the tumor weight of transplanted tumor mice, and 5E is a graph showing H&E staining of the tumor and immunohistochemical staining of Ki67; Figure 6 This is the dose-response matrix diagram for the combination of lomitapide and osimertinib. DETAILED DESCRIPTION

[0035] The definitions of some terms used in this specification are provided below. Unless otherwise defined, all technical and scientific terms used herein generally have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0036] The present invention provides a pharmaceutical composition for treating lung cancer / reversing lung cancer drug resistance, wherein the pharmaceutical composition comprises lomitripadip and / or osimertinib.

[0037] In some embodiments, lomistatin or osimertinib comprises lomistatin or osimertinib (hereinafter also referred to as the compound), or a pharmaceutically acceptable salt, hydrate, enantiomer, diastereomer, solvate or crystalline form thereof.

[0038] In some embodiments, the pharmaceutically acceptable salt refers to an acid salt formed with an inorganic and / or organic acid and a basic salt formed with an inorganic and / or organic base. In addition, when the compound contains a basic portion (such as but not limited to, pyridine or imidazole) and an acidic portion (such as but not limited to, carboxylic acid), zwitterions can be formed and the zwitterions are included in the pharmaceutically acceptable salts described in the present application. Pharmaceutically acceptable (i.e., non-toxic, physiologically acceptable) salts are preferred, but other salts are also useful. The pharmaceutically acceptable salt of the compound can be formed, for example, by reacting the compound with a certain amount of acid or base in a medium, such as a medium in which the salt is precipitated or an aqueous medium (freeze-dried after the reaction).

[0039] Specific pharmaceutically acceptable salts include those salts which are suitable for contact with the tissues of humans and lower animals without excessive toxicity, irritation, allergic reaction, etc., and which are commensurate with a reasonable benefit or risk ratio, within the scope of sound medical judgment. Pharmaceutically acceptable salts (pharmaceutically acceptable salts) are well known in the art. Pharmaceutically acceptable salts of the compounds described herein include salts derived from suitable inorganic and organic acids and inorganic and organic bases.

[0040] The example of pharmaceutically acceptable nontoxic acid addition salt is the salt formed with inorganic acid, for example hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid and perchloric acid, or the salt formed with organic acid, for example acetic acid, oxalic acid, maleic acid, tartaric acid, citric acid, succinic acid or malonic acid. Also include the salt formed using the conventional methods in the art, for example, ion exchange method.Other pharmaceutically acceptable salts include: adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate, camphorate, camphorsulfonate, citrate, cyclopentanepropionate, digluconate, dodecyl sulfate, ethanesulfonate, formate, fumarate, gluconate, glycerophosphate, hemisulfate, enanthate, hydroiodide, 2-hydroxy-ethanesulfonate, lactobionate, lactate, laurate, lauryl sulfate, malate, maleate, malonate, mesylate. Pharmaceutically acceptable salts derived from suitable bases include alkali metal, alkaline earth metal, ammonium salts. Representative alkali metal or alkaline earth metal salts include sodium, lithium, potassium, calcium, magnesium salts and the like.

[0041] In some embodiments, the hydrate refers to a compound combined with water. An organic compound can form a complex with a solvent, in which it reacts or precipitates or crystallizes from the solvent. These complexes are called solvates. When the solvent is water, the complex is called a hydrate.

[0042] In some embodiments, the solvate refers to a form of a compound or salt thereof that is combined with a solvent, usually formed by a solvolysis reaction. This physical association may include hydrogen bonding. Conventional solvents include water, methanol, ethanol, acetic acid, DMSO, THF, ether, etc. In some cases, the solvate will be able to separate, for example, when one or more solvent molecules are incorporated into the crystal lattice of the crystalline solid. Solvates include solvates in solution and separable solvates.

[0043] In some embodiments, the crystalline form refers to the crystalline form of a compound with a specific crystal packing arrangement. Different crystalline forms generally have different X-ray diffraction patterns, infrared spectra, melting points, densities, hardnesses, crystal shapes, photoelectric properties, stability, and solubility. Recrystallization solvents, crystallization rates, storage temperatures, and other factors may lead to a crystalline form dominating. Various polymorphs of a compound can be prepared by crystallization under different conditions.

[0044] The pharmaceutical composition also includes pharmaceutically acceptable excipients.

[0045] In some embodiments, the pharmaceutically acceptable excipients include, but are not limited to, diluents, binders, surfactants, wetting agents, adsorption carriers, lubricants, fillers, and disintegrants.

[0046] In some embodiments, the diluent includes but is not limited to lactose, sodium chloride, glucose, urea, starch, water, etc. The binder includes but is not limited to starch, pregelatinized starch, dextrin, maltodextrin, sucrose, gum arabic, gelatin, methylcellulose, carboxymethylcellulose, alginic acid and alginate, xanthan gum, hydroxypropyl cellulose, etc. The surfactant includes but is not limited to sodium lauryl sulfate, stearic acid monoglyceride, hexadecanol, etc. The humectant includes but is not limited to glycerol, starch, etc. The adsorption carrier includes but is not limited to starch, lactose, bentonite, soap clay, etc. The lubricant includes but is not limited to zinc stearate, talc, calcium and magnesium stearate, polyethylene glycol, polyoxyethylene monostearate, monolauric sucrose acid ester, magnesium lauryl sulfate, etc. The filler includes but is not limited to mannitol, xylitol, sorbitol, maltose, glucose, lactose, sucrose, dextrin, starch, etc. The disintegrant includes but is not limited to cross-linked vinyl pyrrolidone, sodium carboxymethyl starch, low-substituted hydroxypropyl methyl, cross-linked sodium carboxymethyl cellulose, soybean polysaccharide, etc.

[0047] In some embodiments, the pharmaceutical composition comprises a single compound preparation or a combination of two separate single preparations, specifically, the compound preparation is a compound preparation comprising lomitaxepide and osimertinib, and the combination of single preparations is a combination of a single preparation comprising lomitaxepide and a single preparation comprising osimertinib. In a specific embodiment, the two single preparations in the combination of single preparations are administered simultaneously or sequentially.

[0048] In some embodiments, lomitripapine and osimertinib in the pharmaceutical composition can be administered simultaneously, separately or successively. Wherein, simultaneously refers to the synchronous administration of two drugs. If not administered simultaneously, then administered successively within a time range, so that the two can act therapeutically within the same time range. Therefore, sequential administration allows administration of another drug within 5 minutes, 10 minutes, 15 minutes, 30 minutes, 1 hour, 2 hours or several hours after providing a drug, provided that the circulation half-life of the first administered drug allows the two to exist simultaneously in a therapeutically effective amount. The time delay of administration between each component will change according to the exact nature of the component, the interaction therebetween, and their respective half-lives. Different from simultaneously or successively, respectively refers to the significant interval between the administration of one drug and another drug, that is, when the second drug is administered, the first administered drug may no longer be present in the bloodstream in a therapeutically effective amount.

[0049] The present invention provides a pharmaceutical preparation for treating lung cancer / reversing lung cancer drug resistance, and the pharmaceutical preparation comprises the above-mentioned pharmaceutical composition.

[0050] The dosage forms of the pharmaceutical preparation include a dosage form for enteral administration and a dosage form for parenteral administration.

[0051] The dosage forms for administration through the gastrointestinal tract include tablets, granules, capsules, solutions, dry suspensions, powders, sustained-release agents, effervescent agents, emulsions, suspensions, syrups, drops, and chewables.

[0052] The non-intestinal administration dosage forms include injection dosage forms, respiratory tract administration dosage forms, cavity administration dosage forms, mucosal administration dosage forms, and skin administration dosage forms.

[0053] In some embodiments, the injection dosage forms include but are not limited to various injections such as intravenous injections, intramuscular injections, subcutaneous injections, intradermal injections and intracavitary injections; the respiratory tract administration dosage forms include but are not limited to sprays, aerosols, powder aerosols, etc.; the cavity administration dosage forms include but are not limited to suppositories, aerosols, effervescent tablets, drops, pills, etc., which are used in the rectum, vagina, urethra, nasal cavity, ear canal, etc.; the mucosal administration dosage forms include but are not limited to eye drops, nasal drops, eye ointments, gargles, sublingual tablets, adhesive tablets, patches, etc.; the skin administration dosage forms include but are not limited to external solutions, lotions, liniments, ointments, plasters, pastes, patches, etc.

[0054] In some embodiments, the pharmaceutical composition or pharmaceutical preparation may also contain other drugs or agents that can be used for treatment and / or prevention, or auxiliary treatment and / or prevention. The other drugs or agents are not particularly limited. As long as they can produce the expected treatment and / or prevention, or auxiliary treatment and / or prevention effect on lung cancer / lung cancer resistance, they are all within the scope of protection of this application.

[0055] In some embodiments, the other drugs or agents that can be used for treatment and / or prevention, or auxiliary treatment and / or prevention include chemotherapy drugs, targeted therapy drugs, and immunotherapy drugs.

[0056] Among them, chemotherapy drugs include platinum drugs: such as cisplatin and carboplatin; taxanes: such as paclitaxel and docetaxel; other drugs: such as gemcitabine, pemetrexed, vinorelbine, etc.

[0057] Targeted therapy drugs include EGFR mutation inhibitors, ALK inhibitors, KRAS G12C inhibitors, KRAS G12C inhibitors, ROS1 inhibitors, MET inhibitors, RET inhibitors, and HER2 inhibitors.

[0058] EGFR mutation inhibitors include first-generation drugs: gefitinib, erlotinib, icotinib; second-generation drugs: afatinib, dacomitinib; third-generation drugs: osimertinib, ametinib, and vometinib.

[0059] ALK inhibitors include the first-generation drug: crizotinib; the second-generation drugs: alectinib, ceritinib, brigatinib; and the third-generation drug: lorlatinib.

[0060] KRAS G12C inhibitors include sotolagrate and adagrasib.

[0061] ROS1 inhibitors include entrectinib, ceritinib and lorlatinib.

[0062] MET inhibitors include capmatinib, tepotinib, etc.

[0063] RET inhibitors include selpercatinib and pralsetinib.

[0064] HER2 inhibitors include Enhertu.

[0065] Immunotherapy drugs include tislelizumab, cemiplizumab, nivolumab, pembrolizumab, and atezolizumab.

[0066] The invention will be further described below in conjunction with specific examples. It should be understood that the specific embodiments described herein are presented by way of example and are not intended to limit the invention. The main features of the invention may be used in a variety of embodiments without departing from the scope of the invention.

[0067] Example

[0068] 1. Experimental methods Cell lines and culture The human NSCLC cell line NCI-H1975 (parental cell line) was purchased from Wuhan Punosai Biotechnology Co., Ltd. To establish the corresponding osimertinib-resistant cell line (NCI-H1975 / OR), the drug dose of osimertinib (Osim) was increased from 0.1 μM to 8 μM. All cells were cultured in RPMI 1640 medium containing 10% fetal bovine serum and 100 U / mL penicillin / streptomycin and placed in a 37°C, 5% CO2 incubator.

[0069] Cell viability assay The effect of drugs on the viability of NCI-H1975 and NCI-H1975 / OR cells was investigated using the Cell Counting Kit 8 (CCK-8) assay. Exponentially growing cells were seeded in 96-well plates and cultured to a confluence of approximately 70-80%. Cells were treated with different concentrations of drugs. Then, 10 µL of CCK-8 reagent was added to each well and incubated at 37°C for 2 h. The absorbance at 450 nm was recorded using a microplate reader (Molecular Devices).

[0070] Apoptosis analysis Apoptosis detection was performed using the Annexin V-FITC apoptosis detection kit (Beyotime Biotechnology) according to the manufacturer's instructions, and the stained cells were analyzed using a NovoCyte Advanteon flow cytometer (Agilent).

[0071] Colony formation assay The cells were seeded in 6-well plates (2000 cells / well) and incubated with drugs for 14 days, with the medium changed every 3 days. The colonies were fixed with 4% paraformaldehyde and stained with crystal violet solution (0.1%). The stained colonies were washed with PBS and photographed.

[0072] Three-dimensional (3D) spheroid assay Cells were seeded on dishes coated with Matrigel (BD Biosciences) and 50% serum-free medium. Cells were grown in complete medium with or without drug treatment. Medium was changed every three days. 3D structures were imaged using an inverted phase-contrast microscope (Leica Microsystems).

[0073] Cell migration assay The cultured cells were routinely treated by adding 600 μL of medium containing 20% ​​FBS to a 24-well plate, resuspending the cells in 10% FBS medium, and adding them to a Transwell chamber (1×10 4 / well), incubated in a 37°C incubator for 24 h, fixed the invading cells in the upper chamber, stained with 0.1% crystal violet, and photographed under an inverted microscope.

[0074] Western blotting Cell lysates were prepared using RIPA buffer supplemented with 446 protease / phosphatase inhibitors (Biomark), and Western blotting was performed as described previously.

[0075] Immunofluorescence Immunofluorescence staining was performed as described previously. Briefly, cells were fixed with 4% formaldehyde, permeabilized with 0.2% Triton X-100, and blocked with 5% BSA. Then, they were incubated with the indicated antibodies overnight at 4 °C and then stained with fluorescence-conjugated secondary antibodies and DAPI solution. Cells were photographed under a fluorescence microscope (Leica DM6B Thunder). The antibody used was Anti-LC3 (1:100; Proteintech, #81004-1-RR).

[0076] Transmission electron microscopy Transmission electron microscopy (TEM) was used to monitor autolysosome formation after or without drug treatment. Briefly, cells were collected, pre-fixed with 2.5% glutaraldehyde solution and fixed with 1% osmium tetroxide for 2 h at room temperature. Subsequently, samples were gradually dehydrated with increasing concentrations of ethanol and acetone and embedded in epoxy resin. Finally, 50–60 nm sections were prepared on an LKB-1 ultramicrotome, transferred to copper grids, and photographed using a JEM-1400 plus transmission electron microscope (JEOL).

[0077] In vivo subcutaneous tumor model BALB / c nude mice (6 weeks old) were provided by Liaoning Changsheng Biotechnology Co., Ltd. When the tumor volume reached about 100 mm 3 At the same time, the mice were randomly divided into 4 groups. In vivo experiments: osimertinib 10 mg / kg was administered orally once every 2 days, lomitapide 20 mg / kg was administered intraperitoneally once every 2 days, and the control group mice were given an equal amount of 2% DMSO saline. The weight of the mice was recorded during the experiment. The tumor volume was measured with a caliper and calculated as ([width] 2 ×[length] / 2). After the experiment, the mice were killed, the tumors were removed, and the tumor tissues were embedded for immunohistochemical detection. All experiments were approved by the Dalian Medical University Animal Care Committee (AEE23117) and performed in accordance with the institutional guidelines for animal care and handling.

[0078] Histochemical or immunohistochemical staining Tumors were removed for histopathological examination. Tissues were cut into 5-µm-thick sections. Paraffin sections were dewaxed with xylene, rehydrated through decreasing concentrations of ethanol, and stained with hematoxylin and eosin (H&E). Sections were visualized using a DAB kit (Beyotime Biotechnology), and images of three randomly selected fields on each slide were taken under a microscope.

[0079] RNA-seq RNA-seq was performed by LC Biotechnology Co., Ltd. Briefly, pellets of H1975 parental cells, H1975 / OR cells treated with osimertinib alone, and osimertinib and lomitapide were collected, and after total RNA extraction, total RNA was isolated using TRIzol (Invitrogen), quantified using NanoDrop, and assessed for integrity using Bioanalyzer (Agilent).

[0080] Statistical analysis GraphPad Prism software was used to calculate significance. All statistical analyses were performed using data from at least three independent experiments. Two-way or one-way ANOVA with Bonferroni correction was used to compare more than three means, and unpaired Student's t-test was used to compare two means; *P < 0.05 was considered to be significantly different.

[0081] King's formula King's formula was used to analyze the effect of combined use (Q value), Q = E (A + B) / (EA + EB-EA × EB), Q represents the measured combined effect, that is, the ratio of the actual effect to the expected effect when the two drugs are used in combination; E (A + B) represents the effect of a certain amount of drug A and drug B used together. EA represents the effect of drug A when used alone; EB: represents the effect of drug B when used alone. When Q>1.15, it means that the two drugs have a synergistic effect; when 0.85≤Q≤1.15, it means that the two drugs have an additive effect; when Q<0.85, it means that the two drugs have an antagonistic effect.

[0082] 2. Experimental results Construction and characterization of osimertinib-resistant cells in NSCLC NSCLC parental cells (NCI-H1975 with EGFR-L858R / T790M mutation) were exposed to osimertinib by increasing the dose of osimertinib from 0.1 μM to 8 μM, and an osimertinib-resistant cell line (NCI-H1975 / OR cells) was successfully established ( Figure 1 A). To further study the effect of osimertinib on the proliferation of NSCLC cells in vitro, CCK-8 assay was used to detect the cell viability of NSCLC parental cells and NCI-H1975 / OR cells. Figure 1 As shown in B, the IC50 value of osimertinib in NCI-H1975 / OR cells is approximately 6 times that in NSCLC parental cells, indicating that osimertinib significantly improved cell viability. In addition, osimertinib treatment failed to induce apoptosis in NCI-H1975 / OR cells (0, 1, and 6 μM, Figure 1 C) and does not affect its clone formation ( Figure 1 D) 3D tumor spheroid formation ( Figure 1 E) and cell metastasis ( Figure 1 F) Capacity. In summary, the results showed that osimertinib resistance effectively inhibited apoptosis of NCI-H1975 / OR cells but enhanced cell viability, proliferation, and metastasis in vitro.

[0083] Lomitapide reverses osimertinib resistance in vitro The inhibitory effect of lomitapide on NCI-H1975 / OR cells was evaluated by CCK-8 assay. Figure 3As shown in A, lomistatapide (5 μM) significantly increased the sensitivity of NCI-H1975 / OR cells and reduced cell viability in a dose-dependent manner (0, 5, 15, 20, and 25 μM). To further determine the synergistic effect of lomistatapide and osimertinib, NCI-H1975 / OR cells were treated with osimertinib alone (Osim), lomistatapide alone (Lomi), or osimertinib (6-7 μM) and lomistatapide (3-6 μM) combined (Comb). The results showed that the addition of lomistatapide significantly promoted cell apoptosis ( Figure 2 A). 3D tumor spheroids showed that NCI-H1975 / OR cells were more sensitive to Comb than to Osim, thus indicating that osimertinib resistance in NCI-H1975 / OR cells could be reversed by the addition of lomitapide ( Figure 2 B).

[0084] Jin's formula: EA=(6.3%+8.3%+8.9%) / 3=7.8%, EB=(9.3%+7.6%+8.9%) / 3=8.6%; E(A+B)=(41.0+45.7+41.8) / 3=42.8%; Q=E(A+B) / (EA+EB-EA*EB)=42.8% / (7.8%+8.6%-7.8%*8.6%)=2.72.

[0085] Table 1 Evaluation of the combination index of osimertinib and lomitapide

[0086] The inhibitory effect of osimertinib and lomitapide combination on osimertinib-resistant lung cancer cells and the evaluation of the combination index are shown in Table 1 , and the dose-response matrix is ​​shown in Figure 6 As shown, the results showed that the combination of osimertinib and lomitapide had a synergistic effect in reversing osimertinib resistance.

[0087] Lomitapide reverses osimertinib resistance in NSCLC by activating autophagy To verify whether lomitapide triggers the autophagy mechanism to reverse osimertinib resistance in NSCLC, NCI-H1975 / OR cells treated with Comb were collected for RNA sequencing. KEGG analysis showed that the autophagy pathway was mainly enriched in the lomitapide-treated group but not the control group ( Figure 3 B). Fluorescence signal and protein detection showed that after the addition of lomitapide, the classic autophagy-related marker genes LC3 and p62 were significantly upregulated ( Figure 3 C-3D, lomitapide 5 μM, osimertinib 6 μM). In addition, transmission electron microscopy (TEM) data also confirmed that lomitapide inevitably increased the formation of autophagosomes and autolysosomes ( Figure 3E). The autophagy inhibitor chloroquine (CQ) failed to affect LC3 protein levels after lomitapide treatment, indicating a strong complementary effect ( Figure 3 F). In addition, CQ significantly reduced the sensitivity of NCI-H1975 / OR cells treated with lomitapide to osimertinib ( Figure 4 A) and apoptosis ( Figure 4 B). In conclusion, our results suggest that lomistatin reverses osimertinib resistance in NSCLC by activating autophagy.

[0088] Lomitapide inhibits reversal of osimertinib resistance in vivo To further investigate the in vivo therapeutic potential of lomistatin, the NCI-H1975 / OR xenograft model was established in BALB / c nude mice, and the mice were divided into four groups (5 mice in each group), namely, the group injected intraperitoneally with 100 μL saline with equal concentration of DMSO (2%) (control group), the group treated with Osim (10 mg / kg) alone, the group treated with Lomi (20 mg / kg) alone, or the group treated with Comb (10 mg / kg Osim and 20 mg / kg Lomi). Comb significantly inhibited the tumor growth curve better than the treatment with Lomi or Osim alone. The tumor size and weight of the drug-treated groups were significantly smaller than those of the control group, especially the Comb group. There was no effect of lomistatin treatment on the body weight of the animals, indicating that there was no toxic effect on the animals. Hematoxylin and eosin staining (H&E) and Ki-67 assay were performed to evaluate cell proliferation. The results showed that the proliferation rate of xenograft tumor cells in mice treated with lomistatin and osimertinib was reduced, which was consistent with the in vitro results. NCI-H1975 / OR cells are sensitive to the combination of omemetapide and osimertinib, which inhibits cell viability and proliferation ( Figure 5 ).

[0089] The description of the above embodiments is only used to understand the method and core idea of ​​the present invention. It should be pointed out that, for those skilled in the art, several improvements and modifications can be made to the present invention without departing from the principle of the present invention, and these improvements and modifications will also fall within the scope of protection of the claims of the present invention.

Claims

1. A pharmaceutical composition for treating lung cancer / reversing lung cancer drug resistance, characterized in that: The pharmaceutical composition comprises lomitapide and / or osimertinib.

2. The pharmaceutical composition according to claim 1, characterized in that The drug resistance is osimertinib resistance; Preferably, the lung cancer is selected from non-small cell lung cancer; Preferably, the non-small cell lung cancer is non-small cell lung cancer with EGFR mutation; Preferably, the EGFR mutation is EGFR-L858R / T790M mutation.

3. The pharmaceutical composition according to claim 2, characterized in that The pharmaceutical composition also includes pharmaceutically acceptable excipients.

4. A pharmaceutical preparation for treating lung cancer / reversing lung cancer drug resistance, characterized in that: The pharmaceutical preparation comprises the pharmaceutical composition according to any one of claims 1 to 3; Preferably, the dosage form of the pharmaceutical preparation includes a dosage form for enteral administration and a dosage form for parenteral administration; Preferably, the dosage form for administration through the gastrointestinal tract includes tablets, granules, capsules, solutions, dry suspensions, powders, sustained-release agents, effervescent agents, emulsions, suspensions, syrups, drops, and chewable tablets; Preferably, the non-intestinal administration dosage form includes an injection dosage form, a respiratory tract administration dosage form, a cavity administration dosage form, a mucosal administration dosage form, and a skin administration dosage form.

5. Use of lomitapide and / or osimertinib in the preparation of a pharmaceutical composition for treating lung cancer / reversing lung cancer drug resistance; Preferably, the drug resistance is osimertinib resistance; Preferably, the lung cancer is selected from non-small cell lung cancer; Preferably, the non-small cell lung cancer is non-small cell lung cancer with EGFR mutation; Preferably, the EGFR mutation is EGFR-L858R / T790M mutation.

6. Use of lomitapide and / or osimertinib in the preparation of a pharmaceutical preparation for treating lung cancer / reversing lung cancer drug resistance; Preferably, the drug resistance is osimertinib resistance; Preferably, the lung cancer is selected from non-small cell lung cancer; Preferably, the non-small cell lung cancer is non-small cell lung cancer with EGFR mutation; Preferably, the EGFR mutation is EGFR-L858R / T790M mutation.

7. A method for regulating cell autophagy in vitro, characterized in that: The method comprises administering lomitapide.

8. Application of lomitapide in the preparation of drugs for regulating cellular autophagy.

9. A method for regulating apoptosis / metastasis of lung cancer cells / drug-resistant lung cancer cells in vitro, characterized in that: The method comprises administering lomistatapide and / or osimertinib.

10. Use of lomitripadip and / or osimertinib in the preparation of drugs for regulating apoptosis / metastasis of lung cancer cells / lung cancer resistant cells.

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