Preparation for evaluating lung cancer EGFR-TKI treatment drug resistance by utilizing inosine

Abstract

The invention discloses application of a reagent for detecting inosine content in a biological sample in preparation of a lung cancer EGFR-TKI treatment drug resistance evaluation preparation. The drug resistance of a lung cancer patient to an EGFR-TKI drug can be judged through detection of an in-vitro biological sample, traumatic injury to the patient caused by an invasive living body detection mode of secondary biopsy or liquid biopsy is avoided, and individualized precise treatment of the lung cancer patient is facilitated. And a new strategy for non-invasive in-vitro sample detection and evaluation of EGFR-TKI treatment drug resistance is developed.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of biological detection, and relates to a preparation and method for evaluating lung cancer EGFR-TKI treatment drug resistance by using inosine. BACKGROUND

[0002] In non-small cell lung cancer (NSCLC), lung adenocarcinoma is a common pathological type, and epidermal growth factor receptor (EGFR) gene mutation has a high incidence in lung adenocarcinoma patients. In Asian population, the EGFR mutation rate of lung adenocarcinoma patients is as high as 30% - 55%, which makes EGFR a key target for the treatment of lung adenocarcinoma. EGFR belongs to the HER receptor family and is a transmembrane tyrosine kinase receptor that plays an important role in regulating cell division and death. When EGF ligand binds to EGFR, it forms a homodimer or heterodimer, which in turn phosphorylates tyrosine kinase and activates downstream cell proliferation, migration, anti-apoptosis and other signaling pathways.

[0003] EGFR tyrosine kinase inhibitor (EGFR-TKI) as a standard treatment for EGFR mutation-positive lung adenocarcinoma patients, has significantly prolonged the survival of patients and improved the quality of life since its inception. Currently, the EGFR-TKI drugs used in clinical practice have developed to the third generation:

[0004] First generation EGFR-TKI: such as gefitinib, erlotinib, icotinib, etc. They reversibly bind to the ATP binding site of the EGFR tyrosine kinase domain, inhibit the formation of phosphorylated tyrosine residues of EGFR. For patients with EGFR mutations, the first generation of EGFR-TKI can play a good role in anti-tumor cell proliferation, but patients usually develop acquired drug resistance after 8-14 months of treatment.

[0005] Second generation EGFR-TKI: represented by afatinib and dacomitinib, as irreversible inhibitors of EGFR and HER2, they not only can competitively occupy the ATP binding site of EGFR, but also can alkylate or covalently bind with the amino acid residues unique to the vicinity of the opening of the EGFR binding pocket, realizing irreversible inhibition of EGFR. However, the second generation of EGFR-TKI also faces the problem of drug resistance, and the median progression-free survival of patients is about 13-14 months.

[0006] Third-generation EGFR-TKI: osimertinib is a typical drug in it, which contains Michael acceptor structure in the molecule, and forms covalent bond with cysteine-797 residue in ATP binding site through unsaturated acryloyl chain, and irreversibly covalently binds with the catalytic active center of EGFR receptor. Osimertinib has 200 times stronger inhibitory effect on L858R / T790M mutation than on wild-type EGFR, providing a new treatment option for patients with first-generation EGFR-TKI resistance and T790M mutation, and the median progression-free survival of patients can be prolonged to about 19 months. However, even so, patients will inevitably develop drug resistance after a period of treatment with osimertinib.

[0007] The currently known EGFR-TKI resistance mechanisms are complex and diverse, mainly including the following categories:

[0008] Target mutation: the most common T790M mutation in the first and second generations of EGFR-TKI resistance, which changes the conformation of the originally sensitive EGFR receptor to TKI, thereby avoiding the inhibitory effect of the drug; C797S mutation, which is common in third-generation EGFR-TKI osimertinib resistance, will make osimertinib unable to continue to form a covalent bond in the ATP binding domain, losing the effect of inhibiting EGFR activation. In addition, KRAS mutation may be related to the primary resistance of EGFR-TKI, and the deletion of BIM gene polymorphism (the 2nd intron of BIM gene in East Asian population has a deletion polymorphism, which leads to the lack of expression of BIM subtypes with pro-apoptotic activity) will also cause primary resistance to EGFR-TKI or weaken the clinical efficacy of TKI.

[0009] Activation of bypass signaling pathways: MET amplification is one of the most important acquired resistance targets other than T790M. After MET gene amplification, the c-MET protein encoded by it is overexpressed, activating downstream PI3K-AKT and RAS-MAPK signaling pathways, bypassing the EGFR signaling pathway, leading to continuous proliferation of tumor cells and resistance to EGFR-TKI. HER2 amplification can also activate the HER2 itself signaling pathway, promote the survival and proliferation of tumor cells, and cause drug resistance. In addition, the activation of bypass signaling pathways such as PI3K / AKT / mTOR pathway activation, interaction with IGF1R pathway, FGFR1 activation, BRAF mutation, etc. are all related to EGFR-TKI resistance.

[0010] Organizational or phenotypic transformation: some patients will transform from lung adenocarcinoma to small cell lung cancer or from epithelial cells to mesenchymal cells after receiving EGFR-TKI treatment, resulting in tumor cells no longer sensitive to EGFR-TKI. Studies have found that in the mechanism of resistance to first-generation EGFR-TKI, about 11% of patients will transform from lung adenocarcinoma to small cell lung cancer, and EGFR mutant adenocarcinoma with Rb1 and p53 gene inactivation is more prone to such transformation.

[0011] Tumor microenvironment influence: The tumor microenvironment is composed of various cellular components (such as tumor-infiltrating lymphocytes, tumor-associated myeloid cells, natural killer cells, neutrophils, and tumor-associated fibroblasts) and non-cellular components (such as blood vessels, extracellular matrix, cytokines, and exosomes). Tumor-associated macrophages can induce drug resistance by activating multiple signaling pathways (such as AKT, STAT3, and ERBB2) and secreting exosomes as important drug resistance modulators; tumor-associated fibroblasts secrete various cytokines (such as IL-6, IL-8, and HGF) that can activate intracellular signaling pathways in tumor cells, promoting the formation of drug resistance; components such as collagen, integrin-β1, and proteoglycans in non-cellular components can also induce drug resistance by regulating cell pathways.

[0012] However, the current clinical prediction of EGFR-TKI resistance in lung adenocarcinoma patients mainly relies on secondary biopsy or liquid biopsy after disease progression during treatment to detect drug resistance-related gene mutations and signal pathway activation. However, this method has certain limitations. Secondary biopsy is an invasive procedure that may cause pain and complications for patients, and due to tumor heterogeneity, the biopsy sample may not fully reflect the overall drug resistance of the tumor. Although liquid biopsy has the advantages of non-invasiveness and repeatability, the sensitivity and specificity of detection still need to be improved, and false negative or false positive results may occur. Therefore, when patients receive EGFR-TKI treatment, there is a lack of an effective, accurate, and non-invasive method to predict drug resistance to the drug, so as to develop personalized treatment plans in advance, improve treatment effectiveness, and improve patient prognosis. SUMMARY

[0013] In the research of lung cancer EGFR-TKI drug resistance, we conceived a method to determine the lung cancer patient's EGFR-TKI drug resistance by detecting specific components such as proteins, genes or compounds in vitro samples in vitro. First, we used lung adenocarcinoma cell lines to construct EGFR-TKI drug-resistant cell lines, compared the changes of metabolites before and after the lung cancer cells developed drug resistance, and found that inosine, an intermediate product of nucleotide metabolism, was significantly increased in drug-resistant lung cancer cells. Subsequently, we collected a large number of blood samples from lung cancer patients receiving EGFR-TKI, and compared the changes of inosine content in the patient's blood before and after the drug resistance was developed. Unexpectedly, we found that the inosine content in the samples before and after the drug resistance was developed had a significant change, and the inosine content in the samples after the drug resistance was developed was significantly higher than that in the samples just receiving EGFR-TKI treatment. For this new discovery, we confirmed the results and the feasibility of clinical application through lung adenocarcinoma cells and mouse models. Therefore, the present application includes the following technical solutions:

[0014] The first aspect of the present application provides the use of a reagent for detecting the content of inosine in a biological sample in the preparation of a lung cancer EGFR-TKI treatment drug resistance evaluation preparation.

[0015] Alternatively, the above-mentioned EGFR-TKI is a drug selected from the group consisting of gefitinib, erlotinib, icotinib, afatinib, dacomitinib and osimertinib.

[0016] Preferably, the lung cancer is lung adenocarcinoma.

[0017] The above-mentioned biological sample can be selected from the group consisting of whole blood, blood plasma, blood serum, lung tissue sampling, tissue fluid, saliva, oral mucosa, nasopharyngeal secretion, body fluid and urine, and the biological sample is preferably blood.

[0018] In the above-mentioned application, without inosine supplementation or administration to the lung cancer patient, the inosine content detected before the lung cancer patient is treated with EGFR-TKI is the baseline level, and when the ratio of the inosine content detected during treatment to the baseline level is not less than 1.8, 1.9, 2.0, 2.1, 2.2 or 2.3, it is determined that the lung cancer patient has developed resistance to EGFR-TKI, i.e. the lung cancer patient is no longer sensitive to EGFR-TKI treatment, and it is recommended to change the treatment regimen; when the ratio of the inosine content detected during treatment to the baseline level is less than 1.8, it is determined that the lung cancer patient has not developed resistance to EGFR-TKI, i.e. the lung cancer patient is sensitive to EGFR-TKI treatment, and it is recommended to continue to use the present treatment regimen.

[0019] The judgment criteria can be used as a mathematical model for evaluating lung cancer EGFR-TKI treatment drug resistance.

[0020] As a specific application embodiment, the above-mentioned preparation is preferably a kit for evaluating the drug resistance and sensitivity of lung cancer patients to EGFR-TKI drugs by detecting the inosine content in the biological sample.

[0021] In one embodiment, the detection of the inosine content in the biological sample is carried out by the following method (1): the inosine content is detected by using inosine or inosine reaction products such as decomposition products hypoxanthine involved in fluorescence or luminescence reactions; accordingly, the reagent includes reagents for catalyzing inosine reactions and / or subsequent reactions of inosine reaction products (such as PicoProbe, transaminase, and chromogenic enzyme required for the reaction of hypoxanthine converted from inosine by purine nucleoside phosphorylase (PNP), etc., such as the reagents contained in the Inosine Quantification Assay Kit Fluorometric (ab126286) of the American abcam company) and inosine standard.

[0022] In another embodiment, the detection of the inosine content in the biological sample is carried out by the following method (2): the inosine content is detected by using inosine antibodies based on the principle of antigen-antibody affinity, which are monoclonal antibodies or polyclonal antibodies; accordingly, the reagent includes inosine antibodies and inosine standards, and the kit (2) includes the above-mentioned reagents and reagents for pretreatment of biological samples.

[0023] Further, the kit (2) can be a latex immunoturbidimetry kit, a magnetic microparticle chemiluminescence kit, a radioimmunoassay kit, or an immunochromatographic test strip.

[0024] In yet another embodiment, the detection of the inosine content in the biological sample is carried out by the following method (3): the inosine content is detected by using liquid chromatography tandem triple quadrupole mass spectrometry (LC-MS / MS); accordingly, the reagent includes mobile phases used in liquid chromatography, such as aqueous phase A (aqueous solution) and / or organic phase B (organic solution), inosine standards; the kit (3) includes the above-mentioned reagents and reagents for pretreatment of biological samples.

[0025] Further, the above-mentioned kit further includes an instruction manual, which includes a mathematical model for judging the drug resistance of lung cancer EGFR-TKI treatment, i.e., the inosine content / baseline level ratio cutoff standard.

[0026] The above-mentioned instruction manual can be written on bottles, test tubes and the like, plates, or on a separate piece of paper, or on the outside or inside of the container, such as a paper with an operation demonstration video APP download window such as a two-dimensional code, and the instruction manual can also be in the form of multimedia, such as a CD, a U disk, a network disk, etc.

[0027] The second aspect of the present application provides the use of inosine in the screening and development of EGFR-TKI drugs, wherein inosine is used as an inhibition target of EGFR-TKI drugs, that is, the developed new EGFR-TKI drugs can inhibit the expression of inosine in lung cancer patients.

[0028] The present application develops a new method for evaluating the resistance of EGFR-TKI treatment by non-invasive in vitro sample detection, which can determine whether the lung cancer patients have developed resistance to EGFR-TKI drugs by detecting the changes in the content of inosine in in vitro biological samples, avoid the traumatic injury caused by secondary biopsy or liquid biopsy to patients, and help to realize the individualized precision management of lung cancer patients, which has important clinical significance and application value for the evaluation of lung cancer EGFR-TKI treatment resistance and the formulation or adjustment of individualized precision treatment plan for lung cancer. BRIEF DESCRIPTION OF DRAWINGS

[0029] Figure 1 It is shown that the content of inosine in the EGFR-TKI-resistant lung adenocarcinoma cell line constructed by long-term induction treatment with osimertinib is significantly increased. Among them, A: the construction process of osimertinib-resistant cells; B: compared with the wild-type cell line, the drug-resistant cells are resistant to EGFR-TKI drugs represented by osimertinib;

[0030] Figure 2 It is shown that the metabolomics analysis of EGFR-TKI-resistant lung adenocarcinoma cell lines. Among them, A: metabolomics results show that the content of inosine in EGFR-TKI-resistant cell lines is significantly increased; B: the content of inosine in osimertinib short-term treatment, drug persistence survival (DTP) cells and drug-resistant cell lines is significantly increased; C: the content of inosine in the blood of TKI-resistant lung cancer patients is increased by 1.8 times compared with sensitive patients; D: the change of inosine after the withdrawal of osimertinib.

[0031] Figure 3 It is shown that the supplementation of inosine can induce the resistance of lung adenocarcinoma cells to EGFR-TKI drugs. Among them, A: in PC-9 and H1975 cell lines, the supplementation of inosine can weaken the killing effect of osimertinib on lung adenocarcinoma cell lines, and the effect is dose-dependent with the concentration of supplemented inosine; B: cell clone experiment proves that the supplementation of inosine can weaken the killing effect of osimertinib; C: tumor xenograft model tumor picture and weight size, showing that the feeding of inosine to mice can weaken the killing effect of osimertinib on subcutaneous tumors. DETAILED DESCRIPTION

[0032] We first proposed to predict the malignant degree and EGFR-TKI sensitivity of lung adenocarcinoma by detecting inosine expression, which provides a reference for optimizing treatment decisions and improving the prognosis of lung cancer patients. The judgment of EGFR-TKI drug sensitivity of patients can effectively guide the individualized treatment of lung adenocarcinoma patients, improve clinical benefit, and avoid unnecessary waste of medical resources.

[0033] The present application provides a method for accurately predicting the drug resistance of lung adenocarcinoma patients to EGFR-TKI drugs and related products, thereby overcoming the difficulty in effectively, accurately and non-invasively predicting drug resistance before and after patients receive EGFR-TKI treatment in the prior art. By detecting the change of inosine content in the patient sample, such as blood, and combining the inosine content / baseline level ratio to evaluate EGFR-TK drug resistance, the following goals can be achieved:

[0034] Predict drug resistance in advance: By detecting the change of inosine level in the sample of lung adenocarcinoma patients, such as blood, before the patient receives EGFR-TKI drug treatment, it can predict whether the patient will develop drug resistance, providing early guidance for clinical treatment.

[0035] Develop individualized treatment plans: Based on the prediction results, doctors can develop individualized treatment plans for patients. For patients predicted to be drug-resistant, other alternative or combined treatment plans can be selected to avoid blind use of EGFR-TKI drugs, improve the effectiveness of treatment, and reduce unnecessary drug adverse reactions and economic burden. After the patient receives EGFR-TKI treatment, the patient's blood inosine content is followed up to predict the patient's drug resistance, and the treatment strategy is adjusted as soon as possible when the inosine content is found to be significantly increased, reducing tumor residue.

[0036] Improve the prognosis of lung cancer patients: Through early intervention and precise treatment, the occurrence of drug resistance is delayed or prevented, the progression-free survival and overall survival of patients are prolonged, and the quality of life of patients is improved, bringing better treatment outcomes and survival hope to lung adenocarcinoma patients.

[0037] Promote the development of precise treatment of lung cancer: The clinical application of the present application will provide new strategies for the field of precise treatment of lung cancer, help to further understand the mechanism of drug resistance of lung adenocarcinoma to EGFR-TKI drugs, and provide theoretical basis and practical reference for the further development of new anti-tumor drugs and treatment strategies. The kit of the present application can be used to judge whether the lung adenocarcinoma patient is suitable for adopting the EGFR-TKI drug treatment plan, and by synchronously evaluating the malignant degree and EGFR-TKI sensitivity of lung adenocarcinoma, it has clinical guiding significance for optimizing treatment decisions (such as targeted combination of EGFR-TKI induction therapy) and improving patient prognosis.

[0038] As known to those skilled in the art, the preparation for diagnosing the drug resistance of lung cancer patients to EGFR-TKI is usually provided in the form of a kit. It is easily understood that, in addition to the common biological reagents and chemical reagents for detecting the content of inosine, the kit of the present application can also include at least one of the following articles: a carrying tool, the space of which is divided into a defined space that can accommodate one or more containers, 96-well plates or strips, such as kits, medicine bottles, test tubes and the like, each of which contains a separate component for the method of the present application; an instruction, which can be written on the bottle, test tube and the like, or on a separate piece of paper, or on the outside or inside of the container, such as a paper with an operation demonstration video APP download window such as a two-dimensional code, and the instruction can also be in the form of tangible or intangible multimedia, such as a U disk, a network disk and the like.

[0039] The kit of the present application establishes the cut-off value of 1.8 for the ratio of inosine content / baseline level as a mathematical model for evaluating the drug resistance of lung cancer to EGFR-TKI, and when the expression level of inosine in the tumor tissue of a lung adenocarcinoma patient is higher than the predetermined standard, it indicates that the patient's tumor has drug resistance to EGFR-TKI drug treatment, and it is suitable to select other treatment options at the same time as the conventional EGFR-TKI targeted treatment, or to change the treatment options in time to avoid delaying the condition. When the ratio of inosine content / baseline level reaches 1.8 or more, the increase in inosine content indicates that the patient is drug resistant, and the treatment options need to be adjusted.

[0040] In an alternative embodiment, the kit of the present application also includes an inosine detection kit of the prior art, such as the Inosine Quantification Assay Kit Fluorometric (ab126286) of the American abcam company. This is a kit for converting inosine into hypoxanthine, which reacts with a substrate mixture and PicoProbe to produce fluorescence (Ex / Em = 535 / 587 nm) under the action of a conversion enzyme and a chromogenic enzyme, and the detection limit of this detection method is about 100 pmol inosine / well.

[0041] The detection means of inosine content is not limited to the above-mentioned fluorescence or luminescence reaction involving hypoxanthine, the decomposition product of inosine, but also can utilize the fluorescence or luminescence reaction involving inosine itself.

[0042] If conditions permit, liquid chromatography-mass spectrometry such as liquid chromatography-tandem mass spectrometry (LC-MS / MS) can also be used to detect the content of inosine, in which the mobile phase includes aqueous phase A, i.e. an aqueous solution, and organic phase B, i.e. an organic solution, and the biological sample pretreatment reagent can be determined by simple experiments.

[0043] Further, based on the antigen-antibody affinity principle, an inosine antibody can be used to detect the inosine content in a biological sample. The inosine antibody can be prepared by traditional immunization methods, for example, immunizing mice or rabbits with inosine as an antigen, and extracting and separating inosine antibodies from mouse / rabbit ascites or serum; or further fusing the spleen cells of the immunized mice with myeloma cells such as Sp2 / 0 myeloma cells to prepare hybridoma cells, and then producing monoclonal antibodies.

[0044] Correspondingly, the kit for detecting inosine by using an inosine antibody can be in the form of a latex immunoturbidimetry kit, a magnetic microparticle chemiluminescence kit, a radioimmunoassay kit, or an immunochromatographic test strip.

[0045] We verified the feasibility of the scheme of the present application on the cell level and in a mouse model. First, lung adenocarcinoma cells were treated with osimertinib for a long time to construct three lung adenocarcinoma cell lines PC9 / OR, H1975 / OR, and HCC827 / OR resistant to EGFR-TKI drugs. By detecting the metabolome of the EGFR-TKI-resistant lung adenocarcinoma cell lines, it was found that the inosine content in the drug-resistant cell lines was significantly higher than that in the sensitive cell lines. Subsequently, by exogenously supplementing inosine, it was found that excessive inosine could induce lung adenocarcinoma cells to be resistant to EGFR-TKI drugs. Mouse model experiments also showed that there was an intrinsic relationship between inosine upregulation and lung adenocarcinoma cell resistance to EGFR-TKI drugs. The results suggest that the inosine content can be detected to predict the EGFR-TKI drug resistance of lung cancer patients.

[0046] The present application will be further described below in conjunction with specific examples. It should be understood that these examples are for illustrative purposes only and are not limiting to the present application.

[0047] Examples

[0048] In this paper, the addition amount, content and concentration of various substances are involved. Unless otherwise specified, the percentage content refers to the mass percentage content.

[0049] The human lung adenocarcinoma cell lines PC-9 and H1975 were provided by Zhongshan Hospital Affiliated to Fudan University. The culture conditions were as follows: the cells were cultured in DMEM (high glucose) medium (Jiangsu Kaikai Biotechnology Co., Ltd.) containing 10% FBS and 1% double antibody in a 37 ℃, 5% CO2 incubator (Thermo Fisher Scientific, USA).

[0050] The inosine detection kit Fluorometric (ab126286) was provided by Aibio (Shanghai) Trade Co., Ltd. and was operated according to the instructions.

[0051] The mice used in the lung adenocarcinoma tumor xenograft mouse model were purchased from Shanghai Jiesijie Experimental Animal Co., Ltd. The SPF female BALB / c nude mice were 4-6 weeks old and weighed 20-25 g.

[0052] All blood samples were collected from lung adenocarcinoma patients receiving EGFR-TKI treatment in Zhongshan Hospital Affiliated to Fudan University.

[0053] Unless otherwise defined, the technical terms used in the following examples have the same meanings as commonly understood by those skilled in the art to which the present application belongs. The experimental reagents used in the following examples, unless otherwise specified, are conventional biochemical reagents; the experimental methods, unless otherwise specified, are conventional methods.

[0054] Example 1: Construction of EGFR-TKI-resistant lung adenocarcinoma cell lines

[0055] We constructed EGFR-TKI-resistant lung adenocarcinoma cell lines PC9 / OR and H1975 / OR by long-term induction treatment with osimertinib, as shown in Figure 1 As shown in A and B, the steps include:

[0056] PC-9 and H1975 cells were treated with osimertinib, starting from 2.5 nM and gradually increasing the dose to 200 nM, for 2 weeks. Then PC-9 and H1975 were maintained at a dose of 200 nM of osimertinib for 3 months to obtain osimertinib-resistant [OR] cell lines, denoted as PC-9OR and H1975-OR. Subsequently, the drug resistance effect of the cell lines to osimertinib was verified according to Figure 1 It can be found that, compared with wild-type cells, the drug-resistant cell lines are significantly resistant to EGFR-TKI drugs represented by osimertinib.

[0057] Example 2: Metabolomics analysis of EGFR-TKI-resistant lung adenocarcinoma cell lines

[0058] We performed metabolomics detection on cell lines PC9 / OR and H1975 / OR, as shown in Figure 2Specifically, 2 hours before cell collection, cells were cultured with fresh medium. When cells were collected, cells were washed with cold physiological saline, lysed with 80% methanol aqueous solution, and then scraped into a test tube quickly. Centrifugation was performed in a cooled centrifuge at 4°C, and the supernatant was transferred to a new test tube and evaporated to dryness with a SpeedVac concentrator (Thermo Scientific). Metabolites were reconstituted in LC-MS grade water with 0.03% formic acid, vortex-mixed and centrifuged to remove debris. 150 μL of supernatant was filtered using a 0.22 μm organic phase needle filter and then transferred to an LC injection vial, which was stored at -80°C for subsequent LC-MS analysis. Quality control samples (QCs) were prepared by mixing equal volumes of extracts from all samples. Metabolomics data analysis was completed by Shanghai Luming Biotechnology Co., Ltd. The analytical instrument used in this experiment was an LC-MS system consisting of an ACQUITY UPLC I-Class plus ultra-high performance liquid chromatograph and a QE plus high-resolution mass spectrometer, equipped with a heated electrospray ionization (ESI) source (Thermo Fisher Scientific, Waltham, MA, USA) for analyzing metabolite profiles in ESI positive and ESI negative modes. An ACQUITY UPLC HSS T3 column (1.8 μm, 2.1 x 100 mm) was used in both positive and negative modes. The gradient elution system consisted of (A) water containing 0.1% v / v formic acid and (B) acetonitrile containing 0.1% v / v formic acid, with the following gradient: 0.01 min, 5% B; 2 min, 5% B; 4 min, 30% B; 8 min, 50% B; 10 min, 80% B; 14 min, 100% B; 15 min, 100% B; 15.1 min, 5% and 16 min; 5% at a flow rate of 0.35 mL / min, and the column temperature was 45°C. All samples were kept at 10°C during analysis. The mass range was from 100 m / z to 1200 m / z. The first mass spectrometry scan resolution was 70,000, and the second mass spectrometry scan resolution was 17500, with collision energies of 10, 20 and 40 eV, respectively. The mass spectrometer worked as follows: spray voltage, 3800 V (+) and 3200 V (-); sheath gas flow, 35 arbitrary units; auxiliary gas flow, 8 arbitrary units; capillary temperature: 320°C; Aux gas heater temperature, 350°C; s lens RF level, 50.

[0059] After the detection was completed, differential metabolites were analyzed, and the ratio of each metabolite of the drug-resistant cells / control cells was calculated, and several metabolites that were most obviously increased in the drug-resistant cells were selected, such as Figure 2The results of the metabolomics analysis in A showed that the inosine content was significantly increased in the EGFR-TKI-resistant lung adenocarcinoma cell lines, by 20 and 8 times, respectively, compared with the wild-type lung adenocarcinoma cell line.

[0060] Figure 2 The results of the metabolomics analysis in A showed that the inosine content was significantly increased in the EGFR-TKI-resistant lung adenocarcinoma cell lines, by 20 and 8 times, respectively, compared with the wild-type lung adenocarcinoma cell line. Figure 2 The results of the metabolomics analysis in B showed that the inosine content gradually increased with the increase of the treatment time and the drug resistance of the cells after treatment with EGFR-TKI drugs. Figure 2 The results of the metabolomics analysis in C and D showed that the inosine content in TKI-resistant patients was increased by more than 1.8 times compared with TKI-sensitive patients. That is, the ratio of the inosine content in EGFR-TKI-resistant lung adenocarcinoma patients to the inosine content in wild-type lung adenocarcinoma patients (baseline concentration) was not less than 1.8.

[0061] Example 3: Investigation of the effect of inosine supplementation on EGFR-TKI drug resistance

[0062] This example investigates the effect of inosine on the EGFR-TKI drug resistance of lung adenocarcinoma cells by exogenous supplementation of inosine. The experimental steps are as follows. 5000 cells were inoculated in each well of a 96-well culture plate, and 24 hours later, different concentrations of inosine were added to the cell culture medium, together with different concentrations of osimertinib. After 120 hours of treatment, 10 μl of CCK-8 reagent (Shanghai Biyun Tian Company) was added to each well of fresh culture medium, and after 2 hours of incubation at 37°C, the absorbance was detected by an absorbance enzyme marker and the cell activity was calculated. Then the data was input into the Synergyfinder website for drug synergy model calculation, and the Bliss Score score calculated was used as the drug synergy score. An index <0 and a p value <0.05 indicate that there is antagonism between the two drugs.

[0063] In the clonogenic assay, 1000 cells were seeded in a 6-well plate, and then colonies were formed after 14 days of culture in the medium added with (PBS+DMSO; PBS+1.5nM osimertinib; 100µM inosine+DMSO; 100µM inosine+1.5nM osimertinib). The colonies were washed with PBS three times, fixed with 4% paraformaldehyde, and stained with crystal violet (Sigma-Aldrich, USA) for counting. Only colonies containing more than 50 cells were counted. Finally, the ratio of the number of colonies of the osimertinib group to the DMSO control group was calculated for the PBS and inosine groups.

[0064] In the process of nude mice tumorigenesis experiment, 4-6 weeks old female BALB / c nude mice were used. PC-9 cells were injected subcutaneously into the right groin of the mice. When the tumor volume reached 50 mm 3 , the mice were divided into four groups, (control group; oral inosine group; oral osimertinib group; oral inosine + osimertinib group). The mice were orally administered with drug solvents every day for 12 consecutive days; inosine (10 mg / kg); osimertinib (5 mg / kg), inosine + osimertinib (10 mg / kg; 5 mg / kg). The tumor volume was measured every three days (the calculation formula was long x wide 2 x 0.5). After five weeks of injection, the tumors were harvested and the tumor weight was measured.

[0065] See Figure 3 , the experimental results show that after exogenous supplementation of inosine, lung adenocarcinoma cells will develop resistance to EGFR-TKI drugs represented by osimertinib. The hypothesis was verified by cytotoxicity test, cell cloning experiment and lung adenocarcinoma tumor xenograft mouse model. And the effect of inosine on osimertinib resistance is closely related to its concentration. This result suggests that inosine can be used as a target / index for screening and developing new EGFR-TKI drugs, and the developed new EGFR-TKI drugs can inhibit the expression of inosine in lung cancer patients.

[0066] It should be understood that the above description is only a preferred embodiment of the present application, and is not intended to limit the present application. Any modification, equivalent replacement and improvement made within the spirit and principle of the present application shall be included in the protection scope of the present application.

Claims

1. Use of a reagent for detecting inosine content in a biological sample in the preparation of a preparation for evaluating the drug resistance of lung cancer to EGFR-TKI treatment.

2. Use according to claim 1, wherein The EGFR-TKI is a drug selected from the group consisting of gefitinib, erlotinib, icotinib, afatinib, dacomitinib and osimertinib.

3. The use according to claim 1, wherein The lung cancer is lung adenocarcinoma.

4. The use according to claim 1, wherein The biological sample is selected from the group consisting of whole blood, blood plasma, blood serum, lung tissue sampling, tissue fluid, saliva, buccal mucosa, nasopharyngeal secretion, body fluid and urine, and preferably the biological sample is blood.

5. The use according to claim 1, wherein the compound is ###0002### In the case where the lung cancer patient has not received inosine supplementation or administration, the inosine content detected in the lung cancer patient before EGFR-TKI treatment is the baseline level, and when the ratio of the inosine content detected during treatment to the baseline level is not less than 1.8, 1.9, 2.0, 2.1 or 2.2, it is determined that the lung cancer patient has developed resistance to EGFR-TKI; and when the ratio of the inosine content detected during treatment to the baseline level is less than 1.8, it is determined that the lung cancer patient has not developed resistance to EGFR-TKI.

6. The use according to claim 1, wherein The preparation is a kit for evaluating the drug resistance of a lung cancer patient to an EGFR-TKI drug by detecting the inosine content in a biological sample.

7. Use as claimed in claim 6, characterised in that, The detection of the inosine content in the biological sample is carried out by the following method (1): the inosine content is detected by using a fluorescence or luminescence reaction in which inosine or an inosine reaction product participates; accordingly, the reagent comprises reagents for catalyzing the inosine reaction and / or subsequent reactions of the inosine reaction product and an inosine standard, and the kit (1) comprises the above-mentioned reagents and reagents for pre-treatment of the biological sample.

8. Use as claimed in claim 6, characterised in that, The detection of the inosine content in the biological sample is carried out by the following method (2): the inosine content is detected by using an inosine antibody based on the antigen-antibody affinity principle, and the inosine antibody is a monoclonal antibody or a polyclonal antibody; accordingly, the reagent comprises an inosine antibody and an inosine standard, and the kit (2) comprises the above-mentioned reagents and reagents for pre-treatment of the biological sample.

9. Use as claimed in claim 6, characterised in that, The detection of the inosine content in the biological sample is carried out by the following method (3): the inosine content is detected by using liquid chromatography tandem triple quadrupole mass spectrometry (LC-MS / MS); accordingly, the reagent comprises a mobile phase used in liquid chromatography, such as an aqueous phase A (aqueous solution) and / or an organic phase B (organic solution), an inosine standard; and the kit (3) comprises the above-mentioned reagents and reagents for pre-treatment of the biological sample.

10. Use of inosine in the screening of EGFR-TKI drugs, wherein inosine acts as an inhibition marker for EGFR-TKI drugs.

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