Application of an EGFR inhibitor combined with a PARP inhibitor in the treatment of ovarian cancer

Abstract

The present invention relates to an application of an EGFR inhibitor combined with a PARP inhibitor in the treatment of ovarian cancer, and relates to the technical field of ovarian cancer treatment. The present invention obtains the EGFR inhibitor lapatinib and the PARP inhibitor niraparib by screening the FDA / CFDA approved compound library, and finds that lapatinib and niraparib have a good synergistic anti-ovarian cancer effect. In order to further study the molecular mechanism of the synergistic effect of the two, the present invention also detects the cell death pattern after the combined action of the two by apoptosis and protein immunoblotting. The combination of lapatinib and niraparib effectively inhibits the growth of ovarian cancer in an ovarian cancer animal model, showing good clinical research and application value, and is expected to become a new tool for the treatment of ovarian cancer.

Description

Technical Field

[0001] The present invention relates to the technical field of ovarian cancer treatment, and in particular to the use of an EGFR inhibitor combined with a PARP inhibitor in the treatment of ovarian cancer. Background Art

[0002] Ovarian cancer is currently one of the most lethal gynecological cancers worldwide and the second most common malignancy in women over 40, after breast cancer. Compared to other cancers, ovarian cancer has a diverse and complex histological origin, making clinical diagnosis and staging more challenging. According to the World Health Organization, approximately 310,000 new cases and 210,000 deaths from ovarian cancer will occur in 2020. Due to the difficulty in diagnosing ovarian cancer early, over 70% of patients are diagnosed at an advanced stage. Even after surgery and radiotherapy, the recurrence rate remains approximately 70%, resulting in a five-year survival rate of less than 30%. Once platinum resistance develops, patients are treated with other chemotherapies, such as gemcitabine and topotecan, but these are less effective and may even lead to multidrug resistance. Furthermore, the main FDA-approved, widely used targeted drugs are the VEGF inhibitor bevacizumab and PARP inhibitors. Other targeted agents, such as VEGF receptor inhibitors, EGFR tyrosine kinase inhibitors, folate receptor α inhibitors, and immune checkpoint inhibitors, have either been found to be ineffective in some patients, are in the early stages of clinical trials, or have demonstrated significant toxicity. Therefore, in response to the current dilemma in ovarian cancer treatment, it is of great significance to develop new therapies to overcome drug resistance. Although the initial response rate to PARP inhibitor treatment is good and the progression-free survival and overall survival are significantly increased, most cancers will eventually develop drug resistance. The development of new drug combinations is not only conducive to overcoming PARP inhibitor resistance and reducing the toxic side effects of high-dose PARP inhibitors used alone, but also helps to further explore the molecular mechanisms related to the emergence of drug resistance. Summary of the Invention

[0003] To solve the above problems, the present invention provides an application of an EGFR inhibitor combined with a PARP inhibitor in the treatment of ovarian cancer.

[0004] In a first aspect, the present invention provides a use of an EGFR inhibitor combined with a PARP inhibitor in the preparation of a drug for treating ovarian cancer.

[0005] Furthermore, the EGFR inhibitor includes lapatinib, or its tautomers, stereoisomers, prodrugs, pharmaceutically acceptable salts, hydrates or solvates.

[0006] Furthermore, the PARP inhibitor includes niraparib, or a tautomer, stereoisomer, prodrug, pharmaceutically acceptable salt, hydrate or solvate thereof.

[0007] In a second aspect, the present invention provides a drug for treating ovarian cancer, which comprises an EGFR inhibitor and a PARP inhibitor.

[0008] Furthermore, the EGFR inhibitor includes lapatinib, or a tautomer, stereoisomer, prodrug, pharmaceutically acceptable salt, hydrate or solvate thereof; the PARP inhibitor includes niraparib, or a tautomer, stereoisomer, prodrug, pharmaceutically acceptable salt, hydrate or solvate thereof.

[0009] Furthermore, the dosage form of the drug for treating ovarian cancer includes oral solution, capsule, oil drop, powder, tablet or injection.

[0010] Furthermore, the drug for treating ovarian cancer also includes a pharmaceutically acceptable carrier or excipient.

[0011] The above technical solution provided by the present invention has at least the following advantages compared with the prior art:

[0012] The present invention provides an application of an EGFR inhibitor combined with a PARP inhibitor in the treatment of ovarian cancer. The EGFR inhibitor lapatinib and the PARP inhibitor niraparib were obtained by screening the FDA / CFDA approved compound library. The present invention found that lapatinib and niraparib have a good synergistic anti-ovarian cancer effect. In order to further study the molecular mechanism of the synergistic effect of the two, the present invention also detected the cell death pattern after the combined action of the two by apoptosis and protein immunoblotting. The combination of lapatinib and niraparib effectively inhibited the growth of ovarian cancer in an ovarian cancer animal model, showing good clinical research and application value, and is expected to become a new tool for the treatment of ovarian cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate embodiments consistent with the invention and, together with the description, serve to explain the principles of the invention.

[0014] In order to more clearly illustrate the technical solutions in the present invention or the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, for ordinary technicians in this field, other drawings can be obtained based on these drawings without any creative work.

[0015] Figure 1 Figure 3 is a diagram showing that lapatinib is identified as a target drug for combination testing with niraparib in the present invention; Figure 1Middle: (A) Screening scheme; Cell viability of niraparib and non-niraparib drugs in SKOV3 (B) and ES-2 cells (C); (B) and (C) are FDA / CFDA-approved compound libraries used alone in SKOV3 (B) and ES-2 cells (C) as control groups, and in combination with the PARP inhibitor niraparib as a control group. A heat map was drawn based on cell viability to screen for synergistic drug combinations; (DG) After treating ovarian cancer cell lines (SKOV3 / ES-2 / OVCA420 and HEYA8) with gradient niraparib and lapatinib at different concentrations, MTS was added to detect cell proliferation ability; (H) shows the IC of niraparib and lapatinib on ovarian cancer cell lines (SKOV3 / ES-2 / OVCA420 and HEYA8) 50 .

[0016] Figure 2 The effect of lapatinib combined with niraparib on the growth of ovarian cancer cells in the present invention; Figure 2 Middle: (AD) Proliferation plots of SKOV3, ES-2, HeyA8, and OVCA420 cell lines after treatment with niraparib and lapatinib; "Synergy" indicates a CI less than 1, and "Antagonism" indicates a CI greater than 1. Data are presented as mean ± SD (n = 4 per group); (EH) SKOV3, ES-2, HeyA8, and OVCA420 cells were co-treated with niraparib and lapatinib to assess their clonogenic potential and statistical significance; (I) SKOV3 and ES-2 cells were co-treated with niraparib and lapatinib to assess their migration capacity; (J) Western blot analysis of changes in MMP2 and vimentin expression in SKOV3 and ES-2 cells after co-treatment with niraparib and lapatinib.

[0017] Figure 3 The effect of lapatinib combined with niraparib on apoptosis of ovarian cancer cells in the present invention; Figure 3 Middle: (A) Apoptosis of ES-2 cells after 48 h of treatment with niraparib and lapatinib alone or in combination; (BC) Western blot analysis was used to detect the expression changes of apoptosis-, cell cycle-, or proliferation-related proteins in SKOV3 and ES-2 cells after co-treatment with niraparib, and grayscale analysis was used for statistical analysis (DE).

[0018] Figure 4 The present invention is that lapatinib combined with niraparib inhibits ES-2 derived xenograft tumors in vivo; Figure 4 Middle: (A) 1x10 7 Cells were transplanted subcutaneously into mice, and when the tumor volume was ≥100 mm 3Dosing began at 4:00 p.m., and tumor volume was measured every 3 days. (B) Tumors were euthanized and weighed at the end of dosing. (C) Protein extraction was performed on tumor tissues from each group, and the expression of cell proliferation-related proteins was detected by immunoblotting. (D) IHC staining of Ki67. Scale bar, 50 μm. (E) Body weights of mice were measured every 3 days. (F) Mice in each group were stained with hematoxylin and eosin. Drug toxicity in mice in each group was assessed by hematoxylin and eosin staining.

[0019] Figure 5 The results of immunoblotting and statistical analysis are shown in the present invention. Figure 5 Middle: (AC) Immunoblotting was used to detect the expression changes of proteins related to the EGFR / AKT and ERK signaling pathways in SKOV3 and ES-2 cells after co-treatment with niraparib and lapatinib, and grayscale analysis was used for statistical analysis; (DE) Immunoblotting was used to detect the expression changes of proteins related to PARP and γH2AX in SKOV3 and ES-2 cells after co-treatment with niraparib and lapatinib, and grayscale analysis was used for statistical analysis; (F) Immunofluorescence staining of H2AX in cells treated with niraparib and lapatinib; scale bar, 50 μm. DETAILED DESCRIPTION

[0020] To make the objectives, technical solutions, and advantages of the present invention more clear, the technical solutions of the present invention will be clearly and completely described below in conjunction with the accompanying drawings. Obviously, the embodiments described are part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0021] Unless otherwise specified, various raw materials, reagents, instruments and equipment used in the present invention can be purchased from the market or prepared by existing methods.

[0022] In a first aspect, the present invention provides a use of an EGFR inhibitor combined with a PARP inhibitor in the preparation of a drug for treating ovarian cancer.

[0023] The present invention provides an application of an EGFR inhibitor combined with a PARP inhibitor in the treatment of ovarian cancer. The EGFR inhibitor lapatinib and the PARP inhibitor niraparib were obtained by screening the FDA / CFDA approved compound library. The present invention found that lapatinib and niraparib have a good synergistic anti-ovarian cancer effect. In order to further study the molecular mechanism of the synergistic effect of the two, the present invention also detected the cell death pattern after the combined action of the two by apoptosis and protein immunoblotting. The combination of lapatinib and niraparib effectively inhibited the growth of ovarian cancer in an ovarian cancer animal model, showing good clinical research and application value, and is expected to become a new tool for the treatment of ovarian cancer.

[0024] The EGFR inhibitors in the present invention are also called EGFR tyrosine kinase inhibitors (TKIs), which are a class of drugs that can inhibit the activity of EGFR, thereby preventing excessive proliferation of cancer cells. Currently, a number of EGFR inhibitors have been approved for marketing in China, including first-generation, second-generation and third-generation EGFR inhibitors, such as gefitinib, erlotinib, icotinib, afatinib, dacomitinib, osimertinib, ametinib, vumetinib, befortinib and lapatinib.

[0025] The PARP inhibitors of the present invention are medical agents that can affect the self-replication of cancer cells, including niraparib and the like.

[0026] In some specific embodiments, the EGFR inhibitor includes lapatinib, or a tautomer, stereoisomer, prodrug, pharmaceutically acceptable salt, hydrate or solvate thereof; the PARP inhibitor includes niraparib, or a tautomer, stereoisomer, prodrug, pharmaceutically acceptable salt, hydrate or solvate thereof.

[0027] In the present invention, "tautomers" refer to compounds in which one functional group changes its structure to become another functional group isomer, and can rapidly convert into each other, forming two isomers in dynamic equilibrium, and these two isomers are called tautomers.

[0028] The compounds of the present invention may include one or more asymmetric centers, and therefore may exist in a variety of stereoisomeric forms, for example, enantiomers and / or diastereomeric forms. For example, the compounds of the present invention may be individual enantiomers, diastereomers, or geometric isomers (e.g., cis and trans isomers), or may be in the form of a mixture of stereoisomers, including a racemic mixture and a mixture enriched in one or more stereoisomers. Isomers may be separated from the mixture by methods known to those skilled in the art, including chiral high pressure liquid chromatography (HPLC) and the formation and crystallization of chiral salts; or preferred isomers may be prepared by asymmetric synthesis.

[0029] In the present invention, "prodrugs" are also referred to as prodrugs, drug precursors, and prodrugs. They refer to compounds that are inactive or less active in vitro after chemical structural modification of a drug, but release the active drug in vivo through enzymatic or non-enzymatic conversion to exert its pharmacological effect. Prodrugs include, for example, compounds of the present invention in which a hydroxyl, amino, or sulfhydryl group is bonded to any group, which, when administered to a patient, can be cleaved to form a hydroxyl, amino, or sulfhydryl group. Therefore, representative examples of prodrugs include (but are not limited to) acetate / amide, formate / amide, and benzoate / amide derivatives of the hydroxyl, sulfhydryl, and amino functional groups of the compound of formula (I). In addition, in the case of formic acid (-COOH), esters such as methyl esters and ethyl esters can be used. The ester itself can be active and / or can be hydrolyzed under human in vivo conditions. Suitable pharmaceutically acceptable in vivo hydrolyzable ester groups include those that are easily decomposed in the human body to release the parent acid or its salt.

[0030] Those skilled in the art will appreciate that organic compounds can form complexes with solvents in which they react or from which they precipitate or crystallize. These complexes are referred to as "solvates." When the solvent is water, the complex is referred to as a "hydrate." The present invention encompasses all solvates of the compounds of the present invention.

[0031] In the present invention, "pharmaceutically acceptable salts" refer to salts that are suitable for contact with the tissues of humans and lower animals without excessive toxicity, irritation, allergic reactions, etc., within the scope of sound medical judgment, and are commensurate with a reasonable benefit / risk ratio. Pharmaceutically acceptable salts are well known in the art. For example, Berge et al. describe pharmaceutically acceptable salts in detail in J. Pharmaceutical Sciences (1977) 66: 1-19. Pharmaceutically acceptable salts of the compounds of the present invention include salts derived from suitable inorganic and organic acids and inorganic and organic bases. Examples of pharmaceutically acceptable non-toxic 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, dodecylsulfate, ethanesulfonate, formate, fumarate, gluconate, glycerophosphate, gluconate, hemisulfate, heptanoate, hexanoate, hydroiodide, 2-hydroxy-ethanesulfonate, lactobionate, lactate, laurate, lauryl sulfate, malate, maleate, malonate, methanesulfonate, 2-naphthosulfonate, nicotinate, nitrate, oleate, oxalate, palmitate, dihydroxynaphthoate, pectinate, persulfate, 3-phenylpropionate, phosphate, picrate, pivalate, propionate, stearate, succinate, sulfate, tartrate, thiocyanate, p-toluenesulfonate, undecanoate, valerate, and the like. Pharmaceutically acceptable salts derived from suitable bases include alkali metal, alkaline earth metal, ammonium and N (C 4 alkyl) salts. Representative alkali metal or alkaline earth metal salts include sodium, lithium, potassium, calcium, magnesium salts, and the like. Other pharmaceutically acceptable salts include non-toxic ammonium salts, quaternary ammonium salts and amine cations formed with counterions, such as halides, hydroxides, formates, sulfates, phosphates, nitrates, lower alkyl sulfonates and aryl sulfonates, if appropriate.

[0032] In the present invention, "solvate" refers to a form of a compound or its salt that is combined with a solvent, usually formed by a solvolysis reaction. Conventional solvents include water, methanol, ethanol, acetic acid, DMSO, THF, diethyl ether, etc. The compounds described herein can be prepared, for example, in a crystalline form and can be solvated. Suitable solvates include pharmaceutically acceptable solvates and further include stoichiometric solvates and non-stoichiometric solvates. 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. "Solvate" includes solvates in the solution state and separable solvates. Representative solvates include hydrates, ethanolates and methanolates.

[0033] In a second aspect, based on the same inventive concept, the present invention provides a drug for treating ovarian cancer, which includes an EGFR inhibitor and a PARP inhibitor.

[0034] In some specific embodiments, the EGFR inhibitor includes lapatinib, or a tautomer, stereoisomer, prodrug, pharmaceutically acceptable salt, hydrate or solvate thereof; the PARP inhibitor includes niraparib, or a tautomer, stereoisomer, prodrug, pharmaceutically acceptable salt, hydrate or solvate thereof.

[0035] In some specific embodiments, the dosage form of the drug for treating ovarian cancer includes oral solution, capsule, oil drop, powder, tablet or injection.

[0036] In some specific embodiments, the drug for treating ovarian cancer further comprises a pharmaceutically acceptable carrier or excipient.

[0037] In the present invention, "pharmaceutical" can also be called "pharmacologically acceptable", which refers to a substance that is not biologically or otherwise substantially undesirable, that is, the substance can be administered to an individual without causing any undesirable biological effect or interacting in a harmful manner with any other components of the composition containing such substance.

[0038] In the present invention, "carrier" may also be referred to as "drug carrier", which refers to a system that can change the way drugs enter the human body and their distribution in the body, control the release rate of drugs, and deliver drugs to target organs.

[0039] In this context, "excipients," also known as "auxiliary materials," include saline, glucose, vitamin C, and amino acids, and refer to additives in pharmaceutical preparations other than the principal drug. Examples include binders, fillers, disintegrants, and lubricants in tablets; alcohol, vinegar, and medicinal juices in traditional Chinese medicine pills; the base component of semisolid ointments and creams; and preservatives, antioxidants, flavoring agents, fragrances, cosolvents, emulsifiers, solubilizers, osmotic pressure regulators, and colorants in liquid preparations.

[0040] The present invention will be further described below in conjunction with specific examples. It should be understood that these examples are intended to illustrate the present invention only and are not intended to limit the scope of the invention. The experimental methods in the following examples where specific conditions are not specified are generally measured in accordance with national standards. If there are no corresponding national standards, then the methods are carried out in accordance with general international standards, conventional conditions, or the conditions recommended by the manufacturer.

[0041] Example

[0042] This example provides an application of an EGFR inhibitor combined with a PARP inhibitor in the treatment of ovarian cancer. The specific process is as follows:

[0043] Step 1. Cell Lines and Culture Conditions: Ovarian cell lines (SKOV3, ES-2, OVCA420, and HEYA8) were purchased from the American Type Culture Collection (ATCC). SKOV3, ES-2, OVCA420, and HEYA8 cells were cultured in DMEM medium supplemented with 10% fetal bovine serum, 100 U / ml penicillin, and 100 μg / ml streptomycin. Cells were cultured in a fully humidified atmosphere at 37°C with 5% CO2 / 95% air.

[0044] Step 2: Cell viability assay and combination index (CI): SKOV3, ES-2, OVCA420, and HEYA8 cells were plated at 8×10 cells per well. 3 The cells were seeded at a density of 100 μg / mL in a 96-well plate and then treated with the compound for 72 hours. 20 μL of MTS reagent was added to each well, the cells were incubated for 1-2 hours, and then the absorbance was measured at 490 nm. The results of the MTS test were analyzed using GraphPad Prism8 software and were consistent with the half-inhibition rate of cell proliferation (half-inhibitory concentration). The results are expressed as the average of three repeated measurements. Calcusyn software was used to calculate the CI value and affected fraction (FA) of the combined experiment. FA refers to the proportion of cells whose cell viability is affected. Survival plots and scatter plots of CI values were drawn using GraphPad Prism8.

[0045] Step 3: Colony formation rate: SKOV3, ES-2, OVCA420 and HEYA8 cells were plated at 4×10 per well. 3The concentration of cells was inoculated into a 12-well plate. 1 mL of culture medium containing 10% fetal bovine serum was added to each well. After the cells attached, drugs were added to treat the cells. An equal volume of DMSO was added as a control. After 1 week, the cells formed colonies, which were washed with PBS, fixed with 4% paraformaldehyde for 30 minutes, removed with 4% paraformaldehyde, and stained with 2% crystal violet solution for 15 minutes. Finally, the floating color was washed in water and air-dried. The number of cell colonies in each well was counted, and the colony formation rate was calculated: colony formation rate (%) / colony formation rate (control) (%).

[0046] Step 4: Western Blot Analysis: First, lyse drug-treated cells or tumor tissues with RIPA lysis buffer and quantify BCA protein for Western blot analysis. Image J software was used for optical density analysis.

[0047] Step 5: Flow cytometry: Lapatinib, niraparib, and the combination drug were added to ES-2 cells. After 48 hours of treatment with the corresponding drug, the digested cells were collected using the supernatant. 100 μL of binding buffer, 1 μL of RNase (Sigma, USA), 2 μL of Annexin V-FITC (BD, USA), and 2 μL of propidium iodide (PI) (Sigma, USA) were added to each tube and incubated at room temperature away from light. Under these conditions, the cells were incubated for 15 minutes and analyzed by flow cytometry using a FACS Calibur (BD).

[0048] Step 6: Immunofluorescence: Place the glass cover in a 24-well plate and inoculate 8×10 3 Cells. Cells were treated with different concentrations of lapatinib and niraparib and incubated at 37°C, 5% carbon dioxide, and 95% humidity for 48 hours. Fixed cells were permeabilized with 0.2% Triton (Sangon, China) in 1× PBS for 30 minutes. Cells were incubated in 1% BSA (Sangon) and 0.2% Triton / PBS for 30 minutes. They were then incubated with rabbit anti-γH2AX antibody (1:400) at 4°C overnight. The cells were then washed three times with 0.2% triton / PBS for 3 minutes each and incubated with a second anti-rabbit 800 antibody for 1 hour in the dark. Cell nuclei were stained with DAPI (D9542, Sigma) for 5 minutes and washed three times with 0.2% triton / PBS for 5 minutes each. Images were captured using an Olympus inverted fluorescence microscope.

[0049] Step 7: Xenograft tumor growth: 1 × 10 7 ES-2 cells. When the average tumor volume reaches 100 mm 3At 18 days, mice were randomly divided into groups and injected intraperitoneally. Body weight and tumor size were measured. After administration, the mice were removed, and tumors and major organs were dissected for subsequent experiments.

[0050] The test results are as follows:

[0051] Figure 1 Figure 3 is a diagram showing that lapatinib is identified as a target drug for combination testing with niraparib in the present invention; Figure 1 Middle: (A) Screening scheme; Cell viability of niraparib and non-niraparib drugs in SKOV3 (B) and ES-2 cells (C); (B) and (C) are FDA / CFDA-approved compound libraries used alone in SKOV3 (B) and ES-2 cells (C) as control groups, and in combination with the PARP inhibitor niraparib as a control group. A heat map was drawn based on cell viability to screen for synergistic drug combinations; (DG) After treating ovarian cancer cell lines (SKOV3 / ES-2 / OVCA420 and HEYA8) with gradient niraparib and lapatinib at different concentrations, MTS was added to detect cell proliferation ability; (H) shows the IC of niraparib and lapatinib on ovarian cancer cell lines (SKOV3 / ES-2 / OVCA420 and HEYA8) 50 .

[0052] Figure 2 The effect of lapatinib combined with niraparib on the growth of ovarian cancer cells in the present invention; Figure 2 Middle: (AD) Proliferation plots of SKOV3, ES-2, HeyA8, and OVCA420 cell lines after treatment with niraparib and lapatinib; "Synergy" indicates a CI less than 1, and "Antagonism" indicates a CI greater than 1. Data are presented as mean ± SD (n = 4 per group); (EH) SKOV3, ES-2, HeyA8, and OVCA420 cells were co-treated with niraparib and lapatinib to assess their clonogenic potential and statistical significance; (I) SKOV3 and ES-2 cells were co-treated with niraparib and lapatinib to assess their migration capacity; (J) Western blot analysis of changes in MMP2 and vimentin expression in SKOV3 and ES-2 cells after co-treatment with niraparib and lapatinib.

[0053] Figure 3 The effect of lapatinib combined with niraparib on apoptosis of ovarian cancer cells in the present invention; Figure 3 Middle: (A) Apoptosis of ES-2 cells after 48 h of treatment with niraparib and lapatinib alone or in combination; (BC) Western blot analysis was used to detect the expression changes of apoptosis-, cell cycle-, or proliferation-related proteins in SKOV3 and ES-2 cells after co-treatment with niraparib, and grayscale analysis was used for statistical analysis (DE).

[0054] Figure 4 The present invention is that lapatinib combined with niraparib inhibits ES-2 derived xenograft tumors in vivo; Figure 4 Middle: (A) 1x10 7 Cells were transplanted subcutaneously into mice, and when the tumor volume was ≥100 mm 3 Dosing began at 4:00 p.m., and tumor volume was measured every 3 days. (B) Tumors were euthanized and weighed at the end of dosing. (C) Protein extraction was performed on tumor tissues from each group, and the expression of cell proliferation-related proteins was detected by immunoblotting. (D) IHC staining of Ki67. Scale bar, 50 μm. (E) Body weights of mice were measured every 3 days. (F) Mice in each group were stained with hematoxylin and eosin. Drug toxicity in mice in each group was assessed by hematoxylin and eosin staining.

[0055] Figure 5 The results of immunoblotting and statistical analysis are shown in the present invention. Figure 5 Middle: (AC) Immunoblotting was used to detect the expression changes of proteins related to the EGFR / AKT and ERK signaling pathways in SKOV3 and ES-2 cells after co-treatment with niraparib and lapatinib, and grayscale analysis was used for statistical analysis; (DE) Immunoblotting was used to detect the expression changes of proteins related to PARP and γH2AX in SKOV3 and ES-2 cells after co-treatment with niraparib and lapatinib, and grayscale analysis was used for statistical analysis; (F) Immunofluorescence staining of H2AX in cells treated with niraparib and lapatinib; scale bar, 50 μm.

[0056] The present invention found that the combination of niraparib and lapatinib can inhibit EGFR activation, thereby inhibiting the expression of p-AKT and p-ERK, showing a good synergistic effect in inhibiting ovarian cancer cell apoptosis. It can also induce DNA damage and increase PARP cleavage, ultimately inhibiting the progression of ovarian cancer in vitro and in vivo. The results of the present invention provide new ideas for the clinical combination strategy of two different inhibitors and provide a new treatment approach to alleviate the current drug resistance of ovarian cancer patients.

[0057] In summary, the present invention provides an application of an EGFR inhibitor combined with a PARP inhibitor in the treatment of ovarian cancer. By screening the FDA / CFDA approved compound library, the EGFR inhibitor lapatinib and the PARP inhibitor niraparib were obtained. The present invention found that lapatinib and niraparib have a good synergistic anti-ovarian cancer effect. In order to further study the molecular mechanism of the synergistic effect of the two, the present invention also detected the cell death pattern after the combined action of the two by apoptosis and protein immunoblotting. The combination of lapatinib and niraparib effectively inhibited the growth of ovarian cancer in an ovarian cancer animal model, showing good clinical research and application value, and is expected to become a new tool for the treatment of ovarian cancer.

[0058] The foregoing description is intended only to provide specific embodiments of the present invention, which will enable those skilled in the art to understand and implement the present invention. 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 the present invention. Therefore, the present invention is not intended to be limited to the embodiments shown herein, but is intended to be accorded the widest scope consistent with the principles and novel features claimed herein.

Claims

1. Use of an EGFR inhibitor combined with a PARP inhibitor in the preparation of a drug for treating ovarian cancer, characterized in that: The EGFR inhibitor is lapatinib, or a pharmaceutically acceptable salt thereof, and the PARP inhibitor is niraparib, or a pharmaceutically acceptable salt thereof.

2. The use according to claim 1, characterized in that The dosage forms of the drug for treating ovarian cancer include oral liquid, capsule, oil drop, powder, tablet or injection.

3. The use according to claim 1, characterized in that The drug for treating ovarian cancer also includes a pharmaceutically acceptable carrier.

Images