Combination for the treatment of lung cancer

Abstract

The application discloses a combined drug for treating lung cancer. It is found for the first time that lomethixol can be used alone or in combination with other drugs to have a significant anticancer effect on NSCLC patients and NSCLC patients resistant to osimertinib, and in addition, it is found that lomethixol is a potential autophagy inducer and can activate cell autophagy. The application provides a new clinical treatment method for NSCLC patients and NSCLC patients resistant to osimertinib, and has a wide application prospect.

Description

Technical Field

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

[0002] Lung cancer is a highly prevalent cancer 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 approximately 85% of lung cancer patients. Genetic mutations, particularly the L858R and T790M mutations in the epidermal growth factor receptor (EGFR), are the primary cause of NSCLC.

[0003] Osimertinib is an irreversible third-generation EGFR-TKI. Compared to first- and second-generation EGFR-TKIs, osimertinib exhibits stronger EGFR inhibition and can overcome acquired resistance to first- and second-generation drugs caused by the T790M mutation. However, acquired resistance to osimertinib is inevitable and remains the greatest obstacle to its 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 overcome the deficiencies of the prior art, the present invention provides a combined drug for treating lung cancer.

[0006] To achieve the above objectives, the present invention adopts the following technical solutions.

[0007] A first aspect of the present invention provides a pharmaceutical composition for treating lung cancer / reversing lung cancer drug resistance, wherein the pharmaceutical composition comprises lomitapide 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 EGFR mutated non-small cell lung cancer.

[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 gastrointestinal 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 preparations, effervescent preparations, emulsions, suspensions, syrups, drops, and chewable preparations.

[0016] Furthermore, the non-gastrointestinal 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.

[0017] A third aspect of the present invention provides the use of lomitripadipide 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 EGFR mutated non-small cell lung cancer.

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

[0022] A fourth aspect of the present invention provides the use of lomitapide 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 EGFR mutated non-small cell lung cancer.

[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, comprising administering lomitapide.

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

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

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

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

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

[0033] Advantages and beneficial effects of the present invention:

[0034] This application first discovered that lomitapide, used alone or in combination with other drugs, has significant anti-cancer effects in both NSCLC patients and osimertinib-resistant NSCLC patients. Furthermore, lomitapide was found to be a potential autophagy inducer, capable of activating cellular autophagy. This application provides a new clinical treatment method for NSCLC patients and osimertinib-resistant NSCLC patients, with broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 Figure 1 is a diagram showing the establishment and identification of osimertinib-resistant cells. 1A is a schematic diagram showing the establishment of H1975 / OR cells, 1B is a diagram showing cell viability, 1C is a flow cytometry and statistical diagram showing cell apoptosis, 1D is a diagram showing cell clone formation ability, 1E is a diagram showing cell 3D sphere formation ability, and 1F is a diagram showing cell invasion ability.

[0036] Figure 2 Figure 2A shows the in vitro reversal of NSCLC drug resistance by lomitapide and osimertinib. Figure 2B shows the 3D spheroid formation ability of cells after the addition of lomitapide and osimertinib.

[0037] Figure 3 Figure 3A shows the reversal of osimertinib resistance by lomitapide through autophagy. Figure 3A shows cell viability after lomitapide addition, Figure 3B shows KEGG analysis of pathways significantly enriched in cells treated with lomitapide compared with H1975 / OR cells, Figure 3C shows LC3 (green) fluorescence imaging of cells after treatment with the specified drugs, Figure 3D shows immunoblotting and expression statistics of autophagy-related proteins after treatment with the specified drugs, Figure 3E shows representative transmission electron microscopy images of cells treated with and without lomitapide, and Figure 3F shows immunoblotting and expression statistics of autophagy-related proteins induced by lomitapide under the action of CQ (chloroquine, an autophagy inhibitor).

[0038] 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;

[0039] Figure 5 Figure 5 is a graph showing the reversal of osimertinib resistance by lomitapide and osimertinib in vivo, wherein 5A is a graph showing the tumor volume of mice with transplanted tumors, 5B is a graph showing the tumor volume of mice with transplanted tumors, 5C is a graph showing the tumor weight of mice with transplanted tumors, 5D is a graph showing the tumor weight of mice with transplanted tumors, and 5E is a graph showing H&E staining and Ki67 immunohistochemical staining of the tumor;

[0040] Figure 6 Figure 4 is a dose-response matrix for the combination of lomitapide and osimertinib. DETAILED DESCRIPTION

[0041] The following provides definitions of some terms used in this specification. Unless defined otherwise, 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.

[0042] The present invention provides a pharmaceutical composition for treating lung cancer / reversing lung cancer drug resistance, wherein the pharmaceutical composition comprises lomitapide and / or osimertinib.

[0043] In some embodiments, lomitapide or osimertinib comprises lomitapide or osimertinib (hereinafter also referred to as the compound), or a pharmaceutically acceptable salt, hydrate, enantiomer, diastereomer, solvate, or crystalline form thereof.

[0044] In some embodiments, the pharmaceutically acceptable salts include acidic salts formed with inorganic and / or organic acids and basic salts formed with inorganic and / or organic bases. Furthermore, when the compound contains a basic moiety (such as, but not limited to, pyridine or imidazole) and an acidic moiety (such as, but not limited to, a carboxylic acid), zwitterions may form, and such zwitterions are included in the pharmaceutically acceptable salts described herein. Pharmaceutically acceptable (i.e., non-toxic, physiologically acceptable) salts are preferred, but other salts are also useful. Pharmaceutically acceptable salts of the compounds can be formed, for example, by reacting the compound with an amount of an acid or base in a medium, such as a medium in which the salt precipitates or an aqueous medium (followed by lyophilization).

[0045] Specific pharmaceutically acceptable salts include those salts that are, within the scope of sound medical judgment, suitable for contact with the tissues of humans and lower animals without excessive toxicity, irritation, allergic reaction, etc., and are commensurate with a reasonable benefit / risk ratio. 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.

[0046] Examples of pharmaceutically acceptable nontoxic acid addition salts are salts formed with inorganic acids, such as hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid and perchloric acid, or salts formed with organic acids, such as acetic acid, oxalic acid, maleic acid, tartaric acid, citric acid, succinic acid or malonic acid. Salts formed using conventional methods in the art, such as ion exchange methods, are also included. 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, heptanoate, hydroiodide, 2-hydroxy-ethanesulfonate, lactobionate, lactate, laurate, lauryl sulfate, malate, maleate, malonate, methanesulfonate. Pharmaceutically acceptable salts derived from appropriate bases include alkali metal, alkaline earth metal, and ammonium salts. Representative alkali metal or alkaline earth metal salts include sodium, lithium, potassium, calcium, magnesium salts, and the like.

[0047] In some embodiments, the hydrate refers to a compound that combines with water. An organic compound can form a complex with a solvent, in which it reacts or precipitates or crystallizes. These complexes are called solvates. When the solvent is water, the complex is called a hydrate.

[0048] In some embodiments, the solvate refers to a form of the compound or its salt that is associated with a solvent, typically formed by a solvolysis reaction. This physical association may include hydrogen bonding. Conventional solvents include water, methanol, ethanol, acetic acid, DMSO, THF, diethyl ether, and the like. In some cases, the solvate will be capable of separation, for example, when one or more solvent molecules are incorporated into the crystal lattice of the crystalline solid. Solvates include solution-state solvates and separable solvates.

[0049] In some embodiments, the crystalline form refers to a crystalline form of a compound having a specific crystal packing arrangement. Different crystalline forms typically 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 can lead to one crystalline form dominating. Various polymorphs of a compound can be prepared by crystallization under different conditions.

[0050] The pharmaceutical composition further includes pharmaceutically acceptable excipients.

[0051] In some embodiments, the pharmaceutically acceptable excipients include but are not limited to diluents, binders, surfactants, wetting agents, adsorption carriers, lubricants, fillers, and disintegrants.

[0052] 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 alginates, xanthan gum, hydroxypropyl cellulose, etc. The surfactant includes but is not limited to sodium lauryl sulfate, monoglyceride stearate, cetyl alcohol, etc. The humectant includes but is not limited to glycerin, starch, etc. The adsorption carrier includes but is not limited to starch, lactose, bentonite, bentonite, etc. The lubricant includes but is not limited to zinc stearate, talc, calcium and magnesium stearate, polyethylene glycol, polyoxyethylene monostearate, monolaurate sucrose, 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, soy polysaccharide, etc.

[0053] 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 lomitapide and osimertinib, and the combination of single preparations is a combination of a single preparation comprising lomitapide and a single preparation comprising osimertinib. In specific embodiments, the two single preparations in the combination of single preparations are administered simultaneously or sequentially.

[0054] In some embodiments, lomitripapine and osimertinib in the pharmaceutical composition can be administered simultaneously, separately, or sequentially. Simultaneously refers to the simultaneous administration of the two drugs. If not administered simultaneously, they are administered sequentially within a timeframe such that both drugs can act therapeutically within the same timeframe. Thus, sequential administration allows for administration of one drug within 5 minutes, 10 minutes, 15 minutes, 30 minutes, 1 hour, 2 hours, or several hours of one drug being administered, provided that the circulating half-life of the first administered drug allows for both drugs to be present simultaneously in therapeutically effective amounts. The time delay between administration of the components will vary depending on the precise nature of the components, their interactions, and their respective half-lives. Unlike simultaneous or sequential administration, separately refers to administration of one drug with a significant interval between the administration of the other, i.e., when the second drug is administered, the first administered drug may no longer be present in the bloodstream in a therapeutically effective amount.

[0055] 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.

[0056] The dosage forms of the pharmaceutical preparation include a dosage form for enteral administration and a dosage form for parenteral administration.

[0057] The dosage forms for administration through the gastrointestinal tract include tablets, granules, capsules, solutions, dry suspensions, powders, sustained-release preparations, effervescent preparations, emulsions, suspensions, syrups, drops, and chewable preparations.

[0058] The non-gastrointestinal 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.

[0059] In some embodiments, the injection dosage forms include but are not limited to 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.

[0060] 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. There are no special restrictions on the other drugs or agents. 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.

[0061] In some embodiments, the other drugs or agents that can be used for treatment and / or prevention, or adjuvant treatment and / or prevention include chemotherapy drugs, targeted therapy drugs, and immunotherapy drugs.

[0062] 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.

[0063] Targeted therapy drugs include EGFR mutation inhibitors, ALK inhibitors, KRAS G12C inhibitors, KRAS G12C inhibitors, ROS1 inhibitors, MET inhibitors, RET inhibitors, and HER2 inhibitors.

[0064] EGFR mutation inhibitors include first-generation drugs: gefitinib, erlotinib, and icotinib; second-generation drugs: afatinib and dacomitinib; and third-generation drugs: osimertinib, ametinib, and vometinib.

[0065] ALK inhibitors include the first-generation drug: crizotinib; the second-generation drugs: alectinib, ceritinib, brigatinib; and the third-generation drug: lorlatinib.

[0066] KRAS G12C inhibitors include sotolacizumab and adagracilib.

[0067] ROS1 inhibitors include entrectinib, ceritinib, and lorlatinib.

[0068] MET inhibitors include capmatinib, tepotinib, etc.

[0069] RET inhibitors include selpercatinib and pralsetinib.

[0070] HER2 inhibitors include Enhertu.

[0071] Immunotherapy drugs include tislelizumab, cemiplizumab, nivolumab, pembrolizumab, and atezolizumab.

[0072] The present invention will be further described below with reference to 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 present invention. The main features of the present invention may be applied to various embodiments without departing from the scope of the present invention.

[0073] Example

[0074] 1. Experimental methods

[0075] Cell lines and culture

[0076] 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 dose of osimertinib (Osim) was increased from 0.1 μM to 8 μM. All cells were cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum and 100 U / mL penicillin / streptomycin in a 37°C, 5% CO2 incubator.

[0077] Cell viability assay

[0078] The effects of drugs on NCI-H1975 and NCI-H1975 / OR cell viability were investigated using the Cell Counting Kit-8 (CCK-8) assay. Exponentially growing cells were seeded in 96-well plates and cultured to approximately 70–80% confluence. Cells were treated with various drug concentrations. Then, 10 µL of CCK-8 reagent was added to each well and incubated at 37°C for 2 hours. Absorbance at 450 nm was recorded using a microplate reader (Molecular Devices).

[0079] Apoptosis analysis

[0080] Apoptosis was detected 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).

[0081] Colony formation test

[0082] Cells were seeded in 6-well plates (2000 cells / well) and incubated with drugs for 14 days, with the medium changed every 3 days. Colonies were fixed with 4% paraformaldehyde and stained with 0.1% crystal violet solution. After washing with PBS, the stained colonies were photographed.

[0083] Three-dimensional (3D) spheroid assay

[0084] 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).

[0085] Cell migration analysis

[0086] The cultured cells were routinely treated by adding 600 μL of culture medium containing 20% ​​FBS to a 24-well plate, resuspending the cells in 10% FBS culture medium, and then adding them to a Transwell chamber (1×10 4 / well), incubated in a 37°C incubator for 24 h, and the invading cells in the upper chamber were fixed, stained with 0.1% crystal violet, and photographed under an inverted microscope.

[0087] Western blotting

[0088] Cell lysates were prepared using RIPA buffer supplemented with 446 protease / phosphatase inhibitors (Biomark), and Western blotting was performed as previously described.

[0089] Immunofluorescence

[0090] Immunofluorescence staining was performed as previously described. Briefly, cells were fixed with 4% formaldehyde, permeabilized with 0.2% Triton X-100, and blocked with 5% BSA. They were then incubated with the indicated antibodies overnight at 4°C and stained with a fluorescently conjugated secondary antibody 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).

[0091] Transmission electron microscopy

[0092] Transmission electron microscopy (TEM) was used to monitor autolysosome formation following drug treatment or without drug administration. Briefly, cells were harvested, prefixed with 2.5% glutaraldehyde solution, and fixed with 1% osmium tetroxide for 2 hours 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).

[0093] In vivo subcutaneous tumor model

[0094] BALB / c nude mice (6 weeks old) were provided by Liaoning Changsheng Biotechnology Co., Ltd. When the tumor volume reached about 100 mm 3 Mice were randomly divided into four groups. In vivo experiments: osimertinib 10 mg / kg was administered orally every two days, and lomitapide 20 mg / kg was administered intraperitoneally every two days. The control group received an equal volume of 2% DMSO saline. Mouse body weights were recorded during the experiment. Tumor volume was measured with a caliper and calculated using the formula ([width] 2 × [length] / 2). After the experiment, mice were sacrificed, tumors were removed, and tumor tissues were embedded for immunohistochemistry. 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.

[0095] Histochemical or immunohistochemical staining

[0096] Tumors were removed for histopathological examination. Tissues were cut into 5-µm-thick sections. Paraffin sections were dewaxed with xylene, rehydrated with 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 captured under a microscope.

[0097] RNA-seq

[0098] 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 total RNA was extracted and isolated using TRIzol (Invitrogen), quantified using NanoDrop, and assessed for integrity using Bioanalyzer (Agilent).

[0099] Statistical analysis

[0100] Significance was calculated using GraphPad Prism software. 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 three or more means, and unpaired Student's t-test was used to compare two means; *P < 0.05 was considered significantly different.

[0101] King's formula

[0102] King's formula was used to analyze the combined drug effect (Q value): Q = E(A+B) / (EA+EB-EA×EB). Q represents the measured combined effect, which is the ratio of the actual effect to the expected effect of the two drugs when used together. E(A+B) represents the effect of a specific dose of drug A and drug B when used together. EA represents the effect of drug A alone; EB represents the effect of drug B alone. When Q > 1.15, the two drugs have a synergistic effect; when 0.85 ≤ Q ≤ 1.15, the two drugs have an additive effect; and when Q < 0.85, the two drugs have an antagonistic effect.

[0103] 2. Experimental results

[0104] Construction and characterization of osimertinib-resistant cells in NSCLC

[0105] 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 investigate the effect of osimertinib on the proliferation of NSCLC cells in vitro, CCK-8 assay was used to detect cell viability in NSCLC parental cells and NCI-H1975 / OR cells. Figure 1 As shown in Figure B, the IC50 value of osimertinib in NCI-H1975 / OR cells was 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 effectively inhibited apoptosis of NCI-H1975 / OR cells but enhanced cell viability, proliferation, and metastasis in vitro.

[0106] Lomitapide reverses osimertinib resistance in vitro

[0107] The inhibitory effect of lomitapide on NCI-H1975 / OR cells was evaluated by CCK-8 assay. Figure 3 As shown in A, lomitapide (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 lomitapide and osimertinib, NCI-H1975 / OR cells were treated with osimertinib alone (Osim), lomitapide alone (Lomi), or a combination of osimertinib (6-7 μM) and lomitapide (3-6 μM) (Comb). The results showed that the addition of lomitapide 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).

[0108] 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.

[0109] Table 1 Evaluation of the combination index of osimertinib and lomitapide

[0110]

[0111] 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.

[0112] Lomitapide reverses osimertinib resistance in NSCLC by activating autophagy

[0113] 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 in the control group ( Figure 3B). Fluorescence signal and protein detection showed that after the addition of lomitapide, the classic autophagy 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 3 E). The autophagy inhibitor chloroquine (CQ) did not 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 lomitapide reverses osimertinib resistance in NSCLC by activating autophagy.

[0114] Lomitapide inhibits reversal of osimertinib resistance in vivo

[0115] To further investigate the in vivo therapeutic potential of lomitapide, an NCI-H1975 / OR xenograft model was established in BALB / c nude mice. Mice were divided into four groups (n=5 per group): those receiving intraperitoneal injection of 100 μL of saline with an equal concentration of 2% DMSO (control group), those treated with osim (10 mg / kg) alone, those treated with lomi (20 mg / kg) alone, or those treated with Comb (10 mg / kg of osim and 20 mg / kg of lomi). Comb significantly inhibited tumor growth compared to treatment with lomi or osim alone. Tumor size and weight were significantly smaller in the drug-treated groups, particularly in the Comb group, than in the control group. Lomitapide treatment had no effect on animal body weight, indicating a lack of toxicity. Hematoxylin and eosin (H&E) staining and Ki-67 assay were performed to assess cell proliferation. The results showed that xenograft cell proliferation was reduced in mice treated with lomitapide and osimertinib, consistent with the in vitro results. NCI-H1975 / OR cells are sensitive to the combination of omeprazole and osimertinib, which can inhibit cell viability and proliferation ( Figure 5 ).

[0116] The above embodiments are only provided for understanding the method and core concept of the present invention. It should be noted that, without departing from the principles of the present invention, a number of improvements and modifications may be made to the present invention by a person skilled in the art, and such improvements and modifications shall fall within the scope of protection of the claims of the present invention.

Claims

1. A pharmaceutical composition for reversing drug resistance in lung cancer, characterized in that: The pharmaceutical composition comprises lomitapide and osimertinib, the drug resistance is osimertinib resistance, and the lung cancer is non-small cell lung cancer with EGFR-L858R / T790M mutation.

2. The pharmaceutical composition according to claim 1, characterized in that The pharmaceutical composition further includes pharmaceutically acceptable excipients.

3. A pharmaceutical preparation for reversing drug resistance in lung cancer, characterized in that: The pharmaceutical preparation comprises the pharmaceutical composition according to any one of claims 1 to 2.

4. The pharmaceutical preparation according to claim 3, characterized in that The dosage forms of the pharmaceutical preparation include a dosage form for enteral administration and a dosage form for parenteral administration.

5. The pharmaceutical preparation according to claim 4, characterized in that The dosage forms for administration through the gastrointestinal tract include tablets, granules, capsules, solutions, powders, sustained-release preparations, effervescent preparations, emulsions, suspensions, syrups, drops, and chewable preparations.

6. The pharmaceutical preparation according to claim 4, characterized in that The dosage form for administration through the gastrointestinal tract includes a dry suspension.

7. The pharmaceutical preparation according to claim 4, characterized in that The non-gastrointestinal 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.

8. Use of lomitapide and osimertinib in the preparation of a pharmaceutical composition for reversing drug resistance in lung cancer, wherein the drug resistance is osimertinib resistance, and the lung cancer is non-small cell lung cancer with EGFR-L858R / T790M mutation.

9. Use of lomitapide and osimertinib in the preparation of a pharmaceutical preparation for reversing drug resistance in lung cancer, wherein the drug resistance is osimertinib resistance, and the lung cancer is non-small cell lung cancer with EGFR-L858R / T790M mutation.

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